We show here that two structurally innocent-looking 2,3-dibromo-1,1-dimethylindanes violate the rule by means of their values of 3J(cis) = 6.1 Hz and 3J(trans) = 8.4 Hz. The stereoselective formation of the trans diastereomer from 1,1-dimethylindene was improved with the tribromide anion (Br3−) as the brominating agent in place of elemental bromine; the ensuing, regiospecific HBr elimination afforded 3-bromo-1,1-dimethylindene. The addition of elemental bromine to the latter compound, followed by thermal HBr elimination, furnished 2,3-dibromo-1,1-dimethylindene, whose Br/Li interchange reaction, precipitation, and subsequent protolysis yielded only 2-bromo-1,1-dimethylindene.Do not rely on the widely accepted rule that vicinal, sp We. Wetransmixtures . This revailable , whereastrans-1 and cis-1 since the availability of two different mixtures facilitated the NMR assignments and because any cis-1/trans-1 mixture or the ensuing 3/4 mixtures may be used for preparing 2,3-dibromo-1,1-dimethylindene (7). Both 3 and 4 can add elemental bromine and the adducts  eliminated HBr either spontaneously (5) or in the presence of KOt-Bu or NEt3 (6) at rt to furnish the same product 7. We found through NOESY and HMBC analyses that the thermally more stable tribromide 6 populates the conformation which has a pseudoaxial 3-H  orientation (like trans-1). Therefore, the unusually high thermolability of 5 seemed to be due to the unavoidable presence of one pseudoaxial C(3)–Br bond [8) might also be prone to such thermal HBr elimination. For comparison, treatment of 1,1-dimethylindane (9) with one equivalent of N-bromosuccinimide (NBS) furnished the expected 3-bromo-1,1-dimethylindane (see 2). With two equivalents of NBS, however, 9 afforded a mixture of 3 and 7 through the following sequence in the bottom part of 9 → 8 → 3 → 5 → 7. Under these conditions, the spontaneous HBr elimination from 8 had produced 3, which added Br2 to generate the thermolabile tribromide 5, whose HBr elimination gave 7; the required Br2 was visible in the weakly brownish gas phase and had been provided through the well-known reaction of HBr with the accompanying NBS. Consequently, three equivalents of NBS would be necessary for obtaining 7 in a maximum yield. This encouraged us to reflux 9 in CCl4 with NBS (4 equiv), which furnished mainly 7 along with succinimide (3.6 equiv) and remnant NBS (0.4 equiv). As a side-reaction, the slower thermal HBr elimination from the intermediate 3-bromo-1,1-dimethylindane (see 2), whose in situ bromination furnished 1 (ca. 1%) in a trans/cis ratio of ca. 3:2. Since both dibromides trans-1 and cis-1 were stable under the reaction conditions and would distil together with 7, they were destroyed through HBr elimination by KOt-Bu (or KOH in EtOH) to produce the monobromides 3 and 4. It may be noticed that 4 (from cis-1) cannot have been an intermediate in the initial step of the above “base-free” conversion of 9 to 7, since 4 would generate the thermally stable tribromide 6 that was not detected in the initial product mixture of 1 and 7.We abstained from separating –Br bond . If so, dane see , which ddane see  generate7 may be useful as an alternative starting material in place of 2,3-diiodo-1,1-dimethylindene that had been employed [7 as follows for a first specific route to 2-bromo-1,1-dimethylindene (4). The rapid Br/Li interchange reaction of 7 in hexane as the solvent with n-butyllithium (n-BuLi) ocurred predominantly at the 3-position of 7 with formation of 2-bromo-3-lithio-1,1-dimethylindene .For practical purposes, employed –14 in crdene 10, . In the ylindene , 10 did  problem –17, thisCis/trans differentiation in the saturated part of cyclopentene moieties should be based on 1H nuclear magnetic Overhauser enhancements rather than on the magnitudes of vicinal cis and trans3JH,H NMR coupling constants which are not always reliable. The deceivingly high value of 8.4 Hz in trans-2,3-dibromo-1,1-dimethylindane (trans-1) is explained in this work by a preferentially populated envelope conformation with pseudoequatorial 2-Br and hence pseudodiaxial 2-H and 3-H. The alternative (incorrect) stereoassignment (“cis-19” in reference [cis-33” in reference [trans-1) would have misguided us to claim erroneously that we discovered a most unusual, highly cis selective olefin bromination.eference  for the 1 and its congeners? Trans-1 and cis-1 undergo regiospecific HBr eliminations even in the absence of bases, cis-1 does so more rapidly than trans-1. The products 3 and 4, respectively, can serve as precursors for the same product 2,3-dibromo-1,1-dimethylindene (7). Exploiting the unusually high inclination of 3,3-di- (8) and 2,3,3-tribromo-1,1-dimethylindane (5) toward “base-free” HBr elimination at close to rt [9) with NBS directly to 7. The preparation of clean 2-bromo-1,1-dimethylindene (4) became possible through purification and hydrolysis of crystalline 2-bromo-3-lithio-1,1-dimethylindene (10).What else may appear unusual with se to rt , we wereGeneral remark. 1H and 13C NMR chemical shifts δ (ppm) were referenced to internal tetramethylsilane.2,3-Dibromo-1,1-dimethylindane (1). a) Trans-1: Tetraethylammonium bromide  was dissolved with magnetic stirring in a minimum volume of chloroform (120 mL), treated with 1,1-dimethylindene  until alkaline, washed with distilled water until neutral, dried over Na2SO4, and concentrated. The crude material  was a colorless, liquid mixture of trans-1 and cis-1 (9:1) with bp 145–149 °C/11 Torr  δ 1.22 , 1.42 , 4.35 , 5.40 , 7.17 , 7.28 , 7.31 , 7.41  ppm, assigned through HSQC, HMBC (see below), and the following NOESY correlations: 2-H ↔ pseudoequatorial 1-CH3 ↔ 7-H ↔ pseudoaxial 1-CH3 ↔ 2-H (very weak), 4-H ↔ pseudoaxial 3-H ↔ pseudoaxial 1-CH3 (strong); 1H NMR  δ 1.21 and 1.43 , 4.45 and 5.58 , ca. 7.2  ppm; 13C NMR  δ 25.77 , 26.98 , 46.95 (C-1), 56.61 (C-3), 68.31 (C-2), 122.16 (C-7), 125.85 (C-4), 127.94 (C-5), 129.43 (C-6), 138.70 (C-3a), 147.68 (C-7a) ppm, assigned through HSQC and the following 1H/13C HMBC 3J and 2J interactions. 3J: 2-H → both 1-CH3, pseudoaxial 3-H → neither C-7a nor C-1, 4-H → C-6, 7-H → C-5, both 1-CH3 → C-2 and C-7a, C-3a → 5-H and 7-H, C-4 → 6-H, C-7 → 5-H, C-7a → 4-H and 6-H, pseudoaxial 1-CH3 → pseudoequatorial 1-CH3, pseudoequatorial 1-CH3 → pseudoaxial 1-CH3; 2J: 2-H → C-3, 3-H → C-2 and C-3a, both 1-CH3 → C-1. Anal. calcd for C11H12Br2 (304.02): C, 43.46; H, 3.98; found: C, 44.06; H, 3.92.Cis-1: This was analyzed in the 3:7 mixture with trans-1 as obtained from 1,1-dimethylindene (2) through titration with elemental bromine (not Br3−). 1H NMR  δ 1.33 and 1.42 , 4.34 , 5.55 , 7.20 , 7.27 , 7.34 , 7.42  ppm, assigned through HSQC, HMBC (see below), and the following NOESY correlations: 2-H ↔ both 1-CH3 ↔ 7-H, but pseudoequatorial 3-H ↔ 4-H only (not 1-CH3); 1H NMR  δ 4.54 and 5.76  ppm; 13C NMR  δ 26.07 and 26.58 (2 × 1-CH3), 47.23 (C-1), 55.70 (C-3), 62.07 (C-2), 123.00 (C-7), 125.56 (C-4), 127.83 (C-5), 130.00 (C-6), 139.30 (C-3a), 148.67 (C-7a) ppm, assigned through HSQC and the following 1H/13C HMBC 3J and 2J interactions. 3J: 2-H → both 1-CH3, pseudoequatorial 3-H → C-1 (strong) and C-7a (strong), both 1-CH3 → C-2 and C-7a, C-3a → 5-H and 7-H, C-4 → 6-H, C-5 → 7-H, C-6 → 4-H, C-7 → 5-H, C-7a → 4-H and 6-H, uncertain pseudoaxial 1-CH3 → pseudoequatorial 1-CH3, uncertain pseudoequatorial 1-CH3 → pseudoaxial 1-CH3; 2J: 3-H → C-3a but not C-2, both 1-CH3 → C-1; MS (EI) m/z (%): 225.008 , 223.006 ; HRMS (EI) m/z: 223.0164 , no M+ peak.b) 3-Bromo-1,1-dimethylindene (3). A crude sample of the 9:1 mixture of trans-1 and cis-1  was added to a saturated solution of solid KOH  in ethanol (190 mL) and heated to 50 °C for 4 hours. After cautious removal of some ethanol (ca. 130 mL) in vacuo, the residue was poured into distilled water (ca. 400 mL) and extracted with Et2O (4 × 100 mL). The combined Et2O extracts were washed with distilled water until neutral, dried over CaCl2, concentrated, and distilled to yield a pure 92:8 mixture  of 3- and 2-bromo-1,1-dimethylindene (3 and 4) with bp 115–117 °C/12 Torr.1H NMR of 3  δ 1.32 , 6.46 , 7.25 , 7.28 , 7.30 , 7.33  ppm, assigned through HSQC and 1H/13C HMBC (see below); 13C NMR  δ 24.17 (2 × 1-CH3), 49.97 (C-1), 118.19 (C-3), 120.48 (C-4), 120.81 (C-7), 126.41 (C-6), 126.87 (C-5), 140.84 (C-3a), 145.13 (C-2), 151.74 (C-7a) ppm, assigned through HSQC and the following 1H/13C HMBC 3J interactions. 1-CH3 → C-2 and C-7a, 2-H → C-3a and C-7a, C-3 → 4-H → C-6, C-3a → 7-H → C-5, C-4 → 6-H, C-7 → 5-H, C-7a → 2-/4-/6-H; Anal. calcd for C11H11Br (223.13): C, 59.22; H, 4.97; found [t-Bu was rapid and exothermic in diglyme as the solvent.2-Bromo-1,1-dimethylindene (4). A sample of contaminated 2,3-dibromo-1,1-dimethylindene  in pentane (5.0 mL) was treated with n-BuLi (ca. 40 mmol) in hexane (35 mL) at −70 °C under argon gas cover. Since this reaction was run in an acyclic, saturated hydrocarbon as the solvent , a voluminous precipitate began to emerge slowly at rt within ca. one hour. When this colorless powder of 2-bromo-3-lithio-1,1-dimethylindene (10) had settled after 20 hours at rt, the supernatant was withdrawn by syringe, and the residue was washed with dry pentane (3 × 15 mL). This purified, solid material was suspended in pentane at −70 °C under argon gas and quenched with methanol (2.0 mL). After dilution with water and Et2O, the aqueous layer was extracted with Et2O (2×). The combined Et2O phases were washed with water until neutral and dried over Na2SO4. The crude material contained 4 and 1,1-dimethylindene  without any other indene derivative. Pure 4  distilled at 101–103 °C/12 Torr. 1H NMR  δ 1.25 , 6.75 , 7.15 , 7.18 , 7.23 , 7.27  ppm, assigned through comparisons with 3 and 7, HSQC and 1H/13C HMBC (see below), and the NOESY correlation 1-CH3 ↔ 7-H; 1H NMR  δ 1.25 , 6.89  ppm; 13C NMR  δ 23.94 (2 × 1-CH3), 52.05 (C-1), 120.48 (C-4), 121.51 (C-7), 125.11 (C-6), 126.70 (C-5), 128.99 (C-3), 139.86 (C-2), 141.07 (C-3a), 151.66 (C-7a) ppm, assigned through HSQC, comparison with 7, and the following 1H/13C HMBC 3J and 2J interactions. 3J: 1-CH3 → 1-CH3, 1-CH3 → C-2 and C-7a, 3-H → C-1 and C-7a, 7-H → C-3a and C-5, C-6 → 4-H; 2J: 1-CH3 → C-1, 3-H → C-3a; MS  m/z (%) 224 and 222 , 143 , 128 ; HRMS (EI) m/z 224.0026 , 222.0045 ; Anal. calcd for C11H11Br (223.13): C, 59.22; H, 4.97; found: C, 60.55 H, 4.97.2,3,3-Tribromo-1,1-dimethylindane (5): This thermolabile adduct of 3-bromo-1,1-dimethylindene (3) and elemental bromine (see 7) was not purified but recognized through its 1H NMR chemical shifts and its thermolysis product 7. 1H NMR  δ 1.36 , 1.39 , 4.78  ppm; 1H NMR  δ 1.34 , 1.37 , 4.69  ppm.2,2,3-Tribromo-1,1-dimethylindane (6): A crude sample of 2-bromo-1,1-dimethylindene  in CCl4 (4 mL) was overtitrated with elemental bromine in CCl4 solution at rt. After 3 hours, the excess of bromine was swept off in a stream of N2 gas or destroyed with aqueous sodium sulfite. Almost pure 6 distilled at 157–161 °C (bath temp.)/3 mbar as a nearly colorless, viscos liquid . 1H NMR  δ 1.48 , 1.72 , 5.94 , 7.18 , 7.31 , 7.35 , 7.42  ppm, assigned through HMBC (see below) and the NOESY correlations 4-H ↔ pseudoaxial 3-H ↔ pseudoaxial 1-CH3 ↔ 7-H ↔ pseudoequatorial 1-CH3; 13C NMR  δ 27.49 , 27.84 , 55.18 (C-1), 63.97 (C-3), 84.42 (C-2), 122.45 (C-7), 125.64 (C-4), 128.03 (C-5), 129.77 (C-6), 137.56 (C-3a), 146.18 (C-7a) ppm, assigned through HSQC and the following 3J and 2J HMBC cross peaks. 3J: both 1-CH3 → C-2 and C-7a, 3-H → C-4 and C-7a , 4-H → C-6 and C-7a, 7-H → C-3a and C-5, C-4 → 6-H, C-3a → 5-H; 2J: both 1-CH3 → C-1, 3-H → C-3a; HRMS and MS (EI) m/z (%) 385.8143 , 383.8302 , 381.8358 , 379.8274 , 304.98 , 302.98 , 300.98 .2,3-Dibromo-1,1-dimethylindene (7): a) From 3-bromo-1,1-dimethylindene (3): A solution of 3  in CCl4 (10 mL) was cooled and stirred in an ice-bath and titrated with elemental bromine  in CCl4. The initially instantaneous decolorization of bromine became progressively slower with a half-reaction time of roughly 2 min toward the end. After 2 hours at rt, the solution contained some product 7 and mainly 2,3,3-tribromo-1,1-dimethylindane (5). Upon CCl4 evaporation in vacuo up to 80 °C (darkening), the primary product 5 became thermally converted into 7 during distillation that yielded 7  as a colorless, slightly light-sensitive liquid; analytically pure 7 had bp 144–146 °C/13 Torr. 1H NMR  δ 1.31 , 7.26 , 7.30 , 7.31 , 7.36  ppm, assigned through HSQC, comparison with 3, and the NOESY correlation 1-CH3 ↔ 7-H; 13C NMR  δ 24.2 (2 × 1-CH3), 52.6 (C-1), 120.0 (C-3), 120.3 (C-4), 121.3 (C-7), 126.4 (C-6), 127.2 (C-5), 138.9 (C-2), 139.9 (C-3a), 150.0 (C-7a) ppm, assigned through HSQC, comparison with 3, and the following 3J and 2J HMBC cross peaks. 3J: 1-CH3 → C-2 and C-7a, 4-H → C-3 and C-6 and C-7a, 7-H → C-3a and C-5, C-4 → 6-H, C-7 → 5-H; 2J: 1-CH3 → C-1; HRMS (EI) m/z (%) 303.9135 , 301.9119 , 299.9146 , 222.9936 , 220.9974 ; Anal. calcd for C11H10Br2 (302.0): C, 43.75; H, 3.34; Br, 52.92; found: C, 44.06; H, 3.42; Br, 52.00.4) via 2,2,3-tribromo-1,1-dimethylindane (6): A small sample  of distilled tribromide 6 (obtained as above from 4 and containing no trace of 7) in CCl4 was placed in an NMR tube (5 mm) and treated with an excess of solid KOt-Bu, which consumed 6 within less than 2 hours at rt. Aqueous work-up with Et2O afforded the dibromide 7  as the only product . NEt3 as the base in place of KOt-Bu required 12 days at rt.b) From 2-bromo-1,1-dimethylindene (9): A mixture of N-bromosuccinimide , 1,1-dimethylindane  was treated with azobis(isobutyronitrile) (40 mg) and warmed slowly up to 85 °C. After 30 min of vived refluxing, the dark red suspension showed traces of elemental bromine in the gas phase and was cooled in an ice bath (15 min). A 1H NMR spectrum of the solution revealed that the starting material 9 was completely consumed and that a mixture containing three gem-dimethyl compounds had been generated: 2,3-dibromo-1,1-dimethylindene , 1 , and an unknown side-product (20%). The suspension was filtered, and the undissolved portion was washed with CCl4 (2 × 5 mL), affording a colorless, powdery mixture of NBS and succinimide (4:36 by 1H NMR). The dark red CCl4 filtrate became colorless on shaking with an aqueous solution of sodium sulfite (at least 0.6 g) and was washed with distilled water (10 mL), then dried through stirring with granulated CaCl2 (30 min). After removal of CaCl2, the colorless solution became violet on stirring with a sufficient amount of solid KOt-Bu (2.6 g) for 30 min, which destroyed the two dibromides 1 and other small contaminations. The CCl4 solution was shaken with H2O (10 mL), aqueous HCl , and H2O until neutral, then dried as above with CaCl2 (35 min). Evaporation of CCl4 and subsequent distillation in vacuo furnished the colorless liquid 7 .c) From 1,1-dimethylindane (File 12) and congeners; experimental procedures for 2, 1,1-dimehylindane (9), 3-methyl-1-phenylbutan-2-ol, N-hydrazine, and N,N´-bishydrazine.Alternative synthetic routes to 1,1-dimethylindene (


Pseudoalteromonas strains P1-7a, P1-9, P1-13-1a, P1-16-1b, P1-25, and P1-26, isolated from Hydractinia echinata. These genomes will allow detailed studies on bacterial factors mediating interkingdom communication.To gain a broader understanding of the importance of a surface-associated lifestyle and morphogenic capability, we have assembled and annotated the genome sequences of  Pseudoalteromonas strains P1-7a, P1-9, P1-13-1a, P1-16-1b, P1-25, and P1-26 were isolated from the tissue of a feeding polyp of the marine hydroid Hydractinia echinata , 96 tRNAs, and 4 rRNAs.The draft genome of strain P1-9 was sequenced to 47× coverage, and comprises 211 contigs totaling 4,808,111 bases in length and having a G+C content of 40.7%. Its annotation includes 4,321 CDSs, 84 tRNAs, and 3 rRNAs.The draft genome sequence of strain P1-13-1a was sequenced to 51× coverage, and comprises 174 contigs totaling 4,442,776 bases in length and having a G+C content of 40.7%. Its annotation includes 3,930 CDSs, 93 tRNAs, and 3 rRNAs.The draft genome of strain P1-16-1b was sequenced to 57× coverage, and comprises 90 contigs totaling 3,977,637 bases in length and having a G+C content of 40.1%. Its annotation includes 3,562 CDSs, 90 tRNAs, and 4 rRNAs.The draft genome sequence of strain P1-25 was sequenced to 51× coverage, and comprises 163 contigs totaling 4,399,610 bases in length and having a G+C content of 40.7%. Its annotation includes 3,855 CDS, 97 tRNAs, and 3 rRNAs.The draft genome sequence of strain P1-26 was sequenced to 48× coverage, and comprises 219 contigs totaling 4,715,935 bases in length and having a G+C content of 41.2%. Its annotation includes 4,183 CDS, 96 tRNAs, and 4 rRNAs.H. echinata settlement and metamorphosis , biofilm formation  , secondaorphosis , and helLKDU00000000, LKBD00000000, LKDV00000000, LKGQ00000000, LKDW00000000, and LKDX00000000, respectively. The versions described in this paper are the first versions, LKDU01000000, LKBD01000000, LKDV01000000, LKGQ01000000, LKDW01000000, and LKDX01000000.These whole-genome shotgun projects for strains P1-7a, P1-9, P1-13-1a, P1-16-1b, P1-25, and P1-26 have been deposited in DDBJ/EMBL/GenBank under the accession numbers


Their structures were elucidated as (S)-17-hydroxy-18,20-ene-neogrifolin (1), (S)-18,19-dihydroxyneogrifolin (2), (S)-9-hydroxy-10,22-ene-neogrifolin (3), -6,10-epoxy-9-hydroxyneo grifolin (4), -6,9-epoxy-10-hydroxyneogrifolin (5), (−)-13,14-dihydroxyneogrifolin (6), albatrelin G (7), albatrelin H (8), and one grifolin analogue, (S)-10-hydroxygrifolin (9), grifolin (10), neogrifolin (11), and albatrellin (12) by extensive spectroscopic analyses and chemical methods. Compounds 7 and 8 showed weak cytotoxic activity to cell lines HL-60, SMMC-7721, A-549, and MCF-7, in vitro.Nine previously-unreported farnesylphenols, involving eight neogrifolin derivatives (The online version of this article (doi:10.1007/s13659-014-0015-5) contains supplementary material, which is available to authorized users. Albatrellus genus are well known for producing farnesylphenols, such as grifolin, neogrifolin and their derivatives , ne, neAlbatA. caeruleoporus was subjected to silica gel, RP-18, Sephadex LH-20 column chromatography (CC), and semipreparative HPLC purification steps to give compounds 1–12 -17-hydroxy-18,20-ene-neogrifolin (1), (S)-18,19-dihydroxyneogrifolin (2), (S)-9-hydroxy-10,22-ene-neogrifolin (3), -6,10-epoxy-9-hydroxyneogrifolin (4), -6,9-epoxy-10-hydroxyneogrifolin (5), (−)-13,14-dihydroxyneogrifolin (6), albatrelin G (7), albatrelin H (8), were neogrifolin derivatives, (S)-10-hydroxygrifolin (9) was a grifolin analogue, and compound 12 was a violet pigment named albatrellin.The chloroform–methanol 1:1) extract of fruiting bodies of :1 extrac1, colorless oil, displayed a [M]+ ion peak at m/z 344.2348 in positive HREIMS, corresponding to the molecular formula C22H32O3 and seven degrees of unsaturation. The IR spectrum showed absorption at 3421 cm−1 which indicated the presence of OH groups. The 1H NMR spectrum contained signals for two m-coupling aromatic protons at δH 6.25  and 6.17 , four olefinic protons, and four singlet methyls. Combined with 13C NMR (DEPT) experiment, the existence of a 1,2,4,6-tetra-substituted phenyl ring, a terminal double bond, two tri-substituted double bonds, one oxygen-bearing methine, five methylenes, and four methyls were assigned. The 1H and 13C NMR spectroscopic data were close to those of neogrifolin, except that a methylene and a methyl group in neogrifolin were replaced by a hydroxyl methine (δC 75.3) and a terminal double bond (δC 110.3 and 149.3), respectively. According to the observed HMBC correlations from δH 6.25 and 6.17 (H-20) to δC 17.8 (C-19) and 75.3 (C-17), the terminal double bond was located at the end of the farnesyl side chain, and the hydroxyl group was at C-17 (δC 75.3). This conclusion was supported by cross peaks from δH 3.73 (OH-17) to δC 34.5 (C-16), 75.3 (C-17) and 149.3 (C-18), and from 3.97 (H-17) to δC 36.4 (C-15) in HMBC spectrum  with that of (R)-(+)-3-methyl-3-buten-2-ol  +). The 13C NMR (DEPT) spectra showed signals of a tetra-substituted phenyl moiety, a terminal double bond, two tri-substituted double bonds, a hydroxyl methine, four methyls, and five methylenes, which resembled those of compound 1. Extensive 2D NMR  analyses revealed that the locations of the double bond and the oxygen-bearing methine were different with those of 1. COSY correlations from δH 2.82 and 2.68 (H-8) to δH 4.28 (δC = 77.0) suggested that C-9 was the oxygenated carbon. Moreover, δH 5.11 and 4.84  gave HMBC correlations to δC 32.5 (C-11) and 77.0 (C-9), revealing that the Me-22 in compound 1 converted to be a double bond in 3. Therefore, the structure of compound 3 was identified as 9-hydroxy-10,22-ene-neogrifolin. The absolute stereochemistry of the chiral center of C-9 was determined to be S by comparing the optical rotation value of 3  with (S)-3-methyl-1-phenylbutan-2-ol  + ion peak at m/z 344.2348, indicating a molecular formula of C22H32O3 and seven degrees of unsaturation. A comparison of the MS and 1D NMR data of 4 with those of 3 revealed that 4 was another neogrifolin analogue resembled 3 except for the double bond between C-10 and C-22 in 3 being replaced by a methyl (Me-22) and an oxygenated quaternary carbon (C-10) in 4. This structure requires six degrees of unsaturation, and an additional ring was needed to complete the unsaturation. There were two plausible proposals: an epoxy ring between C-6 and C-9 or between C-6 and C-10. HMBC correlations from H-9 (δH 3.85) to C-1 (δC 111.0), C-10 (δC 78.6), C-11 (δC 38.7), and C-22 (δC 17.9), and from the OH at δH 4.14 to C-8 (δC 29.7), C-9 (δC 68.4) and C-10 (δC 78.6) revealed the location of the free OH at C-9, placing the epoxy ring between C-6 and C-10. Therefore, the planar structure of 4 was elucidated as 6,10-epoxy-9-hydroxyneogrifolin. The ROESY spectrum displayed cross peaks of H-9/H-11 and H-9/H-12, suggesting the same orientation of H-9 and the geranyl group. From a biogenetic point of view, compounds 4 and 3 should share the same absolute configuration on C-9. So the absolute stereochemistry of compound 4 was deduced to be 9S, 10R.The HREIMS of compound 5 was proposed to be a neogrifolin derivative on basis of HREIMS which displayed the molecular ion peak at m/z 344.2358. A comparison of the 13C NMR (DEPT) spectra of 5 with those of 4 revealed the resemblance of the two structures, for example, the presence of the 1--phenyl group and the geranyl moiety. The structural difference between compounds 4 and 5 was the fragment from C-8 to C-10, according to the different chemical shifts of the corresponding carbons and protons -6,9-epoxy-10-hydroxyneogrifolin.Compound 6 was obtained as a colorless oil, with a molecular formula of C22H34O4 according to the HREIMS at m/z 362.2432 ([M]+). Inspection of the 1H, 13C (DEPT) and HSQC NMR spectra allowed the assignment of five methyls, five methylenes, five methines, seven quaternary carbons, and four active protons. Comparing the 1H and 13C NMR spectroscopic data of 6 with those of neogrifolin indicated that compound 6 shared the “1--phenyl” partial structure with neogrifolin, but had a different side chain, in which one double bond in the farnesyl group was replaced by two oxygen-bearing sp3 carbons. The COSY cross peaks of H-11/H-12/H-13, and HMBC correlations from OH at δH 3.56 to C-12, C-13 and C-14, and from δH 3.20 to C-21, C-13, C-14, and C-15  and C-14 . In order to identify the relative configuration of the two chiral centers C-13 and C-14, compound 6 was reacted with 2,2-dimethoxypropane in DMF for 30 min at room temperature to yield its di-O-isopropylidene derivative 6a. The observed ROESY correlations of Me-21/H-13 -phenyl group. Combined with MS spectral data, compound 7 was determined to be a neogrifolin derivative unambiguously. Unlike the other neogrifolin analogues (1–6) which had straight-chains as “tails”, compound 7 had a cyclohexane moiety—by C-8 connecting to C-13—in its tail. It was supported by COSY correlations of H-9/H-8/H-13/H-12/H-11, and HMBC cross peaks from Me-22 to C-9, C-10 and C-11  signal of H-8 (δH = 3.22) in its 1H NMR spectrum indicated that H-8 existed as an equatorial bond in the stable boat conformation of the cyclohexane moiety, as shown in Fig. δH 3.22 with coupling constants of 12.5 and 2.7 Hz, respectively. Furthermore, The ROESY correlations of H-13/Hax-9 and Me-22/Hax-9 revealed the same orientation of H-13 and Me-22. Therefore, H-8, H-13 and Me-22 were deduced to be α-, α-, α- orientated. In compound 7, a ring was formed by new C–C bond connection between C-8 and C-13 in side chain.Compound 8 exhibited a molecular ion peak at m/z 344.2345 in HREIMS, indicating the molecular formula of C22H32O3 which required seven degrees of unsaturation. According to the 1H and 13C NMR (DEPT) spectra, 22 carbon signals were recognized as five methyls, four methylenes, five methines, and seven quaternary carbons. Extensive NMR analyses suggested that the structure of 8 resembled that of 7, except for the terminal double bond C-14=C-21 in 7 being saturated to be a methyl and an oxygen-bearing quaternary carbon, which was confirmed by HMBC correlations from δH 1.13 (Me-21) to δC 36.8 (C-15), 55.9 (C-13), and 74.0 (C-14), and from δH 3.30 (OH-14) to δC 36.8 (C-15), 55.9 (C-13), and 74.0 (C-14). So far, six degrees of unsaturation was assigned, and one more ring should be constructed to complete the structure of 8. The only possible ring to be formed was the oxygen bridge between C-6 and C-10. The stereochemistry of C-13 was identified by analysis of 1H NMR spectrum, in which H-13 showed a doublet-of-triplets peak with the coupling constants of 12.8 and 2.0 Hz, respectively, suggesting the axial bond of H-13. Me-22 had the same orientation as H-13 by the observed ROESY correlations of H-13/Hax-11/Me-22, and because of the planar structure of the phenyl group, H-8 and Me-22 should be on the same orientation. Thus, H-8, H-13 and Me-22 were determined to be α-, α-, α- orientated, the same as for compound 7.Compound 9 was proposed to possess a molecular formula of C22H34O3 on basis of HREIMS at m/z 346.2505 ([M]+). Its 13C NMR (DEPT) spectrum showed 20 carbon signals, including two signals at δC 108.4 (CH) and 156.7 (C) which represented two carbons respectively. The overlapped carbon signals indicated that 9 was a grifolin derivative possessing a symmetric aromatic ring, which was confirmed by HMBC cross peaks from δH 2.10 (Me-7) to δC 108.4 (C-3 and -5) and 136.5 (C-4), from δH 2.67 (H-8) to δC 113.9 (C-1) and 156.7 (C-2 and -6), and from δH 8.05 (OH-2 and -6) to δC 108.4 (C-3 and -5), 113.9 (C-1) and 156.7 (C-2 and -6). Besides the aromatic ring, the remaining signals represented an oxygenated farnesyl group with four methyls, six methylenes, two pairs of tri-substituted double bonds, and one oxygenated quaternary carbon. The next problem to be resolved was the position of oxygenation, which was addressed by 2D NMR (HMBC and COSY) experiments. The HMBC correlations from δH 2.67 (H-8) to δC 72.6 (C-10), and from δH 1.20 (Me-22) to δC 41.2 (C-9), 72.6 (C-10) and 42.6 (C-11) revealed the hydroxylation of C-10. Hence, the planar structure of 9 was established as 10-hydroxygrifolin. The absolute stereochemistry of C-10 was deduced to be S by a comparison of the optical rotation value of 9  with that of (S)-3-methyl-1-phenyl-3-pentanol  +, 326 [M–H2O]+, 191, 175, 137; HR-EI-MS m/z: 344.2348 [M]+ .Colorless oil; [α]D21 −9.2 ; UV λmax (MeOH) (log ε) 282 (2.67) nm; IR (KBr) νmax 3423, 2974, 2925, 2855, 1613, 1467, 1141 cm−1; 1H and 13C NMR spectroscopic data, see Tables m/z: 363 [M+H]+, 362 [M]+, 344 [M–H2O]+, 191, 175, 137; HR-EI-MS m/z: 362.2452 [M]+ .Colorless oil; [α]D21 −8.8 ; UV λmax (MeOH) (log ε) 283 (2.90) nm; IR (KBr) νmax 3420, 2966, 2923, 2855, 1614, 1447, 1143 cm−1; 1H and 13C NMR spectroscopic data, see Tables m/z: 344 [M]+, 326 [M–H2O]+, 137; HR-EI-MS m/z: 344.2364 [M]+ .Colorless oil; [α]D22 −6.7 ; UV λmax (MeOH) (log ε) 282 (2.74) nm; IR (KBr) νmax 3422, 3038, 2967, 2923, 2854, 1618, 1601, 1460, 1138 cm−1; 1H and 13C NMR spectroscopic data, see Tables m/z: 344 [M]+, 191, 137; HR-EI-MS m/z: 344.2348 [M]+ .Colorless oil; [α]D22 −8.6 ; UV λmax (MeOH) (log ε) 282 (2.82) nm; IR (KBr) νmax 3513, 3405, 2969, 2922, 2856, 1629, 1602, 1495, 1449, 1128 cm−1; 1H and 13C NMR spectroscopic data, see Tables m/z: 344 [M]+, 191, 175, 150; HR-EI-MS m/z: 344.2358 [M]+ .Colorless oil; [α]−8.6 c 0.9, MeOH; D22 −9.2 ; UV λmax (MeOH) (log ε) 282 (2.65) nm; IR (KBr) νmax 3440, 2969, 2924, 2856, 1628, 1452, 1141 cm−1; 1H and 13C NMR spectroscopic data, see Tables m/z: 362 [M]+, 344 [M–H2O]+, 326 [M–2 × H2O]+, 191, 175, 137; HR-EI-MS m/z: 362.2432 [M]+ .Colorless oil; [α]D22 −15.2 ; UV λmax (MeOH) (log ε) 283 (2.93) nm; IR (KBr) νmax 3441, 2969, 2929, 2855, 1640, 1615, 1495, 1452, 1141 cm−1; 1H and 13C NMR spectroscopic data, see Table m/z: 344 [M]+, 326 [M–H2O]+, 175; HR-EI-MS m/z: 344.2348 [M]+ .Colorless oil; [α]D22 −9.1 ; UV λmax (MeOH) (log ε) 284 (2.63) nm; IR (KBr) νmax 3441, 2968, 2929, 2872, 2855, 1615, 1595, 1452, 1145 cm−1; 1H and 13C NMR spectroscopic data, see Table m/z: 344 [M]+, 175, 137; HR-EI-MS m/z: 344.2345 [M]+ .Colorless oil; [α]) 284 2.6 nm; IR ; UV λmax (MeOH) (log ε) 276 (2.50) nm; IR (KBr) νmax 3441, 2967, 2925, 2856, 1628, 1598, 1452, 1381, 1050 cm−1; 1H and 13C NMR spectroscopic data, see Table m/z: 346 [M]+, 328 [M–H2O]+, 175, 137; HR-EI-MS m/z: 346.2505 [M]+ .Colorless oil; [α]6  in DMF (2 mL) were added 2,2-dimethoxypropane  and p-toluenesulfonic acid monohydrate , and the mixture was stirred for 30 min at room temperature. The reaction mixture was added into water, and then extracted by EtOAc for three times. The organic layer was evaporated and the residue was chromatographed on a column of silica gel eluting with petroleum ether-acetone 40:1 to yield 6a (2.1 mg).To a solution of compound 2. Cell viability was assessed by conducting colorimetric measurements of the amount of insoluble formazan formed in living cells based on the reduction of 3--2,5-diphenyltetrazolium bromide (MTT) . Briefly, 100 μL of adherent cells were seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before drug addition, both with an initial density of 1 × 105 cells/mL in 100 μL of medium. Each tumor cell line was exposed to the test compounds at various concentrations in triplicate for 48 h, with DDP and toxal as positive controls. After the incubation, MTT (100 μg) was added to each well, and the incubation continued for 4 h at 37 °C. The cells lysed with 200 μL SDS after removal of 100 μL of medium. The optical density of lysate was measured at 595 nm in a 96-well microtiter plate reader (Bio-Rad 680). The IC50 value of each compound was calculated by Reed and Muench’s method [The following human tumor cell lines were used: HL-60, SMMC-7712, A-549, MCF-7, and SW480. All the cells were cultured in RMPI-1640 or DMEM medium , supplemented with 10 % fetal bovine serum (Hyclone) at 37 °C in a humidified atmosphere with 5 % COs method .Supplementary material 1 (DOC 8005 kb)Below is the link to the electronic supplementary material.


Acer tegmentosum (3 kg) was extracted using hot water, and the freeze-dried extract powder was partitioned successively using dichloromethane (DCM), ethyl acetate (EA), butyl alcohol (n-BuOH), and water. From the EA extract fraction (1.24 g), five phenolic compounds were isolated by the silica gel, octadecyl silica gel, and Sephadex LH-20 column chromatography. Based on spectroscopic methods such as 1H-NMR, 13C-NMR, and LC/MS the chemical structures of the compounds were confirmed as feniculin (1), avicularin (2), (+)-catechin (3), (−)-epicatechin (4), and 6′-O-galloyl salidroside (5). Moreover, a rapid on-line screening HPLC-ABTS+ system for individual bioactivity of the EA-soluble fraction (five phenolic compounds) was developed. The results indicated that compounds 1 and 2 were first isolated from the A. tegmentosum. The anti-inflammatory activities and on-line screening HPLC-ABTS+ assay method of these compounds in LPS-stimulated murine macrophages were rapid and efficient for the investigation of bioactivity of A. tegmentosum.The  Acer (A.) tegmentosum  is a type of deciduous tree distributed in the Northeast Asia including Korea, Russia, and China . Eluted fractions were monitored by TLC to produce 12 fractions (OS1E-1–12). Fraction OS1E-6 (55.5 mg) was subjected to Sephadex LH-20 c.c.  yielding seven fractions (OS1E-6-1–7) and ultimately produced compounds 1  and 2 . Fraction OS1E-7 (260.3 mg) was subjected to Sephadex LH-20 c.c.  yielding 10 fractions (OS1E-7-1–10). Fraction OS1E-7-6 (53.1 mg) was subjected to ODS c.c. (φ 1.5 × 8 cm) and eluted with MeOH–H2O [1 : 6 (900 mL) → MeOH] to afford compounds 3  and 4 . Fraction OS1E-9 (237.5 mg) was subjected to ODS c.c. (φ 2.5 × 8 cm) and eluted with MeOH–H2O [1 : 3 (450 mL) → 1 : 2 (300 mL) → 1 : 1 (300 mL) → MeOH] yielding 13 fractions (OS1E-9-1–13). Fraction OS1E-9-3 (77.8 mg) was subjected to silica gel c.c. (φ 1.5 × 10 cm) and eluted with CHCl3–MeOH–H2O [10 : 3 : 1 (2.2 L) → 7 : 3 : 1 (1.6 L) → 4 : 3 : 1 (1.4 L) → MeOH] to afford compound 5 . Based on the spectroscopic methods, such as 1H-NMR, 13C-NMR, and LC/MS, the chemical structures of the compounds were confirmed as feniculin (1), avicularin (2), (+)-catechin (3), (−)-epicatechin (4), and 6′-O-galloyl salidroside (5) by comparison of spectral data with those of references. Moreover, the on-line screening HPLC-ABTS+ assay method was rapid and efficient for the investigation of bioactivity of the isolated compounds from Acer tegmentosum. Compounds 1, 2, 3, 4, and 5 were developed on ODS TLC with 10% H2SO4 and the expected yellow or brown color was observed for the phenolic compounds.The stems ofCompound 1 (Feniculin). Yellow amorphous powder (MeOH); ESI-MS m/z 435.1 [M+H]+; 1H-NMR  7.73 , 7.55 , 6.88 , 6.39 , 6.19 , 5.16 , 3.89 , 3.82 , 3.80 , 3.64 , 3.44 ; 13C-NMR ; see  1 were compared to the literature value and combined with quercetin and arabinopyranoside to confirm the structure as feniculin +; 1H-NMR  7.52 , 7.49 , 6.90 , 6.38 , 6.20 , 5.46 , 4.32 , 3.89 , 3.861 , 3.494 ; 13C-NMR ; see 1H-NMR spectrum of compound 2 showed a similar pattern to compound 1. The structure was confirmed by ESI-MS m/z 435.1 [M+H]+ [ 1 and 2 were isolated from the plant for the first time.1 [M+H]+ . CompounCompound 3 [(+)-Catechin]. Brown powder (MeOH); ESI-MS m/z 291.0 [M+H]+; 1H-NMR  6.83 , 6.76 , 6.71 , 5.92 , 5.85 , 4.56 , 3.97 , 2.50 , 2.84 ; 13C-NMR ; see Compound 4 [(−)-Epicatechin]. Brown powder (MeOH); ESI-MS m/z 291.0 [M+H]+; 1H-NMR  6.96 , 6.79 , 6.75 , 5.93 , 5.91 , 4.80 , 4.16 , 2.86 , 2.73 ; 13C-NMR ; see  3 and 4 were compared to the literature data for (+)-catechin and (−)-epicatechin for the structure identification [ m/z at 291.0 [M+H]+ in the ESI-MS positive mode.fication . The strCompound 5 . Pale yellow oil (MeOH); ESI-MS m/z 453.1 [M+H]+; 1H-NMR  7.10 , 6.96 , 6.65 , 6.65 , 4.52 , 4.45 , 4.32 , 3.95 , 3.70 , 3.55 , 3.42 , 3.22 , 2.77 ; 13C-NMR ; see 1H-NMR data were consistent with the literature values + signals at 435.1, 435.1, 291.0, 291.0, and 453.1 m/z values, respectively.μM, indicating that the compounds were not toxic to cells.We evaluated the cytotoxicity of five compounds using the MTT assay to determine the optimal concentration effective for anti-inflammation with minimum toxicity. As shown in μM dexamethasone, which is widely employed as an anti-inflammatory agent. All the compounds, except 6′-O-galloyl salidroside, did not show any inhibitory effect on LPS-induced NO production -Catechin inhibited IL-6 production at 30 μM or more. (−)-Epicatechin and 6′-O-galloyl salidroside did not show any inhibitory effect on LPS-induced IL-6 secretion. However, all the compounds except 6′-O-galloyl salidroside did not inhibit IL-1β . Five phenolic compounds were isolated by the silica gel, octadecyl silica gel, and Sephadex LH-20 column chromatography. The chemical structures of the isolated compounds were determined by spectroscopic methods, such as 1H-NMR, 13C-NMR, and LC/MS and were confirmed as feniculin (1), avicularin (2), (+)-catechin (3), (−)-epicatechin (4), and 6′-O-galloyl salidroside (5) by comparison of spectral data with those of references. The EA extract of A. tegmentosum containing five phenolic compounds exhibiting the best bioactivity was further monitored by an on-line screening HPLC-ABTS+assay method. Compounds 1 and 2 were isolated for the first time, and their anti-inflammatory activities were evaluated. Moreover, the on-line screening HPLC-ABTS+ assay method was rapid and efficient to search for bioactive compound from A. tegmentosum. Furthermore, (+)-catechin and 6′-O-galloyl salidroside exhibited the inhibitory activities on inflammatory mediator production such as TNF-α, IL-6, and IL-1β cytokines. In conclusion, A. tegmentosum can be used as a basic material for the development of new drugs in OMHs.This study showed that among the soluble fractions from the hot water extract of


We also isolated 1 and 2 together with 3 from A. fumigatus OUPS-T106B-5 separated from the marine fish Mugil cephalus. In this study, we re-examined the stereochemistry of 1 and 2 using chemical transformation and the CD spectra, and found the relationship between the CD Cotton effect and the absolute configurations of 1 and 2, which led us to revise the stereostructure of pseurotin A2.Pseurotins A A close inspection of the 1H- and 13C-NMR spectra of 1 (1H-13C correlation spectroscopy (HMQC) revealed the presence of one primary methyl (C-15), one olefinic methyl (C-16), one methoxy group (8-OCH3), one sp3-hybridized methylene (C-14), three oxygen-bearing sp3-methines , two olefin sp2-methines (C-12 and C-13), two oxygen-bearing quaternary sp3-carbons (C-5 and C-8), five aromatic protons , three quaternary sp2-carbons  including one oxygen-bearing quaternary carbons (C-2), two conjugated carbonyl groups (C-4 and C-17), one amido (C-6 and N-7) and one hydroxy group (9-OH). The connection of these units was determined on the basis of 1H–1H COSY and HMBC correlations to reveal the planar structure of 1, which was identified as being the same as that of pseurotin A1 by comparison with data in the literature + . 1H-NMR δ ppm (CDCl3): 0.95 , 1.43 , 1.58 , 1.73 , 2.04 , 2.17 , 2.76 , 3.31 , 4.77 , 5.15 , 5.25 , 5.57 , 5.57 , 7.33 , 7.43 , 7.66 , 8.22 .Acetonide 7: Pale yellow oil; FABMS m/z (rel. int.): HRFABMS m/z 472.1964 [M + H]+ . 1H-NMR δ ppm (CDCl3): 1.02 , 1.49 , 1.64 , 1.67 , 2.13 , 2.20 , 3.29 , 3.36 , 4.52 , 5.17 , 5.26 , 5.57 , 5.72 , 7.49 , 7.64 , 8.29 .Acetonide et al. [2 as 2 from NOESY experiments and a comparison of CD spectra with cephalimysins. We assigned the CD Cotton effect ascribed to the absolute configuration at C-8 by the epimerization of pseurotin A (3), and revised the stereostructure of pseurotin A2 from 2 to 4. In this process, we newly found the Cotton effect ascribed to the absolute configuration at C-5. In addition, we found that the absolute configuration in the side chain of pseurotins could be established positively by detailed analyses of the NOESY experiments of their acetonide derivatives.Q.Q. Gu et al.  deduced


To study the main clinical manifestations and laboratory findings presented by patients with cow's milk enteropathy (CME).Medical records of 40 children and adolescents were studied, 21 males and 19 females with a mean age of 4, 2625 years (maximum: 12 years / Min: 2 months) with the diagnosis of food allergy and CME.Chief Complain: 1 - weightloss (16-40%), 2 - abdominal pain (14-35%), 3 - diarrhea (13 - 32.5%), 4 – associated respiratory complains (12-30% ), 5 - vomiting (7 - 17.5%). Other clinical Picture with less ferquency: constipation and abdominal distension.Malt systemn affected and Homing response: GALT - 40 (100%), BALT - 30 (75%), SALT - 28 (70%), CNSALT - 18 (45%)Clinical main manifestations in each organ of shock: GALT: Diarrhea (19 to 47.5%), abdominal pain (15 - 37.5%), bulkystools (14-35%), lack of appetite (12-30%). Other less frequent: vomiting, reflux, bloating and abdominal distention. BALT: Rhinitis (11-36.66%), asthma / bronchitis (10 - 33.33%), pharyngotonsillitis (9-30%), phlegm (9-30%). Other less frequent: otitis, sinusitis, snoring and coughing. SALT: Facial pale (18 - 64.29%), shiners (9 - 32.14%), prurigoestrófilo (8 - 28.57%), atopic eczema . Other less frequent: urticaria and erythemaperianal. CNSALT: Irritability (9- 50%), sleepdisorder (7- 38.88%), Hyperactivity (4- 22.22%), headache (3- 16.66%). Other less frequent: ADHD and fatigue.IgE: IgE increased: 20 (52.63%), the normal IgE: 18 (47.36%). (2 patients IgE Unknown); Ratio CD4 / CD8: CD4 / CD8 ratio was low in 16 cases (43.24%), CD4 / CD8 ratio was high in 10 cases (27.07%) and CD4 / CD8 ratio was Normal: 11 (29.72% ). (3 patients with CD4 / CD8 ratio unknown) .IGG4 greater than IgG3: normal relationship: IgG3>IgG4 in 20 cases (60.6%); ratio reversed: IgG4>IgG3 in 13 cases 13 (39.4%) and 7 patientswith IgG3 and IgG4 Unknown.This study adds important clinical data to the understanding of cow's milk enteropathy. In our experience this clinical entity, occurs at any age, from infancy to adolescence and affect both sexes.


Some of the newly synthesized analogues were chosen to evaluate their cytotoxic activity against human carcinoma cell lines (HePG2– MCF7– A549). The docking and the cytotoxic activity results revealed that nearly all of the compounds containing N-phenyl aniline showed signiﬁcant inhibition for the three cell lines.Different acid chlorides (2a-d) reacted with anthranilic acid to produce 2-substituted-3, 1-benzoxazin-4-one (3a-d) which was used as starting material to synthesize some condensed and non-condensed heterocyclic compounds by reaction with nitrogen nucleophiles  The synthesis of quinazolinone heterocycles has become the cornerstone for synthetic chemists and gained extensive importance in medicinal chemistry because of their diverse pharmacological activities including anti-mycobacterial -3, anti-H)-ones are among the versatile heterocyclic compounds, as they have a broad spectrum of pharmacological activities like anti-inflammatory . An EGFR expression level often correlates with vascularity, and is associated with poor prognosis in patients. Inhibitors of the EGFR protein tyrosine kinase are, therefore, expected to have great therapeutic potential in the treatment of malignant and nonmalignant epithelial diseases , which is cellular trans-membrane tyrosine kinase, is over-expressed in a signiﬁcant number of human tumors  at the Central Services Laboratory, National Research Center, Dokki, Cairo, Egypt. The mass spectra were measured with a Finnigan MAT SSQ-7000 mass spectrometer at the Central Services Laboratory, Cairo University, Giza, Egypt. Follow up of the reactions and checking the purity of the compounds were made by TLC on silica gel-precoated aluminum sheets  and the spots were detected by exposure to UV analysis lamp at λ 254/366 nm for few seconds. All melting points are uncorrected and were taken on electro-thermal capillary melting point apparatus. Infrared spectra were recorded on a Jasco FT/IR-6100, Fourier transforms, Infrared spectrometer (Japan) at cmGeneral procedure for the preparation of compounds A solution of acid chloride  (0.01 mol) and anthranilic acid (0.01 mol) in dry pyridine (30 mL) was refluxed for 3 h, the reaction mixture was cooled and poured into cold diluted HCl. The precipitate was collected by filtration and recrystallized from a proper solvent to give . Spectroscopic data for all the compounds are given below.2-(Pyridin-3-yl)-4H-3, 1-benzoxazin-4-one (3a)-1): 1700 (C = O). 1H NMR : 7.50-9.03 . MS: (m/z) ≈ 224 (10%). Anal. Calcd for C13H8N2O2 (224.21): C, 69.64; H, 3.60; N, 12.49%. Found: C, 69.43; H, 3.44; N, 12.14%.Yield 80%. Yellow, white crystals. mp. 210-217 ˚C, IR -4H-3, 1-benzoxazin-4-one (3c)-1): 1692 (C = O). 1H NMR : 7.42-9.21 . MS: (m/z) ≈ 224 (15%). Anal. Calcd for C13H8N2O2 (224.21): C, 69.64; H, 3.60; N, 12.49%. Found: C, 69.55; H, 3.51; N, 12.25%.Yield 85%. Yellow crystals. mp >300 ˚C, IR  phenyl]-4H-3, 1-benzoxazin-4-one (3d)-1): 1690 (C = O) and 3170 (NH). 1H NMR : 7.20-8.20 , 11.72 . MS: (m/z) ≈ 314 (5%). Anal. Calcd for C20H14N2O2 (314.33): C, 76.42; H, 4.49; N, 8.91%. Found: C, 76.03; H, 4.20; N, 8.34%.Yield 85%. Yellow crystals. mp. 235-240 ˚C, IR A mixture of 45)) (0.02-(Pyridin-3-yl) quinazolin-4 (3H)-one (4a):-1): 1700 (C = O) and 3299 (NH). 1H NMR : 7.23-8.32 , 12 . MS: (m/z) ≈ 223 (0.13%). Anal. Calcd for C13H9N3O (223.23): C, 69.95; H, 4.06; N, 18.82%. Found: C, 69.62; H, 3.88; N, 18.60%.Yield 65%, White crystals. mp. >300 ˚C, IR -2-(furan-2-yl) ethenyl] quinazolin-4 (3H)-one (4b)2-+ m/z ≈ 301 (3%). Anal. Calcd for C15H10ClN3O2 (299.71): C, 60.11; H, 3.36; N, 14.02%. Found: C, 59.90; H, 2.98; N, 13.90%.Yield 80%. Gray crystals. mp. >300 ˚C, IR -2-[(E)-2-(furan-2-yl) ethenyl] quinazolin-4 (3H)-one (5b)-1): 1690 (C = O) and 1710 (C = O). 1H NMR : 4.90 , 6.23 , 6.70 , 6.95-8.21 . MS: (m/z) ≈ 314 (1.8%), [M+2] + m/z ≈ 316 (1%). Anal. Calcd for C16H11ClN2O3 (314.72): C, 61.06; H, 3.52; N, 8.90%. Found: C, 60.90; H, 3.30; N, 8.67%.Yield 90%. Black crystals. mp. 151-155 ˚C, IR A mixture of  (0.01 mol) and hydrazine hydrate (0.015 mol) was heated in boiling ethanol (30 mL) under reflux for 4 h. Then the mixture was poured into water. The precipitate was collected by filtration, dried and crystallized from the proper solvent to give . Spectroscopic data for all the compounds are given below. 3-(Hydrazinylacetyl)-2-(pyridin-3-yl) quinazolin-4 (3H)-one (6a)-1): 1690, 1700 (2C = O), 3190 (NH) and 3300-3444 (NH2). 1H NMR : 3.55 , 3.80 , 7.58-9.07 , 10.49 . MS: (m/z) ≈ 295 (12%). Anal. Calcd for C15H13N5O2 (295.29): C, 61.01; H, 4.44; N, 23.72%. Found: C, 60.85; H, 4.20; N, 23.50%.Yield 75%. Gray crystals. mp. 106-110 ˚C, IR -2-(furan-2-yl) ethenyl]-3-(hydrazinylacetyl) quinazolin-4 (3H)-one (6b)-1): 1687, 1697 (2C = O), 3174 (NH) and 3320-3400 (NH2). 1H NMR : 3.49 , 3.70 , 6.65, 6.90 , 7.01-8.48 , 11.21 . MS: (m/z) ≈ 310 (3%). Anal. Calcd for C16H14N4O3 (310.30): C, 61.93; H, 4.55; N, 18.06%. Found: C, 61.70; H, 4.35; N, 17.80%.Yield 65%. White crystals. mp. > 300 ˚C, IR A solution of  (44) (0.01 mol) in dry benzene (30 mL) and hydrazine hydrate (0.015 mol) was heated under reflux for 4 h. Then the mixture was poured into water. The precipitate was collected by filtration, dried and crystallized from the proper solvent to give  (44). Spectroscopic data for all the compounds are given below.3-Amino-2-(pyridin-4-yl) quinazolin-4 (3H)-one (7c)-1): 1685 (C = O) and 3311-3420 (NH2). 1H NMR : 7.68-8.66 , 10.08 . MS: (m/z) ≈ 238 (15%). Anal. Calcd for C13H10N4O (238.24): C, 65.54; H, 4.23; N, 23.52%. Found: C, 65.32; H, 4.18; N, 23.40%.Yield 75%, Black crystals. mp. 150-155 ˚C, IR  phenyl] quinazolin-4 (3H)-one (7d)-1): 1700 (C = O), 3172 (NH) and 3300-3434 (NH2). 1H NMR : 3.60 , 6.68-8.54 , 12.01 . MS: (m/z) ≈ 328 (20%). Anal. Calcd for C20H16N4O (328.36): C, 73.15; H, 4.91; N, 17.06%. Found: C, 73.01; H, 4.75; N, 16.90%.Yield 85%. Yellow crystals. mp. 260-265 ˚C, IR A solution of  (44) (0.01 mol), was allowed to react with chloroacetyl chloride (0.01 mol) in refluxing pyridine about 2 h, and then poured over ice/HCl. The precipitate was collected by filtration and crystallized from the proper solvent to give . Spectroscopic data for all the compounds are given below.2-Chloro-N-[4-oxo-2-(pyridin-4-yl) quinazolin-3 (4H)-yl] acetamide (8c)-1): 1698, 1715 (2C = O) and 3175 (NH). 1H NMR : 4.78 , 7.65-8.44 , 11.87 . MS: (m/z) ≈ 314 (8%), [M + 2] + m/z ≈ 316 (4%). Anal. Calcd for C15H11ClN4O2 (314.72): C, 57.24; H, 3.52; N, 17.80%. Found: C, 57.12; H, 3.40; N, 17.60%.Yield 70%. Yellow crystals. mp. > 300 ˚C, IR  phenyl] quinazolin-3 (4H)-yl} acetamide (8d)-1): 1677, 1690 (2C = O) and 3230 (NH). 1H NMR : 4.90 , 6.81-8.20 , 11.90, 12 . MS: (m/z) ≈ 404 (23%), [M + 2] + m/z ≈ 406 (15%). Anal. Calcd for C22H17ClN4O2 (404.84): C, 65.27; H, 4.23; N, 13.84%. Found: C, 65.05; H, 4.18; N, 13.75%.Yield 75%. Black crystals. mp. 190-195 ˚C, IR N, N-dimethylformamide (DMF) (30 mL). Then the mixture was poured into water. The precipitate was collected by filtration, dried and crystallized from the proper solvent to give . Spectroscopic data for all the compounds are given below.A solution of compounds  (44) (0.01 mol) and chloroacetamide (0.015 mol) was refluxed for 3 h in boiling 6-(Pyridin-4-yl)-3, 4-dihydro-2H- triazino  quinazolin-2-one (9c)-1): 1710 (C = O) and 3189 (NH). 1H NMR : 3.76 , 7.33-8.66 , 10.70 . MS: (m/z) ≈ 277 (13%). Anal. Calcd for C15H11N5O (277.28): C, 64.97; H, 4.00; N, 25.26%. Found: C, 64.70; H, 3.88; N, 25.07%.Yield 65%. Black crystals. mp. > 300 ˚C, IR  phenyl]-3, 4-dihydro-2H- triazino  quinazolin-2-one (9d)-1): 1677 (C = O) and 3150 (NH). 1H NMR : 3.65 , 6.87-7.96 , 10.70, 11.30 . MS: (m/z) ≈ 367 (19%). Anal. Calcd for C22H17N5O (367.40): C, 71.92; H, 4.66; N, 19.06%. Found: C, 71.76; H, 4.49; N, 18.89%.Yield 85%. Yellow crystals. mp. 256-260 ˚C, IR A solution of compounds   (0.01 mo1-[4-Oxo-2-(pyridin-4-yl) quinazolin-3 (4H)-yl]-3-phenylthiourea (10c)-1): 1685 (C = O) and 3190 (NH). 1H NMR : 7.33-8.96 , 10.49, 11.01 . MS: (m/z) ≈ 373 (5%). Anal. Calcd for C20H15N5OS (373.43): C, 64.33; H, 4.05; N, 18.75%. Found: C, 64.12; H, 3.90; N, 18.50%.Yield 90%. White crystals. mp. 195-200 ˚C, IR  phenyl) quinazolin-3 (4H)-yl)-3-phenylthiourea (10d)-1): 1700 (C = O) and 3200 (NH). 1H NMR : 7.09-8.24 , 9.77, 9.86, 11.70 . MS: (m/z) ≈ 463 (3%). Anal. Calcd for C27H21N5OS (463.55): C, 69.96; H, 4.57; N, 15.11%. Found: C, 69.69; H, 4.48; N, 14.90%.Yield 80%. Yellow crystals. mp. 200-205 ˚C, IR A solution of   (0.01 moN-[4-oxo-2-(pyridin-4-yl) quinazolin-3 (4H)-yl] benzamide (11c)-1): 1677, 1690 (2C = O) and 3150 (NH). 1H NMR : 7.31-8.42 , 12.01 . MS: (m/z) ≈ 342 (17%). Anal. Calcd for C20H14N4O2 (342.35): C, 70.17; H, 4.12; N, 16.37%. Found: C, 70.02; H, 3.90; N, 16.17%.Yield 70%. Yellow crystals. mp. 180-185 ˚C, IR  phenyl) quinazolin-3 (4H)-yl) benzamide (11d)-1): 1687, 1693 (2C = O) and 3177 (NH). 1H NMR : 7.16-8.45 , 11.01, 12.01 . MS: (m/z) ≈ 432 (10%). Anal. Calcd for C27H20N4O2 (432.47): C, 74.98; H, 4.66; N, 12.95%. Found: C, 74.70; H, 4.50; N, 12.80%.Yield 80%. Yellow crystals. mp. > 300 ˚C, IR A solution of  (0.01 mol) with ammonium acetate (0.01 mol) in acetic acid (30 mL) was heated under reflux for 3 h, then poured into water. The precipitated solid after concentration and cooling was collected by filtration and crystallized from suitable solvent to give . Spectroscopic data for all the compounds are given below.2-Phenyl-5-(pyridin-4-yl)  triazolo  quinazoline (12c)1H NMR : 7.41-8.75 . MS: (m/z) ≈ 323 (33%). Anal. Calcd for C20H13N5 (323.35): C, 74.29; H, 4.05; N, 21.66%. Found: C, 74.11; H, 3.89; N, 21.56%.Yield 65%. Gray crystals. mp. 215-220 ˚C, N-phenyl-2- aniline (12d)-1): 3177 (NH). 1H NMR : 6.69-8.28 , 13 . MS: (m/z) ≈ 413 (11%). Anal. Calcd for C27H19N5 (413.47): C, 78.43; H, 4.63; N, 16.94%. Found: C, 78.22; H, 4.48; N, 16.80%.Yield 85%. Yellow crystals. mp. 240-245 ˚C, IR Cell viability was assessed by the mitochondrial dependent reduction of yellow MTT -2, 5-diphenyl tetrazolium bromide) to purple formazan . 2.Procedure: All the following procedures were done in a sterile area using a Laminar flow cabinet biosafety class II level . Cells were suspended in RPMI 1640 medium for HePG2- MCF7 and DMEM for A549. The media are supplemented with 1% antibiotic-antimycotic mixture , 1% L-glutamine and 10% fetal bovine serum and kept at 37 ºC under 5% CO3 cells/well in fresh complete growth medium in 96-well Microtiter plastic plates at 37 ˚C for 24 h under 5% CO2 using a water jacketed Carbon dioxide incubator . Media was aspirated, fresh medium (without serum) was added and cells were incubated either alone (negative control) or with different concentrations of sample to give a final concentration of (100-50-25-12.5-6.25-3.125-0.78 and 1.56 µg/mL). After 48 h of incubation, the medium was aspirated, 40 µL MTT salt (2.5 μg/mL) were added to each well and incubated for a further four hours at 37 ºC under 5% CO2. To stop the reaction and dissolving the formed crystals, 200 μL of 10% Sodium dodecyl sulphate (SDS) in deionized water was added to each well and incubated overnight at 37 ºC. A positive control which composed of 100 µg/mL was used as a known cytotoxic natural agent who gives 100% lethality under the same conditions  at 595 nm and a reference wavelength of 620 nm. A statistical significance was tested between samples and negative control (cells with vehicle) using independent t-test by SPSS 11 program. DMSO is the vehicle used for dissolution of plant extracts and its final concentration in the cells was less than 0.2%. The percentage of change in viability was calculated according to the formula: 50 and IC90 determination using SPSS 11 program.(Reading of extract/Reading of negative control)-1) x 100. A probit analysis was carried for ICMolecular docking studyAll docking studies were performed using "Internal Coordinate Mechanics" (Molsoft ICM 3.5-0a). Preparation of small molecule*.Mol).Compounds 2d, 3a, 3b, 3d, 4a, 4b, 5a, 5b, 6a, 6b, 7c, 7d, 8d, 9c, 9d, 10c, 10d, 11c, 11d, 12c, 12d were built in Chem Draw Ultra version 11.0 and their energy minimized through Chem3D Ultra version 11.0/MM2, Jop Type: minimum RMS Gradient of 0.100, and saved as MDL Mol File  complex were retrieved from the RCSB Protein Data Bank (http://www.rcsb.org/pdb/ home/home.do). http://www.prosess.ca/)  (ess.ca/) , 2. In oess.ca/) . All bouDocking using Molsoft ICM 3.5-0 a programThe conversion of our PDB file into an ICM object involves the addition of hydrogen bonds, assignment of atom types, and charges from the residue templates, then perform ICM small molecule docking through setup the receptor, review and adjust binding site makes receptor maps, then start docking simulation, followed by displaying the results. ICM stochastic global optimization algorithm attempts to find the global minimum of the energy function that include five grid potentials describing the interaction of the flexible ligand with the receptor and internal conformational energy of the ligand, during this process a stack of alternative low energy conformations is saved. All inhibitors were compared according to the best binding free energy (minimum) obtained among all the run. ChemistryE)-3-(furan-2-yl) prop-2-enoyl chloride, pyridine-4-carbonyl chloride and 2-(phenylamino) benzoyl chloride 2a-d, respectively reacted with anthranilic acid to produce 2-[substituted]-4H-3, 1-benzoxazin-4-one 3a-d (H)-one 4a, b which reacted with chloroacetyl chloride to give 3-(chloroacetyl)-2-[substituted] quinazolin-4 (3H)-one 5a, b (H)-one 6a, b  spectra of compounds 6a, b revealed signals at δ 3.70-3.80 ppm and 10.49-11.21 ppm representing NH2 and NH groups, respectively.Different acid chlorides namely, pyridine-3-carbonyl chloride, -one 7c, d (N-[4-oxo-2-(substituted) quinazolin-3 (4H)-yl] acetamide 8c, d  quinazolin-4 , respectively . Compounn 12c, d .In-vitro Antitumor Screening against A549, HePG2 and MCF7 cell lines50 = 72.2) as shown in 50 53.4 and 66.7 µg/ml, respectively, as shown in 50 81.9 and 90.5 µg/mL, respectively as shown in 50 ,  and  for HePG2, MCF7 and A549 as shown in 50 of 88.4, 92.1 and 45.6, 32.8 µg/mL concentrations, respectively as shown in 50 33.3 and 87.4 µg/mL, respectively. Moreover, Compounds 6a and 12d considered the most potent compounds against the HePG2 cell line, while compounds 7d, 3d, 9d, 10d, 11d and 2d possessed moderate antitumor activity compared to positive control doxorubicin. The cytotoxic potencies of compounds 2d, 3a, 3b, 3d, 4a, 4b, 5a, 5b, 6a, 6b, 7c, 7d, 8d, 9c, 9d, 10c, 10d, 11c, 11d, 12c, 12d against a panel of three human tumor cell lines were investigated and compared with the reference drug doxorubicin . The humDocking analysisCompounds 2d, 3a, 3b, 3d, 4a, 4b, 5a, 5b, 6a, 6b, 7c, 7d, 8d, 9c, 9d, 10c, 10d, 11c, 11d, 12c, 12d were used for docking study. All the calculations were performed using "Internal coordinate Mechanics" (Molsoft ICM 3.5-0a). Molecular modeling docking studies is performed and ICM score values -51 combiAs shown in ConclusionA novel series of some new quinazolin derivatives were synthesized and evaluated as antitumor agents against human carcinoma cell lines (HePG2– MCF7– A549). The antitumor activity results exhibited that, compounds 2d, 3d, 6a, 7d, 10c, 10d showed signiﬁcant and selective inhibition for A549  Figure . On the Structure-activity relationshipN-phenyl aniline showed signiﬁcant inhibition for the tested three cell lines (2). Cyclization of compound 2d afforded compound 3d  are more desirable for achieving the desired activity. Also Certain isothiocyanates have also been shown to bind to the mutated p53 proteins found in many types of tumors, causing an increase in the rate of cell death (4). Compounds which have CO2CH2Cl yielded the least active series of compounds in this study. Which suggests that electron withdrawing groups with lipophilic characteristics like–Cl may not be an ideal substitution to get the good activity of the designed compounds.The activity of the tested compounds could be correlated to structure variation and modiﬁcations. By investigating the variation in the selectivity of the tested compounds over the three cell lines, it was revealed that: (1) the activity of the designed compounds is dependent upon the substituent at the R positions. The obtained screening results showed that, nearly all of the compounds containing pound 3d  with theectively , 3, 4 (3


Scalarispongia sp. The structures of the new compounds were elucidated by 2D-NMR and HRMS analyses. All of the isolated compounds, with the exception of 16-O-deacetyl-12,16-epi-scalarolbutanolide, showed significant in vitro cytotoxicity  against six human cancer cell lines.Eight scalarane sesterterpenoids, including four new compounds, were isolated from the marine sponge  Scalaranes are a group of sesterterpenoids with a characteristic carbon skeleton consisting of four cyclohexane rings joined together, and an optional five-membered heterocycle, which contains an oxygen or a nitrogen . These c1) from marine sponge Scalarispongia scalaris  25D 33.6 ); UV λmax (log ε) 218 (4.05) nm; IR (KBr) νmax 3396, 2941, 1746, 1047, 1047 cm−1; 1H- and 13C-NMR (CDCl3/CD3OD (3:1), 500 and 125 MHz), see m/z 403.2845 [M + H]+ .The collection  was immediately freeze-dried and kept at −20 °C until the time of our investigation. The sponge was extracted using methanol (1 L × 2) and dicholoromethane (1 L × 1) at room temperature. The combined extract (18.8 g) was partitioned between 6: pale yellow amorphous solid; [α]25D 12.3 ; UV λmax (log ε) 229 (2.56) nm; IR (KBr) νmax 2928, 1755, 1237, 1067 cm−1; 1H- and 13C-NMR , see m/z 429.3008 [M + H]+ .Compound 7: pale yellow amorphous solid; [α]25D 9.2 ; UV λmax (log ε) 229 (2.84) nm; IR (KBr) νmax 3400, 2994, 2361, 1765, 1241, 1051 cm−1; 1H- and 13C-NMR , see m/z 387.2902 [M + H]+ .Compound 8: pale yellow amorphous solid; [α]25D −7.6 ; UV (MeOH) λmax (log ε) 220 (4.04) nm; IR (KBr) νmax 2937, 2360, 1744, 1242, 1045 cm−1; 1H-NMR  δ 6.51 , 4.99 , 4.16 , 2.43 , 2.34 , 2.09 , 2.04 , 1.80 , 1.71 , 1.68 , 1.58 , 1.56 , 1.55 , 1.38 , 1.36 , 1.26 , 1.24 , 1.11 , 1.08 , 0.99 , 0.91 , 0.87 , 0.79 , 0.80 , 0.83 , 0.60 ; 13C-NMR  δ 202.0 (C-16), 170.8 (12-OAc), 155.3 (C-18), 135.4 (C-17), 76.3 (C-12), 62.1 (C-19), 56.8 (C-5), 52.8 (C-9), 49.4 (C-14), 42.1 (C-3), 41.0 (C-13), 40.7 (C-7), 39.8 (C-1), 37.4 (C-8), 37.1 (C-10), 34.3 (C-15), 33.5 (C-4), 33.5 (C-20), 22.3 (C-11), 21.6 (C-21), 21.5 (12-OAc), 19.6 (C-24), 18.6 (C-6), 18.1 (C-2), 16.6 (C-23), 16.2 (C-22); (+)-HRFABMS m/z 417.3008 [M + H]+ .Compound The growth inhibition assays against human cancer cell lines, in particular, HCT-15 (colon), NCI-H23 (lung), ACHN , MDA-MB0231 (breast), NUGC (stomach) and PC-3 (prostate), were carried out according to a published protocol [1‒8), including four new compounds (5‒8), were isolated from the Korean marine sponge of Scalarispongia species. Among the new compounds, compounds 6 and 7 are the first isoscalarane derivatives lacking the C-12 oxygen, and compound 8 is diametrically opposed to other reported tetracyclic scalaranes, which have an oxo group at C-19 and a hydroxy or an acetate group at C-16. The isolated compounds showed cytotoxicity against a panel of human cancer cell lines in all tested cases, with the exception of compound 5, which showed no cytotoxicity against the cancer cell lines considered in this study. The results presented in this contribution may provide new insight into the mode of cytotoxicity of scalarane sesterterpenoids.In summary, eight scalarane sesterterpenoids (


N-acylgalactosamine and N-acylglucosamine derivatives enabling the visualization of mucin-type O-glycoproteins and O-GlcNAcylated proteins through Diels–Alder chemistry. Whereas the previously reported cyclopropene-labeled N-acylmannosamine derivative leads to significantly higher fluorescence staining of cell-surface glycoconjugates, the glucosamine derivative gave higher labeling efficiency with protein preparations containing also intracellular proteins.Monitoring glycoconjugates has been tremendously facilitated by the development of metabolic oligosaccharide engineering. Recently, the inverse-electron-demand Diels–Alder reaction between methylcyclopropene tags and tetrazines has become a popular ligation reaction due to the small size and high reactivity of cyclopropene tags. Attaching the cyclopropene tag to mannosamine via a carbamate linkage has made the reaction even more efficient. Here, we expand the application of cyclopropene tags to  The glycan chains of glycoproteins and lipids have been shown to be involved in numerous biological recognition and regulation events . Glycan 4ManNCyc (4) + calcd for C20H27NO11, 480.14763; found, 480.14511.Further purification by semi-preparative HPLC  allowed separation of the anomers. Retention time β-anomer: 15 min, α-anomer: 17.2 min.1H NMR  δ 6.53 , 6.19 , 5.31–5.12 , 4.75 , 4.25 , 4.18 , 4.05 , 4.02–3.96 , 3.96–3.85 , 2.18 , 2.10 , 2.07 , 2.04 , 2.03 , 1.59  ppm; 13C NMR  δ 171.4 (C=O), 170.8 (C=O), 169.3 (C=O), 168.8 (C=O), 156.1 (HNC=O), 120.5, 102.1, 102.0, 91.0 (C-1), 73.1, 73.0, 70.8 (C-3 or C-5), 69.8 (C-3 or C-5), 67.8 (C-4), 61.7 (C-6), 52.9(C-2), 21.0 (OAc), 20.8 (OAc), 20.7 (OAc), 17.22 (CH2CH), 17.18 (CH2CH), 11.70 (=CCH3) ppm.α-Anomer: 1H NMR  δ 6.53 , 5.70 , 5.22–5.15 , 5.11 , 4.73 , 4.28 , 4.11 , 3.97–3.87 , 3.81 , 2.12 , 2.08 , 2.04 , 2.03 , 1.61  ppm; 13C NMR  δ 170.8 (C=O), 169.5 (C=O), 156.3 (HNC=O), 120.64, 120.58, 102.10, 102.07, 92.8 (C-1), 76.8 (C-3 or C-5 or CH2), 73.0 (C-3 or C-5 or CH2), 72.5 (C-3 or C-5 or CH2), 68.1 (C-4), 61.8 (C-6), 55.0 (C-2), 21.0 (OAc), 20.9 (OAc), 20.8 (OAc), 20.7 (OAc), 17.2 (CH2CH), 11.74 (=CCH3), 11.73 (=CCH3) ppm.β-Anomer: 1,3,4,6-Tetra-O-acetyl-2-deoxy-2-((2-methylcycloprop-2-en-1-yl)methoxycarbonylamino)-D-galactopyranose . To a solution of galactosamine hydrochloride  in MeOH (18 mL) NaOMe  was added under nitrogen. After stirring for 90 min at room temperature, the solution was added to activated cyclopropene 7 . After stirring at room temperature overnight the solvent was evaporated under reduced pressure. The residue was dissolved in pyridine (18 mL) and acetic anhydride (9 mL) was added. After stirring for 40 h at room temperature the solvents were evaporated under reduced pressure. The residue was dissolved in CH2Cl2 (230 mL), washed with 10% aq KHSO4 (100 mL), satd aq NaHCO3 (180 mL) and brine (180 mL). The organic layer was dried (MgSO4) and the solvent was evaporated under reduced pressure. The residue was purified by FC  to afford Ac4GalNCyoc (2) as a mixture of anomers . Rf = 0.33 (petroleum ether/ethyl acetate 1:1); HRMS m/z: [M + Na]+ calcd for C20H27NO11, 480.14763; found, 480.14551.Further purification by HPLC  allowed purification of the β-anomer. Retention time β-anomer: 15 min, α-anomer: 16.5 min.1H NMR  δ 6.54 , 5.72 , 5.39 , 5.10 , 4.61–4.48 , 4.14 , 4.02 , 3.93 , 2.16 , 2.14 , 2.12 , 2.04 , 2.02 , 1.61  ppm; 13C NMR  δ (ppm) 170.5 (2 C=O), 170.3 (C=O), 169.5 (C=O), 156.4 (HNC=O), 120.64, 120.55, 102.14, 102.07, 93.1 (C-1), 73.0 (CH2), 71.9 (C-5), 70.4 (C-3), 66.6 (C-4), 61.4 (C-6), 51.5 (C-2), 21.0 (OAc), 20.9 (OAc), 20.8 (OAc), 20.8 (OAc), 17.21 (CH2CH), 17.23 (CH2CH), 11.8 (=CCH3) ppm.β-Anomer: Cell growth conditions. As described in [–1 penicillin and 100 μg mL–1 streptomycin. All cells were incubated in a 5% carbon dioxide, water saturated incubator at 37 °C.ribed in  HEK 293TFluorescence microscopy with Tz–biotin 10. HEK 293T cells  were seeded in 4-well ibiTreat μ-Slides (ibidi) coated with fibronectin (25 µg mL−1) and poly-L-lysine . After 12 h cells were incubated for 48 h with 50 µM cyclopropene-labeled sugar (Ac4GlcNCyoc (1), Ac4GalNCyoc (2), or Ac4ManNCyoc (3)). The sugars were prepared as stock solutions (0.36 mM) in PBS and diluted into media. Only PBS was added as solvent control. Cells were washed two times with PBS and then treated with Tz–biotin 10 [−1) and Hoechst33342 (10 µg mL−1) for 20 minutes at room temperature in the dark. Cells were washed twice with PBS, and DMEM was added for microscopy. A Zeiss LSM 510 Meta equipped with a 40 × 1.3 NA Plan-Neofluar oil DIC immersion objective was employed for imaging. Analysis of the obtained data was performed using Image J software version 1.45 S.2.iotin 10  (1 mM) fWestern blot analysis. HeLa S3 cells were seeded  and after 16 h the media were exchanged with media containing 100 µM of cyclopropene-labeled sugar (Ac4GlcNCyoc (1), Ac4GalNCyoc (2), or Ac4ManNCyoc (3)). Sugars were diluted from a 0.36 mM stock solution in PBS. As a solvent control PBS was added instead of the sugar stock solution. The cells were cultured for 48 h with or without the additional sugar. Cells were trypsinated and re-suspended in PBS (10 mL) and pelleted by centrifugation . The supernatant was discarded and the pellet re-suspended in PBS (1 mL) and pelleted by centrifugation . The cells were lysed in lysis buffer (400 µL) containing Triton X-100 (0.5%) (for permeabilization of membranes and solubilization of proteins and to prevent aggregate formation), DNase (30 µg mL−1), RNase (30 µg mL−1), β-glycerophosphate (20 mM) (Ser/Thr phosphatase inhibitor), sodium fluoride (20 mM) (Ser/Thr phosphatase inhibitor), sodium orthovandadate (0.3 mM) , complete X protease inhibitor (Roche) (1X), NaCl (300 mM), TrisHCl pH 7.4 (25 mM), EDTA (5 mM) , O-(2-acetamido-2-deoxy-D-glucopyranosylidenamino) N-phenylcarbamate (PUGNAc) (Sigma-Aldrich) (100 µM) (O-GlcNAc-β-N-acetylglucosaminidase inhibitor to maintain O-GlcNAcylation during lysis) and incubated at 4 °C for 30 min. The lysate was cleared by centrifugation . Tz–biotin 10 was added to the cleared supernatant to a final concentration of 150 µM. The samples were incubated for 90 min at rt, 3× SDS-sample buffer was added, and the sample was boiled at 90 °C for 15 min. Proteins were separated by SDS-polyacrylamide gel electrophoresis using 10% polyacrylamide gels and transferred to nitrocellulose membranes (BioRad). Transfer efficiency was analyzed with Ponceau S staining. The membranes were blocked in milk (5% in PBS-T) for 1 h at rt, followed by incubation with anti-biotin antibody  at 4 °C overnight or anti-alpha-tubulin antibody  for 1 h at rt. The membranes were washed , incubated with secondary horseradish-peroxidase-conjugated anti-mouse antibody -HRP, 1:50000 dilution in milk, 1 h, rt), and washed again . Blots were developed by an ECL detection system  and visualised with a CCD camera .1H and 13C NMR spectra of Ac4GlcNCyoc (1) and Ac4GalNCyoc (2).Additional MOE experiments and File 1Additional MOE experiments and NMR spectra.


However the alkylation of sodium 1,2-diphospha-3,4,5-tri(p-fluorophenyl)cyclopentadienide with ethyl iodide leads to stable 1-ethyl-3,4,5-tris(p-fluorophenyl)-1,2-diphosphole (1e), which forms the [4 + 2] cycloadduct 2,3,4,4a,5,6-hexa(p-fluorophenyl)-1-ethyl-1,7,7a-triphospha-4,7-(ethylphosphinidene)indene (2e) only upon heating up to 60 °C. With further heating to 120 °C with N-phenylmaleimide, the cycloadducts 2a–c and 2e undergo the retro-Diels–Alder reaction and form only one product of the [4 + 2] cycloaddition reaction 3a–с, 3e with good yields up to 65%.Two different approaches have been employed to enhance the reactivity of 1-alkyl-1,2-diphospholes – the introduction of electron-withdrawing groups either at the phosphorus atoms or in the para-position of the arene ring. The alkylation of sodium 1,2-diphospha-3,4,5-triphenylcyclopentadienide with alkyl halides Hal-CH Phospholes are weakly aromatic heterocycles and demonstrate rather different properties from those of their S, N and O counterparts –2. Due tH-phospholes  cycloaddition reactions leading to a mixture of cycloadducts -1-ethyl-1,2-diphosphole (1e) is stable at room temperature and undergoes the [4 + 2] cycloaddition reaction only upon heating at 60 °C resulting in only one product, 2e  introduction of EWGs at the phosphorus atom and (b) introduction of EWGs at the carbon atoms of aryl substituents. New 1-alkyl-1,2-diphospholes, t 100 °C . CompareFile 1Experimental procedures and characterization data.


The six-membered rings in 2,4,6,8-tetraphenyl-3,7-diazabicyclo[3.3.1]nonan-9-ones, compounds 1 and 2, adopt a chair–boat conformation, while those in 2,4-diphenyl-3-azabicyclo[3.3.1]nonan-9-ones, compounds 3–8, adopt a chair–chair conformation because of stereoelectronic effects. These stereoelectronic effects were analyzed by the 1JC–H coupling constants, which were measured in the 13C satellites of the 1H NMR spectra obtained with the hetero-dqf pulse sequence. In the solid state, these stereoelectronic effects were investigated by measurement of X-ray diffraction data, the molecular geometry , and inter- and intramolecular interactions, and by natural bond orbital analysis, which was performed using density functional theory at the ωB97XD/6311++G level. We found that one of the main factors influencing the conformational stability of 3–8 is the interaction between the lone-pair electrons of nitrogen and the antibonding sigma orbital of C(7)–Heq (nN→σ*C–H(7)eq), a type of hyperconjugative interaction.This paper reports the synthesis of a series of piperidones  Stereoelectronic effects have attracted the attention of many researchers with an interest in organic chemistry because of the major role that conformation plays in molecules and biomolecules; in addition, such effects are related to the spatial orientation of the orbital –3, whichX→σ*C–Y) or positive hyperconjugation (σC–Y→π* or p).Moreover, stereoelectronic effects have been related to the stabilization of carbanions –14, carbHyperconjugation is commonly described as the interaction between electronic orbitals where one filled orbital (donor) interacts with another unfilled orbital (acceptor) with the presence of an additional resonance structure  with another unfilled orbital (acceptor) can be observed several bonds away from the orbitals. This effect is classified as hyperconjugation  and the second-one is the Plough effect, where the lone-pair electrons (LPEs) of O, N, and S, on the pseudo axial position interact with the antibonding σ orbital (nX→σ*Cβ–Y) In the six-membered saturated heterocycles the more stable conformation is the one where the substituent on the β-carbon is equatorial with respect to the heteroatom, this is known as homoanomeric effect, which is a type of homohyperconjugation –36. The eq) is 4 Hz higher  than the axial one . This difference in the 1JC,H values has been explained in terms of delocalization interactions from electron hyperconjugation.For the study of this kind of interaction, the NMR technique is a useful tool as it is a highly sensitive spectroscopical method, and NMR spin–spin coupling constants are used to experimentally investigate stereoelectronic effects 12–3642–43.36*C(7)–Heq orbital (nN→σ*c(7)–Heq), the results are supported by natural bond orbital (NBO) analysis.This study describes the synthesis of a series of 2,4-diphenyl-3-azabicyclo[3.3.1]nonan-9-one compounds  with res1 and 2 were synthesized using ammonium acetate, benzaldehyde, and acetone or 2-butanone (4:4:1), respectively nonan-9-ones were synthesized by the Mannich reaction –49. As sectively –52.2 and 3-methyl-2,6-diphenylpiperidin-4-one in a ratio of 9:1  for the cis compounds the energy of the 1H,3H-allylic strain  is lower in comparison with the TS trans,1Ar,3H-allylic strain nonan-9-ones.The 1–8 were recorded at 21 ± 1 °C using a Jeol ECA 500 spectrometer equipped with a 5 mm multinuclear pulse-field gradient probe. All of the spectra were recorded in CDCl3 solution (0.9 mmol of the compound per 0.5 mL solvent). The chemical shifts were referenced to tetramethylsilane (CH3)4Si, which served as an internal standard .The NMR spectra of 1H NMR spectra were recorded at 500.159 MHz using a spectral width of 9384.3 Hz, acquisition time of 6.98 s, 65536 points, 512 scans, and a recycle delay of 1 s. Fourier transformation was applied using an exponential function (line width = 0.2). 13C satellites were directly determined from the 1H NMR spectra or using the same spectral condition as the heteronuclear-double-quantum filter experiment nonan-9-one was performed by the following method. First, 5.10 mmol of ketone, 10.2 mmol of ammonium acetate, and 10.2 mmol of benzaldehyde or 3–5 are in agreement with previous reports nonan-9-one (6). White solid, Yield: 73%, by using the general procedure 5.10 mmol of cyclohexanone and 10.2 mmol of ammonium acetate, and 10.2 mmol of m-nitrobenzaldehyde were added to the reaction. ESIMS–TOF: m/z for C20H19N3O5 [M + H]+ calcd: 382.1397, found: 382.1395; 1H NMR  δ 8.41 , 8.20 , 7.92 , 7.64 , 4.58 , 2.82 , 2.56 , 1.86 , 1.78 , 1.49 ; 13C NMR  δ 21.18 (C7), 28.92 , 53.24 , 64.02 , 121.90 , 123.08  129.94 , 133.05 , 142.82 , 148.70 (C12-C18), 214.92 (C=O).1-Methyl-2,4-bis(3-nitrophenyl)-3-azabicyclo[3.3.1]nonan-9-one (7). Yellow solid, Yield: 72%, by using the general procedure 5.10 mmol of 2-methylcyclohexanone and 10.2 mmol of ammonium acetate, and 10.2 mmol of m-nitrobenzaldehyde were added to the reaction. ESIMS–TOF: m/z for C21H21N3O5 [M + H]+ calcd: 396.1553, found: 396.1558; 1H NMR  δ 8.39 , 8.34 , 8.19 , 8.15 , 7.94 , 7.58–7.63 , 7.87 , 4.56 , 4.12 , 3.11 , 2.63 , 2.01 , 1.84 , 1.76 , 1.46–1.45 , 0.82 ; 13C NMR  δ 20.25 (Me), 21.45 (C7), 29.01 (C6), 36.74 (C8), 50.55 (C1), 53.68 (C5), 70.42 (C2), 121.77 (C17), 122.92 (C19), 123.35 (C13), 123.85 (C11), 129.42 (C20), 129.86 (C14), 133.03 (C21), 135.21 (C15), 141.37 (C16) 142.93 (C10), 148.26 (C18), 148.61 (C12), 215.46 (C=O).1,5-Dimethyl-2,4-bis(3-nitrophenyl)-3-azabicyclo[3.3.1]nonan-9-one (8). White solid, Yield: 64%, by using the general procedure 5.10 mmol of 2,6-dimethylcyclohexanone and 10.2 mmol of ammonium acetate, and 10.2 mmol of m-nitrobenzaldehyde were added to the reaction. ESIMS-TOF: m/z for C22H23N3O5 [M + H]+ calcd: 410.1710, found: 410.1713; 1H NMR  δ 7.53 , 7.36 , 7.30 , 3.35 , 3.03 , 1.61 , 1.11 (H6ax ) 0.81 (H8ax), 0.69 ; 13C NMR  δ 20.26 (Me), 21.10 (C7), 36.40 , 50.15 , 70.22 , 123.35 , 123.85 , 129.42 , 135.21 , 142.93 , 148.61 , 215.46 (C=O).1), 928315 (6), and 933224 (7). These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/data_request/cif.Crystallographic data of the structures reported in this paper have been deposited with the Cambridge Crystallographic Data Centre with supplementary publication numbers CCDC 928314 (File 1Additional schemes, figures, theoretical, spectra, and crystallographic data.


The structures of the new compounds were elucidated as -2-(hexadecanoylamino)-4,8,10-octadecatriene-l,3,3′-triol (1), -2-(hexadecanoylamino)-4,8,10-octadecatriene-l,3,2′-triol (2), -2-(octadecanoylamino)-4,8,10-octadecatriene-l,3,2′-triol (3), -2-(hexadecanoylamino)-4,8-octadecadiene-l,3,3′-triol (4) and -2-(hexadecanoylamino)-4-octadecene-l,3,3′-triol (5) on the basis of extensive spectral analysis and chemical evidences. The characteristic C-3′S hydroxyl group in the fatty acid moiety in compounds 1, 4 and 5, was a novel structural feature of ceramides. The rare 4E,8E,10E-triene structure in the sphingoid base of compounds 1–3, was found from marine bryozoans for the first time. The new ceramides 1–5 were evaluated for their cytotoxicity against HepG2, NCI-H460 and SGC7901 tumor cell lines, and all of them exhibited selective cytotoxicity against HepG2 and SGC7901 cells with a range of IC50 values from 47.3 μM to 58.1 μM. These chemical and cytotoxic studies on the new neritinaceramides A–E (1–5) added to the chemical diversity of B. neritina and expanded our knowledge of the chemical modifications and biological activity of ceramides.Five new ceramides, neritinaceramides A ( A close scrutiny of the 1H-NMR and 13C-NMR spectra of 1 (δ 173.0 (C-1′), δ 6.65 ], a nitrogen-linked methine , a hydroxylated methylene  and two hydroxylated methines , which together with the long aliphatic carbon signals at 29.2–29.7, demonstrated a trihydroxyl ceramide structure of compound 1. The observation of 1H-1H COSY correlations from H2-1 to H-2, and in turn, from H-2 to NH and H-3, from H-3′ to H-2a′ (δ 2.30), H-2b′ (δ 2.43), H-4a′ (δ 1.43) and H-4b′ (δ 1.51), and the HMBC correlations from NH to C-1′, and from H2-2′ to C-1′ and C-3′, indicated the three hydroxyl groups were located at C-1, C-3 an C-3′, respectively. The 1H-, 13C-NMR and HSQC spectra of 1 suggested the presence of three double bonds  on the basis of six proton signals at δ 5.54 , 5.77 , 5.59 , 5.99 , 6.00  and 5.52 , as well as the corresponding carbons at δ 129.3 (C-4), 133.2 (C-5), 133.3 (C-8), 130.0 (C-9), 131.2 (C-10) and 130.7 (C-11). The positions of the three double bonds were confirmed to be C-4, C-8 and C-10 on the basis of 1H-1H COSY correlations of H-3/H-4, H-4/H-5, H-5/H2-6, H2-6/H2-7, H2-7/H-8, H-8/H-9, H-9/H-10 and H-10/H-11, and HMBC correlations of H-4/C-3, H-5/C-3, H-5/C-6, H2-6/C-4, H2-6/C-5, H2-6/C-7, H2-7/C-8, H2-7/C-9, H-8/C-10, H-11/C-9 and H-11/C-12 , J8,9 (15.3 Hz) and J9,10 (15.3 Hz). Moreover, the trans configuration (E) for those alkenyl bonds was further confirmed by the chemical shifts of the allylic carbons C-6 (δ 31.9), C-7 (δ 32.1) and C-12 (δ 32.1), since allylic carbon signals of trans- and cis-isomers were observed at δ 32–33 and 27–28, respectively D = −3.0°) D = +2.94°) D +12.5°) was in good agreement with the synthetic (S)-methyl 3-hydroxyhexadecanoate ([α]D = +13.8°), while the specific rotation [α]D for (R)-methyl 3-hydroxyhexadecanoate was −16.6° + (positive mode) and 584 [M + Cl]− (negative mode), and the HR-ESI-MS pseudomolecular ion peak at m/z 572.4652 [M + Na]+ . By comparison of the 13C-NMR data of 2 with those of 1, it could be confirmed that 2 and 1 shared the same LCB structural skeleton, while the hydroxyl group at C-3′ in ceramide 1 was replaced to be at C-2′ in 2, as 2 showed different chemical shifts at C-1′ (δ 175.4), C-2′ (δ 72.5) and C-3′ (δ 35.8) in its FAB. The methene protons H2-3′ (δ 2.03 and 2.22) showed coupling relationships with H2-4′ (δ 1.23) and H-2′ (δ 4.59) in the 1H-1H COSY spectrum, which together with HMBC correlations from NH  to C-1′, from H-2′ to C-1′ and from H2-3′ to C-2′, confirmed that the hydroxyl group was attached at C-2′  and C5D5N , which were close to analogue -2-(2-hydroxyhexadecanoylamino)-16-methyl-4-octadecene-1,3-diol  [2 ([α]D = +9.8°) which was consistent with that of the synthetic ceramide with the 2S,3R,2′R-configuration ([α]D = +7.4°) D = –6.7°) was similar to (R)-methyl 2-hydroxyhexadecanoate ([α]D = –5.7°) [S)-methyl 2-hydroxyhexadecanoate ([α]D = +1.15°) +, and negative ESI-MS pseudomolecular ion peak at m/z 576 [M − H]−, with the help of NMR spectral data  for the methyl ester obtained after methanolysis of 3 with 5% mehtanolic HCl. Therefore, neritinaceramide C (3) was determined to be -2-(octadecanoylamino)-4,8,10-octadecatriene-l,3,2′-triol.Neritinaceramide C  was assigned as C34H65NO4 on the basis of HR-ESI-MS pseudomolecular ion peak at m/z 574.4815 [M + Na]+  and the results of 1H and 13C NMR spectral interpretations , 131.3 (C-8), 129.0 (C-9) and 128.9 (C-4) were determined to be C-4 and C-8 mainly by 1H-1H COSY correlations from H-3 (δ 4.28) to H-4 (δ 5.51), and in turn, from H-4 to H-5 (δ 5.77), H-5 to H2-6 (δ 1.96), H2-6 to H2-7 (δ 2.11), H2-7 to H-8 (δ 5.41), H-8 to H-9 (δ 5.38) and H-9 to H2-10 (δ 2.07), and further confirmed by HMBC correlations from H-3 to C-4 and C-5, H-5 to C-6 (δ 32.6), H2-6 to C-4 and C-5, H-8 to C-6, C-7 (δ 32.4) and C-9, and H-9 to C-10 (δ 32.2)  was elucidated as -2-(hexadecanoylamino)-4,8-octadecadiene-l,3,3′-triol.The molecular formula of neritinaceramide D  A. Cerami336 of 1 B. This a5) was obtained as a white amorphous powder with a molecular formula of C34H67NO3, determined by positive HR-ESI-MS m/z 576.4967 [M + Na]+ , and the negative ESI-MS m/z 552 [M − H]−, with the help of NMR data. Comparison of the NMR spectra of 5 with 4 indicated that they possessed the same ceramide skeleton, except for the absence of a double bond between C-8 and C-9 of 5. The fragment ions of 5 were almost identical with those of 4 in the negative ESI-MS, but the fragmet ion m/z 338 in 4 was replaced by fragmet ion m/z 340 in 5 -2-(hexadecanoylamino)-4-octadecene-l,3,3′-triol.Neritinaceramide E -2-(hexadecanoylamino)-4-octadecene-l,3,2′-triol (6) + in the positive ESI-MS, along with the analysis of NMR spectral data enabled the determination of the molecular formula of 12 as C19H40O3, and 16 carbons for the alkyl long chain accordingly. Thus, the structure of 12 was determined as 1-O-palmityl glycerin ether + in the positive ESI-MS, with the help of NMR spectral data Bugula neritina was collected in March 2008 in Daya Bay, Shenzhen, China. The specimen identification was conducted by Prof. H.-W. Lin. A voucher specimen numbered 20080312, was deposited in the Herbarium of Pharmacy, Changzheng Hospital, Second Military Medical University, Shanghai, China.Marine bryozoan B. neritina  was extracted with 95% EtOH (4 × 500 L) at room temperature with each extraction taking one week. The concentrated EtOH extract (2.0 kg) was suspended in water, and then partitioned successively with EtOAc to obtained EtOAc extract (600 g). One third of the EtOAc extract was subjected to CC over silica gel  eluting with the CHCl3-MeOH  gradient to afford nine major fractions (A−I) based on TLC analysis. Fr. E (15.0 g) was subject to CC on silica gel  eluting with petroleum ether-EtOAc (10:1 to 1:1) gradient to give four major fractions (Fr. E1−E4). The Fr. E3 (2.5 g) was recrystallisated with CH3COCH3 to obtained a crude white powder (900 mg), and then further purified by semi-preparative reverse HPLC with MeOH-H2O (97:3) as the mobile phase at a flow rate of 8.0 mL/min to afford five new ceramides 1 , 2 , 3 , 4  and 5 . Fr. E4 (1.3 g) was eluted with CHCl3-MeOH (1:1) on Sephadex LH-20 to remove pigments, and then further purified by semi-preparative reverse HPLC using MeOH-H2O (97:3) as mobile phase at a flow rate of 8.0 mL/min to afford ceramides 6  and 7 . Similarly, the remainder four known ceramides 8 , 9 , 10  and 11  were obtained from Fr. E2 (176.5 mg) by the same method with Fr. E4. The two known alkyl glycerylethers 12  and 13  were isolated from Fr. D via CC on silica gel column with 5:1 ether-EtOAc as eluting solvent, and finally further purified by semi-preparative reverse HPLC using MeOH-H2O (97:3) as mobile phase at a flow rate of 8.0 mL/min. The known nucleoside 14  was purfied from Fr. H via CC over repeated sephadex LH-20 with CHCl3-MeOH (1:1) as eluting solvent, and then further purified by semi-preparative HPLC with MeOH-H2O (90:10) as mobile phase at a flow rate of 7.0 mL/min.The marine bryozoan 20D −4.5° ; 1H-NMR  and 13C-NMR  data see m/z 572 [M + Na]+, 437, 409, 330, 312, 303, 284, 275, 256; Negative ESI-MS m/z 548 [M − H]−, 584 [M + Cl]−, 336, 325, 311, 297, 283, 255, 227, 165, 113; Positive HR-ESI-MS m/z 572.4658 [M + Na]+ .White amorphous powder, mp 109−110 °C; [α]20D +9.8° ; 1H-NMR  and 13C-NMR  data see m/z 572 [M + Na]+, 437, 409, 312, 284, 274, 256, 166; Negative ESI-MS m/z 548 [M − H]−, 584 [M + Cl]−, 283, 255, 227, 113; Positive HR-ESI-MS m/z 572.4652 [M + Na]+ .White amorphous powder, mp 111−112 °C; [α]20D +10.1° ; 1H-NMR  and 13C-NMR  data see m/z 600 [M + Na]+, 437, 409, 330, 311, 283, 274, 264, 256, 219, 166; Negative ESI-MS (−) m/z 576 [M − H]−, 340, 324, 312, 298, 283, 271, 255, 253, 237, 225, 113.White amorphous powder, mp 105−106 °C; [α]20D −4.3° ; 1H-NMR  and 13C-NMR  data see m/z 574 [M + Na]+, 1125 [2M + Na]+, 437, 409, 274, 262, 256; Negative ESI-MS m/z 550 [M − H]−, 586 [M + Cl]−, 338, 283, 255, 228, 113; Positive HR-ESI-MS m/z 574.4815 [M + Na]+ .White amorphous powder, mp 104−105 °C; [α]20D −4.1° ; 1H-NMR  and 13C-NMR  data see m/z 576 [M + Na]+, 437, 409, 311, 300, 283, 274, 264, 256, 228; Negative ESI-MS m/z 552 [M − H]−, 588 [M + Cl]−, 340, 283, 255, 227, 113; Positive HR-ESI-MS m/z 576.4967 [M + Na]+ .White amorphous powder, mp 101−102 °C; [α]1−5 are available in The copies of the original spectra of the new compounds 1H-NMR  δ 0.88 , 1.26 , 1.58 , 3.52 , 3.65 , 3.72 , 3.86 ; 13C-NMR  δ 64.5 (C-1), 70.6 (C-2), 72.0 (C-3), 14.3 (C-1′), 23.1 (C-2′), 32.1 (C-3′), 29.1-29.8 (C-4′~13′), 26.2 (C-14′), 29.8 (C-15′), 72.7 (C-16′); Positive ESI-MS m/z 339 [M + Na]+, 655 [2M + Na]+; Negative ESI-MS m/z 631 [2M − H]−, 351 [M + Cl]−, 361 [M + COOH]−.White amorphous powder, mp 60–62 °C. 1H-NMR  δ 0.86 , 1.27 , 1.57 , 3.49 , 3.63 , 3.72 , 3.86 ; 13C-NMR  δ 64.4 (C-1), 70.6 (C-2), 72.0 (C-3), 14.3 (C-1′), 22.8 (C-2′), 32.1 (C-3′), 29.5-29.9 (C-4′~15′), 26.2 (C-16′), 29.9 (C-17′), 72.6 (C-18′); Positive ESI-MS m/z 367 [M + Na]+, 711 [2M + Na]+.White amorphous powder, mp 62–63 °C. n-hexane for three times. The n-hexane layer was washed with H2O and concentrated in vacuo to yield the corresponding fatty acid methyl ester (FAME −51) 20D about −2.8° .Each ceramide (about 2 mg) was dissolved in 5% HCl-MeOH (2 mL) and refluxed for 10 h at 80 °C. The reaction mixture was extracted with AME 1−5) . The remS)-Methyl 3-hydroxylhexadecanoate : [α]20D +12.5° ; EI-MS m/z286 [M]+ (4), 268 [M − H2O]+ (9), 236 (6), 211 (4), 194 (7), 111 (9), 103 (100), 83 (10), 74 (22), 55 (19).(R)-Methyl 2-hydroxylhexadecanoate (FAME 2): [α]20D− 6.7° ; EI-MS m/z 286 [M]+ (9), 254 [M − OCH3−H]+ (4), 227 (67), 208 (6), 145 (13), 111 (50), 103 (21), 97 (96), 83 (100).(R)-Methyl 2-hydroxyloctadecanoate (FAME 3): [α]20D− 6.5° ; EI-MS m/z 314 [M]+ (26), 296 [M − H2O]+ (4), 282 [M − OCH3− H]+ (6), 269 (5), 255 (76), 236 (6), 145 (14), 111 (52), 103 (19), 97 (93), 83 (85), 57 (100).(1−5) were evaluated for their cytotoxic activity against human hepatoma HepG2, pulmonary carcinoma NCI-H460 and gastric carcinoma SGC7901 tumor cells by microculture tetrazolium (MTT) assay. The cells were obtained from American Type Culture Collection (ATCC), and were seeded to RPMI-1640 medium with 10% fetal bovine serum and 100 U/mL benzyl penicillin-streptomycin solution at 37 °C in a humidified atmosphere containing in 5% CO2/air for 24 h. Then the test samples were added and incubated at 37 °C for another 72 h. The detailed experiments procedure has been described in our previous published literature [50 value with adriamycin as positive control.Neritinaceramides A−E , six known ceramides (6–11), two known alkyl glycerylethers (12 and 13) and a known nucleoside (14). The characteristic C-3′S hydroxyl group in the fatty acid moiety in compounds 1, 4 and 5, was a novel structure feature in ceramides. The rare 4E,8E,10E-triene structure in the sphingoid base of compounds 1–3, was found from marine bryozoans for the first time. Bioacitve studies indicated that those new ceramides (1−5) exhibited selective cytotoxicity against HepG2 and SGC7901 cells with IC50 values range from 47.3 μM to 58.1 μM. These chemical and cytotoxic studies on the new neritinaceramides A–E (1–5) added to the chemical diversity of B. neritina and expanded our knowledge of the chemical modifications and biological activity of ceramides.The present chemical study of the marine bryozoan


Salt (I) crystallizes with two independent 6-chloro-5-ethyluracil anions and two 1-phenylpiperazine cations in the asymmetric unit.In the crystals of the two novel piperazinium salts with a 6-chloro-5-ethyl-2,4-dioxopyrimidin-1-ide anion in (I) and a 6-chloro-5-isopropyl-2,4-dioxopyrimidin-1-ide anion in (II), the anions and cations are linked  10H15N2+·C6H6ClN2O2−, (I), and C10H15N2+·C7H8ClN2O2−, (II), consist of 4-phenylpiperazin-1-ium cations with a 6-chloro-5-ethyl-2,4-dioxopyrimidin-1-ide anion in (I) and a 6-chloro-5-isopropyl-2,4-dioxopyrimidin-1-ide anion in (II). Salt (I) crystallizes with two independent cations and anions in the asymmetric unit. In the crystal structures of both salts, the ions are linked via N—H⋯O and N—H⋯N hydrogen bonds, forming sheets which are parallel to (100) in (I) and to (001) in (II). In (I), the sheets are linked via C—H⋯Cl hydrogen bonds, forming a three-dimensional framework.The title molecular salts, C In both compounds, the piperazine rings adopt a distorted chair conformation with a positively charged protonated N atom. In compound (I)A, and 39.7 (2)° in cation B. The equivalent dihedral angle is 39.61 (9)° in the cation of compound (II)C and D) of compound (I)The molecular structures of the title salts (I)A and anion C, and cation B and anion D, via N—H⋯O and C—H⋯O hydrogen bonds. These units are linked via N—H⋯N hydrogen bonds, forming separate A/B and C/D sheets parallel to the bc plane e Table 1. The sherk Fig. 3.b-axis direction. The chains are linked via N—H⋯N hydrogen bonds, forming sheets lying parallel to the ac plane e Table 2.et al., 2014A search of the Cambridge Structural Database : A mixture of 6-chloro-5-ethyluracil , 1-phenylpiperazine  and anhydrous potassium carbonate , in ethanol (8 ml), was heated under reflux for 6 h. On cooling, the precipitate, thus formed was separated by filtration to yield 306 mg (51%) of 5-ethyl-6-(4-phenyl-1-piperazinyl)uracil. The filtrate was concentrated by vacuum distillation to 5 ml and allowed to stand at room temperature overnight to yield compound (I)1H NMR : δ 0.93 , 2.35 , 3.25 , 3.45 , 6.83–6.95 , 7.21 , 8.15–8.17 , 10.83 . 13C NMR : δ 13.80 (CH3), 19.55 (CH2), 44.18, 47.86 (piperazine-C), 116.32, 119.62, 128.44, 150.70 (Ar—C), 108.88, 153.90, 155.94, 164.80 (pyrimidine-C),Compound (II): 6-Chloro-5-isopropyluracil , 1-phenylpiperazine  and anhydrous potassium carbonate , in ethanol (8 ml), was heated under reflux for 6 h. On cooling, the precipitate thus formed was separated by filtration to yield 566 mg (90%) of 5-isopropyl-6-(4-phenyl-1-piperazinyl)uracil. The filtrate was concentrated by vacuum distillation to 5 ml and allowed to stand at room temperature overnight to yield compound (II)1H NMR : δ 1.20 , 2.52–2.56 , 3.18 , 3.24 , 6.88–7.02 , 7.20–7.22 , 8.04–8.08 , 11.02 . 13C NMR : δ 19.98 (CH3), 27.0 (CH), 44.50, 47.98 (piperazine-C), 116.16, 119.80, 129.04, 150.0 (Ar—C), 110.82, 152.30, 154.04, 164.06 (pyrimidine-C).Uiso(H) = 1.5Ueq(C) for methyl H atoms and 1.2Ueq for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989015013298/su5162sup1.cifCrystal structure: contains datablock(s) I, II, Global. DOI: 10.1107/S2056989015013298/su5162Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989015013298/su5162IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989015013298/su5162Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015013298/su5162IIsup5.cmlSupporting information file. DOI: 1412124, 1412123CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: