The estrogen receptor (ER) β variant ERβ2 is expressed in aggressive castration-resistant prostate cancer and has been shown to correlate with decreased overall survival. Genome-wide expression analysis after ERβ2 expression in prostate cancer cells revealed that hypoxia was an overrepresented theme. Here we show that ERβ2 interacts with and stabilizes HIF-1α protein in normoxia, thereby inducing a hypoxic gene expression signature. HIF-1α is known to stimulate metastasis by increasing expression of Twist1 and increasing vascularization by directly activating VEGF expression. We found that ERβ2 interacts with HIF-1α and piggybacks to the HIF-1α response element present on the proximal Twist1 and VEGF promoters. These findings suggest that at least part of the oncogenic effects of ERβ2 is mediated by HIF-1α and that targeting of this ERβ2 – HIF-1α interaction may be a strategy to treat prostate cancer. Prostate cancer is a slowly progressing disease, initially treatable with androgen- deprivation therapy (ADT) , but usuA proliferating tumor is often exposed to hypoxic condition, because of its higher metabolic needs and lack of neo-vascularization to keep up with its demands. Hypoxia promotes neuro-endocrine differentiation of the prostate tumor, which increases its aggressiveness . The traThe 22Rv1 and PC3 cell lines were obtained from the American Type Culture Collection (ATCC). 22Rv1 cells were maintained in RPMI-1640  medium supplemented with 10% fetal bovine serum (FBS) , 25 mM HEPES buffer and 2 mM L-glutamine , while PC3 cells were maintained in RPMI-1640  medium supplemented with 10% fetal bovine serum (FBS) . All experiments used cells below passage 30.HIG2 luciferase plasmid have been described earlier , PXP2-TwEscherichia coli biotin holoenzyme synthetase (BirA) from the actin promoter and selected with G418 at 500 μg/ml. After two weeks clones were isolated and assayed for biotinylation activity.A transposon-based system mediating doxycycline-regulated expression of ERβ2 was used to stably transfect 22Rv1 and PC3 prostate cancer cells. These cell lines are described elsewhere . For cong for 10 minutes.To prepare whole-cell extracts, cells were washed twice with PBS, lysed in 10 times packed cell volume of lysis buffer  for 15 minutes on ice and then centrifuged at 14,000 x Twenty micrograms of protein were loaded on an SDS-PAGE 10% Bis-Tris gel with Tris running buffer and transferred to a nitrocellulose membrane after electrophoretic separation. Membranes were blocked with 5% non-fat powdered milk in 0.1% TBST buffer and probed with anti-ERβ2 (produced in the lab), Twist1 (sc-15393), and HIF-1α (sc-10790) . Primary antibodies were used at 1:200–1000 dilutions, and secondary antibody was used at 1:10,000.5′-CCT GCG GCT TAA TTT GAC TCA-3′ and reverse (R), 5′-AGC TAT CAA TCT GTC AAT CCT GTC C-3; glyceraldehyde-3-phosphate dehydrogenase F, 5′-TGA CAA CTT TGG TAT YCG TGG AAG G-3′ and R, 5′-AGG CAG GGA TGA TGT TCT GGA GAG-3′ (reference genes); ERβ2 F, 5-ACT TGC TGA ACG CCG TGA CC-3 and R, 5′-CCA TCG TTG CTT CAG GCA A-3′; Twist1 F, 5′-GGA GTC CGC AGT CTT ACG AG-3′ and R, 5′-TCT GGA GGA CCT GGT AGA GG-3′. qPCR reactions were performed with a 7500 Fast Real-Time PCR System (Applied Biosystems) using optimized conditions for SYBR Green I dye system: 50 C for 2 minutes, 95 C for 10 minutes, followed by 40–50 cycles at 95 C for 15 seconds and 60 C for 50 seconds. Optimum primer concentration was determined in preliminary experiments, and amplification specificity confirmed by dissociation curve analysis.RNA extraction was performed with Qiagen mRNA extraction kit according to standard protocol. cDNA was synthesized from 1μg of total RNA with First Strand System according to standard protocol (Invitrogen Inc. NY). Real-time PCR was performed with SYBR Green I dye master mix . Primers  were: 18s rRNA forward (F), HIF-1α and ERβ2 were translated using the TNT Quick Coupled Transcription/Translation Systems . Briefly, 0.2 μg of HIF-1α or ERβ2 expression vectors (T7 promoter) were added to an aliquot of the TNT Quick master mix and incubated in a volume of 50 μl for 60 minutes at 30°C. The in vitro synthesis of protein was verified by SDS-PAGE. For bacterial expression BL21-DE3 bacterial cells were used to express His-LBD-ERα, His-LBD-ERβ1, His-LBD-ERβ2 and His-LBD-ERβ2ΔCX expression plasmids.et al and Yan et al. [PC3 cells were transfected with 3 μg BirA (biotin ligase) expression plasmid, 3 μg plasmid containing biotinylation consensus tagged receptors B7TEV-ERα, B7TEV-ERβ1, B7TEV-ERβ2 and B7TEV-ERβ5 together with 3 μg pcDNA3 HA-HIF-1α “Addgene plasmid 18949” or pcDNA3 HA-HIF-1α P405/A-P564/A “Addgene plasmid 18955” or pcDNA3 HA-HIF-1α Δ401–603 (pcDNA3 HA-HIF-1αΔODD) described by Kondo n et al. , 28 in ain vitro translated HIF-1α protein was incubated with 25 μl of bacterial lysate containing His-ERβ2 (LBD) overnight, and complexes were adsorbed onto nickel agarose beads for 2 h. Beads were washed three times with ice-cold NETN (20 mM Tris (pH = 8.0), 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40). Complexes were boiled with 20 μl of 2× sample buffer, subjected to SDS-PAGE and transferred to nitrocellulose membrane for Western analyses. The blot was probed with anti-HIF-1α antibody (Santa Cruz). Primary antibodies were at 1:1000 dilution, and secondary antibody at 1:10,000.2μl ERβ2 was cloned into the Gal4DBD expressing plasmid PM2 in frame with the Gal4DBD domain using BamHI and HindIII restriction sites making PM2-ERβ2. The pVP16 plasmid (Clontech) was used to clone the N-terminal HIF-1α (amino acids 1–401), oxygen destabilization domain (amino acids 401–603) and the C-terminal domain (amino acids 603–826) in frame with the VP16 activation domain using BamHI and PstI restriction sites. For interaction assays in PC3 cells the Gal4 responsive reporter FR-LUC 300 ng, PM2-ERβ2 500 ng and VP16 fused HIF-1α domains 200 ng was transfected using PEI method of transfection as described by Longo et al. et al. [The two-color comparative microarray analysis covers the fully known protein-coding transcriptome of 39,600 transcripts and variants by using Human OpArray arrays purchased from Microarrays Inc. . The array was complemented with 133 oligonucleotides specifically synthesized for detailed and robust analysis of nuclear receptors, splice variants, and coregulators. The microarray analysis was performed essentially as in Richter et al. . For eac5’-CGGGGGAGGGGGACTGGAAAGC-3’; (R) 5’-AGGCCTCCTGGAAACGGTGCCG-3’, VEGF promoter: (F) 5’-ACAGACGTTCCTTAGTGCTGG-3’; (R) 5’-AGCTGAGAACGGGAAGCTGTG-3’.Sub-confluent PC3 and 22Rv1 cells (90%) were grown in a 150 mm dish and washed twice with cold PBS and then fixed in PFA (1%) for 10 minutes. Cells were washed twice with cold PBS + protease inhibitor, scraped with 150 μl of resuspension buffer, and centrifuged . Pellets were resuspended in 750 μl ChIP lysis buffer  and cells kept on ice for 30 minutes. Cell lysates were sonicated  with 30 seconds break at 4°C. Following sonication, 25 μl samples were collected and stored at -20°C (input). The rest of the 125 μl cell lysate fraction was used for the following steps. Samples were pre-absorbed in 20 μl of G protein coupled FLAG tagged magnetic beads at 4°C for 2 hours and supernatant collected. Samples were then divided into two fractions and antibodies against FLAG (2.5 μg) or IgGs (2.5 μg) were added and incubated overnight. The next day, the beads were washed three times with ChIP lysis buffer, once with ChIP wash high salt buffer , and finally once with ChIP wash buffer . Beads were resuspended in 40 μl elution buffer  and incubated at 65°C overnight with a small hole on the cap. The input collected previously was also placed at 65°C. The next day, samples were purified using PCR purification kit (Qiagen) and used for qPCR analysis using the following primers. pHRE-Twist1 promoter (F) t-test was used to compare the differences between two groups. The significance is presented as *p<0.05, **p<0.005, and ***p<0.001, and non-significant differences are presented as NS.The values are expressed as the mean with 95% confidence intervals. An unpaired two-tailed response to hypoxia’ was the second most overrepresented function (p = 8.0e-6), second only to ‘gene expression’ , EGLN3 (PHD3), SMAD3, HSD11B2, CAT, CCL2, CDKN1B, MMP13, PLOD2, IL1B, PLAU, SCNN1G, CRYAB, IL18, ITPR1, SOD2, ADM). In addition, the enriched sub-network, defined as the genes being expression target of the respective node, ‘Neighbors of HIF-1α’ was the most overrepresented one (p = 1.5e-09) with 41 regulated members , including 9 genes . Thus, the microarray analysis clearly indicated hypoxia and HIF-1α function as major themes of ERβ2 function (See We have previously shown that ERβ2 increased expression of EMT-associated genes Twist1 and Slug (SNAI2) in prostate cancer cells . Here, wression’ . The ‘Re members . Further members , which wTo validate these data, we analyzed the expression of HIF-1α in PC3 and another prostate cancer cell line-22Rv1, which are both over-expressing ERβ2. We observed an increase in HIF-1α protein level in ERβ2 expressing versus control cells, but no changes in corresponding mRNA level –1D. Thisin vitro translated HIF-1α. HIF-1α strongly bound to ERβ2, but not to equivalent amounts of wild type ERα or ERβ LBDs. This interaction was independent of ERβ2 specific sequences because truncation of amino acids encoded by the ERβ2-specific exon did not eliminate HIF-1α binding (ERβ2ΔCX)  . Pull-doERβ2ΔCX) . Since ETo investigate, if the oxygen destabilizing domain of HIF-1α was required for interaction with ERβ2 we constructed an expression plasmid of HIF-1α with amino acids 401–603 deleted as described earlier . We thenIt is well known that prolines 405 and 564 of HIF-1α under normoxia are hydroxylated by proline hydroxylases (PHD’s) and then recognized by the VHL factor and subsequently targeted for degradation . We wantFinally, we explored whether ERβ2 can bind to the HIF-1α response elements using ChIP-qPCR. We detected increased levels of ERβ2 recruited at the HIF-1α response elements on the HIF-1α dependent Twist1 and VEGF promoters in both PC3 and 22Rv1 cells. However, ERβ2 does not bind to the promoters of the classical estrogen response element in the pS2 gene Fig  and 7B.In conclusion, our findings indicate that ERβ2 binds to and stabilizes HIF-1α and furthermore, is co-recruited to key HIF-1α elements in HIF-1α regulated genes thereby enhancing the activity of HIF-1α during normoxic conditions in prostate cancer cells.Response of normal tissues to hypoxia is crucial for proper vascularization and for subsequent blood supply to oxygenate and provide nutrients to the tissue. A growing tumor becomes hypoxic when reaching more that 1mm in size . The immExpression of ERβ2 correlates with worse prognosis in prostate cancer  and ERα-Surprisingly, although we found that ERβ1 does not interact efficiently with HIF-1α, correlating with its inability to induce HIF-1α responsive genes, the unique last exon of ERβ2 is not required for HIF-1α binding. Apparently, truncation of the ERβ1 C-terminus exposes a new protein surface mediating interaction with HIF-1α.It remains to be shown if ERβ2 expression correlates with levels of Twist 1 or other HIF-1α target genes in clinical samples. A recent study by Ragnum et al.  shows thIn conclusion, expression of the ERβ variants ERβ2 and ERβ5 has previously been shown to correlate with aggressive prostate cancer. Here we show that ERβ2 and ERβ5 stabilize the HIF-1α protein and induce hypoxic gene expression under normoxic conditions, proposing a mechanism for the oncogenic effect of ERβ2 and ERβ5.S1 FigA. Western blot for HIF-1α on extracts from control 22Rv1 cells and two clones expressing ERβ2. (B) Validation of genes from the microarray.(TIF)Click here for additional data file.


In addition to regulating cytoplasmic Ca2+ signals, these responses also affect the concentration of Ca2+ within the ER and mitochondria. Single fluorescent protein-based Ca2+ indicators, such as the GCaMP series based on GFP, are powerful tools for imaging changes in the concentration of Ca2+ associated with intracellular signalling pathways. Most GCaMP-type indicators have dissociation constants (Kd) for Ca2+ in the high nanomolar to low micromolar range and are therefore optimal for measuring cytoplasmic [Ca2+], but poorly suited for use in mitochondria and ER where [Ca2+] can reach concentrations of several hundred micromolar. We now report GCaMP-type low-affinity red fluorescent genetically encoded Ca2+ indicators for optical imaging (LAR-GECO), engineered to have Kd values of 24 μM (LAR-GECO1) and 12 μM (LAR-GECO1.2). We demonstrate that these indicators can be used to image mitochondrial and ER Ca2+ dynamics in several cell types. In addition, we perform two-colour imaging of intracellular Ca2+ dynamics in cells expressing both cytoplasmic GCaMP and ER-targeted LAR-GECO1. The development of these low-affinity intensiometric red fluorescent Ca2+ indicators enables monitoring of ER and mitochondrial Ca2+ in combination with GFP-based reporters.Ca 2+ indicators with low affinities. These indicators enable monitoring of mitochondrial and ER Ca2+ dynamics in combination with a green fluorescent indicator.This paper describes the engineering of two red fluorescent genetically encoded Ca Spectra mCherry  was usedeviously . For Ca22. Cells were seeded on fibronectin-coated, glass-bottomed culture dishes and transiently transfected after 24 h with plasmids using Transit-LT1 (Mirus Bio). Cells were imaged 24–48 h after transfection, using an Olympus IX83 microscope with a ×100/1.49 numerical apesture (NA) objective, at 20°C in Hepes-buffered saline (HBS). HBS had the following composition: 132 mM NaCl, 5 mM KCl, 2 mM MgCl2, 10 mM D-glucose, 10 mM Hepes and 2 mM CaCl2, pH 7.2. Ca2+ was omitted from nominally Ca2+-free HBS. Cells were illuminated alternately, via a 405/488/561/647-nm quad band dichroic/emitter , with a 559-nm light-emitting diode (LED)  for visualizing ER-LAR-GECO1 and a 488-nm diode laser (Coherent) for visualizing ER-GFP and CatchER. Ratiometric measurements of ER-GAP1 fluorescence were achieved by illuminating cells alternately with a 395-nm LED  or a 488-nm laser and measuring fluorescence emission at 520–550 nm using a 535/30 nm emission filter (Chroma Technology). With the filters used there was no significant cross-talk between blue, green and red channels. Images were acquired with an iXon Ultra electron multiplying charge-coupled device (EMCCD) camera (512×512 pixels) (Andor Technology) and images were processed using MetaMorph (Molecular Devices). All records were corrected for background fluorescence determined from regions outside cells. Fluorescence changes from regions of interest were expressed as F/F0, where F0 and F denote the average fluorescence at the start of the experiment (F0) and at each time point (F), respectively. Ratios of the ER-GAP1 fluorescence with alternating excitation at 488 nm and 395 nm were expressed as R (fluorescence intensity with 488-nm excitation/fluorescence intensity with 395-nm excitation), and R0 was defined as the average value of R at the start of the experiment.HeLa, human embryonic kidney (HEK)-293 and U2-OS cells were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 medium (Life Technologies) supplemented with FBS  at 37°C in air containing 5% CO2 flask, split with PBS, and plated in 100-mm tissue culture dishes at ~10% confluence for 18–20 h before transfection with cDNA encoding D1ER or ER-LAR-GECO1 using the calcium phosphate precipitation method. After 24 h, the growth medium was changed to an induction medium containing 1 μg/ml tetracycline (Sigma). After induction for ~22 h, the cells were perfused continuously with KRH buffer  containing various concentrations of CaCl2  and tetracaine (1 mM) for estimating the store capacity or caffeine (20 mM) for estimating the minimum store level by depleting the ER Ca2+ stores at room temperature (23°C). For D1ER recordings, images were captured with Compix Simple PCI 6 software every 2 s using an inverted microscope (Nikon TE2000-S) equipped with an S-Fluor ×20/0.75 NA objective. The filters used for D1ER imaging were excitation, 436±20 nm for CFP, and 500±20 nm for YFP; and emission, 465±30 nm for CFP, and 535±30 nm for YFP, with a dichroic mirror (500 nm). FRET was determined from the ratio of the light emission at 535 nm and 465 nm. For ER-LAR-GECO1 recordings, images were captured using a Nikon A1R confocal system. The cells were excited using a diode laser (561 nm) and fluorescence emission was detected at 570–620 nm.HEK-293 cells stably expressing wild-type murine type 2 ryanodine receptors (RyR2) (GenBank® accession number: AF295105) were grown to 95% confluence in a 75-cm2+ concentration within mitochondria ([Ca2+]mt) and in the cytosol ([Ca2+]i). Cells were then placed in a chamber for flowthrough perfusion and mounted on an inverted IX-71 microscope (Olympus). Cells were perfused with Hepes-buffered Hanks balanced salt solution (HBSS) composed of 140 mM NaCl, 5 mM KCl, 1.3 mM CaCl2, 0.4 mM MgSO4, 0.5 mM MgCl2, 0.4 mM KH2PO4, 0.6 mM NaHPO4, 3 mM NaHCO3, 10 mM glucose and 10 mM Hepes, pH 7.4 with NaOH (310 mOsm/kg with sucrose). Fluorescence was excited sequentially at 340 nm (12 nm bandpass) 380 nm (12 nm bandpass), and 550 nm (12 nm bandpass) using a Polychrome V monochromator (TILL Photonics) and focused on the cells via a ×40/1.35 NA oil-immersion objective (Olympus). Fluorescence emission was separated from excitation by using a dual fluorophore beamsplitter FF493/574-Di01 (Semrock) and collected with a dual band emission filter FF01-512/630 (Semrock) using an IMAGO charge-coupled device (CCD) camera . 2×2 binning was used for acquisition (1 pixel ~500 nm). A series of 340 nm, 380 nm and 550 nm images was acquired every 2 s. [Ca2+]i was calculated from the fluorescence ratio (R=F340/F380) using the formula [Ca2+]i=Kdβ(R−Rmin)/(Rmax−R), where Kd is dissociation constant and β is the scaling factor. Rmin, Rmax and β were calculated by application of 10 μM ionomycin in either Ca2+-free buffer (1 mM EGTA) or in HBSS (1.3 mM Ca2+). [Ca2+]mt changes were quantified as ΔF/F0=(F−F0)/F0×100% where F is the fluorescence intensity at each time point (λex=550 nm) and F0 is the fluorescence intensity in the resting cell before experimental manipulations. [Ca2+]i data were analysed using TILLvisION (TILL Photonics) software. To stimulate neurons by depolarization, the extracellular KCl concentration was elevated by replacing equimolar amounts of extracellular NaCl with KCl.Primary dorsal root ganglion (DRG) neuron cultures were prepared from adult C57BL/6J mice (8–12 weeks of age) and transfected with cDNA plasmids using an Amaxa nucleofection system (Lonza) as described previously ,28. Prim2. Prior to imaging, culture medium was changed to HBSS with 25 mM Hepes. Widefield imaging was performed on an inverted Nikon Eclipse Ti microscope equipped with a 200 W metal halide lamp (PRIOR Lumen), ×20/0.45 and ×60/1.40 NA objectives (Nikon), and a 16-bit QuantEM 512SC EMCCD camera (Photometrics). A filter set of 470/40 nm (excitation), 525/50 nm (emission), and 495 nm (dichroic) was used for Cyto-GCaMP3. Another filter set of 545/30 nm (excitation), 620/60 nm (emission) and 570 nm (dichroic) was used for ER-LAR-GECO1. For time-lapse imaging, HeLa cells were treated with 100 μM histamine.For dual-colour imaging of ER-LAR-GECO1 and Cyto-GCaMP3, HeLa cells  were cultured on collagen-coated 35-mm glass-bottomed dishes (Matsunami) until they reached 40–60% confluency. Transfection was performed by incubating HeLa cells with a mixture of 1 μg of plasmid DNA and 3 μl of Lipofectamine 2000 (Life Technologies) for 2 h. After incubation, the medium was changed to DMEM supplemented with FBS (10%), GlutaMAX™ , penicillin-G potassium salt (50 units/ml) and streptomycin sulfate (50 μg/ml). The cells were incubated for 24 h at 37°C in air containing 5% CO2+-free HBS. Cells were illuminated with 488 nm (for Cyto-G-GECO1.1) or 561 nm (for ER-LAR-GECO1) diode lasers (Olympus), using U-MNIBA  and LF561A  filters, respectively. With the filters used there was no significant cross-talk between green and red channels. Images were acquired with an iXon 897 EMCCD camera (Andor Technology) and processed using cellR software (Olympus). All records were corrected for background fluorescence determined from regions outside cells.For characterizing ER-LAR-GECO1 with Cyto-G-GECO1.1 in cultured human cells, cells were seeded on fibronectin-coated glass-bottomed culture dishes and transiently transfected after 24 h with plasmids using Transit-LT1 (Mirus Bio). Cells were imaged 24–48 h after transfection using an Olympus IX81 microscope with a ×60/1.45 NA objective, at 20°C in HBS or nominally Ca2+ sensors with decreased affinity, we initially considered two single RFP-based Ca2+ indicators, R-GECO1 (Kd=0.48 μM) mt during neuronal activity mt and the cytosolic Ca2+ concentration [Ca2+]i, respectively. The results showed that depolarization evoked coincident and rapidly reversible increases in [Ca2+]mt and [Ca2+]i , slowed the recovery of the increase in [Ca2+]mt, and this effect was reversible . In most cells, the fluorescence intensity of ER-LAR-GECO1 declined transiently and then recovered, mirroring the time course of the cytosolic Ca2+ signals reported by Cyto-G-GECO1.1 . The recovery of the fluorescence intensity of ER-LAR-GECO1 was observed in nominally Ca2+-free medium  and it was inhibited by thapsigargin . These observations are consistent with re-uptake of Ca2+ from the cytosol occurring via the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA).To explore the possibility of using LAR-GECO1 and a green fluorescent indicator for dual-colour imaging of Ca, GCaMP3  (hencefo-GECO1.1  (hencefo2+ indicators, LAR-GECO1 and LAR-GECO1.2, with Kd values for binding to Ca2+ of 24 μM and 12 μM respectively. These indicators enable robust detection of Ca2+ dynamics in organelles containing relatively high Ca2+ concentrations, as we demonstrate for the mitochondria in primary neurons and the ER in cultured human cells. Furthermore, comparisons of LAR-GECO1 with other FP-based low-affinity Ca2+ indicators demonstrate that LAR-GECO1 is a sensitive single-wavelength Ca2+ indicator for detecting Ca2+ dynamics within the ER of mammalian cells. In addition, LAR-GECO1 enables simultaneous dual-colour imaging with GFP-based fluorescent probes.By utilizing rational design and directed evolution, we have developed two low-affinity red fluorescent Ca


In the title compound, the tautomer present in the solid state is that in which the immediately exocyclic N atom bears the H atom. The central five-membered ring is planar, but both its N atoms are significantly pyramidalized. 14H15N5OS, the tautomer present in the solid state is that in which the immediately exocyclic N atom bears the H atom. The central five-membered ring is almost planar (r.m.s. deviation = 0.025 Å), but both its N atoms are significantly pyramidalized. A classical hydrogen bond from the N—H group to the cyanide N atom forms inversion-symmetric dimers, which are further linked by C—H⋯O interactions.In the title compound, C The title compound (1) was synthesized by the condensation of 4-aminoantipyrine and N-cyanoimido-S,S-dimethyldithiocarbonate in a simple one-step reaction. Compound (1) can exist in two tautomeric forms: (1a) and (1b). The 1H and 13C NMR spectra cannot differentiate between the two structures. The X-ray structure determination was undertaken to establish the exact nature of the product.The pyrazolone 4-amino-2,3-dimethyl-1-phenyl-3-pyrazolin-5-one (‘4-aminoantipyrine’) and its derivatives represent some of the most important compounds used as analgesic, antipyretic and anti-inflammatory drugs (Santos b) in the solid state. The central five-membered ring is effectively planar (r.m.s. deviation 0.025 Å), but both its nitrogen atoms are significantly pyramidalized, with C6 lying 0.635 (2) and C11 0.271 (2) Å out of the plane in opposite directions. The imidothiocarbamate group is also roughly planar (r.m.s.d. 0.05 Å) and almost perpendicular to the central ring [interplanar angle 83.38 (3)°]; the interplanar phenyl/dihydropyrazole angle is 44.82 (5)°.The molecule of (1) is shown in Fig. 1a-axis direction by a pair of weak C—H⋯O hydrogen bonds to the same acceptor atom (C6—H6C⋯O1 and C16—H16⋯O1), forming a layer structure parallel to the ab plane  in ethanol (20 ml) was added to a solution of 4-aminoantipyrine (0.01 mol) in ethanol (30 ml) containing catalytic amounts of piperidine (0.5 ml). The reaction mixture was heated at reflux for 30 min and then evaporated under reduced pressure. The yellow solid product was collected by filtration and recrystallized from ethanol, yield 85%, m.p. 489–491 K.A solution of Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989014027601/pk2542sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989014027601/pk2542Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989014027601/pk2542Isup3.cmlSupporting information file. DOI: 1040102CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Herein, we developed a 64Cu-1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA)-(AF)SAv/biotin-PEG-RGD2 was prepared by formation of a complex comprising DOTA-(AF)SAv and biotin-PEG-RGD2, followed by radiolabeling with 64Cu. Receptor binding studies of DOTA-(AF)SAv/biotin-PEG-RGD2 were performed using U87MG cells and 125I-RGDyK as the radioligand, and cellular uptake studies of 64Cu-DOTA-(AF)SAv/biotin-PEG-RGD2 were also performed. MicroPET imaging followed by optical imaging of U87MG tumor-bearing mice was acquired after injection of the hybrid probe, and region of interest (ROI) analysis of tumors was performed. Ex vivo PET/optical imaging and biodistribution studies of the major tissues were performed after the in vivo imaging, and immunofluorescence staining of the tumor tissue sections was carried out.64Cu-DOTA-(AF)SAv/biotin-PEG-RGD2 was prepared in 52.1 ± 5.4 % radiochemical yield and with specific activity of 1.0 ± 0.1 GBq/mg. Receptor binding studies showed that DOTA-(AF)SAv/biotin-PEG-RGD2 had higher binding affinity for integrin αVβ3 than RGD2, reflecting a possible polyvalency effect. Moreover, the hybrid probe revealed time-dependent uptake by U87MG cells. In a microPET/optical imaging study, the hybrid probe demonstrated high accumulation in tumors; ROI analysis revealed 2.7 ± 0.2 % ID/g at 1 h and 4.7 ± 0.2 % ID/g at 21 h after injection, and subsequently acquired optical images showed tumors with strong fluorescence intensity. Ex vivo PET/optical images of the major tissues confirmed the in vivo imaging data, and biodistribution studies demonstrated high and specific uptake in tumors (4.8 ± 0.1 % ID/g). Immunofluorescence staining showed the formation of new blood vessels in tumor tissues, suggesting that the tumor uptake was due to specific binding of the hybrid probe to integrin αVβ3 expressed on tumor cells.64Cu-DOTA-(AF)SAv/biotin-PEG-RGD2 is able to provide quantitative information on hybrid PET/optical imaging of integrin αVβ3 expression.These results indicate that a  Demand for hybrid imaging probes has been growing with advances in hybrid imaging systems such as PET/CT, SPECT/CT, and PET/MRI, and expansion of their clinical uses . PET proVβ3, have been radiolabeled with various radioisotopes. The most well-characterized ligand, 18F-labeled galacto-RGD peptide, has shown promise for tumor imaging of melanoma M21 tumor-bearing mice and diagnosis of cancer patients 2 was purchased from Peptide International , biotin-PEG(3400)-NHS ester was from Nanocs , (AF)SAv was from Life Technologies , and DOTA-NHS ester was from Macrocyclics . 64CuCl2 was kindly provided by KIRAMS . PD-10 columns were purchased from Amersham Biosciences , Amicon filters were from Millipore , and spin columns were from Thermo Scientific . Chelex 100 resin (50–100 mesh) and other chemicals were purchased from Sigma-Aldrich , and BCA protein assay kits were from Pierce . All buffers used for synthesis and radiolabeling were pretreated with Chelex 100 resin to ensure that they were metal-free. Matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry was performed on a Voyager-DETM STR Biospectrometry Workstation . Purification and analysis of products were performed by HPLC , and eluates were monitored using a UV detector (218 nm).RGDRadioactivity was measured using a dose calibrator , and tissue radioactivity was counted using an automatic gamma counter . MicroPET and optical images were acquired at the Center for Molecular and Cellular Imaging, Samsung Biomedical Research Institute  using an Inveon microPET/CT scanner  and a Xenogen IVIS Spectrum , respectively.(AF)SAv (1.3 mg) and 20 equivalents of DOTA-NHS ester  were dissolved in 800 μL of 0.1 M sodium carbonate buffer (pH 8.5), which was then stirred at room temperature for 18 h in the dark. At the end of the reaction, the reaction mixture was purified using a PD-10 column, concentrated using an Amicon filter (cut-off: 10 kDa), and then lyophilized. DOTA-(AF)SAv was obtained at 97.3 % yield, quantified using a BCA protein assay kit, and then analyzed by MALDI-TOF mass spectrometry.2  and biotin-PEG(3400)-NHS ester  were dissolved in 500 μL of 0.1 M sodium carbonate buffer (pH 8.5), which was then stirred at room temperature for 19 h. At the end of the reaction, the reaction mixture was purified by HPLC using a C18 column  with sequential programs using a mixture of 0.1 % trifluoroacetic acid (TFA) in water and CH3CN. The first isocratic program was a 90:10 mixture over 10 min, and the second gradient program was from a 90:10 mixture to a 35:65 mixture over 30 min. The flow rate was 3 mL/min. After lyophilization, biotin-PEG-RGD2 was analyzed by MALDI-TOF mass spectrometry.RGD2  dissolved in acetate buffer (pH 6) was added to DOTA-(AF)SAv  in the same buffer. The reaction mixture  was stirred at room temperature for 1 h, and then incubated with 64CuCl2 (377 MBq) at 40 °C for 30 min with constant shaking. Unreacted 64CuCl2 and biotin-PEG-RGD2 in the reaction mixture were removed by centrifugation using a spin column (molecular weight (mw) cut-off 7 kDa).Biotin-PEG-RGD2 was purified using a spin column (cut-off 7 kDa) and its purity was determined by HPLC using a Superdex™ 75 GL column  and 0.01 M phosphate buffer as the eluent at a flow rate of 0.7 mL/min.In a separate experiment, an aliquot of DOTA-(AF)SAv/biotin-PEG-RGD64Cu-DOTA-(AF)SAv/biotin-PEG-RGD2 (98.4 MBq) in 0.01 M PBS (pH 7.4) was added to 50 % fetal bovine serum  and incubated at 37 °C for 0, 1, 3, 16, and 24 h. At the indicated time points, each sample was loaded onto a PD-10 column, and fractions were eluted using PBS (pH 7.4) and counted using a dose calibrator.Vβ3 receptor binding study was performed using a previously reported procedure with a slight modification + calcd. 5023.77, C225H413N22O98S; found 5024.57.DOTA-(AF)SAv prepared from (AF)SAv and DOTA-NHS ester was readily purified using a size-exclusion column. MALDI-TOF mass spectrometric analysis indicated that the average number of DOTA molecules conjugated to (AF)SAv was 3.8 ± 0.6. In contrast, to prepare biotin-PEG-RGD2 complex after spin column purification was confirmed by HPLC using a Superdex™ 75 GL column SAv/biotin-PEG-RGD2 purification using a PD-10 column, the hybrid probe was prepared in a two-step reaction and finally purified; DOTA-(AF)SAv and biotin-PEG-RGD2 were mixed at a ratio of 1 to 5, which was then labeled with 64Cu, and finally the hybrid probe was purified using a spin column. Non-decay-corrected radiochemical yield of the 64Cu-DOTA-(AF)SAv/biotin-PEG-RGD2 was 52.1 ± 5.4 % and its specific activity was 1.0 ± 0.1 GBq/mg.Purity of a DOTA-(AF)SAv/biotin-PEG-RGDumn Fig. , b. To r64Cu-DOTA-(AF)SAv/biotin-PEG-RGD2 was measured by incubating the hybrid probe in 50 % serum for 24 h. The results demonstrated that 64Cu remained stable on the hybrid probe after the 24-h incubation, showing 96.4 % radioactivity at 1 h, 97.8 % at 3 h, 98.1 % at 16 h, and 97.6 % at 24 h relative to a value of 100 % radioactivity at 0 h SAv/biotin-PEG-RGD2 and RGD2 were 12.1 ± 4.2 nM and 37.5 ± 18.3 nM, respectively, indicating that the former had higher binding affinity for integrin αVβ3 than RGD2 SAv/biotin-PEG-RGD64Cu-DOTA-(AF)SAv/biotin-PEG-RGD2 increased in a time-dependent manner from 100.0 % at 1 h to 298.5 % at 21 h , indicating specific binding of the hybrid probe to the integrin αVβ3 expressed on tumor cells SAv/biotin-PEG-RGD2 in the liver, spleen, and tumor  at 16 h after injection  × 108 photons/s/cm2/sr at 2 h, (6.0 ± 0.2) × 108 photons/s/cm2/sr at 16 h, and (6.7 ± 0.3) × 108 photons/s/cm2/sr at 21 h. In the presence of RGD2, signal intensity decreased to 4.9 ± 0.2 × 108 photons/s/cm2/sr at 21 h  × 107 photons/s/cm2/sr in the spleen, and (3.0 ± 0.2) × 107 photons/s/cm2/sr in the tumor SAv/biotin-PEG-RGD2 for integrin αVβ3.The biodistribution study performed after ex vivo imaging confirmed the in vivo and ex vivo microPET/optical imaging data; 30.6 ± 1.7 % ID/g in the liver, 26.2 ± 1.1 % ID/g in the spleen, and 4.8 ± 0.1 % ID/g in the tumor SAv/biotin-PEG-RGD2 confirmed expression of β3 (green) and VEGFR2 (red), and the fluorescence signals from β3 overlapped with those of VEGFR2 . Different methods were applied to purify these two molecules due to differences in their molecular weights from reactants; DOTA-(AF)SAv was purified using a PD-10 column, in which the unconjugated DOTA was easily removed because of its low molecular weight (mw 501.49), whereas biotin-PEG-RGD2 was purified using a centrifugal filter to remove unreacted RGD2 peptide (mw 1350.43), followed by a reverse-phase HPLC column because of the small difference in molecular weight between biotin-PEG-NHS ester (mw 3790.48) and biotin-PEG-RGD2 (mw 5025.83). As a result, there were no molecular ion peaks corresponding to both biotin-PEG-NHS ester and RGD2 detected on MALDI-TOF mass spectrometry, except for the molecular ion peak of biotin-PEG-RGD2. The key to preparing a SAv/biotin complex is to remove unbound biotin-PEG-RGD2 after the complex formation. In this study, we used a spin column (mw cut-off 7 kDa) to rapidly remove molecules with a molecular weight less than 7 kDa from the 64Cu-DOTA-(AF)SAv/biotin-PEG-RGD2.DOTA-(AF)SAv and biotin-PEG-RGD121 and 64Cu-DOTA-(AF)SAv/biotin-PEG-VEGF121 were shown to have potential for NIRF and PET/optical imaging of VEGF receptor expression, respectively 2 in U87MG tumor-bearing mice (<4 % ID/g at 4 h after injection) [64Cu-DOTA-E[(RGDyK)]2 showed better in vivo kinetics than the D-Phe derivative in MDA-MB-435 tumor-bearing mice 2 2 . This hiat 22 h) . A similanalysis . 64Cu-laarticles . Similar peptide . The lon peptide . Therefoime Fig. . While tmor Fig. , d.3 receptors were highly expressed on endothelial cells (VEGFR2) of blood vessels SAv/biotin-PEG-RGD2 in U87MG tumor-bearing mice were in good agreement for monitoring integrin αVβ3 expression. This hybrid probe provided real-time detection of tumor as well as quantitative information on tumor and major organs with high specificity and sensitivity. In addition, this hybrid probe may have advantages, because hybrid PET/optical probes may allow for preoperative detection, intraoperative image-guided surgery, and postoperative evaluation of tumors.Our study showed that both microPET and optical imaging data of 64Cu-DOTA-(AF)SAv/biotin-PEG-RGD2 efficiently using a SAv/biotin complex as a platform. As shown by a receptor binding study, DOTA-(AF)SAv/biotin-PEG-RGD2 exhibited higher binding affinity for the integrin αVβ3 than RGD2 peptide, which we attributed to a possible polyvalency effect by four flexible biotin-PEG-RGD2 arms complexed with one SAv core. PET/optical images of U87MG tumor-bearing mice indicated that the hybrid probe accumulated in tumor with prolonged retention over 21 h. These results demonstrate that 64Cu-DOTA-(AF)SAv/biotin-PEG-RGD2 provides specific and sensitive hybrid PET/optical images for monitoring integrin αVβ3 expression, and that a SAv/biotin complex is a useful platform for developing hybrid imaging probes.We prepared a


The β-secretase enzyme, β-site amyloid precursor protein-cleavingenzyme 1 (BACE1), cleaves amyloid precursor protein (APP) in the first step inβ-amyloid (Aβ) peptide production. Thus, BACE1 is a key target for candidatedisease-modifying treatment of Alzheimer’s disease. In a previous exploratory Aβbiomarker study, we found that BACE1 inhibitor treatment resulted in decreasedlevels of Aβ1-34 together with increased Aβ5-40, suggesting that these Aβ speciesmay be novel pharmacodynamic biomarkers in clinical trials. We have now examinedwhether the same holds true in humans.n =18) were randomly assignedto receive a single dose of 30 mg of LY2811376 (n =6), 90 mg of LY2811376 (n =6),or placebo (n =6). We used hybridimmunoaffinity-mass spectrometry (HI-MS) and enzyme-linked immunosorbent assays tomonitor a variety of Aβ peptides.In an investigator-blind, placebo-controlled and randomized study,healthy subjects  Aβ1-34, Aβ5-40 and Aβ5-X after treatment with the BACE1-inhibitor LY2811376.Aβ5-40 and Aβ5-X increased dose-dependently, as reflected by two independentmethods, while Aβ1-34 dose-dependently decreased.Using HI-MS for the first time in a study where subjects have beentreated with a BACE inhibitor, we confirm that CSF Aβ1-34 may be useful inclinical trials on BACE1 inhibitors to monitor target engagement. Since it is lesshydrophobic than longer Aβ species, it is less susceptible to preanalyticalconfounding factors and may thus be a more stable marker. By independentmeasurement techniques, we also show that BACE1 inhibition in humans is associatedwith APP-processing into N-terminally truncated Aβ peptides via aBACE1-independent pathway.NCT00838084. Registered: First received: January 23, 2009, Last updated: July14, 2009, Last verified: July 2009.ClinicalTrials.gov  Alzheimer’s disease (AD) is a slowly progressing brain diseasemanifesting several neuropathological characteristics including accumulation ofextracellular plaques, mainly composed of amyloid-β (Aβ) peptides of various lengths,2. Aβ isTo evaluate the biochemical effects of novel BACE1 inhibitorcandidates, biomarkers that reflect target engagement are needed . AnalyziHere, for the first time with a peptidomics approach, we havedemonstrated changes in CSF levels of Aβ1-34 and Aβ5-40 in humans treated with theBACE1 inihibitor LY2811376 . Thetranslation of these findings from preclinical models to man indicates that CSFAβ1-34 and Aβ5-40 have potential utility as markers of BACE1 inhibition in clinicalresearch. Furthermore, the results strongly suggest that Aβ peptides starting atamino acid 5 are produced through a non BACE1-dependent pathway in humans.The study, conducted at PAREXEL International Early Phase LosAngeles, CA, USA, from February to June 2009, was previously reported in detail. In brieImmunoaffinity capture of Aβ species was combined withmatrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS foranalyzing a variety of Aβ peptides in a single analysis as described in detailelsewhere . In brieSH-SY5Y cells obtained5-40 andAβ5-x using ELISA, microtiter plates were coated with10 μg/mL 2G3 [x-40; epitope including valine at position 40,Eli Lilly & Company, Indianapolis, IN, USA) or 266 [1-x; epitope13-28, Eli Lilly & Company) overnight at 4°C. After blocking plates in 2%bovine serum albumin (BSA), dilutions of Aβ5-40 standards(Anaspec) and CSF samples were incubated on plates in 1% BSA, 0.55 Mguanidine-HCL, 5 mM Tris in phosphate buffered saline (PBS) with completeethylenediaminetetraacetic acid (EDTA)-free protease inhibitor  overnight at 4°C. After washing in PBS-0.05% Tween 20, biotinylated 5H5 was used to detect the truncated Aβ beginning at thearginine at position 5. The 5H5 monoclonal antibody was developed in micefollowing standard methods and the specificity for the truncatedAβ5-x was investigated by acid urea gel (a technique thatseparates Aβ peptides by mass and charge) and ELISA methods. Acid urea gelseparation of synthetic Aβ peptides followed by Western blotting with 5H5 revealedcomplete selectivity for the truncated Aβ5-42 as comparedto full-length Aβ1-42. Additionally, acid urea gel/5H5Western blotting analysis of human cortical tissue from multiple Alzheimer’ssubjects resulted in a single identifiable band that co-migrated at the sameposition as the synthetic Aβ5-42 standard. Note, themigration of the Aβ peptides in this gel system completely separates theAβ5-42 from all other Aβ peptides (truncated orfull-length). ELISA analyses to investigate the 5H5 epitope selectivitydemonstrated a 20,000-fold selectivity for the Aβ5-xepitope versus the full-length peptide (Aβ1-x). Followingadditional washes in PBS-0.05% Tween 20, plates were incubated withstreptavidin-horseradish peroxidase (HRP)  andsubsequently, 3,3´,5,5´-Tetramethylbenzzidine (TMB) color development was monitored at 650 nm in a spectrophotometer.For quantification of Aβg/mL 2G3 (anti-Aβ) or 266  .The time series for each treatment were analyzed using Friedman’stest . A dose-dependent effect was consideredsignificant if As expected, SH-SY5Y cells treated with the BACE1-inhibitorLY2811376 or BACE IV secreted less Aβ1-40 and Aβ1-42 to the cell medium while therelative levels of Aβ5-40 (relative to the other Aβ peptides detected) increased,as compared to vehicle-treated cells  and 57% in the 90-mg group (P <0.001) respectively, 24 hours after drugadministration  and the90-mg (P <0.001) groups, respectively. The ELISA-determinedconcentrations of Aβ5-42 were too low to yield an accurate assessment, which is inagreement with the mass spectrometric data where Aβ5-42 could not be detected inany treatment group.The increase in Aβ5-40 detected by mass spectrometry in response totreatment with the BACE1 inhibitor LY2811376 was further confirmed by aproprietary ELISA. While the placebo concentrations were low, in the range ofapproximately 100 pg/mL and approximately 50 pg/mL for Aβ5-X and Aβ5-40,respectively, there were clear increases in the LY2811376 high dose (90 mg) groupover time for both Aβ5-X and Aβ5-40  while Aβ5-X was negatively correlated with sAPPβ as presented inFigure In the 90-mg dose group, there was a compensatory increase in theconcentrations of both Aβ5-X and sAPPα rs =0.94, , P =0.02In the present study, we show marked effects on CSF Aβ5-40 (whichincreases) and Aβ1-34 (which decreases) in response to BACE1 inhibitor treatment.These findings confirm earlier pre-clinical data  and suggSince the discovery and molecular cloning of BACE1 in 1999 by severalindependent groups, this enzyme has been a tempting target for pharmacologicallowering of cerebral Aβ levels with the intent of treating or preventing AD. Todate, there are only a few reports of BACE1 inhibitors that have demonstratedsufficient access to the brain. In a recent paper, oral administration of thenon-peptidic BACE1 inhibitor LY2811376 to healthy subjects (same patients asincluded in the present study) dose-dependently lowered CSF Aβ1-40, Aβ1-42 and sAPPβlevels and dose-dependently increased CSF sAPPα, providing evidence of desirablecentral pharmacodynamic effects on APP processing . In anotin vitro,data from the CNS showing which enzyme that cleaves between Phe4 and Arg5 inhibitionof BACE1 is lacking.LY2811376 treatment consistently increased CSF levels of Aβ5-40. Theincrease of Aβ5-40 in response to BACE1 inhibition clearly suggests that productionof Aβ peptides starting at position 5 is formed via a BACE1-independentAPP-processing pathway . In agreRecently, we showed in pre-clinical models that CSF Aβ1-34 is asensitive marker for BACE1 inhibition . We haveAβ5-40 has been found in AD brains , but theWe found a positive correlation between sAPPα and Aβ5-X. Thiscorrelation may reflect a compensatory increase in APP cleavage at the α-site andbetween amino acid 4/5, or that there might be more substrate for these enzymes dueto inhibition of BACE. We also found a negative correlation between sAPPβ and Aβ5-X,clearly showing that while the amyloidogenic pathway is affected, the (as yet)unknown enzyme generating Aβ5-X cleaves its substrate more.There are several non-quantitative aspects of HI-MS. The relativequantification using mass spectrometry cannot be interpreted as a direct reflectionof an absolute or relative abundance. However, in the present study we have verifiedthe mass spectrometric data showing increased relative levels of Aβ5-40 with aproprietary ELISA showing increased concentrations of both Aβ5-40 and Aβ5-X inresponse to inhibition of BACE1. What also should be noted is that the ELISAmeasures an absolute concentration while MS reports the relative change of Aβ5-40relative to all other Aβ peptides detected in the same spectra. A previous study onthe same patients as those included in the present study showed a marked decrease inCSF Aβ1-40 in response to LY2811376 treatment . Due to In summary, our results confirm that CSF Aβ1-34 may be useful inclinical trials on BACE1 inhibitors to monitor target engagement. By independentmeasurement techniques, we show that BACE1 inhibition in humans is associated withAPP-processing into N-terminally truncated Aβ peptides via a BACE1-independentpathway. The data presented also provide evidence for CSF Aβ1-34 and Aβ5-40 astranslatable pharmacodynamic markers for BACE1-inhibition from cell and animalmodels to humans.


The crystal of the isomorphous anhydrous ammonium salts of phenoxyacetic acid and (4-fluorophenoxy)acetic acid and that of the hemihydrate ammonium salt of 4-chloro-2-methylphenoxy)acetic acid show two-dimensional layered structures based on conjoined cyclic hydrogen-bonded motifs. 4+·C8H6O3−, (I), (4-fluorophenoxy)acetic acid, NH4+·C8H5FO3−, (II), and the herbicidally active (4-chloro-2-methylphenoxy)acetic acid (MCPA), NH4+·C9H8ClO3−·0.5H2O, (III) have been determined. All have two-dimensional layered structures based on inter-species ammonium N—H⋯O hydrogen-bonding associations, which give core substructures consisting primarily of conjoined cyclic motifs. The crystals of (I) and (II) are isomorphous with the core comprising R12(5), R12(4) and centrosymmetric R42(8) ring motifs, giving two-dimensional layers lying parallel to (100). In (III), the water molecule of solvation lies on a crystallographic twofold rotation axis and bridges two carboxyl O atoms in an R44(12) hydrogen-bonded motif, creating two R43(10) rings, which together with a conjoined centrosymmetric R42(8) ring incorporating both ammonium cations, generate two-dimensional layers lying parallel to (100). No π–π ring associations are present in any of the structures.The structures of the ammonium salts of phenoxyacetic acid, NH The crystal packing and hydrogen-bonding in (I)In the crystals of (I)iii, extending the structure down b   or carboxyl-phenoxy  acceptors such as are present in (I)et al., 2009a = 37.338 (8), b = 4.388 (9), c = 12.900 (3) Å, β = 103.82 (3)°, V = 2074.7 (8) Å3, Z = 8, space group C2/c].In the crystal of (III)b Table 3. The fou0) Fig. 6. In (IIIb unit-cell parameter)].No π–π interactions are found in any of the structures reported here [minimum ring centroid separation = 4.8849 (16) (I)M aqueous ammonia solution to 1 mmol of either phenoxyacetic acid [150 mg for (I)], (4-fluorophenoxy)acetic acid [170 mg for (II)] or (4-chloro-2-methylphenoxy)acetic acid [200 mg for (III)] in 10 mL of 10% ethanol–water. Room-temperature evaporation of the solvent gave colourless plate-like crystals of (I)The title compounds were prepared by the addition of excess 5 Uiso(H) = 1.2Ueq(N) or = 1.5Ueq(O). Other H atoms were included at calculated positions  and also treated as riding, with Uiso(H) = 1.5Ueq(C) for methyl H atoms and = 1.2Ueq(C) for other H atoms. In (III)Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S160053681402488X/su5018sup1.cifCrystal structure: contains datablock(s) global, I, II, III. DOI: 10.1107/S160053681402488X/su5018Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S160053681402488X/su5018IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S160053681402488X/su5018IIIsup4.hklStructure factors: contains datablock(s) III. DOI: Click here for additional data file.10.1107/S160053681402488X/su5018Isup5.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S160053681402488X/su5018IIsup6.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S160053681402488X/su5018IIIsup7.cmlSupporting information file. DOI: 1033945, 1033946, 1033947CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The molecular structure of a non-symmetric structure based on a tetrasubstituted ethylenediamine backbone consists of three hydroxybenzyl groups and one 2-hydroxy-5-methylbenzaldehyde group bonded to the N atoms of the diamine unit. The ethylenediamine skeleton shows a regular extended conformation, while the phenol arms are randomly oriented but governed by hydrogen bonds. 32H34N2O5, is based on a tetrasubstituted ethylenediamine backbone. The molecular structure consists of three hydroxybenzyl groups and one 2-hydroxy-5-methylbenzaldehyde group bonded to the N atoms of the diamine unit. The ethylenediamine skeleton shows a regular extended conformation, while the spatial orientation of the phenol arms is governed by hydrogen bonds. In the 2-hydroxy-5-methylbenzaldehyde group, an intramolecular S(6) O—H⋯O hydrogen bond is observed between the alcohol and aldehyde functions, and the neighbouring phenol arm participates in an intramolecular S(6) O—H⋯N hydrogen bond. The third phenol group is involved in a bifurcated intramolecular hydrogen bond with graph-set notation S(6) for O—H⋯N and O—H⋯O intramolecular hydrogen bonds between neighbouring amine and phenol arms, respectively. Finally, the fourth phenol group acts as an acceptor in a bifurcated intramolecular hydrogen bond and also acts as donor in an intermolecular hydrogen bond, which connects inversion-related molecules into dimers with R44(8) ring motifs.The non-symmetric title molecule, C The appds Fig. 3. No π–π et al., 2009et al., 2011aet al., 2011bpara and ortho positions with respect to the O–H function. As observed in (I)A search for similar structures in the current version of the Cambridge Structural Database -1,2-diaminoethane -1,2-diaminoethane  in tetrahydrofuran (40 ml) containing triethylamine . The reaction was kept cooled during addition time, and the resulting solution stirred for 24 h. Yellow mixture oil/solid was obtained after evaporation of the solvent. A solution of this mixture in CH2Cl2 (50 ml) was washed with a saturated solution of NaHCO3 (3 × 50 ml) and filtered off in the presence of NaSO4. The solvent was removed, and a straw-yellow solid was obtained. This solid was refluxed in n-hexane/CHCl3 . After cooling the solid was filtered off, washed with n-hexane (80 ml), dried and recrystallized from an ethyl acetate solution to afford 3-[({2-[bis(2-hydroxybenzyl)amino]ethyl}(2-hydroxybenzyl)amino)methyl]-2-hydroxy-5-methylbenzaldehyde, (I)The title compound was obtained from a nucleophilic substitution reaction between −1 in the IR spectrum, which is typical for stretching vibrations ν(C=O) of free aldehyde. In the 1H NMR spectrum, the signal at 9.81 p.p.m. related to one aldehyde proton is further evidence for product formation. Yield 90%, m.p. 444.8–445.4 K. IR : ν(O—H) 3273, ν 3042–2718, ν(C=O)1655, ν(C=C) 1615–1457, δ(O—H) 1365, δ(C—O) 1252, δ(C—Har) 757; 1H NMR  : 2.29 , 2.78 , 3.58 , 3.61–3.77 , 6.69–6.87 , 6.91 , 6.99 , 7.07–7.19 , 7.24 , 9.81 ; 13C NMR : 20.0, 48.6, 48.8, 53.5, 54.3, 115.2, 121.7, 122.7, 123.2, 124.5, 127.6, 128.4, 128.7, 129.8, 130.8, 136.7, 156.2, 156.5, 158.7, 191.6. Negative HPLC/ESI–MS (m/z): [M−H] calculated for C32H35N2O5−, 527.25; found, 527.19. Colourless blocks were grown by slow evaporation of the solvent from a saturated solution of (I)The formation of (I)Ar), 0.99 (CH2) or 0.98 Å (CH3) with Uiso = 1.2Ueq(C) or 1.5Ueq(Cmethyl). The hydrogen atoms of the alcohol groups were located from a Fourier difference map and treated with a riding-model approximation with Uiso(H) = 1.5Ueq(O).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S1600536814024465/lh5739sup1.cifCrystal structure: contains datablock(s) general, I. DOI: 10.1107/S1600536814024465/lh5739Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536814024465/lh5739Isup3.molSupporting information file. DOI: Click here for additional data file.10.1107/S1600536814024465/lh5739Isup4.cmlSupporting information file. DOI: 1033129CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


A combination of O—H⋯N hydrogen bonds and C—Cl⋯π(pyridyl) interactions links the molecules of the title compounds into (100) sheets. 12H10ClNO, the dihedral angle between the benzene and pyridine rings is 74.34 (6)°. In the crystal, the molecules are linked by O—H⋯N hydrogen bonds, forming zigzag C(5) [001] chains in which alternating R- and S-configuration molecules are related by c-glide symmetry. In addition, inversion-related pairs of molecules are linked into dimers by pairs of weak C—Cl⋯π(pyridyl) interactions, which link the hydrogen-bonded chains into (100) sheets. Structural comparisons are drawn with a number of related compounds.In the title racemic compound, C RPh2COH, exhibit a very rich diversity of supramolecular arrangements, including isolated molecules, hydrogen-bonded dimers, trimers, tetramers and hexamers, as well as continuous hydrogen-bonded chains -4-chlorophenyl(pyridin-2-yl)methanol (I)et al., 2015Simply substituted diphenylmethanols, R-configuration at atom C1. The centrosymmetric space group confirms that compound (I)The molecules of compound (I)R-enantiomer of (II)  direction. The chain containing the reference molecule at  consists of molecules which are related by the c-glide plane at y = R-configuration and S-configuration alternate along the chain in Fig. 2. Two chaCgi = 3.5280 (10) Å, C⋯Cgi = 5.1785 (19) Å and C—Cl⋯Cgi = 157.79 (7)°  where Cg represents the centroid of the pyridine ring. This Cl⋯Cg distance is slightly shorter than the average distance, 3.6 Å, deduced c-glide plane at y = y = −y = The overall effect of the C—Cl⋯π interaction in (I)0) Fig. 4, but theIt is of interest briefly to compare the supramolecular assembly in compound (I)C(5) chains by O—H⋯N hydrogen bonds R-configuration or only from molecules having the S-configuration: in (I)cf. Fig. 2The molecules of compound (II) are linked into C(5) chains are formed in each of the three isomeric carborane derivatives (III)–(V)  type, seems plausible.Similar homochiral Z′ = 2 in space group P22121  symmetry , which differs from (I)A sample of the title compound (I)Uiso(H) = 1.2Ueq(C). For the hydroxyl H atom H1A, the atomic coordinates were refined with Uiso(H) = 1.5Ueq(O), giving an O—H distance of 0.84 (2) Å. The analysis of variance reported a large value of K, 3.187, for the group of 252 very weak reflections having Fc/Fc(max) in the range 0.000 < Fc/Fc(max) < 0.005.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989015023154/hb7553sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989015023154/hb7553Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989015023154/hb7553Isup3.cmlSupporting information file. DOI: 1440028CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Their structures were characterized based on extensive spectroscopic data. Compound 1 showed certain cytotoxicities against four human tumor cell lines.Three rare triterpenoids, saponaceolides Q–S (The online version of this article (doi:10.1007/s13659-015-0071-5) contains supplementary material, which is available to authorized users. Tricholoma terreum as a hitherto unknown poisonous European mushroom [–F and saponaceolides H–P have been isolated. Terreolides A–F possessed novel frameworks, while saponaceolides B and M were the main toxins in the mushroom. The structural diversity, as well as important bioactivity discovery, prompted us to make a further study on this mushroom. According to an investigation on chloroform extract of T. Terreum collected in Arcachon in southwestern France, three new triterpenoids, saponaceolides Q–S (1–3), have been obtained  indicated the molecular formula C30H46O6, requiring eight degrees of unsaturation. The IR absorption bands at 3436 and 1723 cm−1 suggested the presence of hydroxy and carbonyl groups, respectively. In the 1H NMR spectrum  revealed the existence of a double bond that might have an α,β-unsaturated keto moiety. The 13C NMR and DEPT spectra displayed thirty carbon resonances that was classified into five CH3, thirteen CH2, four CH, and eight C, as shown in Table 1 was that C-7 was identified as a methylene  rather than a carbonyl carbon in saponaceolide O, which was supported by 1H–1H COSY fragments of H-7 with δH 1.68  and 2.48 , as well as HMBC correlations from H-7 to δC 53.5  and 24.7    indicated the molecular formula C31H48O7, requiring eight degrees of unsaturation. The 1H and 13C NMR spectra displayed similar patterns to those of 1 except for the signals at δH 4.13  and 3.30  and δC 75.2  and 57.2 , suggesting the existence of a methoxy substituent. The HMBC correlation from δH 3.30  to C-7 indicated that a methoxy group placed at C-7, which was also supported by 1H–1H COSY correlations of H-7 with δH 1.60  and 2.01 . Detailed analyses of 2D NMR data suggested that other parts of 2 were the same to those of 1. The coupling constant of H-7  suggested an S configuration of C-7 referring to the data of the literature [2 was determined to be saponaceolide R.Compound 3 were almost the same to those of 2, which informed that 3 might have the same framework to that of 2. Detailed analyses of 2D NMR data suggested that 3 did possess the same planar structure to that of 2. However, tiny changes of NMR data of CH-6, CH-7, and CH2-8 suggested that the stereoconfiguration of C-7 was different from that of 2. The coupling constant of H-7  was also significantly different to those reported previously (less than 8 Hz) [3, that was also in agreement with those described in the literature [3 was established and named saponaceolide S.The 1D NMR Tables  and HRES1–3 were, therefore, evaluated for their cytotoxicities to five human cancer cell lines. As a result, compound 1 showed moderate activities as shown in Table Many triterpenoids in this type, such as saponaceolides B, E, and F, have been reported to possess cytotoxicities to several cancer cell lines –4. Compo2SO4 in EtOH, in combination with the Agilent 1200 series HPLC system .Optical rotations were measured on a Jasco-P-1020 polarimeter. IR spectra were obtained using a Bruker Tensor 27 FT-IR spectrometer with KBr pellets. NMR spectra were acquired with instrument of a Bruker DRX-600 with tetramethylsilane (TMS) used as an internal standard at room temperature. HRESIMS were recorded on an API QSTAR pulsar spectrometer. Silica gel (200–300 mesh), Sephadex LH-20 and RP-18 gel (20–45 µm) were used for column chromatography (CC). Fractions were monitored by thin layer chromatography and spots were visualized by heating silica gel plates immersed in HT. terreum, were collected from Arcachon in southwestern France in December 2012 and identified by Prof. Zhu-Liang Yang of Kunming Institute of Botany, Chinese Academy of Sciences. A specimen (No. KIB20121205.2) was deposited at the Kunming Institute of Botany, Chinese Academy of Sciences. For details of this mushroom please see that reported previously [Wild mushrooms, T. Terreum (3 kg) were extracted with chloroform (24 h × 3), and then partitioned with water (1:1). Finally, a chloroform extract (12 g) was obtained, which was submitted to silica gel CC using petroleum-acetone (from 1:0 to 0:1) to give six fractions (A–F). Fraction B (1.8 g) was separated by reverse-phased CC eluted with gradient mixture of MeOH and H2O  to afford five sub-fractions (B1–B5). Fraction B2 (32 mg) was purified by Sephadex LH-20 (MeOH) to give compound 1 (2.2 mg), while fraction B4 (57 mg) was also purified by Sephadex LH-20 (MeOH) to give compounds 2 (2.3 mg) and 3 (1.3 mg).The fresh fruiting bodies of c 0.12 MeOH); IR (KBr) νmax 3436, 2937, 1723, 1448, 1367, 1201, 1068, 991 cm−1; for 1H (600 MHz) and 13C NMR (150 MHz) data (CDCl3), see Tables m/z 525.3193 .Colorless oil, mentclass2pt{minimc 0.10 MeOH); IR (KBr) νmax 3443, 2926, 1726, 1457, 1381, 1065, 998 cm−1; for 1H (600 MHz) and 13C NMR (150 MHz) data (CDCl3), see Tables m/z 555.3290 .Colorless oil, mentclass2pt{minimc 0.11 MeOH); IR (KBr) νmax 3441, 2926, 1724, 1452, 1382, 1120, 997 cm−1; for 1H (600 MHz) and 13C NMR (150 MHz) data (CDCl3), see Tables m/z 555.3290 .Colorless oil, mentclass2pt{minim2 at 37 °C. The cytotoxicity assay was performed according to the MTT -2,5-diphenyl tetrazolium bromide) method in 96-well microplates [Human myeloid leukemia HL-60, hepatocellular carcinoma SMMC-7721, lung cancer A-549 cells, breast cancer MCF-7 and colon cancer SW480 cell lines were used in the cytoxic assay. All cell lines were cultured in RPMI-1640 or DMEM medium , supplemented with 10 % fetal bovine serum  in 5 % COSupplementary material 1 (PDF 8569 kb)


The supramolecular structure of the title 1:1 co-crystal consists of (100) sheets linked by O—H⋯N and N—H⋯O hydrogen bonds. 12H10N2·C8H7NO4, the bipyridine moiety shows whole-molecule disorder over two sets of sites in a 0.588 (3): 0.412 (3) ratio. In the crystal, the components form hydrogen-bonded sheets linked by N—H⋯O and O—H⋯N interactions, which stack along the a axis. A comparison to a related and previously published co-crystal of 5-amino-isophthalic acid and the shorter 4,4′-bipryidine is presented.In the title 1:1 co-crystal, C The occupancy of the major and minor components was refined to be 0.588 (3) and 0.412 (3), respectively.The 5AIA–BE co-crystal crystallizes with one molecule of 5AIA and one molecule of BE in the asymmetric unit Fig. 1. Both moa axis to produce a layered structure , a shorter analogue of BE, was reported (Zhang −5 mol) and 5AIA  were added to a 25 ml scintillation vial. To this was added approximately 15 ml of ethyl acetate followed by gentle heating. An additional 2 ml of methanol was added and all remaining solids dissolved. The loosely capped vial was then placed into a dark cabinet. After two weeks, yellow block-shaped crystals of the title compound suitable for single-crystal X-ray diffraction measurements were obtained.Solid BE  = 1.2Ueq(C). The BE was found to be disordered over two sets of sites in a 0.588 (3): 0.412 (3) ratio.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016005259/hb7561sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989016005259/hb7561Isup2.hklStructure factors: contains datablock(s) I. DOI: 1471029CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


KD/KD) mice expressing the kinase-inactive PI3Kγ protein. We showthat CD4+ and CD8+ T cells fromPI3KγKD/KD mice exhibit impaired TCR/CD28-mediated activation that could not berescued by exogenous IL-2. The defects in proliferation and cytokine production were also evident innaïve and memory T cells. Analysis of signaling events in activatedPI3KγKD/KD T cells revealed a reduction in phosphorylation of proteinkinase B (AKT) and ERK1/2, a decrease in lipid raft formation, and a delay in cell cycleprogression. Furthermore, PI3KγKD/KD CD4+ T cellsdisplayed compromised differentiation toward Th1, Th2, Th17, and induced Treg cells.PI3KγKD/KD mice also exhibited an impaired response to immunization and a reduceddelayed-type hypersensitivity to Ag challenge. These findings indicate that PI3Kγ kinaseactivity is required for optimal T-cell activation and differentiation, as well as for mounting anefficient T cell-mediated immune response. The results suggest that PI3Kγ kinaseinhibitors could be beneficial in reducing the undesirable immune response in autoimmunediseases.Phosphatidylinositol-3-kinase gamma (PI3Kγ) is a leukocyte-specific lipid kinase withsignaling function downstream of G protein-coupled receptors to regulate cell trafficking, but itsrole in T cells remains unclear. To investigate the requirement of PI3Kγ kinaseactivity in T-cell function, we studied T cells from PI3Kγ kinase-dead knock-in(PI3Kγ Additional supporting information may be found in the online version of this article at thepublisher's web-siteβγPhosphatidylinositol-3-kinase gamma (PI3Kγ) is a member of the PI3K family thatphosphorylates phosphatidylinositol 4,5-diphosphate to generate phosphatidylinositol3,4,5-triphosphate (PIP3) at the plasma membrane PI3Kγ is involved in T-cell development in the thymus but its role in T-cell activationhas been controversial Upon TCR engagement, multiple signaling mechanisms including NFAT, NF-κB, and MAPKpathways are activated, which result in gene induction and cell cycle progression. Generation ofPIP3 is one of the earliest signals observed in activated T cells + CD4+ T cells can beinduced by IL7 and this induction was blocked by PI3K inhibitors + T cells PI3K has been shown to play a role in T-cell differentiation through TORC1/TORC2 signalingpathways and transcription factors Forkhead box (FOXO) and Krueppel-like factor 2 (KLF2) -deficient mice KD/KD)) have beengenerated by introducing a point mutation into the p110γ gene The scaffolding function of PI3Kγ independent of its kinase activity has been demonstratedin the cardiovascular system KD/KD mice in different activation conditions. Expressionof the kinase-inactive PI3Kγ protein in CD4+ T cells was comparableto wild-type (WT) T cells . The results suggest a functional defect in chemotactic response ofPI3KγKD/KD T cells.The requirement of PI3Kγ kinase activity in CD4KD/KD mice were lesseffective in activation, differentiation, and chemotaxis. The physiological importance ofPI3Kγ kinase activity was further investigated in two immunization models.PI3KγKD/KD and WT mice were immunized with ovalbumin/CFA at the tail base and theimmune response in mice was examined 10 days later. The typical response in immunized mice is anenlargement of draining lymph nodes with increased cellularity as a result of expansion ofAg-specific T and B cells. Although lymph nodes from PI3KγKD/KD mice were enlargedcompared to unimmunized mice, their cellularity was 36% less than that from WT immunized mice/CFAat the tail base. Eleven days postimmunization, the animals were injected at the right hind pawswith mBSA to elicit a DTH response, whereas the left hind paws were injected with saline as negativecontrols. The swelling of Ag-injected paws was measured at different time points up to 48 hpostchallenge. PI3KγKD/KD mice exhibited an impaired DTH response with a25% reduction in edema by comparing the area under curve of edema scores with WT mice. PI3KγKD/KD mice expressing the OT-IItransgenic TCR were generated by cross-breeding OT-II transgenic mice (the Jackson Lab) withPI3KγKD/KD mice in C57BL/6J genetic background. Mice were maintained underspecific pathogen-free condition at the facility of Janssen Research & Development and theJackson Lab. Mice were studied following protocols approved by the Institutional Animal Care and UseCommittee of Janssen Research and Development.PI3Kγ+ T cells, CD4+ T cells, and naïve ormemory CD4+ T cells were isolated with Ab-coated magnetic bead kits(Miltenyi Biotech). T cells at 2 × 105 cells/well were stimulated overnightwith 1 μg/mL plate-bound anti-CD3 (Biolegend) with or without 1 μg/mL solubleanti-CD28 (Biolegend).CD8+ T cells purified from splenocytes of ovalbumin/CFA-immunized micewere expanded ex vivo by stimulation with 5 μg/mL ovalbumin in the presence of mitomycinC-treated syngeneic splenocytes. Highly enriched ovalbumin-specific CD4+T cells after three rounds of Ag stimulation were used in proliferation assays at 2 ×105 cells/well and the addition of ovalbumin and 106 cells/well mitomycinC-treated splenocytes.CD4+ T cells isolated from mice of C57BL/6background were set up at 2 × 105 cells/well and cocultured for 3 days with 2× 106 cells/well of mitomycin C-treated BALB/c allogeneic splenocytes.Proliferation of T cells was measured by overnight pulsing of cells with 1 μCi/well of3H-thymidine (Perkin Elmer) followed by counting radioactivity of harvested cells inscintillant (Perkin Elmer) using a Topcount (Packard).For MLR to allogeneic cells, CD4Cytokines in T-cell culture supernatants were measured in ELISA assays using Ab pairs for IL-2,IL-4, and IFN-γ (BD Biosciences) and IL-17A ELISA kit (R&D) following the kitprotocols.T cells were coated with 1 μg/mL of antimouse CD3ε (Biolegend) alone or incombination with anti-CD28 (Biolegend), followed by coupling with 1 μg/mL of goat antihamsterIgG (Pierce). T cells before and after stimulation were lysed with NP-40 lysis buffercontaining protease inhibitors (Invitrogen). Lysates were separated by electrophoresed and blottedonto polyvinylidene difluoride (PVDF) filters (Bio-Rad). AKT, ERK, and their phosphorylated forms,as well as PI3Kγ p110 were detected and quantitated in Western blots using specificantibodies  following the protocols of reagent kits.T cells were stained with fluorescent dye conjugated antibodies specific for differentcell surface markers (eBioscience) according to standard staining procedures. Intracellular stainingwas performed by stimulating T cells for 4 h with a leukocyte activation cocktail containingGolgiPlug (BD Biosciences), followed by fixing with paraformaldehyde (Biolegend) andpermeabilization with saponin buffer (Biolegend), and then staining with antibodies (eBioscience)according to kit protocols. Cell division was detected by staining T cells with 1 μMCFSE (Invitrogen) prior to activation or differentiation assays. Samples were acquired withFACSCalibur (BD Biosciences) and analyzed with FlowJo software (Tree Star).+ T cells were activated with plate-bound anti-CD3 (1μg/mL) and soluble anti-CD28 (2.5 μg/mL) under different polarizing conditions. Th1differentiation was driven by 20 U/mL IL-12, 100 U/mL IL-2, and 10 μg/mL anti-IL-4. Th2differentiation condition was set up with 200 U/mL IL-4, 100 U/mL IL-2, and 10 μg/mL each ofanti-IL12 and anti-IFN-γ. Th17 polarization was prepared with 5 ng/mL TGF-β, 20 ng/mLIL-6, and 10 μg/mL each of anti-IL-4 and anti-IFN-γ .Treg-cell differentiation condition was set by addition of 5 ng/mL TGF-β1 and 100 U/mL IL-2in medium. After 6 days of differentiation, T cells were characterized by intracellularstaining of cytokines and transcription factors as described in the earlier section on T-cellphenotyping.Naive CD4+ T cells from OT-II transgenic mice. Transgenic T cells werestimulated for 6 days with 0.5 μM ovalbumin peptide OVA323–339  in the presence of mitomycin C-treated splenocytes under Th1 or Th17 differentiationcondition.Differentiation of Ag-specific T cells was performed with naïveCD4+ T cells were incubated with anti-CD3- or anti-CD3-coated plusanti-CD28-coated polystyrene beads (Spherotech) at 1:5 cell/bead ratio at 37°C for 30 minfollowed by cell attachment onto poly-l-lysine-coated slides . Cells onslides were treated with 3.7% paraformaldehyde in PBS for 10 min, permeabilized with0.1% Triton X-100 in PBS for 10 min, and then blocked with 1% BSA in PBS for 30 minu.Cells were then incubated with FITC-conjugated cholera toxin B (Sigma) for 1 h, washed with PBS, andmounted onto slides with Vectashield containing 4′, 6′-diamidino-2-phenylindole(Vector Laboratories). Cells on slides were examined using a Leica SPE2 confocal microscope systemwith Zeiss 63 × 1.4 oil objectives and SlideBook 4.2 software (Intelligent ImagingInnovations). Cholera toxin B-stained lipid rafts at cell/bead contact areas were evaluated visuallyon ∼150 cell/bead conjugates.CD46cells/well and chemokine CCL3 (0.27 μg/mL), CCL19 (0.2 μg/mL), CCL21 (0.5μg/mL), CXCL12 (3 μg/mL), or RANTES  was addedin the lower chamber. T cells entered in the lower chamber after 1 h of incubation werequantitated by acquisition with FACSCalibur.Chemotaxis assays were performed in transwell plates with uncoated filters of 5 μm poresize (Corning Coastar). Splenocytes were placed at the upper chamber at 2 × 10KD/KD mice backcrossed to C57BL/6 background and WT C57BL/6 mice wereimmunized at tail base with 200 μg ovalbumin  emulsified with CFA (BDDiagnostics) at 1:1 volume ratio. For ovalbumin/alum immunization, BALB/cJ mice andPI3KγKD/KD mice of BALB/cJ background were immunized at tail base with 50μg ovalbumin /alum mixture. Ovalbumin/alum mixture was prepared bymixing 200 μg/mL ovalbumin in PBS with 4% Imject Alum (Pierce) at 1:1 volume ratio andincubated for 1 h at room temperature. On day 10, postimmunization, mice were sacrificed and thedraining inguinal lymph nodes were collected and cell suspensions were prepared for cell count,intracellular cytokine detection, and proliferation assays.PI3Kγ5 cells/well for 3 days. Cell proliferation and cytokine productionwere measured with methodologies as described in T-cell proliferation section.Draining lymph node cells from ovalbumin/CFA immunized mice were stimulated with ovalbumin exvivo at 5 × 10KD/KD mice were immunized at tail base with 100μL of 1 mg/mL mBSA (Sigma) in 1:1 volume CFA emulsion . On day 7 micewere challenged by injecting 50 μL of 180 μg/mL mBSA into left footpads and 50μL of saline into right footpads as negative controls. Footpad thickness was measured with acaliper at 0, 6, 26, 29.5, and 48 h time points. On day 11, mice were sacrificed and inguinal nodeswere collected to prepare cells for proliferation assays. Paws were collected and fixed in10% formalin, decalcified with formic acid, processed, and longitudinally embedded inparaffin. Serial 5 μm sections were prepared and stained with H&E for histologyevaluation. Tissue was scored for inflammation, edema, hemorrhage, and subcutaneous necrosis. Eachparameter was scored as follows: 0 = Normal, 1 = Mild, 2 = Moderate, 3 =Moderately severe, 4 = Markedly Severe. A summary histological score for severity ofinflammation was accounted as summary of all the parameters.C57BL/6J mice and PI3Kγ5 cells/well for 3 days. Cell proliferation andcytokine production was measured as described previously.Draining inguinal lymph node cells from mBSA immunized mice were stimulated with differentconcentrations of mBSA at 5 × 10


N-(Substituted phenyl)-4-oxo-4H-chromene-3-carboxamides have very similar conformations but show different inhibition activities against h-MAO-B so it may be assumed that the electronic environment provided by the substituents on the phenyl ring is the primary condition for the pharmacological activities displayed by these molecules. N-substituted-phenyl 4-oxo-4H-chromene-3-carboxamides, namely N-(2-nitrophenyl)-4-oxo-4H-chromene-3-carboxamide, C16H10N2O5 (2b), N-(3-methoxyphenyl)-4-oxo-4H-chromene-3-carboxamide, C17H13NO4, (3a), N-(3-bromophenyl)-4-oxo-4H-chromene-3-carboxamide, C16H10BrNO3, (3b), N-(4-methoxyphenyl)-4-oxo-4H-chromene-3-carboxamide, C17H13NO4, (4a), N-(4-methylphenyl)-4-oxo-4H-chromene-3-carboxamide, C17H13NO3, (4d), and N-(4-hydroxyphenyl)-4-oxo-4H-chromene-3-carboxamide, C16H11NO4, (4e), have been structurally characterized. All compounds exhibit an anti conformation with respect to the C—N rotamer of the amide and a trans-related conformation with the carbonyl groups of the chromone ring of the amide. These structures present an intramolecular hydrogen-bonded network comprising an N—H⋯O hydrogen bond between the amide N atom and the O atom of the carbonyl group of the pyrone ring, forming an S(6) ring, and a weak Car—H⋯O hydrogen bond in which the carbonyl group of the amide acts as acceptor for the H atom of an ortho-C atom of the exocyclic phenyl ring, which results in another S(6) ring. The N—H⋯O intramolecular hydrogen bond constrains the carboxamide moiety such that it is virtually coplanar with the chromone ring.Six  Recent data  and –CH3 (4d) substituents favours the potency while an –OH (4e) substituent has the opposite effect. The data acquired so far point out the importance of a structure–activity relationship study to optimize the potency vs selectivity of this type of inhibitor, namely performing structural and electronic changes in the substituents.Chromones are a group of natural and synthetic oxygen heterocyclic compounds having a high degree of chemical diversity that is frequently linked to a broad array of biological activities. The chromone-3-(phenyl)carboxamide derivatives, depicted the scheme, have emerged as promising compounds for the management of neurodegenerative diseases such as Alzheimer’s and Parkinson’s since they display selective inhibition activities against N-phenyl-4-oxo-4H-chromene-3-carboxamide : N-(2-methoxyphenyl)-4-oxo-4H-chromene-3-carboxamide : N-(2-nitrophenyl)-4-oxo-4H-chromone-3-carboxamide (CCDC 1025354); (3a): N-(3-methoxyphenyl)-4-oxo-4H-chromene-3-carboxamide (CCDC 102353); (3b): N-(3-bromophenyl)-4-oxo-4H-chromene-3-carboxamide (CCDC 1025352); (4a): N-(4-methoxyphenyl)-4-oxo-4H-chromene-3-carboxamide (CCDC 1025355); (4b): N-(4-bromophenyl)-4-oxo-4H-chromene-3-carboxamide : N-(4-chlorophenyl)-4-oxo-4H-chromene-3-carboxamide : N-(4-methylphenyl)-4-oxo-4H-chromene-3-carboxamide; (4e): N-(4-hydroxyphenyl)-4-oxo-4H-chromene-3-carboxamide (CCDC 102524). Compounds with CCDC numbers given were deposited by the current authors, Gomes, Borges and Low, in the Cambridge Structural Database : Conformations and intramolecular hydrogen-bond networkb), (3a), (3b), (4a), (4d) and (4e), the structurally characterized compounds in this work. As seen, the molecules exhibit an anti conformation with respect to the C–N rotamer of the amide following a pattern given by compound (1), which was previously described by Cagide et al. –(4) exhibit a trans relation between these bonds as can be seen in Figs. 1ortho position of the phenyl ring, forming another S(6) ring. This hydrogen-bonding network probably enhances the planarity of the molecules and may prevent them from adopting some other possible conformations by restraining their geometries. Compounds (2a) and (2b) have substituents located at the ortho position on the benzyl ring with oxygen atoms  that act as acceptors for the amide nitrogen atom of the carboxamide residue, hence forming a third intramolecular hydrogen bond (see scheme).The structural analysis confirms that the molecules are 4-chromone derivatives with a phenylamide substituent on position number 3 of the pyrone ring. Fig. 1 al. 2015. Due to Molecular geometriessp3—Csp3 bond , θChr-amide, and between the exocyclic phenyl ring and the amide, θPhe-amide. Those dihedral angles are primarily due to the rotation of the rings around the C3—C31 and N3—C311 bonds with exception of (3a) that assumes mainly a bent conformation between the rings. The structural analysis of (1) performed previously  which, simultaneously, cause steric hindrance and act as acceptors for the hydrogen atom of the amide, thus forming an intramolecular hydrogen bond, suggest that a tricky balance between those two factors allows the formation of several energetically accessible rotated conformations. This fact is especially noticeable in the various conformation polymorphs of (2a).Table 1ortho-substituents but they still present a wide range of values for the θPhe-amide dihedral angles (between 3 and 24°). The θChr-Phe values may be used as a measure of the relative positioning of the two aromatic rings which may define the primary conformation for the molecules. The aromatic rings are usually rotated or co-planar, with exception of (3a) where they are bent with respect to each other. The chromones with halogen substituents assume the most planar conformations, probably related to the typical positive mesomeric effects on the π system. Considering the fact that the para-substituent on the exocyclic phenyl ring for chromone-3-phenylcarboxamides has a positive effect on their activity, and the requirement of establishing the factors that can modulate the enzyme–ligand interaction, it can be assumed their h-MAO-B activity is strongly dependent on the electronic environment of the substituent. This is not a preferred conformation that reduces or enhances the activity, so it may be assumed that the electronic environment provided by the substituent is the primary condition for the pharmacological activities displayed by those molecules.The remaining compounds are not constrained by steric hindrance of the b) there are two molecules in the asymmetric unit. A calculation using Molfit with Quaternion Transformation Method , the molecules are linked by C8—H8⋯O32, the molecules are linked by the C2—H2⋯O3 hydrogen bond, forming centrosymmetric dimers across the inversion centre at  (Table 3In (3) Table 3.b), independent ladders of molecule 1 and molecule 2 are propagated along the a-axis direction by unit translation. These are formed by chains of x2—Hx2⋯Ox4 and Cx36—Hx36⋯Ox3 interactions, where x = 1 or 2 –(4d) is the formation of a ladder structure composed of molecules propagated by unit axial translations involving intermolecular hydrogen bonds between C2 and O4 of the chromone ring and the C atom located at the ortho position of the exocyclic phenyl ring and the carboxamide O atom. This is also found in (1) and in compound (3b), which has a Br substituent located at the meta position, in which the ladder structure is supplemented by an intermolecular hydrogen bond between C5 and O1 of the chromone moiety. In (4a), the molecules are linked by C2—H2⋯O4  and C316—H316⋯O3  hydrogen bonds, forming b-axis direction by unit translation (Table 5d), the molecules are linked by C2—H2⋯O4 and C316—H316⋯O3 hydrogen bonds, forming a-axis direction by unit translation (Table 6A common feature found for compounds with n Table 5. In (4d)n Table 6.e), the molecules in the asymmetric unit are linked by the O114—H114⋯O23 hydrogen bond, forming a dimer. These dimers are linked by the O214—H214⋯O13 and weak C16—H16⋯O114, C18—H18⋯O24, C26—H26⋯O214 and C28—H28⋯O14 hydrogen bonds, which link the molecules into sheets that form a chequerboard pattern and which lie parallel to the (In the hydroxyl compound (4s Table 7.b), (3b), (4a) and (4d) are listed in Table 8a).Selected π–π contacts, with centroid-to-centroid distances less than 4.0 Å and with angles between planes of less than 10° for compounds  oxychloride (POCl3) in dimethylformamide, react with the different substituted anilines. Crystals were recrystallized from ethylacetate forming colourless plates whose dimensions are given in Table 9The compounds were obtained by synthetic strategies described elsewhere  there are two molecules in the asymmetric unit. The largest difference map peaks are associated with the Br atoms.In (3Uiso(H) = 1.2Ueq(C); C—H(methyl), = 0.98 Å with Uiso= 1.5Ueq(C). In all compounds, the amino H atoms were refined with the exception of (3b) where these atoms were refined as riding atoms with N—H = 0.88 Å with Uiso = 1.2Ueq(C) and in (4e) in which the positional parameters of the amino and hydroxyl H atoms were refined but their Uiso values were constrained to be Uiso(N) = 1.2Ueq(N) and Uiso(O)b= 1.5Ueq(O). The final positions of these atoms were checked in a difference Fourier map, as were the positions of the H atoms in any methyl groups. The quality of the crystals for (4e) was poor and the crystals were twinned. The completeness is 97%. The crystal studied was refined as a two-component twin [twin law: 2-axis (001) [In all compounds, H atoms attached to C atoms were treated as riding atoms with C—H(aromatic) = 0.95 Å with 10.1107/S2056989015007859/lh5762sup1.cifCrystal structure: contains datablock(s) 2b, 3a, 3b, 4a, 4d, 4e, global. DOI: 10.1107/S2056989015007859/lh57622bsup2.hklStructure factors: contains datablock(s) 2b. DOI: 10.1107/S2056989015007859/lh57623asup3.hklStructure factors: contains datablock(s) 3a. DOI: 10.1107/S2056989015007859/lh57623bsup4.hklStructure factors: contains datablock(s) 3b. DOI: 10.1107/S2056989015007859/lh57624asup5.hklStructure factors: contains datablock(s) 4a. DOI: 10.1107/S2056989015007859/lh57624dsup6.hklStructure factors: contains datablock(s) 4d. DOI: 10.1107/S2056989015007859/lh57624esup7.hklStructure factors: contains datablock(s) 4e. DOI: Click here for additional data file.10.1107/S2056989015007859/lh57622bsup8.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015007859/lh57623asup9.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015007859/lh57623bsup10.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015007859/lh57624asup11.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015007859/lh57624dsup12.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015007859/lh57624esup13.cmlSupporting information file. DOI: 1025354, 1025353, 1025352, 1025255, 1025257, 1025254CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Mangifera mekongensis (Anacardiaceae) is cultivated for its edible fruit and has been used in traditional Vietnamese medicine for its anti-aging properties and for treating diabetes, vermifuge, and dysentery. As part of a search for biologically active compounds with reduction of the rate of glucose absorption, a screening has been initiated to evaluate natural product extracts for the inhibition of enzyme α-glucosidase. A n-hexane extract of the bark of M. mekongensis showed strong α-glucosidase inhibitory activity with IC50 value of 1.71 µg/mL. Thus, the constituents of this plant were examined.1) and mekongsterol B (2), were isolated from the n-hexane extract of the bark of M. mekongensis (Anacardiaceae), together with seven known compounds (3–9). Their chemical structures were elucidated on the basis of spectroscopic data. All compounds possessed significant α-glucosidase inhibitory activity in a concentration-dependent manner, except for 3 and 4. Compounds 1, 2, 5–9 showed more potent inhibitory activity, with IC50 values ranging from 1.2 to 112.0 µM, than that of a positive control acarbose .Two new steroids named mekongsterol A  contains supplementary material, which is available to authorized users. Mangifera mekongensis (Anacardiaceae), commonly known as mango, is widely distributed in tropical and subtropical regions of Asia. In Vietnam, M. mekongensis is called as “Xoai Thanh Ca”, and this plant is cultivated for its edible fruit and has been used in traditional Vietnamese medicine for treating anti-aging, diabetes, vermifuge, dysentery +, corresponding to the molecular formula C48H86O2K in HR-ESI–MS. The IR spectrum of 1 showed absorption of ester carbonyl (1720 cm−1), double bond (1610 cm−1), and methyl, methylene, and methine (2950 and 2870 cm−1) groups. The 1H NMR spectrum of 1 , three methyl doublets , a methyl triplet , an oxymethine , and trisubstituted olefinic bond , together with many aliphatic methylene and aliphatic methine groups (δH 0.95–2.30). The 13C NMR , an oxymethine (δC 73.8), and two olefinic carbons (δC 122.7 and 139.9). These data closely resembled those of β-sitosterol (3) +, corresponding to the molecular formula C38H64O4Na in HR-ESI–MS. Absorption bands at 3500, 1710, 1730, 1600, 2960 and 2860 cm−1 in the IR spectrum of 2 indicated the presence of hydroxyl, acid carbonyl, ester carbonyl, double bond, methyl, methylene, and methine groups. The 1H NMR spectrum of 2 , three methyl doublets , a methyl triplet , an oxymethine , and trisubstituted olefinic bond , together with many aliphatic methylene and aliphatic methine groups (δH 0.95–2.30). The 13C NMR , an oxymethine (δC 73.8), two olefinic carbons (δC 122.7 and 139.9), an ester carbonyl carbon (δC 173.4), and an acid carbonyl carbon (δC 178.5). These 1H and 13C data were similar to those of β-sitosterol (3) + ; 1H NMR  and 13C NMR , see Table sterol A : white csterol A : white cMekongsterol B (2): white crystal; IR νmax (CHCl3) 3500, 2960, 2860, 1730, 1710, 1600 cm−1; HR-ESI–MS positive m/z 607.4719 [M + Na]+ ; 1H NMR  and 13C NMR , see Table p-nitrophenyl-α-d-glucopyranoside (25 μL) and 0.2 U/mL α-glucosidase (25 μL) in 0.01 M phosphate buffer (pH = 7.0) were added to the sample solution (625 μL) to start the reaction. Each reaction was carried out at 37 °C for 30 min and stopped by adding 0.1 M Na2CO3 (375 μL). Enzymatic activity was quantified by measuring absorbance at 401 nm. One unit of α-glucosidase activity was defined as amount of enzyme liberating p-nitrophenol (1.0 μM) per min. The IC50 value was defined as the concentration of α-glucosidase inhibitor that inhibited 50 % of α-glucosidase activity. Acarbose, a known α-glucosidase inhibitor, was used as positive control.The inhibitory activity of α-glucosidase was determined according to the modified method of Kim et al. . 3 mM p-1) and mekongsterol B (2), together with seven known compounds isolated from the bark of M. mekongensis. Seven compounds possessed α-glucosidase inhibitory activity. This is the first report on α-glucosidase inhibitory activity of the bark of this plant. These results suggested that the traditional use of the bark of M. mekongensis for the treatment of diabetes diseases in Vietnam may be attributable to the α-glucosidase inhibitory activity of its steroid and cycloartane constituents.In this paper, we have reported two new compounds, mekongsterol A (


The presence of N—H⋯S hydrogen bonds lead to layers in the bc plane which are connected by methyl-C—H⋯π interactions.The title twisted thiosemicarbazone molecule has, respectively,  11H15N3S, the p-tolyl-N—H and imino-N—H groups are anti and syn, respectively, to the central thione-S atom. This allows for the formation of an intramolecular p-tolyl-N—H⋯N(imino) hydrogen bond. The molecule is twisted with the dihedral angle between the p-tolyl ring and the non-hydrogen atoms of the N=CMe2 residue being 29.27 (8)°. The crystal packing features supramolecular layers lying in the bc plane whereby centrosymmetric aggregates sustained by eight-membered thioamide {⋯HNCS}2 synthons are linked by further N—H⋯S hydrogen bonds. Layers are connected via methyl-C—H⋯π interactions. The supramolecular aggregation was further investigated by an analysis of the Hirshfeld surface and comparison made to related structures.In the title thiosemicarbazone, C N-alkyl- or N-aryl-isothiocyanides usually results in the formation of thiocarbamides. For example, in the case of reactions involving a monofunctional alcohol, the reaction proceeds in the following manner: R—OH + R′N=C=S → ROC(=S)N(H)R′ =N(aryl)] compounds exhibit significant cytotoxicity against a variety of cancer cell lines and mechanistic studies show that they can kill cancer cells by initiating a variety of apoptotic pathways, both extrinsic and intrinsic =N(p-tolyl)], exhibit potency against Gram-positive bacteria  complexes  with (p-tolyl)N=C=S, conducted in acetone solution, is described, namely the thiosemicarbazone, (I)et al., 2013et al., 2013The reaction between an alcohol or amine (primary or secondary) with anti- and syn-dispositions of the N1- and N2-bound H atoms, respectively, with respect to the central thione-S1 atom. The N1-bound H atom is syn to the imino-N3 atom allowing for the formation of a five-membered loop via an N1—H⋯N3 hydrogen bond, Table 12; r.m.s. deviation for the C3N atoms = 0.0002 Å) and 43.30 (5)° with the p-tolyl ring. Overall, the molecule is twisted as quantified by the dihedral angle between the outer planes of 29.27 (8)°.The molecular structure of (I)P21/c polymorphs have been reported for the parent compound, i.e. having a phenyl rather than a p-tolyl substituent , b = 7.618 (2), c = 11.639 (3) Å, β = 102.660 (4)° reported for the former thiourea residue has a locked configuration, consistent with the persistence of the intramolecular N1—H⋯N3 hydrogen bond in CDCl3 solution.The conformation of (I)2 synthons. These are connected into supramolecular layers in the bc plane by amide-N1—H⋯S1 hydrogen bonds so that the S1 atom accepts two hydrogen bonds, Fig. 3p-tolyl groups protrude to either side of each layer and inter-digitate along the a axis with adjacent layers allowing for the formation of methyl-C8—H⋯π(C2–C7) interactions, thereby consolidating the three-dimensional architecture, Fig. 4In the crystal, N—H⋯S and C—H⋯π interactions provide identifiable points of contact between molecules; these interactions are quantified in Table 1CrystalExplorer  confirm their role as an acceptor and donor in the hydrogen-bonding scheme, respectively. This is also evident from the dark-red and blue regions, respectively, on the Hirshfeld surface mapped over the calculated electrostatic potential . The diminutive red spots near S1 and N1  indicate their involvement in the intermolecular N—H⋯S hydrogen bond.Two views of the Hirshfeld surface mapped over ms Fig. 5a confiral Fig. 5c. The dN1 Fig. 5b indicaa) and those delineated into H⋯H, S⋯H/H⋯S, N⋯H/H⋯N and C⋯H/H⋯C H⋯H  interactions operating in the crystal structure of (I)de + di = 2.45 Å; Fig. 6b) with a 15.2% contribution due to S⋯H/H⋯S contacts to the Hirshfeld surfaces also suggest the presence of these interactions in the crystal packing. The light-red region near N3  and diminutive red spot near N1—H  are consistent with relatively smaller contributions from N⋯H/H⋯N contacts, i.e. 2.5 and 3.0%, respectively, and are indicative of the weak intramolecular hydrogen bond. The strength of such an interaction can be visualized from the 2D fingerprint plot corresponding to N⋯H/ H⋯N contacts . The bright-orange spot in the surface mapped with de . The wing at the top, left belongs to C—H donors, while that at the bottom, right corresponds to the surface around π-acceptors with 11.3 and 7.8% contribution from C⋯H and H⋯C contacts, respectively. The H⋯H contacts reflected in the middle of scattered points in Fig. 6e provide the most significant contribution, i.e. 57.0%, to the Hirshfeld surface arising from a side-ways approach. The small, flat segments delineated by the blue outline in the surface mapped with curvedness  contribution from C⋯C contacts to the surface indicates the absence of π–π stacking interactions in the structure.The overall two-dimensional fingerprint plot Fig. 6a and thN3 Fig. 5a and di—H Fig. 5b are cots Fig. 6c. The b7C Fig. 7, suggest7C Fig. 7. This is7C Fig. 7d. The wss Fig. 8 and the Structural commentary, the parent compound has been characterized in two polymorphic forms 2] 2]Br RuCl(LH)]Cl °.According to a search of the Cambridge Structural Database  in acetone (20 ml) was added hydrazine monohydrate . The resulting mixture was stirred for 4 h at room temperature. Both chloroform (20 ml) and acetonitrile (20 ml) were then added, and the resulting mixture left for slow evaporation. Light-brown crystals were obtained after 2 weeks. Yield: 2.012 g (91%). M.p. 412–413 K. 1H NMR : 9.19 , 8.56 , 7.49 , 7.17 , 2.34 , 2.05 , 1.94 . 13C NMR : 176.4 [CS], 149.6 [C(CH3)2], 135.8 [Cipso], 135.4 [Cpara], 129.3 [Cmeta], 124.5 [Cortho], 25.3 [CH3], 21.0 [aryl-CH3], 16.9 . IR (cm−1): ν(N—H) 3240, 3168 (m), ν(C=N) 1514 (vs), ν(C—N) 1267 (s), ν(C=S) 1188 (vs).To Uiso(H) set to 1.2–1.5Ueq(C). The N-bound H atoms were located in a difference Fourier map but were refined with a distance restraint of N—H = 0.88±0.01 Å, and with Uiso(H) set to 1.2Ueq(N).Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989015017624/hb7507sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989015017624/hb7507Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989015017624/hb7507Isup3.cmlSupporting information file. DOI: 1425975CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: