Interleukin-1β (IL-1β), a master regulator of neuroinflammation produced by activated inflammatory cells of the myeloid lineage, in particular microglia, plays a key role in the pathogenesis of acute and chronic diseases of the peripheral nervous system and CNS. IL-1β release is promoted by ATP acting at the purinergic P2XPurified (> 99%) microglia cultured from neonatal rat cortex and cerebellum were first primed with the putative TLR4/TLR2 agonist SAA (recombinant human Apo-SAA) or the established TLR4 agonist lipopolysaccharide (LPS) followed by addition of ATP. Expression of genes for the NLRP3 inflammasome, IL-1β, tumor necrosis factor-α (TNF-α), and SAA1 was measured by quantitative real-time polymerase chain reaction (q-PCR). Intracellular and extracellular amounts of IL-1β were determined by ELISA.7R antagonist A740003 blocked ATP-dependent release of IL-1β. Microglia prepared from rat cerebellum displayed similar behaviors. As with LPS, Apo-SAA upregulated SAA1 and TLR2 mRNA, and downregulated that of TLR4. LPS was less efficacious than Apo-SAA, perhaps reflecting an action of the latter at TLR4 and TLR2. The TLR4 antagonist CLI-095 fully blocked the action of LPS, but only partially that of Apo-SAA. Although the TLR2 antagonist CU-CPT22 was inactive against Apo-SAA, it also failed to block the TLR2 agonist Pam3CSK4.Apo-SAA stimulated, in a time-dependent manner, the expression of NLRP3, IL-1β, and TNF-α in cortical microglia, and produced a concentration-dependent increase in the intracellular content of IL-1β in these cells. A 2-h ‘priming’ of the microglia with Apo-SAA followed by addition of ATP for 1 h, resulting in a robust release of IL-1β into the culture medium, with a concomitant reduction in its intracellular content. The selective P2X7R-triggered IL-1β maturation and export is thus likely to represent an important contributor to this cytokine pool. Given that SAA is detected in Alzheimer disease and multiple sclerosis brain, together with IL-1β-immunopositive microglia, these findings propose a link between P2X7R, SAA, and IL-1β in CNS pathophysiology.Microglia are central to the inflammatory process and a major source of IL-1β when activated. P2XThe online version of this article (10.1186/s12974-018-1205-6) contains supplementary material, which is available to authorized users. Inflammatory conditions are marked by the production of mediators such as cytokines, chemokines, reactive oxygen species, and acute phase proteins that are key elements of the accompanying physiological and metabolic changes. C-reactive protein, complement proteins, and serum amyloid A protein (SAA) are some of the principal acute phase proteins, mainly generated in the liver and released into the systemic circulation in response to inflammation , 2. SAA Saa1.Central nervous system (CNS) disorders are characterized by central activation of innate immunity and activation of a potent peripheral acute phase response that influences central inflammation and contributes to poor outcome . Syrian 7 (P2X7R), an ATP-gated ion channel that chiefly acts through the recruitment of the NLRP3 inflammasome-caspase-1 complex [7R-triggered IL-1β maturation and export may thus represent a major contributor to this cytokine pool [Interleukin-1β (IL-1β), a master regulator of neuroinflammation  mainly p complex , 19. Thi complex –22. P2X7ine pool , 23.7R-dependent manner. This is especially important, given the growing body of data indicating that genetic or pharmacological manipulation of P2X7Rs alters responsiveness in animal models of CNS neurological disorders [7Rs regulate the pathophysiology of psychiatric disorders, including mood disorders [7R, TLR4, and TLR2.SAA appears to be an endogenous ligand for both TLR4 –27 and Tisorders . Recent isorders . The preE. coli 0111:B4 strain; only activates TLR4), Pam3CSK4, and ethyl-(6R)-6-(N-(2-chloro-4-fluorophenyl)sulfamoyl)cyclohex-1-ene-1-carboxylate (CLI-095 or TAK 242) were from InvivoGen ; A740003 from Tocris Bioscience, Pittsburgh, PA, USA); poly-L-lysine hydrobromide , papain, DNase I (bovine pancreas), trypsin inhibitor, L-leucyl-L-leucine methyl ester (L-LME), protease inhibitor cocktail, Pefabloc® SC (100 mM), CU-CPT22, and all other biochemicals were purchased from Sigma-Aldrich  unless noted otherwise; recombinant human Apo-SAA  from Peprotech ; QIAzol from Qiagen  were obtained from Sarstedt .Tissue culture media, antibiotics, fetal calf serum (FCS), and NP40 cell lysis buffer (10×) were purchased from Life Technologies ; lipopolysaccaride (LPS) (Ultra-Pure LPS-EB from 2 poly-L-lysine-coated tissue culture flasks (1.5 brains per flask) and grown in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) with 2 mM glutamine, 50 units/ml penicillin/50 μg/ml streptomycin, 50 μg/ml gentamycin, and 10% FCS (‘growth medium’). Culture medium was changed after 24 h. The cultures reached confluence by 7 days at which time microglia were recovered by shaking the flasks on an orbital shaker at 200 rpm for 1 h (37 °C). The remaining cell monolayers were highly enriched in astrocytes  [2, 95% air) to allow adhesion of microglia. The adherent microglial cells  [Microglia were isolated from mixed glial cell cultures as previously described . All expibodies) . The culibodies)  were detibodies) , as desc for 1 h  °C. The ibodies) . Astrocy260/280 ratio > 1.8). Reverse transcription was performed with Superscript III reverse transcriptase (Invitrogen). The q-PCR reaction was performed as described previously [Total RNA was extracted from cells by QIAzol, according to the manufacturer’s instructions. RNA integrity and quantity were determined by RNA 6000 Nano assay in an Agilent BioAnalyser  in growth medium and allowed to adhere overnight. These plating densities do not affect glial cell vitality/function , 40. Celt test, or by one-way analysis of variance followed by Dunnett’s or Bonferroni’s post hoc tests for comparisons involving more than two data groups. Significance was taken at p < 0.05.Data are given as mean ± sem, unless stated otherwise. Statistical analyses to determine group differences were performed either by two-sample equal variance Student’s Nlrp3 expression that peaked at 3 h  of contaminating microglia . A numbe7R in the Apo-SAA/ATP-mediated IL-1β release, microglia were pretreated 30 min with the selective P2X7R antagonist A740003 [7R-dependent cleavage of IL-1β precursor to the releasable mature form of the cytokine.To interrogate a role for P2X A740003 , then pr7R-dependent manner [Purified microglia cultured from rat cerebellum also respond to ATP-dependent IL-1β release upon priming with TLR2, TLR3, and TLR4 isoform agonists, in a P2Xt manner .To determine if this is also the case for priming with Apo-SAA, cerebellar microglia were incubated for 2 h with 0.5 μg/ml recombinant human Apo-SAA followed by addition of ATP to 5 mM or culture medium only. As Fig. Tlr2 and Tlr4 expression in cortical microglia. Apo-SAA treatment of microglia time-dependently increased Tlr2, with a peak at 3 h and a decline over 24 h  was significantly reduced at 3, 6, and 24 h  binding to TLR1/2 and repress downstream signaling from IL-1β and TNF-α [Several studies suggest that SAA is capable of activating TLR4 –27 and Tnd TNF-α .3CSK4 (0.3 μg/ml) produced significant increases in the intracellular content of IL-1β after both 3 and 24 h  that of Apo-SAA at both time points. However, the stimulatory effect of Pam3CSK4 was unaffected by the TLR4 antagonist at 3 h, and modestly so at 24 h. CU-CPT22 failed to alter the stimulatory effect of Apo-SAA or LPS; surprisingly, this TLR1/2 antagonist also was ineffective when tested on cells challenged with the TLR1/2 agonist Pam3CSK4—in contrast to earlier findings [3CSK4, 322 ± 9; LPS + CLI-095, 0; Apo-SAA + CLI-095, 224 ± 3; Pam3CSK4 + CLI-095, 242 ± 12; LPS + CU-CPT22, 399 ± 23; Apo-SAA + CU-CPT22, 822 ± 76; 420 ± 3; CLI-095 fully eliminated the effect of LPS and partially, but significantly, that of Apo-SAA (p < 0.001).Subsequent addition of LPS (0.1 μg/ml), Apo-SAA (0.5 μg/ml), or the TLR2 agonist Pamfindings . Similar7R-dependent manner. The rise in extracellular release of IL-1β in the presence of ATP was accompanied by a fall in the intracellular content, consistent with NLRP3/caspase 1-complex 1 activation and cleavage of the pro-form of IL-1β to the mature, active secreted species [Growing evidence indicates that CNS disorders are characterized by central activation of innate immunity, as well as activation of a potent peripheral acute phase response that influences central inflammation and leads to poor disease outcome . The acu species –22. The  species  that con species –17. It mp = 0.030 vs. control) in neuromyelitis optica patients [A comparison of the SAA concentrations used in the present investigation with levels of SAA previously detected in human cerebrospinal fluid and plasma suggests the potential for physiological relevance to the in vivo setting. In one report, SAA levels in cerebrospinal fluid of AD subjects were found to be much higher than in normal controls , and genpatients . These v7Rs alters their responsiveness in animal models of CNS neurological disorders [7R has been suggested to also regulate the pathophysiology of psychiatric disorders, including mood disorders [7R-triggered IL-1β maturation and export is thus likely to represent an important contributor to this cytokine pool [7R-positive microglia surrounded amyloid plaques in a mouse transgenic AD model [7R, Apo-SAA, and IL-1β in AD pathophysiology. In addition, the ability of Apo-SAA to regulate its own gene expression suggests the potential for autocrine/paracrine effects of SAA. Since microglia in the AD brain adopt distinct functional and molecular phenotypes, it is conceivable that the response of “AD microglia” to SAA would differ from that of wild-type microglia.An expanding body of data demonstrates that pharmacological or genetic manipulation of P2Xisorders , 68. Theisorders . P2X7R-tine pool , 23. SAAine pool . P2X7R-pAD model , and micAD model . Collect3CSK4 leads to the upregulation of Tlr2 and downregulation of Tlr4 in cortical microglia [Tlr2 expression in cortical microglia, with a concomitant reduction in the relative level of Tlr4. Conceivably, this action of SAA could result in a ‘feed-forward’ mechanism, whereby SAA increases expression of its receptor and amplification of a priming response. Attempts at using pharmacological tools to dissect participation of TLR4 and TLR2 in the actions of Apo-SAA were equivocal. The selective TLR4 antagonist CLI-095 completed blocked the ability of LPS to synthesize/release IL-1β and partially, but significantly, that of Apo-SAA. While the TLR2 antagonist CU-CPT22 failed to alter the stimulatory effect of Apo-SAA or LPS, it also proved ineffective on microglia treated with the TLR1/2 agonist Pam3CSK4. Our inability to confirm the earlier report for CU-CPT22 action against Pam3CSK4 [3CSK4 binding to TLR1/2, thus disrupting formation of the TLR1/TLR2 heterodimer [−/− animals could provide a tool to address this question.A number of reports indicate the capability of SAA to act as an agonist for both TLR4 –27 and Ticroglia . ConsistPam3CSK4  could bePam3CSK4 ), even tPam3CSK4 . Anothererodimer . AlthougSaa1 expression in cultured OPCs [7R in the development of experimental autoimmune encephalomyelitis [A failure of remyelination is responsible, in large part, for the long-term neurologic consequences of MS. An intriguing study by Sloane et al.  describered OPCs . Our finmyelitis  and micrmyelitis , propose7R-dependent manner. Apo-SAA upregulated expression of its own gene and that of TLR2, suggesting a potential ‘feed-forward’ mechanism, whereby SAA increases expression of its receptor and amplification of a priming response. The effects of Apo-SAA on gene expression and cytokine production were of a far greater magnitude than those observed with the classical TLR4 agonist lipopolysaccharide, highlighting the pro-inflammatory potency of this acute phase protein. Given the evidence for P2X7Rs involvement in CNS neurological disorders and expression of SAA in AD and MS brain, the findings presented here propose a link between P2X7R, SAA, and IL-1β in CNS pathophysiology.CNS disorders are characterized by central activation of innate immunity and activation of a potent peripheral acute phase response that influences central inflammation and contributes to poor outcome. Our data show that in microglia, the acute phase protein Apo-SAA upregulates NLRP3 inflammasome and IL-1β mRNA expression and intracellular production of IL-1β and stimulates release of IL-1β in the presence of ATP in a P2XAdditional file 1:Figure S1. Treatment of rat cortical microglia with Apo-SAA or LPS upregulates, in a time-dependent manner mRNA for TNF-α. Cultures were treated the day after plating with 0.5 μg/ml recombinant human Apo-SAA or 0.1 µg/ml LPS and processed 1, 3, 6, and 24 h later for q-PCR, as detailed in the “n = 3. Control values (which were at the limit of detection) were omitted for clarity. Apo-SAA (■); LPS (▓). For LPS, *p < 0.05 vs 1 and 24 h; for Apo-SAA: °°°p < 0.001 vs 1, 6, and 24 h. (TIF 468 kb)Additional file 2:Figure S2. Treatment of rat cortical microglia with Apo-SAA or LPS upregulates, in a time-dependent manner, intracellular content of IL-1β. Cultures were treated the day after plating with 0.5 μg/ml recombinant human Apo-SAA or 0.1 µg/ml LPS and processed 1, 3, 6, and 24 h later for measurement of intracellular IL-1β, as detailed in the “n = 3. The quantity of intracellular IL-1β in unstimulated cells was below the detection limit of the ELISA assay kit. For LPS, **p < 0.01 vs 3 and 6 h; for Apo-SAA, °°°p < 0.001 vs 1 h, ***p < 0.001 vs 3 and 24 h. (TIF 485 kb)Additional file 3:Figure S3. Treatment of rat cortical microglia with LPS, in a time-dependent manner, upregulates mRNA for TLR2 and downregulates that for TLR4. Cultures were treated the day after plating with 0.1 μg/ml LPS and processed 1, 3, 6, and 24 h later for q-PCR, as detailed in the “n = 3. Control  cultures. Data are expressed as mean ± sem, n = 3 (n = 9 for 3 h). (a) **p < 0.01 vs CTRL (3 h) and ***p < 0.001 vs CTRL (6 h). (b) *p < 0.05 vs CTRL (3 h), ***p < 0.001 vs CTRL for that time point. Note the difference in expression levels between TLR2 and TLR4 mRNA. (TIF 957 kb)


In both compounds, there is an intramolecular hydrogen bond forming an S(6) ring motif; an O—H⋯O hydrogen bond in (I), but a charge-assisted N+—H⋯O− hydrogen bond in (II).The title Schiff base compounds, (I) and (II), were synthesized  20H16ClNO2 (I) and C24H22N2O2 (II), were synthesized via the condensation reaction of 2-amino-4-chlorophenol for (I), and 2-ethan-1-amine for (II), with 4-benzyloxy-2-hydroxybenzaldehyde. In both compounds, the configuration about the C=N imine bond is E. Neither molecule is planar. In (I), the central benzene ring makes dihedral angles of 49.91 (12) and 53.52 (11)° with the outer phenyl and chlorophenyl rings, respectively. In (II), the central benzene ring makes dihedral angles of 89.59 (9) and 72.27 (7)°, respectively, with the outer phenyl ring and the mean plane of the indole ring system (r.m.s. deviation = 0.011 Å). In both compounds there is an intramolecular hydrogen bond forming an S(6) ring motif; an O—H⋯O hydrogen bond in (I), but a charge-assisted N+—H⋯O− hydrogen bond in (II). In the crystal of (I), molecules are linked by C—H⋯π interactions, forming slabs parallel to plane (001). In the crystal of (II), molecules are linked by pairs of N—H⋯O hydrogen bonds, forming inversion dimers. The dimers are linked by C—H⋯O hydrogen bonds, C—H⋯π interactions and a weak N—H⋯π interaction, forming columns propagating along the a-axis direction. The antioxidant capacity of the synthesized compounds was determined by cupric reducing antioxidant capacity (CUPRAC) for compound (I) and by 2,2-picrylhydrazyl hydrate (DPPH) for compound (II).The title Schiff base compounds, C RR′C=NR′′ exhibit a wide structural diversity and have found a wide range of applications 1H NMR spectra revealed the presence of an imino group (N=CH) in the range δ = 8.5–8.6 p.p.m. Cupric reducing antioxidant capacity (CUPRAC) of (I)in vitro 2,2-diphenyl-1-picrylhydrazil hydrate (DPPH) radical scavenging.Schiff bases of the general type +—H⋯O− in (II)S(6) ring motifs as shown in Figs. 1Section 6, Refinement) and the C1—O13 (C—O−) bond length is 1.281 (2) Å, compared to the C9—O1 (C—OH) bond length of 1.343 (3) Å in (I)E in both compounds and the C=N bond lengths are 1.286 (3) Å for C7=N1 in (I)The molecular structures of compounds (I)a-axis direction. The different hydrogen bonds and X—H⋯π  interactions are illustrated in Fig. 4In the crystal structures of both compounds C—H⋯π interactions predominate; see Table 1et al., 2010et al., 2016Z)-3-benzyloxy-6-[(2-hydroxyphenylamino)methylene]cyclohexa-2,4-dienone -5-benzyloxy-2-[(4-nitrophenyl)carbonoimidoyl]phenol The structures of Schiff bases derived from hydroxyaryl aldehydes have recently been the subject of a general survey, in which a number of structural errors, often involving misplaced H atoms, were pointed out (Blagus II–Nc) (CUPRAC) method Nc2–Cu+2 complex. Indeed, in the presence of an antioxidant agent, the copper–neocuproene complex is reduced and this reaction is quantified spectrophotometrically at a wavelength of 450 nm.The antioxidant activity profile of the synthesized compound (I)0.50 > 100) for a 4 mg dosage (see Table 350 > 100) for a 1 mg dosage, by comparison with buthylated toluene (BHT) IC50 = 22.32 ±1.19, used as a positive control (see Table 3The current results indicate that Schiff base compound (I)Note: Compound (I)Compound (I)in vacuo. The residue was recrystallized from methanol, yielding green block-like crystals of (I)3). In the 1H NMR spectrum, the azomethine proton appears in the 8.5–8.6 p.p.m. range, while the imine bond is characterized in the 13C MNR spectrum with the imine C signal in the 158–162 p.p.m. range. 1H NMR: δ 6.5–7.6 , 13.8–14.0 . 13C NMR: 70.22, 127.6, 128.8, 129.5 133.8, 136.2, 147.1.2-Amino-4-chlorophenol (1 equiv.) and 4-benzyloxy-2-hydroxybenzaldehyde (1 equiv.) in ethanol (15 ml) were refluxed for 1 h. On completion of the reaction (monitored by thin layer chromatography), the solvent was evaporated Compound (II)H-indol-3-yl)ethan-1-amine (1 equiv.) and 4-benzyloxy-2-hydroxybenzaldehyde (1 equiv.) in methanol (15 ml) were refluxed for 1 h. On completion of the reaction (monitored by thin layer chromatography), the solvent was evaporated in vacuo and the residue recrystallized from methanol, yielding orange block-like crystals of (II)1H NMR spectrum, the azomethine proton appears in the 8.5–8.6 p.p.m. range, while the imine bond is characterized in the 13C NMR spectrum with the imine C signal in the 163.3–168.4 p.p.m. range. 1H NMR: δ 6.5–7.7 , 13.8–14.0 . 13C NMR: 56.9, 128.2, 128.7, 132.9, 136.4, 163.3.2-= 1.5Ueq(O). In compound (II)Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989018003687/su5426sup1.cifCrystal structure: contains datablock(s) global, I, II. DOI: Click here for additional data file.10.1107/S2056989018003687/su5426Isup2.cmlSupporting information file. DOI: 1827172, 1827171CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Gigantol is a bibenzyl compound derived from several medicinal orchids. This biologically active compound has been shown to have promising therapeutic potential against cancer cells, but its mechanism of action remains unclear.The inhibitory effect of gigantol on Wnt/β-catenin signaling was evaluated with the SuperTOPFlash reporter system. The levels of phosphorylated low-density lipoprotein receptor related protein 6 (LRP6), total LRP6 and cytosolic β-catenin were determined by Western blot analysis. The expression of Wnt target genes was analyzed using real-time PCR. Cell viability was measured with a MTT assay. The effect of gigantol on cell migration was examined using scratch wound-healing and transwell migration assays.Gigantol decreased the level of phosphorylated LRP6 and cytosolic β-catenin in HEK293 cells. In breast cancer MDA-MB-231 and MDA-MB-468 cells, treatment with gigantol reduced the level of phosphorylated LRP6, total LRP6 and cytosolic β-catenin in a dose-dependent manner, resulting in a decrease in the expression of Wnt target genes Axin2 and Survivin. We further demonstrated that gigantol suppressed the viability and migratory capacity of breast cancer cells.Gigantol is a novel inhibitor of the Wnt/β-catenin pathway. It inhibits Wnt/β-catenin signaling through downregulation of phosphorylated LRP6 and cytosolic β-catenin in breast cancer cells. The Wnt/β-catenin signaling pathway plays a crucial role in embryonic development and tumorigenesis , 2. In tGigantol is a bibenzyl-type phenolic compound isolated from several medicinal orchids. This biologically active compound has been shown to have significant antioxidative –13, antiv12 and β-gal have been described previously [Gigantol Fig.  was purceviously .Fig. 1Gi2 at 37 °C. Human breast cancer MDA-MB-231 and MDA-MB-468 cells were cultured in Leibovitz’s L-15 medium (Gibico) supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C without CO2. Non-tumorigenic human mammary epithelial MCF-10A cells were maintained in DMEM/F12 medium containing 5% horse serum, 10 mg/mL insulin, 20 ng/mL epidermal growth factor (EGF), 100 mg/mL choleratoxin, 0.5 mg/mL hydrocortisone, 100 U/mL penicillin, and 100 μg/mL streptomycin and incubated in 5% CO2 at 37 °C.The human embryonic kidney HEK293T cells, mouse fibroblast L-cells, and L-cells stably expressing Wnt3A (L-Wnt3A) were grown in Dulbecco’s modified Eagle’s media (DMEM) containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin in 5% COHEK293T cells were grown in 24-well plates for 16–20 h and then transfected with 250 ng of reporter plasmid, 50 ng of pCMXβgal plasmid, and 50–200 ng of the indicated expression plasmids using TransIn EL Transfection Reagent (Transgene) according to the manufacturer’s instructions. At 24 h after transfection, cells were incubated with either DMSO or various indicated concentrations of gigantol. The control conditioned medium and Wnt3A conditioned medium (Wnt3A-CM) were collected as previously described [Cells were first seeded onto a 6-well plate. After overnight incubation, cells were treated with various concentrations of gigantol for 24 h. Cells were then lysed with 0.015% digitonin in PBS supplemented with 2.5 mM sodium pyrophosphate, 1 mM β-glycerol phosphate, 1 mM sodium orthovanadate, 2 μg/mL leupeptin, and 1 mM PMSF. After being centrifuged at 3000 rpm for 3 min, the supernatant was collected as the cytosolic extracts.Cell lysates were prepared with buffer containing 20 mM Tris·HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerol phosphate, 1 mM sodium orthovanadate, 2 μg/mL leupeptin, and 1 mM PMSF. Equal amounts of protein from each sample were separated by SDS-PAGE followed by immunobloting with the following antibodies: anti-LRP6 , anti-phosphorylated LRP6 (Ser1490) , anti-β-catenin (Santa Cruz Biotechnology), and anti-GAPDH (Transgene).Total RNA was extracted using RNAiso Plus (TaKaRa) and subsequently reverse-transcribed into cDNA using the Primescript RT Reagent Kit (TaKaRa) according to the manufacturer’s instructions. Quantitative PCR analysis was performed with FastStart Universal SYBR Green Master (Roche). The primers were as follows: Axin2: sense, 5’-TACACTCCTTATT-GGGCGATCA-3′; antisense, 5’-TTGGCTACTCGTAAAGTTTTGGT-3′; Survivin: sense, 5′- AGGACCACCGCATCTCTACAT-3′; antisense, 5’-AAGTCTGG-CTCGTTCTCAGTG-3′; GAPDH: sense, 5’-CCAGAACATCATCCCTGCCTCTACT-3′; antisense, 5′-GGTTT-TTCTAGACGGCAGGTCAGGT-3′.4 cells per well) were seeded onto 96-well plates and incubated overnight. Cells were then treated with DMSO or various concentrations of gigantol for 48 h before being cultured with fresh medium containing MTT (5 mg/mL) for another 4 h. The formazan crystals were dissolved in DMSO and the absorbance of the formazan solution measured at 570 nm using a microplate reader.Cells  were trypsinised and resuspended in serum-free medium and then seeded onto 24-well transwell chambers with 8-μm pore membrane in 100 μL serum-free medium contained DMSO or various concentrations of gigantol. The lower chamber contained medium supplemented with 20% FBS. After incubation for 6 h, the unmigrated cells on the upper side of membrane were removed with a cotton swab and the migrated cells stained with crystal violet and photomicrographed.As previously described , cells (P < 0.05 were considered statistically significant.Statistical analyses were performed using two-way ANOVA GraphPad Prism 5.0. Results are presented as mean ± SD. Differences at We explored the effects of some natural products on Wnt/β-catenin signaling using the SuperTOPFlash cell reporter system. Gigantol exhibited an inhibitory effect on Wnt reporter gene expression. To confirm its inhibitory effect on Wnt/β-catenin signaling, the SuperTopFlash reporter was transfected into HEK293 cells along with Wnt1, LRP6, Wnt1/LRP6, or β-catenin expression plasmids. Gigantol treatment inhibited Wnt signaling activated by Wnt1 Fig. , LRP6 F and Wnt1v12 were used to activate the NFAT and AP1 signaling pathways in HEK293 cells, respectively. As shown in Fig. To test the effect of gigantol on other signaling pathways, transfection assays were also performed using NFAT-Luc and AP1-Luc reporters. The expression plasmids encoding NFATc and RasTo explore the molecular mechanism underlying Wnt signaling inhibition by gigantol, we examined the effect of gigantol on LRP6 phosphorylation. As expected, Wnt1 expression or specifically Wnt3A-CM induced endogenous LRP6 phosphorylation at serine 1490 and increased cytosolic β-catenin in HEK293 cells Fig. . TreatmeThe MDA-MB-231 and MDA-MB-468 cell lines were used to assess the effect of gigantol on Wnt/β-catenin signaling in breast cancer cells. In both cell lines, gigantol markedly decreased the levels of phosphorylated LRP6 and total LRP6 dose-dependently Fig. . FurtherTo further confirm the inhibitory effects of gigantol on Wnt/β-catenin signaling in breast cancer cells, we investigated the effect of gigantol on the expression of Axin2 and Survivin, both of which are well-known target genes in the Wnt/β-catenin pathway. The results of real-time PCR showed that mRNA expression of Axin2 and Survivin was significantly decreased after gigantol treatment in breast cancer MDA-MB-231 and MDA-MB-468 cells Fig. .Fig. 5Gi50 values of 115.2 ± 6.7 μM in MDA-MB-231 cells and 103.6 ± 10.9 μM in MDA-MB-468 cells (Fig. 50 value for the non-tumorigenic human mammary epithelial MCF10A cells was 192.1 ± 17.0 μM, indicating that gigantol has selective cytotoxicity in cancer cells.To assess the potential cytotoxic effects of gigantol on breast cancer cells, MDA-MB-231 and MDA-MB-468 cells were treated with varying concentrations of gigantol ranging from 1.25 to 500 μM for 48 h. Gigantol reduced the viability of breast cancer cells, with IClls Fig. . In contWe further tested the effect of gigantol on cell migration with wound-healing and transwell assays. As shown in Fig. Gigantol has been shown to have promising therapeutic potential in cancer cells –22, but LRP6 is an essential coreceptor in the Wnt/β-catenin signaling pathway and thus a potential therapeutic target in breast cancer treatments. Several studies have shown that LRP6 is up-regulated in human breast cancer cells . LRP6 si50 values of 247.55 ± 4.94 μM [A recent study showed that gigantol inhibited proliferation and induced apoptosis of liver cancer HepG2 cells . In non- 4.94 μM . Giganto 4.94 μM . Unahabh 4.94 μM , 36. Int 4.94 μM . Conside


Uncaria rhynchophylla is commonly recognized as a traditional treatment for dizziness, cerebrovascular diseases, and nervous disorders in China. Previously, the neuro-protective activities of the alkaloids from U. rhynchophylla were intensively reported. In current work, three new indole alkaloids (1–3), identified as geissoschizic acid (1), geissoschizic acid N4-oxide (2), and 3β-sitsirikine N4-oxide (3), as well as 26 known analogues were isolated from U. rhynchophylla. However, in the neural stem cells (NSCs) proliferation assay for all isolated compounds, geissoschizic acid (1), geissoschizic acid N4-oxide (2), isocorynoxeine (6), isorhynchophylline (7), (4S)-akuammigine N-oxide (8), and (4S)-rhynchophylline N-oxide (10) showed unexpected inhibitory activities at 10 μM. Unlike previous neuro-protective reports, as a warning or caution, our finding showcased a clue for possible NSCs toxicity and the neural lesions risk of U. rhynchophylla, while the structure–activity relationships of the isolated compounds were discussed also.The online version of this article (doi:10.1007/s13659-017-0141-y) contains supplementary material, which is available to authorized users. Uncaria rhynchophylla, named as Gou-teng or Cat’s claw, was used for treatment of dizziness, cerebrovascular diseases, and nervous disorders + . The UV spectrum showed absorption maxima characteristic of an indolenine chromophore  + , 16 mass units more than 1. Comparison the 1H and 13C NMR spectroscopic data of 1 with those of 2 , C-5 , and C-21  in 2, which suggested 2 was an N4-oxide derivative of 1 [2 shared the same configurations with 1 from their same ECD spectral curves  was established a molecular formula C21H26N2O4 on the basis of HRESIMS ion at m/z 371.1960 [M+H]+  and the comprehensive analysis of 13C NMR data (Table 1H NMR and 13CNMR spectroscopic data (Table 5)  and H-17b [δH 3.52 ] were similar to those of sitsirikine (5).3Table 5) . The viskine (5) , 26. Con1 and 2 showed cytotoxicity in NSCs proliferation assay, while none of the other isolated tetracyclic yohimbine-type alkaloids   did not work. Interestingly, may due to the configuration change of C-7, compound 14 was not active, while its N4-oxide derivative (10) inhibited NSCs proliferation at tested concentration.By structural analysis, we found only compounds U. rhynchophylla were purchased from the Luo-Si-Wan Chinese herbal medicine market, Kunming, Yunnan province, China, in April 2016, and identified by Dr. Zhang Jun, Kunming Plant Classification Biotechnology Co., Ltd. A voucher specimen (No. WEI_20160418) was deposited in the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences.The hook-bearing branches of 3/MeOH  as developing solvents and spots were visualized by Dragendorff’s reagent. High performance liquid chromatography (HPLC) was performed using waters 600 equipment with semi-preparative and preparative C18 columns .Optical rotations were performed on a JASCO P-1020 polarimeter. IR spectra were measured on a Bruker FT-IR Tensor 27 spectrometer with KBr pellets. UV spectra were obtained on Shimadzu UV-2401A spectrometer. 1D-NMR and 2D-NMR spectra were recorded on an AV-600 MHz or a Bruker DRX-400 MHz spectrometer. Coupling constants were expressed in Hz and chemical shifts were given on a ppm scale with tetramethylsilane as internal standard. HRESIMS were recorded on an API QSTAR Pulsar 1 spectrometer. CD spectra were obtained on a JASCO 810 spectrometer. Column chromatography (CC) was performed on silica gel , Sephadex LH-20 , and MCI-gel CHP 20P . Thin-layer chromatography (TLC) was carried out on silica gel H-precoated plates  with CHClU. rhynchophylla (10 kg) were extracted with MeOH (50 L × 3) under reflux conditions at 70 °C, 3 h for each time. After removal of the organic solvent under reduced pressure, the residue was dissolved in 0.3% aqueous hydrochloric acid (v/v); the solution was subsequently basified to pH 9–10 using ammonia and then extracted with EtOAc (3 L × 4) to give an alkaloidal extract. The extract (52 g) was subjected to a silica gel column  to afford fractions (A-F). Fr. A (4.8 g) was subjected to silica gel column chromatography (CC) using a petroleum ether/acetone gradient (10:1–1:9) to afford sub-fractions (Fr. 1–4). Fr. 3 (1.2 g) was further purified on Sephadex LH-20 using MeOH under isocratic conditions to afford isocorynoxeine (6) (12 mg), augustine (29) (12 mg), and corynoxeine (13) (8 mg). Fr. 4 (1.5 g) purified on Sephadex LH-20 column by isocratic elution with MeOH to get a mixture (400 mg), which was then further separated on a semi-preparative C18 HPLC column with a gradient of MeOH/H2O (1:1–1:0) to obtain isorhynchophylline (7) (5 mg) and rhynchophylline (14) (6 mg). Fr. B (9.8 g) was chromatographed on silica gel column  to yield seven fractions (Fr. 5–11). Fr. 6 (0.8 g) was purified on Sephadex LH-20 column with MeOH under isocratic elution and further purified on a semi-preparative C18 HPLC column  to afford corynantheine (20) (12 mg), dihydrocorynantheine (21) (6 mg), sitsirikine (5) (11 mg), and geissoschizine methyl ether (4) (5 mg). Fr. C (10 g) was fractionated on MCI-gel CHP 20P column by eluting with a gradient of MeOH (30–100%) in H2O to yield four fractions (Fr. 12–15). Fr. 14 (200 mg) was subjected to Sephadex LH-20 CC using MeOH under isocratic elution and was further purified on a semi-preparative C18 HPLC column  to yield akuammigine (18) (3 mg), 3-epi-geissoschizine methyl ether (17) (1.2 mg), hirsuteine (22) (7 mg), and nitrocadambine B (28) (9 mg). Fr. D (12 g) was subjected to silica gel CC using a CHCl3/MeOH gradient (9:1–0:1) to give six fractions (Fr. 16–21). Fr. 20 (800 mg) was subjected to MCI-gel CHP 20P column using MeOH/H2O gradient (3:7–1:0) and further separated on Sephadex LH-20 column using MeOH with isocratic elution to yield (4S)-akuammigine N-oxide (8) (2 mg) and hirsuteine N-oxide (26) (3 mg). Fr. 21 (2.5 g) was subjected to Sephadex LH-20 column using MeOH with isocratic elutionand was further purified on a semi-preparative C18 HPLC column  to obtain 3α-dihydrocadambine (27) (8 mg), (4S)-rhynchophylline N-oxide (10) (7 mg), and (4S)-corynoxeine N-oxide (15) (6 mg). Fr. E (7 g) was chromatographed over silica gel column using a CHCl3/MeOH gradient (4:1–0:1) to obtain seven fractions (Fr. 22–28). Fr. 24 (200 mg) was further subjected to Sephadex LH-20 column using MeOH to yield geissoschizine methyl ether N4-oxide (16) (10 mg) and hirsutine N-oxide (23) (9 mg). Fr. 25 (400 mg) was further purified on MCI-gel CHP 20P column with MeOH/H2O gradient (1:4–1:0) to afford (4R)-akuammigine N-oxide (19) (17 mg) and hirsutine (24) (13 mg). Fr. 26 (400 mg) was subjected to MCI-gel CHP 20P column using MeOH/H2O gradient (1:4–1:0) and was further purified on a semi-preparative C18 HPLC column  to yield dihydrocorynantheine N-oxide (25) (18 mg) and 3β-sitsirikine N4-oxide (3) (7 mg). Fr. 27 (300 mg) was repeatedly chromatographed over Sephadex LH-20 column with MeOH to afford isorhynchophylline N4-oxide (11) (5 mg), (4S)-isocorynoxeine N-oxide (12) (4 mg), and cadambine (9) (10 mg). Fr. 28 (300 mg) was subjected to MCI-gel CHP 20P column using MeOH/H2O gradient (1:4–1:0) and was further purified on a semi-preparative C18 HPLC column  to yield geissoschizic acid (1) (20 mg) and geissoschizic acid N4-oxide (2) (2 mg).The air-dried and powdered hook-bearing branches of Geissoschizic acid(1): colorless amorphous solid; [α]D26 +31.9 ; UV (MeOH) λmax (log ε): 269 (4.05), 223 (4.74), 207 (4.60) nm; ECD  λ (Δε): 218 (−7.19), 234 (+3.71), 267 (+2.48); IR (KBr) νmax 3421, 2933, 1644, 1382, 1238, and 1132 cm−1; HRESIMS m/z 353.1852 [M + H]+ ; 1H and 13C NMR data, see Table Geissoschizic acid N4-oxide(2): colorless amorphous solid; [α]D21 +101.8 ; UV (MeOH) λmax (log ε): 269 (3.42), 223 (4.11), 208 (4.00) nm; ECD  λ (Δε): 219 (−8.02), 231 (+4.21), 266 (+2.03); IR (KBr) νmax 3393, 2943, 1636, 1448, 1246, and 1113 cm−1; HRESIMS m/z 369.1806 [M+H]+ ; 1H and 13C NMR data, see Table 4-oxide: colorle3β-sitsirikine N4-oxide(3): colorless amorphous solid; [α]D24 +104.3 ; UV (MeOH) λmax (log ε): 273 (4.04), 221 (4.72) nm; ECD  λ (Δε): 213 (+7.41), 234 (+1.88), 262 (−2.07); IR (KBr) νmax 3414, 2930, 1727, 1633, 1454, 1319, and 1063 cm−1; HRESIMS m/z 371.1960 [M+H]+ ; 1H and 13C NMR data, see Table 2 supplement (Gibco), 1 × B27 (Gibco) and 10 μg/mL heparin as previously described [Neural stem cells (NSCs) were grown in serum-free growth medium  containing 20 ng/mL human epidermal growth factor , 20 ng/mL human fibroblast growth factor , 1% penicillin/streptavidin, 1% Nescribed –39. The 2 for 48 h, without removing the cell culture supernatant, cells were fixed with 16% TCA and incubated at 4 °C for 1 h. Plates were washed 5 times with water and air dried. A 100 μL of sulforhodamine B solution 0.4% (w/v) in 1% acetic acid was added into each well. After 10 min at room temperature, the plates were rinsed five times with 1% acetic acid quickly to remove unbound dye and then air dried. Then 50 μL 10 mM un-buffered Tris-base was added to solubilize the protein-bound dye. The absorbance was read on automated plate reader  at 515 nm. Each compound was treated in 3 independent wells per experiment, and the assay was repeated at least 3 times. The value of DMSO was set to 1, and the other values were normalized to DMSO.Cell clusters generated by adult neural stem cells (NSCs) proliferation were trypsinized into single cell and evenly plated into 96 well plate overnight. NSCs proliferation rate was measured by SRB assay according to the standard protocol . Briefly1–3 are available as Supplementary Information. Supplementary material 1 (PDF 929 kb)1D and 2D NMR spectra, ECD, HRESIMS, and UV spectra of compounds Below is the link to the electronic supplementary material.


O-β-d-apiofuranosylapterin (1), 4′-O-isobutyroylpeguangxienin (2), 6-(3-methyl-2-oxobutyroyl)-7-methoxycoumarin (3), and 6-hydroxycoumurrayin (4), were isolated from the ethanol extract of Heracleum stenopterum, Peucedanum praeruptorum, Clausena lansium, and Murraya paniculata, respectively. Their chemical structures were established on the basis of extensive spectroscopic analysis. Compound 2 exhibited in vitro cytotoxic activity against five human cancer cell lines  with IC50 values ranging from 15.9 to 23.2 μM.Four hitherto unknown prenylated coumarins, namely 6″-The online version of this article (doi:10.1007/s13659-016-0107-5) contains supplementary material, which is available to authorized users. Heracleum and Peucedanum from Apiaceae can be characterized by the dominance of furanocoumarins + , requiring 10 degrees of unsaturation. The characteristic UV absorptions at 221 (sh), 248, 258, 325 nm indicated the presence of a 7-oxygenated coumarin chromophore , and the pentose as a terminal apiofuranosyl moiety . In the HMBC spectrum  of glucose, which unambiguously established an api(1 → 6)glc sugar chain at C-4′. The same linkage pattern also occurred in the structure of heraclenol 3′-O-β-d-apiofuranosyl-(1 → 6)-β-d-glucopyranoside, isolated from this plant in our current research + . The 1H NMR spectrum , 7.59 , 7.34 , and 6.80 ], two vicinal oxymethines bearing ester function at δH 5.36  and 6.56 , a gem-dimethyl group at 1.44  and 1.40 , a 3-methyl-2-butenoyl unit , and an isobutyroyl group . The 13C NMR spectrum , 68.7 , 60.8 , 22.9  and 24.5 ], as well as two ester carbonyl carbons [δC 165.2  and 175.7 ]. Comparison of its NMR data with those of hyuganin D + . The NMR data , 7.69 , 7.97 , and 6.83 ], a methoxy group , and a set of signals originated from a prenyl unit , which were further established as a 3-methyl-2-oxobutyroyl unit by the HMBC correlations of H-4′ and H-5′ with C-2′ as well as comparison with literature data + . The NMR spectra  and 7.92 ; δC 161.2 , 114.5 , 138.3 , 140.9 , 138.4 , 149.4 , 118.9 , 110.0 , and 146.0 ], two methoxys , a prenyl , and a phenolic hydroxy group [δH 5.56 ]. All of the above spectroscopic data were generally consistent with those of 6-methoxycoumurrayin +; HRESIMS (pos.): m/z 579.1681 [M + Na]+ .White amorphous powder; UV (MeOH) λmax: 218, 256 (sh), 296 (sh), 323 nm; c 0.20, MeOH); 1H NMR and 13C NMR data: see Table m/z 437 [M + Na]+; HRESIMS (pos.): m/z 437.1573 [M + Na]+ .White amorphous powder; UV (MeOH) λmax: 215 (sh), 227 (sh), 261, 308, 340 nm; 1H NMR and 13C NMR data: see Table m/z 297 [M + Na]+; HRESIMS (pos.): m/z 297.0737 [M + Na]+ .Yellow amorphous powder; UV (MeOH) λmax: 232 (sh), 307, 357 (sh) nm; 1H NMR and 13C NMR data: see Table m/z 313 [M + Na]+; HRESIMS (pos.): m/z 313.1042 [M + Na]+ .White amorphous powder; UV (MeOH) 2. Cell viability was assessed by conducting colorimetric measurements of the amount of insoluble formazan formed in living cells based on the reduction of MTS . Briefly, 100 μL of adherent cells were seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before drug addition, both with an initial density of 1 × 105 cells/mL in 100 μL medium. Each cell line was exposed to the test compound at various concentrations in triplicate for 48 h, with cisplatin and paclitaxel as positive controls. After the incubation, 20 μL MTS and 100 μL medium was added to each well after removal of 100 μL medium, and the incubation continued for 2–4 h at 37 °C. The optical density was measured at 492 nm using a Multiskan FC plate reader . The IC50 value of each compound was calculated according to the Reed and Muench method.Five human tumor cell lines  obtained from ATCC  were used in the cytotoxicity assay. All cells were cultured in RPMI-1640 or DMEM medium , supplemented with 10 % fetal bovine serum (Hyclone) at 37 °C in a humidified atmosphere containing 5 % COSupplementary material 1 (DOCX 541 kb)Below is the link to the electronic supplementary material.


Hibiscus sabdariffa  are described. Hibiscus acid dimethyl sulfoxide monosolvate forms a two-dimensional hydrogen-bonded motif, while hibiscus acid dimethyl ester (Z′ = 2) forms a one-dimensional hydrogen-bonded motif.The isolation and crystal structures of the title compounds from  Hibiscus sabdariffa plants, hibiscus acid as a dimethyl sulfoxide monosolvate , C6H6O7·C2H6OS, (I), and hibiscus acid dimethyl ester , C8H10O7, (II). Compound (I) forms a layered structure with alternating layers of lactone and solvent molecules, that include a two-dimensional hydrogen-bonding construct. Compound (II) has two crystallographically independent and conformationally similar molecules per asymmetric unit and forms a one-dimensional hydrogen-bonding construct. The known absolute configuration for both compounds has been confirmed.The biologically active title compounds have been isolated from  Hibiscus sabdariffa , have been documented to have significant potential in the traditional treatment of various diseases. H. sabdariffa Linn is a species of hibiscus from the Malvaceae family, commonly known as ‘Karkade’ or ‘red sorrel’. It is used in traditional medicine in the form of herbal teas or cold drinks for its hypotensive and diuretic effects and to lower body temperature and blood viscosity , and on their purification and characterization. The crystal structures of the acid, as the dimethyl sulfoxide monosolvate, (I)Lactone acid producing plants, including R (R = H or Me) groups lie in equatorial positions on their rings and the absolute configuration of both species is confirmed by the Flack parameter values ,C1(S), 0.00 (4)] and both arbitrarily named equivalent atoms in (II)R),C4(S) and C11(R),C12(S), 0.08 (17)]  solvate of hibiscus acid, (I)] Table 1. The abs al. 1969 for hibiA and B) (Z′ = 2), whose molecular geometries differ only by small deviations in torsion angles, for example, C3—C5—O5—C6 in A is 175.1 (4)°, whilst the equivalent angle in B (C11—C13—O12—C—14) is 180.0 (4)°. As with structure (I)A and B, respectively.The structure of (II)ROH and C=O functions (i.e. O4i and O6i) of one neighbour, whilst the other two donors, the second carboxylic acid (O5—H) and the hydroxy group (O4—H), form hydrogen bonds with atoms O8ii and O8 of DMSO solvent molecules, respectively es Fig. 3. The H ay Table 2. These ion Fig. 4.A) acts as a hydrogen-bond acceptor is Fig. 5.et al., 2016et al., 1972et al., 2007et al., 1997A search of the Cambridge Structural Database  and extracted in a Soxhlet apparatus using 2500 ml each of hexane, ethyl acetate and methanol. The methanol extract was dried and concentrated at 313 K by rotatory evaporation, yielding about 125 g (25%) of crude extract. The methanol extract (2 g) was dissolved in about 2 ml of methanol and subjected to gel filtration chromatography (GFC) using a glass column packed with a wet slurry of 30 g of Sephadex LH20 in methanol. Vials were collected (5 ml each) after elution with 100% methanol, which led to isolation of pure hibiscus acid (0.5%). Crystals of (I)1H NMR [OC(CD3)2]: 5.31 , 3.23 , 2.77 . HRMS: found 189.0000; calculated 189.0035.Dried 1H NMR [OC(CD3)2]: 5.35 , 3.23 , 2.77 , 3.87 , 3.76 . HRMS: found 218.000; calculated 218.035.Hibiscus acid dimethyl ester, (II)Uiso(H) = 1.5Ueq(C) for methyl C atoms and 1.2Ueq(C) for the other H atoms. The absolute configuraion was determined for the molecules in both acid (I)R),C1(S), Flack parameter 0.00 (4)] and both arbitrarily named equivalent atoms in (II)R),C4(S) (molecule A) and C11(R),C12(S) (molecule B), Flack parameter 0.08 (17)]  I, II, global. DOI: 10.1107/S2056989017011902/zs2386Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989017011902/zs2386Isup4.cmlSupporting information file. DOI: 10.1107/S2056989017011902/zs2386IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989017011902/zs2386IIsup5.cmlSupporting information file. DOI: 1569231, 1569230CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Polymers with moderate molecular weights and relatively broad dispersity (Ð) were obtained. Kinetic studies revealed that the polymerization followed first-order kinetics.The preparation, structural characterization, luminescence, and catalytic activity of three palladium(II) complexes bearing imino phenoxide ligands are reported. The X-ray studies revealed that the complexes are mononuclear with palladium centres coordinated in a square-planar coordination environment. Two of the complexes are emissive in solution at room temperature. The catalytic activities towards the ring-opening polymerization of The online version of this article (10.1007/s00706-017-2119-1) contains supplementary material, which is available to authorized users. Tetradentate salen-type ligands are omnipresent in coordination chemistry. They are easy to synthesize and they exhibit a high structural variability due to a large number of commercial available building blocks which can be used as starting materials. In many cases, they form highly stable metal complexes throughout the whole periodic table and a plethora of metal complexes have been reported in the past , 2. Rela8 configuration and hence prefers a square-planar coordination. It is well known that such complexes form polymeric columnar structures, often with infinite and close Pd–Pd bonds. Contrary to their platinum congeners + and [L2PdNa]+ are detected. The IR spectra of the free ligands show intense signals in the region from 1623 to 1632 cm−1 . In the complexes, the spectra are shifted towards lower frequencies (1607–1624 cm−1), which eventually revealed the coordination of imine nitrogen atoms to the metal . In the 1H NMR spectra, the formation of the metal complexes is shown by the disappearance of the phenol –OH signals of the ligands. In addition, the signals corresponding to the protons of the CH=N groups appear low field shifted (7.52–7.59 ppm) compared to those of the ligands (8.4–8.6 ppm) which indicates the coordination of nitrogen and oxygen atoms of the ligands to the metal.Complexes 1 crystallizes in the monoclinic space group P21/n, complex 2 in the triclinic space group P2 is isostructural to the homologues copper(II) complex . This might be due to steric reasons and packing effects. The palladium atoms in all complexes are not engaged in any further Pd–Pd interactions.Single crystals suitable for X-ray diffraction of all three complexes were grown by slow evaporation of the solvent from their solutions in dichloromethane over a period of 1 week. All complexes were obtained as red crystalline solids. The crystallographic data are summarized in Table mentclasspt{minimaractions , 47. Sim2 carries the moderately electron-withdrawing bromine substituent, whereas the ligand in the non-emissive complex 1 is substituted by a strongly electron-donating methoxy group. For 3, the ligand is substituted with a very bulky t-butyl group which leads not only to a strongly distorted coordination geometry as elucidated by the solid state structure, but also to a more rigid structure with respect to a distortion in the excited state. Minimizing the excited state structural distortion has been found to be effective in increasing the luminescence quantum yield. It should be noted that analogous Pt-complexes often feature intensive luminescence [The electronic spectra of all the complexes were recorded in solution at room temperature Fig. . The datnescence .Fig. 2UV1–3 were investigated as catalysts for ROP of rac-LA (Scheme 1H NMR spectroscopy. The polymerization was performed in a 200:1 ratio ([rac-LA]0/[Cat]0 = 200:1) catalysed by complexes 1–3. The plot of % conversion of rac-LA against time gives a sigmoid curve meaning that initially the conversion rate is high but decreases significantly at later stage  vs. time indicative for a first-order dependency on the monomer concentrations with the absence of any induction period  were extracted from the linear plot of ln([rac-LA]0/[rac-LA]t) vs. time and were found to be 3.47 × 10−2 min−1, 2.14 × 10−2 min−1, and 2.84 × 10−2 min−1 for 1–3, respectively  and relatively broad dispersities (Mw/Mn = 1.91–1.98). The large difference between the experimental and theoretic values and broad Ð compared to literature values +) 637.13, found 637.33.Yield: 0.07 g (79%); 1H NMR : δ = 1.17–1.20 2, 12H), 1.29–1.32 2, 12H), 3.52–3.66 2, 4H), 7.15–7.16 , 7.18-7.19 , 7.21 , 7.28–7.29 , 7.44 , 7.52–7.53  ppm; 13C{1H} NMR : δ = 23.48 (CH(CH3)2), 24.42 (CH(CH3)2), 28.81 (CH(CH3)2), 105.27 (Ar–C), 114.32 (Ar–C), 120.93 (Ar–C), 124.70 (Ar–C), 127.54 (Ar–C), 135.44 (Ar–C), 140.31 (Ar–C), 142.02 (Ar–C), 145.07 (Ar–C), 159.98 (Ar–O), 163.25 (CH=N) ppm; IR (ATR): −1; MS (ESI): m/z calculated for C38H41Br4N2O2Pd ([M + H]+) 983.780, found 983.497.Yield: 0.07 g (73%); 1H NMR : δ = 1.37 3, 18H), 2.18 , 3.84 , 4.98 , 6.79–6.80 , 6.86–6.89 , 7.06–7.07 , 7.48 , 7.53–7.56  ppm; 13C{1H} NMR : δ = 20.40 (CH3), 29.52 (C(CH3)3), 35.22 (C(CH3)3), 55.38 (Ar–O–CH3), 60.87 (Ar–CH2), 114.31 (Ar–C), 122.32 (Ar–C), 123.14 (Ar–C), 129.35 (Ar–C), 130.23 (Ar–C), 131.16 (Ar–C), 131.83 (Ar–C), 133.35 (Ar–C), 139.33 (Ar-C), 159.12 (Ar–O), 165.95 (CH=N) ppm; IR (ATR): −1; MS (ESI): m/z calculated for C40H49N2O4Pd ([M + H]+) 727.27, found 727.60.Yield: 0.06 g (75%); λ = 0.71073 Å). The structures were solved by direct methods  [F2 (SHELXL-97) [http://www.ccdc.cam.ac.uk. The crystal data are given in Table Suitable single crystals for X-ray diffraction were grown from concentrated dichloromethane solution of the respective compounds over a period of 7 days. Single crystal analysis were carried out on a Bruker SMART APEX and a Bruker Smart X2S diffractometer operating with Mo Kα radiation ( SIR-97) , 54 and ELXL-97) . The H aSupplementary material 1 (PDF 122 kb)Supplementary material 2 (PDF 150 kb)Supplementary material 3 (PDF 208 kb)Supplementary material 4 (CIF 14 kb)Supplementary material 5 (CIF 16 kb)Supplementary material 6 (CIF 48 kb)Supplementary material 7 (DOCX 1576 kb)Below is the link to the electronic supplementary material.


The predominant LTα1β2 binds to LTβ receptor (LTβR) primarily expressed in epithelial and stromal cells. Most studies on LTβR signaling have focused on the organization, development, and maintenance of lymphoid tissues. However, the roles of LTβR signaling in the nervous system, particularly in neurogenesis, remain unknown. Here, we investigated the role of LTβR-mediated NFκB signaling in regulating neural lineage differentiation.Lymphotoxin (LT) is a lymphokine mainly expressed in lymphocytes. LTα binds one or two membrane-associated LTβ to form LTαfirefly-luciferase reporter assay and p65/RelB/p52 nuclear translocation assay. LTβR mRNA expression was evaluated by quantitative RT-PCR and LTβR protein expression was determined by immunohistochemistry and Western blot analysis. Multilabeled immunocytochemistry or immunohistochemistry followed by fluorescent confocal microscopy and quantitative analysis of neural lineage differentiation were performed. Graphing and statistical analysis were performed with GraphPad Prism software.The C57BL/6J wild-type and GFAP-dnIκBα transgenic mice were used. Serum-free embryoid bodies were cultured from mouse embryonic stem cells and further induced into neural stem/progenitor cells (NSCs/NPCs). Primary neurospheres were cultured from embryonic and adult mouse brains followed by monolayer culture for amplification/passage. NFκB activation was determined by adenovirus-mediated NFκB-1β2 stimulation induced an activation of classical and non-classical NFκB signaling. The expression of LTβR-like immunoreactivity in GFAP+/Sox2+ NSCs was identified in well-established neurogenic zones of adult mouse brain. Quantitative RT-PCR and Western blot analysis validated the expression of LTβR in cultured NSCs/NPCs and brain neurogenic regions. LTβR expression was significantly increased during neural induction. LTα1β2 stimulation in cultured NSCs/NPCs promoted astroglial and oligodendrocytic lineage differentiation, but inhibited neuronal lineage differentiation. Astroglial NFκB inactivation in GFAP-dnIκBα transgenic mice rescued LTβR-mediated abnormal phenotypes of cultured NSCs/NPCs.In cultured NSCs/NPCs, LTαThis study provides the first evidence for the expression and function of LTβR signaling in NSCs/NPCs. Activation of LTβR signaling promotes glial lineage differentiation. Our results suggest that neurogenesis is regulated by the adaptive immunity and inflammatory responses.The online version of this article (10.1186/s12974-018-1074-z) contains supplementary material, which is available to authorized users. Neurogenesis involves the proliferation, migration, and lineage differentiation of neural stem/progenitor cells (NSCs/NPCs) during development and adult life. Defects or impairments in embryonic and adult neurogenesis have been implicated in a large array of neurodevelopmental disorders –6 and neThe nuclear factor κB (NFκB) is well known for its critical role in inflammation, immunity, cancer, and neural plasticity. In most cell types, NFκB maintains cellular growth (proliferation) and survival. However, little is known about the role of NFκB signaling in regulating neural differentiation of NSCs. Activation of NFκB by Toll-like receptor 2 (TLR2) induces neuronal differentiation whereas TLR4 activation inhibits neuronal differentiation of NSCs . In p50 Inflammatory mediators such as cytokines and chemokines have been shown to regulate neurogenesis –23. Tumo3 that binds to TNFR  was from EMD Chemicals and dissolved in DMSO. Antibodies against LTβR(M-110), Calretinin(N-18), Doublecortin , Myelin basic protein , Sox2(Y-17), p65(C-20), GAPDH and Lamin A/C(636) were obtained from Santa Cruz Biotechnology. RelB and p100/p52 were from Cell Signal Technology. Mouse anti-NeuN antibody was from Millipore. Chicken anti-glial fibrillary acidic protein (GFAP), anti-Neuron-specific class III β-tubulin (Tuj1), and anti-Nestin antibodies were from Aves. Rabbit anti-GFAP (Z0334) was from Dako. All other reagents were from Sigma.TNFα, LTαAll procedures for animal usage were complied with the guidelines of the National Institutes of Health and approved by the Institutional Animal Care and Use Committee (IACUC) at Temple University. The transgenic (TG) mice carrying the NFκB super-inhibitor dominant-negative IκBα (dnIκBα) transgene driven by the promoter of Glial fibrillary acidic protein (GFAP) were obtained from Dr. Bethea’s Lab at the University of Miami and maintained by breeding heterozygous GFAP-dnIκBα males with wild-type (WT) females . WT littn = 3) as described previously [5 cells/ml in DMEM/F12 NSC/NPC proliferation medium containing × 1 B27 Supplement, 20 ng/ml epidermal growth factor (EGF), 10 ng/ml basic fibroblast growth factor (bFGF), and 0.2% Heparin (StemCell Technologies). Medium was changed every other day, and primary neurospheres were formed within 3–7 days.Primary neurospheres were cultured from the subventricular zone (SVZ) of adult mice , 10% fetal bovine serum (FBS), × 1 Penicillin/Streptomycin/Glutamine (Invitrogen), and 0.1 mM 2-Mercaptoethanol. After initial culture on 60-mm dish for 2–4 h to allow the attachment of mouse embryonic fibroblast, the suspension media containing ESCs were transferred to tissue culture 6-well plate, and cultured at 37 °C, 5% CO2 with daily media change until ESC colonies were formed. Serum-free EB was generated following previously described protocol . Brieflyfirefly-luciferase reporter (Vector Biolabs) at a multiplicity of infection of 10 for 24 h. The cells in 6 wells per group were treated with or without indicated cytokines for 24 h, including TNFα (10 ng/ml), IL1β (10 ng/ml), LTα1β2 (100 ng/ml), BAFF (100 ng/ml), CD40L (100 ng/ml), and LIGHT (100 ng/ml). The cell lysate was prepared for measuring of firefly-luciferase activity using the ONE-Glo luciferase assay system (Promega). Luminescence was quantified in a 2104 EnVision® Multilabel Reader (PerkinElmer).NSCs/NPCs were cultured in a 96-well plate and infected with adenovirus carrying the NFκB-Cultured ESCs, EBs, NSCs/NPCs, or snap-frozen brain tissues were processed with an RNeasy Mini kit (Qiagen) according to the manufacturer’s instruction for total RNA extraction. RNase-Free DNase Set (Qiagen) was used to remove any potentially residual genomic DNA through on-column DNase digestion. High Capacity cDNA Reverse Transcription Kit  was used for cDNA synthesis from 2 μg of RNA for each sample using random hexanucleotide primer. Quantitative PCR (qPCR) was performed in a BioRad CFX qPCR instrument using an SYBR® Green PCR Master Mix Kit (BioRad). One pair of mouse LTβR primers targeted the range 786–923 nucleotide (5′-gcagctccaggtacctcctactcg-3′ on exon-6 and 5′-cctcatccaggcacaggccaggac-3′ on exon-7) of mouse LTβR (NM_020736.3). The primers for mouse housekeeping gene Ppia were forward 5′-gcccagtatgcttgggtatc-3′ and reverse 5′-tgctgactcccagaacaga-3′, or β-actin were forward 5′-aagagctatgagctgcctga-3′ and reverse 5′-tacggatgtcaacgtcacac-3′. Each sample was tested in triplicate. Cycle threshold (Ct) values were obtained graphically for LTβR and Ppa2 or β-actin. Their difference (ΔCt) was used to obtain ΔΔCt values by subtracting the ΔCt values of control samples from those of experimental samples. Relative fold change in gene expression was calculated as 2-ΔΔCt.g for 20 min at 4 °C, the supernatant was collected for protein concentration determination with a Pierce BCA Protein Assay Kit (cat# 23225). An equal amount of protein lysate (20 μg) was resolved by the SDS-polyacrylamide gel electrophoresis system and transferred to nitrocellulose membrane (BioRad). The SeeBlue prestained protein standards (Invitrogen) were used as a molecular weight reference. The Odyssey CLx Infrared Fluorescent Western Blot system  was used according to the manufacture’s instruction. Briefly, after blocking with Odyssey blocking buffer containing 0.1% (v/v) Tween 20, the membranes were incubated overnight at 4 °C with goat anti-LTβR polyclonal antibody  and mouse anti-GAPDH monoclonal antibody . After washing three times, the membrane was incubated with fluorescently conjugated secondary antibodies (IRDye 680LT-conjugated anti-mouse or IRDye 800CW-conjugated anti-rabbit) for 1 h at room temperature. The membranes were scanned and analyzed using the Odyssey Infrared Imaging System. Relative signal intensities were determined using the LI-COR imaging software, which is independent of the image intensity adjustment.Culture NSCs/NPCs or snap-frozen brain tissues were lysed at 4 °C for 30 min in Triton X-100-based lysis buffer (20 mM Tris-HCl (pH 7.4), 1% Triton X-100, 5 mM ethylenediaminetetraacetic acid, 5 mM dithiothreitol, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, × 1 proteinase inhibitor cocktail , 1 mM sodium orthovanadate and 30 mM NaF). After centrifugation at 20,000Cultured NSCs/NPCs upon differentiation in 16-well chambered slides were fixed for 20 min in 2% paraformaldehyde and 25% sucrose in PBS. After three rinses, the cells were treated with 0.5% Triton X-100 PBS for 20 min and blocked by 10% donkey serum or 2% bovine serum albumin/PBS for 2 h. Cells were incubated overnight at 4 °C with indicated primary antibodies. After washing three times, cells were incubated with corresponding secondary antibodies for 1 h and with DAPI for 5 min. After three rinses with PBS, the cells were mounted with a coverslip using antifading aqueous mounting media  and analyzed under fluorescence microscope and confocal imaging analysis.For multilabeled immunohistochemistry, mice were euthanized with an overdose of pentobarbital solution and transcardially perfused with 4% paraformaldehyde. The brains were dissected, postfixed overnight in the same fixative, and cryopreserved with buffered 30% sucrose. A series of coronal sections of brain at 40 μm thickness were cut on a freezing sliding microtome and collected in a serial manner and stored at − 20°C. Then, standard multiple-labeled immunofluorescent staining was performed. Briefly, sections were permeated for 30 min in PBS containing 0.5% Triton X-100 and blocked with 10% normal donkey serum for 30 min. The sections were incubated with indicated primary antibodies in PBS with 0.1% Triton X-100 overnight at 4 °C. After washing three times each for 10 min, the sections were incubated with corresponding Alexa Fluor® conjugated donkey secondary antibody  for 1 h at room temperature. DAPI was used for nuclear counterstaining. After three rinses with PBS, sections were mounted on a slide and coverslipped with antifading aqueous mounting media .For confocal imaging analysis, samples were viewed on a Leica SP8 confocal microscope system. Differential visualization of three or four fluorophores Alexa-488, Alexa-594, Alexa-680 and DAPI was obtained via specific filter combinations. Samples were scanned sequentially to avoid any potential for fluorophore bleed-through. The Z-stack scanning images (1024 × 1024 pixels) through 0.5 or 1.0 μm optical sections were obtained under identical exposure conditions and processed through the build-in processing tools. For quantification analysis, at least 8 images were collected for each condition and the Z-stack images for equal number of Z-stacks were maximized for further identification and manual counting of indicated positive cells. The percentage of indicated positive cells over total DAPI-positive nucleus was calculated.t test was performed between two groups of different treatments. The p value at < 0.05 and < 0.01 were used for statistical significance.All statistical analysis was performed using Prism GraphPad 6.0. Significance was determined between multiple genotypes using a one-way ANOVA with Tukey post-hoc analysis. An unpaired two-tailed Student’s 1β2 stimulates activation of NFκB-luciferase reporter in mouse embryonic/neonatal SVZ NSCs/NPCs and enteric neuronal cell line [1β2 (for both pathway) [1β2 v.s. TNFα in adult SVZ NSCs/NPCs  –66 inducpathway) , with loPCs Fig. . InteresPCs Fig. ; thus, bdow Fig. . Howeverdow Fig. , consistdow Fig. . HoweverPCs Fig. , d, whic1β2-induced activation of NFκB signaling in NSCs/NPCs indicates the presence of its receptor LTβR in these cells. To validate this, we first determined whether LTβR mRNA was expressed in cultured NSCs/NPCs from adult mouse SVZ. The melting peak curve of RT-qPCR reaction attested to the presence of LTβR mRNA expression in cultured NSCs/NPCs  using LTβR antisense probe generated by PCR with primer forward 5′-cctcctactcggataccatctg-3′ and reverse 5′-atcctagtgtctctgtctcggc-3′. LTβR mRNA was extensively expressed in the mouse brain  is expressed primarily in epithelial and stromal cells , 70. ThePCs Fig. , which wPCs Fig. . Our prePCs Fig. . The prePCs Fig. . To detePCs Fig. . When usPCs Fig. –d. LTβR PCs Fig. , d. To aain Fig. –d, predoain Fig. . Then, wain Fig. , f. To vain Fig.  and Sox2ain Fig. , c, but ain Fig.  and DCX-ain Fig. . These d1β2 at the initiation of neural differentiation increased the number at day 1 after treatment of those three lineage neural cells, including DCX-positive neuroblasts/neuronal cells, MBP-positive oligodendrocytes and GFAP-positive astrocytes . It significantly reduced the number of DCX-positive neuroblasts/neuronal cells  mouse line expressing a dominant-negative IκBα under the GFAP promoter . This moent Fig. , d. LT-sent Fig. . Howeverent Fig. . Interesent Fig. , b. ThesThe salient finding in this study is the first identification of LTβR signaling and function in NSCs/NPCs both in vitro and in vivo. Activation of LTβR signaling promotes astroglial and oligodendrocytic, but inhibits neuronal, lineage differentiation. Astroglial inactivation of NFκB signaling compromises astroglial, but promotes neuronal, lineage differentiation Fig. . Our finIntensive attention has been paid on inflammatory mediators including cytokines, chemokines, growth factors, and adhesion molecules due to their critical and diverse roles in the pathophysiology related to the disorders of nervous system , 72–74. 1β2 induced activation of NFκB-luciferase reporter in a similar manner as TNFα and IL-1β; (2) LTα1β2 dramatically stimulates p65 phosphorylation [There are three NFκB activation pathways described: classical , non-classical , and atypical pathways . LTβR acrylation , IκBα derylation , and p65rylation , 68, 92.NSC transplantation has been extensively studied in both animal models and clinical trials , 94. A mIn conclusion, this is the first report to show LTβR effects on regulating neural differentiation by demonstrating evidence of the expression and function of LTβR signaling in neural stem/progenitor cells. Activation of LTβR signaling promotes lineage differentiation of neural stem/progenitor cells preferentially through classical NFκB pathway. Understanding neuroinflammatory responses and neuroimmunity modulating neurogenesis might help understand the underlying pathophysiology relative to neuro-plasticity and relevant neurological disorders. LTβR may become a potential therapeutic target in the future in treating various neurodevelopmental and neurodegenerative diseases , 16.Additional file 1:Figure S1. Representative micrographs showing transgenic inactivation of astroglial NFκB pathway inhibits constitutive and LT-induced astroglial lineage differentiation but favors neuronal lineage differentiation in SVZ NSCs/NPCs from littermated wild-type  and GFAP-dnIκBα transgenic mice . At the 1st, 3rd and 6th day of differentiation, GFAP (green) and DCX (red)-positive cells were determined by multi-labeled fluorescent immunocytochemistry and confocal image analysis. Scale bars= 50 µm. Figure S2. LTα1β2 promoted astrocytic and oligodendrocytic differentiation but inhibited neuronal differentiation in mouse embryonic NSCs/NPCs.  Representative micrographs showing 3 lineage differentiation of cultured NSCs/NPCs from E14 mouse brain. White arrows (a) indicate representative Tuj1-positive typical neurons with various degrees of neurites and branches. The small red dots in GFAP staining (b) derived from non-specific debris for chicken anti-GFAP antibody. Scale bars = 75 µm. (c) Quantitative analysis of lineage differentiation after treatment with LTα1β2. Figure S3. Effects of NFκB inhibition on neural lineage differentiation in mouse NSCs/NPCs.  Representative micrographs showing complete loss of DCX (neuroblasts) and MBP (oligodendrocytes) and dramatic reduction of Tuj1 (immature neurons) and GFAP (astrocytes) after NFκB activation inhibitor APQ (10 µM) pretreatment 30 min before LTα1β2 (100 ng/ml) treatment under differentiation condition for 3 days. White arrows indicate representative Tuj1-positive typical neurons and green arrows show the nuclear location of Tuj1 expression (a). Scale bars = 75 µm. (c) Quantitative analysis of Tuj1 and GFAP positive cells after LTα1β2 treatment in the presence or absence of APQ. (d) Adenovirus-mediated NFκB-firefly-luciferase reporter assay showing a significant reduction in cytokine-induced NFκB activation in SVZ NSCs/NPCs from TG mice. Data represent mean ± SEM. * p<0.05 and ** p<0.01 indicate significant changes after cytokine treatment as compared to corresponding control group. ++ p<0.01 indicates significant decrease in APQ group compared with corresponding DMSO group. (PPTX 5201 kb)


Candidatus Profftella armatura’ a bacterial endosymbiont of D. citri, biosynthesizes diaphorin, which is a hybrid polyketide–nonribosomal peptide comprising two highly substituted tetrahydropyran rings joined by an N-acyl aminal bridge. The relative configurations of three out of its nine stereogenic centers, which could not be determined by NMR, were assigned based on the crystal structure.‘ 22H39NO9·CH3OH -4-hydroxy-5,5-dimethyltetrahydro-2H-pyran-2-yl}(hydroxy)methyl)-2-hydroxy-2-acetamide methanol monosolvate], was isolated from the Asian citrus psyllid, Diaphorina citri Kuwayama, and crystallizes in the space group P21. ‘Candidatus Profftella armatura’ a bacterial endosymbiont of D. citri, biosynthesizes diaphorin, which is a hybrid polyketide–nonribosomal peptide comprising two highly substituted tetrahydropyran rings joined by an N-acyl aminal bridge . The crystal structure of the title compound establishes the complete relative configuration of diaphorin, which agrees at all nine chiral centers with the structure of the methanol monosolvate of the di-p-bromobenzoate derivative of pederin, a biogenically related compound whose crystal structure was reported previously . Thus, the absolute configuration of diaphorin is proposed by analogy to that of pederin.The title compound C al. 2013. Curr. B al. 1968. Tetrah All three bacteria are spread within a grove by psyllids – sap-sucking insects in the order Hemiptera. In North America, ‘Ca. L. asiaticus’ is transmitted by the invasive citrus pest, the Asian citrus psyllid, Diaphorina citri Kuwayama. A complex community of vertically transmitted endosymbiotic bacteria colonizes D. citri, whether or not the psyllids are infected with ‘Ca. L. asiaticus’ /nonribosomal peptide synthetase (NRPS) gene and associated tailoring genes dedicated to the biosynthesis of diaphorin. Because ‘Ca. P. armatura’ is unculturable, diaphorin is extracted directly from its D. citri host , also known as citrus greening disease, which destroys the marketability of citrus fruit and eventually kills the tree, is a major threat to world citrus production  has a chair conformation with puckering parameters: amplitude Q = 0.541 (4) Å, θ = 173.0 (5)°, φ = 265 (4)°. Ring B (O11/C11–C15) substitutions comprise a hydroxyl group at C13, a geminal pair of methyl groups at C14 and a 2,3 dihydroxypropyl group at C15. It also has a chair conformation with puckering parameters: amplitude Q = 0.559 (4) Å, θ = 8.1 (4)°, φ = 258 (3)°. The mean planes of rings A and B are inclined to each other at an angle of 80.1 (2)°. For the plane including the central amide bond, (C7/O7/C8/O8/N9/C10), the r.m.s. deviation from the plane for those atoms is 0.045 Å. This planar conformation is likely influenced by a hydrogen bond in which the amide proton H9 is the donor and O7 is the acceptor with an interatomic distance of 2.16 Å between the participants  at the US Horticultural Research Laboratory, Fort Pierce, FL 34945, USA. Insects were allocated to 2 ml microcentrifuge tubes, then flash frozen in liquid N2 and cryoground for 3.5 min at 30 Hz using 3 × 3.2 mm metal beads per tube in a ball mill apparatus (Retsch Mixer Miller MM-400). Ground insects in each tube were then extracted three times in MeOH for 45 min at 298 K. After agitation, the tubes were centrifuged for 2 min at 16,000 g, and the supernatants were pooled, filtered through two layers of Whatman #1 paper and dried in vacuo. The residue was taken up in 90% MeOH and partitioned against cyclohexane. The methanolic phase was then fractionated by repetitive semi-preparative reversed phase HPLC using a Thermo Fluophase® column , eluted at 4 ml min−1 with 20% MeCN, and 1 ml fractions were collected. Following detection by UV absorption at 215 nm, selected fractions were monitored for the presence of diaphorin by syringe pump infusion (5 µL min−1) into a Waters-Micromass ZQ single quadrupole mass spectrometer . Fractions showing the pseudomolecular ion of diaphorin (M + Na+ at m/z 484) were recombined and dried in vacuo to afford ca 4.0 mg of diaphorin. Crystals of the title compound were obtained by slow evaporation from MeOH. A single crystal measuring approximately 0.01 × 0.02 × 0.20 mm was harvested using a needle dipped in a drop of oil for adhesion  and mounted in a small nylon loop (Hampton). The identity and purity of diaphorin was confirmed by comparing 1H NMR data acquired using the sample that afforded crystals with published data  and accurate mass estimates were compared with those of authentic diaphorin using a Waters Acquity UPLC system with a Waters C18 BEH column , eluted at 0.3 ml min−1 using a gradient formed from 0.1% formic acid (A) and acetonitrile (B) with 0.1% formic acid . Spectra were acquired on a Waters Xevo G-2 QTOF mass spectrometer operated in positive ion mode scanning the mass range from m/z 50 to 1200 in 0.1 sec with capillary and cone voltages set at 3.5 V and 25 k V, respectively. The spectrometer was calibrated in the range m/z 50–1200 using sodium formate. Spectra were calibrated in real-time using the M + H+ of co-infused leucine encephalin (m/z 556.2771) as the reference and were further processed by centering using the proprietary ‘automatic peak detection’ tool supplied with Waters MassLynx® 4.1 software.Diaphorin was isolated using a liquid–liquid extraction scheme with semi-preparative HPLC with modifications from the published method , referenced to the center of the residual CHD2OD pentet at δH 3.31. δH (p.p.m.) 5.60 , 4.80 , 4.64 , 4.26 , 3.882 , 3.880 , 3.76 , 3.61 , 3.49 , 3.40 , 3.25 , 2.50 , 2.31 , 2.20 , 2.04 , 1.76 , 1.67 , 1.17 , 0.99 , 0.95 , 0.88 . HRESIMS m/z 484.2521 ; tR = 8.61 min.Uiso(H) = 1.5Ueq and 1.2Ueq for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 2p-bromobenzoate methanol monosolvate . The synchrotron beamline available to us (CHESS F1) is normally used for macromolecular data collection. It is a fixed-wavelength line and it is not possible  to move the area detector (Pilatus 6M) close enough to the sample to record data beyond 0.95 Å . This explains the lack of high-resolution data, and the large s.u.’s on the cell dimensions, which may also be related to the use of the program 10.1107/S2056989018002992/su5424sup1.cifCrystal structure: contains datablock(s) I, Global. DOI: 10.1107/S2056989018002992/su5424Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989018002992/su5424Isup3.cmlSupporting information file. DOI: 1824900CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Dendritic epidermal T cells (DETCs) and dermal Vγ4 T cells engage in wound re-epithelialization and skin inflammation. However, it remains unknown whether a functional link between Vγ4 T cell pro-inflammation and DETC pro-healing exists to affect the outcome of skin wound closure. Here, we revealed that Vγ4 T cell-derived IL-17A inhibited IGF-1 production by DETCs to delay skin wound healing. Epidermal IL-1β and IL-23 were required for Vγ4 T cells to suppress IGF-1 production by DETCs after skin injury. Moreover, we clarified that IL-1β rather than IL-23 played a more important role in inhibiting IGF-1 production by DETCs in an NF-κB-dependent manner. Together, these findings suggested a mechanistic link between Vγ4 T cell-derived IL-17A, epidermal IL-1β/IL-23, DETC-derived IGF-1, and wound-healing responses in the skin. The results showed that mice with Vγ4D compared to isotype controls displayed markedly improved wound healing  and co-cultured them with rIL-17A. The results showed that rIL-17A failed to inhibit the production of IGF-1 in eDETCs  on wound beds of WT mice, while the expression of IGF-1 was decreased when adding rIL-1β plus rIL-23. Blocking IL-17A could not rescue the declined production of IGF-1  to deplete and detect Vγ4 T cells, which cannot distinguish cells that have had their TCR downregulated from depletion. Therefore, we could still not exclude the possibility that Ab treatment does not deplete γδ T cells but rather reduces their TCR complexes on the cell surface.Wound healing is a complex process divided into four phases of hemostasis, inflammation, proliferation, and remodeling . Murine lization . TherefoTcrδ−/− mice exhibit wound healing defects, which are rescued by the reconstitution of DETCs or addition of recombinant IGF-1 . Whether epidermal-infiltrating Vγ4 T cells that produce IL-17A are associated with some ligands expressed on stressed epidermal cells is still unclear. With the exception of TCR signaling, NKG2D has been demonstrated to activate γδ T cells . TCRδ-GFP, Tcrδ−/− and Ifn-γ−/− mice on C57BL/6 background were purchased from Jackson Laboratory . Sex-matched mice aged 6–8 weeks were used for all experiments. All experiments were performed under specific pathogen-free conditions in conformity with ethical guidelines and approved by the Animal Ethics Committee of the Third Military Medical University.C57BL/6 WT and Excision wounds were cut down to the musculus panniculus carnosus with a diameter of 6-mm sterile punch. For wound model without contraction, glue the biological membrane  with the adhesive dressings immediately onto the surface of the wound before contraction and single-house the mice. Wound area was recorded daily by macroscopic digital photographs and estimated at each time point relative to day 0 until completely closure.Mouse model of Vγ1D was implemented by intraperitoneal injection of 200 μg anti-Vγ1 Ab  3 days before wound excision. Vγ4D was generated by anti-Vγ4 Ab . Control mice were intraperitoneally administrated isotype control (armenian hamster IgG) . Neutralization mouse models were conducted by injecting subcutaneously into wound margin 0, 1, and 2 days following excision with 20 μg/wound of anti-IGF-1 Ab , anti-IL-17A Ab , anti-IFN-γ Ab , anti-IL-1β Ab , anti-IL-23 Ab , anti-CCL20 Ab , respectively. IL-17A neutralization of low and high doses was performed by 2 and 200 μg/wound of anti-IL-17A Ab, respectively. Isotype control mice received equivalent doses of isotype Abs subcutaneously. Isotype control Abs of IGF-1, IL-17A, IL-1β, IL-23, CCL20 are poly goat IgG, mouse IgG1, armenian hamster IgG, rat IgG2, and rat IgG1 (BioXcell), respectively. Mouse models of recombinant cytokine addition were generated by subcutaneously injecting rIGF-1/rIL-17A/rIL-1β/rIL-23  with the doses of 2, 20, 200 ng/wound, respectively, in the wound margin. Control mice were administrated with sterile PBS.Il-17a−/−, and Ifn-γ−/− mice by the EasySep positive cell isolation systems . The purity of isolated Vγ4 T cells was identified by FACS analysis. 1 × 104 cells/wound of WT, Il-17a−/−, and Ifn-γ−/− Vγ4 T cells were placed onto the wound bed immediately following excision.Vγ4 T cells were isolated from lymph node cells and splenocytes of C57BL/6 WT, 2. Epidermal sheets were torn off carefully from wound margins after incubated with 5 mg/ml dispase II at 37°C for 2 h. Epidermal single-cell suspensions were isolated by dissociating epidermal sheets with 0.3% Trypsin/GNK solution for 30 min at 37°C, then washed by PBS and filtered through a 70-μm strainer. Epidermal cells were cultured in the concentration of 5 × 105–1 × 106/ml with 10% FBS 1640 medium. Primary keratinocytes were isolated from skin of newborn mice in the same method as above. According to the manufacturer’s instructions , CD11c+ cells were removed from primary keratinocytes. The purity of isolated keratinocyte population was >95%.Skin that is less than 5 mm away from wound edge was regarded as wound margin. Each group of epidermis tissue around wound was collected from 3–7 mice (4 wounds/mouse) for further analysis. Subcutaneous tissue of skin was wiped off and then skin was cut into small pieces of 0.5 × 0.5 cm4 cells/wound). For in vitro assays, DETC population was expanded in 48-well plates (1–2 × 106/ml) with RPMI containing 10% FBS, 1 μg/ml Con A , 10 ng/ml mouse rIL-2, 1 mM Na Pyruvate , 2 mM glutamine, 25 mM HEPES, 50 μM 2-ME , 100 M non-essential amino acids , 100 μg streptomycin and 100 U penicillin for 2–4 weeks. The purity of expanded DETCs (eDETCs) was > 95% by means of FACS analysis. eDETCs were rested without ConA for 2 weeks before used for further experiments.After epidermal single-cell suspension was prepared, lympholyte-M  was used to enrich DETCs (purity = 15–20%). For reconstitution assays, DETCs were further purified by the EasySep positive cell isolation systems . The purity of isolated DETCs was identified by FACS analysis (purity > 90%). Then, DETCs were transferred to wound bed immediately following excision , and 100 ng/ml Brefeldin A  at 37°C, 5% CO2 for 6 h. For in vitro stimulation, eDETCs or eDETCs plus primary keratinocytes cultured with murine rIL-1β/rIL-23/rIL-17A , IL-1β/IL-23/IL-17A neutralizing Ab , or signal inhibitors NFκB inhibitor , STAT3 inhibitor   for 6–24 h. Cells were stained as above. Stained cells were detected on Attune Acoustic Focusing Cytometer  and analyzed with FlowJo software .The following fluorochrome-labeled murine monoclonal antibodies (mAbs) were purchased from BD, Biolegend, eBioscience, and Sungene Biotech: CD16/32(2.3G2), γδTCR (GL3), Vγ1 (2.11), Vγ4 (UC3-10A6), Vγ5 (536), CD25 (3C7), CD44 (IM7), CD62L (MEL-14), CD69 (H1.2F3), NKG2D (CX5), JAML (4E10), CCR6 (29-2L217), IL-17A (TC11-18H10.1), IFN-γ (XMG1.2). IGF-1  was stained followed by fluorescent-labeled secondary reagents FITC goat-anti-rabbit mAb , PE donkey-anti-rabbit mAb . For surface staining, cells were first blocked with anti-CD16/32 mAb for 15 min and then incubated with different cellular surface mAbs for 30 min at 4°C. For further intracellular staining, cells were fixed and permeabilized according to the manufacturer’s recommendations . For g for 15 min. Concentration of protein extraction was determined by BCA protein assay kit . 25 μg/sample was run on 10% or 12% SDS-PAGE, then transferred to polyvinylidenedifluoride (PVDF) membranes . PVDF membranes were incubated with 3% bull serum albumin (BSA)  at room temperature for 2 h, then with following Abs at 4°C overnight: IL-17A, IGF-1 , CCL20 , IL1β/IL23 Ab , NF-κB p65 (D14E12), phospho-NF-κB p65 , STAT3 (D3Z2G)/phospho-STAT3 , phospho-STAT3  . Anti-GAPDH Ab  was as loading control. The membranes were visualized with goat-anti-rabbit/mouse/rat-HRP IgGs  at room temperature for 1 h and chemiluminescence liquid for seconds . ChemiDoc™ XRS detection system  was used to analyze blots.Tissue protein extracts of whole skin, dermis, and epidermis were prepared by grinding the tissue into powder in liquid nitrogen. Then, the powder was lysed in lysis buffer  with 1 μg/ml protease inhibitor, 5 μg/ml PSMF, and 10 μg/ml phosphatase inhibitor, followed by low-speed rotation at 4°C for 20 min and centrifugation at 14,000 × Specimens of wound margin were embedded in paraffin by routine methods. The sections were stained with H&E and new epithelial length was measured under the microscope in a blinded manner. For immunohistochemistry, skin sections were deparaffinized and hydrated in xylene and graded alcohol series. Antigen retrieval was conducted by incubating sections in 10 mM citric acid (pH 6) at 95°C for 15 min. Sections were blocked and stained with goat-anti-rabbit serum or 3% BSA with 0.3% Triton X-100  at room temperature for 60 min and followed by primary antibodies: anti-IL-1β, anti-IL-23, anti-IGF-1, and anti-IL-17A  overnight at 4°C. Samples were washed and incubated with secondary antibodies at room temperature for 60 min. Slides were incubated with drops of diaminobenzidine solution  and counterstained with hematoxylin . Stained sections were examined under Olympus BX51 microscope .6/ml. 20 μl cell suspension was dropped to slide and went dry at room temperature. Then cells were fixation with acetone at 4°C for 30 min and washed by PBS for 5 min × 3 times. Cells were permeabilized with 0.5% Triton X-100 at 4°C for 15 min and washed three times with PBS. Cells were then incubated in 5% BSA at room temperature for 1 h. Cells were washed with PBS and incubated with anti-NF-kB p65 or anti-STAT3 Ab  in PBS at 4°C overnight. Cells were stained with DAPI  for 30 s and washed in PBS for 1 min. Cells were covered by mounting solution  and preserved at 4°C. Epidermis sheets isolated from TCRδ-GFP mice were lay on slice and directly covered by mounting solution. Slices were photographed using Zeiss Axioplan fluorescent microscope.eDETCs were stimulated with anti-CD3ε Ab (5 μg/ml), rIL-1β, or/and IL-23 (100 ng/ml) for 30 min, then were washed with PBS. eDETCs were re-suspended by PBS to concentration of 1 × 10F = 2−ΔΔCt, ΔCt = Cttarget gene−CtGAPDH and ΔΔCt = ΔCtinduced−ΔCtreference.RNAs were extracted by a Qiagen RNeasy kit or TRIzol reagents . cDNA was reverse transcripted by 0.5 mg total RNA with a TaqMan reverse transcription kit . qRT-PCR was detected on Bio-Rad RT-PCR system using SYBR Green Supermix and gene-specific primer pairs: IL-1β: 5′—3′(forward) ACCTTCCAGGATGAGGACATGA, 5′—3′(reverse) CTAATGGGAACGTCACACACCA; IL-23p19: 35′—3′(forward) CTGAGCCACCCAGGAAAGTA, 5′—3′(reverse) TGAGAAAACCCAGAGCATCA; GAPDH: 5′—3′(forward) CGTGCCGCCTGGAGAAAC, 5′—3′(reverse) AGTGGGAGTTGCTGTTGAAGTC. Gene expression level was normalized to GAPDH and represented as fold differences by the method where t-test or one-way ANOVE with Bonferroni comparison test on SPSS19.0 software , and graphs were manufactured by GraphPad Prism software . P < 0.05 was considered significant.All data were shown as mean ± SD. Statistical differences were calculated by two-tailed unpaired Student’s All experiments were performed under conventional animal raising conditions in conformity with ethical guidelines and approved by the Animal Ethics Committee of the Third Military Medical University.Conceptualization, WH; methodology, YL, YW, ML, LZ, and XH; validation, RY, JH, XZ, XH, and YH; formal analysis, YL, LZ, and YW; investigation, YL and YW; resources, GL, YJ, RW, and ZY; writing-original draft, WH, YL, and YW. Writing-reviews and editing, WH, GL, and JW; funding acquisition, WH, GL, and JW; supervision, WH, GL, and JW.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Poecillastra compressa  led to the identification of seven new steroidal saponins named poecillastrosides A–G (1–7). All saponins feature an oxidized methyl at C-18 into a primary alcohol or a carboxylic acid. While poecillastrosides A–D (1–4) all contain an exo double bond at C-24 of the side-chain and two osidic residues connected at O-2′, poecillastrosides E–G (5–7) are characterized by a cyclopropane on the side-chain and a connection at O-3′ between both sugar units. The chemical structures were elucidated through extensive spectroscopic analysis (High-Resolution Mass Spectrometry (HRESIMS), 1D and 2D NMR) and the absolute configurations of the sugar residues were assigned after acidic hydrolysis and cysteine derivatization followed by LC-HRMS analyses. Poecillastrosides D and E, bearing a carboxylic acid at C-18, were shown to exhibit antifungal activity against Aspergillus fumigatus.The first chemical investigation of the Mediterranean deep-sea sponge  Asteropus spp. − . Poecillastroside A (2): Yellow, amorphous solid; c 0.1, CH3OH); UV (DAD) λmax 210 nm; 1H NMR and 13C NMR data, see m/z 769.4743 [M − H]− . Poecillastroside B (3): Yellow, amorphous solid; c 0.1, CH3OH); UV (DAD) λmax 212 nm; 1H NMR and 13C NMR data, see m/z 769.4745 [M − H]− . Poecillastroside C (4): Yellow, amorphous solid; c 0.1, CH3OH); UV (DAD) λmax 222 nm; 1H NMR and 13C NMR data, see m/z 791.4567 [M + Na]+ . Poecillastroside D (5): White, amorphous solid; c 0.1, CH3OH); UV (DAD) λmax 220 nm; 1H NMR and 13C NMR data, see m/z 845.4307 [M + Na]+ . Poecillastroside E (6): White, amorphous solid; c 0.1, CH3OH); UV (DAD) λmax 222 nm; 1H NMR and 13C NMR data, see m/z 789.4405 [M + Na]+ . Poecillastroside F (7): White, amorphous solid; c 0.1, CH3OH); UV (DAD) λmax 225 nm; 1H NMR and 13C NMR NMR data, see m/z 831.4518 [M + Na]+ . Poecillastroside G (v/v/v) with 0.1% formic acid (B). The analysis was performed in isocratic mode at 13% B and at a flow rate of 360 µL/min. The injection volume was set at 3 µL. The identity of all monosaccharide derivatives was confirmed after extraction of the ion [M + H]+ at m/z 433.1098 , and S. aureus MB5393 (MRSA)) and Gram negative bacteria , and fungi (A. fumigatus ATCC46645), following previously described methodologies [Compounds dologies . Cytotoxdologies .1–7) share an unusual oxidized methyl at C-18, and they are the first saponins exhibiting this feature. The structures of poecillastrosides E–G (5–7) also incorporate a terminal methylated cyclopropyl ring already known in some sponge steroids and already investigated for biosynthetic studies [4) and E (5), bearing a carboxylic acid at C-18, were found to be the most bioactive compounds in the antimicrobial bioassays with an interesting antifungal activity against Aspergillus fumigatus.Poecillastrosides A–G ( studies . This cy studies , but to


The synthesis, spectroscopic and crystal structure of 2-chloro-1,3-bis-1,3,2-diazaphospholidine 2-oxide are reported. 26H38ClN2OP, was synthesized by reacting phosphoryl chloride with N,N′-bisethane-1,2-diamine in the presence of N-methylmorpholine which acted as an auxilliary base to quench the HCl released as a by-product. The resultant N-heterocyclic phosphine five-membered ring adopts a half-chair conformation and features a tetracoordinate P atom ligated by the chelating diamine [P—N = 1.6348 (14) and 1.6192 (14) Å], one double-bonded O atom [P1—O1 = 1.4652 (12) Å] and one Cl atom [P1—Cl1 = 2.0592 (7) Å]. The sterically hindered 2,6-diisopropylphenyl (Dipp) groups twist away from the central heterocycle, with torsion angles of −75.66 (19) and 83.39 (19)° for the P—N—Car—Car links. A number of intramolecular C—H⋯N, C—H⋯O and C—H⋯Cl close contacts occur. In the crystal, molecules are linked by C—H⋯O hydrogen bonds to generate [010] chains. C—H⋯π interactions are also observed.The title compound, C N-heterocyclic phosphines (NHPs) that feature an N—P—N moiety bridged by a C2H4 fragment, thus forming a five-membered ring. Derivatives are often substituted by alkyl, aryl, or halogen groups at the phosphorus position (denoted as position 2), allowing them to serve as both ligands and/or precursors in organometallic chemistry  Å and P1—N2 = 1.6192 (14) Å] and is likely caused by the half-chair (or envelope) conformation of the heterocycle at the C2 position. The N—P—N bond angle of 95.60 (7)° deviates significantly from an ideal tetrahedral geometry. Bond lengths between P1—Cl1 and P1—O1 are 2.0592 (7) and 1.4652 (12) Å, respectively, with a bond angle of 105.51 (5)° for the O—P—Cl atoms. The isopropyl groups are oriented away from the central five-membered ring and lead to intramolecular short-contact D—H⋯A interactions between methine atoms H9, H12, H21, and H24, and N1 and N2. Intramolecular short-contact D—H⋯A interactions are also present for Cl1 and O1 atoms and are summarized in Table 1The molecular structure of the title compound is shown in Fig. 1D—H⋯A π-interactions between C6—H6 and the centroid of the C3–C8 ring of a neighboring molecule , with an H⋯centroid distance of 2.740 (3) Å. The isopropyl groups of the flanking aromatic rings also display short contacts for Cl1 and O1, with H⋯Cl distances measuring 2.950 (5) and 3.086 (6) Å between H14A⋯Cl1 and H23B⋯Cl1, respectively. A significantly short contact of 2.357 (2) Å occurs for H2A⋯O1. A distance this small is likely indicative of C—H⋯O hydrogen bonding .The crystal of the title compound contains intermolecular short-contact ng Fig. 2 acceptedet al., 2016p-tolyl)-2-chloro-1,3,2-diazaphospholidine-2-oxide (p-tolyl = 4-methylphenyl), and the analogous fluorine derivative ethane-1,2-diamine were dissolved in 45 ml of THF producing a colourless solution. Separately 0.478 g  of phosphoryl chloride and 0.959 g  of N-methylmorpholine were dissolved in 75 ml of THF producing a colourless solution, and transferred to a 125 ml pressure-equalizing dropping funnel. The diamine solution was cooled to 195 K using a liquid nitrogen/acetone bath and monitored using a thermocouple, and once cold (ca 10 minutes) the phosphoryl chloride mixture was added dropwise to the diamine solution over 30 minutes. Once the addition was complete, the colourless reaction mixture was left to stir at 195 K for 60 minutes, after which it was allowed to warm to room temperature and left to stir for two days at room temperature. The reaction was monitored by 31P{1H} NMR spectroscopy, and became pale yellow in colour with a slight amount of colourless precipitate as it proceeded. Once the starting material was completely consumed, the reaction mixture was dried in vacuo to give a pale-yellow coloured solid. Extraction of this solid with 50 ml of a 3:2 mixture of pentane:THF produced the desired product as a pale-yellow coloured solution following filtration through Celite, which when dried in vacuo afforded 0.919 g (66%) of the desired product as a faintly yellow coloured powder. Crystals of the product in the form of colourless blocks were obtained by concentrating the filtrate and storing in a 238 K freezer overnight.The synthesis of the title compound was achieved using a similar method as used for 2-chloro-1,3-bis-1,3,2-diazaphospholidine (Caputo 1H NMR (CDCl3): δ 7.32 , 7.21 , 3.86–3.50 , 1.38 , 1.35 , 1.28 , 1.26 ppm . 13C{1H} NMR (CDCl3): δ 149.8, 149.6, 131.8, 129.1, 125.0, 124.9, 48.8, 29.0, 25.9, 24.5, 23.9 ppm. 31P{1H} NMR (CDCl3): δ 15.1 ppm. IR (KBr pellet): ν 3068 (w), 2967 (s), 2929 (m), 2869 (m), 1681 (w), 1588 (w), 1464 (s), 1448 (s), 1383 (w), 1368 (w), 1348 (w) 1323 (m), 1268 (s), 1217 (w), 1194 (w), 1106 (m), 1093 (m), 1077 (m), 1056 (m), 1043 (w), 934 (w), 860 (w), 803 (s), 756 (w), 747 (w), 733 (w), 648 (w), 592 (w), 575 (w), 558 (w), 544 (w), 496 (s) 466 (w), 437 (w), 412 cm−1 (w). m.p. 509.7–511.0 K.Uiso(H) = 1.2–1.5Ueq(C)]. The methyl H atoms were allowed to rotate, but not to tip, to best fit the electron density.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989017005825/hb7671sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989017005825/hb7671Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989017005825/hb7671Isup3.cmlSupporting information file. DOI: 1544709CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: