The title compound is nearly planar with a dihedral angle of 10.6 (4)° between the two benzene rings. 13H8Br2FN3O3, is nearly planar with a dihedral angle of 10.6 (4)° between the two benzene rings. Intramolecular N—H⋯O and O—H⋯N hydrogen bonds occur. In the crystal, the molecules are linked by weak C—H⋯O and C—H⋯Br hydrogen bonds. The roles of the intermolecular interactions in the crystal packing were clarified using Hirshfeld surface analysis.The title compound, C If the value of dnorm is negative, the intermolecular contacts are shorter than the van der Waals radius; these are shown as red regions. A positive value of dnorm, shown in blue, indicates that the intermolecular contacts are longer than the van der Waals radius . The 20.2% contribution from the O—H⋯O hydrogen bond is seen as a pair of sharp spikes at de + di = 2.3 Å) in Fig. 5a. The distribution of positive and negative potential over the Hirshfeld surface is represented in Fig. 6The two-dimensional fingerprint (FP) plots are used to analyse significant differences between the intermolecular interaction patterns -N′-methyl-N-(quinolin-4-ylmethylene)hydrazine [1.367 (2) and 1.386 (3) Å; Karadayı et al., 2005N-(4-bromo-2-nitrophenyl)-N-methyl-N′-(quinolin-4-ylmethylene)hydrazine [1.359 (3) and 1.393 (4) Å; Öztürk et al., 2003There are no direct precedents for the structure of C3): λmax 340, 430 nm; IR (KBr): υ 3610 (–OH), 3285 and 1155 (N—H), 3120–2985 (=C—H), 2915 (C—H), 1608 (C=N), 1558 (C=C), 1515 (N—N), 1475 and 1310 (N=O), 1195, 690 and 665 (C—X) cm−1; MS (ESI+): 434.01 .5-Bromo-4-fluoro-2-hydroxybenzaldehyde (0.5 mmol) was dissolved in hot absolute ethanol (10 mL) and an equimolar amount of 5-bromo-2-nitrophenylhydrazine, dissolved in a minimum volume of absolute ethanol, was slowly added. The product appeared in the first minute. The reaction mixture was refluxed for an additional hour to complete the condensation and then allowed to cool in room temperature. The separated solid was then filtered and washed with ethanol and diethyl ether. The crude product was recrystallized from toluene as pink needle-shaped crystals, 96% yield, m.p. 569–570 K (dec.). The reaction scheme is shown in Fig. 7Uiso(H) = 1.2Ueq(C)Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989018014627/xu5944sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989018014627/xu5944Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989018014627/xu5944Isup3.cmlSupporting information file. DOI: 1864935CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The zinc atom is coordinated by two bdippza ligands resulting in a six-coordinate distorted octahedral geometry.The compound [(bdippza) nBuLi and reaction with carbon dioxide yields lithium bisacetate (1). Treatment of 1 with ZnCl2 results in the compound bis[bisacetato]zinc(II), [Zn(C20H31N4O2)2] (2), whose structure has monoclinic (P21/c) symmetry. The ZnII ion resides on an inversion center and is coordinated by two bisacetate (bdippza) ligands. Each ligand facially coordinates the zinc center via κ3N,N′,O coordination modes to form a distorted octahedral complex with four pyrazole N atoms in the basal plane and two carboxylate O atoms in the axial sites.Deprotonation of the methylene group in bismethane with  Formation of 2 occurs regardless of the stoichiometric ratio and indicates that the steric environment of the bdippza ligand is too small to prevent complexation of two ligands per zinc ion. Spectroscopic characterization of 2 is consistent with the solid-state structure. For instance, identification of the acetate group is evident by a strong IR absorption at 1687 cm−1 and a 13C NMR signal at 165.8 ppm (the carbon peak of the carboxylate was identified by an HMBC experiment that showed a two-bond correlation between the proton of the bridging C atom and the C atom of the carboxylate). Furthermore, the positive-ion ESI–MS spectrum of 2 shows the presence of the [M + Na]+ ion, whose isotope pattern is in good agreement with the theoretical isotope pattern of the compound (see supporting information for ESI–MS spectra and 1D and 2D NMR spectra).In an attempt to model the two histidine and glutamate binding motifs present in thermolysin and carboxypeptidase A, the coordination chemistry of bisacetate (bdippza) with zinc chloride was explored to determine if the steric demands of the anionic heteroscorpionate ligand were suitable to form a zinc complex of the form [(bdippza)ZnCl]. However, structural determination of the title compound identified the product not as the target compound but instead as the homoleptic zinc compound . The Zn—O bond length is 2.0471 (10) Å. The carbonyl oxygen of the carboxylate donor is tilted away from the zinc carboxylate plane, as indicated by the Zn1—O1—C8—C4 torsion angle of 20.61 (16)°. Complexation of the two bdippza ligands to the ZnII ion results in the formation of six six-membered metallocycles  that are all nonplanar. A ring-puckering analysis  is consistent with each of the metallocycles being described as having a twist-boat conformation  is 118.36°, while the dihedral angle between the mean planes of the imidazole rings Cg1 and Cg2i, which are on different bdippza ligands, is 61.64°.The molecular structure of the title complex is shown in Fig. 1a axis. The weak C—H⋯O intermolecular contacts consist of the carboxylate oxygen (O2) at  acting as a hydrogen-bond acceptor to three C—H bonds  on an adjacent complex at , as shown in Fig. 2Cg1⋯Cg2) on the same bdippza ligand are observed. Furthermore, a weak slipped-parallel C—H⋯π  interaction is present. Full details of the hydrogen-bonding geometries and π–π interactions are provided in Table 2Within the crystal, close intermolecular C—H⋯O contacts are present between molecules, which result in the molecules being packed in columns along the II compounds containing different substituted bisacetate supporting ligands  have been characterized crystallographically  Å] is shorter compared to [(bdmpza)2Zn]·3H2O 2Zn] 2Zn] having a distorted square-pyramidal environment instead of a distorted octahedral coordination due to one of the 3,5-di-tert-butylpyrazol-1-yl groups having a weak interaction with the zinc ion.Three related homoleptic Zn2. NMR spectra were recorded on a Bruker AVANCE III 600 NMR. Chemical shifts are expressed in parts per million (ppm) and referenced to residual solvent as the internal reference for 1H  and 13C . IR spectra were measured using a PerkinElmer Spectrum 100 spectrometer. Electrospray mass spectra were recorded on a Bruker HCTultra ETD II mass spectrometer. Bismethane was prepared according to a previously reported procedure  solvent was distilled from sodium/benzophenone ketyl, while methanol was distilled from CaHnBuLi  in hexane at 195 K. After 1 h of stirring, carbon dioxide was bubbled through the solution at 233 K for 30 min. The solution then was allowed to reach ambient temperature and stirred for 2 h before the volume was reduced to 3 ml under reduced pressure. Addition of hexane (10 ml) resulted in the formation of a white solid, which was filtered off, washed with hexane (2 × 5 ml) and dried under reduced pressure . 1H NMR (CDCl3): δ 6.59 , 5.80 , 3.06 , 2.83 , 1.31 , 1.23 , 1.05 , 0.98 . FT–IR : 2966 (m), 2930 (m), 2870 (m), 1676 (m), 1643 (s), 1551 (m), 1458 (m), 1408 (m), 1373 (m), 1310 (m), 1284 (m), 1226 (m), 1181 (m), 1104 (m), 1073 (m), 1060 (m), 1012 (m), 912 (m), 861 (m), 792 (s), 771 (m), 738 (m), 723 (m), 686 (m). MS : m/z found for [C20H31N4O2 −Li]−, 359; [C19H31N4 − Li − CO2]−, 315.To a solution of bismethane  dissolved in dry THF (40 ml) was added 2  was added to [Li(bdippza)] (1)  in dry MeOH (15 ml). The reaction was stirred for 24 h, during which time a white solid formed. The solvent was removed under reduced pressure, dichloromethane (15 ml) was added, and the solution filtered through celite. The volume was reduced (∼3 ml) and addition of hexane (10 ml) caused the formation of a white solid. The solid was collected, washed with hexane (2 × 5 ml), and dried under vacuum . Colorless crystals suitable for crystallographic characterization were obtained by hexane diffusion into THF at room temperature. 1H NMR (CDCl3): δ 6.56 , 6.00 , 3.59–3.47 , 3.02 , 1.37 , 1.30 , 1.19 , 1.02 . 13C NMR (CDCl3): δ 165.8 (CO2−), 163.9 (Cpz), 154.6 (Cpz), 99.6 (Cpz), 67.0 (CH), 27.2 (CH-iPr), 25.9 (CH-iPr), 23.3(CH3-iPr), 22.8 (CH3-iPr), 22.4 (CH3-iPr), 22.1 (CH3-iPr). FT–IR : 2966 (m), 2932 (m), 2871 (m), 1687 , 1552 , 1475 (m), 1460 (m), 1409 (m), 1356 (s), 1315 (m), 1292 (m), 1252 (m), 1184 (m), 1088 (m), 1059 (m), 1024 (m), 910 (m), 854 (m), 798 (s), 778 (s), 724 (m), 692 (s). MS : m/z found for [C40H62N8O4Zn + Na]+, 805.ZnClCrystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989018011246/jj2201sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989018011246/jj2201Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989018011246/jj2201sup3.pdfNMR  and ESI-MS spectra for the titled compound are provided.. DOI: 1860544CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


P21/c) and triclinic (space group Pī) forms from N,N-dimethylformamide and isopropyl alcohol solutions, respectively. The planar structures of the molecules in both crystals are essentially the same as each other, with maximum deviations of 0.0969 (11) and 0.209 (4) Å for the monoclinic and triclinic forms, respectively, from the mean planes of all non-H atoms.The title compound crystallizes in monoclinic  and triclinic (space group Pī) forms, obtained from N,N-dimethylformamide and isopropyl alcohol solutions, respectively. The molecular structures and conformations in the two forms are essentially the same as each other. The naphthoquinone ring systems are essentially planar with r.m.s. deviations of 0.015 and 0.029 Å for the monoclinic and triclinic forms, respectively. The O-propargyl groups are coplanar with the naphthoquinone units with r.m.s deviations ranging from 0.04 to 0.09 Å. In the monoclinic crystal, molecules are linked via pairs of C—H⋯O hydrogen bonds, forming a tape structure running along [120]. The tapes are further linked by a C—H⋯π interaction into a layer parallel to the ab plane. Adjacent layers are linked by another C—H⋯π interaction. In the triclinic crystal, molecules are linked via C—H⋯O and π–π interactions, forming a layer parallel to the ab plane. Adjacent layers are linked by a C—H⋯π interaction.The title compound, C In the monoclinic crystal, molecules are linked via pairs of C—H⋯O hydrogen bonds ]. In the layer, molecules are arranged parallel to each other and adjacent layers are linked by another C—H⋯π interaction ]. In the triclinic crystal, molecules are linked via C—H⋯O interactions ]. Adjacent layers are linked by a C—H⋯π interaction methoxy}naphthalene-1,4-dione  and propargyl bromide . The mixture was stirred at 363 K for 24 h. Then hydrochloric acid  was added and the resulting solution was extracted with dichloromethane (3 × 25 ml). The organic layers were washed with water (60 ml), dried over anhydrous sodium sulfate and concentrated. The solid obtained was purified by column chromatography using silica gel and hexane–ethyl acetate (9:1) and furnished the title compound in 70% yield. Yellow single crystals of the monoclinic and triclinic forms (m.p. 420.0–423.1 K) suitable for X-ray diffraction were obtained by slow evaporation of DMF and isopropyl alcohol solutions (about 0.5 mg ml-1), respectively, at room temperature.The synthesis of the title compound was achieved in one step according to the literature method (Raja max (cm−1): The spectrum show the characteristic absorption bands of the main functional groups for title compound at IR (ν max/cm−1): 3250 , 3053 (C—H aromatic), 2130 (C≡C) 1649, 1680 (C=O quinone), 1575–1604 (C—C aromatic), 1016, 1208 and 1245 (C—O).1H NMR : δH 8.12 , 8,07 , 7.74 , 7.70 , 6.33 , 4.78 , 2.63 . 13C NMR : δC 184.6 (C-4), 179.8 (C-1), 158.1 (C-2), 134.3 (C-6), 133.4 (C-7), 131.9 (C-10), 131.1 (C-9), 126.7 (C-5), 126.2 (C-8), 111.6 (C-3), 78.2 (C-13), 75.5 (C-12), 56.7 (C-11).Spectrometric data. IR νUiso(H) = 1.2Ueq (C).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989018015438/is5501sup1.cifCrystal structure: contains datablock(s) Monoclinic, Triclinic, OPLAU. DOI: Click here for additional data file.10.1107/S2056989018015438/is5501Monoclinicsup2.cmlSupporting information file. DOI: 1876486, 1876485CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


CircMYO10 is a circular RNA generated by back-splicing of gene MYO10 and is upregulated in osteosarcoma cell lines, but its functional role in osteosarcoma is still unknown. This study aimed to clarify the mechanism of circMYO10 in osteosarcoma.CircMYO10 expression in 10 paired osteosarcoma and chondroma tissues was assessed by quantitative reverse transcription polymerase chain reaction (PCR). The function of circMYO10/miR-370-3p/RUVBL1 axis was assessed regarding two key characteristics: proliferation and endothelial–mesenchymal transition (EMT). Bioinformatics analysis, western blotting, real-time PCR, fluorescence in situ hybridization, immunoprecipitation, RNA pull-down assays, luciferase reporter assays, chromatin immunoprecipitation, and rescue experiments were used to evaluate the mechanism. Stably transfected MG63 cells were injected via tail vein or subcutaneously into nude mice to assess the role of circMYO10 in vivo.CircMYO10 was significantly upregulated, while miR-370-3p was downregulated, in osteosarcoma cell lines and human osteosarcoma samples. Silencing circMYO10 inhibited cell proliferation and EMT in vivo and in vitro. Mechanistic investigations revealed that miR-370-3p targets RUVBL1 directly, and inhibits the interaction between RUVBL1 and β-catenin/LEF1 complex while circMYO10 showed a contrary effect via the inhibition of miR-370-3p. RUVBL1 was found to be complexed with chromatin remodeling and histone-modifying factor TIP60, and lymphoid enhancer factor-1 (LEF1) to promote histone H4K16 acetylation (H4K16Ac) in the vicinity of the promoter region of gene C-myc. Chromatin immunoprecipitation methods showed that miR-370-3p sponge promotes H4K16Ac in the indicated region, which is partially abrogated by RUVBL1 small hairpin RNA (shRNA) while circMYO10 showed a contrary result via the inhibition of miR-370-3p. Either miR-370-3p sponge or ShRUVBL1 attenuated circMYO10-induced phenotypes in osteosarcoma cell lines. MiR-370-3p inhibition abrogated the inhibition of proliferation, EMT of osteosarcoma cells in vitro and in vivo seen upon circMYO10 suppression via Wnt/β-catenin signaling.CircMYO10 promotes osteosarcoma progression by regulating miR-370-3p/RUVBL1 axis to promote chromatin remodeling and thus enhances the transcriptional activity of β-catenin/LEF1 complex, which indicates that circMYO10 may be a potential therapeutic target for osteosarcoma treatment. Osteosarcoma (OS) originates from mesenchymal stem cells. It is the most common malignant bone tumor, mainly occurring during childhood and adolescence , 2. OS fNon-coding RNAs, including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), play crucial roles in cell development and disease –9. CircRglycogen synthase kinase 3 (GSK3), and casein kinase 1α (CK1α) -8 assays) or 6-well plates (for colony formation assays) at a density of 3 × 10TOPFLASH plasmids (GeneChem) contain 6 TCF/LEF binding sites which are mutated in FOPFLASH plasmids. Stably transfected MG63 cells were seeded in 24-well plates at 40% confluence. After 24 h, MG63 cells were transiently transfected with 2 μg FOPFLASH or TOPFLASH plasmids, 0.2 μg Renilla luciferase plasmids. 48 h after transfection, activities of both firefly luciferase (LUC) and Renilla luciferase (RLUC) were measured with a Dual Luciferase Reporter Gene Assay Kit . The firely luciferase activity was normalized to the Renilla luciferase activity and expressed as the fold change compared with cells transfected with empty vector alone.6 to 6 × 106 cells were treated with 1% formaldehyde to cross-link proteins to DNA. The chromatin was then prepared, sonicated and immunoprecipitated with indicated antibodies. The recovered DNA was detected by PCR using a primer flanking the LEF1-binding site at − 1804 in the human MYC promoter. The Sequence for primers is as follows: 5′-tgagagcaattaaagtagttagg-3′ (forward primer) and 5′-gcccagcatcttataattagtaa-3′ (reverse primer). The input sample contains 2% of the total input chromatin as PCR template in the detection of DNA pulled down with RUVBL1 and 0.5% of the total input in the detection of DNA pull downed with H4K16 (Acetylated). The band intensity of immunoprecipitated DNA in DNA-blot was normalized to that of input.The ChIP assay was performed according to the manufacturer’s recommendation , with an average size of sheared fragments of about 200 to 1000 base pairs. In brief, 4 × 10MG63 cells were co-transfected with indicated plasmids with lipofectamine 3000 (Invitrogen). In brief, cells were lysed by RIPA Lysis Buffer (Cwbio) and lysates were incubated with indicated antibodies to capture the target protein at 4 °C overnight with gentle mixing. Next, lysates were incubated with Protein A/G agarose  at 4 °C for 2-4 h so as to collect Protein A/G-Antibody-protein complexes. Protein A/G agarose was recollected via centrifuge and was washed to remove non-specifically bounded protein. The bounded protein was eluted with elution buffer for western blot analysis.5 cells/well) were seeded in 6-well plates and incubated at 37 °C with 5% CO2 saturation overnight. Pipette tips (200 μl) were used to scrape a straight scratch in the confluent cell layer, and marker lines were drawn under the plate to mark the scratch position. Cells were washed twice with PBS, and images from specific scratched positions were captured by microscopy as a baseline. Fresh DMEM was added and, 24 h later, images from the same position were captured. The ratio of the area of wound-healing was quantified by ImageJ software .Transfected cells  was added to the upper chamber. DMEM (300 μl) containing 10% FBS was added to the lower chamber. After 24 h, cells in the upper chamber were removed and the lower side of the chamber was gently washed twice with PBS and fixed with 4% paraformaldehyde for 30 min. Cells were then stained with 1% crystal violet solution for 10 min and images were captured by microscopy.At 48 h post-transfection, cells were digested, washed twice with PBS, re-suspended, and seeded in 12-well chamber-containing plates. Chambers for the invasion assay were pre-coated with Matrigel according to the manufacturer’s instructions . Serum-free DMEM (200 μl) containing cells  for 1 h. Primary antibodies were diluted 1:100 in 5% BSA and incubated with cells overnight at 4 °C. The following day, the cells were gently washed several times in PBS, followed by incubation with CL594- or CL488-conjugated secondary antibodies  and diluted 1:300 in PBS for 1 h at 25 °C. Finally, cells were washed several times in PBS and immunofluorescence images were obtained using a Colibri epifluorescence microscope  and processed with Image J software. Detailed information about the antibodies is provided in the Supplementary Materials.Cy3-labeled circMYO10 probes and fluorescein amidite-labeled miR-370-3p probes were designed and synthesized by RiboBio. A FISH Kit (RiboBio) was used to detect probe signals according to the manufacturer’s instructions. Nuclei were stained with 4,6-diamidino-2-phenylindole. All images were acquired on a LSM880 NLO (2 + 1 with BIG) confocal microscope system (Carl Zeiss).7 cells from each group were subjected to an equal pellet volume of RIP lysis Buffer (100 ml) plus protease inhibitors cocktail and RNase inhibitors. The cell lysates were incubated with IgG or anti-Ago2 antibody-coated beads (Millipore) and rotated at 4 °C overnight. The immunoprecipitated RNAs were extracted by a RNeasy MinElute Cleanup Kit  after treatment with proteinase K buffer, and subjected to reverse transcription (CWBio). The circMYO10 levels were measured by quantitative reverse transcription PCR (qRT-PCR).RNA immunoprecipitation (RIP) experiments were performed using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit . MG63 cells stably expressing vector or shcircMYO10 were constructed. Approximately 1 × 107) were harvested, lysed, and sonicated. C-1 magnetic beads were incubated with circMYO10 probe (Life Technologies) at 25 °C for 2 h to generate probe-coated beads. The cell lysates were incubated with circMYO10 or oligo probe-coated C-1 magnetic beads to pull-down circMYO10. The RNA complexes bound to the beads were eluted and extracted with a RNeasy Mini Kit (Qiagen) for RT-PCR or real-time PCR. The biotinylated-circFAT1 probe was designed and synthesized by RiboBio.OS cells  as reported  and is aP-values.Statistical analysis was carried out using GraphPad Prism 5.0 . Most graphs contain plots with each data point represented and the mean ± SD is shown. To test significance, t tests were performed and results are presented with asterisks indicating CircMYO10 was first reported in A549 and other cell lines  and was We first analyzed the microarray data downloaded from GEO and drew a heatmap of the top 30 most differentially upregulated and downregulated circRNAs. This revealed the significant upregulation of hsa_cirRNA_103801 (circMYO10) in OS cell lines , which target the junction site of circMYO10, significantly decreased circMYO10 expression as detected by qRT-PCR , a negative form of RUVBL1 without ATPase activity but still competing with wild type RUVBL1 for β-catenin , 43, wasSince we had shown that circMYO10 acts as a sponge for miR-370-3p, we wondered whether circMYO10 is involved in the regulation of Wnt/β-catenin signaling via miR-370-3p/RUVBL1 axis. By applying TCF/LEF activity assats, we found that TCF/LEF activity was significantly increased in circMYO10 group when compared to Vector group Fig. f. MoreovSince reduced interaction between RUVBL1 and β-catenin/LEF1 complex was not sufficient to explain the reduced Wnt/β-catenin signaling, we sought to find further mechanistic insight into circMYO10/miR-370-3p/RUVBL1 axis.Previous study showed that RUVBL1 is a coactivator of TIP60 and enhances the transcription activity of TCF4 via histone H4 acetylation and thus the expression of ITF-2, a Wnt target gene . More spNext, we sought to investigate whether circMYO10 and miR-370-3p had a role in H4K16Ac via RUVBL1. First, we transfected MG63 cells with vector, pre-miR-370-3p or pre-miR-370-3p and RUVBL1. Interestingly, as illustrated in Fig. Next, we sought to find whether circMYO10 was involved in the H4K16Ac at the indicated region since circMYO10 was shown to activate Wnt/β-catenin signaling via miR-370-3p/RUVBL1 axis. We transfected MG63 cells with vector or shcircMYO10 or shcircMYO10 and miR-370-3p sponge or shcircMYO10 and RUVBL1 to construct stable cell lines. Consistent with our hypothesis, knockdown of circMYO10 inhibited the presence of RUVBL1 at the promoter region of C-myc and inhibited H4K16Ac  Expression of circMYO10 upon transfection with Si-circMYO10. (b) Efficiency of miR-370-3p mimics and inhibitors were measured in both MG63 and U2OS cells. (a-b) Data represents the mean ± SD (n = 3). Three independent assays were performed in the above assays. * P < 0.05, ** P < 0.01, *** P < 0.001 (Student’s t-test).Additional file 3: Figure S2. The seed regions between miR-877-3p and circMYO10. (a) Schematic illustration showing complementarity to the miR-370-3p sequence in circMYO10. (TIF 1399 kb)Additional file 4: Figure S3. MiR-370-3p showed a strong effect on the migration, invasion, and EMT process than miR-877-3p in MG63 and U2OS cells. (a) MiR-370-3p and miR-877-3p inhibited MG63 and U2OS cells to migrate and invade through transwells. Scale bars = 50 μ m. Data represents the mean ± SD. (b) Western blot analysis of Vimentin, N-cadherin and E-cadherin in cells transfected with either miR-370-3p or miR-877-3p. Three independent assays were performed in the above assays. (a) * P < 0.05, ** P < 0.01, *** P < 0.001 (Student’s t-test).Additional file 5: Figure S4. RUVBL1 is an oncogene in osteosarcoma. (a) CCK-8 assays for MG63 and U2OS cells transfected with either NC or RUVBL1 SiRNA. Data represents the mean ± SD (n = 18) Data represents the mean ± SD. (b) Anchorage -independent colony formation of MG63 and U2OS cells was inhibited upon RUVBL1 knockdown. Scale bars = 50 μ m. (c) RUVBL1 inhibited compromised the migration ability of MG63 and U2OS cells. Scale bars = 200 μ m. Data represents the mean ± SD (n = 3) Data represents the mean ± SD. Three independent assays were performed in the above assays.  * P < 0.05, ** P < 0.01, *** P < 0.001 (Student’s t-test).Additional file 6: Figure S5. RUVBL1 positively regulates Wnt/β-catenin signaling. (a) Downregulation of C-myc, cyclinD1 and β-catenin was detected in RUVBL1-inhibited MG63 and U2OS cells by (a) qRT-PCR and (b) immunofluorescence analysis. (a) Data represents the mean ± SD. (b) Scale bars = 50 μ m. Three independent assays were performed in the above assays. (a-b) * P < 0.05, ** P < 0.01, *** P < 0.001 (Student’s t-test).Additional file 7: Figure S6. MiR-370-3p inhibits Wnt/β-catenin signaling. (a-c) Transfection with miR-370-3p mimics significantly downregulated the expression of β-catenin, C-myc and cyclinD1 as revealed by (a) qRT-PCR, (b) western blot analysis and (c) immunofluorescence analysis, while inhibition of miR-370-3p showed a contrary result. (a) Data represents the mean ± SD. (c) Scale bars = 50 μ m. Three independent assays were performed in the above assays. (a) * P < 0.05, ** P < 0.01, *** P < 0.001 (Student’s t-test).Additional file 8: Figure S7. Either overexpression of RUVBL1 or inhibition of miR-370-3p partially restored the phenotypes caused by the circMYO10 knockdown. (a-c) Transfection with either miR-370-3p sponge or RUVBL1 partially abrogated the inhibited proliferation ability of MG63 and U2OS cells induced by shcircMYO10. (a) CCK-8 analysis, (b) colony formation assays, (c) cell cycle analysis. (a) Data represents the mean ± SD (n = 18). (b-c) Data represents the mean ± SD (n = 3). Three independent assays were performed in the above assays. (a-b) * P < 0.05, ** P < 0.01, *** P < 0.001 (Student’s t-test).Additional file 9: Figure S8. High degree colocalization of miR-877-3p and circMYO10 was detected. (a) FISH assays showed high degree colocalization of circMYO10 and miR-877-3p.Additional file 10: Figure S9. MiR-877-3p inhibits OS progression. (a) The expression of miR-877-3p in ten paired chondroma (n = 10) and osteosarcoma tissues (n = 10) was measured by qRT-PCR and FISH assays. (a) Data represents the mean ± SD (n = 90 per group). (b) FISH assays showed miR-877-3p expression is lower in human OS tissue than in chondroma tissue. Representative images are shown. Scale bars = 100 μm. (c) The expression of miR-877-3p in hFOB1.19, 143B, U2OS, HOS, MG63, and SJSA-1 was measured by qRT-PCR. Data represents the mean ± SD (n = 9). (d) The efficiency of miR-877-3p mimics and miR-877-3p inhibitor was detected by qRT-PCR. Data represents the mean ± SD (n = 9). (e) Transwell migration and invasion assays revealed enhanced migration and invasion ability of MG63 and U2OS cells after transfection with miR-877-3p inhibitors. Scale bars = 50 μ m. Data represents the mean ± SD. (f-g) Proliferation ability of MG63 and U2OS cells transfected with either miR-877-3p mimics or miR-877-3p inhibitor was evaluated by CCK-8 assays and colony formation assays. Data represents the mean ± SD. (h) The changes of EMT markers, N-cadherin, E-cadherin, and Vimentin upon miR-877-3p overexpression and inhibition were detected by western blot analysis. Three independent assays were performed in the above assays.  * P < 0.05, ** P < 0.01, *** P < 0.001 (Student’s t-test).Additional file 11: Figure S10. LEF1 is a direct target of miR-877-3p and miR-877-3p inhibits Wnt/β-catenin signaling. (a) Pathway enrichment analysis was conducted for miR-877-3p via mirPath v. 3. (b) Complementary sequences of the LEF1 3′ UTR for miR-877-3p. (c) 293 T cells were co-transfected with miR-877-3p and a luciferase reporter plasmid containing either wild type or mutated LEF1 3′ UTRs. Data represents the mean ± SD (n = 9). (d) The changes in levels of LEF1, β-catenin, C-myc, and CyclinD1 were detected by western blot. (e) Relative mRNA expression of LEF1, β-catenin, C-myc, and cyclinD1 was measured by qRT-PCR. Data represents the mean ± SD (n = 9). Three independent assays were performed in the above assays.  * P < 0.05, ** P < 0.01, *** P < 0.001 (Student’s t-test).Additional file 12: Figure S11. MiR-877-3p is involved in the process where circMYO10 promotes osteosarcoma progression via activated Wnt/β-catenin signaling. (a) The expression of miR-877-3p in MG63 cells and U2OS cells stably transfected with shcircMYO10 or shcircMYO10 plus miR-877-3p sponge. Data represents the mean ± SD. (b) Migration and invasion assay in transwells showed that miR-877-3p inhibition promoted the migration and invasion ability of cells stably transfected with shcircMYO10 and miR-877-3p overexpression induced contrary results. Scale bars = 50 μm. Data represents the mean ± SD. (c-d) Inhibition of miR-877-3p partially abrogated the suppressive effect of shcircMYO10 on proliferation ability of both MG63 and U2OS cells. Data represent the mean ± SD. (e) The effect of miR-877-3p on EMT and Wnt/β-catenin signaling in cells stably transfected with shcircMYO10 was measured by western blot. Three independent assays were performed in the above assays. (a-d) * P < 0.05, ** P < 0.01, *** P < 0.001 (Student’s t-test).Additional file 13: Figure S12. (a-c) Nude mice were injected subcutaneously with 107 stable MG63 control cells, cells transfected with shcircMYO10, or cells co-transfected with circMYO10 shRNA and miR-877-3p sponge. Tumors were dissected, photographed, and weighed after 5 weeks. (b) Tumor volume was measured every week. Data represent the mean ± SD (n = 5). (c) Average wet tumor weight in each group at the end of the experiment (day 35). Data represents the mean ± SD (n = 5). (d) Western blot analysis of LEF1, β-catenin, C-myc, CyclinD1, Vimentin, E-cadherin, and N-cadherin of protein extracted from tumors. Sh: shcircMYO10. Sh + sponge: shcircMYO10 + miR-877-3p sponge. (e) Immunohistochemistry analysis of LEF1, β-catenin, C-myc, CyclinD1, Vimentin, N-cadherin, and E-cadherin in tumors. Scale bars = 100 μm. Three independent assays were performed in the above assays. (c-d) * P < 0.05, ** P < 0.01, *** P < 0.001 (Student’s t-test).


In the crystal, weak C—H⋯π interactions connect the molecules into zigzag chains along the a-axis direction.The essentially planar chalcone unit adopts an  18H12O3S2, synthesized by the Claisen–Schmidt condensation method, the essentially planar chalcone unit adopts an s-cis configuration with respect to the carbonyl group within the ethylenic bridge. In the crystal, weak C—H⋯π interactions connect the molecules into zigzag chains along the b-axis direction. The molecular structure was optimized geometrically using Density Functional Theory (DFT) calculations at the B3LYP/6–311 G++ basis set level and compared with the experimental values. Molecular orbital calculations providing electron-density plots of HOMO and LUMO molecular orbitals and molecular electrostatic potentials (MEP) were also computed both with the DFT/B3LYP/6–311 G++ basis set. The experimental energy gap is 3.18 eV, whereas the theoretical HOMO–LUMO energy gap value is 2.73 eV. Hirshfeld surface analysis was used to further investigate the weak interactions present.In the title compound, C In principle, the intermolecular charge-transfer (ICT), HOMO–LUMO gap and optical properties can be tailored by attaching electron donors and acceptors of various electronic nature, assuring efficient D⋯A interactions and planarization of the entire molecule  and two thiophene rings, B (S1/C11–C14) and C (S2/C15–C18), these substituent rings representing a donor–linker–acceptor conjugated system. Ring A [maximum deviation of 0.011 (4) Å at C3] forms dihedral angles of 1.88 (15) and 5.37 (16)°, respectively, with rings B  and C [maximum deviations of −0.009 (3) and 0.009 (3) Å for C15 and C16 respectively], respectively. The enone moiety  forms dihedral angles of 3.3 (2), 4.3 (2) and 7.4 (2)° with rings A, B and C, respectively. This planar conformation for the molecule indicates that the 1,3-benzodioxole group and the thiophene rings have stabilized the conjugated π-bond system.The experimental and optimized structures of (I)s-cis configuration with respect to the C8=O3 [experimental = 1.229 (4) Å and DFT = 1.227 Å] bond length within the enone moiety (O3/C8–C10). The molecule is observed to be essentially planar  about the C9—C10 bond with a C8—C9—C10—C11 torsion angle of 178.5 (3)°, whereas the corresponding DFT value is 179.6°. The slight difference is the result of the optimization being carried out in an isolated gaseous state whereas the experimental molecular structure could easily be affected by its normal environment ] with the 6-311++G basis set as implemented in the via C3—H3A⋯Cg are represented by a pair of short spikes at de + di = 2.60 Å. The significant contributions by H⋯H, H⋯C/C⋯H and H⋯O/O⋯H interactions suggest that weak hydrogen bonding and van der Waals interactions do play relevant roles in the crystal packing , while the simulated value is observed at 422 nm . The absorption maximum is assigned to the π–π* transitions that arise from the carbonyl group (C=O) of the compound. The slight difference in wavelength is due to the fact that the experimental study is conducted in solution whereas the theoretical study is performed for a gaseous environment  with an energy band gap of 2.73 eV.The experimental UV–Vis absorption spectrum consists of one major band that lies in the visible region at 400 nm Fig. 5a, whilenm Fig. 5b. The aet al., 2018EHOMO – ELUMO gap is calculated to be 3.18 eV. Generally, the value of the energy gap characterizes the chemical stability of the molecule  acts as an electron donor and represents the ability to donate electrons while the lowest unoccupied molecular orbital (LUMO) acts as the electron acceptor, representing the ability to accept electrons  are useful in investigating the relationship between the molecular structure and its physicochemical properties, visualizing the molecular size and shape, along with the charge distributions in molecules in terms of colour grading -1-(2-aminophenyl)-3-(thiophen-2-yl)prop-2-en-1-one -3-(5-bromo-2-thienyl)-1-(4-hydroxyphenyl)prop-2-en-1-one -1-(4-bromophenyl)-3-(thiophen-2-yl)prop-2-en-1-one -1--3-(4-chlorophenyl)prop-2-en-1-one -1--3-(3-bromophenyl)prop-2-en-1-one -1-(2-aminophenyl)-3-(thiophen-2-yl)prop-2-en-1-one -3-(5-bromo-2-thienyl)-1-(4-hydroxyphenyl)prop-2-en-1-one -1--3-(4-chlorophenyl)prop-2-en-1-one acetophenone (0.5 mmol) and 2,2′-bithiophene-5-carboxaldehyde (0.5 mmol) was dissolved in methanol. A catalytic amount of NaOH was added dropwise with vigorous stirring. The reaction mixture was stirred for about 5 h at room temperature and then poured into ice-cold water. The resulting crude solid was collected by filtration. Single crystals were grown from an acetone solution by slow evaporation.Uiso(H) = 1.2 Ueq(C). One outlier (104) was omitted from the final refinement.Crystal data collection and structure refinement details are summarized in Table 210.1107/S2056989019004912/jj2209sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989019004912/jj2209Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989019004912/jj2209sup3.docxComparison between calculated (DFT) and X-ray of selected geometrical data. DOI: Click here for additional data file.10.1107/S2056989019004912/jj2209Isup4.cmlSupporting information file. DOI: 1899823CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


O–H···Br– hydrogen bonding gives water-stable hexagonal nanotubes. Hydrogen bonding between bromide anions and a tetrahydroxytriptycene ligand was used to assemble crystalline hexagonal tubes with nanometer diameters in good yield. Use of a hexahydroxytriptycene ligand again gave hexagonal nanotubes, but containing the spontaneously-oxidised quinone–tetrahydroxy ligand. The surprisingly robust nanotubes are stable to heat, vacuum and water, and represent an unprecedented use of O–H···anion coordination to assemble complex three-dimensional structures. Inspired by Nature, directed self-assembly7ii) chloride gives a pentanuclear system containing a central chloride anion, while iron(ii) sulfate yields a hexanuclear product.9Anion templation has been exploited within the field of transition metal chemistry, where the choice of counteranion to the metal cation can dramatically influence the structure of the product. A number of elegant examples of this approach have been reported,While the field is still very much in its infancy, recently a few studies have reported the use of anion templation to prepare self-assembled systems. Notably, the groups of Wu, and Kruger and Gunnlaugsson have prepared “tetranuclear” cages assembled through hydrogen bonding between four phosphate or sulfate anions and four bis-urea ligands.111 1  salts were added to 1, resulting in downfield shifts of the molecule's O–H resonance  experiments showed that all of the crystals had extremely similar unit cells, despite the crystals being grown from a range of solvents 2]n (– hydrogen bonds n . The nan1 and TBA·Br in acetonitrile. The product was isolated as single crystals in 67% yield after drying in vacuo.The nanotubes could be prepared in bulk by simply diffusing diethyl ether vapour into a 1 : 2 stoichiometric mixture of 1H NMR spectroscopy confirmed the purity of the product, and powder X-ray diffraction (PXRD) of the dried bulk crystalline sample , but disappointingly nitrogen adsorption measurements showed a negligible nitrogen-accessible surface area.24Despite the large number and large size of the cations, there is still significant void space in the crystalline structure. In the solid state, this space appears to be occupied by poorly-defined diffuse solvent molecules , even though it is held together only by apparently-weak O–H···Brree days . This is2 could be used to prepare anion-templated framework materials– hydrogen bonding 2]n, where 3 is a partially-oxidized form of 2 containing one quinone ring 2]n; 1H NMR spectroscopy, elemental analysis and PXRD showed that the bulk material was pure and had the nanotube structure observed by SCXRD. It is noteworthy that the nanotubes crystallize from the highly competitive hydrogen-bonding solvent methanol.We have found that hexahydroxytriptycene Ka ∼ 80 M–1 for Br– in CD3CN) can be used to assemble complex three-dimensional nanotube structures. Even though these interactions are weak in solution, in the solid state, they act in concert to give stable materials that can be grown from methanol solution, or retain their structure even after soaking in water for several days. Importantly, these results demonstrate that interactions involving anions that are weak in solution can be used to prepare practically-useful solid-state materials. Work is continuing in our laboratories to expand this new strategy to prepare porous anion-templated materials.In this work, we have demonstrated that relatively weak O–H···anion hydrogen bonding interactions  and TBA·Br  were dissolved in acetonitrile (5 mL) and subjected to diethyl ether vapour diffusion. Over 2–4 days, large brown crystals developed; these were isolated by filtration, washed with copious diethyl ether and thoroughly dried in vacuo to give [1·(TBA·Br)2]n as brown single crystals. Yield: 66 mg . 1H NMR : 7.26–7.30 , 6.97–7.01 , 6.85 , 6.822O.‡Peak disappears on addition of D , 3.05–3.10 , 2.21 , 1.56–1.64 , 1.30–1.39 , 0.97 .Dimethyltetrahydroxytriptycene 1·(TBA·Br)2], C54H90N2O4Br2: C 65.4, H 9.2, N 2.8%. Mp: 140.0–141.5 °C. IR: ∼3180 cm–1 .EA: C 65.7, H 9.4, N 2.7%; calc. for n as very dark brown single crystals. Yield: 17 mg .The tetrahydroxy quinone ligand 1H NMR : 7.34J = 7.3 Hz, 24H).3·(TBA·Br)2], C54H88N2O6Br2: C 63.5, H 8.7, N 2.7%. Mp: 193–195 °C. IR: ∼3160 , 1652  scale" fill="currentColor" stroke="none"><path d="M0 1440 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z M0 960 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z"/></g></svg>O stretch) cm–1.EA: C 63.2, H 8.7, N 2.6%; calc. for [Product identity was elucidated using single crystal X-ray diffraction; powder X-ray diffraction on the bulk sample showed that the identity of the bulk product was consistent with the single crystal structure.The synthesis could also be performed using acetonitrile instead of methanol as solvent to give product of indistinguishable purity in similar yield.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file.


Salix daphnoides VILL. bark is described. Procyanidin B1 (1), B2 (2), B3 (3), B4 (4), C1 (5), epicatechin-(4β→8)-epicatechin-(4β→8)-catechin (6) and epicatechin-(4β→8)-epicatechin-(4β→8)-epicatechin-(4β→8)-catechin (7) have been isolated by a combination of different chromatographic separations on Sephadex® LH-20-, MCI®-, Diol-and RP-18-phases. Mass spectrometry, 1D- and 2D-NMR, circular dichroism and polarimetry were used for their structure elucidation and verification by comparison with the literature. Additionally, two fractions of very polar flavan-3-ols were compared: “regular” polymeric PAs received at the very end of the Sephadex® LH-20 chromatography showing no mobility on silica TLC and “unusual” PAs with the same RF-value but already eluting together with flavonoids in the Sephadex® LH-20 system. These “unusual” PAs were subsequently enriched by centrifugal partition chromatography (CPC). 13C-NMR, polarimetry, thiolysis, acid hydrolysis and phloroglucinol degradation were used to characterize both fractions. Differences in the composition of different flavan-3-ol units and the middle chain length were observed.In the present study, a qualitative analysis of proanthocyanidins (PAs) from an aqueous-methanolic extract of  Salicis cortex, Salix spp., Salicaceae) is an herbal drug monographed in the European Pharmacopoeia (Ph. Eur.) -HSQC, [1H-13C]-HMBC, [1H-1H]-COSY and [1H-1H]-ROESY experiments were performed. The 1D-1H-NMR- and 1D-13C-NMR-spectra of the peracetates were performed on a AVANCE 300 (Bruker Corporation) operating at 300.13 MHz (1H-NMR) and 75.48 MHz (13C-NMR).All shift values (δESI-HRMS for structure elucidation and verification were performed on a Q-TOF 6540 UHD (Agilent).1 to 6 were concentrated at 175.0 µmol/L, compound 7 was concentrated at 36.6 µmol/L. The concentrations of the fractions S2 W and S3.8 were 0.06 mg/mL and 0.12 mg/mL respectively. The spectra were smoothed by the Savitzky-Golay algorithm with a convolution width of 15.CD-spectra were acquired by a J-715 spectropolarimeter  operating at 22 °C. Area of measurement was 200–400 nm in 0.5 nm steps with 200 nm/min and 10 cycles. 1 mm Quartz cuvettes were used. Samples were all solved in methanol for spectroscopy, but the fraction containing the “unusual” PAs was solved in a mixture of methanol/water (80 + 20). Compounds -cis to 2,3-trans ratio of the oligomeric and polymeric PAs, Equation (4) was used after determinating cis = amount of 2,3-cis configurated flavan-3-ols included in a PA; Polarimetry of the isolated compounds was achieved by a UniPol L 1000 polarimeter . The samples were solved in methanol for spectroscopy with a concentration of 0.1% and were measured at 589.30 nm in a micro tube . To estimate the 2,3raction) ,31:(4)χEpicatechin-(4β→8)-catechin, procyanidin B1 (1): 70.9 mg; m/z 577.1370 [M − H]−.Epicatechin-(4β→8)-epicatechin, procyanidin B2 (2): 11.7 mg; m/z 577.1358 [M − H]−.Catechin-(4α→8)-catechin, procyanidin B3 (3): 45.8 mg; m/z 577.1369 [M − H]−.Catechin-(4α→8)-epicatechin, procyanidin B4 (4): 7.4 mg; m/z 577.1364 [M − H]−.Epicatechin-(4β→8)-epicatechin-(4β→8)-epicatechin, procyanidin C1 (5): 8.7 mg; m/z 865.2001 [M − H]−.Epicatechin-(4β→8)-epicatechin-(4β→8)-catechin (6): 20.5 mg; m/z 865.1988 [M − H]−.Epicatechin-(4β→8)-epicatechin-(4β→8)-epicatechin-(4β→8)-catechin (7): 15.3 mg; m/z 1153.2624 [M − H]−.Fraction S2 W : CD  205.5 (−92820.8), 221.5 (+86787.4), 227 (+95101.3), 280.5 (−8054.69).Fraction S3.8 (“regular” PAs): S. daphnoides revealed the presence of four dimeric, two trimeric as well as a tetrameric procyanidin. In contrast to Pobłocka-Olech et al. [-cis configuration predominates in both fractions. The fraction with the “regular” PAs consists of slightly more tannins with more trihydroxylated constituent flavan-3-ol units and longer chains compared to the fraction with the “unusual” PAs. The identification of B-ring vicinal trihydroxylated flavan-3-ol units in polymeric PAs is in accordance with Foo and coworkers [Salix caprea L. However, that these observed slight differences of both fractions are responsible for the different chromatographic behavior on Sephadex® LH-20 seems to be questionable. Further studies are necessary to satisfactorily describe chemical differences of the “unusual” and “regular” PA fractions.A systematic investigation of the proanthocyanidin pattern of h et al. , who puroworkers  who foun


E isomer of a nicotinamide derivative to its Z isomer. The chemical and molecular structures of the product was established crystallographically. The surprising synthesis, chromatographic purification and comprehensive characterization of this compound including single crystal structural analysis are reported and its structure including crystal packing is discussed in detail.A benzoquinone compound was unintentionally synthesized by photocyclization and subsequent oxidation in air while attempting to transform the  14H10N2O, crystallizes in the monoclinic space group P21/c with four molecules in the unit cell. All 17 non-H atoms of one molecule lie essentially in one plane. In the unit cell, two pairs of molecules are exactly coplanar, while the angle between these two orientations is close to perfectly perpendicular at 87.64 (6)°. In the crystal, molecules adopt a 50:50 crisscross arrangement, which is held together by two nonclassical and two classical intermolecular hydrogen bonds. The hydrogen-bonding network together with off-centre π–π stacking interactions between the pyridine and outermost benzene rings, stack the molecules along the b-axis direction.The title compound, C Their multifaceted biological efficacy is outstanding and ranges from cardiovascular  and there is one molecule in the asymmetric unit. Benzo[h]quinoline-3-carboxamide consists of a nicotinamide unit being fused with a benzo[h]quinoline moiety, while the pyridine ring is shared between these two common structural building blocks , while for benzoquinoline, there are over 500; Groom et al., 2016P21/c or P21/a, the angles between the aromatic plane (here C2/C3/N2/C4/C13/C14) and the amide substituent (here O1/N1/C1) range from 22.1 to 23.3° °. This points toward an extension of the aromatic resonance systems to include the amide substituent. In the parent nicotinamide scaffolds, this does not occur. Similarly, the comparatively long C1=O1 distance of 1.238 (3) Å (average 1.23 Å) and the comparatively short C1—C2 distance of 1.491 (3) Å (average 1.50 Å in other nicotinamide structures) indicate some involvement of these atoms in resonance effects. In support of this extended resonance, in the nicotinamide structures, the aromatic C—C bonds are much less diverse  than in the structure reported here. In fact, the C—C [range 1.376 (3)–1.414 (3) Å] and C—N [1.321 (3) and 1.360 (3) Å] bond lengths here are much more similar to the two known structures of 2-unsubstituted and 3-substituted benzo[h]quinolines (refcodes JAFVEU and SUDVES), with ranges of average C—C and C—N bond lengths of 1.38–1.42 and 1.32–1.36 Å, respectively °]. This forms a crisscross pattern when viewed along the al Fig. 2. Classicfs Fig. 3. Each mofs Fig. 3, and theon Fig. 4. The obson Fig. 4. These con Fig. 6.E)-5-styrylnicotinamide  in methanol (350 ml) was treated with a solution of iodine  in methanol (50 ml). A slow stream of compressed air was bubbled through the reaction mixture while it was irradiated with UV light . After complete consumption of the starting material (24 h), the solvent was removed under reduced pressure. Purification of the residue by silica-gel column chromatography  gave pure benzo[h]quinoline-3-carboxamide as a colourless solid . Crystallization was accomplished by slow evaporation of a solution in THF (5 mg ml−1) and yielded the title compound as colourless needles: RF = 0.32 ; m.p. 549.8 K (decomposition); 1H NMR, H,H-COSY : δ (ppm) 9.48 , 9.26–9.19 , 8.89 , 8.36 , 8.12–8.07 , 8.03 , 7.95 , 7.85–7.78 , 7.74 ; 13C NMR, DEPT135, HSQC, HMBC : δ (ppm) 166.4 (C1), 147.8 (C3), 146.7 (C4), 135.5 (C14), 133.8 (C13), 130.3 (C5), 129.0 (C8), 128.1 (C9/C11), 128.0 (C9/C11), 127.8 (C2), 127.3 (C7), 125.8 (C12), 124.9 (C10), 124.1 (C6); IR (ATR): ν (cm−1) 3336, 3136, 1686, 1482, 1395, 1295, 801, 691, 539, 489; ESI–HRMS calculated for [C14H10N2O + H]+ 222.0793, found 222.0796; compound purity (220 nm): 100%.A solution of (Uiso(H) = 1.2Ueq(C). The two amine H atoms were found and refined without any constraints or restraints.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989019014440/sj5580sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989019014440/sj5580Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989019014440/sj5580Isup3.cmlSupporting information file. DOI: 1960760, 1960760CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


A triclinic polymorph of 1,4-bisbenzene was obtained under solvothermal conditions. 36H24N6, was formed in the presence of the Lewis acid yttrium trichloride in an attempt to obtain a coordination compound. The crystal structure of the orthorhombic polymorph (Form II), has been described previously , forming a three-dimensional structure.The title triclinic polymorph (Form I) of 1,4-bisbenzene, C al. 2010. Acta Cr Opposite central pyridine rings (N1/C1–C5 and N1′/C1′–C5′) are coplanar by symmetry, and the angle between them and the central benzene ring (C16–C18/C16′–C18′) is 49.98 (8)° . The nitrogen atoms of the pyridine rings inside the 2,2′:6′,2′′-terpyridine (terpy) entities, N3⋯N1⋯N2, lie in trans–trans positions.The molecular structure of the title triclinic polymorph (Form I) is illustrated in Fig. 1viz. 24.86 (12) and 5.10 (12)° on one side and 6.30 (11) and 8.21 (12)° on the opposite side. The dihedral angles between the central pyridine rings of the terpy units and the central benzene ring are 34.95 (11) and 36.17 (11)°. A structural overlay of the molecules of the two polymorphs (r.m.s. deviation = 0.0705 Å), illustrating the differences in their conformation, is given in Fig. 2Mercury; Macrae et al., 2008In the orthorhombic polymorph, Form II, all the angles between side and central pyridine rings of the terpy units are different (because of the lack of symmetry elements inside the molecule), a-, b- and c-axis directions  through white to blue .The Hirshfeld surfaces are colour-mapped with the normalized contact distance, dnorm are given in Fig. 4a and 5a, respectively, where short interatomic contacts are indicated by the faint red spots. The π–π stacking is confirmed by the small blue regions surrounding bright-red spots in the various aromatic rings  on the Hirshfeld surface mapped over the shape-index, and by the flat regions around the aromatic regions in Fig. 4c and 5c, the Hirshfeld surface mapped over the curvedness.The Hirshfeld surface of Forms I and II, mapped over gs Fig. 4b and 5bb), C⋯H/H⋯C at 24.7% , C⋯C at 9.6% , N⋯H/H⋯N at 9.4%  and C⋯N at 6.2% .The fingerprint plots for Forms I and II, are given in Figs. 67% Fig. 6c, C⋯C a6% Fig. 6d, N⋯H/H4% Fig. 6e and C⋯2% Fig. 6f.b), C⋯H/H⋯C at 30.6% , N⋯H/H⋯N at 13.3% , C⋯C at 8.3%  and C⋯N at 4.3% . Here, the C⋯H/H⋯C and N⋯H/H⋯N contacts at 30.6 and 13.3%, respectively, are more important than those in Form I at 24.7 and 9.4%, respectively.The principal intermolecular contacts in the crystal of Form II are H⋯H at 43.3% Fig. 7b, C⋯H/H6% Fig. 7c, N⋯H/H3% Fig. 7d, C⋯C a3% Fig. 7e and C⋯3% Fig. 7f. Here,et al., 20063  was purchased and used as received. Solvents  were dried using standard techniques and stored with molecular sieves in flasks with a J. Young valve.1,4-Bisbenzene was synthesized according to the literature procedure , 1,4-bisbenzene  and 1 ml DMF were filled together under inert conditions in a self-made Duran(R) glass ampoule . The ampoule was sealed under vacuum and placed in a resistance heating oven with a thermal control (Eurotherm 2416). The heating program was as follows: heating up to 503 K in 30 min, holding temperature for 8 h, cooling down to RT uncontrollably. The ampoule was then taken out of the oven and a star-like net of needle-shaped single crystals was observed. The ampoule was heated again as previously but up to 523 K and then cooled down to RT uncontrollably. Now only a few plate-shaped single crystals were present. The ampoule was unsealed, the solution removed and the remaining single crystals were washed with toluene (1 ml).YClUiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989019015810/su5525sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989019015810/su5525Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989019015810/su5525sup3.pdfCSD search. DOI: Click here for additional data file.10.1107/S2056989019015810/su5525Isup4.cmlSupporting information file. DOI: 1967605CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The molecules are held together by moderate N—H⋯O and O—H⋯N, and weak O—H⋯O and C—H⋯Ocarbonyl hydrogen bonds in which the primary and secondary amine and water H atoms are involved. The H atom directly bonded to the P atom avoids hydrogen bonding, as usual.Two of the constituent molecules in bis(3-carbamoylpyridin-1-ium) phosphite monohydrate,  6H7N2O+·HPO32−·H2O, i.e. the phosphite anion and the water molecule, are situated on a symmetry plane. The molecules are held together by moderate N—H⋯O and O—H⋯N, and weak O—H⋯O and C—H⋯Ocarbonyl hydrogen bonds in which the amide and secondary amine groups, and the water molecules are involved. The structural features are usual, among them the H atom bonded to the P atom avoids hydrogen bonding.Two of the constituent molecules in the title structure, 2C Wald, 1991et al., 1967Nicotinamide (pyridine-2-carboxamide) is a biologically important molecule, being the active part of vitamin B3 and nicotinamide adenine dinucleotide (NAD)  − pKa(acid) , respectively  would make a salt or a co-crystal with phosphorous acid, Hn1 is involved in the strongest hydrogen bond present in the structure (N1—H1n1⋯O3i). Its parameters indicate that this hydrogen bond is situated on the boundary between strong and moderate hydrogen bonds  Å for each hydrogen, while ξ2 = 12.6.The C—NHet al., 1990n2iv—N2iv—H1n2iv⋯Ow⋯H1n2vii—N2vii—H2n2vii⋯O3i  of equimolar amounts of nicotinamide (1.49 g) and phosphorous acid (1 g). Colourless crystals were isolated after two months.Uiso(H) = 1.2Ueq(C). Water hydrogen H2ow was refined freely, while H1ow was restrained with a distance restraint of 0.84 Å with elasticity 0.02 Å  = 1.5Ueq(O). The hydrogens of the primary amine N2 group and the secondary amine N1 group were constrained by Uiso(H) = 1.2Ueq(N). The P—H hydrogen was refined isotropically. Three reflections, i.e. 95I(obs) − I|/σ(I) > 20.Crystal data, data collection and structure refinement details are summarized in Table 2Robs = 0.0242, Rwobs = 0.0773, Rall = 0.0243, Rwall = 0.0774 and S = 3.13, with number of parameters = 127. With application of anharmonic approximation, Robs = 0.0188, Rwobs = 0.0535, Rall = 0.0188, Rwall = 0.0535 and S = 2.21, with number of parameters = 181. The respective values of the third- and fourth-order components of the displacement tensor are given in the CIF.)Since the phosphite oxygens revealed large displacement ellipsoids, the anharmonic displacement parameters upto the fourth grade were included for atoms P1, O2 and O3. (The refinement with the harmonic approximation resulted in Refinement with the assumption of the presence of inversion twinning resulted in a Flack parameter of 0.012 (13) (726 Friedel pairs used in the refinement). Therefore, the crystal was considered as single-domained in the final stage of the refiement, the results of which are presented here.10.1107/S2056989018011192/eb2010sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989018011192/eb2010Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989018011192/eb2010Isup3.smiSupporting information file. DOI: Click here for additional data file.10.1107/S2056989018011192/eb2010Isup4.cmlSupporting information file. DOI: 1860376CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Spodoptera exigua, uses -9,12-tetradecadienyl acetate as the major component of its sex pheromone. Previous isotope-labeling experiments demonstrated an unusual ∆12 desaturase activity involved in the biosynthesis of this compound; however, the putative ∆12 desaturase gene has not been reported to date. In the present study, we confirmed this ∆12 desaturation pathway by in vivo labeling experiments, and characterized candidate desaturase genes in a yeast heterologous expression system. We demonstrated that a pheromone gland-specific desaturase, SexiDes5, uses palmitic acid and the subsequently chain-shortened product (Z)-9-tetradecenoic acid as substrates to produce (Z)-11-hexadecenoic and -9,12-tetradecadienoic acids, respectively. In addition, the homologous desaturase SlitDes5 from the congeneric Spodoptera litura had analogous functions.The beet armyworm, The online version of this article (10.1007/s10886-019-01067-3) contains supplementary material, which is available to authorized users. Upon hatching, larvae were reared at 23 ± 1 °C under a 17:7 L:D photoperiod and 70% RH, and were fed on a modified artificial diet based on the formula of Hinks and Byers  and  hexadecanoic acid (D3–16:acid) were purchased from Larodan AB . (Z)-11- Hexadecenoic acid (D9-Z11–16:acid) was synthesized as described in Löfstedt et al. (Z)-9- tetradecenoic acid (D9-Z9–14:acid) was synthesized as described in Zhu et al. (E)-12- tetradecenoic acid (D3-E12–14:acid) and Z9–14:acid were obtained from Prof. Cheng-Hua Zhao as gifts. Other fatty acids and synthetic pheromone components were available in our laboratory collection of compounds and were of various origins.[14,14,14-t et al. , and (Z)u et al. . (E)-12-3–16:acid, D3–14:acid, D3-E12–14:acid, D9-Z9–14:acid, D9-Z11–16:acid, as well as unlabeled Z9–14:acid, were dissolved in dimethylsulphoxide (DMSO) at 40 μg/μl. About 1 h into the scotophase, 0.4 μl of a solution was topically applied to the pheromone gland of a female. The same volume of DMSO was applied as a control to another group. After a 5 h incubation period, pheromone glands were excised, with five glands pooled in a 250 μl insert in a 1.5 ml glass vial to which 20 μl heptane was added. The glands were allowed to extract at room temperature for 30 min and the solvent, containing the pheromone components, was transferred to a new vial. The remaining lipids in the glandular residue were subsequently extracted with 100 μl chloroform:methanol (2:1 v:v) at room temperature overnight in order to analyze the fatty acyl precursors. This was accomplished by base methanolysis, which converted fatty acyl moieties to methyl esters  or non-polar column . Helium was the carrier gas.−1, held for 10 min, and finally increased to 230 °C at 4 °C.min−1 and held for 10 min. Deuterium label incorporation into the pheromone components was detected using selected ion monitoring (SIM): m/z 61, 192 and 210 for Z9,E12–14:OAc and Z9,E11–14:OAc, m/z 61, 195 and 213 for D3-Z9,E12–14:OAc and D3-Z9,E11–14:OAc, m/z 61 and 194 for (Z)-9-tetradecenyl acetate (Z9–14:OAc), m/z 31 and 194 for (Z)-9-tetradecenol (Z9–14:OH), m/z 61 and 197 for D3-Z9–14:OAc, m/z 31 and 197 for D3-Z9–14:OH, m/z 61, 194 and 203 for D9-Z9–14:OAc, m/z 31, 194 and 203 for D9-Z9–14:OH, m/z 61 and 222 for (Z)-11-hexadecenyl acetate (Z11–16:OAc), m/z 61 and 225 for D3-Z11–16:OAc, m/z 31 and 222 for (Z)-11-hexadecenol (Z11–16:OH), and m/z 31 and 225 for D3-Z11–16:OH.For analyzing pheromone gland extracts, the oven temperature was set at 80 °C for 1 min, then increased to 190 °C at 10 °C.min−1 and held for 10 min. Deuterium label incorporation into putative pheromone precursors was detected by characteristic ions using SIM: m/z 242 and 245 for methyl myristate (14:Me) and D3–14:Me, respectively, m/z 208 and 240 for monounsaturated 14:Me, m/z 211 and 243 for monounsaturated D3–14:Me, m/z 164, 206 and 238 for methyl -9,12-tetradecadienoate , m/z 164, 209 and 241 for D3-Z9,E12–14:Me, m/z 236 and 268 for methyl (Z)-11-hexadecanoate (Z11–16:Me), m/z 239 and 271 for D3-Z11–16:Me, m/z 234 and 266 for diunsaturated methyl palmitate (16:Me), m/z 237 and 269 for diunsaturated D3–16:Me.For fatty acid methyl esters (FAMEs), the oven temperature was set at 80 °C for 1 min, then increased to 230 °C at 10 °C.min−1 to 210 °C, held for 15 min, and then increased at 10 °C.min−1 to 230 °C for 20 min. Full scan mode was used to record mass spectra and compounds were identified by comparison of retention times and mass spectra with those of reference compounds.For analysis of yeast extracts, the oven temperature was set to 80 °C for 1 min, then increased at 10 °C.min−1 to 140 °C, then increased at 5 °C.min−1 to 260 °C, and held for 30 min.DMDS derivatization , and DNaseI digestion was included to avoid genomic DNA contamination. The quality of total RNA was checked with a spectrophotometer . The single-stranded cDNA templates were synthesized from 1 μg of total RNA from S. exigua and S. litura pheromone gland and abdomen tissue samples using the PrimeScript RT Master Mix (Thermo Scientific™).Twenty pheromone glands of 1–4-d-old female 2O. The PCR products were analyzed by electrophoresis on 1.5% w/v agarose gel in TAE buffer  and were then gel purified with a GeneJET Gel Extraction Kit (Thermo Scientific™), cloned into pDONR221 by BP reaction to generate entry clone for each gene  and sequenced by M13+ and M13- primers. The correct entry clones were cloned into destination vector pYEX-CHT-DEST (Ding and Löfstedt The desaturase-like SexiDes5 (GenBank accession number: KU755471), SlitDes5 (GenBank accession number: XP_022827905.1), SexiDes7 (GenBank accession number: AFO38465.1) and SexiDes11 (GenBank accession number: AFO38464.1), previously described to show pheromone gland-biased expression  glucose, 0.7% YNB , synthetic complete amino acid drop-out lacking uracil and leucine , 1% tergitol , 0.01% adenine (Sigma-Aldrich) and 0.5 mM oleic acid (Sigma-Aldrich) for a week. For the functional assay, individual yeast colonies were inoculated in 5 ml of the same selective liquid medium for 48 h at 30 °C and 300 rpm, diluted to an OD600 of 0.4 in 250 ml flasks containing 20 ml selective medium and 2 mM CuSO4 and added supplementation of a FAME precursor . Each FAME precursor was prepared at a concentration of 100 mM in 96% ethanol and added to reach a final concentration of 0.5 mM in the culture medium  and hexadecanol  monounsaturated Z9–14:OAc, Z9–14:OH, Z11–16:OAc and Z11–16:OH, and doubly unsaturated Z9,E12–14:OAc and -9,12-tetradecadienol , were found in gland extracts of S. exigua based on GC/MS analysis. In addition to various ubiquitous fatty acids, including myristic, palmitic, palmitoleic, stearic, oleic, linoleic and linolenic acids, the following fatty acyl compounds  were found in the pheromone gland extracts in relatively abundant amounts: monounsaturated Z11–16:acid and Z9–14:acid, and a doubly unsaturated Z9,E12–14:acid. In addition to these acids, traces of -9,11-tetradecadienoic acid  and an unidentified doubly unsaturated C16 acid were detected, but no monounsaturated tetradecenoic or hexadecenoic acids with a double-bond at Δ12 or Δ14 locations were detected in the present study  identity of 90% to ∆11 desaturase of S. litura, of which GenBank accession number was AGH12217.1. SexiDes7 and SexiDes11 encoded proteins with aa identity of 90% and 98% to Acyl-CoA desaturase 4 from Helicoverpa armigera (GenBank accession number: AKU76403.1) and ∆9 desaturase from Spodoptera littoralis (GenBank accession number: AAQ74258.1), respectively. For the desaturase candidate gene from S. litura, SlitDes5 shared 96% aa identity to the Acyl-CoA ∆11 desaturase of S. littoralis of which GenBank accession number was AY362879.Reverse transcription-PCR was used to amplify desaturase candidate genes by their gene specific primers from the pheromone glands of The four full-length sequences of SexiDes5 (1017 bp), SexiDes7 (1116 bp), SexiDes11 (1062 bp) and SlitDes5 (1017 bp) were obtained from the pheromone gland cDNA library transcripts , as well as the Δ11-monounsaturated E11–14: Me, Z11–14:Me and Z11–18:Me, both with and without above substrates added  was subcloned into the CuS. exigua -10,12-tetradecadienoic acid , and non-conjugated Z9,E12–14:acid. It is interesting that SexiDes5 and SlitDes5 make a “cis” double bond in saturated 16C and 18C substrates but both “cis” and “trans” double bonds in 14C substrates. The difference in chain length and structural difference between saturated and monounsaturated substrates might influence the acyl chain entry into the ¨kink¨ in the substrate-binding tunnel. These differences between substrates and the geometry of the tunnel might influence the regioselectivity and stereospecificity of the desaturation reaction  there might be additional mechanisms to ensure the directed biosynthesis of the Z9,E12- and Z9,E11–14:acid precursors and corresponding pheromone components.In the heterologous expression system, SexiDes5 and its homolog SlitDes5 from Acheta domesticus and Tribolium castaneum, ∆12 desaturases with ancestral ∆9 desaturase origin have been found. These desaturases are, however, not involved in sex pheromone biosynthesis but in the production of linoleic acid  the mass spectrum of the DMDS adduct of the minor doubly unsaturated C16 acid in the pheromone gland of S. exigua exhibited m/z 84, 87, 189, 277 and 360, implying the positions of the two double bonds at ∆7 and ∆10, possibly through chain-shortening product of linoleic acid, 2) no monounsaturated hexadecenoic acids with a double-bond at C14 were found in the pheromone gland, 3) no deuterium label was incorporated from D3–16:acid into the above-mentioned hexadecadienoic acid, and 4) no Z11,E14–16 or Z9,E12–14 compound was observed after supplementing the SexiDes5-pYEX-CHT yeast with Z11–16:Me.In two other insect species, Spodoptera species, S. exigua and S. litura. They have a high ∆11 desaturase activity on palmitic acid, and they are also able to catalyze production of the doubly-unsaturated pheromone precursors Z9,E12–14:acid and Z9,E11–14:acid by interaction with Z9–14:acid in the respective species.In conclusion, we report that the multi-functional desaturase homologs SexiDes5 and SlitDes5 are active in sex pheromone biosynthesis in two ESM 1(DOCX 144 kb)


Nature Communications; 10.1038/s41467-019-08747-4; published online 15 February 2019Correction to: Mtb-SecBTA sequence was displayed incorrectly. In the inset panel within Fig. 4c, the y-axis of the graph incorrectly read (Q.Rg)2 × I(Q)//(0), and should have read (Q.Rg)2 × I(Q)/I(0). These errors have been corrected in both the PDF and HTML versions of the Article.The original version of this Article contained errors in Figures 1 and 4. In Fig. 1b, the


X) non-bonded contacts in crystals of halogenated dicyanobenzenes. Each Br atom accepts one C≡N⋯Br non-bonded contact, and each N atom is bisected by two, forming a nearly planar sheet structure.The title compound is the first bromo analog in a study of cyano–halo  non-bonded contacts in crystals of halogenated dicyanobenzenes. The complete molecule is generated by a crystallographic center of symmetry. In the extended structure, each Br atom accepts one C≡N⋯Br interaction, and each N atom is bisected by two. This contact network forms a nearly planar sheet structure propagating in the , C X  non-bonded contacts in substituted benzonitriles. The question is whether these contacts will form for a given nitrile, and whether they are isolated or extended to create ribbon or sheet structures in their crystals. The prevailing trend is that C≡N⋯F contacts do not form  halogenated terephthalodinitriles have followed this trend. The crystal of 2,3,5,6-tetrafluoroterephthalodinitrile (F4TN) does not contain any C≡N⋯F contacts, with molecules adopting a sawtooth formation . In co-crystals of Cl4TN with anthracene  , adapted from the work of Schäfer et al.  was added and then the mixture was stirred for 18 h. Water (50 mL) and an Na2CO3 solution  were added, and then the mixture was stirred for 24 h. A precipitate was collected by suction filtration, and then washed with water, giving a white powder . A trace of ammonium chloride could not be removed, based on the MS results. M.p. 627 K  δ 8.085 , 7.936 , 7.889 ; 13C NMR  δ 166.8 (2C), 143.4 (2C), 122.2 (4C); IR  3292, 3158, 2966, 2907, 2853, 1679, 1427, 1315, 1287, 1252, 1114, 1089, 866; MS  [M+35Cl]− calculated for C8H479Br281Br2N2O2 514.6660, found 514.6672. al. 2017: Tetrab2,3,5,6-Tetrabromoterephthalodinitrile (Br4TN), adapted from the work of Schäfer et al.  δ 129.6 , 123.5 , 116.0 ; IR  2236, 1364, 1330, 1293, 1229, 1156, 1121, 732; MS  [M]+ calculated for C879Br281Br2N2 443.6749, found 443.6764. al. 2017  in bis(2-methoxyethyl) ether (10 mL) at 425 K was cooled by 30 K h−1 until a precipitate began to form. The temperature was held for 1 h, and then cooled by 10 K h−1 to ambient temperature. After 24 h, colorless, highly twinned, prismatic crystals were collected by decantation and then washed with methanol. A monocrystalline tip similar to the one indicated in Fig. 5Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989019005486/hb7811sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989019005486/hb7811Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989019005486/hb7811Isup3.cmlSupporting information file. DOI: 1911575CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


O-β-d-glucopyranosyl-l-ascorbic acid (AA-2βG), a unique AA (or vitamin C) derivative identified in Lycium barbarum, exhibited enhanced free radical scavenging activity compared with AA and its synthetic derivative AA-2αG. AA-2βG protected hydrogen peroxide-induced cell death in murine macrophage RAW264.7 cells. Treatment with AA-2βG eliminated oxidative stress and the ratio of cellular glutathione to glutathione disulfide more effectively than AA and AA-2αG. AA-2βG also significantly reduced the fluorescent intensity of DCFH-DA triggered by chemotherapeutic agent camptotehcin-11 but not fluorouracil. AA, AA-2αG, and AA-2βG significantly decreased Keap-1expression, and increased the expression levels of nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1. All compounds triggered the nuclear translocation of Nrf2, while the ability of AA-2βG to enhance the Nrf2-DNA binding affinity was approximately two fold as those of AA and AA-2αG. Sodium ascorbate cotransporters (SVCT) inhibitors, sulfinpyrazone, phloretin, and 3-O-methyglucose, potently abrogated the free radical scavenging activities of AA, AA-2αG, and AA-2βG. The cellular uptake efficacy of AA-2αG and AA-2βG was less than 10% of AA, while the inhibition of SVCT with sulfinpyrazone considerably diminished the uptake efficacy of these compounds. AA-2αG and AA-2βG are more stable in the Fenton reagents than AA. In summary, AA-2βG from L. barbarum with excellent free radical scavenging activity is a promising natural AA derivative for further pharmacological evaluation.Reducing agents are crucial for the management of maladaptive inflammation-induced macrophage death and hematopoietic toxicity of chemotherapy. 2- The accumulation of free radicals is associated with maladaptive inflammation-induced macrophage death that leads to the subsequent necrotic death of plaque cells . Chronicl cancer ,6. Therel-Ascorbic acid . Fluorescent probe 2′,7′-dichlorofluorescin diacetate (DCFH-DA), dihydroethidium (DHE), 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), AA, AA-2αG, camptothecin-11 (CPT-11), 5-fluorouracil (5-FU), and 3-O-methylglucose were obtained from Sigma-Aldrich . AA-2βG was separated and purified as previously reported [O-methy glucose (3-OMG) were purchased from Cayman Chemical . Phosphate-buffered saline (PBS), Dulbecco's modified Eagle medium (DMEM), fetal bovine serum (FBS), and 100 × penicillin and streptomycin were purchased from Gibco-Invitrogen . AA-2αG and AA-2βG were dissolved in PBS prior to use. Superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and glutathione disulfide (GSSG) detection kits were purchased from Beyotime Biotechnology .Hreported . Sodium-2.22 at 37 °C.Murine macrophage RAW 264.7 cell line was acquired from the American Type Culture Collection , and cultured in DMEM medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin and streptomycin with humidified atmosphere of 5% COαG, or AA-2βG for 4 h, and then stimulated with 400 μM H2O2 for 6 h.For detection of SOD, CAT, GSH, GSSG, DHL, TNF-α, and western blot analysis, RAW264.7 cells were pre-incubated with AA, AA-2αG, or AA-2βG for 4 h. ROS was then stimulated with 400 μM H2O2 for 30 min.For co-treatment with SVCT inhibitors, RAW264.7 cells were pre-incubated with 5 μM SU, 10 μM PH, or 10 μM 3-OMG for 20 min and pre-treated with AA, AA-22.3αG, AA-2βG, or PBS as vehicle for 4 h. MTT was then added at 5 mg/mL for an addition 4 h at 37 °C. At the end of the incubation, the formazan crystals were dissolved with DMSO. The absorbance was determined at 570 nm using MultiLabel Counter Victor . Cell viability was calculated by the ratio of absorbance between treated and vehicle groups.RAW 264.7 cells were seeded in 96 well plates for 18 h and then treated with AA, AA-22.4αG, or AA-2βG for 1 h, incubated with 10 μM DCFH-DA or DHE for 30 min in serum free DMEM, and then stimulated with H2O2 for 30 min. For detection of ΔΨm, RAW264.7 cells were pre-treated with AA, AA-2αG, or AA-2βG for 1 h, incubated with 10 μM JC-1 at 37 °C for 30 min, and then stimulated with H2O2 for 30 min. Changes in the fluorescent intensity of DCFH-DA, DHE, and JC-1 was determined using BD Accuri™ C6 flow cytometer . At least 10,000 cells were recorded for each sample. The results were analyzed by using FlowJo software .For ROS detection, RAW 264.7 cells were pre-treated with AA, AA-22.5LDH released into the culture medium was determined using LDH Diagnostic kit . The absorbance was measured at 490 nm following the manufacture's recommendation .2.6The level of secreted TNF-α in culture supernatant was measured with Multi-Analyte Flow Assay Kit  using BD Accuri™ C6 flow cytometer according to the manufacturer's instructions.2.7SOD, CAT, GSH, and GSSG activities in RAW264.7 cells were measured using commercially available assay kits from Beyotime according to the manufacturer's instructions.2.8® BCA Protein Assay Kit (Thermo Fisher Scientific). The samples were denatured by boiling in SDS loading buffer. Twenty μg of total protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by electro blotting onto a polyvinylidene difluoride (PVDF) membrane . After blocking in 5% nonfat milk dissolved in Tris-buffered saline-Tween 20 (TBS-T) buffer  for 1 h at room temperature, the membrane was probed with primary antibodies against Kelch-like ECH-associated protein-1 (Keap-1), nuclear factor E2-related factor 2 (Nrf2), heme oxygenase (HO)-1, and β-actin at 1:1,000 dilution in TBS-T overnight at 4 °C and specific secondary antibodies for 2 h at room temperature. Specific protein bands were visualized using ECL advanced Western blot detection kit .At the end of the treatment, cells were lysed with ice-cold RIPA lysis buffer (Beyotime Biotechnology) supplemented with 1% PMSF and 1% protease inhibitor cocktail  on ice for 20 min. Cell lysate were cleared by centrifugation at 12,000 rpm for 15 min at 4 °C. Protein concentration was determined using Pierce2.9Immunofluorescent analysis was performed as previously reported . In brie2.10αG, or AA-2βG for 4 h, and then treated with 400 μM H2O2 for 6 h. Nuclear extract of RAW264.7 cells were prepared following the method reported previously [AM Nrf2 DNA-binding ELISA assay from Active Motif  following the manufacture's recommendation.RAW264.7 cells were pre-treated with AA, AA-2eviously . Nrf2 pr2.11αG, or AA-2βG were quantitatively analyzed using ESI-Q-TOF-MS/MS on a Bruker Impact HD Q-TOF MS system operating in ESI positive ion mode. The mass detection range was set as m/z 100–1500 in the full scan mode. Samples were injected into the system and quantify with external standard dissolved in methanol-water .Cells were washed with PBS and harvested with methanol-water . Equal number of cells were lysed by sonication and cleared by centrifugation at 15,000 rpm for 20 min at 4 °C. The intracellular content of AA, AA-22.12In vitro redox reaction was conducted with Fenton reagents [αG, or AA-2βG in PBS. The change of absorbance was measured at 300 nm using FlexStation 5 multi-mode microplate reader  every 3 min for a total of 30 min.reagents . In brie2.13p values less than 0.05 was considered statistically significant.Data were presented as mean ± SD from at least three independent experiments. Statistical significance between different experimental groups was determined by one-way analysis of variance (ANOVA) followed by Dunnett's post hoc test, and 33.12O2-induced oxidative stress in murine M1/M2 macrophage RAW264.7 cells to determine the antioxidant activities of AA, AA-2αG, and AA-2βG. None of these compounds alone affect cell viability as detected using MTT assay  or 12% (12.5 μM vs 100 μM) required for AA  is required to achieve the antioxidant capacity in overcoming its fast oxidation rate in the clinic . High AApathways . AA-2αG,roblasts ,36. Thesd for AA , Fig. 3CTT assay B but abr release F–G. MTT  with AA F–G.αG activates the SIRT1 signaling pathway to delay the pathogenesis of dermal fibroblasts from H2O2-induced oxidative damage and cellular senescence [αG maintains the integrity of gastric epithelial cells from Helicobacter pylori-initiated mitochondrial death signaling [αG, and AA-2βG treatment  AA-2αG and AA-2βG were not metabolized into AA in RAW264.7 cells, thereby suggesting that their antioxidant activity may not require AA as intermediate; (3) AA-2αG and AA-2βG shared the same cellular uptake transporters as AA; and (4) the glucosyl moiety and its configuration on AA-2βG increased stability and enhanced the Nrf2-DNA-binding affinity. Here, AA-2βG can be used as a highly reactive antioxidant for the prevention of chemotherapeutic agents-induced damage and other free radical-triggered diseases.In summary, the study showed the following results: (1) the reactivity against free radical followed the order of AA-2The authors declare no conflict of interests.


Two asymmetrically substituted cobaltocenium carboxylic acid compounds were synthesized and their crystal structures determined. Both crystallize as hydrates and exhibit an extended hydrogen-bonding network. 5H6N)(C6H5O2)]Cl·H2O, (3), and its azo derivative 1′-[2-diazen-1-yl]cobaltocenium-1-carboxylic acid hexafluoridophosphate, [Co(C13H14N3)(C6H5O2)]PF6·H2O (5) were obtained from cobaltocenium-1,1′-dicarboxylic acid hexafluoridophosphate by converting one carboxyl group to its chlorocarboxyl derivative followed by chloride/azide exchange, Curtius rearrangement, diazotiation and azo coupling with 2,6-dimethylaniline. Both title compounds crystallize as their monohydrates. In the crystal structure of 3, both functional groups lie in the same direction, with the Cp rings being nearly eclipsed, and participate in an extended hydrogen-bonded supramolecular network including the counter-ion and the water molecule of crystallization. Although the functional groups in 5 are somewhat further apart, bearing a greater torsion angle with the Cp rings now staggered, a similar supramolecular network is observed with not only the carboxylic acid and azo groups, but also with the more remote amino group participating in a hydrogen-bonded network, again including the counter-ion and the water molecule. The hexafluoridophosphate ion shows positional disorder. Compound 3 was refined as an inversion twin. In 5, each of the six F atoms is disordered over two sets of sites in a 1:1 ratio.1′-Aminocobaltocenium-1-carboxylic acid chloride, [Co(C In comparison to its known iron analogue, 1′-aminoferrocene-1-carboxylic acid -2-]-cobaltocenium-1-carboxylic acid hexafluoridophosphate, 5.One of the title compounds, 1′-aminocobaltocenium-1-carboxylic acid chloride, 3 and 5 are reported in this communication.The molecular and crystal structures of compounds 3 and 5 both crystallize as their monohydrates. Compound 3 forms crystals with one formula unit per asymmetric unit  Å] to Co1 than the other four carbon atoms in this ring [Co—Caveraged = 2.031]. In addition, the formal C—N single bond [C1—N1 = 1.343 (4) Å] of the amino substituent is considerably shortened, as has also been observed in aminocobaltocenium tetraphenylborate . This bond is slightly elongated but not as much as the corresponding bond to the amino group in the structure of 3. The azo group features a trans-configuration with distances typical for asymmetric azo compounds.In the cobaltocenium cation of )° Fig. 3. The Co—3 are part of an extended hydrogen-bonding network in the crystal , ppm: δ = 5.16 , 5.48 , 5.51 , 5.97 . ESI-MS showed a signal at 248.0139 m/z in accordance to the molecular cation.Compound 5: 1′-Aminocobaltocenium-1-carboxylic acid chloride hydrate (3)  was dissolved in 5 ml of concentrated HCl and the mixture was cooled to 273 K. Then NaNO2  was added and the yellow solution was stirred for 15 min. After addition of 2,6-dimethylaniline , the solution immediately turned red and was stirred for a further 30 min. When neutralized with saturated Na2CO3 solution, the reaction mixture again changed color to a darker red. The mixture was concentrated on a rotary evaporator and the salts were precipitated with ethanol. The solution was filtered, evaporated to dryness, the residue taken up in acetonitrile and after filtering and evaporating to dryness the product was dissolved in small amounts of water, and a few drops of aqueous HPF6 (60%) were added. The solution was extracted three times with dichloromethane, the combined dark-violet-colored organic phases were evaporated to dryness and the product (5) was dried in vacuo. Yield: 92.1 mg (52.2%) as a dark orange–red powder. Slow concentration of a solution in ethanol yielded single crystals suitable for X-ray analysis. 1H NMR (CD3OD), ppm: δ = 2.3 , 5.80 , 5.89 , 6.15 , 6.29 , 7.52 . ESI-MS showed a signal at 380.0836 m/z in accordance with the molecular cation.Compound Uiso(H) = 1.2Ueq(C) or 1.5Ueq(Cmethyl). For the refinement of 3, H atoms bound to N1, O2 and O3 were found in difference-Fourier maps and were treated with restraints on bond lengths (d = 0.89 Å for N and d = 0.83 Å for O) and refined with isotropic displacement parameters. The crystal studied was refined as an inversion twin. For 5, H atoms bound to N3 and O2 were treated in the same way as for 3 while the H atoms of the water molecule  were refined with Uiso(H) = 1.2Ueq(O3). The hexafluoridophosphate ion shows positional disorder. Each of the six F atoms was refined with two sets of sites in a 1:1 ratio.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989019000562/wm5478sup1.cifCrystal structure: contains datablock(s) global, 3, 5. DOI: 10.1107/S2056989019000562/wm54783sup4.hklStructure factors: contains datablock(s) 3. DOI: 10.1107/S2056989019000562/wm54785sup5.hklStructure factors: contains datablock(s) 5. DOI: 1884166, 1884167CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The structures of two sterically hindered silicon compounds feature intermolecular C—H⋯π interactions in the solid state. The silapropan-2-ol compound also features an intermolecular O—H⋯π interaction. 33H30Si (I), was prepared via the reaction of two equivalents of diphenylmethyllithium (benzhydryllithium) and dichloromethylphenylsilane. This bisbenzhydryl-substituted silicon compound was then reacted with trifluoromethanesulfonic acid, followed by hydrolysis with water to give the silanol 2-methyl-1,1,3,3-tetraphenyl-2-silapropan-2-ol, C27H26OSi (II). Key geometric features for I are the Si—C bond lengths that range from 1.867 (2) to 1.914 (2) Å and a τ4 descriptor for fourfold coordination around the Si atom of 0.97 (indicating a nearly perfect tetrahedron). Key geometric features for compound II include Si—C bond lengths that range from 1.835 (4) to 1.905 (3) Å, a Si—O bond length of 1.665 (3) Å, and a τ4 descriptor for fourfold coordination around the Si atom of 0.96. In compound II, there is an intramolecular C—H⋯O hydrogen bond present. In the crystal of I, molecules are linked by two pairs of C—H⋯π interactions, forming dimers that are linked into ribbons propagating along the b-axis direction. In the crystal of II, molecules are linked by C—H⋯π and O—H⋯π interactions that result in the formation of ribbons that run along the a-axis direction.The sterically hindered silicon compound 2-methyl-1,1,2,3,3-pentaphenyl-2-silapropane, C The addet al., 2010et al. (2006i-Pr3Si)3SiOH exists as a monomer while (t-BuMe2Si)3SiOH is a hydrogen-bonded dimer. Our observation that compound II is monomeric indicates that the silicon atom is very hindered by the presence of the two benzhydryl groups.The steric bulk of the benzhydryl group has been used to advantage in several silyl reagents. It has been reported that the benzhydryldimethylsilylgroup is readily synthesized and undergoes facile oxidation with hydrogen peroxide to form alcohols , with the aromatic rings gauche. For the benzhydryl group containing C3, the hydrogen atom H3 is gauche to the methyl group (C1) with a C1—Si1—C3—H3 torsion angle of 69°, with the aromatic ring (C22–C27) occupying the anti position.The molecular structure of compound II is shown in Fig. 34 descriptor for fourfold coordination around Si1 is 0.96, again indicating an almost perfect tetrahedral geometry around this silicon atom. The orientation of the C2 benzhydryl group is such that the hydrogen atom H2 is anti to the methyl group (C1) with a C1—Si1—C2—H2 torsion angle of −165°. For the benzhydryl group containing C3, the hydrogen atom H3 is again gauche to the methyl group (C1) with a C1—Si1—C3—H3 torsion angle of 55°, and the aromatic ring C22–C27 occupies the anti position. An intramolecular C—H⋯O hydrogen bond is present between H27 and O1 with an H⋯A distance of 2.55 Å bis(diphenylmethyl)silane (dimethyl)silyl]-5′-fluoro-2′-oxo-1′,2′-dihydrospiro-2-carboxylate -4-[dimethyl(diphenylmethyl)silyl]-5-{[dimethyl(phenyl)silyl]methyl}-3-methyl-tetrahydrofuran-2-one silyl]methane  coordinated to the central silicon atom.A search of the Cambridge Structural Database : Diphenylmethane  was added to an oven-dried, argon-flushed 100 ml Schlenk flask along with a magnetic stirbar. Anhydrous tetrahydrofuran (10 ml) was then added to the flask to dissolve the solid and the solution was cooled to 273 K. After the solution had cooled for 10 min, n-butyllithium  was added and the solution was stirred for 1 h. The reaction mixture was then cooled further to 195 K and dichloromethylphenylsilane was added . After warming to room temperature and stirring for 12 h, the solution was poured into hexanes (20 ml) and the organic layer was washed with water (20 ml), dilute hydrochloric acid , water (10 ml) and finally brine (10 ml). The hexanes solution was dried over sodium sulfate, filtered and concentrated in vacuo. The product was purified by dissolving it in 20 ml hexane, cooling to 195 K and isolating the white crystals by filtration. The crystals were then washed with pentane and dried in vacuo . Colorless block-like crystals suitable for analysis by X-ray diffraction were grown by recrystallization of compound I (0.1 g) from hexanes (2 ml) with heating (0.08 g isolated yield). FT–IR : 3057, 3019, 2869, 1597, 1493, 696; 1H NMR  δ 0.39 , 3.87 , 6.8–7.4 ; 13C NMR  δ −4.77, 42.70, 125.28, 125.67, 127.37, 128.14, 128.49, 129.21, 129.46, 129.81, 134.62, 135.99, 142.02, 142.14; 29Si NMR  δ −3.12.Synthesis of 2-methyl-1,1,3,3-tetraphenyl-2-silapropan-2-ol (II): Bis(diphenylmethyl)methylphenylsilane  was added to an oven-dried, argon-flushed 50 ml Schlenk flask along with a stirbar. Anhydrous toluene (5 ml) was added to dissolve the solid and the solution was cooled to 273 K. Trifluoromethanesulfonic acid was weighed in a vial  and then added to the Schlenk flask using a Pasteur pipette, at which point the solution went from colorless to a bright yellow. The solution was stirred for 2 h at room temperature after which time the solution went from cloudy to clear. At this point a mixture of water  and triethylamine  in ether (2 ml) was prepared and added to the rapidly stirring solution of the triflate in toluene, which caused the yellow solution to immediately turn colorless. After stirring for 1 h, the mixture was poured into hexanes (20 ml) and the organic layer was washed with water (20 ml), dilute hydrochloric acid , water (10 ml) and finally brine (10 ml). The hexanes solution was dried over sodium sulfate, filtered and the solvent was removed in vacuo. The crude product was then dissolved in 5 ml hexane and cooled to 195 K. The white crystals were isolated by vacuum filtration, washed with pentane and dried in vacuo . Colourless block-like crystals suitable for analysis by X-ray diffraction were grown by recrystallization of compound II (0.4 g) from hexanes (5 ml) with heating  375.8–376.2 K). FT–IR : 3591, 3057, 3024, 1597, 1491, 696; 1H NMR  δ 0.15 , 3.49 , 7.22 . 13C NMR  δ −2.34, 44.44, 125.64, 125.68, 128.62, 129.06, 129.28, 141.20, 141.43. 29Si NMR  δ 7.98.Uiso(H) = 1.5Ueq(C-methyl) and 1.2Ueq(C) for other H atoms in compound I, and C—H = 0.93–0.98 Å with 1.2Ueq(C) in compound II. The hydrogen atom bonded to O1 (H1) in compound II was located in an electron-density difference map and freely refined.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989019011265/su5509sup1.cifCrystal structure: contains datablock(s) Global, II, I. DOI: 10.1107/S2056989019011265/su5509Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989019011265/su5509IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989019011265/su5509Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989019011265/su5509IIsup5.cmlSupporting information file. DOI: 1946744, 1946743CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: