Dapk−/− Th17 cells and prevents experimental autoimmune encephalomyelitis development. Our results reveal a mechanism involving DAPK-mediated degradation of cytoplasmic HIF-1α, and suggest that raising DAPK levels could be used for treatment of Th17-associated inflammatory diseases.Death-associated protein kinase (DAPK) is a tumour suppressor. Here we show that DAPK also inhibits T helper 17 (Th17) and prevents Th17-mediated pathology in a mouse model of autoimmunity. We demonstrate that DAPK specifically downregulates hypoxia-inducible factor 1α (HIF-1α). In contrast to the predominant nuclear localization of HIF-1α in many cell types, HIF-1α is located in both the cytoplasm and nucleus in T cells, allowing for a cytosolic DAPK–HIF-1α interaction. DAPK also binds prolyl hydroxylase domain protein 2 (PHD2) and increases HIF-1α-PHD2 association. DAPK thereby promotes the proline hydroxylation and proteasome degradation of HIF-1α. Consequently, DAPK deficiency leads to excess HIF-1α accumulation, enhanced IL-17 expression and exacerbated experimental autoimmune encephalomyelitis. Additional knockout of HIF-1α restores the normal differentiation of  HIF-1α is critical for Th17 differentiation. Here the authors show that DAPK (Death-Associated Protein Kinase) inhibits Th17 differentiation and immunopathology in a mouse model of multiple sclerosis by promoting HIF1-α binding to its negative regulator PHD2. Upon activation, the T helper 17 (Th17) subset of immune cells plays critical roles in modulating tissue inflammation and combating microbial infections. However, due to their inflammatory nature, Th17 cells also contribute to autoimmune diseases121047121314152, HIF-1α is hydroxylated at Pro402 and Pro564 by prolyl hydroxylase domain protein 2 (PHD2)/PHD3, followed by ubiquitination by the von Hippel–Lindau (VHL)-containing E3 complex that promotes proteasome degradation18192021Hypoxia-inducible factor-1α (HIF-1α) is an oxygen tension sensor widely expressed in different cell types, including Th17 cells. In the presence of OHif1a transcript is constitutively expressed in T lymphocytes, and the HIF-1α protein is detected after T-cell receptor (TCR) stimulation under hypoxic conditions242626Rorc (RORγt), and it helps recruit CBP/p300 to the RORγt transcription complex but does not directly bind to the IL-17 promoter26The HIF-1α promotes carcinogenesis and is a prominent cancer target18181931Death-associated protein kinase (DAPk/DAPK) is a multi-domain serine/threonine kinase regulated by calcium333637383940In the present study, we found that DAPK negatively regulates Th17 differentiation. DAPK deficiency leads to preferential Th17 differentiation and exacerbated EAE induction. During the differentiation of Th17, the presence of DAPK is accompanied by downregulation of HIF-1α. We further found that, in contrast to the exclusive nuclear localization of HIF-1α in most other cells, HIF-1α is located in both the cytoplasm and nucleus of T cells, allowing the cytosolic binding of DAPK and subsequent HIF-1α degradation. Our results illustrate a novel mechanism of Th17 regulation by downregulating cytoplasmic HIF-1α, and suggest the therapeutic potential of increasing DAPK levels in Th17-mediated inflammatory diseases.Dapk−/− mouseDapk−/− mouse (Dapk−/− T-cell proliferation was enhanced relative to the wild-type (WT) T cells  peptide, disease onset was earlier in Dapk−/− mice than their WT littermates  and Ifng was not affected by the deficiency of DAPK  were not affected in Dapk−/− nTregs 27− nTregs . However T cells . The in T iTregs .To isolate the mechanism underlying DAPK-dependent HIF-1α degradation, we next examined whether there was a direct interaction between DAPK and HIF-1α. DAPK is a cytosolic protein whereas HIF-1α is known for its predominant nuclear localization, as shown in hypoxic HeLa cells . HIF-1α in vitro  with Dapk−/− mice. Deletion of HIF-1α and/or DAPK in T cells from each genotype was confirmed by immunoblotting , leupeptin, cycloheximide (CHX), DFX, anti-DAPK  1:1,000, immunofluorescence (IF) 1:50), anti-HA  1 μg per test), and anti-FLAG  were purchased from Sigma . Anti-p70S6K , anti-GAPDH , anti-HDAC2  and anti-p65  were obtained from Santa Cruz Biotech . Anti-RORγt , anti-CD4-PE-Cy7  1:100), anti-CD62L-APC , recombinant mouse IL-6 (14–8061) and IL-23 (14–8231), anti-CD69-PE , anti-Foxp3-APC , mouse IL-2 and mouse IFN-γ ELISA kit were purchased from eBioscience . STAT3 antibody  was obtained from GeneTex . Anti-phospho STAT3Y705 , anti-phospho mTORS2448 , anti-phospho p70S6KT389 , anti-phospho 4E-BP1T70 , Alexa 647-conjugated anti-S6S235/236 , Alexa 647-conjugated anti-S6S240/244 , anti-mTOR , anti-4E-BP1 , anti-PHD2 , anti-Myc , and anti-hydroxy HIF-1αP564  were purchased from Cell Signaling Technology . Anti-actin  and anti-tubulin  were purchased from Merck Millipore . Anti- mouse HIF-1α  was purchased from Cayman Chemicals . Anti-human HIF-1α , and anti-HSP90  were obtained from BD-Biosciences . Anti-CD4-FITC , anti-CD8a-PE , anti-CD44-FITC , anti-IL-17A-PE , anti-TCRβ-FITC , anti-CD25-PE , anti-IFN-γ-APC , anti-IFN-γ , LEAF purified anti-mouse IFN-γ  and LEAF purified anti-mouse IL-4  were purchased from BioLegend . Anti-PHD1  and anti-IL-17  were purchased from Abcam . Anti-mouse CD3 (2C11), anti-mouse CD28 (37.51), anti-mouse IL-2, and anti-mouse CD4 (RL172.4) were purified in our laboratory. Recombinant mouse IL-12 and IL-21, mouse IL-17A ELISA kit, and mouse IL-4 ELISA kit were purchased from R&D . Recombinant mouse IL-2 and human TGF-β were purchased from PeproTech . Anti-HIF-1α  was purchased from Bethyl Laboratories . Mouse anti-Lamin B1 , Alexa Flour488-labelled goat anti-mouse IgG , Alexa Flour555-labelled goat anti-mouse IgG , Alexa Flour488-labelled goat anti-rabbit IgG  and Alexa Flour555-labelled goat anti-rabbit IgG  were purchased from Thermo Fisher Scientific . siRNA control (siCtrl), siRNAs specific for PHD2 (EGLN1 siRNA SMARTpool) and for human DAPK (siDAPK) were purchased from GE Dharmacon . The sequence of siDAPK is: UCU GGG AAG CGG AGC UGA AUU.Calcium ionophore (A23187), phorbol 12-myristate 13-acetate (TPA), MG132, chloroquine, ammonium chloride . HEK293T and HeLa cells were cultured in DMEM medium with the same supplements as in complete RPMI medium. For hypoxia (1% O2) experiments, T cells were cultured in the cell incubator containing an oxygen controller ProOx-110 (BioSpherix) using gas of 5% CO2 and 95% N2 to reduce the oxygen content. Expression of DAPK or its mutants was performed by transfection of T cells with DAPK, [K42A]DAPK, or [ΔCAM]DAPK through electroporation using the MP-100 system (Life Technologies).HEK293T (ATCC CRL-3216), HeLa (ATCC CCL-2) and Jurkat T lymphoma  were obtained from ATCC. Cell lines were examined for mycoplasma contamination using Mycoplasma Detection Kit (R&D). Normal T cells and Jurkat cells were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum (Invitrogen Life Technology), 10 mM glutamine, 100 U mlDapk−/− miceHif1atm3Rsjo/J (Hif1af/f) miceRag1−/− mice (in C57BL/6 background), and C57BL/6-Tg1Kuch/J (2D2 TCRMOG) transgenic miceCd4-Cre mice (in C57BL/6 background) were purchased from Taconic Farms . Mice were maintained in the SPF mouse facility of the Institute of Molecular Biology, Academia Sinica. All mouse experiments were performed using 8–16-week-old mice. For in vivo experiments, sex-matched male and female mice were used. Donor and recipient mice were also sex-matched in T cells transfer assay. Because mouse experiments are limited by the availability of the knockout mice and their littermate control, no randomization was used. Mouse experiments were not blinded. All mouse experiments were conducted with approval from the Institutional Animal Care & Utilization Committee, Academia Sinica.Production of + T cells were purified by positive selection using a rat anti-mouse CD4 antibody (RL172.4) panning method. Naive CD4 T cells (CD4+CD25−CD44lowCD62Lhigh) were then sorted by FACSVantage SE (BD Bioscience). Naive CD4 T cells were then activated by 2 μg ml−1 plate-bound anti-CD3 (2C11) plus 1 μg ml−1 anti-CD28 (37.51). IL-2 (20 U ml−1) was added for Th0 differentiation; IL-12 (10 ng ml−1), IL-2 (5 ng ml−1) and anti-IL-4 (10 μg ml−1) were added to induce Th1 development; and TGF-β (2 ng ml−1), IL-6 (2 ng ml−1), anti-IL-2 (5 μg ml−1), anti-IL-4 (5 μg ml−1) and anti-IFN-γ (5 μg ml−1) were added to induce Th17 differentiation. Th17 cells were also induced by replacing IL-6 with IL-21 (100 ng ml−1) in the same mixture. After 3-to-5 days, Th1 and Th17 cells were re-stimulated with anti-CD3/CD28 or TPA/A23187. For intracellular staining, monensin (2 nM) was added 1 h after activation, and intracellular content of IL-17 and IFN-γ was determined after another 3 h. For mRNA expression, cells were harvested 3 h after TPA/A23187 stimulation and RNA was prepared. For ELISA, cell culture supernatants were collected 24 h after TPA/A23187 activation. Induced Treg (iTreg) cells were generated from CD4+CD25− T cells and activated by IL-2 (20 ng ml−1), TGF-β (5 ng ml−1), and plate-bound anti-CD3 (2 μg ml−1) plus anti-CD28 (1 μg ml−1) for 1-to-5 days.Mouse T cells were isolated from spleen and peripheral lymph nodes by negative selection using an anti-mouse Ig panning method. Total CD4Mycobacterium tuberculosis (Sigma), followed by intraperitoneal injection of 200 ng pertussis toxin  at day 0 and day 2. Mice were monitored daily for clinical signs and then clinically scored by the following criteria: 0, no syndrome; 1, limp tail; 1.5, tail paralysis and waddling gait; 2, weak tail and partial hind limb paralysis; 2.5, paralysis of one hind limb; 3, total hind limb paralysis; 3.5, complete hind limb and partial fore limb paralysis; 4, moribund state; 5, complete paralysis and death. Numbers of mice used in EAE experiments were based on those used in documented EAE studies275960DAPK[ΔCAM]-transgenic, DAPK-knockout, and their WT littermate control mice were immunized subcutaneously with 200 μg of MOG peptide 35–55  emulsified in CFA containing 400 μg et al.Dapk−/− mice were differentiated into Th17 cells by incubation with MOG35-55 peptide (20 μg ml−1), hTGF-β (3 ng ml−1), mIL-6 (20 ng ml−1), anti-IL-4 (5 μg ml−1), anti-IFN-γ (5 μg ml−1) and threefold γ-irradiated T-cell-depleted splenic cells. IL-23 (10 ng ml−1) was added 48 h after differentiation. Five days after initial Th17 differentiation, Th17 cells were re-stimulated with plate-bound anti-CD3/anti-CD28 (2 μg ml−1each) in the absence of exogenous cytokines for 48 h. 2D2 or 2D2 Dapk−/− naïve T cells were differentiated into Th1 by incubation with MOG35-55 peptide (20 μg ml−1), mIL-12 (10 ng ml−1), anti-IL-4 (10 μg ml−1) and threefold γ-irradiated T-cell-depleted splenic cells for 5 days. Th17 and Th1 cells were washed three times with cold PBS, and 5 × 106 Th17 cells or 3 × 106 Th1 cells were transferred intravenously into each Rag1−/− mouse. Recipient mice were injected i.p. with 200 ng of pertussis toxin at day 0 and day 2 after T cells transfer. The classical EAE scores were described as above. The non-classical  EAE was scored by the following criteriaEAE induced by adoptive transfer of 2D2 T cells was performed according to the protocol of Jäger Mice were perfused with phosphate buffer saline (PBS) followed by 4% (v/v) paraformaldehyde (PFA) in PBS. Spinal cords were isolated and fixed with 4% (v/v) PFA in PBS overnight at 4 °C. Spinal cords were dehydrated with 30% sucrose in PBS for 48 h at 4 °C. Spinal cords were embedded in optimal cutting temperature compound and serial cryosections (30 μM thick) were acquired. Tissue sections were stained with hematoxylin & eosin or luxol fast blue. Images were obtained on a Zeiss Axio Imager Z1 microscope  with an objective lens of plan-Apochromat 10 × /0.45 M27 and the camera of Axiocam 506. The channel was TL Brightfield and the depth of focus was at 5.43 μM.For staining with anti-IFNγ and anti-IL-17, PFA-fixed optimal cutting temperature-embedded sections were pretreated with sodium citrate buffer  at 55 °C for 30 min for antigen retrieval. Sections were washed and permeabilized with 0.3% (v/v) Triton X-100 in PBS at room temperature for 30 min. Sections were incubated in blocking buffer  at room temperature for 30 min. Sections were then incubated with primary antibodies diluted in blocking buffer for 48 h at 4 °C. The primary antibodies were anti-IL-17  and anti-IFNγ . Sections were washed and incubated with Alexa Flour 488 or 555 secondary antibodies  at room temperature for 2 h. Samples were then washed and mounted with DAPI Fluoromount-G buffer .Rorc, forward, 5′-CGG AGT TTT CCT TTG GGTG-3′ and reverse, 5′-CCG AGG TAT CTC AGT CAC GAA-3′; Rora, forward, 5′-GAA CAC CTT GCC CAG AAC AT-3′ and reverse, 5′-AGC TGC CAC ATC ACC TCT CT-3′; Il17a, forward, 5′-CTC ACA CGA GGC ACA AG-3′ and reverse, 5′-CTC AGC AGC AGC AAC AG-3′; Il17f, forward, 5′-GGG AAG AAG CAG CCA TTG-3′ and reverse, 5′-TCC AGG GGA GGA CAG TT-3′; Il21, forward, 5′-AAA CTC AAG CCA TCA AAC CC-3′ and reverse, 5′-GGT GTC CTT TTC TCA TAC GAA TC-3′; Il23r, forward, 5′-GCA GGA AGT ATT TGG TAT GGG -3′ and reverse, 5′-GAA ATG ATG GAC GCA GAA GG-3′; Actb, forward, 5′-GCC CCC CTG AAC CCT AA-3′ and reverse, 5′-GAC AGC ACA GCC TGG AT-3′; Tbx21, forward, 5′-CGT GGA GGT GAA TGA TGG-3′ and reverse, 5′-GTG ATC TCT GCG TTC TGG TAG-3′; Ifng, forward, 5′-ATC AGC AAC AAC ATA AGC GT-3′ and reverse, 5′-GAC CTC AAA CTT GGC AAT ACTC-3′; Hif1a, forward, 5′-CGG CGA GAA CGA GAA GAA-3′ and reverse, 5′-GCA ACT GAT GAG CAA GCT CATA-3′.T cells were harvested at the time indicated and total RNA was isolated using Trizol (Invitrogen). cDNAs were prepared and analysed for the expression of the gene of interest by real-time PCR using a SYBR-Green PCR master mix kit (Roche). The expression of each gene was normalized to the expression of β-actin. The sequences of the primers were: 2) containing 0.1% (primary T cells) or 0.2% NP-40 (for HeLa cells) for 4 min on ice. Cell lysates were centrifuged at 5,000 r.p.m. for 3 min and the supernatants obtained represented cytoplasmic extracts. Pellets were washed and lysed with RIPA buffer . The supernatants obtained after centrifuging at 13,200 r.p.m. for 10 min represented the nuclear extracts.Cells were harvested and lysed in cytoplasmic extract (CE) buffer  using transfer buffer  at 4 °C for 2 h at 400 mA. The membrane was treated with block buffer ) at room temperature for 1 h. The membrane was then incubated with primary antibodies overnight at 4 °C, followed by horseradish peroxidase-conjugated secondary antibodies in blocking buffer at room temperature for 1.5 h. The membrane was developed using ECL western blotting detection kit , and the chemiluminescence detected by X-ray film. Western blot images have been cropped for presentation. Full size images are presented in +CD25+) were purified by FACS sorting. For in vitro suppressive assays, CD4+CD25− cells (Teff) were placed into round-bottom 96-well plates at 4 × 104 cells per well (for nTreg) or 1 × 105 cells per well (for iTreg), together with anti-CD3 (1 μg ml−1), 3 × 105 γ-irradiated autologous presenting cells, and different ratios of Treg cells. Cells were incubated for 72 h, and the incorporation of [3H]thymidine was determined.nTregs and iTregs (CD4Cells were fixed with 4% (v/v) PFA in PBS, and permeabilized with 0.1% (v/v) Triton X-100. The fixed cells were incubated with blocking buffer (5% BSA in PBS) at room temperature for 1 h, followed by primary antibodies in blocking buffer overnight at 4 °C. The primary antibodies were anti-FLAG , anti-Myc , anti-human HIF-1α , anti-mouse HIF-1α , anti-DAPK  and anti-PHD2 . Cells were washed and incubated with Alexa Flour-labelled secondary antibodies, and were then mounted in DAPI Fluoromount-G buffer . Confocal images were obtained with a Zeiss LSM 780 confocal microscope  with an objective lens of Plan-Apochromat 63 × /1.4 Oil DIC M27 at room temperature. DAPI-bound DNA was excited at 405 nm and collected at 420 to 475 nm. Alexa Fluor 488 dye was excited at 488 nm and collected at 500 to 550 nm. Alexa Fluor 555 dye was excited at 555 nm and collected at 568 to 629 nm. The pinhole was at 53 μm, with a 9.8 μm section.+ T cells were activated with plate-bound anti-CD3 (2 μg ml−1) and anti-CD28 (1 μg ml−1) with the addition of IL-2 (100 U ml−1) for 48 h. Activated T cells were infected with retrovirus in the presence of 8 μg ml−1 polybrene by spinning at 2,000 r.p.m. for 1 h at room temperature. Infected T cells were cultured in complete RPMI medium with IL-2 (100 U ml−1) for 48 h. GFP+ cells were sorted by FACSVantage SE, and were cultured in complete RPMI medium plus IL-2 (100 U ml−1) for 2 days before hypoxic treatment.DAPK cDNA was cloned into retroviral vector GFP-RV and then transfected into HEK293T cells. Retroviruses were isolated from the culture supernatants of the transfected HEK293T cells by centrifugation at 26,000 r.p.m. for 2 h at 4 °C on a Beckman SW28 rotor. The viruses were resuspended in PBS containing 5% BSA overnight at 4 °C and stored at −80 °C. Purified WT CD4Escherichia coli, and recombinant GST-HIF-1α fusion proteins purified on a glutathione Sepharose 4B column. RK5F-DAPK (DAPK-FLAG) and pcDNA4-PHD2 (FLAG-PHD2) were transfected into HEK293T cells. Transfected cells were collected and lysed with WCE buffer . Recombinant FLAG fusion proteins were purified with anti-FLAG M2 agarose. Purified recombinant proteins were incubated in PBS at 4 °C for 4 h, and then incubated with anti-GST antibody overnight at 4 °C. Samples were incubated with protein G magnetic beads (GE) at 4 °C for 1 h. The immune complexes were washed with WCE buffer and examined by western blot.pGEX-4T3-HIF-1α (GST-HIF-1α) was transformed in BL21 (DE3) 2, 2 mM MnCl2, 0.5 mM CaCl2, 0.1 mg ml−1 bovine serum albumin (BSA), 1 μM bovine CaM (Sigma), 50 μM ATP, 10 μCi [γ-32P]ATP) at 25 °C for 15 min. The reactions were stopped by adding SDS sample buffer, and resolved on SDS–PAGE. The phosphorylation was detected by autoradiography.DAPK kinase assays were performed as previously describedt-tests were used to compare results from between two groups. Data were presented as mean with standard deviation (s.d.) or standard error of the mean (s.e.m.), as indicated in the figure legend.Our data were randomly collected but were not blinded. We did not exclude any data in this study. Our data mostly meet the assumption of the tests . Microsoft Office Excel and Prism 5.0 (GraphPad software) were used for data analysis. Unpaired two-tailed Student All relevant data are available from the authors upon request.How to cite this article: Chou, T.-F. et al. Tumour suppressor death-associated protein kinase targets cytoplasmic HIF-1α for Th17 suppression. Nat. Commun. 7:11904 doi: 10.1038/ncomms11904 (2016).Supplementary Figures 1-17


Soft set theory, proposed by Molodtsov, has been regarded as an effective mathematical tool to deal with uncertainties. Recently, uncertainty measures of soft sets and fuzzy soft sets have gained attentions from researchers. This paper is devoted to the study of uncertainty measures of fuzzy soft sets. The axioms for similarity measure and entropy are proposed. A new category of similarity measures and entropies is presented based on fuzzy equivalence. Our approach is general in the sense that by using different fuzzy equivalences one gets different similarity measures and entropies. The relationships among these measures and the other proposals in the literatures are analyzed. To solve complicated problems in economics, engineering, environmental science, and social science, methods in classical mathematics are not always successful because of various types of uncertainties presented in these problems. In 1999, Molodtsov –5.Works on soft set theory are progressing rapidly. Maji et al.  defined The measurement of uncertainty is an important topic for the theories dealing with uncertainty. The similarity measure, distance measure, entropy in fuzzy set theory, and the relationships among these measures have been extensively studied for their wide applications in image processing, clustering, pattern recognition, case-based reasoning, and many other fields –40. MajuThe aim of this paper is to propose a new category of similarity measures and entropies for fuzzy soft sets based on fuzzy equivalences. Our approach is general in the sense that by using different fuzzy equivalences one gets different similarity measures and entropies. The paper is organized as follows. In In this section, we recall some fundamental notions of soft sets, fuzzy soft sets, and similarity measures for fuzzy sets. See especially , 37, 47 U be the universe set and E the set of all possible parameters under consideration with respect to U. Usually, parameters are attributes, characteristics, or properties of objects in U.  will be called a soft space.Let F, A) is called a soft set over U, where A⊆E and F is a mapping given by F : A → P(U).A pair . For e ∈ A, F(e) may be considered as the set of e-approximate elements of . For illustration, we consider the following example of soft set.In other words, a soft set over U given by U = {h1, h2, h3, h4, h5, h6} and E = {e1, e2, e3, e4, e5} is the set of parameters. e1, e2, e3, e4, and e5 stand for the parameters “expensive,” “beautiful,” “wooden,” “cheap,” and “in the green surroundings,” respectively.Suppose that there are six houses in the universe F, E) may describe the “attractiveness of the houses” which Mr. X is going to buy. Suppose that F(e1) = {h2, h4}, F(e2) = {h1, h3}, F(e3) = {h3, h4, h5}, F(e4) = {h1, h3, h5}, and F(e5) = {h1}. Then the soft set  is a parameterized family {F(ei); 1 ≤ i ≤ 5} of subsets of U and gives us a collection of approximate descriptions of an object. F(e1) = {h2, h4} means “houses h2 and h4” are “expensive.”In this case, to define a soft set means to point out expensive houses, beautiful houses, and so on. The soft set  essentially specifies the degree to which x belongs to the fuzzy set μ. There are many different definitions for fuzzy set operations. With the min-max system proposed by Zadeh  is called a similarity measure if it satisfies the following properties.N1) = 0 and N = 1 for any A∈F(U).N2) = N for any A, B∈F(U).N3), N ≤ min, N) whenever A⊆B⊆C.For all Let Li et al.  proposedU = {x1,…, xn}, suppose Nθ and Nδ are functions defined for all A, B ∈ F(U) bya ≥ 0, b ≥ 0; then Nθ and Nδ are similarity measures.Given a discrete universe a and b, we can obtain some typical similarity measures for fuzzy sets 2 →  is called a fuzzy equivalence if it satisfies the following properties.E1) = E for all x, y∈ .E2) = 1 for all x∈ .E3) = E = 0.E4) ≤ E.For all A function E4) is equivalent to the following: for all x, y, z ∈ , if x ≤ y ≤ z, then E ≤ min, E).It is trivial to prove that  = x.M2) = (1/n)∑i=1nxi.The arithmetic mean: M = λ · min+(1 − λ)max.The convex linear combinations: M = max/ + max).As examples of aggregation operators, we can take the following.U = {x1,…, xn}, let M be an aggregation operator, E a fuzzy equivalence. Suppose Nτ : F(U) × F(U)→ is a function defined for all A, B ∈ F(U) byNτ is a similarity measure.Given a discrete universe S : F(U) × F(U)→ to be a similarity measure between fuzzy sets, we give a definition of a similarity measure for fuzzy soft sets as follows.Similarity measures quantify the extent to which different patterns, images, or sets are alike. Such measures are used extensively in the application of fuzzy sets. Also, it is essential to the application of fuzzy soft set theory. Based on the axioms  for a maS : FS × FS→ is called a similarity measure for fuzzy soft sets if it satisfies the following properties.S1) = 0 for any A⊆E, and S, ) = 1 for any  ∈FS.S2), ) = S, ) for any ,  ∈FS.S3), ,  ∈FS, if ⊆⊆, then S, ) ≤ min, ), S, )).For any , ) is a similarity measure, whereF, A),  ∈ FS.S1) For any A⊆E and  ∈ FS, we have (S2) is trivial.(S3) Let , ,  ∈ FS, and ⊆⊆. It follows that A⊆B⊆C and F(e)⊆G(e)⊆H(e) for each e ∈ A. Consequently, Nθ(F(e), H(e)) ≤ Nθ(F(e), G(e)), Nθ(F(e), H(e)) ≤ Nθ(G(e), H(e)). Thus we have, ) = 1 − (1/4n)∑j=1n(|SF(ei)(xj) − SG(ei)(xj)| + |tF(ei)(xj) − tG(ei)(xj)| + |fF(ei)(xj) − fG(ei)(xj)|), SF(ei)(xj) = tF(ei)(xj) − fF(ei)(xj), and SG(ei)(xj) = tG(ei)(xj) − fG(ei)(xj). In this similarity measure, if  and  are fuzzy soft sets, that is, tF(ei)(xj) = 1 − fF(ei)(xj), tG(ei)(xj) = 1 − fG(ei)(xj), then we haveWang and Qu  proposedS2, ) if A = B = E.We note that this similarity measure is a special case of F, E) and  be two fuzzy soft sets. The similarity measure M, ) is defined asMajumdar and Samanta  proposedE = {e1, e2}, F(e1) = U, G(e1) = ∅, F(e2) = G(e2). ByM, ) = 1. On the other hand, F(e1) and G(e1) are quite different. Clearly, S3 is an improved version of this measure.This measure does not conform to the idea of similarity measure. For example, let Sδ, ) is a similarity measure, whereF, A),  ∈ FS.It can be proved in the same manner with a = 0, b = 1; then we haveIn , let a =a = 0, b = 2; then we haveLet Sτ, ) is a similarity measure, whereF, A),  ∈ FS.The proof is similar to that of Sτ is determined by a fuzzy equivalence and an aggregation operator. Fuzzy equivalences can be constructed from biresiduations, automorphisms, t-norms, and t-conorms 2 →  is called a t-norm . The residuation of a t-norm T is the function x, y ∈  by ET of a t-norm T is a fuzzy equivalence 2 →  with boundary conditions φ(0) = 0 and φ(1) = 1 is called an automorphism . Then the function Eφ : 2 →  is a fuzzy equivalence . Taking the automorphisms φ1(x) = 2x/(1 + x),  φ2(x) = x2 and the fuzzy equivalence ETL, we obtain the following fuzzy equivalences.(4)E1 = 1 − |2x/(1 + x) − 2y/(1 + y)| = 1 − 2|x − y|/((1 + x)(1 + y)).(5)E2 = 1 − |x2 − y2|.A continuous, strictly increasing function morphism  of the iivalence , where EM and the fuzzy equivalences ETM, ETP, ETL, E1, and E2, respectively, then we have the following similarity measures for fuzzy soft sets:S9 = S2.If we take the arithmetic mean aggregation operator Sθ, Sδ, and Sτ, for fuzzy soft sets , , we only compare F(e) and G(e) for common parameter e ∈ A∩B. The parameters e ∈ (A − B)∪(B − A) have not been taken into account. According to the idea of soft equality  for any e∈A, where [0.5] is the fuzzy set with the membership function [0.5](x) = 0.5 for each x∈U.E3) be crisper than ; that is, for any e∈A and x∈U, F(e)(x) ≤ G(e)(x) if G(e)(x) ≤ 0.5 and F(e)(x) ≥ G(e)(x) if G(e)(x) ≥ 0.5. Then E ≤ E.Let (E4) = E, where  is the complement of fuzzy soft set  given by Fc(e) = (F(e))c for each e∈A.A real function Eθ is an entropy, whereF, A) ∈ FS.E1) We assume that  is a soft set. It follows that, for each e ∈ A and xi ∈ U, F(e)(xi) = 0 or F(e)(xi) = 1. Consequently,(Thus we haveE2) We assume that F(e) = [0.5] for any e ∈ A. It follows that F(e) = Fc(e) and hence Nθ(F(e), Fc(e)) = 1. Thus we have Eθ = Sθ, ) = 1.(E3) Let  be crisper than . By the definition, we have if yj ∈ , and f is decreasing with respect to yj   if yj ∈ . We note that F(e)(xi) ≤ G(e)(xi) if G(e)(xi) ≤ 0.5 and F(e)(xi) ≥ G(e)(xi) if G(e)(xi) ≥ 0.5. Thus we have Nθ(F(e), Fc(e)) ≤ Nθ(G(e), Gc(e)) and, consequently,Therefore, we can conclude that E4) is trivial. = Nθ. It follows that Nδ = Nθ and hence Sδ, ) = Sθ, ) for any  ∈ FS. Thus we have the following corollary.By the definition, we have Eδ is an entropy, whereF, A) ∈ FS.Eτ is an entropy, whereF, A) ∈ FS.E1) We assume that  is a soft set. It follows that, for each e ∈ A and xi ∈ U, F(e)(xi) = 0 or F(e)(xi) = 1. Consequently,Eτ = 0.(E2) We assume that F(e) = [0.5] for any e ∈ A. It follows that F(e) = Fc(e) and hence F(e)(xi) = Fc(e)(xi) = 0.5. Thus we have = Sτ, ) = 1.Consequently, E3) Let  be crisper than . For any e ∈ A, we haveG(e)(xi) ≤ 0.5, then F(e)(xi) ≤ G(e)(xi) and henceG(e)(xi) ≥ 0.5, then F(e)(xi) ≥ G(e)(xi) and henceE(F(e)(x1), 1 − F(e)(x1)) ≤ E(G(e)(x1), 1 − G(e)(x1)). We note that M is increasing in all of its arguments. Thus Nτ(F(e), Fc(e)) ≤ Nτ(G(e), Gc(e)) and in consequence Eτ ≤ Eτ.(E4) is trivial.(Si  (1 ≤ i ≤ 11), we can obtain the corresponding entropies Ei  (1 ≤ i ≤ 11). For example, we haveE2 = E11.Using similarity measures Soft set theory was originally proposed as a general mathematical tool for dealing with uncertainties. Majumdar and Samanta , 42 init


In the title solvate, the oxamide derivative presents the same conformation as in the unsolvated molecule, but the lattice solvent introduces disorder for various functional groups. et al. :0.304 (4); one tert-butyl group is disordered by rotation about the C—C bond, and was modelled with three sites for each methyl group, each one with occupancy 1/3. Finally, the methanol solvent, which lies on a twofold axis, is disordered by symmetry. The disorder affecting hydroxy groups and the solvent of crystallization allows the formation of numerous supramolecular motifs using four hydrogen bonds, with N—H and O—H groups as donors and the oxamide and methanol molecule as acceptors.The here crystallized oxamide was previously characterized as an unsolvated species [Jímenez-Pérez  al. 2000. J. Orga The involved OH group is disordered over two chemically equivalent positions on the benzene ring, C7 and C3. However, the most populated site, O3A, which has a site occupancy factor of 0.696 (4), is that forming this contact  motif are stabilizing the twisted conformation.The molecular conformation is not planar, and can be described using the dihedral angle between the oxamide core C1/O1/N1 and the benzene ring C2–C7. In the title solvate, this angle is 32.4 (2)°, slightly smaller than the same angle observed in the unsolvated crystal, 38.4°. A comparison of conformations stabilized for this molecule shows that a planar conformer is obtained only if amine and hydroxy groups are deprotonated to form a tetraanion, which is then able to coordinate a metal centre  with the amine groups. As a result, The introduction of methanol changes the original c Table 2. The meted Fig. 2. The lased Fig. 2, entry 4tert-butyl-2-hydroxyphenyl)oxamidate oxamido and 81 mg (0.342 mmol) of NiCl2·6H2O in methanol with a molar ratio 1:2 afforded a dark-brown solution. An amount of maleic acid , intended as a bridging ligand, was then added, changing the colour of the solution to light green. After a few minutes under stirring, a cottony precipitate formed. The solution was filtered and the filtrate allowed to crystallize by solvent evaporation, affording needle-shaped green crystals. The green colour is due to a thin layer of nickel chloride covering the crystals. Some of these crystals were washed with methanol, giving colourless crystals (m.p. 496-497 K), used for X-ray crystallography.The reaction of 100 mg (0.171 mmol) of disodium bis: 3501, 3355, 3274 , 1651 (C=O). 1H NMR  δ, p.p.m.: 9.54 , 7.57 , 7.32 , 7.15 , 3.51 , 1.61 , 1.48 , 1.33 . 13C NMR  δ, p.p.m.: 157.28 , 145.82 , 143.31 , 139.77 , 123.99 (C—N), 123.17 , 117.71 , 35.40 , 34.41 , 31.45 , 30.97 (CH3OH), 29.83 .Spectroscopic data: FT–IR  and 0.304 (4), respectively. Finally, the methanol molecule is disordered by symmetry over a twofold axis, and its occupancy was fixed at 1/2. The geometry for this molecule was restrained with bond length C21—O21 = 1.45 (1) Å. All H atoms bonded to C atoms were placed in idealized positions and refined as riding atoms, with C—H bond lengths fixed to 0.93 (aromatic) or 0.96 Å (methyl groups) and Uiso(H) = xUeq(C) with x = 1.2 (aromatic) or 1.5 (methyl groups). H atoms bonded to heteroatoms were found in difference maps and refined with restraints applied to the O—H bond lengths: 0.90 (1) Å (methanol) and 0.85 (2) Å (hydroxy). For these H atoms Uiso(H) = xUeq(carrier atom), with x = 1.2 (NH) or x = 1.5 (OH).Crystal data, data collection and structure refinement details are summarized in Table 2/3 Fig. 1, inset. 10.1107/S205698901600880X/su5302sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S205698901600880X/su5302Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S205698901600880X/su5302Isup3.cmlSupporting information file. DOI: 1482681CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Artocarpus heterophyllous (AH) potently inhibited tyrosinase activity.Tyrosinase is an oxidoreductase that is very important in medicine and cosmetics because the excessive production of melanin causes hyperpigmentation. The development of novel, effective tyrosinase inhibitors has long been pursued. In preliminary tests, we found that an extract of the wood of 1) and artocaepin F (2), were isolated from the wood of AH, together with norartocarpetin (3), artocarpanone (4), liquiritigenin (5), steppogenin (6), and dihydromorin (7). Their structures were elucidated using one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) and mass spectrometry. The absolute configuration of 2 was determined from the circular dichroism (CD) spectrum. Artocarpanone (4) had the most potent tyrosinase inhibitory effect, with an IC50 of 2.0 ± 0.1 μM, followed by artocaepin E (1) and steppogenin (6), with IC50 values of 6.7 ± 0.8 and 7.5 ± 0.5 μM, respectively. A kinetic investigation indicated that 1 showed competitive inhibition, with an inhibition constant (Ki) of 6.23 μM.Two new flavonoids, artocaepin E  contains supplementary material, which is available to authorized users. This o-quinone is a highly reactive compound that can polymerize spontaneously to form melanin + , consistent with the molecular formula C29H24O9. The infrared (IR) spectrum suggested the presence of hydroxy (3410 cm−1), phenyl , and carbonyl  groups. The 1H NMR spectrum of 1 included signals due to two sets of ortho-coupled aromatic protons at δH 7.53  and 6.78 , three aromatic protons of the typical ABX system at δH 6.49 , 7.73 , and 6.44 , an isolated aromatic proton at δH 6.51 , two trans olefinic protons at δH 7.54  and 6.39 , and two isolated olefinic protons at δH 6.98  and 5.55 , together with one methyl, one methylene, one oxymethylene, and a characteristic signal of a hydrogen-bonded hydroxyl proton at δH 13.34   and heteronuclear multiple bond correlation (HMBC) spectra  with the C-9′″ (δC 166.5) ester carbonyl carbon of the trans-p-coumaroyl group. The nuclear Overhauser effect spectroscopy (NOESY) correlations of H-1″ with H-5″ and of H-2″ with H-4″ indicated an E-configuration for the C-2″/C-3″ double bond of 1. Therefore, the structure of artocaepin E was concluded to be 1.Compound 2) was isolated as a yellow amorphous solid. The molecular formula of 2 was determined to be C20H20O5 by HR-ESI–MS. The IR spectrum showed absorption bands of hydroxy (3400 cm−1), phenyl , and ketone (1705 cm−1) groups. The 1H NMR spectra of 2  and 6.90 , and others at δ 7.59  and 6.63 , together with two methylene signals at δH 2.70  and 3.00 , and one oxymethine at δH 5.44 , which are typical of the flavanone skeleton D25 −10.0° ; IR νmax (CHCl3) 3365, 1630, 1600, 1510 cm−1; 1H and 13C NMR (acetone-d6 500 MHz) see Table m/z 363.1224 .Artocaepin F -1 µg/mL (or µM for pure compounds). The tyrosinase inhibitory activity assay was performed as previously described by Arung et al. [l-DOPA in 0.1 M phosphate buffer pH 6.8). The assay mixture was incubated at room temperature for 7 min, and the absorbance at 475 nm was measured with a Shimadzu UV-1800 spectrophotometer. Kojic acid, a known tyrosinase inhibitor, was used as positive control. Tyrosinase inhibitory activity was expressed as the percentage inhibitory of enzyme tyrosinase in the above assay system, calculated as (1 − B/A) × 100, where A and B are the activities of the enzyme without and with test material. IC50 values were calculated from the mean values of data from four determinations.All the samples were first dissolved in DMSO and used for the actual experiment at concentrations of 100g et al. . The ass50, except that uninhibited and inhibited reactions were observed for three different concentrations of l-DOPA  at 30 °C in 0.1 M phosphate buffer pH 6.8. The dependence of absorbance (475 nm) on time was measured, and the reaction rate was calculated for all reactions (uninhibited and inhibited). Then, a Lineweaver–Burk plot was constructed, and Km and Vm values were calculated. Each measurement was performed in duplicate.The procedure for determination of the inhibition mechanism was similar to that for determination of IC1) and artocaepin F (2), together with five known compounds: norartocarpetin (3), artocarpanone (4), liquiritigenin (5), steppogenin (6), and dihydromorin (7). Regarding tyrosinase inhibition, artocarpanone (4) had the greatest inhibitory effect, followed by artocaepin E (1) and steppogenin (6). Liquiritigenin (5) also showed significant concentration-dependent inhibition. Kinetic studies indicated that the new active compound artocaepin E (1) displayed competitive inhibition. These results suggest that these compounds may serve as structural templates for the design and development of novel tyrosinase inhibitors as effective anti-browning agents in cosmetics.In this study, we identified two new flavonoids from the wood of AH, artocaepin E (


The neuropeptide substance P (SP) exhibits cytokine-like properties and exerts different effects in autoimmune inflammation. Various immune cells express SP and its neurokinin-1 receptor (NK1R) isoforms. A role for SP has been demonstrated in a number of autoimmune conditions, including multiple sclerosis (MS). In this work, we studied the role of SP and NK1R in human immune cells with a focus on their relationship with IL-12/IL-23 family cytokines and the associated IFN-γ/IL-17. AIMS: (1) To determine the role of SP mediated effects on induction of various inflammatory cytokines in peripheral blood mononuclear cells (PBMC); (2) to investigate the expression of SP and its receptor in T cells and the effects of stimulation with IL-12 and IL-23. Quantitative real-time PCR, flow cytometry, ELISA, promoter studies on PBMC and primary T cells from healthy volunteers, and Jurkat cell line. Treatment with SP significantly increased the expression of IL-12/IL-23 subunit p40, IL-23 p19 and IL-12 p35 mRNA in human PBMC. Expression of NK1R and SP in T cells was upregulated by IL-23 but a trend was observed with IL-12. The IL-23 effect likely involves IL-17 production that additionally mediates IL-23 effects. Mutual interactions exist with SP enhancing the cytokines IL-23 and IL-12, and SP and NK1R expression being differentially but potentially synergistically regulated by these cytokines. These findings suggest a proinflammatory role for SP in autoimmune inflammation. We propose a model whereby immunocyte derived SP stimulates Th1 and Th17 autoreactive cells migrating to the central nervous system (CNS), enhances their crossing the blood brain barrier and perpetuates inflammation in the CNS by being released from damaged nerves and activating both resident glia and infiltrating immune cells. SP may be a therapeutic target in MS.The online version of this article (doi:10.1007/s11481-015-9589-x) contains supplementary material, which is available to authorized users. Substance P (SP) is an ubiquitously found 11-amino acid peptide with a number of biological functions. It belongs to the family of tachykinins, which also includes neurokinin A, neurokinin B, neuropeptide K, neuropeptide Y, and hemokinin-1. SP is generated through the enzymatic cleavage of pre-pro-tachykinin A, which is encoded by the TAC1 gene. Splice variants of TAC1 also generate neurokinin A and neuropeptide K. The role of SP in pain pathways and neurogenic inflammation has been well established, including effects of vasodilatation and plasma extravasation. With its effects on smooth muscle, SP is known to be involved in motility of different organ tracts as well as stimulation of glandular secretion and modulating autonomic reflexes.SP originates from several cellular sources. It is released by peripheral nerve endings and central terminals of sensory neurons in the central nervous system (CNS). In addition, it is produced by a variety of cells of the immune system. This, and the presence of its receptor, the neurokinin-1 receptor (NK1R) on cells of the immune system, suggest a role for SP in immune responses. Immunomodulatory properties and its role in autoimmune inflammation have only recently been investigated in more detail. A role for SP has been demonstrated in neuroimmune and systemic autoimmune/inflammatory conditions, including multiple sclerosis (MS), rheumatoid arthritis and inflammatory bowel disease , expressed in monocytes and other peripheral cells .SP and NK1R are involved in neuroimmune communication, being abundantly expressed in the nervous system and its sensory nerve projections into peripheral immune organs, and immune cells themselves . SP is involved in both innate and adaptive immune responses. The functions of SP involve induction of cytokines (see below), activation of immune cells  in 10 % FCS/RPMI media. After media change, cells were stimulated with 1000 U/ml IL-2 (PeproTech) at 37 °C for 24 h. The resulting cells “blasts” are typically >95 % CD3+.6 cells/well).CD4+ cells were magnetically isolated using the human CD4+ T cell Isolation Kit , following the manufacturer’s protocol. These cells were then transferred into a 24-well plate pre-coated overnight with 0.5 ml/well of a mixture of anti-CD3 (0.5 μg/ml) (BD Pharmingen) and anti-CD28 (2.5 μg/ml) (BD Pharmingen) in PBS. The coated wells were washed with 1 ml RPMI medium prior to CD4+ cells transfer  was used for NK1R promoter-reporter assay.6/ml were stimulated for 24 and 48 h with 10−6–10−12 M SP (Sigma-Aldrich) with and without the presence of NK1R antagonist CP-96345 (Pfizer) 10−5–10−6 M, its inactive enantiomer CP-96344 (Pfizer) 10−5–10−6 M, anti-NK1R antibody  1:500 or 1:1000, lipopolysaccharide  1 μg/ml. After stimulation, the supernatants were removed and stored at -80 °C for ELISA. Stimulations with cytokines were carried out at the following final concentrations: 10 ng/ml IL-12 (R&D Systems) with and without 2.5 μg/ml anti-IFN-γ antibody (PeproTech EC), 10 ng/ml IL-23 (R&D Systems) with and without 2.5 μg/ml anti-IL-17 antibody (PeproTech), 10 ng/ml IL-17 (PeproTech), 10 ng/ml IFN-γ (PeproTech) for 8 and 24 h for mRNA-level, and 24 and 48 h for protein-level expression.PBMC 10Flow cytometry was performed for protein level expression of NK1R in PHA-stimulated PBMC (T cell blasts). After overnight incubation, stimulated T cell blasts were transferred into FACS tubes and 20 μl EDTA 100 mM (Sigma-Aldrich) were added to each tube and incubated for 5 min at room temperature, the cells were then fixed using formaldehyde 4 % (1 ml/tube) and left for 5–10 min at room temperature prior to washing three times, each with 1 ml PBA , stained with 2.5 μl of monoclonal surface antibodies  and incubated for 40 min in the dark at 4 °C. The cells were then washed once with PBA and twice, each with 1 ml perm/wash buffer (BD Biosciences), stained with 10 μl anti-NK1R-APC antibody  and its isotype control  and incubated in the dark at room temperature for 30 min. Cells were washed once with perm/wash buffer, fixed with 0.4 ml of 0.5 % formaldehyde and analysed.Regarding CD4+ cells staining, 20 μl EDTA (100 mM) and 1 ml formaldehyde 4 % were added as above. The cells were washed three times with 1 ml perm/wash buffer and 10 μl anti-NK1R-APC antibody were added. The cells were incubated for 30 min in the dark at room temperature, washed once with 1 ml perm/wash buffer, and fixed with 0.4 ml formaldehyde 0.5 %. For flow cytometry experiments, FC500 (Beckman Coulter) flow cytometer and Weasel 3.1 software were used. For data presentation, GraphPad Prism 6.0 software was used. The quadrants were set in flow cytometry plots using single colour control tubes, fluorescence minus one (FMO) tubes, and isotype control tubes.Commercial kits were used for measuring concentrations of IL-1β (R&D Systems) and the different IL-12 and IL-23 subunits in stimulated PBMC culture supernatants, as well as SP in T blast supernatants. IL-12p40 (Diaclone), IL-12p70 (eBioscience) and IL-23 p19/p40 (eBioscience) kits were used following manufacturer’s instructions with assay sensitivities specified as follows: below 2.58 pg/ml for IL-1β, below 20 pg/ml for IL-12p40, below 15 pg/ml for IL-23 p19/p40 and 4 pg/ml for IL-12p70. For measuring SP in T blast supernatants, Parameter™ SP ELISA kit (R&D Systems) was used according to the instructions with 31.5 pg/ml as the mean minimum detectable concentration of SP.Quantitative real-time reverse transcriptase PCR was used to assess mRNA abundance of IL-12, IL-23 subunits in PBMC on stimulation with SP, and mRNA abundance of SP precursor and NK1R isoforms in PHA/IL-2 T blasts that had been stimulated with IL-12, IL-23, IL-17, IFN-γ. Total-RNA was extracted by column extraction using RNeasy Miniprep Kit® (Qiagen) following the manufacturer’s instructions: the homogenization step was performed by needle homogenization. First strand cDNA synthesis was initiated from 0.5 μg total RNA using random primers  and MMLV reverse transcriptase (Promega) using conditions specified by the manufacturer in a final volume of 25 μl. The following specific primer sequences were used for IL-23p19 sense: 5′ctccctgatagccctgtg3′, antisense: 5′gactgaggcttggaatct3′; for IL-12p40 sense: 5′ggagtaccctgacacctg3′, antisense: 5′agatgaccgtggctgagg3′; for IL-12p35 sense: 5′ccactccagacccaggaatg3′, antisense: 5′gacggccctcagcaggt3′; for NK1R-F (full length): sense 5′gaatgaggacagtgacgaac3′, antisense 5′ttgtggaacttgcagtagaac3′; for NK1R-T (truncated): sense 5′tcttcttcctcctgccctacatc3′; antisense 5′tggagagctcatggggttggatcct3′; for TAC1: sense 5′tcaatgggcaatgacaggta3′, antisense 5′tccgcagtagctgacacaac3′. As an internal standard, β2-microglobulin (β2MG) with the following primers was used: sense 5′ctccgtggccttagctgtg3′, antisense 5′tttggagtacgctggatagcct3′ . For primer design Primer 3 software was employed. Real-time PCR reactions were carried out using the SYBR Green fluorescence method with SYBR Green Mastermix (Stratagene) as specified by the manufacturer. All real-time PCR reactions were carried out in triplicate on multiplex qPCR system Mx4000® (Stratagene) using default thermal cycling conditions. Dissociation curves were included routinely for each reaction for assessing product homogeneity. For quantitation of transcripts, the relative standard curve method, described by Applied Biosystems (AppliedBiosystems 7 cells/400 μl ice-cold PBS were transferred into each electroporation cuvette (Invitrogen) and kept on ice for 10 mins. Subsequently, 3 μg prepared NK1R promoter-reporter plasmid  and 0.3 μg Renilla reniformis luciferase reporter vector (Promega) as an incorporation control were added per cuvette and pulsed on Gene Pulser X cell™ (Biorad) with 250 V and 950 μF. After electroporation, cells were kept in 10 % FCS/RPMI overnight at 37 °C, 5 % CO2. Following stimulation of transfected cells with cytokines as above for 24 h, cells were washed with PBS and passively lysed according to the Dual-Luciferase Reporter Assay protocol (Promega), whilst kept on a shaker for 15 min. The lucifearase activities of each lysate were sequentially read on TD-20/20 single-sample luminometer (Turner Designs) by adding initiation/stop reagents (Promega) per protocol. Background luminescence of non-transfected control was subtracted from individual sample values and ratios of Photinus/Renilla luciferase signals were calculated.The vector for the NK1R promoter activation assay was prepared by transforming E coli DH5α –T1 competent cells (Invitrogen) with human NK1R gene promoter 1,837-bp fragment, cloned into luciferase expression vector pGL3-Basic (Promega) . For incorporation of the vector into Jurkat cells, 2 × 10p was ≤0.05.Data were analysed using nonparametric tests: Friedman test for comparisons of means of ordinal data, or Wilcoxon test for continuous variables. SPSS 16.0 software was employed for analysis. Differences were considered statistically significant when 6/ml) were stimulated with SP 10−6, 10−9 and 10−12 M, and IL-1β and IL-12p40 were measured by ELISA after 36–48 h. SP alone was unable to induce detectable IL-1β, unless co-stimulation with bacterial lipopolysaccharide (LPS) was used (data not shown). SP 10−6 M enhanced IL-12p40 from 53 (±36) pg/ml to 135 (±2) pg/ml (p = 0.02). Lower concentrations of SP failed to induce IL-12p40 in the absence of co-stimulation with LPS (data not shown). Because the amounts of IL-12p40 are likely to be too low to correspond to detectable levels of IL-12p70 or IL-23 protein by ELISA, we measured the mRNA expression of the IL-12 family cytokines.PBMC (10p = 0.043), followed by p19 and p35 induction with a 2.7-fold (p = 0.068) and 2.3-fold (p = 0.043) increase in their expression respectively.To assess IL-12 and IL-23 induction by SP in PBMC, experiments with a 24-h induction of the IL-12/23p40, IL-12p35 and IL-23p19 subunit mRNA were done on 5 healthy volunteers. The expression of the three IL-12 and IL-23 subunits showed a similar response to stimulation with SP and suppression by CP-96345 (specific NK1R antagonist), but not by CP-96344 (control with no antagonist properties). SP strongly upregulated the expression of the subunits to a degree similar to LPS Fig. . The indThe product homogeneity and size for each of the conditions was confirmed on agarose gel electrophoresis with a product size of 131 bp for IL-12/23p40, 62 bp for IL-12p35, 109 bp for IL-23p19 and 148 bp for β2MG.The expression of the NK1R receptor on subgroups of human immune cells is not well known. We determined this expression by flow cytometry using a directly conjugated antibody. The percentages of NK1R+ cells in PHA-stimulated T cells (“blasts”), and subgroups of blasts based on expression of cell surface markers CD3, CD8, and CD56, unstimulated or stimulated with IL-12 or IL-23 for 24 h, and gated for CD3  can be found in supplemental Table Both IL-12 and IL-23 appeared to modestly  increase NK1R in PHA-stimulated T cells (“blasts”), and subgroups expressing CD3, CD8, and CD56 .The results shown so far, regarding NK1R, refer to the full-length transcript. We have examined the NK1R isoform expression in T cell blasts with similar expression of the truncated (NK1R-T) versus full-length NK1R  isoform in unstimulated T cell blasts  and TAC1 transcripts (p = 0.022) .The effects of different cytokine stimuli on SP precursor and NK1R mRNA level expression were studied in PHA/IL-2-stimulated T cell blasts from 10 healthy volunteers. IL-23 significantly upregulated both NK1R (22) Fig. . There wp = 0.037) but not TAC1 (p = 0.203) expression was also significantly upregulated by IL-17A (here referred to as IL-17). Anti-IL-17 abolished the effects of IL-23 in inducing TAC1 (p = 0.041), but not NK1R (p = 0.262) , and induction by IL-12 showed only a trend (p = 0.063).However, in the experiments done on CD4+ cells, a slightly greater tendency for NK1R induction by IL-12 was seen at 24 h as compared to 8 h, and in SP induction by IL-23 at 8 h as opposed to 24 h , lower than the individual ELISA, had been also tested before ELISA measurements and found insufficient to detect the heterodimeric cytokines in our series (data not shown). Small effects of SP on IL-12 secretion are consistent with the published report  may play a role.As pointed out in a recent review on SP  . In the CNS, SP stimulating cytokines, produced by infiltrating T cells and monocytes, and SP itself further stimulate both CNS-intrinsic cells with immune functions such as microglia and astrocytes. Consistent with this, SP-immunoreactive astrocytes are found in MS lesions . The results are given as means and SEM of the percentage of cells expressing NK1R. (DOCX 13 kb)Supplemental Figure 1NK1R expression in: PHA/IL-2 stimulated PBMCs (“blasts”) (Histograms A-H) and magnetically isolated CD4+ lymphocytes (Histogram I). Cells Gating: (A), non-gated; (B), CD56+ cells; (C), CD3+CD8- lymphocytes; (D), CD3+CD8+CD56-lymphocytes; (E), CD3-CD56+ cells; (F), CD3+CD56+ lymphocytes; (G), CD3+ lymphocytes; (H), CD8+ lymphocytes; (I), Lymphocytes. Colour legend: Black: Unstimulated cells; Red: IL-12-stimulated; Blue: IL-23-stimulated; Pink: Isotype control-bound cells. NK1R-APC: Allophycocyanin-conjugated mouse monoclonal anti-human NK1R (GIF 299 kb)High Resolution Image(TIFF 320 kb)Supplemental Figure 2Dual-luciferase reporter assay with activation of NK1R promoter-reporter construct in Jurkat cells. The results represent mean ratios of Photinus/Renilla luminescence of two experiments after 24 h stimulation with different conditions. (GIF 104 kb)High Resolution Image(TIFF 36 kb)Below is the link to the electronic supplementary material.


Ischemic preconditioning (IPC) induced cardioprotection has been reported to be blunted in hyperlipidemic subjects. Dopamine, via its D2 receptor signaling, appears to mimic the signaling cascade involved in myocardial preconditioning and is also involved in the inhibition of hyperlipidemia induced mediators. The present study was designed to investigate the possible involvement of D2 receptors in IPC and to see whether dopamine preconditioning can offer cardioprotection in hyperlipidemic rat hearts.Wistar albino rats were divided into 8 groups and fed on normal or high fat diet for 4 weeks. Hyperlipidemia was confirmed after 4 weeks by serum lipid estimations. Isolated perfused hearts were subjected to ischemic preconditioning or dopamine induced pharmacological preconditioning followed by 30-min ischemic insult and 60-min reperfusion. Clozapine was administered as D2 antagonist. Coronary perfusate  was collected for the estimations of LDH (Lactate dehydrogenase) and CKMB (Creatine kinase MB). Hearts were then removed and frozen for infarct size measurement.A significant increase body weight, serum lipids except HDL was noted in high fat diet fed rats, as compared to normal rats. The level of LDH, CKMB in coronary effluent and infarct size were found to be decreased in preconditioned normal hearts, as compared to hearts treated with ischemia reperfusion. This effect was found to be blunted in hyperlipidemic animals. Dopamine (10 μM) alone and in combination with ischemic preconditioning significantly reduced the levels of LDH, CKMB and infarct size in hyperlipidemic hearts, as compared to preconditioned and non-preconditioned hyperlipidemic hearts. This effect was abolished significantly by Clozapine (D2 antagonist).The present study reveals possible involvement of D2 receptors in ischemic preconditioning and suggests that dopamine preconditioning may offer significant cardioprotection in hyperlipidemic rat hearts. ATP channels and mitochondrial permeability transition pore [Ischemic Preconditioning (IPC) is a protective phenomenon which provides resistance to myocardium against ischemic insult after brief episodes of sub-lethal ischemia and reperfusion . IPC reqion pore –9 therebion pore . Howeverion pore .Hyperlipidemia is a pathological state which is characterized by elevated levels of serum lipids and TGs, subsequently resulting atherosclerosis, coronary heart disease, hypertension, myocardial infarction and stroke. The clogged arteries (due to the deposition of lipids) and released endogenous cell mediators in hyperlipidemic state exaggerate the myocardial ischemic injury . The relin-vitro report revealed the involvement of D2 receptors in protection of cardiomyocytes against ischemia reperfusion induced apoptotic damage [1-adrenergic receptors [Dopamine is a monoamine neurotransmitter having diverse effects on human physiology. It is used intravenously to treat congestive heart failure. The dopamine receptors are of 5 subtypes: D1, D2, D3, D4 and D5 which are metabotropic G protein-coupled receptors (GPCRs). All the 5 subtypes are reported to be present in human heart, except D3 . The invc damage . A reporeceptors . Dopamineceptors –22 whicheceptors , 13. Thead libitum. The animals were allowed to acclimatize to the environment for 7 days prior to the experiment. The experiment was conducted in accordance with the guidelines of Committee for the purpose of control and supervision of experiments on animals (CPCSEA) and was approved by Institutional animal ethical committee (IAEC) vide ref. no. IAEC/SU-PHARM/12/016.Wistar albino rats of either sex weighing 160-180 g were obtained from the animal house, Shoolini University, Solan, HP, India (Reg. No. 1541/PO/a/11/CPCSEA) and maintained at 25 ± 2 °C temperature, 45 ± 5 % humidity, food and water The animals were divided into 8 groups, each comprising eight animals (n = 8) except two groups which were fed on normal chow diet and comprising six animals in each group (n = 6). The animals were fed on high fat diet (HFD) for 4 weeks. The success rate of hyperlipidemia using HFD in Wistar rats is 80-90 %. High fat diet was prepared using following ingredients: Powdered Normal chow (365 g/Kg), Butter (310 g/Kg), Casein (250 g/Kg), Cholesterol (10 g/Kg), Vitamin and mineral mix (60 g/Kg), DL-Methionine (03 g/Kg), Yeast powder (01 g/Kg) and Sodium chloride (01 g/Kg) i.p.) 20 min. prior to the experimentation. The animals were then sacrificed by cervical dislocation. Thorax was surgically opened; hearts were rapidly excised and placed immediately in cold perfusion buffer before being mounted on Langendorff’s apparatus. The isolated heart preparation on Langendorff’s apparatus was provided with retrograde perfusion of oxygenated (95 % O2 - 5 % CO2) normothermic (37 °C) Krebs-Henseleit (KH) buffer solution at a constant pressure of 100 cm H2O. The heart was then mounted over assembly within the time interval of 1 min after excision. The composition of KH buffer used was (g/L): 2.1 NaHCO3, 0.35 KCl, 0.28 NaCl, 1.28 MgSO4, 0.16 KH2PO4, 0.28 CaCl2 and 2.0 Glucose; (pH 7.4). The hearts were allowed to stabilize for 10 min before undergoing any treatment. The period of 30 min of ischemia followed by 120 min of reperfusion was used to induce ischemia-reperfusion injury.After the induction of hyperlipidemia for 4 weeks, rats were administered with heparin   Fig. . The firAfter 4 weeks of HFD treatment, % change in body weight and level of serum lipid markers: total cholesterol, triglycerides, LDL, HDL and VLDL were estimated as an index of hyperlipidemia using biochemical kits from Transasia Bio-medical Ltd., Baddi, India.Coronary effluent was collected after the stabilization period, and during first 5 min of period of reperfusion and the level of lactate dehydrogenase (LDH) and creatine kinase MB (CKMB) were estimated using biochemical kits from Transasia Bio-medical Ltd., Baddi, India.Coronary flow Rate (CFR) was assessed as a parameter for cardiac function by collecting the coronary effluent during the stabilization period and at the end of 120 min of reperfusion. It was expressed in mL/min.After the experiment, the hearts were removed and frozen to facilitate slicing into 2 mm thick transverse sections across the long axis. The slices were incubated in 1 % triphenyl tetrazolium chloride (TTC) in phosphate buffer (pH 7.4) for 30 min at 37 °C. The slices were then immersed in 10 % formalin solution to enhance the contrast between unstained and stained cardiac tissue. The stained slices were observed for cardiac injury. The tissue stained brick red was considered viable and pale coloured tissue was considered as damaged. The slices were photographed and infarcted area was calculated by planimetry using ‘ImageJ’ (version 1.47) software from the National Institute of Health (NIH), USA. Infarct size was expressed as a percentage of left ventricular volume.post hoc analysis to determine statistical difference between groups using ‘GraphPad Prism’ (version-6.02) software. P value < 0.05 was considered to be statistically significant.Results were expressed as mean ± SD analysed by Student t-test (for body weight and lipid profile), One-way analysis of variance (ANOVA) (for infarct area) and Two way analysis of variance (ANOVA)  followed by Bonferroni’s multiple comparison test as p < 0.05) in % increase in body weight in HFD treated animals (53.52 ± 4.54) as compared to animals treated with normal chow diet (19.63 ± 3.78)  in the levels of total cholesterol and triglycerides in the HFD treated animals (182.39 ± 7.9 and 153.16 ± 7.7 respectively) as compared to animals treated with normal chow diet (68.91 ± 5.05 and 80.96 ± 4.96 respectively) Fig. .Fig. 4(p < 0.05) in the serum levels of LDL and VLDL whereas a significant decrease (p < 0.05) in the levels of HDL was observed in the HFD treated animals  as compared to normal diet treated animals   Fig. .p < 0.05) in LDH level (408.69 ± 23.66), as compared to its basal value (47.50 ± 5.27). IPC group showed significant decrease (p < 0.05), whereas HL-IR group showed significant increase (p < 0.05) in LDH levels (193.32 ± 9.03 and 463.96 ± 20.18 respectively), as compared to IR treated group (408.69 ± 23.66). HL-IPC group showed significant decrease in LDH level (370.30 ± 14.70), as compared to HL-IR group (463.96 ± 20.18) and a significant increase, as compared to IPC (193.32 ± 9.03)(p < 0.05). However, HL-DA and HL-DA-IPC groups showed significant decrease (p < 0.05) in LDH levels (180.30 ± 8.47 and 166.05 ± 7.81 respectively), as compared to both HL-IR (463.96 ± 20.18) and HL-IPC groups (370.30 ± 14.70). A significant elevation in LDH level was also observed in HL-CZ-IPC (393.85 ± 14.46), as compared to HL-IPC group (370.30 ± 14.70) (p < 0.05). HL-CZ-DA-IPC group also produced a significant increase (p < 0.05) in LDH level (381.43 ± 12.47), as compared to HL-DA-IPC group (166.05 ± 7.81), however the reduction in LDH levels in HL-CZ-DA-IPC group (381.43 ± 12.47), as compared to HL-CZ-IPC (393.85 ± 14.46) was insignificant (p > 0.05). The levels of LDH in HL-CZ-IPC and HL-CZ-DA-IPC groups were also found to be significantly lesser (p < 0.05), as compared to HL-IR  in CKMB level (145.12 ± 8.89) as compared to its basal value (21.57 ± 1.95). IPC group showed significant decrease (p < 0.05), whereas HL-IR group showed significant increase (p < 0.05) in CKMB level (69.74 ± 5.66 and 164.92 ± 11.82 respectively), as compared to IR treated group (145.12 ± 8.89). HL-IPC group showed significant decrease in CKMB levels (117.12 ± 7.64), as compared to HL-IR group (164.92 ± 11.82) and a significant increase, as compared to IPC (69.74 ± 5.66)(p < 0.05). However, HL-DA and HL-DA-IPC groups showed significant decrease (p < 0.05) in the levels of CKMB (66.51 ± 4.89 and 61.48 ± 3.64 respectively), as compared to both HL-IR (164.92 ± 11.82) and HL-IPC groups (117.12 ± 7.64). A significant elevation in CKMB levels was also observed in HL-CZ-IPC group (134.01 ± 8.05), as compared to HL-IPC (117.12 ± 7.64) (p < 0.05). HL-CZ-DA-IPC group also showed a significant increase (p < 0.05) in CKMB level (126.13 ± 7.12), as compared to HL-DA-IPC (61.48 ± 3.64), however the reduction in CKMB levels in HL-CZ-DA-IPC group (126.13 ± 7.12), as compared to HL-CZ-IPC (134.01 ± 8.05) was insignificant (p > 0.05). The levels of CKMB in HL-CZ-IPC and HL-CZ-DA-IPC groups were also found to be significantly lesser (p < 0.05), as compared to HL-IR  in coronary flow rate levels (6.85 ± 0.59 and 6.11 ± 0.45 respectively), as compared to their basal values (10.48 ± 0.78 and 11.43 ± 0.67 respectively). However, a significant increase in CFR of IPC (9.91 ± 0.57), as compared to IR (6.85 ± 0.59) and in CFR of HL-IPC (7.46 ± 0.83), as compared to HL-IR (6.11 ± 0.45) were observed (p < 0.05). HL-DA and HL-DA-IPC groups also showed significant increase in CFR levels (10.93 ± 0.69 and 11.18 ± 0.69 respectively), as compared to HL-IR (6.11 ± 0.45) and HL-IPC groups (7.46 ± 0.83) (p < 0.05). A significant decrease (p < 0.05) in coronary flow rate was observed in HL-CZ-IPC group (6.16 ± 0.59), as compared to HL-IPC (7.46 ± 0.83) and in HL-CZ-DA-IPC group (6.88 ± 0.56), as compared to HL-DA-IPC (11.18 ± 0.69)  and hyperlipidemic hearts (HL-IR) showed significant decrease (69) Fig. .p < 0.05) in infarct size (21.22 ± 4.36) as compared to IR treated group (45.64 ± 5.93). HL-IPC group showed significant decrease in infarct size (37.65 ± 4.88) as compared to HL-IR group (50.78 ± 4.62) and a significant increase as compared to IPC (21.22 ± 4.36) (p < 0.05). However, HL-DA and HL-DA-IPC groups showed significant fall (p < 0.05) in the infarct size (17.66 ± 2.73 and 11.87 ± 2.61 respectively) as compared to both HL-IR (50.78 ± 4.62) and HL-IPC groups (37.65 ± 4.88). A significant rise in infarct size was also produced in HL-CZ-IPC group (47.54 ± 5.45) as compared to HL-IPC (37.65 ± 4.88) (p < 0.05). HL-CZ-DA-IPC group also produced a significant increase (p < 0.05) in infarct size (47.04 ± 4.66) as compared to HL-DA-IPC (11.87 ± 2.61), however the fall in infarct size in HL-CZ-DA-IPC group (47.04 ± 4.66) as compared to HL-CZ-IPC (47.54 ± 5.45) was insignificant (p > 0.05)  Fig.  & b.Fig.Ischemic preconditioning (IPC) is a well known phenomenon which limits the infarct size and offers cardioprotection to myocardium . PrimariHyperlipidemia subsequently results in atherosclerosis, coronary heart disease, hypertension, myocardial infarction and stroke. The elevated levels of serum lipids, TGs, clogged arteries and release of endogenous cell mediators in hyperlipidemic state exaggerate the myocardial ischemic injury . The car1 receptors [in-vitro has been found to inhibit apoptosis of cardiomyocytes encountered during ischemia/reperfusion through various pathways and also the expression of D2 receptor was reported to increase during ischemia/ reperfusion [Dopamine is reported to offer cardioprotection by pharmacological preconditioning in normal rat hearts via αeceptors . Intereserfusion . Dopaminerfusion –22 that erfusion , 13. TheIn the present study, treatment with high fat diet induced hyperlipidemia in the animals, characterized by significantly elevated LDL, VLDL, TC and TG with reduced HDL levels and also increased body weights after 4 weeks which conforms to previously reported studies . IschemiThe attenuated cardioprotective effect of preconditioning in hyperlipidemic condition was also reproduced against I/R injury in the present study as evidenced by increased level of LDH, CKMB and infarct size in hyperlipidemic preconditioned rat hearts, as compared to IPC treated normal rat hearts . This efA combined cardioprotective effect of ischemic preconditioning and dopamine preconditioning was also explored in the hyperlipidemic hearts and the levels of LDH, CKMB and infarct size were found to be significantly decreased as compared to non-preconditioned and preconditioned hyperlipidemic hearts. This effect was further abolished with the use of clozapine administration as the infarct size, LDH and CKMB were significantly elevated. This confirms the availability of dopamin and role of D2 signaling in both ischemic preconditioning and dopamine induced pharmacological preconditioning in the pathological state of hyperlipidemia.The coronary flow rate (CFR) was found to be decreased against I/R in both normal and hyperlipidemic rat hearts, as compared to basal values. CFR also decreased in preconditioned hyperlipidemic hearts and clozapine treated preconditioned hearts. The levels of CFR were restored in groups treated with ischemic preconditioning alone and dopamine preconditioned groups with or without ischemic preconditioning, as compared to normal rat hearts and ischemic preconditioned hyperlipidemic hearts respectively.Therefore, the findings may conclude for the involvement of D2 signalling in ischemic preconditioning and dopamine induced pharmacological preconditioning in hyperlipidemic rat hearts. Further, research can be targeted on the role of pharmacological preconditioning in other pathological states like Diabetes or in aged subjects for it to be rendered clinically applicable.


The structures of two butenolide derivatives are reported. The conformations are differ largely in the orientation of the amide carbonyl atom. 22H19F4NO4, (I), and C25H22F3NO5, (II), each contain a central nearly planar dihydrofuranone ring. The r.m.s. deviation from planarity of these rings is 0.015 Å in (I) and 0.027 Å in (II). The molecules are T-shaped, with the major conformational difference being the O—C—C—O torsion angle [−178.9 (1) in (I) and 37.7 (2)° in (II)]. In the crystal of (I), molecules are linked by N—H⋯O hydrogen bonds, forming chains along [001] while in (II) molecules are linked by N—H⋯O hydrogen bonds, forming chains along [010]. In (II), the trifluoromethyl substituent is disordered over two sets of sites, with refined occupancies of 0.751 (3) and 0.249 (3).The title compounds, C H)-ones or furanones, are a recurrent moiety in more than 13,000 natural products -5-oxo-4-[2-(trifluoromethoxy)phenyl]-2,5-dihydrofuran-2-carboxamide (I)H-1,3-benzodioxol-5-yl)-N-cyclohexyl-5-oxo-3-[4-(trifluoromethyl)phenyl]-2,5-dihydrofuran-2-carboxamide (II)The molecular structures of A, with contact distances to O2 of 2.738 (1) Å and to H1A of 2.54 Å. The central, dihydrofuranone ring is nearly planar in both compounds. The r.m.s. deviation of these central rings is 0.015 Å in (I)p-fluoro substituted benzene ring is 44.66 (4)° and with the trifluoromethoxy-substituted benzene ring it is 48.71 (3)°. In (II)p-trifluoromethyl substituted benzene ring is 40.03 (5)° and the dihedral angle with the benzene ring of the benzodioxol-5-yl ring system is 43.06 (6)°. The cyclohexane ring of (II) In (II)3 substituent of (II)A has a close contact to atom O1 [2.772 (2) Å] of a neighbouring molecule, which is lengthened to 3.093 (6) Å in the alternate configuration i.e. the minor component. Two hydrogen atoms from symmetry-equivalent molecules flank F3B  and prevent rotational disorder from alleviating the close contact. In the minor component, the –CF3 group deviates from the plane of the aromatic ring, with C25B displaced by 0.36 (1) Å from the mean plane of the aromatic ring.The –CFc-axis direction (Table 1b-axis direction (Table 2C(4) in both (I)In both crystals, N—H⋯O hydrogen bonds connect the molecules into chains which run, in (I)n Table 1, and in n Table 2. The hydH)-ones. A search . For TIFXIP, the O1—C—C—O2 torsion angles for the two molecules in the asymmetric unit are −40.1 and 40.4°, similar to that found in (II)The Cambridge Structural Database (CSD) contains few examples of 3,4,5-substituted furan-2(5Compound (I): 4-fluorophenylglyoxal , trifluoromethoxyphenylacetic acid  and tert-butyl isocyanide  were dissolved in DCM (2 mL) and stirred at room temperature for 1 h. After confirming the exclusive formation of the Passerini product (via TLC and LC/MS), the solvent was removed and the crude product was dissolved in DMF (2 mL). Diisopropylamine (DIPEA)  was added and the reaction mixture was heated at 393 K using microwave irradiation for 20 minutes. After cooling and verifying reaction completion (TLC and LC/MS), the crude mixture was directly purified by flash chromatography (EtOAc/hexane 0–100%) using an ISCO TM flash chromatography system to afford N-tert-butyl-3-(4-fluorophenyl)-5-oxo-4-[2-(trifluoromethoxy)phenyl]-2,5-dihydrofuran-2-carboxamide as a beige solid (67% yield).Compound (II): 4-trifluoromethylphenylglyoxal , 3,4-methylenedioxyphenylacetic acid  and cyclohexyl isocyanide  were dissolved in DCM (2 mL) and stirred at room temperature for 1 h. After confirming the exclusive formation of the Passerini product (via TLC and LC/MS), the solvent was removed and the crude product was dissolved in DMF (2 mL). Diisopropylamine (DIPEA)  was added and the reaction mixture was heated at 393 K using microwave irradiation for 20 minutes. After cooling and verifying reaction completion (TLC and LC/MS), the crude mixture was directly purified by flash chromatography (EtOAc/hexane 0–100%) using an ISCO TM flash chromatography system to afford 4--N-cyclohexyl-5-oxo-3-[4-(trifluoromethyl)phenyl]-2,5-dihydrofuran-2-carboxamide as a yellow solid (61% yield).For both compounds, crystals suitable for X-ray structure elucidation were obtained by slow evaporation of a solution of the compound in a mixture of ethyl acetate/hexanes (1:3).Uiso(H) = 1.5Ueq(C) for methyl H atoms and = 1.2Ueq(C) for other atoms.Crystal data, data collection and structure refinement details are summarized in Table 33 group, but rather consists of a deviation, in the minor component, of the central carbon atom out of the plane of the aromatic ring.In (II)10.1107/S2056989015000936/lh5745sup1.cifCrystal structure: contains datablock(s) I, II, New_Global_Publ_Block. DOI: 10.1107/S2056989015000936/lh5745Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989015000936/lh5745IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989015000936/lh5745Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015000936/lh5745IIsup5.cmlSupporting information file. DOI: 1043839, 1043838CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Two biogenetically related isoflavone intermediates, pruinosanones B and C (2 and 3), were also isolated, together with five known analogs identified as 3-hydroxy-9-methoxypterocarpan (4), 7,2′-dihydroxy-4′-methoxyisoflavanol (5), retusin-8-methylether (6), 7,2′-dihydroxy-8,4′-dimethoxy isoflavone (7) and 7,3′-dihydroxy-8,4′-dimethoxy isoflavone (8). The structures of 1–3 were elucidated based on extensive spectroscopic methods. Notably, 1 is the first example of a spirochromone possessing an unprecedented pentacyclic skeleton containing a spiro[benzo[d]dioxole-2,3′-chroman]-4′-one motif, which was confirmed by X-ray diffraction analysis. A plausible biosynthetic pathway for 1 was also proposed. Compounds 1–8 were tested for their ability to inhibit nitric oxide (NO) production in LPS-induced RAW 264.7 macrophages, and compounds 1–3 were the most potent inhibitors of NO production, with IC50 values of 1.96, 1.93 and 1.58 μM, respectively. A structure-activity relationship analysis revealed that the fused 2-isopropenyl-2,3-dihydrofuran moiety plays a vital role in the potency of these compounds. Moreover, 1 was found to significantly inhibit inducible nitric oxide synthase (iNOS) protein expression, which accounts for the potent inhibition of NO production by this spirochromone.Pruinosanone A ( Caragana pruinosa (Leguminosae) is a dwarf shrub that is found primarily in the Xinjiang Province of China and Central Asia. Its roots are widely used for the treatment of inflammatory disorders in the folk medicine of northwestern ChinaCaragana plants have confirmed the presence of stilbenoids246Caragana species4910C. pruinosa, which led to the characterization of a novel spirochromone called pruinosanone A (1). This compound possesses a novel spiro[benzo[d]dioxole-2,3′-chroman]-4′-one ring system within its pentacyclic skeleton (2 and 3), together with five known analogs (4–8), were also isolated. To the best of our knowledge, this is the first phytochemical report on this medicinal species. Herein, we report the isolation, structural elucidation and determination of the absolute configuration of the new compounds (1–3). Furthermore, a pathway for the biosynthesis of 1 from chalcone, involving two epoxidation steps, is proposed. Finally, we investigated the inhibition of nitric oxide (NO) production by compounds 1–8 in LPS-induced RAW 264.7 cells. Compounds 1–3, which bear a fused 2-isopropenyl-2,3-dihydrofuran moiety, exhibited much better activity, with IC50 values ranging from 1.58 to 1.96 μM. Compound 1 was found to be a potent NO production inhibitor, remarkably suppressing NO release with an IC50 value of 1.96 μM while causing no cytotoxicity. This NO suppression was probably due to down-regulation of inducible nitric oxide synthase (iNOS) expression.As part of our continuing efforts focused on skeleton . Two bioC. pruinosa roots was subjected to a succession of chromatographic procedures to yield three new isoflavone derivatives, named pruinosanones A-C (1–3), along with five known analogs (4–8). The structures of the known compounds were elucidated by comparing their NMR data with those reported in the literature and were identified as 3-hydroxy-9-methoxypterocarpan (4)5)6)7)8)The EtOH extract of 1), obtained from C. pruinosa as optically active, colorless needles (22H20O7 (13 degrees of unsaturation), which was deduced from HRESIMS analysis ([M + H]+ at m/z 397.1269), 13C NMR data, and DEPT spectra  and δH 6.64 ; δH 6.49  and δH 6.37 ], an isopropenyl group , an oxygenated methine [δH 5.37 ], an oxygenated methylene [δH 4.64  and δH 4.63 ], a benzylic methylene , and two methoxy groups [δH 4.02  and δH 3.82 ]. The 13C and DEPT spectra revealed the presence of 22 carbon resonances, which were divided into 3 methyls, 3 methylenes, 5 methines, and 11 quaternary carbons. The 1H–1H COSY spectrum , C-3″ (δC 142.8), C-4″ (δC 112.9), suggesting the presence of an 3-oxygenated 1-isopentenyl group. In addition to two methoxy groups, the remaining 15 carbon resonances were observed to be fairly close to those of isoflavanones, with two characteristic signals at δC 70.5  and δC 180.7 , except for the distinguished geminal coupling of the H-2 [δH 4.64  and δH 4.63 ] neighboring the quaternary carbon C-3 (δC 106.0), with a large down-field shift in 1. C-3 was assigned to be an acetal carbon deduced from two more oxygen atoms that remained according to the molecular formula, which led to the formation of a spiro system between rings C and B. The HMBC experiment also indicated correlations between H-2/C-3, C-4, H-5′/C-1′, and H-6′/C-2′. Furthermore, the HMBC correlations  over 7 days. The resulting crystals were suitable in size and quality for single-crystal X-ray analysis using anomalous dispersion with Cu Kα radiation, which revealed an absolute structure parameter (Flack’s x) of 0.03(7)171 as a 3S and 2″S configuration (1) was therefore defined as -4,5-dimethoxy-8′-(prop-1-en-2-yl)-8′,9′-dihydrospiro[benzo[d]dioxole-2,3′-furochromen]-4′(2′H)-one.Fortunately, guration . The str2, named pruinosanone B, was obtained as a yellow, amorphous powder. The positive HRESIMS data  indicated the molecular formula of 2 to be C22H20O6. Its 1H and 13C NMR spectra  and the appearance of several slightly shifted signals from the B-ring. These data indicated that 2 was an isoflavone derivative of 1, which was confirmed by detailed HMBC analysis, particularly the HMBC correlations from H-2  to C-4 (δC 175.3), C-1′ (δC 113.9) and from H-6′  to C-3 (δC 122.2)  in 2; δH 5.37  in 1] and C-2″ . The structure of pruinosanone B (2) was therefore defined as (2″S)-8-(2-methylbut-1-en-4-yl)-7,2″-epoxy-2′-hydroxy-3′,4′-dimethoxyisoflavone.Compound  spectra  were chaC 122.2) . The sig3, named pruinosanone C, was obtained as a yellow powder. The molecular formula of 3 was determined to be C22H22O6 by positive HRESIMS . Its 1H NMR and 13C NMR data are summarized in 2, except that compound 3 has one less degree of unsaturation than 2. Considering the molecular weights of 3 and 2, compound 3 was considered to be a 2,3-hydrogenation derivative of 2, which was further confirmed by detailed HMBC, COSY and NOESY examination. The equatorial orientation of the B-ring was verified by the NMR coupling constant between the trans-diaxial H-2β and H-3 of ca. 11 HzR based on the positive cotton effect (CE) of the n → π* carbonyl absorption band at 329 nm3) was concluded to be -8-(2-methylbut-1-en-4-yl)-7,2″-epoxy-2′-hydroxy-3′,4′-dimethoxyisoflavanone.Compound 1–3 were proposed to start from a chalcone derivative  was a potent inhibitor of NO production, remarkably suppressing NO release in a dose-dependent manner .NO acts as a host defense mechanism by damaging pathogenic DNA and is also a regulatory molecule with homeostatic activitiest manner , with anCell viability was determined using the CCK-8 method to evaluate whether the inhibition of NO production was due to the cytotoxicity of the tested compounds. It was found that none of the concentrations used in this experiment were cytotoxic . Thus, t1) was selected to investigate the effect of the compound on iNOS protein expression in LPS-induced RAW 264.7 cells. According to the results, pruinosanone A was found to significantly down-regulate the expression of iNOS in a dose-dependent manner, which accounts for the potent inhibitory activity of compound 1 against NO production (Nitric oxide synthases (NOSs) play a very important role in catalyzing the production of NO from L-arginine. Inflammatory mediators, such as IL-1, TNF-α, and LPS, stimulate the expression of the inducible isoform of NOS (iNOS) in rodent macrophages, which leads to the prolonged production of large amounts of NO, a characteristic of many inflammatory diseases262728oduction .1) is the first pentacyclic spirochromone containing a spiro[benzo[d]dioxole-2,3′-chroman]-4′-one motif, which has never been observed before for any chromone. Therefore, this structure represents a new carbon skeleton. The structure of 1 provides not only an interesting synthetic target but also a potent inhibitor of NO release. Further studies on this molecule will provide valuable insights into the development of anti-inflammatory drugs.In conclusion, pruinosanone A . Preparative TLC (0.4–0.5 mm) was conducted on glass plates precoated with silica gel GF254 (Yantai).Optical rotations were acquired with a Perkin-Elmer 341 polarimeter. The UV spectra were acquired using a Varian Cary Eclipse 300 spectrophotometer, while the IR spectra were recorded on a Bruker Vector 22 spectrometer with KBr pellets. The NMR spectra were recorded on a Bruker Avance 600 NMR spectrometer with TMS as an internal standard. The HRESIMS measurements were obtained with a Q-TOF Micromass spectrometer . X-ray crystallographic analysis was performed with a Bruker SMART APEX (II)-CCD diffractometer with Cu Kα radiation (λ = 1.54178 Å). The materials for the CC were silica gel , silica gel H , Sephadex LH-20 , and YMC-GEL ODS-A . Semi-preparative HPLC was conducted on an Agilent 1200 instrument using an Eclipse XDB-CC. pruinosa roots were collected from Urumuchi, Xinjiang, P. R. China and authenticated by Prof. Xiao-Guang Jia, Xinjiang Institute of Chinese Materia Medica and Ethnodrug . A voucher specimen of this plant was kept at the Herbarium of the Department of Pharmacognosy, School of Pharmacy, Second Military Medical University, Shanghai, P.R. China (No. *201203).C. pruinosa (11.5 kg) were extracted with 80% EtOH (×3), with each extraction period lasting 2 h. The solvent was removed under reduced pressure, and the residue (1.2 kg) was suspended in H2O and partitioned sequentially with petroleum ether, EtOAc, and n-butanol to afford four fractions, a petroleum ether fraction , an EtOAc fraction , an n-butanol fraction  and a remaining water fraction (WF). In our preliminary study, the EtOAc-soluble fraction exhibited notable anti-inflammatory activity in vitro and therefore was selected for investigation in the present study.The air-dried and pieced roots of 2O  followed by preparative TLC to yield compounds 2 (7 mg) and 3 (6 mg). Fraction C.4 (500 mg) was further separated on Sephadex LH-20 resin with MeOH-H2O  followed by semi-preparative HPLC with MeOH-H2O  as the eluent to yield compound 1 (8 mg), which was further crystallized by slow evaporation from a solution of a CH3OH/CH2Cl2 (1:1) mixture over 7 days.The EtOAc fraction  was subjected to CC on silica gel  and eluted successively with a gradient of petroleum ether-EtOAc mixtures  to afford fractions A-G. Fraction C (5.0 g) was further fractionated by column chromatography on silica gel (200–300 mesh) employing a petroleum ether-EtOAc mixture (20:1) as the eluent to provide five fractions (C.1-C.5). Fraction C.3 (400 mg) was rechromatographed on Sephadex LH-20 resin with MeOH-HPruinosanone A (1): colorless needles; m.p. 123–124 °C; c 0.08, methanol); IR (KBr) vmax 2924, 2852, 1610, 1511, 1464, 1384, 1084; 1H-NMR  and 13C-NMR  spectra . spectra ; HRESIMSPruinosanone B (2): yellow amorphous powder; c 0.06, methanol); IR (KBr) vmax 3394, 2925, 2854, 1610, 1509, 1458, 1243, 1105, 1036, 829; 1H-NMR  and 13C-NMR  spectra . spectra ; HRESIMSPruinosanone C (3): yellow amorphous powder; c 0.04, methanol); CD  [Δε]329 = +9.6; IR (KBr) vmax 3421, 2924, 2853, 1654, 1608, 1508, 1466, 1258, 1094; 1H-NMR  and 13C-NMR  spectra . spectra ; HRESIMS3OH/CH2Cl2 (1:1) using the vapor diffusion method, colorless crystals were obtained for 1. A crystal was separated from the sample and mounted on a glass fiber. X-ray crystallographic analysis was carried out on a Bruker SMART APEX (II)-CCD diffractometer with Cu Kα radiation (λ = 1.54178 Å). Structure solution and refinement were performed with the SHELXL-97 program. The crystal data for pruinosanone A (1) are as follows: Empirical formula: C22H20O7; Formula weight: 396.38; Crystal system: monoclinic; Space group: P21; Crystal size: 0.250 mm × 0.200 mm × 0.100 mm; Unit cell dimensions: a = 9.58860(10) Å, b = 9.43910(10) Å, c = 10.7943(2) Å, α = 90°, β = 109.7170(10)°, γ = 90°, V = 919.69(2) Å3; Index ranges: −11 ≤ h ≤ 11, −11 ≤ k ≤ 9, −13 ≤ l ≤ 13; θ range for the data collection was from 4.351° to 69.814°; Z = 2; Dc = 1.431 g/cm3; F (000) = 416; Refinement method: Full-matrix least-squares on F2; Goodness-of-fit on F2: 1.183; Final R indices [I > 2σ (I)]: R1 = 0.0324, wR2 = 0.0834; R indices : R1 = 0.0338, wR2 = 0.0927; Largest differences in peak and hole: 0.377 and −0.247 e/Å−3; absolute structure parameter (Flack’s x) of 0.03(7).Upon crystallization from CH1) reported in this paper have been deposited at the Cambridge Crystallographic Data Center as supplementary publication number CCDC 1058372. Copies of the data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif  1223-336-033; e-mail: deposit@ccdc.cam.ac.uk).The crystallographic data for the structure of pruinosanone A (5 cells/well). The cells were co-incubated with the isolated compounds and LPS (1 μg/mL) for 24 h. The amount of NO was assessed by determining the nitrite concentration in the cultured RAW 264.7 macrophage supernatants with the Griess reagent. Aliquots of the supernatants (100 μL) were incubated in sequence with 50 μL of 1% sulfanilamide and 50 μL of 0.1% naphthylethylenediamine in a 2.5% phosphoric acid solution. The absorbance at 548 nm was read using a microplate reader (POLARstar).RAW 264.7 macrophages were seeded in 96-well plates  for 24 h in the presence or absence of the tested compounds. These compounds were dissolved in DMSO at an initial concentration of 10 mM and diluted to an appropriate concentration using culture medium; the final concentration of DMSO was adjusted to ≤0.01%. Beta-actin (β-actin) was used to ensure that the amounts of protein were equal in each lane. Protein samples were collected and prepared as described previouslyThe murine RAW 264.7 cell line was seeded at an initial density of 2 × 10How to cite this article: Zheng, C. et al. Pruinosanones A-C, anti-inflammatory isoflavone derivatives from Caragana pruinosa. Sci. Rep. 6, 31743; doi: 10.1038/srep31743 (2016).


H-indol-3-yl)propenoic acid (a 1:1:1 d6-DMSO solvate) show, respectively, polymeric and hexamolecular hydrogen-bonded and π–π-bonded structuresThe crystal structures of the co-crystal adducts of 3,5-dinitrobenzoic acid with 4-aminosalicylic acid (a 2:2:0.4-hydrate) and with 2-hydroxy-3-, and with 2-hydroxy-3-(1H-indol-3-yl)propenoic acid (HIPA), the 1:1:1 d6-dimethyl sulfoxide solvate, C7H4N2O6·C11H9NO3·C2D6OS, (II), are reported. The crystal substructure of (I) comprises two centrosymmetric hydrogen-bonded R22(8) homodimers, one with 3,5-DNBA, the other with PASA, and an R22(8) 3,5-DNBA–PASA heterodimer. In the crystal, inter-unit amine N—H⋯O and water O—H⋯O hydrogen bonds generate a three-dimensional supramolecular structure. In (II), the asymmetric unit consists of the three constituent molecules, which form an essentially planar cyclic hydrogen-bonded heterotrimer unit [graph set R32(17)] through carboxyl, hydroxy and amino groups. These units associate across a crystallographic inversion centre through the HIPA carboxylic acid group in an R22(8) hydrogen-bonding association, giving a zero-dimensional structure lying parallel to (100). In both structures, π–π interactions are present [minimum ring-centroid separations = 3.6471 (18) Å in (I) and 3.5819 (10) Å in (II)].The structures of the co-crystalline adducts of 3,5-dinitrobenzoic acid  with 4-aminosalicylic acid (PASA), the 1:1 partial hydrate, C With (II)2D6OS resulted from recrystallization from d6-dimethylsulfoxide.3,5-Dinitrobenzoic acid  has been an important acid for the formation of crystalline materials, which have allowed structural characterization using single crystal X-ray methods. Most commonly proton-transfer salts are formed with organic Lewis bases, A and B), two 3,5-DNBA molecules (C and D) and a partially occupied water molecule of solvation (O1W), with site occupancy = 0.4. However, what is most unusual in this structure is the presence of not four homodimers in the unit cell, but two homodimers , as well as two heterodimer B–D pairs B) with 2-hydroxy-3-(1H-indol-3-yl)propenoic acid (A) and d6-dimethylsulfoxide (C), (II)A and the benzene ring of B. With the HIPA molecule there is a maximum deviation from the least-squares plane of the 15-atom molecule of 0.120 (2) Å (C6A). The planar conformation of the acid side chain in this molecule is maintained by the presence of delocalization extending from C2A of the ring to O14A of the carboxylic acid group [torsion angle C11A—C12A—C13A—O14A = −177.43 (16)°]. This is also found in the parent acid, which has the similar enol configuration as in (II)E orientation and in the crystal forms a centrosymmetric homodimer with an In the ternary co-crystal of 3,5-DNBA B molecule in (II)et al., 1991et al., 2012et al., 2001et al., 2010et al., 2009In the adducts (I)A⋯Cvi, 3.7542 (16); A⋯Cvii, 3.6471 (16); B⋯Dviii, 3.6785 (14) Å] . The B⋯D heterodimers in the π–π association are not only related by inversion but are cyclically linked by the amine N4B—H⋯O52iiiD hydrogen bond, forming an enlarged et al., 1993B molecule [the phenolic O2B acceptor] and the C molecule [the nitro O32Civ acceptor]. Also present in the structure are two very weak C—H⋯Onitro interactions i in a cyclic d6-DMSO methyl C—H⋯O interactions are present between these units interactions  .In (II)0) Fig. 4. Only twH-indol-3-yl)propenoic acid [for (II)] in ethanol and heating under reflux for 5 min after which room-temperature evaporation of the solutions gave for (I)d6-deuterated DMSO and solvent diffusion of water into this solution gave red prisms of (II)The title co-crystalline adducts (I)A and O2B groups and on the disordered water molecule O1W, with riding restraints . The phenolic and water H atoms were set invariant with Uiso(H) = 1.2Ueq(O). Other H atoms were included in the refinement at calculated positions [C—H (aromatic) = 0.95 or (methylene) 0.99 Å] , with Uiso(H) = 1.2Ueq(C), using a riding-model approximation. The site-occupancy factor for the disordered water molecule of solvation was determined as 0.403 (4)  and was subsequently fixed as 0.40. In the structure of (I)−3) was located 0.80 Å from H6B.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S1600536814019898/sj5421sup1.cifCrystal structure: contains datablock(s) global, I, II. DOI: 10.1107/S1600536814019898/sj5421Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S1600536814019898/sj5421IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S1600536814019898/sj5421Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S1600536814019898/sj5421IIsup5.cmlSupporting information file. DOI: 1022646, 1022647CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


H-1,2,4-triazole, the ReI atom has a distorted octahedral coordination environment. Mutual N—H⋯Br hydrogen bonds arrange the molecules into centrosymmetric dimers. Additional stabilization within the crystal structure is provided by C—H⋯O and C—H⋯Br hydrogen bonds, as well as by slipped π–π stacking interactions.In this rhenium carbonyl complex of 5-phenyl-3-(pyridin-2-yl)-1 13H10N4)(CO)3], the ReI atom has a distorted octahedral coordination environment. Two N atoms of the 5-phenyl-3-(pyridin-2-yl)-1H-1,2,4-triazole ligand and two of the three carbonyl groups occupy the equatorial plane of the complex, with the third carbonyl ligand and the bromide ligand in the axial positions. The three carbonyl ligands are arranged in a fac configuration around the ReI atom. Mutual N—H⋯Br hydrogen bonds arrange molecules into centrosymmetric dimers. Additional stabilization within the crystal structure is provided by C—H⋯O and C—H⋯Br hydrogen bonds, as well as by slipped π–π stacking interactions [centroid-to-centroid distance = 3.785 (5) Å], defining a three-dimensional network.In the title compound, [ReBr(C When NN is a ligand with low π* orbitals, the corresponding [Re(CO)3(NN)] unit is luminescent -1H-1,2,4-triazole, [Re(CO)3(C13H10N4)Br]. Its luminescent properties will be reported in a forthcoming article.In this communication, we report the synthesis and crystal structure analysis of a novel ReI atom is in a slightly distorted octahedral coordination environment , 1.915 (4), and 1.922 (6) Å, respectively, and the Re—N bonds lengths are 2.201 (3) and 2.164 (3) Å. The CO ligands are almost linearly coordinated with O—C—Re bond angles of 178.4 (4), 175.6 (3) and 179.0 (4)°. The C—Re—C bond angles between CO carbon atoms are 87.78 (17), 90.4 (2) and 89.18 (19)°, close to ideal values, whereas the cis equatorial bite angle [N1—Re1—N2] is 74.33 (11)°. All other bond lengths and angles are comparable to those found for related ReI complexes   also appear to be involved in the stabilization of the crystal structure  and one acceptor (Br) site for hydrogen-bonding interactions, which arrange molecules into dimers Table 1. Weak hyre Fig. 2.H-1,2,4-triazole  in benzene at 353 K, with stirring, under a steady stream of argon for five h. The dark-yellow solution was removed from the heat and allowed to cool overnight. The yellow product was collected by suction filtration, washed with a 50 ml portion of petroleum ether and dried. Yield = 0.107g, (76.4%). Crystals suitable for X-ray diffraction were obtained by slow diffusion of hexane into a methanol solution of the complex. IR : νas(CO) 2028 (s), νs(CO) 1912 (s).Pentacarbonylrhenium(I) bromide  was reacted with 5-phenyl-3-(pyridin-2-yl)-1Uiso(H) = 1.2Ueq.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S1600536814025604/wm5089sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S1600536814025604/wm5089Isup2.hklStructure factors: contains datablock(s) I. DOI: 1031232CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: