In the present study, a series of 4-(4-substituted aryl) semicarbazones were synthesized from substituted anilines and subsequently evaluated for their anticonvulsant activities. The anticonvulsant activities were established by the anticonvulsant drug development (ADD) programme NIH, USA using experimental animal, adult male FCM mice (20–25 g) and adult Sprague-Dawley rats (100–150 g) and screened against electroshock seizure, subcutaneous metrazole and minimal neurotoxicity tests in mice. Compound 7 was found equipotent to carbamazepine in both MES and ScPTZ tests. This study has highlighted the importance of distal alkyl chain which influences the anticonvulsant activity. The antfunctions.et al.[2 group. In continuation, Pandeya et al.[The lipophilic aryl ring with chloro, bromo or nitro groups have been found to be essential for anticonvulsant activity. The distal aryl ring is also implicated at the binding site. The hydrogen bonding in semicarbazone series has been suggested by Dimmock et al.7 to be tya et al.9 suggestya et al.. In thismax, nm) and IR  spectra were consistent with the assigned structures recorded on Jasco Model 7800 and Jasco FT/IR-5300 instruments respectively and 1H NMR  spectra were recorded Jeol FX 90Q FT spectroscope using TMS as internal standard. Chemical shift are quoted in δ (ppm) units relative to TMS. Purity of the synthesized compounds were checked by TLC using silica gel G  and chloroform methanol (9:1) solvent system. 4-substituted semicarbazone derivatives were prepared in three steps as follows:Melting points of the synthesized compounds were taken by the open capillary method and are uncorrected. UV (EtOH-Band λ4-substituted aniline (0.1 mol) was dissolved in 10 ml of glacial acetic acid (GAA) and diluted to 100 ml with water. Equimolar quantity (0.1 mol) of sodium cyanate in 50 ml of warm water was added in previous solution with stirring. The reaction mixture was allowed to stand for 30 min, and then it was filtered, washed, dried and recrystallized from boiling water.To a solution of 4-substituted phenyl urea (0.1 mol) in 20 ml ethanol, an equimolar (0.1 mol) quantity of hydrazine hydrate was added. The reaction mixture was made alkaline by adding 4 g of NaOH, was refluxed for 1-2 h and cooled in ice. The product was filtered under suction and recrystallised from ethanol.A solution of p-phenyl semicarbazide (0.01 mol) and an equimolar (0.01 mol) quantity of appropriate carbonyl compound was refluxed for 30 min in the presence of glacial acetic acid (1-1.5 ml). The product obtained after cooling was filtered and recrystallised from 95% ethanol to give pure compounds . The chaf 0.56; UV  230, 337, 376; IR  3350 (Ar-NH), 1700(C=O), 1610 (C=N), 820 (Ar-H); 1H NMR  5.8 , 6.3-6.5 , 7.6-7.9 , 8.0 .1-(diPhenylmethylene)-4-(4-nitrophenyl)semicarbazone (1); m.p. 123-125°; Yield (%) 70; Rf 0.63; UV  231, 373, 379; IR  3365 (Ar-NH), 1635 (C=O), 1475 (Ar-NO2), 845 (Ar-H); 1H NMR  5.9 , 3.4-3.7 , 6.4-6.7 , 7.6-8.0.1--4-(4-nitrophenyl)semicarbazone (2); m.p. 145-147°; Yield (%) 65; Rf 0.59; UV  283, 372, 380; IR  3610 (Ar-NH), 1638(C=O), 1470(Ar-NO2), 850(Ar-H); 1H NMR  5.7 , 6.9-7.1 , 2.8-2.9 , 8.1 .1-Methylene-4-(4-nitrophenyl) semicarbazone (3); m.p. 230-235°; Yield (%) 60; Rf 0.48; UV  ;225, 376; IR  3370 (CONH), 3270 (Ar-NH), 1720 (C=O), 1610 (C=N), 1525 (Ar-NO2), 855(Ar-H); 1H NMR  6.0 , 4.01 , 3.76 , 3.75 , 8.1 .1--4-(4-nitrophenyl)semicarbazone (4); m.p. 125-130°; Yield (%) 92; Rf 0.76; UV  280, 236; IR  3430(CONH), 3320(Ar-NH), 1735(C=O), 1570(C=N), 1245(Ar-F), 830 (Ar-H); 1H NMR  3.48 , 3.95 , 6.0 , 7.23-7.33 , 7.25-7.59 , 10.09 .1-(4-Methoxybenzylidene)-4-(4-fluorophenyl) semicarbazone (5); m.p. 178-180°; Yield (%) 74; Rf 0.74; UV  328, 228; IR  3370 (sec. NH), 2815 (CH3-N), 1682 (C=O), 1560 (C=N), 1312 (C-N amine), 1035 (Ar-Cl), 845 (Ar-H); 1H NMR  3.42 , 3.92 , 7.85-8.05 , 8.83 , 7.23-7.26 , 8.06 .1-Benzylidene-4-(4-chlorophenyl) semicarbazone (6); m.p. 160-163°; Yield (%) 55; Rf 0.77; UV  228, 251, 374; IR  3420 , 3320 sec. NH, amide), 1735 (C=O), 1570 (C=N), 1245 (Ar-Cl), 830 (Ar-H); 1H NMR  2.29 , 5.8 , 7.2-7.3 , 8.8 .1-(Butan-2-ylidene)-4-(4-chlorophenyl) semicarbazone (7); m.p. 154-157°; Yield (%) 65; Rf 0.49; UV  251, 283, 372, 380; IR ; 3372 (Ar-NH), 1630(C=O), 1575(C=N), 1472 CH2 def.), (840 (Ar-H); 1H NMR  6.1 , 7.35-7.9 , 7.12-7.56 , 8.88 .4-(4-Bromophenyl)-1-(9-oxoanthracen-10(9H)-ylidene) semicarbazone (8); m.p. 207-211°; Yield (%) 30; Rf 0.52; UV  251, 271, 324; IR  3365(Ar-NH), 1572 (C=N), 1478 (CH2 def.), 800 (Ar-H); 1H NMR  5.9 , 6.4-6.6 , 8.80 .4-(4-Bromophenyl)-1-(diphenylmethylene) semicarbazone (9); m.p. 150-156°; Yield (%) 37; Rf0.53; UV  260, 266, 272; IR  3372 (Ar-NH), 1635 (C=O), 1572(C=N), 820(Ar-H); 1H NMR  6.0 , 6.5-6.9 , 8.1 , 9.8 .4-(4-Bromophenyl)-1-semicarbazones (10); m.p. 147-150°; Yield (%) 35; RThe anticonvulsant activities were established by the anticonvulsant drug development (ADD) programme. Adult mInitially all the compounds dissolved in polyethylene glycol were administered intraperitoneally (0.01 ml /g body weight in mice and 0.004 ml /g body weight in rats) at a dose of 30, 100, and 300 mg/kg to all the animals respectively. Compounds were screened for anticonvulsant activity by maximal electroshock seizure (MES), subcutaneous metrazole (ScMet) and Minimal neurotoxicity (TOX) test in mice.MES or maximal seizure pattern test, in which an electrical stimulus of 0.2 s in duration (50 mA in mice and 150 mA in rat at 60Hz) was delivered via corneal electrodes primed with an electrolyte solution containing an anesthetic agent. Mice are tested at 30 min and 4 h following doses of 30, 100 and 300 mg/kg of test compound. Abolition of the hindlimb tonic extensor component indicates the test compound’s ability to inhibit MES-induced seizure spread.The subcutaneous pentylenetetrazol (metrazol) seizure test (scPTZ) was performed using a dose of pentylenetetrazol (70 mg/kg in Sprague Dawley rats) to produce clonic seizures lasting for a period of at least five seconds in 97 per cent (CD97) of animals tested. At the anticipated time of testing the convulsant is administered subcutaneously. The test compound is administered intraperitoneally in mice and orally in rats. Animals are observed over a 30 min period. Absence of clonic spasms in the observed time period indicates a compound’s ability to abolish the effect of pentylenetetrazol on seizure threshold.Minimal neurotoxicity (TOX) was evaluated in rats by examining them for behavioral toxicity using the positional sense test and a gait and stance test. The mice were trained to stand on an accelerating rotarod of diameter 3.2 cm, which rotated at 10 rev/min. Animals were given intraperitoneal injections of the test compounds in the doses of 30, 100 and 300 mg/ kg body weight. Neurotoxicity was indicated by the inability of the animal to maintain equilibrium on the rod for at least one minute in each of the three trials.*transition. K-band of substituted p-nitroaniline was very strong λmax 375–380 nm. The halo substituted derivatives absorbed at 265 nm. The formation of the substituted hydrazones and semicarbazones were confirmed with the formation of C=N band in all spectra from 1500–1620 cm−1. The CONH stretching for amides was observed in the 4-substituted aryl/alkyl semicarbazones at about 3180-3320 cm−1. The chemical shift for aromatic amino group (Ar-NH) was found at 5.82 to 5.89 ppm and 8.86-8.91 ppm for -CONH group present in semicarbazone. The signals for aromatic protons were observed at 7.2-7.6 ppm.The UV spectrum shows presence of chromophores in the 4-substituted aryl/alkyl semicarbazones. The benzene ring absorption is in the range of 200-205 nm pertaining to K-band. The π- πThe series of 4-subsituted phenyl semicarbazones were found to possess significant anticonvulsant activities . The comGenerally MES active compounds are considered to exhibit activity in grandmal epilepsy, contain larger hydrophobic center (phenytoin) and ScMe


The structures of all compounds were unambiguously elucidated by spectroscopic analyses, as well as by comparison with the literature data. Compounds 1–2 are characterized with triazapyrene lactam skeleton, whereas compounds 4–5 share an imidazole-fused aaptamine moiety. These compounds were evaluated in antifungal and anti-HIV-1 assays. Compounds 3, 7, and 8 showed antifungal activity against six fungi, with MIC values in the range of 4 to 64 μg/mL. Compounds 7–8 exhibited anti-HIV-1 activity, with inhibitory rates of 88.0% and 72.3%, respectively, at a concentration of 10 μM.Five new alkaloids of aaptamine family, compounds ( H-benzo[de]-1,6-naphthyridine skeleton + peak at m/z 330.1220, suggesting a molecular formula of C18H17N3O2 and 12 degrees of unsaturation. The IR spectrum showed bands due to lactam carbonyl functions (1663 cm−1) +) in combination with extensive NMR analyses. The NMR data for 2 indicated an overall structure similar to 1, with the only difference at the side chain substituted to C-11 /H3-15 (δH 0.91) and H-13 (δH 3.51)/H3-16 (δH 1.31), as well as the HMBC correlations from H2-14 and H3-15 to C-13 (δC 37.7), and from H-13, H2-14, and H3-16 to C-11 (δC 157.8).Compound  to C-11 . The 1H 3 was obtained as a yellow solid. The molecular formula was determined as C15H16N2O3 by an HRESIMS ion peak at m/z 295.1058 [M + Na]+. The 1H NMR spectrum revealed the resonances for two sets of coupled protons at δH 8.93  and 7.87 , as well as 8.91  and 7.14 , one singlet at δH 6.14, and two methoxy groups at δH 3.96 and 3.22, indicating the characteristic patterns for aaptamine congener naphthyridine skeleton pyridoquinoline (6) naphthyridinoquinoxalin-3-one (1): yellow solid; UV (MeOH) (log ε) λmax 208 (4.31), 224 (4.36), 273 (3.96), 282 (4.22), 412 (4.18), 434 (4.12); IR (KBr) νmax 3404, 2955, 2926, 2856, 1730, 1663, 1638, 1597, 1527, 1472, 1387, 1320, 1256, 1174, 1098, 1010, 849 cm−1; 1H and 13C NMR data, see m/z 330.1220 [M + Na]+ .2-Isobutyl-11-methoxy-3Sec-butyl)-11-methoxy-3H-naphthyridinoquinoxalin-3-one (2): yellow solid; c 0.10, MeOH); UV (MeOH) (log ε) λmax 204 (3.82), 224 (3.78), 273 (3.56), 282 (3.59), 391 (3.35), 410 (3.53), 432 (3.49); IR (KBr) νmax 3371, 2960, 2927, 2855, 1728, 1665, 1637, 1597, 1527, 1469, 1383, 1320, 1257, 1176, 1100, 1032, 846 cm−1; 1H and 13C NMR data, see m/z 308.1400 [M + H]+ .2-: yellow solid; c 0.15, MeOH); UV (MeOH) (log ε) λmax 202 (3.59), 229 (3.67), 352 (3.23); IR (KBr) νmax 3064, 2928, 2855, 1716, 1673, 1641, 1570, 1490, 1458, 1413, 1371, 1349, 1305, 1276, 1223, 1201, 1176, 1075, 694, 917, 869, 656, 575 cm−1; 1H and 13C NMR data, see m/z 295.1058 [M + Na]+ . 9-Ethoxy-8,9-dimethoxy-9Sec-butyl)-10-methoxyimidazopyridoquinolone (4): yellow solid; c 0.12, MeOH); UV (MeOH) (log ε) λmax 210 (4.00), 225 (3.98), 239 (4.03), 272 (3.71), 360 (3.55); IR (KBr) νmax 3315, 3065, 2960, 2926, 2854, 1731, 1688, 1607, 1573, 1515, 1465, 1419, 1366, 1307, 1279, 1167, 1020, 840, 799 cm−1; 1H and 13C NMR data, see m/z 280.1452 [M + H]+ .2-: yellow solid; UV (MeOH) (log ε) λmax 211 (4.10), 225 (4.09), 239 (4.14), 272 (3.82), 361 (3.66); IR (KBr) νmax 3342, 3050, 2957, 2925, 2869, 2853, 1729, 1688, 1607, 1572, 1542, 1515, 1465, 1420, 1348, 1324, 1307, 1279, 1194, 1167, 1020, 840 cm−1; 1H and 13C NMR data, see m/z 280.1448 [M + H]+ .2-Isobutyl-10-methoxyimidazo[4,5,1-3–8 were determined against six indicators , following the National Center for Clinical Laboratory Standards (NCCLS) methods [3–8 prepared in RPMI 1640 were next inoculated with the microorganisms and incubated 24 h for C. parapsilosis, and C. glabrata at 35 °C, 46 h for C. albicans at 35 °C, 72 h for C. neoformans at 35 °C, and 4–7 days for T. rubrum and M. gypseum at 30 °C. The MIC values were evaluated in triplicate for each compound. Concentration (MIC) values of compounds  methods ,18. Fluc4 cells per well one day prior to infection. Compounds with the concentrations of 10 μM were incubated with target cells for 15 min prior to adding VSVG/HIV-1. The same amount of solvent alone was used as blank control. After postinfection for 48 h, cells were lysed in 50 μL Cell Lysis Reagent . Luciferase activity of the cell lysate was measured by a FB15 luminometer .The antiviral activity against HIV-1 was evaluated by using a cell-based VSVG/HIV-1 pseudotyping system as described ,20. For A. aaptos from the South China Sea led to the isolation and structure elucidation of five new aaptamine derivatives (1–5), together with three related known aaptamines (6–8), demonstrating an excellent example of chemical diversity. Structurally, compounds 1–2 possesses an uncommon triazapyrene lactam fused to aaptamine moiety, compounds 4–5 share an imidazole-fused aaptamine skeleton. Unfortunately, due to the lack of compounds 1 and 2, the bioactivity of 1–2 could not be evaluated. These compounds displayed different levels of in vitro antifungal and anti-HIV-1 activities. Especially, according to our research, compounds 7 and 8 exhibited better antifungal and anti-HIV-1 activities than the rest compounds.Chemical investigation of the sponge


A new group of 4-methyl) 7,8,9,10-tetrahydro-2-(4-methylsulfonylphenyl)-benzo[h]quinoline (9d)was identified as the most potent andselective COX-2inhibitor as well as the most cytotoxic agent against MCF-7 cells. Breast cancer is one of the leading causes of cancer-related mortality among women worldwide . In mostpara position C-2 phenyl ring ..para pospara position C-2 phenyl ring) with the imidazolering, the main pharmacophore for anti-aromatase activityn was used to acquire 1H NMR spectra with TMS as internal standard. Chloroform-D and DMSO-d6 were used as solvents. Coupling constant (J) values are estimated in hertz (Hz) and spin multiples are given as s (singlet), d (double), t (triplet), q (quartet), m (multiplet), and br (broad). The mass spectral measurements were performed on an 6410Agilent LCMS triple quadrupole mass spectrometer(LCMS) with an electrospray ionization (ESI) interface. Microanalyses, determined for C and H, were within ± 0.4% of theoretical values.General procedure for preparation of 2-(4-(methylthio)phenyl)-7,8-substituted-quinoline-4-carboxylic acid (4a-4e)These compounds were synthesized according to our pervious methods, 11.General procedure for preparation of 2-(4-methylthio-phenyl)-7,8-substituted-quinoline-4-yl)methanol (7a-7e)4  was suspended in dry THF (20 ml) under a nitrogen atmosphere. Under vigorous stirring a solution of appropriate acid(4a-4e) (5.67 mmol) in dry THF was added dropwise to keep the reaction mixture slightly boiling. After stirring for 2 h at room temperature, the suspension was carefully hydrolyzed with NaOH solution (10%) till no more hydrogen was produced. The solid was filtered off and washed thoroughly with chloroform (yield: 62-79 %).LiAlH 0.45 g,  mmol was2-(4-Methylthio-phenyl)-8-phenylquinoline-4-yl)methanol (7a)-1), 3745-2371(OH);LCMS (ESI): 380.6 (M+23) +100.Yield: 77%; yellow crystalline powder; mp=131-133˚C; IR (KBr): ν (cm2-(4-Methylthio-phenyl)-7,8-dimethylquinoline-4-yl)methanol (7b)-1), 3406(OH);Yield: 62%; pale yellow crystalline powder; mp=140-141˚C; IR (KBr): ν (cm +100.LCMS (ESI): 310.2 (M+1)2-(4-Methylthio-phenyl)-benzo[h]quinoline-4-yl)methanol (7c)-1), 3290(OH);Yield: 68%; yellow crystalline powder; mp=111-113˚C; IR (KBr): ν (cm +100.LCMS (ESI): 332.9 (M+1)7,8,9,10-Tetrahydro-2-(4-methylthio-phenyl)-benzo[h]quinoline-4-yl)methanol (7d)ºC; IR (KBr): ν (cm-1), 3234(OH);Yield: 79%; pale yellow crystalline powder; mp=139-140 +100.LCMS (ESI): 336.9 (M+1)2-(4-Methylthio)phenyl- quinoline-4-yl)methanol (7e)ºC; IR (KBr): ν (cm-1), 3224(OH);Yield: 63%; yellow crystalline powder; mp=120-122 +100.LCMS (ESI): 282.1 (M+1)General procedure for preparation of 2-(4-methylsulfonyl-phenyl)-7,8-substituted-quinoline-4-yl)methanol (8a-8e)One gram of 2-(4-methylthio)phenyl-7,8-substituted-quinoline-4-yl)methanol (7a-7e)was dissolved in 10 ml THF and 5 g oxone in THF/water was added. The mixture was stirred at room temperature for 3-5 h, after evaporation of THF, the residue was extracted with chloroform and dried with sodium sulfate and then evaporated, the pro2-(4-Methylsulfonyl-phenyl)-8-phenylquinoline-4-yl)methanol (8a)ºC; IR (KBr): ν (cm-1), 3442(OH) 1312, 1157(SO2);LCMS (ESI): 390.8 (M+1) +100.Yield: 48%; yellow crystalline powder; mp=178-1792-(4-Methylsulfonyl-phenyl)-7,8-dimethylquinoline-4-yl)methanol (8b)-1), 3241(OH), 1303, 1149(SO2);LCMS (ESI): 342.1(M+1) +100.Yield42%; orange crystalline powder; mp=148-150ºC; IR (KBr): ν (cm2-(4-Methylsulfonyl-phenyl)-benzo[h]quinoline-4-yl)methanol (8c)-1), 3485 (OH), 1296, 1145(SO2);1HNMR (DMSO-d6 ): δ(ppm) 3.28, 5.18 , 5.71 , 7.74-7.80 , 7.96-7.80, 7.99  8.11 , 8.37 , 8.63 , 9.4 ; LCMS(ESI): 364.1 (M+1)+100.Yield: 65%; yellow crystalline powder; mp=210-211ºC; IR (KBr): ν -benzo[h]quinoline-4-yl)methanol (8d)-1), 3365 (OH), 1309, 1159(SO2);LCMS (ESI): 368.9 (M+1) +100.Yield: 57%; yellow crystalline powder; mp=162-163ºC; IR (KBr): ν (cm2-(4-Methylsulfonyl)phenyl-quinoline-4-yl)methanol (8e)-1), 3340(OH), 1301, 1151(SO2);LCMS (ESI): 314.9 (M+1) +100.Yield: 75%; yellow crystalline powder; mp=164-166ºC IR (KBr): ν methyl)-2-(4-methylsulfonyl-phenyl)-7,8-substituted-quinoline2SO4, and evaporated under reduced pressure  in 15 ml NMP (N-methyl-2-pyrrolidone), CDI   was added. Then the solution was heated to reflux for 20 h at 170 ºC. After cooling to ambient temperature, it was diluted with water (50 ml) and extracted with ethyl acetate. The combined organic phases was washed with brine and water, dried over Napressure . Then th4-((1H-Imidazol-1-yl)methyl)-2-(4-methylsulfonyl phenyl)-8-phenyl-quinoline (9a)-1), 1310, 1150 (SO2);1HNMR (CDCl3 ):δ(ppm) 3.03  5.73 , 7.04 , 7.23, 7.34, 7.45 , 7.52 , 7.69-7.76, 7.86 ,7.92 , 7.95 , 8.14 ; LCMS(ESI): 440.6 (M+1)+100.Yield: 25%; cream crystalline powder; mp=196-198ºC;IR (KBr): ν (cm4-((1H-Imidazol-1-yl)methyl)-2-(4- methylsulfonyl phenyl)-7,8-dimethyl-quinoline (9b)-1), 1301, 1150 (SO2); 1HNMR (CDCl3 ): δ(ppm) 2.58, 2.91, 3.12, 5.76 , 7.05 , 7.26, 7.39, 7.50 , 7.73 , 8.01 , 8.08 , 8.26 ; LCMS(ESI): 392.1 (M+1)+100.Yield: 61%; yellow crystalline powder; mp=218-219ºC; IR (KBr): ν (cm4-((1H-Imidazol-1-yl)methyl)-2-(4- methylsulfonyl phenyl)-benzo[h]-quinoline (9c)-1) 1303, 1148 (SO2);1HNMR (DMSO-d6 ): δ(ppm) 3.26, 5.90 , 7.01 , 7.36, 7.79 , 7.93, 8.03-8.05, 8.10 , 8.14 , 8.50 , 9.4 ; LCMS(ESI): 414.6 (M+1)+100.Yield: 25%; cream crystalline powder; mp=216-217ºC; IR (KBr): ν (cm4-((1H-Imidazol-1-yl)methyl)-7,8,9,10-tetrahydro-2-(4-methylsulfonylphenyl)-benzo[h]-quinoline (9d)-1) 1318, 1163(SO2);1HNMR (CDCl3 ): δ(ppm) 1.9-2.01, 2.96 , 3.08 , 3.46 ,5.67 , 7.0 , 7.2, 7.29 , 7.35 , 7.66 , 7.70 , 8.03 , 8.29 ; LCMS(ESI): 418.7 (M+1)+100.Yield: 35%; cream crystalline powder; mp=247-248ºC; IR (KBr): ν (cm4-((1H-Imidazol-1-yl)methyl)-2-(4- methylsulfonyl phenyl)-quinoline (9e)-1) 1300, 1150(SO2);1HNMR (CDCl3 ): δ (ppm) 3.1, 5.76 , 7.07 , 7.26, 7.29, 7.69-7.72 , 7.87 , 7.98 , 8.09 , 8.26 , 8.3 ; LCMS(ESI): 364.8 (M+1)+100.Yield: 15%; cream crystalline powder; mp=189-190ºC; IR (KBr): ν (cmMolecular modeling (docking) studiesDocking studies were performed using Autodock software Version 3.0. The coordinates of the X-ray crystal structure of the selective COX-2 inhibitor SC-558 bound to the murine COX-2 enzyme was obtained from the RCSB Protein Data Bank (1cx2) and hydrogens were added. The ligand molecules were constructed using the Builder module and were energy minimized for 1000 iterations reaching a convergence of 0.01 kcal/mol.The energy minimized ligands were superimposed on SC-558 in the PDB file 1cx2 after which SC-558 was deleted. The purpose of docking is to search for favorable binding configuration between the small flexible ligands and the rigid protein. Protein residues with atoms greater than 7.5 A from the docking box were removed for efficiency. These docked structures were very similar to the minimized structures obtained initially. The quality of the docked structures was evaluated by measuring the intermolecular energy of the ligand–enzyme assembly , 16.In-vitro cyclooxygenase (COX) inhibition assaysThe assay was performed using an enzyme chemiluminescentkit  according to our previously reportedmethodThe Cayma(ovine) inhibitor screening assay utilizes the heme-catalyzedhydroperoxidase activity of ovine cyclooxygenases to generateluminescence in the presence of a cyclic naphthalene hydrazideand the substrate arachidonic acid. Arachidonate-induced luminescencewas shown to be an index of real-time catalytic activity anddemonstrated the turnover inactivation of the enzyme. Inhibitionin-vitro and whole cell methods.of COX activity, measured by luminescence, by a variety of selectiveand non-selective nhibitors showed potencies similar to those observedwith other Cytotoxicity assayCell line and culture conditions-1 of penicillin and 100 ng Ml-1 of streptomycin  at 37 C in 5% CO2 incubator. All reagent and chemicals used in this experiment were of cell culture or molecular biology grade, purchased from different international sources.The human breast cancer T47D and MCF-7 cell lines were obtained from Pasteur Institute Cell Bank of IRAN . Cells were maintained in RPMI-1640 culture medium supplemented with 10% fetal bovine serum  and 100 U MlGeneral procedure-1) were added to each well and further incubated at 37ºC for 3h. At the end of incubation, formazan crystals were dissolved in 100 µL of DMSO and plates were read in a plate reader  at 540 nm. This experiment was performed in triplicate determination each time  based assay was performed by seeding 5000 cells (T47D and MCF-7) per 180 µL RPMI complete culture medium in each well of 96-well culture plates. The day after seeding, culture medium was changed with medium containing standard anti-tumor drug Doxorubicin as well as different concentrations of newly synthesized compounds and RPMI control (no drug). Cells were then incubated at 37ºC in 5% Co2 incubator for 48h and 72h. Then 25 µL of MTT solution  acquired by determination of the in-vitro ability of the title compoundsto inhibit the COX-1 and COX-2 isozymes showed thatthe COX inhibition was sensitive to the lipophilic nature of substituents.As shown in 50 = 0.063 µM; SI =547.6) thatwasas potent as the reference drug celecoxib and more selective COX-2 inhibitor than celecoxib .SAR data also showed that the COX inhibition was sensitive to the nature of substituents on the C-4 quinoline ring. All of the 4-imidazolylmethylquinoline derivatives were less potent but more selective COX-2 inhibitors than their corresponding 4-carboxyl derivatives. Our molecular modeling studies showed that the carboxyl group can interact with Arg120 in COX-2, so replacement of carboxyl group withimidazolylmethyl may decrease COX-2 inhibitory activity. In addition, carboxyl group can also interact with Arg120 as a key amino acid in COX-1 enzyme, so 4-imidazolylmethylquinoline derivatives have less affinity to bind to COX-1 than the 4-carboxyl derivatives and as a consequence are more selective COX-2 inhibitors.SAR data . These observations together with experimental results provide a good explanation for design of potent and selective COX-2 inhibitors possessing 4-((1H-imidazol-1-yl)methyl)-2-(4-methylsulfonylphenyl)quinoline framework.The binding interactions of the three most potent and selective COX-2 inhibitor compound  within the COX-2 binding site were investigated. They all were docked well in the COX-2 binding site. The most stable enzyme-ligand complex of whichpossessing a MeSOing site  shows th50< 5μM). The IC50 of 9a was less than 10 μM and the IC50 of9c was 10 μM but the IC50of 9b and 9e were more than 10μMwhichwere the least potent compounds. The most potent compound against T47D cells was9a which exhibited 33.8% of survival (after 2 days exposure) (IC50 ≤ 25μM). Compounds 9c and 9e were the least potent compounds with IC50 = 50μM. The calculated IC50 values of all tested compounds after two days exposure showed that the order of the cytotoxicity against MCF-7 cells were 9d>9a >9c >9b = 9e and in T47D cells were 9a >9d>9e>9b = 9c. These data showed that the increase of lipophilic properties of substituents on the C-7 and C-8 quinoline ring increased their cytotoxicity on MCF-7. This may be due to their ability to penetrate the cell membrane or their ability to inhibit or suppress some factors and enzymes such as COX-2.As our result showed that the order of the cytotoxicity of quinolines 9a-9eagainst MCF-7 cells were the same as that of their COX-2 inhibitory so one of their cytotoxic mechanism of these compoundsmay be through their COX-2 inhibitory activity. In addition, thesequinolines9a-9e were more cytotoxic against MCF-7 cells in comparison with those of T47D. So this may be due to their different mechanism of action in these two breast cancer cellssuch as more anti-aromatase activity in MCF-7 cells in comparison with T47D cells which express aromatase mRNA less than MCF-7 cells.The cytotoxicity of quinolines9a-e against human breast cancer MCF-7 and T47D cells by MTT assayafter 2 and 3 days of exposure was also evaluated. After some initial evaluations concentrations of 10 and 25 μM of quinolines were used for evaluation and comparison of cytotoxicity of these compounds with doxorubicin at concentration of 250 nM against MCF-7 cells and concentrations of 25 and 50 μM of quinolines were used for evaluation and comparison of cytotoxicity of these compounds with doxorubicinat concentration of 100 nM against T47D cells. Results for each compound as the percentage of growth of the treated cells in comparison to untreated cells are shown in


Their structures were determined on the basis of spectroscopic data , HR-MS and chemical methods. The absolute configuration of briaviolide A (1) was determined by X-ray crystallographic analysis. Compounds 5, 9 and derivative 11 showed moderate inhibitory activities on superoxide-anion generation and elastase release by human neutrophils in response to N-formyl-methionyl-leucyl-phenylalanine/Cytochalasin B (fMLP/CB).Ten new briarane diterpenoids, briaviolides A–J ( Briareum asbestinum in 1977  + and [M + Na + 2]+ peaks for pseudo-molecular ions at m/z 521 and 523 (3:1) and HRESIMS at m/z 521.1554 [M + Na]+, indicating a molecular formula C24H31O9Cl, which contains one chlorine atom and accounts for nine degrees of unsaturation. The IR spectrum of 1 showed absorption frequencies at 3524, 1767 and 1737 cm−1, indicating the presence of hydroxyl, γ-lactone and carbonyl ester functionalities, respectively. The 1H- and 13C-NMR spectroscopic data (H 2.09 (δC 21.1) and 2.17 (δC 22.1), with corresponding carbonyl signals at δC 170.3, 170.2, respectively. The carbonyls showed respective HMBC connectivity . The carbonyl signal of a γ-lactone (δC 174.9) was connected with a secondary methyl doublet at δH 1.17 (δC 6.3) by the HMBC (H-19/C-18) and COSY (H-19/H-17) correlations. The connection between C-17 and C-8 (δC 83.9) was elucidated by the HMBC correlation from H-19 to C-8. The hydroxyl proton (δH 3.52) of C-8 was correlated with the adjacent lactonide carbon C-7 and C-9 by virtue of the HMBC correlations from 8-OH to C-7 and C-9. These correlations along with the H-6/H-7 (COSY) correlation fixed the position of the lactone ring. The signals of an exocyclic double bond were observed at δH 6.24 (br s) and 5.95  (δC 118.7) and were conjugated with an endocyclic double bond at δH 5.63 , 5.90  by related HMBC and COSY correlations. The correlations of H-6/H-7 (COSY) and H-16/C-6 (HMBC) thus connected the conjugated double bond with the γ-lactone ring. The signals of a pair of methine protons at δH 3.23 (H-13) and δH 2.99 (H-14) were assigned for an epoxide ring. This moiety was neighbored on a quaternary carbon (δC 40.6) and a hydroxylated carbon  by the HMBC correlation of H-12/C-13 and H-14/C-1. The COSY correlations of H-12/H-11/H-20 and H-9/H-10 indicated the presence of two proton sequences. These two sequences were connected by the HMBC correlations of H-20/C-10 and H-9/C-11. By deducing the unsaturation of two acetates, conjugated diene, a γ-lactone ring and the epoxide ring, the remaining two degrees of unsaturation strongly suggested that Compound 1 possesses two additional rings belong to briarane-type diterpenoids.Briaviolide A  diagram stereo-drawing in 1 was similar to that of briaexcavatolide I + in the HRESIMS suggested that briaviolide D (4) had the molecular formula of C24H31O10Cl with nine degrees of unsaturation. The presence of a chlorine atom was supported by an isotope peak at m/z 539 in the LRESI spectrum, having one third of the intensity relative to m/z 537. The NMR spectroscopic data . This finding was confirmed from 13C-NMR data and HMBC correlations between H-10/C-11, H-12/C-11 and Me-20/C-11. The NOESY correlations of H-10/Me-11, H-12 required that the methyl group at C-11 be α-oriented. The configurations of other chiral centers are similar to Compound 1 as ascertained by NOESY experiments (4) was established as -6-chloro-13,14-epoxy-2,9-diacetoxy-8,11,12-trihydroxybriaran-3(4),5(16)-dien-18,7-olide.A pic data  and IR seriments . Thus, t5 agreed with the molecular formula C29H39O11Cl containing a chlorine atom and 10 degrees of unsaturation. The IR bands at 3429, 1779 and 1738 cm−1 suggested the presence of hydroxyl group, ester groups and a γ-lactone moiety. A detailed inspection of 1H and 13C-NMR spectroscopic data ;  in Compound 5. The configurations of 5 was deduced by the NOESY analysis  was established as -6-chloro-3(4),13(14),-diepoxy-2,9-diacetoxy-12-isovaleryloxy-8-hydroxybriaran-5(16)-dien-18,7-olide.The HRESIMS data of pic data  of 5 indianolide . Howeveranalysis  and was 6) showed a pseudo-molecular ion peak at m/z 593.2125 [M + Na]+, consistent with a molecular formula of C28H39O10Cl (Δ = 10). Cross comparison of 1H- and 13C-NMR spectroscopic data of 6  in 6. Signals of an acetyl group  located at C-9 (δC 68.7) and one hexanoate group  located at C-12 (δC 69.7) were determined by their HMBC correlations  had the molecular formula C26H33O11Cl, as determined by HRESIMS analysis. It was found that the 1H-, 13C-NMR /carbonyl carbon (δC 170.0) and H-12 /carbonyl carbon (δC 169.4). Comparing the 1H- and 13C-NMR data of 7 with those of solenolide D +, 523 [M + Na + 2]+; HRESIMS m/z 521.1554 [M + Na]+ .Briaviolide A (2): Colorless amorphous gum; c 0.1, CH2Cl2); IR νmax 3453, 2965, 2942, 1783, 1738, 1732, 1278 cm−1; 1H-NMR data , see 13C-NMR data , see m/z 577 [M + Na]+, 579 [M + Na + 2]+; HRESIMS m/z 577.1812 [M + Na]+ .Briaviolide B (3): Colorless powder; c 0.1, CH2Cl2); IR νmax 3429, 2965, 2928, 1776, 1738, 1373, 1213 cm−1; 1H-NMR data , see 13C-NMR data , see m/z 587 [M + Na]+; HRESIMS m/z 587.2101 [M + Na]+ .Briaviolide C (4): Colorless amorphous gum; c 0.4, CH2Cl2); IR νmax 3433, 3015, 2989, 2938, 1776, 1734, 1373, 1220, 1019, 756 cm−1; 1H-NMR data , see 13C-NMR data , see m/z 537 [M + Na]+, 539 [M + Na + 2]+; HRESIMS m/z 537.1506 [M + Na]+ .Briaviolide D (5): Colorless amorphous powder; c 0.1, CH2Cl2); IR νmax 3429, 2965, 2928, 1779, 1738, 1373, 1213 cm−1; 1H-NMR data , see 13C-NMR data , see m/z 612 [M + Na]+, 614 [M + Na + 2]+; HRESIMS m/z 612.2075 [M + Na]+ .Briaviolide E (6): Colorless amorphous gum; c 0.4, CH2Cl2); IR νmax 3410, 2981, 2938, 1768, 1742, 1369, 1216, 1019, 756 cm−1; 1H-NMR data , see 13C-NMR data , see m/z 593 [M + Na]+, 595 [M + Na + 2]+; HRESIMS m/z 593.2125 [M + Na]+ .Briaviolide F (7): Colorless amorphous gum; c 0.6, CH2Cl2); IR νmax 3544, 3447, 2981, 2937, 1782, 1739, 1375, 1223, 1021, 737 cm−1; 1H-NMR data , see 13C-NMR data , see m/z 579 [M + Na]+, 581 [M + Na + 2]+; HRESIMS m/z 579.1605 [M + Na]+ .Briaviolide G (8): Colorless amorphous gum; c 0.2, CH2Cl2); IR νmax 3440, 2923, 1732, 1712, 1644, 1375, 1260, 1034, 953, 733 cm−1; 1H-NMR data , see 13C-NMR data , see m/z 489 [M + Na]+; HRESIMS m/z 489.2106 [M + Na]+ .Briaviolide H (9): Colorless amorphous powder; c 0.4, CH2Cl2); IR νmax 3425, 2928, 1738, 1715, 1373, 1259 cm−1; 1H-NMR data , see 13C-NMR data , see m/z 505 [M + Na]+; HRESIMS m/z 505.2046 [M + Na]+ .Briaviolide I (10): Colorless amorphous gum; c 0.1, CH2Cl2); IR νmax 3426, 2927, 2853, 1732, 1675, 1375, 1257, 1044, 737 cm−1; 1H-NMR data , see 13C-NMR data , see m/z 531 [M + Na]+; HRESIMS m/z 531.2204 [M + Na]+ .Briaviolide J (11): Colorless amorphous gum; c 0.2, CH2Cl2); IR νmax 3463, 2985, 2938, 1779, 1734, 1270, 1224 cm−1; 1H-NMR data , see 13C-NMR data ; see m/z 625 [M + Na]+, 627 [M + Na + 2]+; HRESIMS m/z 625.1820 [M + Na]+ .Benzyl briaviolide A (1 (3.0 mg) was stirred with 0.1 mL of benzoyl chloride in pyridine (1.0 mL) for 20 h at room temperature. After evaporation, the residue was separated by a C18 reversed-phase HPLC column  to give pure Compound 11 (2.8 mg).Compound 3) of 1 for diffraction was obtained by simple evaporation from methanol solution. Crystal data: C24H31ClO9, orthorhombic, a = 10.4634(2) Å, b = 13.9191(3) Å, c =17.1825(3) Å, V = 2502.48(8) Å3, space group P212121, Z = 4, Dcalcd = 1.324 Mg/m3, λ= 1.54178 Å, μ(Mo Kα) 1.783 mm−1, F(000) = 1056, T = 295(2) K. A total of 17,948 reflections were collected, of which 4536 unique reflections (Rint = 0.0427) with I > 2σ(I) were used for the analysis. The data was solved using the direct method, and the structure was refined by a full-matrix least-squares procedure on F2 values. All non-hydrogen atoms were refined with anisotropic thermal parameters. The hydrogen atom positions were geometrically idealized and allowed to ride on their parent atoms. The final indices were R1 0.0499, wR2 0.1420 with goodness-of-fit = 1.149. The final X-ray molecular model is shown in A suitable colorless crystal , neutrophils (6 × 105 cell/mL) were equilibrated at 37 °C for 2 min and incubated with each test compound for 5 min. Cells were activated by fMLP (100 nM)/CB (0.5 μg/mL), and changes in absorbance at 405 nm were monitored continuously for elastase release. The results are expressed as the percentage of the initial rate of elastase release in the fMLP/CB-activated, test compound-free (DMSO) control system.Degranulation of azurophilic granules was determined by elastase release, as described previously . Experimc [c and 1.0 mM Ca2+, neutrophils were equilibrated at 37 °C for 2 min and incubated with drugs for 5 min. Cells were activated with 100 nM fMLP for 10 min. When fMLP was used as a stimulant, CB (1 μg/mL) was incubated for 3 min before activation by the peptide (fMLP/CB). Changes in absorbance with the reduction of ferricytochrome c at 550 nm were continuously monitored in a double-beam, six-cell positioner spectrophotometer with constant stirring . Calculations were based on differences in the reactions with and without SOD (100 U/mL) divided by the extinction coefficient for the reduction of ferricytochrome c.Human neutrophils were obtained by means of dextran sedimentation and Ficoll centrifugation. Superoxide anion production was assayed by monitoring the superoxide dismutase-inhibitable reduction of ferricytochrome c . In brie1–10), were successfully isolated from the Taiwanese soft coral, Briareum violacea, and their structures determined. The inhibitory effects of the isolates and new derivative 11 on superoxide-anion generation and elastase release by human neutrophils in response to fMLP/CB were evaluated. Compounds 5 and 9 showed moderate anti-inflammatory activities at a concentration of 10 μg/mL. Compound 11, derived from Compound 1, showed better inhibition of elastase release than that of 1. Further comparison of the activities of those compounds may suggest that β-orientation and the chain length of ester groups at C-12 are important for the anti-inflammatory activities in briarane-type diterpenoids.Sixteen briarane diterpenoids, including ten new compounds briaviolides, A–J (


The structures of compounds 1–5 were elucidated through extensive spectroscopic analysis, including HRMS, 1D, and 2D NMR experiments. The stereochemistry, including absolute configurations of these compounds, was determined by spectroscopic, chemical, and computational methods. Compounds 1 and 5 showed moderate protein tyrosine phosphatase 1B (PTP1B) inhibitory activities with IC50 values of 5.2 μM and 8.7 μM, respectively, more potent than previously reported hippolides.Five new sesterterpenoids, compounds  Ircinia (unknown species), marine sponges have proven to be a valuable source of structurally diverse molecules with PTP1B inhibitory activity + at 392.2567, consistent with a molecular formula of C24H35NO2, implying eight degrees of unsaturation. The 1D and 2D NMR spectra ; ten olefinic carbons , correlated with five olefinic protons at δH 7.13 , 5.66 , 5.11 , 5.10 , and 5.08 ; two oxygen-bearing sp3 methines ; seven methylenes, and four methyl carbons  with no correlation in HSQC spectrum. The COSY correlations for H-8/H-9/H-10, H-12/H-13/H-14, and H-16/H-17/H-18, together with the HMBC correlations of four methyl groups, H3-20/C-18, C-19, and C-21, H3-21/C-18, C-19, H3-22/C-14, C-15, and C-16, and H3-23/C-10, C-11, and C-12, delineated the presence of a farnesyl moiety  spectrum was experimentally recorded, which showed positive Cotton effect at 219 and 297 nm. The theoretical ECD of 1a and its enantiomer 1b were then calculated with a time-dependent density function theory (TD-DFT) method at the b3lyp/aug-cc-pvdz level, and the polarizable continuum model (PCM) was adopted to consider solvent effects using the dielectric constant of methanol. The calculated ECD spectra were produced by SpecDis software in 1b was in good agreement with the experimental one. Thus, the absolute configurations at C-4, and C-24 of 1 were determined as R and S, respectively.The relative configuration of elations  and compelations . The NOEoisomers ,19,20. Oomers +m/z 438.2619. The 13C NMR and DEPT spectra indicated 25 resonances for four methyls, eight methylenes, six methines (two oxygen-bearing sp3 methines), and seven quaternary carbons (two carbonyl carbons) , and 5.09 , two oxygenated methine protons at δH 5.14 , and 4.21 , four methyl groups at δH 1.68 , and 1.60 , in addition to two NH2 protons at δH 7.56  and 7.26 , and one OH proton at δH 4.99 . Inspection of the 1H and 13C NMR, HMBC and COSY spectra of 2 suggested that it was structurally related to compound 1, possessing a farnesyl moiety. Six of the eight degrees of unsaturation of 2 were accounted for by four double bonds and two carbonyl carbons, implying that the structure retained two rings as well. Interpretation of the COSY correlations of H-4/H-5a, 5b/H-6, and H-4 with an exchangeable proton at δH 4.99 , together with the HMBC correlations of H-24/C-6 and C-3, H-5a/C-3, and C-4, indicated the presence of a 4-hydroxycyclohexenyl moiety. The connection of the farnesyl group to the 4-hydroxycyclohexenyl moiety at C-7 was supported by the HMBC correlations of H-8/C-6 and C-7. The remaining ring structure was assigned to a butanolide moiety attached to the 4-hydroxycyclohexenyl moiety via an ester linkage at C-24 and a quaternary carbon at C-3, which was delineated by the HMBC correlations of H-24/C-1 and C-3, H-2a, H-2b/C-1 and C-3 and C-24. The last unassigned substructure CONH2 was determined as an acetamide motif based on the chemical shift of the carbonyl carbon (δC 175.2), and two NH2 protons at δH 7.56, 7.26 in DMSO-d6, as well as the IR absorption bands . Furthermore, the 3JCH correlations from H-2, H-4, and H-24 to C-25 indicated that the acetamide group was attached to the bicyclo-moiety at C-3. The NOESY correlations for H-10/H-12, H-9/H3-23, H-14/H-16, and H-13/H3-22 - and (R)-MTPA esters of 2 were prepared by treatment with (R)- and (S)-MTPA chloride, respectively. The ∆δS–R values of the Mosher ester observed for the protons near the secondary C-4 hydroxy group indicated the R configuration for the carbinol stereogenic center in 2  . The 1H 13/H3-22  suggesteH-24/NH2 . The abster in 2 . On the ter in 2 , the abs3 exhibited the same molecular formula of C25H37NO4 ([M + Na]+m/z 438.2622) as that of 2. A detailed analysis of the 1D and 2D NMR spectroscopic data confirmed that 3 possessed the farnesyl and 4-hydroxycyclohexenyl moieties as well. However, careful comparison of 1H NMR spectra of compounds 2 and 3 revealed that they are isomers, in which the δH 5.14 of H-24 in 2  changed to δH 4.58  in 3, and displayed clear correlation with NH proton at δH 5.42  in the COSY spectrum of 3. In addition, the resonance of 1H NMR for 3 indicated the presence of one carboxyl proton at δH 11.04 . The HMBC of H-2/C-1, C-3, C-4, C-24, and C-25, and COSY correlations of NH/H-24, H-6/H-5a/H-4 (3 possessed a pyrrolidin-2-one moiety and a carboxy group (C-25) at C-3 instead of butenolide moiety and carbamoyl group in 2. The NOESY correlations for H-10/H-12, H-9/H3-23, H-14/H-16, and H-13/H3-22 , 5.11 , and 5.10 . In addition, the analysis of HSQC and DEPT data delineated the presence of a substituted benzene ring with three signals at δC 117.0/δH 6.78, at δC 128.2/δH 6.93, and at δC 130.9/δH 6.89. The HMBC correlations of H-5/C-3, C-4, and C-7, H-6/C-4, C-5, and C-24, H-8/C-6, C-7, and C-24, as well as the COSY correlations of H-5/H-6 revealed that this benzene ring is substituted at three positions, C-3, C-4 and C-7, with the connection of the farnesyl group at C-7. The 13C NMR and DEPT data indicated C-4 is an oxygenated quaternary carbon, which characterized the phenolic identity of compound 4. Furthermore, an acetic acid moiety was elucidated to be attached to the phenolic ring at C-3 using the assemblage of 1D and 2D experiments, in which methylene protons of H-2  have displayed HMBC correlations with C-1 , C-3, C-4, and C-24. The NOESY correlations for H-10/H-12, H-9/H3-23, H-14/H-16, and H-13/H3-22  and NMR spectral data  and 9.37 , seven olefinic protons at δH 7.70 , 6.86 , 6.66 , 6.64 , 5.16 , 5.11 , and 5.09 , and four methyl groups at δH 1.68 , 1.60 , and 1.58 . The HMBC and COSY correlations , as well as the presence of IR band at 3379 cm−1. In addition, the olefinic proton at δH 7.70 (H-2) displayed the 4JH-1/H-2 and 3JH-3/NH as doublets of triplets , revealing the NH2 motif to be attached at C-3 position. Furthermore, the HMBC correlations of H-1/C-2, H-4/C-1, C-2, C-3, and H-24/C-6, C-7, and C-8, together with the COSY correlations of H-1/H-3 (W effect), and H-4/H-5/H-6 confirmed the presence of an 2-(aminomethylene) hepta-3,5-dienedial moiety connected with the farnesyl group at C-7. Therefore, the eight degrees of unsaturation were accounted for by six double bonds, two aldehyde moieties. The NOESY correlations for H-3/H-4, H-5/H-8, H-6/H-24, H-10/H-12, H-9/H3-23, H-14/H-16, and H-13/H3-22 . Reversed-Phase HPLC was performed on a YMC-Pack Pro C18 RS column  using a Waters 600 HPLC instrument  with Waters 996 UV detector. Column chromatography (CC) was performed on Sephadex LH-20  and YMC ODS-A (50 μm) . Vacuum liquid chromatography (VLC) was performed on silica gel ; the fractions were monitored by TLC  and spots were visualized by heating silica gel plates sprayed with 10% H2SO4 in H2O.IR spectra were recorded on a Bruker vector 22 spectrometer  with KBr pellets. Optical rotation data were recorded on a Perkin-Elmer model 341 polarimeter  with 1 dm cell. The CD spectra were obtained with a JASCO J-715 spectropolarimeter . The NMR experiments were measured on Bruker AMX-400 MHz and AMX-500 MHz instruments  in CDClH. lachne was collected off Yongxing Island and seven connected islets in the South China Sea in June 2007, and was identified by Jin-He Li . A voucher sample (No. B-2) was deposited in Laboratory of Marine Drugs, Department of Pharmacy, Changzheng Hospital, Second Military Medical University, Shanghai, China.A specimen of 2O and extracted with EtOAc and n-BuOH to afford the EtOAc- and n-BuOH-soluble extracts . The EtOAc-soluble extract was partitioned between MeOH–H2O (9:1) and petroleum ether to yield a brownish red oil (84 g). The MeOH–H2O (9:1) phase was diluted 3:2 with H2O and extracted with CH2Cl2 to afford the CH2Cl2-soluble extract (110 g). This CH2Cl2-soluble extract was subjected to VLC on silica gel using CH2Cl2/MeOH  as eluent to give nine sub-fractions (A-G). Sub-fraction C with PTP1B inhibitory activity was subjected to CC on Sephadex LH-20 (CH2Cl2:MeOH = 1:1) and silica gel using Petroleum ether/Acetone  to give eight sub-fractions (a–i). Sub-fractions (b–d) were further purified by HPLC  eluting with MeOH/H2O (80:20) to yield pure compounds 1 (6.4 mg), 4 (8.9 mg), and 5 (12.0 mg) at 56, 85, and 93 min, respectively. Similarly, compounds 2 (3.9 mg), and 3 (5.1 mg) were purified from sub-fractions h and e using HPLC  in MeOH/H2O (83:17) at 28 and 23 min, respectively.The sponge  was extracted with 95% aq. EtOH and combined extracts were concentrated under reduced pressure to yield the crude extract (671 g). This extract was suspended in HCompound 1: colorless oil;  +67° ; CD (MeOH) λmax (∆ε) 219 (3.40), 266 (−0.80), 297 (2.77) nm; UV (MeOH) λmax (log ε) 210 (1.68), 233 (1.64), 282 (1.56) nm; IR (KBr) νmax 3369, 2926, 1596, 1448, 1212, 1067, 840, 673 cm−1; 1H and 13C NMR data, see m/z 392.2567 [M + Na]+ .Compound 2: colorless oil;  −15° ; CD (MeOH) λmax (∆ε) 189 (−3.48), 196 (5.71) nm; UV (MeOH) λmax (log ε) 190 (3.30) nm; IR (KBr) νmax 3427, 2924, 1773, 1671, 1377, 1189, 1097, 981 cm−1; 1H and 13C NMR data, see 1H NMR data (in DMSO): δH 7.56 , 7.26 , 5.54 , 4.99 , 5.14 , 4.10 ; HRESIMS m/z 438.2619 [M + Na]+ .Compound 3: colorless oil;  −10° ; CD (MeOH) λmax (∆ε) 184 (−3.30), 198 (4.39) nm; UV (MeOH) λmax (log ε) 195 (3.84), 223 (1.48) nm; IR (KBr) νmax 3434, 2923, 1712, 1374, 1199, 1038, 742 cm−1; 1H and 13C NMR data, see 1H NMR data (in DMSO): δH 11.04 , 5.42 , 5.22 , 4.30 , 4.02 , 3.86 ; HRESIMS m/z 438.2622 [M + Na]+ .Compound 4: yellow oil; UV (MeOH) λmax (log ε) 228 (3.20), 282 (3.16) nm; IR (KBr) νmax 3415, 2923, 1566, 1439, 1377, 1248, 1015, 823, 651 cm−1; 1H and 13C NMR data, see m/z 393.2404 [M + Na]+ .Compound 5: yellow oil; UV (MeOH) λmax (log ε) 365 (2.56) nm; IR (KBr) νmax 3379, 2925, 1582, 1441, 1380, 1234, 1188, 1126 cm−1; 1H and 13C NMR data, see 1H NMR data (in DMSO): δH 9.33 , 9.04 , 8.20 , 7.70 , 7.29 , 6.97 , 6.92 ; HRESIMS m/z 392.2564 [M + Na]+ .pNPP. Dephosphorylation of pNPP generates product pNP, which was monitored at an absorbance of 405 nm. In a typical 100 μL assay mixture containing 50 mmol/L 3-[N-morpholino] propanesulfonic acid (MOPs), pH 6.5, 2 mmol/L pNPP, and 30 nmol/L recombinant PTP1B, activities were continuously monitored and the initial rate of the hydrolysis was determined using the early linear region of the enzymatic reaction kinetic curve.PTP1B inhibitory activity was determined using a PTP1B inhibitory assay as described in a previous report . The enzAll quantum-chemical calculations were performed by the Gaussian 03 program. The TD calculations were calculated by b3lyp/aug-cc-pvdz method under Self-Consistent Reaction Field model of solvent (MeOH). Details of the DFT calculation see S)- and (R)-MTPA esters of 2  and 3  were obtained by treatment of 2  and 3  with (R)- and (S)-MTPA chlorides (10 μL) in dry pyridine (0.5 mL), and stirred at room temperature overnight. The MTPA esters were purified by mini-column chromatography on silica gel .A previously described modified Mosher’s method was used . The (S)1 and 5, with IC50 values of 5.2 μM and 8.7 μM, are the most potent compounds isolated from marine sponges of the genus Hippospongia relevant to PTP1B inhibitory activity. Biogenetically, all the hippolides discovered so far are sesterterpenoid derivatives. The sesterterpenoids are a group of pentaprenyl terpenoids whose structures are derivable from geranylfarnesyl diphosphate [1–5 could be biosynthetically formed via multiple reactions involving oxidations, decarboxylations, aminations, dehydrations and double bond formations and shifts, etc. with the same precursor, acyclic carboxylic sesterterpenoid.Protein tyrosine phosphatase 1B (PTP1B), one of the protein tyrosine phosphatases (PTPases), is known to be a negative regulator of insulin signal transduction by dephosphorylating the insulin receptor as well as its substrate, insulin receptor substrates . The PTPhosphate . Hypothe


The biotransformation products, 7β-hydroxycholesterol, 7-oxocholesterol, and 20-droxyl-16α,17α-epoxypregn-1,4-dien-3-one, were purified by silica gel and Sephadex LH-20 column chromatography and identified by nuclear magnetic resonance and mass spectroscopy. The hydroxylase was isolated from the bacterium and the partial sequences of the hydroxylase, which belong to the catalases/peroxidase family, were analyzed using MS/MS analyses. The enzyme showed activity toward cholesterol and had a specific activity of 37.2 U/mg of protein at 30°C and pH 7.0.The metabolism of cholesterol is critical in eukaryotes as a precursor for vitamins, steroid hormones, and bile acids. Some steroid compounds can be transformed into precursors of steroid medicine by some microorganisms. In this study, the biotransformation products of cholesterol and 16 Rhizopus nigricans could transform progesterone into 11α-hydroxyl progesterone, it has attracted considerable attention + at m/z 425 in the ESI-MS spectra of compound 1 indicated a formula of C27H46O2. The analysis of the 13C spectral data showed 27 carbon signals . A signal of δ143.5 and δ125.5 with sp2 character suggested a double-doublet at C-5 and C-6 in DEPT data . This was affirmed by hydrogen shifts at δ5.28  in the 1H spectrum data . The carbon signal of δ71.4 (C-3) and 73.4 (C-7) showed that a hydroxyl group linked the A ring at C-3 and another hydroxyl group linked the B ring at C-7. We compared the shift value at δ125.5 (C-6) and 40.9 (C-8) of the product with δ121.6 (C-6) and 31.9 (C-8); β-OH can cause the chemical shift to increase greater than 4.8–7.4 downfield than α-OH. Based on this information from MS, 1H NMR, and 13C NMR, product 1 was characterized as 7β-hydroxycholesterol. This also agreed with the data in the literature terature . The che 2 showed the ion peak [M + H]+ at m/z 401.6. The analysis of the 13C spectrum data of it showed 27 carbon signals. The data indicated a formula of C27H44O2 . The signal of δ165.0 and δ126.2 with sp2 character suggested a double-doublet at C-5 and C-6 in DEPT data . This was affirmed by hydrogen shifts at δ5.69  of the 1H spectrum data . The carbon signal of δ70.6 (C-3) showed that a hydroxyl group linked the A ring at C-3. Based on the information from MS, 1H NMR, and 13C NMR, compound 2 was characterized as 7-oxocholesterol. This was in accord with the data in literature [ 2 were seen in 1H and 13C NMR spectrum data of compound 2 was in Figures S14 and S15.The analysis of the ESI-MS spectrum data of productterature . The che 3 showed the ion peak [M + H]+ at m/z 329.2 and [M + Na]+ at m/z 351.2 and [M + K]+ at m/z 367.1 in the ESI-MS spectra agreed with the formula C21H28O3. The 13C spectrum revealed 21 carbon signals : these were sorted by DEPT experiments into CH3 × 3, CH2 × 5, CH × 8, and C × 5. The signal of δ155.1 , 127.7 , 124.1 , and δ168.3 with sp2 character suggested a double-doublet at C-1 and C-2 and another double-doublet at C-4 and C-5 in DEPT data . The signal at δ186 showed a carbonyl group. These formed a conjugate system. This was affirmed by HSQC and HMBC experiments. The signal at δ7.01 , 6.22 , and 6.06  correlated with the δ155.1 , 127.7 , and 124.1 , respectively . The signal at δ7.01  correlated with the δ168.3  and 21.1  and δ6.06  correlated with the δ127.4 , 33.1 , and 45.2  in HMBC spectrum . The carbon signal of δ69.2  showed a hydroxyl group at C-20. Based on the information from MS, 1H NMR, and 13C NMR, compound 3 was characterized as 20-droxyl-16α,17α-epoxypregn-1,4-dien-3-one. The TLC plate showed a red spot that indicated that 16α,17α-epoxypregn-1,4-dien-3-one has been transformed into 20-droxyl-16α,17α-epoxypregn-1,4-dien-3-one  (lesterol . The enzDS-PAGE) . Its molBurkholderia vietnamiensis AU4i; the MS/MS data with Mascot accession number gi|543285001, protein sequence coverage: 24%), the heme catalase/peroxidase , catalase/hydroperoxidase HPI(I) , and catalase/hydroperoxidase HPI(I)  (http://www.matrixscience.com/cgi/master_results.pl?file=../data/20130930/FTntOicSm.dat#Hit1).The amino acid sequence of the purified enzyme was analyzed by MALDI-TOF/TOF. The MS data of pure enzyme is seen in Figure S17 and the MS/MS data of oligopeptides (Fr1–Fr3) in Figure S18 and those of oligopeptides (Fr4–Fr7) are seen in Figure S19. A trypsin digest of hydroxylase generated oligopeptides and the amino acid sequences of seven peptides (Fr1–Fr7) of the enzyme were determined. The sequences of Fr1–Fr7 were DWWPNQLNLNILHR, RFYENPAEFADAFAR, FYENPAEFADAFAR, HLFSYEW ELTK, IWLELSGGPNSR, VMNLDRFDLA, and SPAGAHQWVAK, respectively. These sequences are similar to those of catalases/peroxidases, for example, the catalase/peroxidase , 5-androsten-3β,17β-diol, and 17α-methyl-5-androsten-3β,17β-diol were hydroxylated entirely at the 7α-axial, allylic position by Fusarium culmorum, which showed 7α-hydroxylase activity [ Botryodiplodia malorum showed the 7β-hydroxylation of DHEA and 15β,16β-methylene-dehydroepiandrosterone [ Fusarium moniliforme could almost entirely hydroxylate DHEA at the 7α-axial, allylic position but could not hydroxylate cholesterol [α-hydroxylase was a cytochrome p450 enzyme (Cyp7b), which occurred in humans and animals [+ as a coenzyme. Our experiments showed a requirement for NAD+ in the generation of 7β-hydroxycholesterol. Appleby reported that they found cytochrome P450 in bacteria in 1967 [ Burkholderia cepacia SE-1, contained hydroxylase.In the present study, the morphology of strain SE-1 showed different colony characteristics when grown on different media. It was pale yellow on the Beef Extract Peptone medium but was white and smaller in size on the screening medium. Doukyu reported thatactivity . Botryodosterone . Morfin lesterol . It was  animals . This en in 1967 . Such prα,17α-epoxypregnenolone were transformed into useful products and hydroxylase was isolated from Burkholderia cepacia SE-1 successfully. These studies suggest that the steroid hydroxylase possesses 7β-site-selectivity and has latency value in production of the steroid medicine.Hydroxylation at position 7 of steroids with controlled stereoselectivity allows for the production of important pharmaceutical intermediates. Therefore, it was necessary that the microbial strains were screened which can transform steroids effectively. In our study, cholesterol and 161H and 13C NMR spectral data for the product 1 was as follows: 1H NMR δ  0.69, 1.05. 0.86, 0.87, 0.93, 1.15–1.58, 2.05, 2.30, 3.54, 3.84, 5.28; 13C NMR δ: 36.9 (C-1), 31.6 (C-2), 71.4 (C-3), 41.7 (C-4), 143.5 (C-5), 125.5 (C-6), 73.4 (C-7), 40.9 (C-8), 48.3 (C-9), 36.4 (C-10), 21.l (C-11), 28.6 (C-12), 42.9 (C-13), 55.5 (C-14), 26.4 (C-15), 39.6 (C-16), 54.9 (C-17), 11.8 (C-18), 19.2 (C-19), 35.7 (C-20), 18.7 (C-21), 36.2 (C-22), 23.9 (C-23), 39.5 (C-24), 28.0 (C-25), 22.6 (C-26), 22.8 (C-27).The 1H and 13C NMR spectrum data of compound 2 was as follows: 1H NMR δ  0.68 , 0.86, 0.87 0.92  1.12 , 5.69 ; 13C NMR δ: 36.4 (C-1), 31.3 (C-2), 70.6 (C-3), 41.8 (C-4), 165.0 (C-5), 126.2 (C-6), 202.2 (C-7), 45.5 (C-8), 50.0 (C-9), 38.3 (C-10), 21.3 (C-11), 28.6 (C-12), 43.1 (C-13), 54.9 (C-14), 26.3 (C-15), 38.8 (C-16), 54.9 (C-17), 12.0 (C-18), 18.9 (C-19), 35.8 (C-20), 18.7 (C-21), 36.2 (C-22), 23.9 (C-23), 39.5 (C-24), 28.0 (C-25), 22.6(C-26), 22.8 (C-27).The


Further, (-)-2-O-(E)-caffeoyl-D-malic acid (12) was isolated from C. submollis and also identified for C. pentagyna and C. nigra by co-chromatography. The isolates 10 and 11 were not found in the authentic fresh specimen, indicating that they may be formed during extraction by acyl migration from the 2-O-acylderivatives. Also, 9 and 11 are described here for the first time. All structures were assigned on the basis of their spectroscopic data ( Crataegus monogyna Jacq. (Lindm.), C. laevigata (Poiret) D.C. (syn. C. oxyacantha Thuill.), C. pentagyna Waldst. et Kit. ex Willd., C. nigra Waldst. et Kit., and C. azarolus L. . More recently, 5-p-coumaroylquinic acid was also detected from Crataegus monogyna cell suspension cultures D20 value  corresponded to a sample of commercially available L-threonic acid. Thus, 10 was identified as (-)-4-O-(E)-caffeoyl–L-threonic acid.The ESI-MS of ure data , 17. Aft11 showed a relative molecular mass of m/z 282, the HR-ESI-MS of [M + H]+ = 282.0814 . The AA’ XX’ spinsystem and the UV spectrum revealed a p-coumaric acid derivative. Loss of m/z 16 in comparison to 10 and 9 indicated an analog trihydroxybutyric acid derivative with a p-coumaroyl instead of a caffeoyl moiety. All signals of the trihydroxy butyric acid moiety were assigned by HMBC experiments. After hydrolysis of 11 and isolation of the products on polyamide, the [α]D20 was in good agreement to the corresponding commercially available L-threonic acid. Thus, 11 was identified as (-)-4-O-(E)-p-coumaroyl–L-threonic acid; its NMR data are shown in The ESI-MS of 13C- and 1H-NMR data and the optical rotation value of compound 9 coincided with the data obtained for 2-O-(E)-caffeoyl-L-threonic acid lactone . In view of the relatively high amount of HCAs in Crataegus crude drugs of c. 1% to more than 3%, it seems worthwhile to investigate whether they participate in the therapeutical efficacy of Crataegus preparations.Further investigations of the flowers and leaves of the different species separately and in more detail showed that the 3-11 was determined with a Bruker micrOTOF-qII mass spectrometer coupled to a Dionex Ultimate 3000 RS UHPLC System. Separation was achieved over an RP18 column  eluted by a binary water-acetonitrile gradient containing 0.1% formic acid at 0.8 mL/min, +10% acetonitrile/min beginning at 5% acetonitrile; the mass spectrum was acquired in positive mode over a m/z range of 50–1500 with a nebulizer pressure of 4.0 bar (N2), dry gas flow of 9.0 L/min at 220°C (N2), capillary voltage of 4500 V and 8 eV collision energy. 1H- and 13C-NMR data were recorded at 200 and 50 MHz, respectively, with a Varian Gemini 200 spectrometer or at 600 MHz and 150 MHz with a Varian Unity 600 . Offline data processing and 1H-NMR simulation was done with the Nuts programme package .Optical rotations were measured on a Perkin-Elmer 241 apparatus, mass spectra on a Finnigan LC-Q and Quattro LC Z (ESI-MS), and on a Finnigan MAT 8230 combined with a Varian 3400 GC using an HP-5 column (25 m) (GC-MS). The mass spectra were calibrated using a reference spectrum of sodium formate. The exact mass for 3 solution.Analytical TLC was carried out on silica gel aluminum plates  using ethyl acetate/formic acid/water (90:5:5), ethyl acetate/methanol/water/formic acid (100:4:8:5), and ethyl acetate/formic acid/acetic acid/water (100:11:11:27). Spots were visualized with Naturstoff reagent (1% MeOH) or 1% ethanolic FeClThe analytical HPLC (system 1) was performed with a Waters 600 controller, Waters 990 PAD; software: millennium; column: Prosep C18 (Latek), 5 µm, and 2 mL/min. flow rate. Mobile phase A: acetonitrile, B: 0.1% TFA. Elution gradient: 0–20 min. 0% A 𠆒8% A, 20–35 min. 8% A→ 21% A, 35–40 min. 21% A→ 40% A, 40–45 min. 40% A→ 100 % A, 45–60 min. 100% A→ 0% A.The semi-preparative HPLC (system 2) was carried out with a Waters 600 controller, Waters 515 pump, and Waters Lambda-Max 481 LC spectrophotometer on the Hypersil ODS , 5 µm, 250 × 16 mm, or preparative (system 3) on the Eurospher 100-C18 .The quantitative HPLC (system 4) was performed with a Waters 600 controller, Waters 996 PAD; software: Millennium; column: Discovery C18, 5 µm, 150 × 4.6 mm and 2 mL/min. flow rate. Mobile phase A: acetonitrile, B: methanol C: 0.1% TFA. Elution gradient: 0–12 min. 5% A →12% A, 0 % B, 95% C→ 88% C; 12–20 min. 12% A→ 15% A, 0% B→ 15% B, 88% C → 70% C; 20–30 min. 15% A→ 23% A, 15% B, 70% C→ 62% C; 30–35 min. 23% A→ 5% A, 15% B→ 0% B, 62% C→ 95% C.Crataegus extract LI 132 (Ch.-Nr. 96970100 and 97020100) was obtained from Lichtwer AG, Berlin. It was standardized on 2% flavonoids calculated as hyperoside by the method of DAB 10 2D0 -2.1° ; ESI-MS (m/z): 337.4 [M - H]- Rt: 7.9 min (system 4); 1H]- and 13C-NMR data see Tables 1S and 3S in “Supporting Information.”[α]2D0 -42.5° ; ESI-MS (m/z): 337.4 [M - H]-; Rt: 12.9 min (system 4); 1H- and 13C-NMR data see Tables 1S and 3S in “Supporting Information.”[α]2D0 -70.8° ; ESI-MS (m/z): 337.4 [M - H]-; ESI-MS/MS (m/z) (rel. int. in %) (337.4): 173 [M – 164 - H]- (73); 119 [M – 218 - H]- (71); 93 [M – 218 - H]- (100); Rt: 13.9 min (system 4); 1H- and 13C-NMR data see Tables 1S and 3S in “Supporting Information.”[α]2D0 +7° ; ESI-MS (m/z): 353.4 [M - H]-; LC/MS (m/z) (rel. int. in %): 371.8 [M + NH4]+ (100); 725.7 [2xM + NH4]+ (18); 355 [M + NH4]+ (27); Rt: 5.4 min (system 4); 1H- and 13C-NMR data see Tables 2S and 3S in “Supporting Information.”[α]2D0 -60.3° ; ESI-MS (m/z): 353.4 [M - H]-; Rt: 10.7 min (system 4); 1H- and 13C-NMR-data see Tables 2S and 3S in “Supporting Information.”- (100); 820,6 [2x M + TFA - H]- (4); Rt: 9 min (system 4); 1H- and 13C-NMR data see Tables 2S and 3S in “Supporting Information.”[α]D20 -139° ; LC/MS (m/z) (rel.int. in %): 628.9 [M + TFA - H]- (100); Rt: 22.2 min (system 4); 1H- and 13C-NMR data see Table 4S in “Supporting Information.”[α]D20 -170° ; LC/MS (m/z) (rel.int. in %): 628.9 [M + TFA - H]- (100); Rt: 24.6 min (system 4); 1H- and 13C-NMR data see Table 4S in “Supporting Information.”[α]D20 -17.6° ; ESI-MS (m/z) (rel. int. in %): 297 [M - H]- (100); 279 [M - 18 - H]- (4); 135 [threonic acid - H]-; 117 [threonic acid - H2O]”; ESI- MS/MS (m/z) (rel. int. in %) (297): 179 [caffeoyl - H]- (10); 135 [threonic acid - H]” (100); ESI-MS/MS/MS (m/z) (rel. int. in %) (135): 117 [threonic acid - H2O]- - H]- (100); 75 (13) (a-splitting of m/e 117); Rt: 8.3 min (system 4); 1H- and 13C-NMR data see [α]D20 -18.8° ; -24.7 ; ESI-MS (m/z) (rel. int. in %): 297 [M - H]- (100); 319 [M + Na - 2H]- (90); ESI-MS/MS (m/z) (rel. int. in %) (297): 179 [caffeoyl - H]- (7), 135 [threonic acid - H]- (100); GC-MS of the silylated compound: GC-MS (m/z) (rel. int. in %): 658 (32); 643 (12); 439 (92); 307 (100); 219 (18); Rt: 12.5 min (system 4); 1H- and 13C-NMR data see Tables 5S and 6S in “Supporting Information.”[α]D20 -34° ; ESI-MS (m/z): 281 [M - H]-; 563 [2M - H]-; 305 [M + Na]+; 587 [2M + Na]+; HR-ESI-MS (m/z): 283.0814 [M + H]+ ; Rt: 16.4 min (system 4); 1H- and C-NMR data see [α]Crataegus submollis: [α]D20 -27.9° ; ESI-MS (m/z):281 [M - H]-; 563 [2M - H]-; 305 [M + Na]+; 587 [2M + Na]+; Rt: 15.8 min (system 4); 1H- and 13C-NMR data see Tables 5S and 6S in “Supporting Information.”from 11 and 10 mg of 10 were hydrolyzed enzymatically  and the optical rotation was measured after purification on polyamide  and checked for purity by TLC. The L-threonic acid thus obtained from 11 and 10 showed an optical rotation value of [α]D20 +18.4°  and [α]D20 +23.1° , respectively. L-Threonic acid was prepared from its hemicalcium salt × 1 H2O  using cation exchange on DOWEX 50 WX 8  and purified in the same way showed [α]20D +31.1° .The hydrolysis and analysis of free L-threonic acid were performed according to Hahn . Amounts9 at position 4 (see discussion), γ-L-threonic acid lactone was synthesized from L-threonic acid lactone hemicalcium salt x 1 H2O (Sigma-Aldrich) by a modified method according to Angelotti [2O5 under vacuum for five days. The resulting 1H- and 13C-NMR spectra were compared with the data of 11 and free L-threonic acid lactone (To demonstrate a missing acylation shift of ngelotti . After c lactone  and 7S.


H-indole-4,7-dione and 1-tosyl-1H-indole-4,7-dione with 2-aminoethanesulfinic acid afforded a regioisomeric series, which was subsequently deprotected and oxidized to yield the tricyclic core scaffolds present in the thiaplakortones. All compounds were fully characterized using NMR and MS data. A single crystal X-ray structure was obtained on one of the N-tosyl derivatives. All compounds were screened for in vitro antiplasmodial activity against chloroquine-sensitive (3D7) and multidrug-resistant (Dd2) Plasmodium falciparum parasite lines. Several analogues displayed potent inhibition of P. falciparum growth (IC50 < 500 nM) but only moderate selectivity for P. falciparum versus human neonatal foreskin fibroblast cells.Six regioisomers associated with the tricyclic core of thiaplakortones A–D have been synthesized. Reaction of 1 The. The1–4)ertained  2]. Whi. Whi1–4)11–16 commenced with the generation of 1-tosyl-1H-indole-4,7-dione (10), which was accessible via known procedures (10 with 2-aminoethanesulfinic acid .,5,6.11–1 1 ratio . Separat11  in the thiazine dioxide ring .Subjection of the mixture of tosyl derivatives 10 and acquire suitable amounts of isomer 14, the parent, non-tosyl protected system, 1H-indole-4,7-dione 17 was prepared according to literature procedures +; (−)-LRESIMS m/z (rel. int.) 405 (100) [M − H]−; (+)-HRESIMS m/z 429.0200 [M + Na]+ .Compound 12: Bright orange amorphous solid; 1H-NMR  δH 2.41 , 3.26–3.28 , 3.75–3.77 , 6.81 , 7.48 , 7.98 , 8.12 , 9.13 ; 13C-NMR  δC 21.1 (C-15), 39.2 (C-3), 47.9 (C-2), 108.0 (C-9a), 108.8 (C-8), 125.9 (C-8a), 128.3 , 130.0 , 133.1 (C-11), 133.2 (C-7), 133.3 (C-5a), 146.4 (C-14), 147.4 (C-4a), 166.3 (C-5), 172.9 (C-9); (−)-LRESIMS m/z (rel. int.) 405 (100) [M − H]−; (+)-LRESIMS m/z (rel. int.) 407 (100) [M + H]+; (+)-HRESIMS m/z 429.0167 [M + Na]+ .Compound 12 and 11  in a saturated solution of NaHCO3 (5 mL) and MeOH (50 mL) was heated to reflux for 2.5 h. The mixture was acidified with HCl (32% aqueous) to pH 6 then concentrated and subjected to flash chromatography  to afford a 1:26 mixture of compounds 14 and 13 . This material (59 mg) was pre-adsorbed to C18-bonded silica (1 g) overnight, then packed into a guard cartridge that was attached to a C18-bonded silica semi-preparative HPLC column. Application of the same reversed-phase HPLC purification method described above  as an orange powder.A 1:11 mixture of ed above  afforded13: Orange amorphous solid; 1H-NMR  δH 3.31–3.33 , 3.81–3.84 , 6.57 , 7.13 , 9.05 , 12.76 ; 13C-NMR  δC 39.4 (C-3), 48.2 (C-2), 107.5 (C-6), 120.7 (C-5a), 125.4 (C-7), 132.9 (C-8a), 148.2 (C-4a), 169.8 (C-9), 174.1 (C-5); (+)-LRESIMS m/z (rel. int.) 253 (100) [M + H]+; (−)-LRESIMS m/z (rel. int.) 251 (100) [M − H]−; (+)-HRESIMS m/z 275.0104 [M + Na]+ .Compound H-indole-4,7-dione  in MeCN (160 mL) was treated with a solution of 2-aminoethanesulfinic acid  in H2O (50 mL) in one portion and the mixture stirred for 20 h under an atmosphere of O2. The MeCN and H2O were removed by rotary evaporation to afford an orange solid, which was purified by flash chromatography  to afford a 3.3:1 mixture of regioisomers 14 and 13, respectively . A portion of this material (40 mg) was pre-adsorbed to C18-bonded silica (1 g) overnight, then packed into a guard cartridge that was attached to a C18-bonded silica semi-preparative HPLC column. Isocratic HPLC conditions of 95% H2O (0.1% TFA)/5% MeOH (0.1% TFA) were employed for the first 10 min, then a linear gradient to 50% H2O (0.1% TFA)/50% MeOH (0.1% TFA) was run over 40 min, followed by a linear gradient to MeOH (0.1% TFA) in 1 min, then isocratic conditions of MeOH (0.1% TFA) for a further 9 min, all at a flow rate of 9 mL/min. Sixty fractions (60 × 1 min) were collected by time from the start of the HPLC run. All UV active fractions were analyzed by 1H-NMR spectroscopy and MS, and identical fractions were combined. This yielded 14  as a bright orange powder.A solution of 114: Bright orange amorphous solid; 1H-NMR  δH 3.28–3.33 , 3.79–3.82 , 6.53 , 7.40 , 8.89 , 12.81 ; 13C-NMR  δC 39.3 (C-3), 48.4 (C-2), 108.4 , 127.2 (C-8a), 128.2 (C-5a), 130.1 (C-7), 147.5 (C-4a), 167.9 (C-5), 174.7 (C-9); (+)-LRESIMS m/z (rel. int.) 253 (100) [M + H]+; (−)-LRESIMS m/z (rel. int.) 251 (100) [M − H]−; (+)-HRESIMS m/z 275.0087 [M + Na]+ .Compound 14 and 13  from the synthesis described above  and then the mixture was concentrated in vacuo to afford a residue that was subjected to flash chromatography through a small plug of silica (1:10 v/v MeOH/CH2Cl2 elution) and after concentration of the eluent in vacuo, the residue (40 mg) was pre-adsorbed to C18-bonded silica (1 g) overnight, then packed into a guard cartridge that was attached to a C18-bonded silica semi-preparative HPLC column. Application of same reversed-phase HPLC purification method described above  and 15  as yellow and orange powders, respectively.A 3.3:1 regioisomeric mixture of ed above  in a soled above  resulted15: Orange amorphous solid; 1H-NMR  δH 6.49 , 6.66 , 7.07 , 7.27 , 11.07 , 12.99 ; 13C-NMR  δC 108.0 (C-6), 111.7 (C-2), 113.4 (C-9a), 121.8 (C-5a), 126.8 (C-7), 130.2 (C-3), 131.3 (C-8a), 140.5 (C-4a), 172.2 (C-9), 174.1 (C-5); (+)-LRESIMS m/z (rel. int.) 251 (100) [M + H]+; (−)-LRESIMS m/z (rel. int.) 249 (100) [M − H]−; (+)-HRESIMS m/z 272.9939 [M + Na]+ .Compound 16: Yellow amorphous solid; 1H NMR  δH 6.42 , 6.60 , 7.04 , 7.43 , H-4 and H-6 not observed; 13C-NMR  δC 108.5 (C-8), 111.6 (C-2), 113.9 (C-9a), 126.9 (C-8a), 127.6 (C-5a), 129.8 (C-7), 130.1 (C-3), 140.2 (C-4a), 167.9 (C-5), 177.3 (C-9); (+)-LRESIMS m/z (rel. int.) 251 (100) [M + H]+; (−)-LRESIMS m/z (rel. int.) 249 (100) [M − H]−; (+)-HRESIMS m/z 272.9933 [M + Na]+ .Compound http://www.ccdc.cam.ac.uk.Intensity data were collected with an Oxford Diffraction SuperNova CCD diffractometer using Cu-Kα radiation, the temperature during data collection was maintained at 100.0(1) using an Oxford Cryosystems cooling device. The structure was solved by direct methods and difference Fourier Synthesis . Hydroge11: C17H14N2O6S2, M = 406.42, T = 100.0(2) K, λ = 1.5418 Å, Triclinic, space group 21/cP, a = 11.6802(7), b = 28.0975(14), c = 10.3047(6) Å, β = 91.539(5)° V = 3380.6(3) Å3, Z = 8, Z′ = 2, cD = 1.597 Mg·M−3, μ = 3.230 mm−1, F(000) = 1680, crystal size 0.49 mm × 0.38 mm × 0.31 mm. θmax = 67.6°, 10,831 reflections measured, 5907 independent reflections (Rint = 0.051) the final R = 0.0559  and wR(F2) = 0.1577  GOOF = 1.027.Crystal data for compound P. falciparum growth inhibition assays were carried out using an isotopic microtest, as previously described  or test compounds and incubated under standard P. falciparum culture conditions with 0.5 μCi [3H]-hypoxanthine. The final concentration of DMSO vehicle was <0.5% in all assay wells (non-toxic). After 48 h cells were harvested onto 1450 MicroBeta filter mats  and [3H] incorporation determined using a 1450 MicroBeta liquid scintillation counter. Percentage inhibition of growth compared to matched DMSO controls was determined and IC50 values were calculated using linear interpolation of inhibition curves [50 or % inhibition (±SD) was calculated for three independent experiments, each carried out in triplicate.escribed . Brieflyn curves . The meaH-indole-4,7-dione derivatives, and isolated in low to moderate yields. Regiochemistry was moderated by substitution of the indole nitrogen. All compounds were tested for in vitro antiplasmodial activity towards two P. falciparum parasite lines (3D7 and Dd2). Compound 16 showed the best antiparasitic activity with IC50 values of 252 and 127 nM towards 3D7 and Dd2 lines, respectively. The moderate toxicity (IC50 4600 nM) of compound 16 towards NFF cells equates to a selectivity index of 18.2–36.2. These studies have identified that while the ethylamine side-chain present in the marine natural products, thiaplakortones A and B, translates to more potent and selective antiplasmodial compounds, this functionality is by no means essential for activity. Furthermore, the truncated thiaplakortone molecules (11–16) synthesized during this work has allowed delineation of a minimum antiplasmodial pharmacophore for the thiaplakortone chemotype.In summary, six analogues associated with the tricyclic core of thiaplakortones were synthesized from readily accessible and known 1


Phallusia fumigata, afforded two new sulfated sterols, phallusiasterols A (1) and B (2). The structures of the new compounds have been elucidated using mass spectrometry and NMR experiments. The effects of phallusiasterols A and B as modulators of pregnane-X-receptor (PXR) have been investigated. These studies revealed that phallusiasterol A induces PXR transactivation in HepG2 cells and stimulates the expression of the PXR target genes CYP3A4 and MDR1 in the same cell line. Molecular docking calculations suggested the theoretical binding mode of phallusiasterol A with hPXR and revealed that phallusiasterol A fitted well in the LBD of PXR.Purification of the apolar extracts of the marine ascidian  The MS/MS fragmentation pattern of 1 was compatible with the presence of a sulfate group, displaying the peak at m/z 425.3392 [M − NaHSO4 + Na]+. Consistent with the MS data, the 1H and 13C-NMR spectra of 1 contained two hydroxymethine signals  and a highly deshielded unprotonated carbon (δ 87.8), presumably the locus of the sulfate group. This assumption was verified by acetylation of phallusiasterol A (1) which gave the corresponding diacetate 3 , assisted by TOCSY information, allowed the sequential assignment of all the protons of the tetracyclic system  and the methilene protons at δ 1.38 and 1.03 (H-22a and H-22b). The sequence was extended to the H3-26 and 27 methyls, taking also advantage of HSQC and TOCSY information. The steroidal skeleton of and C-17 . Informa1) was established through analysis of ROESY data (3 − δ pyridine-d5) observed for some key signals in the 1H-NMR spectrum of 1. The equatorial orientation of the hydroxyl group at C-3, assigned to the β face, was easily deduced by the presence of an axial-axial coupling constant (J = 13.1 Hz) found for the vicinal H-4β. Furthermore, a large pyridine-induced downfield shift (−0.46 ppm) was observed for the H-3α signal, which resonates at δ 4.33 when the proton spectrum is recorded in CDCl3 . According to Fujimoto et al. [3-19 (−0.32 ppm) and H-8 (−0.31 ppm), which supported the axial orientation of the hydroxyl group at C-6. The orientation of substituents at C-17 and C-20 in phallusiasterol A, as shown in The small coupling constants showed by H-6  suggested a β-orientation of the OH group at C-6. This was confirmed by the signal large pyridine-induced downfield shift observed for H-4o et al. , the res27H47SO6Na established for the second metabolite phallusiasterol B (2) by HRESIMS and NMR data was identical to that of 1, indicating that the compounds were isomers. The 1H and 13C-NMR resonances of 2 closely resembled those of 1, except for some signals surrounding C-6  when compared to that of H-6 in 1 . The upfield shift of C-6 in the 13C-NMR spectrum of 2 (δ 66.1) relative to that observed for 1 (δ 75.3) added support to this assignment [3-19 and the β orientation of H-6 is the only position according with this demand. In addition, on comparison of the proton spectra of 2 recorded in CDCl3 and pyridine-d5, a pyridine-induced deshielding was observed for H-4α (−0.64 ppm), comparable to that observed for H-4β in 1, indicating a 1,3-diequatorial interaction of this proton with the 6α OH group [The molecular formula Cding C-6 . Interprsignment ,19,20. COH group . Thus, p1) and B (2) in regulating the PXR activity. A transactivation assay on HepG2 cells, a human hepatocarcinoma cell line, has been performed, as described in the Experimental Part. As shown in 1 acted as PXR agonist at concentration of 10 μM; its activity was comparable to that of rifaximin, a well characterized ligand for the human PXR. As shown in 1 and 2 failed to reverse the induction of luciferase activity caused by rifaximin, indicating that they were not PXR antagonists. Similar results have been obtained by analyzing the effect exerted by 1 and 2 in terms of regulation of PXR mediated induction of two PXR target genes, CYP3A4 . For an accurate measurement of the coupling constants, the one-dimensional 1H-NMR spectra were transformed at 64K points . Homonuclear 1H connectivities were determined by COSY experiment. Through-space 1H connectivities were evidenced using a ROESY experiment with a mixing time of 500 ms. Two and three bond 1H–13C connectivities were determined by gradient 2D HMBC experiments optimized for a 2,3J of 8 Hz. 3JH–H values were extracted from 1D 1H-NMR. High performance liquid chromatography (HPLC) separations were achieved on a Shimadzu LC-10AT apparatus equipped with a Knauer K-2301 refractive index detector.High-resolution ESI-MS spectra were performed on a Thermo LTQ Orbitrap XL mass spectrometer. The spectra were recorded by infusion into the ESI source using MeOH as the solvent. Optical rotations were measured at 589 nm on a Jasco P-2000 polarimeter using a 10-cm microcell. NMR spectra were determined on Varian Unity Inova spectrometers at 700 and 500 MHz; chemical shifts were referenced to the residual solvent signal . The samples were frozen immediately after collection and stored at −20 °C until extraction. A reference specimen is deposited at the Dipartimento di Farmacia, University of Naples. The fresh thawed animals (424 g of dry weight after extraction) were homogenized and extracted twice with methanol and then twice with chloroform (4 × 200 mL). The combined extracts were concentrated in vacuo, and the resulting aqueous residue was extracted with EtOAc and subsequently with n-BuOH. Separation of the EtOAc soluble material (1.04 g) was achieved by gradient silica gel MPLC (hexane→EtOAc→MeOH). The fraction eluted with hexane/EtOAc 3:7 v/v, (37.0 mg) was chromatographed by HPLC on a SiO2 column  eluting with hexane/EtOAc 55:45 (v/v), yielding a fraction (3.3 mg) which has been further purified by HPLC on a SiO2 column , eluting with hexane/propan-2-ol 93:7, thus affording phallusiasterol A (2.0 mg) and B (1.1 mg) as pure compounds.Specimens of  −3.5 ; HRESIMS  m/z 545.2858 ; 1H and 13C-NMR (C5D5N): see 1H-NMR (CDCl3): δ 4.33 , 3.96 , 2.34 , 2.02 , 1.99 , 1.98 , 1.90 , 1.83 , 1.74 , 1.68 , 1.60 , 1.57 , 1.57 , 1.54 , 1.51 , 1.48 , 1.41 , 1.37 , 1.35–1.33 , 1.29 , 1.28 , 1.26 , 1.25 , 1.24 , 1.18 , 1.15–1.10 , 1.07 , 1.00 , 0.91 , 0.87 , 0.86 , 0.67 . 13C-NMR (CDCl3): δ 84.2 (C-5), 75.8 (C-6), 67.8 (C-3), 56.0 (C-17), 55.8 (C-14), 46.0 (C-9), 42.8 (C-13), 41.3 (C-4), 39.8 (C-10 and C-12), 39.6 (C-24), 36.2 (C-22), 35.8 (C-20), 34.0 (C-7), 33.6 (C-1), 30.5 (C-2), 30.4 (C-8), 28.2 (C-16), 28.0 (C-25), 24.0 (C-15), 23.8 (C-23), 21.3 (C-11), 22.7 (Me-26), 22.5 (Me-27), 18.8 (Me-21), 18.2 (Me-19), 12.1 (Me-18).Colorless amorphous solid,  +7.9 ; HRESIMS  m/z 545.2870 ; 1H and 13C-NMR (C5D5N): see 1H-NMR (CDCl3): δ 4.05 , 3.84 , 2.25 , 1.99 , 1.96 , 1.67 , 1.87–1.84 , 1.68 , 1.56-1.54 , 1.51 , 1.42–1.41 , 1.37 , 1.33 , 1.30 , 1.28 , 1.27-1.24 , 1.12 , 1.20-1.11 , 1.07 , 1.00 , 0.91 , 0.87 , 0.86 , 0.70 . 13C-NMR (CDCl3): δ 77.2 (C-5), 67.3 (C-3), 63.5 (C-6), 56.4 (C-17), 55.6 (C-14), 46.1 (C-9), 42.9 (C-13), 41.6 (C-4), 40.2 (C-12), 39.6 (C-24), 39.4 (C-10), 36.1 (C-22), 35.8 (C-20), 35.2 (C-7), 32.6 (C-1), 30.6 (C-2), 29.7 (C-8), 29.1 (C-16), 27.9 (C-25), 24.0 (C-15), 23.8 (C-23), 21.1 (C-11), 22.7 (Me-26), 22.5 (Me-27), 18.5 (Me-21), 18.2 (Me-19), 12.0 (Me-18).Colorless amorphous solid, 1 in 0.5 mL of dry pyridine was added 0.3 mL of Ac2O. After the mixture was stirred for 12 h at room temperature, evaporation under vacuum gave 0.7 mg of 3 as a white solid: HRESIMS  m/z 629.3089 ; 1H-NMR (C6D6): δ 0.61 , 0.94 , 0.98 , 1.14 , 1.52 , 1.70 , 5.43 , 5.72 ; 13C-NMR (C6D6): δ 11.9 (C-18), 17.2 (C-19), 18.5 (C-21), 20.1 (COMe), 20.4 (COMe), 22.4 (C-26 and C-27), 69.9 (C-3), 75.1 (C-6), 168.8 (CO), 169.4 (CO).To a stirred solution of 0.5 mg of pure 4 cells/well, and transfected with 75 ng of pSG5-PXR, 75 ng of pSG5-RXR, 125 ng of pCMV-β-galactosidase, and with 250 ng of the reporter vector pCYP3A4promoter-TKLuc, using Fugene HD transfection reagent (Roche). At 24 h post-transfection, cells were primed with Rifaximin, 1 and 2 (10 μM) or with the combination of Rifaximin (10 μM) plus compounds 1 and 2 (50 μM). After treatments, cells were lysed in 100 μL Lysis Buffer  and 20 μL cellular lysate was assayed for Luciferase activity using the Luciferase Assay System (Promega). Luminescence was measured using an automated luminometer . Luciferase activities were normalized for transfection efficiencies by dividing the Luciferase relative light units (RLU) by β-galactosidase activity  expressed from cells co-transfected with pCMVβgal. All experiments were performed in triplicate.HepG2 cells were plated in a 24-wells plate, at 5 × 10l-glutamine and 1% penicillin/streptomycin. To evaluate PXR target genes expression, serum starved HepG2 cells were stimulated for 18 h with Rifaximin and compound 1 and 2 (10 μM). Total RNA was extracted using the TRIzol reagent (Invitrogen), purified of the genomic DNA by DNAase I treatment (Invitrogen) and random reverse-transcribed with Superscript II (Invitrogen). 10 ng template was amplified using the following reagents: 0.2 μM of each primer and 10 μL of KAPA SYBR FAST Universal qPCR Kit (KAPA BIOSYSTEMS). All reactions were performed in triplicate and the thermal cycling conditions were: 3 min at 95 °C, followed by 40 cycles of 95 °C for 15 s, 58 °C for 20 s and 72 °C for 30 s. The relative mRNA expression was calculated and expressed as 2−(ΔΔCt). Primers used for qRT-PCR were:hGAPDH: GAAGGTGAAGGTCGGAGT and CATGGGTGGAATCATATTGGAA;hCYP3A4: CAAGACCCCTTTGTGGAAAA and CGAGGCGACTTTCTTTCATC;hMDR1: GTGGGGCAAGTCAGTTCATT and TCTTCACCTCCAGGCTCAGT.HepG2 cells were maintained at 37 °C in E-MEM supplemented with 10% FBS, 1% P < 0.05.All values are expressed as means ± standard error (SE) of n observations/group. Comparisons of two groups were made with a one-wayANOVA with post hoc Tukey’s test. Differences were considered statistically significant at values of Molecular docking of phallusiasterol A in the three-dimensional X-ray structures of the PXR LBD  without the co-crystallized inhibitor and waters were carried out using the AutoDock software package  . LigandsIn vitro assays demonstrated that, in spite of their close structure similarity , only phallusiasterol A is endowed with PXR agonistic activity, indicating a crucial role in the ligand-receptor binding of phallusiasterols of the configuration at C-6. The activity of 1 is comparable to that of rifaximin, a well characterized ligand for the human PXR. Supporting the role of phallusiasterol A as PXR regulator, we have observed that it effectively increases the expression of two PXR target genes, CYP3A4 and MDR1, in a human hepatocyte cell line, whereas 2 failed to induce them. Both compounds 1 and 2 were not PXR antagonists. The experimental biological results have been also validated by docking studies which allowed rationalization of the activity of phallusiasterol A. Phallusiasterols A (1) and B (2) to our knowledge are the first sulfated sterols isolated from tunicates. These unusual marine steroids have been recognized as new potential nuclear receptor ligands and their possible role in regulating the PXR activity has been investigated.