The complex has two redox couples at −1.50 and −1.80 V ( N){N-benzyl-N,N′,N′-tris[(6-methylpyridin-2-yl)methyl]ethane-1,2-diamine}nickel(II) bis(tetrafluoridoborate), [Ni(C30H35N5)(CH3CN)](BF4)2, was prepared from the reaction of Ni(BF4)2·6H2O with N-benzyl-N,N′,N′-tris[(6-methylpyridin-2-yl)methyl]ethane-1,2-diamine (bztmpen) in acetonitrile at room temperature. With an open site occupied by the acetonitrile molecule, the nickel(II) atom is chelated by five N-atom sites from the ligand and one N atom from the ligand, showing an overall octahedral coordination environment. Compared with analogues where the 6–methyl substituent is absent, the bond length around the Ni2+ cation are evidently longer. Upon reductive dissociation of the acetronitrile molecule, the title complex has an open site for a catalytic reaction. The title complex has two redox couples at −1.50 and −1.80 V (versus Fc+/0) based on nickel. The F atoms of the two BF4− counter-anions are split into two groups and the occupancy ratios refined to 0.611 (18):0.389 (18) and 0.71 (2):0.29 (2).The mononuclear nickel title complex (acetonitrile-κ The latter can easily be dissociated from nickel. The axial positions are occupied by two pyridine nitrogen atoms . The Ni—N bond lengths for the two axial pyridine–nitrogen atoms [Ni—N5 = 2.209 (3) and Ni—N6 = 2.187 (3) Å] are significantly longer than that for the other four nitrogen atoms . The presence of the 6-methyl substituent hinders the approach of the pyridine group to the Ni2+ core. A a result of the steric hindrance from the methyl substituent, the three atoms N5, Ni1 and N6 are not completely linear in the axial direction, with a contact angle of 170.89 (9)°. Two intramolecular C—H⋯N contacts occur  based on nickel, assigned to NiII/I and NiI/0 respectively ethylenediamine (tpen) adopts a normal octahedral geometry  complex, the Ni—N1, Ni—N2, Ni—N3, Ni—N4, Ni—N5 and Ni—N6 bonds  are shorter than the corresponding bond lengths in the title complex. Among the earliest reports, the nickel complex with N-benzyl-N,N′,N′-tris(2-pyridylmethyl)ethylenediamine (bztpen) ligand is most similar to the title complex  displays a high activity on electro-catalytic water reduction. The title complex possesses a higher steric hindrance than Ni2+(bztpen), which affects evidently the bond lengths, especially in the axial direction. The bonds lengths in the title complex  are longer than those in Ni2+(bztpen) . The nickel complex with a PY5 ligand {PY5 = 2,6-bispyridine} displays a similar configuration to the title complex, but the labile ligand is at the axial site  has been found to act as an electro-catalyst for oxidizing water to dioxygen in an aqueous phosphate buffer solution.There are three published nickel complexes with poly-pyridine groups methyl]ethane-1,2-diamine (bztmpen) was prepared according to literature procedures : δ 7.44 , 7.25 , 6.96 , 3.74 , 3.58 , 2.75 , 2.49 . ESI–MS: calculated for [M + H]+: m/z 466.63; found: 466.27.The tripyridine-diamine ligand bztmpen)(CH3CN)](BF4)2. Compound Ni(BF4)2·6H2O  was added to an acetonitrile solution (5 mL) of bztmpen . The mixture was stirred at room temperature for 6 h. The purple solution was then transferred to tubes, which were placed in a flask containing ether. Block-shaped blue crystals were obtained in a yield of 85% (0.25 g). Analysis calculated for C32H38B2F8N6Ni (%): C, 50.01; H, 5.18; N, 11.37; found: 50.01; H, 5.19; N, 11.36; MS (TOF–ES): m/z = 282.6341 {[M−2BF4−]/2}+, 599.3015 [M – 2BF4− + Cl−]+.Preparation of [Ni(4− counter-anions were split into two groups and the occupancies refined to 0.611 (18)/0.389 (18) and 0.71 (2)/0.29 (2). The hydrogen atoms were refined in a riding mode with C—H = 0.93–0.97 Å and Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989017006764/vn2128sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989017006764/vn2128Isup4.hklStructure factors: contains datablock(s) I. DOI: 1548052CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Tfh derived from p110α−/−ΔT cells also have enhanced responses when compared to normal mice, and IL-2 expanded p110α−/−ΔT CD8+ T cells had enhanced levels of LAMP-1 and Granzyme B. By contrast, the expansion of p110α−/−ΔT iTreg cells was diminished. Also, p110α−/−ΔT mice had enhanced anti-keyhole limpet hemocyanin (KLH) IFN-γ, or IL-4 responses and IgG1 and IgG2b anti-KLH antibodies, using CFA or Alum as adjuvant, respectively. When compared to WT mice, p110α−/−ΔT mice inoculated with B16.F10 melanoma showed delayed tumor progression. The percentage of CD8+ T lymphocytes was higher and the percentage of Treg cells lower in the spleen of tumor-bearing p110α−/−ΔT mice. Also, IFN-γ production in tumor antigen-activated spleen cells was enhanced. Thus, PI3K p110α plays a significant role in antigen activation and differentiation of CD4+ and CD8+ T lymphocytes modulating antitumor immunity.Class IA phosphatidylinositol 3-kinase (PI3K) catalytic subunits p110α and p110δ are targets in cancer therapy expressed at high levels in T lymphocytes. The role of p110δ PI3K in normal or pathological immune responses is well established, yet the importance of p110α subunits in T cell-dependent immune responses is not clear. To address this problem, mice with p110α conditionally deleted in CD4 Generation of PtdIns(3)P in the inner leaflet of membrane bilayers allow the recruitment of cytosolic proteins that participate in distinct signaling cascades [reviewed in Ref –5]. Thre therapy –8.PIKAB regulatory subunit and one p110γ catalytic subunit. Class IB, but also class IA p110β kinases, are recruited and activated upon ligand binding to cell surface G-protein coupled receptors  and one regulatory subunit  that characteristically bind to proteins phosphorylated at the tyrosine of YxxM motifs , 5, incleceptors , 5. Whereceptors , 13–15. eceptors , 17].The abundant expression of class IA p110δ catalytic subunits by leukocytes, and the functional association of p110δ to the antigen receptors of T and B lymphocytes might point to these catalytic subunits as the main source of class IA PI3-kinase activity in lymphocytes , 18, 19.+ T lymphocytes, or T cell lines at levels comparable to those of p110δ subunits  have enhanced effector function and hampered expansion of Treg cells. Particularly, they show higher IFN-γ secretion in vitro that correlate with higher T-bet levels and early Erk and P38 activation. Using protein antigens and experimental models of melanoma we show that p110α−/−ΔT mice have altered humoral responses and inhibited growth of melanoma linked to enhanced antigen-specific IFN-γ responses and lower Treg cell numbers.Here, we have used T-lymphocyte-specific deletion of p110α PI3-kinase to further analyze the role of p110α in T cell activation, CD28-dependent costimulation, and T-dependent responses. Our data show that CD4pik3ca gene in T cells were generated by crossing CD4-Cre mice and mice with a floxed pik3ca gene (p110αflox/flox) . PI3-kinase p110α was efficiently removed from peripheral CD4+ and CD8+ T lymphocytes of p110α−/−ΔT mice; however, the PI3-kinase p110δ subunit or other proteins like CD4, or Erk were unaffected . Most subpopulations in the peripheral lymphoid organs of WT and p110α−/−ΔT mice were not significantly changed, including the percentage of total T (CD3+) cells, CD8 T lymphocytes, B lymphocytes, γδ, and NKT lymphocytes, or NK cells . However, p110α−/−ΔT mice showed a slightly lower number of spleen cells and a lower percentage of CD4+ cells , even though the percentage of naive and memory or Treg cells within the CD4+ T cell population was not significantly different. Analysis of thymus cells indicated that this was not due to a deficient development of mature CD4+ T lymphocytes .PI3-kinase p110α-null mice are embryonic lethal . So, to ox/flox) . CD4-Cre+ T lymphocytes in vitro was determined. Secretion of IL-10 and, particularly, IFN-γ were significantly enhanced in p110α−/−ΔT cells activated with anti-CD3 plus anti-CD28 antibodies, as compared to WT littermates . In both cases, a p110δ-specific inhibitor  blocked ICOS-induced elongation.In differentiated Tfh cells, the production of IL-21 (Tfh) was significantly enhanced in p110α-deficient cells , in agreement with the results obtained under neutral conditions . Furthermore, similar amounts of IL-10 were secreted on a per-cell basis  and Granzyme B . Mice were first immunized s.c. with KLH in Freund’s adjuvant (KLH/FCA). Cytokine release upon antigen re-stimulation “in vitro” of cells from draining lymph nodes was then determined , even though the levels of the inflammatory cytokines IL-17A and TNF-α were higher in p110α−/−ΔT cells . In these conditions, the level of anti-KLH-specific antibodies of the IgG1 and IgG2b isotypes was significantly higher in the sera of p110αe Figure B, as wase Figure C. Spleend Figure D or in I−/−ΔT mice had enhanced effector mechanisms including IFN-γ secretion, and might have altered Treg proliferation, the effect of PI3K p110α deletion in T cells on the growth of s.c. injected B16.F10 melanoma was examined. Tumor growth was significantly lower in p110α−/−ΔT mice, as shown by tumor size or by the survival rate  were significantly diminished in p110α−/−ΔT mice, and the level of Foxp3 in these cells was lower than in their WT counterparts  to obtain mice (p110α−/−ΔT) whose T cells lacked the PI3K p110α isoform.Class IA PI3Ks are essential to normal development and function of adaptive immune reactions. In T and B lymphocytes, the p110δ isoform has a major role in ontogeny and adaptive immunity. Intriguingly, the p110α isoform is abundant in lymphocytes and can bind to and participate in the signals of costimulatory molecules like CD28 or ICOS , 25. Stuflox/flox to delete PI3K p110α in lymphocytes indicated a role for p110α in pre-B cell receptor and tonic B-cell receptor signaling, contributing to B lymphocyte differentiation and B cell survival (+ cells in these p110α-deficient mice was apparently unaffected (+) lymphocytes in the spleen of p110α−/−ΔT animals. However, a closer analysis of lymphocyte subpopulations indicated a minor but significant decrease in the percentage of CD4+ T cells as well as in the number of spleen cells.Previous data using CD2-Cre mice and p110αsurvival . In contaffected . Indeed,+ or CD8+ p110α−/−ΔT cells activated through TCR/CD3 and CD28 show augmented secretion of certain cytokines, particularly IFN-γ. Enhanced signaling is observed very early in the activation of naive T cells at the Tyr phosphorylation of cell substrates, or in the activation of Akt and the MAP kinases Erk and P38. Furthermore, the level of the Th1 master transcription factor T-bet is also higher in activated p110α−/−ΔT T cells.In the absence of additional cytokines or signals, CD4−/−ΔT mouse T cells have significantly enhanced Tfh and effector CD8 cell responses “in vitro.” “In vivo,” injection of KLH in FCA induces enhanced IFN-γ responses, whereas immunization with the same antigen in Alum induces enhanced production of the Th2 cytokine IL-4. Although this suggests a widespread enhancing effect of p110α deletion, the absence of significant changes in some cases like IL-2 point to a selective effect on specific signal pathways and transcription factors.Although these data suggest that Th1 responses are favored in the absence of p110α, our results also indicate that, depending on the conditions and/or the presence of additional cytokines, p110αTaken together, our results with p110α deletion, plus previous data using inhibitors suggest that p110α contribute to PI3K signaling upon T lymphocyte activation, but at the same time might compete with other catalytic PI3K subunits  and down-modulate their signals. Since some molecules of the Ras family (including Ras) are activators of p110α but not p110δ  and are −/−ΔT mice have interesting consequences in antitumor immunity. When compared to WT mice, tumor-bearing p110α−/−ΔT mice have enhanced numbers of CD8+ T lymphocytes and enhanced production of IFN-γ. Furthermore, the percentage of Treg cells was lower in tumor-bearing p110α−/−ΔT mice. Overall, these features should delay melanoma growth. In our p110α−/−ΔT mice, p110δ is left as the main PI3K class IA subunit present in T lymphocytes. Interestingly, PI3K p110δ activity is necessary for efficient Treg expansion and function, and Tregs have a particular sensitivity to p110δ inactivation or inhibition , suggesting that, in our conditions, p110α is needed for iTreg expansion and/or survival in vitro. Whereas the percentage of Treg in the spleen of tumor-bearing mice was elevated compared to normal mice, p110α−/−ΔT tumor-bearing mice had significantly lower Treg than WT mice. Previous data showing that Akt/mTOR activation fosters Th1 but hamper TGF-β signaling and Treg differentiation , or the European Mouse Mutant Archive Repository (CD4-Cre), and used at 8–12 weeks of age. CD4-Cre and p110αflox/flox mice were crossed to obtain CD4-Cre+/− p110αflox/flox (p110α−/−ΔT) mice, and these crossed with CD4-Cre−/− p110αflox/flox (WT) mice to obtain p110α−/−ΔT and WT littermates used in the experiments. All experimental procedures were performed according to established institutional and national guidelines under project licenses PROEX 181/15  and PROEX 330/15  from the Consejeria de Medio Ambiente y Ordenación del Territorio de la Comunidad de Madrid. Mice were genotyped for Cre, p110α, p110αflox, or Cre-mediated removal of floxed p110α using the oligonucleotides and conditions described in Ref. , and anti-Foxp3 (FJK-16s) antibodies; APC conjugates of anti-CD62L-, anti- ICOS- (C398.4A), anti-PD-1- (J43) antibodies were from eBioscience. Anti-mouse CXCR5-Biotin (2G8) was from BD-Pharmingen. Anti-CD107a (LAMP-1) (eBio1D4B) Alexa Fluor 488 conjugate and anti-Granzyme B-PE (NGZB-PE) were from eBioscience; anti-Granzyme A-FITC (3G8.5-FITC) was from Santa Cruz Biotechnology.Antibodies used were monoclonal rat anti-mouse CD3ε [YCD3-1 ]; rat anAnti-phosphotyrosine antibody (PY-20) was from GE Healthcare. Polyclonal rabbit anti-ZAP-70 and anti-CD4 antibodies were obtained and purified as described previously , 50. RabHorseradish peroxidase-coupled polyclonal goat antibodies specific for mouse IgM, IgG1, IgG2b, IgG2c, and IgG3 immunoglobulin were from Southern Biotech.Rabbit polyclonal anti-ERK antibody was from Upstate Biotechnology. Rabbit anti-PI3K p110δ (H-219) was from Santa Cruz Biotechnology; the rabbit anti-p110α monoclonal antibody C73F8 was from Cell Signaling Technology.Recombinant human IL-2 and mouse IL-4 and IL-6 were from Preprotech; recombinant human TGF-β was from R&D. PI3Kα inhibitor (A66) was from Selleckchem; the PI3K PI3Kδ Inhibitor IC87114 was from Symansis Pty. The P38 inhibitor SB203580 was from Sigma; the MEK1 inhibitor U0126 was from Calbiochem.+ (CD4+CD62L+) T cells were isolated using mouse naive CD4+ T cell isolation kit II . CD8+CD62L+ T cells were obtained with the CD8a T Cell Isolation Kit (130-104-075) and anti-CD62L beads (Miltenyi Biotech). Where required, CD4+ T cells were purified to >98% CD4+ T cells by sorting using a FACS Vantage-DIVA flow cytometer (B-D Bioscience).Spleen or thymus cell suspensions were prepared in culture medium  and passed through 30-µm filters to remove clumps. Naive CD4+CD62L+ or CD8+CD62L+ T cells were cultured at 0.5–1 × 106 cells/ml in 24-well culture plates  or 96-well culture plates  previously coated with anti-CD3  plus soluble anti-CD28 (2.5 µg/ml), as described in the Section “+ T cells into distinct functional effector subsets was performed by activation with anti-CD3 and anti-CD28 antibodies plus cytokines and antibodies as follows: for Th1 differentiation, 20 ng/ml IL-12 and 5 µg/ml anti-IL-4 (11B11); for Th2 differentiation, 10 ng/ml IL-4 and 10 µg/ml anti-IFN-γ (XMG.1); for Th17 differentiation, 20 ng/ml IL-6, 5 ng/ml human TGF-β1 plus anti-IL-4 and anti-IFN-γ; for Tfh differentiation, cells were cultured in the presence of anti-IL-4 and anti-IFN-γ antibodies plus 20 ng/ml IL-6. Treg differentiation from naive CD4+ T cells was achieved with anti-CD3  plus huIL-2 (20 ng/ml), 5 ng/ml TGF-β1, 5 µg/ml anti-IL-4, and 10 µg/ml anti-IFN-γ. At 48 h, cells were stained for Foxp3 with the PE anti-mouse/rat FoxP3 staining set (eBioscience), according to the manufacturer’s instructions.CD4+CD62L+ T cells were first activated for 72 h with anti-CD3 plus anti-CD28, washed, adjusted to 15 × 104 cells/ml in culture medium and then expanded for further 96 h in the presence of 20 ng/ml huIL-2.Where needed, CD8+ cells were first differentiated into Tfh cells that express high levels of ICOS. Elongation assays were performed in 24-well culture plates containing glass coverslips previously coated with anti-ICOS antibody or Poly-l-Lysine (10 µg/ml in PBS), as described . The cells were analyzed in an Axioplan Universal microscope  and a Leica DFC 350 FX CCD at a 40× magnification to acquire images. Cell elongation was expressed as the Axis Ratio of the major and minor axis of the ellipse fitting the cell using the shape descriptors of the ImageJ 1.51j8 public domain software .Naive CD4escribed . After wCytokines in culture supernatants  were determined with Ready-SET-Go! kits (eBioscience).+CD62L+ T cells from the spleen of WT or p110α−/−ΔT mice were washed with PBS and activated for 20 min at 37°C in serum-free medium by mixing the cells at 50 × 106/ml with an equal number of latex beads coated with anti-CD3 or control antibodies (5 µg/ml) in the presence of anti-CD28 antibodies (5 µg/ml), as described in Ref. , according to the manufacturer’s instructions. Staining of CD107a was performed as described in detail in Ref. . Flow cy6 cells/ml plus 100 µg/ml KLH in 1 ml of culture medium. Culture supernatants were taken at 96 h to determine KLH-induced cytokine secretion. The titer of anti-KLH-specific antibodies of the IgM, IgG1, IgG2b, IgG2c, and IgG3 subclasses were determined in the serum of individual mice, as described previously (Keyhole limpet hemocyanin (Sigma) was injected at 100 μg/mouse. It was either emulsified v:v in Freund’s complete adjuvant  and injected s.c. in two sites in the base of the tail, or mixed with Alum  and injected i.p. Mice were bled and sacrificed on day 7 (FCA) or day 10 (Alum) after immunization. Cells from inguinal lymph nodes (FCA) or the spleen (Alum) of individual mice were analyzed by flow cytometry, or cultured in round-bottom 5 ml polystyrene tubes at 5 × 10eviously .5 B16.F10 melanoma cells in one flank of mice. Tumor size was periodically measured with calipers by experimenters blind to phenotype. Mice were sacrificed when tumor diameter reached 10 mm, or on day 21 after inoculation, and spleen was obtained.Mouse melanoma B16.F10 cells from a mycoplasma-free stock were maintained by serial passage in culture medium. Primary B16.F10 tumors were induced by s.c. inoculation of 106 cells/culture) in 1 ml of culture medium in round-bottom 5 ml polystyrene tubes containing 100 µg/ml of protein extract from B16.F10 cell lysates. Supernatants were collected after 6 days of culture and cytokine content determined by ELISA.Spleen cells from individual mice were analyzed by flow cytometry, or cultured .The statistical analyses were performed with GraphPad Prism 6.05 using the Student’s All experimental procedures were performed according to established institutional, local, and national guidelines. The studies were first approved by the Animal Welfare Commitees of the Centro de Investigaciones Biológicas (OEBA-CIB) and the Centro Nacional de Microbiología (OEBA-Majadahonda). Then, the study was approved by the Ethics Commitee, Subcommiteee of Bioethics, of the Consejo Superior de Investigaciones Científicas as well as by the Ethics and Animal Welfare Commitee of the Instituto de Salud Carlos III (CEYBA). The study was eventually authorized by the “Consejeria de Medio Ambiente y Ordenación del Territorio de la Comunidad de Madrid” under project licenses PROEX 181/15 (to JR) and PROEX 330/15 (to PP).JR, PP, and UD conceived, designed, and supervised the study, and wrote, reviewed, and revised the manuscript. LA-F, GO, MM-C, YA-A, and JR performed the experiments and acquired the data, analyzed and interpreted the results.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The hexanuclear copper pyrazolato complex has a trigonal prismatic shape and contains an encapsulated chloride ligand. 3(μ3-OCH3)(μ-C3H2ClN2)3}2(μ-C3H2ClN2)3(μ6-Cl)] or [Cu6(C3H2ClN2)9(CH3O)2Cl], crystallizes in the space group Pbcn, with individual molecules being located on a twofold rotation axis. The molecule adopts a trigonal prismatic shape, with two trinuclear units linked by three 4-chloropyrazolate ligand bridges by encapsulating a Cl− anion in a μ6-coordination mode. In the crystal, individual molecules are stacked into rods parallel to [1-10] that are arranged in a pseudo-hexagonal packing. Cohesion between molecules is accomplished through weak C—H⋯Cl interactions.The hexanuclear title compound, [{Cu N-donor ligands have coordination plasticity and large affinity for transition metals, and their employment has provided structures of various nuclearities and dimensionalities, which have been shown to be of interest in catalysis, bio-inorganic chemistry and molecular magnetism. There have been several reports concerning multinuclear copper(II) complexes supported by pyrazolato (pz−) bridging ligands. In this context, we have investigated a family of redox-active Cu6-pyrazolato complexes with trigonal prismatic shapes (μ-C3H2N2Cl)3}2((μ-C3H2N2Cl)3(μ6-Cl)], has been prepared recently; it contains an encapsulated μ6-Cl ligand at the center of the hexanuclear complex.Multinuclear transition metal ion complexes often have interesting properties, such as magnetic, electrochemical, and catalytic functions. 3(μ3-OMe)(μ-4-Cl-pz)3]2+  units bridged by three μ-4-Cl-pz− ligands; the complete molecule adopts .2. point group symmetry. The six CuII ions form a trigonal prismatic array and a chloride ion is located at the center of the cage, coordinating to the two {Cu}3 units in a μ6 mode. All six CuII atoms are five-coordinate with distorted square-pyramidal N3OCl coordination sets with the Cl atom occupying the apical position. Each Cu3 triangle is capped by an OMe group with the O atom 0.8472 (1) Å above the Cu3 plane, a somewhat smaller deviation from the Cu3 plane than the one found in the previously reported structure of [{Cu3(μ3-OMe)(μ-pz)3}2(μ-pz)3(μ6-Cl)], where μ3-bridging methoxy groups are located ca 1.0 Å above this plane  Å. The six Cu—O bond lengths range from 2.033 (2)–2.044 (2) Å, while the Cu—O—Cu angles are in the 102.70 (10)–105.62 (10)° range. The Cu⋯Cu distances within each triangle, 3.1801 (9)–3.2526 (9) Å, are slightly shorter than those between the triangles, 3.356 (2)–3.401 (2) Å). The μ6-Cl ligand is located close to the center of the trigonal prism defined by the six Cu atoms and non-equidistant from the three pairs of CuII ions. The longest Cu—Cl distance of 2.6222 (13) Å (Cu2) is longer than the sum of the ionic radii (2.38 Å), indicating that the two [Cu3(μ3-OMe)(μ-4-Cl-pz)3]2+ units are not templated by the encapsulated chloride. The other two Cu—Cl bond lengths are 2.424 (2) (Cu1) and 2.4859 (13) Å.The crystal structure of the title compound Fig. 1 consists6-pyrazolato} complexes with trigonal prismatic shape are compiled in Table 16Cl] compound reported earlier with 4-H-pz as a ligand  and 3.6059 (6) Å. These distances are comparable to the ones found in the Cu6-pyrazolato complex with no encapsulated anion, where the pyrazolato ring centroids are 3.741 (6), 3.700 (6) and 3.680 (6) Å apart (μ-pz)3}2(μ-pz)3(μ6-Cl)] 3}2(μ6-F)(μ2-OH)] (μ-pz)3}23(μ6-F)] , 4-Cl-pzH  and ethylamine  were stirred in 10 ml CH2Cl2 for 24 h at ambient temperature. The green solution was transferred to a test tube after filtration. A 4 ml 1:1 mixture of CH2Cl2:MeOH (v/v) was layered over the CH2Cl2 layer, 1,2-di(4-pyridyl)ethylene   in 4 ml MeOH was added as the third layer on top of the lower two. Suitable crystals for X-ray diffraction were obtained one week later. Yield: 29%. Analysis calculated/found for C29H24Cl10Cu6N18O2: C, 25.15/25.22; H,1.75/1.76; N, 18.22/18.17.The complex was formed in an one-pot reaction when CuCl−3) near the μ6-Cl atom (Cl6), which decreases if the structure is refined with a free site-occupation factor for this atom. This can be explained if some of the Cu6-cages (< 10%) are vacant. Such a discrepancy is within the experimental error of the CHN elemental analysis, and we decided to refine the model with full occupancy for this Cl atom. In the final cycles, restraints were applied to obtain acceptable ijU parameters for Cl6.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989017001189/wm5353sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989017001189/wm5353Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989017001189/wm5353Isup3.cdxSupporting information file. DOI: 1529271CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The molecular structure of the title compounds may be visualized as two octahedral metal coordinated units, fused through μ-oxide bridges across symmetry centres, leading to edge-sharing dimers. 2(C13H8NOS)2(CO)6], crystallizes in two polymorphs where the 2-phenolate ligands and two carbonyl groups are trans- (I) or cis-arranged (II) with respect to the [Re2O2(CO)4] core. Polymorphs I and II exhibit a crystallographically imposed centre of symmetry and a twofold rotation axis, respectively. The structures may be described as being formed by two octahedrally distorted metal-coordinating units fused through μ-oxido bridges, leading to edge-sharing dimers. The crystal packing is governed by C—H⋯O hydrogen-bonding interactions, forming chains parallel to the c axis in I and a three-dimensional network in II.The title dinuclear complex, [Re The N1—Re01—O1i—C9i torsion angle associated with the Re—O bridging of symmetry-related molecules in trans polymorph I [137.1 (5)°] is distinctly different from the corresponding value in the cis polymorph II [−59.4 (3)°]. The Re⋯Re and O1⋯O1 separations in the Re2O2 core are 3.4799 (5) and 2.581 (8) Å, and 3.4332 (5) and 2.535 (4) Å in I and II, respectively.The title compound,  involving centrosymmetrcally related carbonyl groups C15=O4 [2.792 (10) Å] is present, linking the chains along the b axis to form layers parallel to the bc plane.The crystal structures of is Fig. 3. In addiII, the oxygen atom of the equatorial carbonyl group C14=O2 links the molecules across the glide planes into a three-dimensional network  was made. The search returned 45 crystal structures with three-dimensional coordinates determined, excluding duplicate structure determinations and having an R factor less than 0.075. Out of these 45 crystal structures, 25 crystallize in the monoclinic, nine in the triclinic, eight in the orthorhombic and three in the trigonal systems. In these compounds, the Re⋯Re distance ranges from 3.330 to 3.501 Å, the O⋯O separation within the Re2O2 core ranges from 2.485 to 2.701 Å, and Re—O bond lengths from 2.065 to 2.215 Å.A search in the Cambridge structural Database 10 , 2-phenol  and 2-phenyl-2-imidazoline  in toluene (10 ml) in a Teflon flask was placed in a steel bomb. The bomb was placed in an oven maintained at 433 K for 48 h and then cooled to 298 K. Pale-yellow crystals were obtained and separated by filtration.A mixture of ReII:For 2(CO)10 , 2-phenol  and 2-(pyridin-4-yl)-1--1H-benzo[d]imidazole  in toluene (10 ml) in a Teflon flask was placed in a steel bomb. The bomb was placed in an oven maintained at 433 K for 48 h and then cooled to 298 K. Pale-yellow crystals were obtained and separated by filtration.A mixture of ReI and II, the H atoms were placed in calculated positions (C—H = 0.93–0.97 Å) and were included in the refinement in the riding-model approximation, with Uiso(H) set at 1.2–1.5Ueq(C). In I, two outliers  were omitted in the last cycles of refinement.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989017001347/rz5204sup1.cifCrystal structure: contains datablock(s) I, II. DOI: 10.1107/S2056989017001347/rz5204Isup6.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989017001347/rz5204IIsup7.hklStructure factors: contains datablock(s) II. DOI: 1529701, 1529700CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The bile acid activated transcription factor farnesoid X receptor (FXR) regulates numerous metabolic processes and is a rising target for the treatment of hepatic and metabolic disorders. FXR agonists have revealed efficacy in treating non-alcoholic steatohepatitis (NASH), diabetes and dyslipidemia. Here we characterize imatinib as first-in-class allosteric FXR modulator and report the development of an optimized descendant that markedly promotes agonist induced FXR activation in a reporter gene assay and FXR target gene expression in HepG2 cells. Differential effects of imatinib on agonist-induced bile salt export protein and small heterodimer partner expression suggest that allosteric FXR modulation could open a new avenue to gene-selective FXR modulators. As ligand-activated transcription factor, FXR controls metabolic and inflammatory pathways as monomer or as a permissive heterodimer with the retinoid X receptor (RXR)4. Bile acids, such as chenodeoxycholic acid  which lacks a DNA-binding domain. SHP regulates the expression of several key genes within gluconeogenesis (e.g. phosphoenolpyruvate carboxykinase 1 (PEPCK1)) or lipid metabolism (e.g. sterol regulatory element binding protein 1c (SREBP-1c))8. Therefore, modulation of FXR may hold therapeutic potential for type 2 diabetes mellitus, dyslipidemia and liver disorders such as non-alcoholic steatohepatitis (NASH)9. Accordingly, the first-in-class FXR agonist 6α-ethyl-CDCA  has gained approval for primary sclerosing cholangitis and is in late-stage clinical trials for NAFLD and NASH underlining the value of FXR as drug target11. For the receptor’s extraordinary therapeutic potential, FXR drug discovery is intensive and several compounds including descendants of the widely-used reference compound GW4064 (3) are succeeding in clinical trials.Farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily predominantly found in liver, intestine and kidney4) with low intrinsic efficacy12. Since 4 still possesses high therapeutic relevance in the treatment of chronic myeloid leukemia (CML) such side activity seems important especially because it might be connected to anti-diabetic effects that were observed under treatment with 415. In our consecutive in silico and in vitro screening campaign12., 4 significantly transactivated a full-length FXR reporter gene assay at 30 µM concentration and modulated FXR dependant gene expression. Here we report in-depth evaluation of its mode of FXR activation and characterize it as allosteric modulator of FXR with moderate micromolar potency. Furthermore, we prepared a series of close structural analogues and therein identified an optimized allosteric FXR activator with remarkably enhanced potency.Recently, we reported FXR modulatory potency of the kinase inhibitor imatinib  of 10.7 ± 0.1% compared to FXR full agonist 3 (3 µM) indicating that 4 has partial FXR agonistic activity. To gain evidence that the observed activity is mediated by direct interaction with FXR, we studied binding of imatinib (4) to FXR by saturation-transfer difference NMR (STD-NMR) which is particularly suitable to determine binders with low affinity. We recorded NMR spectra of imatinib (4) alone and in presence of the FXR ligand binding domain (LBD) as well as an STD spectrum of this mixture  did not affect FXR activation but in a concentration (20 µM) above its EC50 value of 6.6 µM, 4 remarkably enhanced FXR activation by the orthosteric ligands instead of reducing FXR-mediated transactivation which would be expected for an orthosteric partial agonist. We studied this effect further by recording dose-response curves of 4 and 1 in presence of a constant concentration of the respective other compound  excluding that RXR activation contributed to FXR modulatory activity of 4.To examine whether modulation of proteins interacting with FXR might contribute to the observed effects, we determined the activity of ion Fig. . However4 is a potent kinase inhibitor, kinase-mediated effects may affect transactivation of the FXR/RXR heterodimer in our reporter gene assay. Therefore, we determined the inhibitory activity of imatinib (4) on kinases modifying FXR or RXR in conventional radiometric assays or homogeneous time-resolved fluorescence (HTRF) technology based test systems  and AMPK has been characterized as a negative regulator of many FXR target genes17. Inhibition of AMPK could therefore potentially cause suppression of this negative regulatory effect leading to FXR activation. However, 4 did not markedly inhibit AMPKα1 or AMPKα2 at 20 µM. Moreover, FXR activity might be affected by the AMPK upstream kinases calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2), transforming growth factor β activated kinase-1 (TAK1) and ataxia-telangiectasia mutated (ATM) kinase that regulate the activity of AMPK by an activating phosphorylation at Thr-172. Inhibition of these AMPK kinases (AMPKK) might reduce activity of AMPK and consequently reduce inhibitory FXR phosphorylation increasing FXR activity, as well19. High concentrations of 4 (20 µM) caused slight inhibition of the AMPKKs, CaMKK2 and TAK1, whereas no activity was observed on ATM. Another prominent AMPKK liver kinase B1 (LKB1) is not expressed in HeLa cells in which we studied the FXR activating activity of 419. Hence, an influence of LKB1 could be excluded. PKC-mediated phosphorylations of FXR could be excluded as well, because 4 containing the flag-methyl-group at position 2 of the central 1,5-diaminophenyl partial structure is inactive at kinases of the PKC-class20. Additionally, known PKA-mediated phosphorylations of RXR could be excluded, since 4 is known as inactive on PKA21.According to literature, a number of kinases can directly or indirectly affect the phosphorylation state of the FXR/RXR-heterodimer4. The observed inhibitory activity of 4 on the AMPKKs, CaMKK2 and TAK1, was very weak especially compared to the efficacy of 4 on its main targets Abl, c-Kit and PDGF-Rα. Cascadic kinase signalling is self-enhancing and known to require strong inhibition for a significant effect. Therefore, enhanced FXR activation by 4 or by 1–3 in presence of 4 does not seem to be affected by kinase inhibition but arise alone from direct modulation of the nuclear receptor.Taken together, it is unlikely that kinase-mediated effects are significantly contributing to FXR modulation by 4 also interacts with this membrane bile acid receptor. In vitro evaluation of 4 on TGR5  endogenously recognizes bile acids. It was therefore possible that GR5 Fig.  revealed27–36 from precursor 6 and carboxylic acids 18–26 using CDI in DMF and subsequently transformed them to mesylate salts 7–17 for improved solubility and comparability with 4 mesylate  increased potency by almost 10-fold, indicating an optimal substitution site for optimization22. When we enlarged the methyl residue of 11 to an ethyl group (12), single-digit nanomolar activity was achieved but further enlargement of the lipophilic para-substituent to tert-butyl (13) or phenyl (14) caused a significant loss of potency indicating that only small 4-substituents were well tolerated but further optimization potential could lie in the electronic characteristics of the benzoyl residue. Following the Topliss’ strategy23, we therefore introduced a 4-methoxy group (15) and a 4-chlorine atom (16) which showed pronounced effects on potency. Electron releasing methoxy substitution (15) was poorly tolerated whereas the electron withdrawing chlorine atom (16) further increased activity to an EC50-value of 1.9 nM. With this high FXR modulatory potency, 16 seemed sufficiently optimized as potential tool compound mimicking the activity of 4.As a starting point for structural optimization Table , we char16 as we observed for 4, 16 was equally characterized in vitro. However, STD-NMR is not suitable to detect high-affinity binders because dissociation of magnetized ligand from the protein is required to observe saturation transfer25. Binding of 16 to the FXR-LBD was therefore confirmed by thermal shift  or 16 (0.3 µM) alone or in presence of 1 (20 or 50 µM) and determined the expression of FXR target genes bile salt export protein (BSEP) and small heterodimer partner (SHP) on mRNA level by quantitative real-time polymerase chain reaction  concentration of 1 -induced SHP and BSEP expression. Surprisingly, 4 markedly diminished whereas 16 strongly promoted CDCA (1)-induced BSEP expression. Regarding SHP expression, 4 and 16 both enhanced CDCA (1)-induced expression although with different efficacy. Such gene-selective activity might be due to differential recruitment of distinct co-activators as it has been described e.g. for some selective PPAR modulators27. Our results indicate that FXR activity can be modulated via an allosteric mechanism in both, synergistic ago-positive and allosteric antagonistic fashion. Moreover, minor structural changes seem capable of affecting the mode of allosteric modulation since exchanging the N-methylpiperazinylmethyl moiety of 4 by a chlorine atom in 16 changed the allosteric antagonism that 4 exhibits on BSEP expression to synergistic ago-positive activity. Understanding of the structural basis for this gene selective allosteric modulation requires further studies but will be of great value for the development of safer and more efficacious FXR targeting agents.Allosteric FXR modulators 4 on SHP mRNA levels in presence of CDCA (1) provides another potential link between the low partial agonistic potency of 4 and significant effects on glucose homeostasis that were observed under treatment with 4. Physiologically, bile acids such as 1 govern FXR activity and our results indicate that 4 and 16 are capable of enhancing their FXR agonistic activity. Consequently, the expression of SHP is strongly increased and by SHP-dependent repression of PEPCK1 and G-6-Pase, gluconeogenesis is reduced. Altogether, this mechanism provides a possible new explanation for reduced glucose levels upon imatinib treatment and in light of an EC50-value of 6.6 ± 0.3 µM for FXR activation, reported plasma levels for 4 of up to 8.1 µM are sufficient to cause FXR modulation under treatment with 428.The effect of 4 and descendant 16 as allosteric modulators of FXR30. This novel mode of FXR modulation might hold considerable pharmacological potential by fueling the efficacy of endogenous FXR agonists. Additionally, distinctly pronounced additive effects on the expression of different genes suggest that allosteric FXR modulation is feasible and could open a new avenue towards gene-selective FXR modulation. Still, allosteric FXR modulators without kinase inhibitory potency are required to promote this concept towards clinical development.In conclusion, we have classified N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)benzamide (36): Preparation according to general procedure (a) using 4-chlorobenzoic acid (26). Yield: 19%. 1H-NMR 2SO): δ = 10.28 , 9.28 , 8.99 , 8.69 , 8.52 , 8.49 , 8.08 , 7.99 , 7.61 , 7.53 , 7.48 , 7.44 , 7.22 , 2.23 ; 13C-NMR 2SO) δ: 164.71 (C=O), 162.08 (Pyrim-4-C), 161.62 (Pyrim-2-C), 159.96 (Pyrim-6-C), 151.88 (Pyrid-4-C), 148.67 (Pyrid-2-C), 138.30 (Phen-NH-1-C), 137.41 (Phen-NH-5-C), 136.75 (Phen-CO-4-C), 134.90 (Pyrid-6-C), 134.22 (Phen-CO-1-C), 132.66 (Pyrid-1-C), 130.55 (Phen-NH-3-C), 130.06 , 128.92 , 128.27 (Phen-NH-2-C), 124.27 (Pyrid-5-C), 117.68 (Phen-NH-6-C), 117.21 (Phen-NH-4-C), 108.03 (Pyrim-5-C), 18.14 (-CH3); Rf (PE/THF = 1/1): 0.17; C23H18ClN5O; MS (ESI+): m/z = 416.1 [M + H]+; colourless solid2.4-Chloro-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)benzamide dimesylate-sesquihydrate (16): Preparation according to general procedure (b) using 36. Yield: 43%. 1H-NMR 2SO): δ = 10.29 , 9.38 , 9.09 , 8.82 , 8.77 , 8.58 , 8.12 , 8.00 , 7.78 , 7.62 , 7.51 , 7.46 , 7.23 , 2.32 , 2.24 ; 13C-NMR 2SO) δ: 164.75 (C=O), 161.47 (Pyrim-2-C), 160.28 (Pyrim-6-C), 160.17 (Pyrim-4-C), 149.15 (Pyrid-4-C), 146.22 (Pyrid-2-C), 138.20 (Pyrid-6-C), 137.70 (Phen-NH-1-C), 137.39 (Phen-NH-5-C), 136.78 (Phen-CO-4-C), 134.18 (Phen-CO-1-C), 133.86 (Pyrid-1-C), 130.73 (Phen-NH-3-C), 130.20 , 129.02 , 128.25 (Phen-NH-2-C), 125.59 (Pyrid-5-C), 117.78 (Phen-NH-6-C), 117.42 (Phen-NH-4-C), 108.28 (Pyrim-5-C), 40.24 (S-CH3), 18.10 (-CH3); C23H18ClN5O × 2 CH4O3S × 1.5 H2O; combustion analysis: measured : C 47.36 (47.28); H 4.40 (4.60); N 11.28 (11.03); orange solid.4-Chloro-7–15 as well as their precursors, please refer to supporting information.For general synthetic procedures and analytical characterization of 12. pGL3basic  was used as a reporter plasmid, with a shortened construct of the promotor of the bile salt export protein (BSEP) cloned into the SacI/NheI cleavage site in front of the luciferase gene31. pRL-SV40 (Promega) was transfected as a control for normalization of transfection efficiency and cell growth. pSG5-hRXR was already published elsewhere as well32.pcDNA3-hFXR contains the sequence of human FXR and was already published elsewhere2. 24 h before transfection, HeLa cells were seeded in 96-well plates with a density of 8000 cells per well. 3.5 h before transfection, medium was changed to DMEM high glucose, supplemented with sodium pyruvate (1 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and 0.5% charcoal-stripped FCS. Transient transfection of HeLa cells with BSEP-pGL3, pRL-SV40 and the expression plasmids pcDNA3-hFXR and pSG5-hRXR was carried out using calcium phosphate transfection method. 16 h after transfection, medium was changed to DMEM high glucose, supplemented with sodium pyruvate (1 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and 0.5% charcoal-stripped FCS. 24 h after transfection, medium was changed to DMEM without phenol red, supplemented with sodium pyruvate (1 mM), penicillin (100 U/mL), streptomycin (100 µg/mL), L-glutamine (2 mM) and 0.5% charcoal-stripped FCS, now additionally containing 0.1% DMSO and the respective test compound or 0.1% DMSO alone as untreated control. Each concentration was tested in triplicate wells and each experiment was repeated independently at least three times. Following 24 h incubation with the test compounds, cells were assayed for luciferase activity using Dual-Glo™ Luciferase Assay System (Promega) according to the manufacturer’s protocol. Luminescence was measured with a Tecan Infinite M200 luminometer . Normalization of transfection efficiency and cell growth was done by division of firefly luciferase data by renilla luciferase data multiplied by 1000 resulting in relative light units (RLU). Fold activation was obtained by dividing the mean RLU of the tested compound at a respective concentration by the mean RLU of untreated control. Relative activation was obtained by dividing the fold activation of the tested compound at a respective concentration by the fold activation of FXR full agonist 3 at 3 µM. EC50 and standard error of the mean values were calculated with the mean relative activation values of at least three independent experiments by SigmaPlot 10.0  using a four-parameter logistic regression. The assay was validated with FXR agonists 1 , 2  and 3 33.HeLa cells were grown in DMEM high glucose supplemented with 10% FCS, sodium pyruvate (1 mM), penicillin (100 U/mL) and streptomycin (100 µg/mL) at 37 °C and 5% CO34, pFA-CMV-hPPARγ-LBD34, pFA-CMV-hPPARδ-LBD34, pFA-CMV-hLXRα-LBD35, pFA-CMV-hLXRβ-LBD35, pFA-CMV-hRXRα-LBD36, pFA-CMV-hRXRβ-LBD36, pFA-CMV-hRXRγ-LBD36, pFA-CMV-hRARα-LBD36, pFA-CMV-hRARβ-LBD36, pFA-CMV-hRARγ-LBD36, pFA-CMV-hVDR-LBD36 and pFA-CMV-hCAR-LBD36 coding for the hinge region and ligand binding domain (LBD) of the canonical isoform of the respective nuclear receptor have been reported previously. pFR-Luc (Stratagene) was used as reporter plasmid and pRL-SV40 (Promega) for normalization of transfection efficiency and cell growth.The Gal4-fusion receptor plasmids pFA-CMV-hPPARα-LBD2. The day before transfection, HEK293T cells were seeded in 96-well plates (2.5·104 cells/well). Before transfection, medium was changed to Opti-MEM without supplements. Transient transfection was carried out using Lipofectamine LTX reagent (Invitrogen) according to the manufacturer’s protocol with pFR-Luc (Stratagene), pRL-SV40 (Promega) and pFA-CMV-hNR-LBD. 5 h after transfection, medium was changed to Opti-MEM supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL), now additionally containing 0.1% DMSO and the respective test compound or 0.1% DMSO alone as untreated control. Each concentration was tested in duplicates and each experiment was repeated independently at least three times. Following overnight (12–14 h) incubation with the test compounds, cells were assayed for luciferase activity using Dual-Glo™ Luciferase Assay System (Promega) according to the manufacturer’s protocol. Luminescence was measured with an Infinite M200 luminometer (Tecan Deutschland GmbH) or a Tecan Spark 10 M luminometer (Tecan). Normalization of transfection efficiency and cell growth was done by division of firefly luciferase data by renilla luciferase data and multiplying the value by 1000 resulting in relative light units (RLU). Fold activation was obtained by dividing the mean RLU of a test compound at a respective concentration by the mean RLU of untreated control. Relative activation was obtained by dividing the fold activation of a test compound at a respective concentration by the fold activation of a respective reference agonist at 1 µM . All hybrid assays were validated with the above-mentioned reference agonists which yielded EC50 values in agreement with literature.HEK293T cells were grown in DMEM high glucose, supplemented with 10% FCS, sodium pyruvate (1 mM), penicillin (100 U/mL) and streptomycin (100 μg/mL) at 37 °C and 5% CO2O with 10 mM Tris-d11 (DCl), 100 mM NaCl, 2 mM DTT-d10, pD = 8.3). The storage buffer of the purified FXR-LBD protein was exchanged against STD-NMR buffer by 10 cycles of diluting the protein solution with STD-NMR buffer 1:2, before concentrating it to the starting volume via Vivaspin2 5 kDa MWCO centricons . STD NMR samples were prepared in a 550 µL scale with 50 µM FXR-LBD (or STD-NMR buffer in case of the reference measurement of the test compounds) and 1.5% DMSO-d6 with either 1  or 4 . Samples were measured with a 700 MHz Bruker AvIIIHD spectrometer equipped with a 1H{31P/13C/15N} cryogenic quadruple-resonance probe (QCI) at 298 K. Pulse programs, described by A. D. Gossert and W. Jahnke were downloaded from the Bruker database and adjusted for the 700 MHz Bruker AvIIIHD spectrometer37. To suppress the residual water resonances the excitation sculpting method was used in all measurements. For STD-experiments the frequencies for ON and OFF irradiation were set at +0.5 and −30 ppm saturation for 2 s using a 50-ms Gaussian pulse train (10 µW) truncated at 1% with a 50 ms spin-lock to suppress protein signals . The standard 1D 1H-NMR experiments were performed with a perfect echo sequence with the following parameters .STD NMR experiments were performed in STD-NMR buffer  in assay buffer (10 mM TRIS (pH 8.3), 5 mM DTT, 0.5 mM EDTA, 100 mM NaCl) were mixed with 10 μL of FXR-LBD  in assay buffer and 5 µL of SYPRO Orange . Temperature-dependent fluorescence increase reporting protein denaturation was measured in duplicates in an ICycler  from 20 to 90 °C in steps of 0.5 °C per minute at 300 nm excitation and 570 nm emission wavelength. The first derivative of the melting curve was calculated using the Graph Pad Prism 5 software. All curves were determined at least four times in independent experiments. Results : 3: 100 µM: 3.75 ± 0.25 °C; 16: 100 µM: −1.0 ± 0.0 °C; 3: 100 µM + 16: 100 µM: 6.00 ± 0.33 °C.Thermal shift experiments were performed in clear 96-well plates (Invitrogen) using SYPRO Orange (Invitrogen) as dye as described previously39. In brief, HepG2 cells were incubated with test compound 4 (10 µM), 16 (0.3 µM) or 1 (20 or 50 µM), or 0.1% DMSO alone as untreated control for 24 h, harvested, washed with cold phosphate buffered saline (PBS) and then directly used for RNA extraction. Two micrograms of total RNA were extracted from HepG2 cells by the Total RNA Mini Kit . RNA was reverse-transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit  according to the manufacturer’s protocol. FXR target gene expression was evaluated by quantitative real time PCR analysis with a StepOnePlus™ System  using PowerSYBRGreen . The primers have been described previously39. Each sample was set up in duplicates and repeated in at least three independent experiments. The expression was quantified by the comparative ∆∆Ct method and glycerinealdehyde 3-phosphate dehydrogenase (GAPDH) served as reference gene. Results (expressed as mean fold activation ± SEM compared to DMSO (0.1%); n ≥ 3): BSEP: 1 (20 µM): 2.85 ± 0.80; 1 (50 µM): 11.32 ± 0.93; 4 (10 µM): 2.25 ± 0.16; 16 (0.3 µM): 2.64 ± 0.07; 1 (20 µM) + 4 (10 µM): 1.19 ± 0.18; 1 (20 µM) + 16 (0.3 µM): 4.56 ± 0.77; 1 (50 µM) + 4 (10 µM): 4.73 ± 0.61; 1 (50 µM) + 16 (0.3 µM): 29.37 ± 3.32. SHP: 1 (20 µM): 1.56 ± 0.12; 1 (50 µM): 3.83 ± 0.14; 4 (10 µM): 3.49 ± 0.32; 16 (0.3 µM): 2.32 ± 0.18; 1 (20 µM) + 4 (10 µM): 1.33 ± 0.04; 1 (20 µM) + 16 (0.3 µM): 1.64 ± 0.23; 1 (50 µM) + 4 (10 µM): 4.72 ± 0.52; 1 (50 µM) + 16 (0.3 µM): 12.70 ± 1.04.FXR target gene quantification was performed as described previouslySupporting Information


Since activation of vascular α1/α2-adrenoceptors results in systemic vasopressor responses, the purpose of this study was to investigate the specific role of α1- and α2-adrenoceptors mediating DHE-induced vasopressor responses using several antagonists for these receptors.Dihydroergotamine (DHE) is an acute antimigraine agent that displays affinity for dopamine D2 receptor-mediated systemic vasoconstriction). Then, the vasopressor responses to i.v. DHE  were determined after i.v. administration of some α1/α2-adrenoceptor antagonists.For this purpose, 135 male Wistar rats were pithed and divided into 35 control and 100 pretreated i.v. with ritanserin ; (ii) slightly, but significantly, blocked after rauwolscine ; and (iii) markedly blocked after prazosin (30 μg/kg) plus rauwolscine (300 μg/kg). In contrast, after pretreatment with ritanserin, the vasopressor responses to DHE were: (i) attenuated after prazosin  or rauwolscine ; (ii) markedly blocked after prazosin (30 μg/kg) plus rauwolscine (300 μg/kg); (iii) attenuated after 5-methylurapidil , L-765,314 , BMY 7378 , BRL44408 , imiloxan  or JP-1302 ; and (iv) unaffected after the corresponding vehicles (1 ml/kg).In control animals (without ritanserin pretreatment), the vasopressor responses to DHE were: (i) unaffected after prazosin  and α2-  adrenoceptors. These findings could shed light on the pharmacological profile of the vascular side effects (i.e. systemic vasoconstriction) produced by DHE and may lead to the development of more selective antimigraine drugs devoid vascular side effects.These results suggest that the DHE-induced vasopressor responses in ritanserin-pretreated pithed rats are mediated by α All compounds were dissolved in physiological saline. When needed, 1% ascorbic acid was used to dissolve ritanserin or 20% PPG (dissolved in bidistilled water) to dissolve DHE. Initially, DHE (3100 μg/ml) was dissolved in 20% PPG and the subsequent solutions were finally diluted with physiological saline. Fresh solutions were prepared for each experiment.P > 0.05; as compared with the corresponding untreated control group) in the groups of animals pretreated with all doses of the antagonists or their vehicles  after i.v. administration of 1 ml/kg saline  after 30 μg/kg prazosin  after the combination 30 μg/kg prazosin plus 300 μg/kg rauwolscine   slHE) Fig. .1- and α2-adrenoceptor antagonists).Figure Figures P < 0.05) in animals pretreated with the antagonists prazosin  than that produced by each antagonist given individually  in the DHE vasopressor responses. In keeping with this view: (i) in animals without ritanserin-pretreatment the vasopressor responses to DHE remained unchanged after 30 μg/kg prazosin  [1-adrenoceptors by prazosin. Certainly, prazosin has higher affinity (approximately 1 to 2 logarithmic units) than DHE for α1-adrenoceptors (Table i) of prazosin for 5-HT2 receptors (if any) is <<4 [2 receptors. This suggestion is reinforced when considering that the blockade produced by the combination 30 μg/kg prazosin plus 300 μg/kg rauwolscine in the absence of ritanserin was more pronounced than that produced by rauwolscine alone  is a cosin Fig. , a dose hed rats ; and  , may hav) is <<4 , whereasone Fig. . This lione Fig.  as compaone Fig. . These f1-  and α2-  adrenoceptors, although some caution should be exerted when interpreting the “subtype selectivity” of the compounds used (see below and Table 1A), L-765,314 (α1B) or BMY 7378 (α1D) , imiloxan (α2B) or JP-1302 (α2C)  produced dose-dependent increases in diastolic blood pressure Fig.  without 1-adrenoceptors (pKi: 8.0) and rat α2-adrenoceptors (pKi: 8.0) [1- and α2-adrenoceptor subtypes  blocked by prazosin . This suggestion may help partly explain why Roquebert and Grenié [2-adrenoceptors, but not of α1-adrenoceptors, in the DHE responses in Wistar rats without 5-HT2 receptor blockade. Admittedly, Roquebert and Grenié [1-adrenoceptor subtypes depends on several factors, including age [DHE displays affinity for a wide variety of receptors , with thKi: 8.0) . Interesoceptors  and α2-aoceptors . Nonethed Grenié  could shd Grenié : (i) didding age .Interestingly, the failure of the combination prazosin plus rauwolscine to abolish  the DHE responses in ritanserin-pretreated rats Fig.  cannot c2-adrenoceptors and, apparently to a lesser extent, by prazosin-sensitive α1-adrenoceptors. Nevertheless, these antagonists do not display selective affinities for distinguishing amongst their corresponding α1- and α2-adrenoceptor subtypes  and the α2-adrenoceptor subtypes   to completely block the vasopressor responses mediated by the α1A/α1B/α1D subtypes and the α2A/α2B/α2C subtypes in pithed rats [The fact that the DHE responses were blocked after administration of each of these antagonists for α1- Fig.  and α2-aors Fig.  basicallhed rats , 13; andhed rats  (see Tab1A (pKi: 9.0) and α1D (pKi: 9.0) subtypes, but they also display moderate affinity for the other α1 subtypes A-61603; and (ii) 100–1000 μg/kg BMY 7378, which dose-dependently blocked the vasopressor responses to phenylephrine, failed to block those to (R)A-61603. Thus, it would seem logical to suggest that 5-methylurapidil  for the α1B subtype and its moderate affinity for the α1D subtype (Table 1B subtype (relative to that of the α1A- and α1D- subtypes) in the systemic vasculature, as suggested by Daly et al. [On the one hand, 30–100 μg/kg of 5-methylurapidil Fig.  and BMY d Vargas  showed idil Fig.  and BMY dil Fig.  are reass et al. . In conty et al. .2-adrenoceptor subtypes, BRL44408 and JP-1302 are “relatively selective” for, respectively, the α2A (pKi: 8.7) and α2C (pKi: 7.6) subtypes , the vasopressor responses to i.v. bolus injections of 0.1, 0.3, 1, 3, 10 and 30 μg/kg phenylephrine : (i) remained unaltered after an i.v. bolus of 100 μg/kg BRL44408 ; and (ii) were attenuated (at the highest doses) after an i.v. bolus of 300 μg/kg BRL44408  (unpublished observations). The latter finding may explain why the blockade produced by BRL4408  from that produced by the combination prazosin plus rauwolscine , but its affinity for the α2B (pKi: 7.3) and α2C (pKi: 6.0) subtypes  adrenoceptor subtype. Hence, we considered it unnecessary to explore the effect of more antagonist combinations.On the other hand, as to the role of the αes Table  and the es Table  to block408 Fig. : (i) didine Fig. ; and  the nature of our pithed rat model ; (ii) the relative “selectivity” of the antagonists used -adrenoceptors and, apparently to a lesser extent, by α1 -adrenoceptors. Admittedly, this conclusion is based on the assumption that all antagonists used are relatively selective  responses to DHE in ritanserin pretreated pithed rats could be mediated by activation of α


The crystal structure of the enantiopure sulfoxide of a 2,3,5,6-tetrahydro-1,3-thiazin-4-one exhibits a twisted half-chair pucker for the thiazine ring. Intermolecular N—H ⋯O hydrogen-bonding interactions form a two-dimensional layered structure lying parallel to (001). 18H18N2O3S, comprises two independent molecules (A and B) having almost identical conformations. When overlayed, the alignment–r.m.s. deviation value is 0.30 Å. The six-membered heterocycle has a twisted half-chair conformation in both molecules. The O atom on the S atom of the ring is pseudo-axial on the thiazine ring and trans to both a phenyl group substituent and the acetamide group in each case. The two benzene rings in each molecule are almost orthogonal to each other, with interplanar dihedral angles of 83.79 (17) and 86.95 (16)°. The acetamide group is pseudo-equatorial and a phenyl ring is pseudo-axial on the thiazine ring. Both molecules show a weak intramolecular C—H⋯O interaction between H-atom donors of one of the phenyl rings and the acetamide group. In the crystal, an intermolecular N—H⋯O(thiazine) hydrogen bond links B molecules along the 21 (b) screw axis and, in addition, an N—H⋯O(acetamide) hydrogen bond links A and B molecules across a. A two-dimensional layered structure lying parallel to (001) is generated, also involving weak intermolecular C—H⋯O interactions.The asymmetric unit of the enantiomerically pure title compound, C The acetamide group is pseudo-equatorial and the 2-phenyl group is pseudo-axial on the thiazine ring. A weak intramolecular C—H⋯O hydrogen bond between the 2-phenyl ring and the O atom of the acetamide group is seen in both molecules (C10A—H⋯O3A and C10B—H⋯O3B), as detailed in Table 1The crystal structure of the title compound has two independent homochiral molecules -4-oxo-2,3-diphenyl-1,3-thiazinan-5-yl]acetamide 0.375-hydrate  −x, y + z; Table 1A and B interact, wherein the O atom in the 4-position of molecule B accepts a proton from the acetamide N atom of molecule A . The sulfoxide O atom of molecule A does not participate in any hydrogen bonding. A two-dimensional sheet structure lying parallel to (001) is generated. No benzene ring in either of the molecules participates in face-to-face π–π stacking interactions.In the crystal, the et al., 2010et al., 1983et al., 2011et al., 2008et al., 2015H-1,3-thiazin-4-one 1,1-dioxide] has also been reported -4-oxo-2,3-diphenyl-1,3-thiazinan-5-yl]acetamide 0.375-hydrate, whose configuration was established previously . The solids were dissolved by the addition of 5 ml of distilled water. The solution was extracted with 10 ml of dichloromethane. The organic layer was washed with 5 ml of distilled water and then with 5 ml of saturated sodium chloride. The solution was dried over Na2SO4 and concentrated under vacuum giving a crude solid. This was chromatographed on flash silica gel, eluting with a gradient of 0–60% acetone in ethyl acetate, giving 55.8 mg of product . Crystals suitable for X-ray crystallography were grown by slow evaporation from propan-2-ol.A 5 ml round-bottomed flask was charged with 53.9 mg of Uiso(H) = 1.2Ueq(aromatic or methylene C) or 1.5Ueq(methyl C). H atoms on N atoms were located in a difference Fourier map and were refined isotropically. The absolute configuration for the chiral centres in the molecule was determined as  , with a Flack absolute structure parameter  I. DOI: 10.1107/S2056989017012488/zs2387Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989017012488/zs2387Isup3.molSupporting information file. DOI: 10.1107/S2056989017012488/zs2387sup4.pdfAnalysis of short ring interactions.. DOI: 1571357CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


This work shows that hmta only protonates once, even in the presence of excess acid.Four molecular salts made from hexamethylenetetraminium and 2-chloro-4-nitrobenzoate have been synthesized and are reported. All four molecular salts show N 4+·C6H13N4+·2C7H3ClNO4−, (I), hexamethylenetetraminium hydrogen bis(2-chloro-4-nitrobenzoate), 0.5C6H13N4+·C7H3.50ClNO4−, (II), hexamethylenetetraminium 2-chloro-4-nitrobenzoate, C6H13N4+·C7H3ClNO4−, (IIIa) and (IIIb). All four molecular salts show N+—H⋯O− hydrogen bonding. Salt (I) crystallized out with an NH4+ counter-ion which came from decomposition of 50% of the hexamethylenetetraminium cation in solution. (II) shows an unusual asymmetric unit, with both a hexamethylenetetraminium cation and a partially deprotonated 2-chloro-4-nitrobenzoate anion. Salts (IIIa) and (IIIb) are polymorphs of each other. This work shows that hexamethylenetetramine only protonates once, even in the presence of excess acid.Four molecular salts made from hexamethylenetetraminium and 2-chloro-4-nitrobenzoate have been synthesized and are reported, namely ammonium hexamethylenetetraminium bis(2-chloro-4-nitrobenzoate), NH However, the carboxylic acid group of 2c4nH has bond lengths typical of being neutral and clearly shows an acidic H atom, H2, located near O1 in the difference-Fourier map. Combined, this means that H1 acts as a bifurcated donor to two 2-chloro-4-nitrobenzoic molecules (Table 2a) and (IIIb) both have a 1:1 ratio and are polymorphs of each other. Both have charge-assisted N+—H⋯O− hydrogen bonds . The NH4+ cation forms a hydrogen-bonded ring using two carboxylate groups and this ring repeats along the b-axis direction. The ring can be described as − anion that hydrogen bonds to the hmta+ cation. Overall, the hydrophilic layer consists of the cationic NH part of hmtaH+, NH4+ and the carboxylate CO2− part of 2c4n− . Salt (II)+ and 2c4n− anion in a 1:2 ratio. However, it appears crystallographically that only one complete proton transfer has taken place, and that on average, each of the 2c4n anions has released half a proton each to the N atom (labelled H1) and that the other half proton (labelled as H2) is located in between the two anions. Hence, only one N atom on hmta has been protonated, and subsequently, two 2c4n− anions are behaving as acceptors from a single N—H group  and (IIIb) have identical asymmetric units with a 2:1 ratio of hmtaH+ and 2c4n−, in contrast to the previous two salts. The only significant difference is in the relative packing of these ion pairs. In (IIIa), the pairs pack anti-parallel , and in (IIIb), parallel .The packing of salt (I)n− Fig. 3a. Salt up Fig. 1. Overallel Fig. 3c, and iel Fig. 3d.+ molecular salts in the Cambridge Structural Database a) and (IIIb), concomitantly from a 1:1 molar ratio. The morphology of the yellow-tinted crystals are shown in Fig. 4a) and (IIIb): hexamethylenetetramine  and 2-chloro-4-nitrobenzoic acid acid  in ethanol (5 mL).All chemicals were purchased from commercial sources (Sigma Aldrich) and used as received without further purification. Crystals were grown 2), and 0.95 (Ar—H) Å) and refined as riding with Uiso(H) = 1.2Ueq(C). The N–bound H atoms were located in difference-Fourier maps and their coordinates and isotropic displacement parameters allowed to refine freely. The O–bound H atom in (II)Uiso(H) = 1.5Ueq(O).Crystal data, data collection and structure refinement details are summarized in Table 510.1107/S2056989017014359/eb2001sup1.cifCrystal structure: contains datablock(s) I, II, IIIa, IIIb, shelx. DOI: 10.1107/S2056989017014359/eb2001Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989017014359/eb2001IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989017014359/eb2001IIIasup4.hklStructure factors: contains datablock(s) IIIa. DOI: 10.1107/S2056989017014359/eb2001IIIbsup5.hklStructure factors: contains datablock(s) IIIb. DOI: 1578099, 1578098, 1578097, 1578096CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Whereas much is known about the physiological effects of 17 β-E2, much less is known about 17 α-E2. For example, 17 β-E2 exerts anti-inflammatory effects in neurons and adipocytes through binding and activation of estrogen receptor alpha (ERα); however, if 17 α-E2 has similar effects on inflammation is currently unknown.To begin to address this, we analyzed the ability of 17 α-E2 and 17 β-E2 to suppress lipopolysaccharide (LPS)-induced inflammation in vitro using embryonic fibroblast cells (MEF) from wild type and total body ERα (ERKO) male and female mice. Additionally, we further probed if there were sex differences with respect to the effects of E2s using primary pre-adipocyte cells from C57BL/6J male and female mice. Also, we probed mechanistically the effects of E2s in fully differentiated 3T3-L1 cells.Tnf-α and Il-6, and increased the anti-inflammatory markers Il-4 and IL-6 receptor (Il-6ra) in MEF cells. To begin to understand the mechanisms by which both E2’s mediate their anti-inflammatory effects, we probed the role of ERα using two methods. First, we used MEF cells from ERKO mice and found reductions in ERα diminished the ability of 17 α-E2 to suppress Tnf-α in female but not in male cells, demonstrating a sexual dimorphism in regard to the role of ERα to mediate 17 α-E2’s effects. Second, we selectively reduced the expression of ERα in 3T3-L1 cells using siRNA and found reductions in ERα diminished the ability of both E2s to suppress Tnf-α and Il-6 expression. Lastly, to determine the mechanisms by which E2s reduce inflammation, we explored the role of NFκB-p65 and found both E2s decreased NFκB-p65 expression.Both E2s decreased LPS-induced markers of inflammation In conclusion, we demonstrate for the first time that 17 α-E2, as well as 17 β-E2, suppresses inflammation through their effects on ERα and NFκB-p65.The online version of this article (10.1186/s13293-017-0151-9) contains supplementary material, which is available to authorized users. Estrogens play a crucial role in suppressing the development of chronic inflammatory diseases  and mitiTnf-α and Il-6 genes [Il-6 [Estrogens are a family of compounds that include 17 alpha-estradiol (17 α-E2) as well as 17 beta-estradiol (17 β-E2). 17 β-E2 is the main circulating and biologically most active form of estrogen , 7, and -6 genes , 12. In es [Il-6 . Furtheres [Il-6 .17 α-E2 is a natural, non-feminizing isomer of 17 β-E2 , and itsIn contrast to 17 α-E2, 17 β-E2 also has feminizing effects and can lead to uterine and ER-positive breast cancer in postmenopausal women –30. ReceBriefly, mouse embryonic fibroblast (MEF) cells were generated from C57BL/6J WT and total body ERα knockout (ERKO) mice. ERKO mice were a gift from Pierre Chambon . EmbryosMEF cells were cultured in DMEM no-phenol red , supplemented with 10% charcoal: dextran stripped fetal bovine serum , 1% pen-strep 10,000 U/ml (Thermo Fisher), and 1% sodium pyruvate 100 mM (Thermo Fisher). Primary pre-adipocytes were cultured in DMEM/F-12 with GlutaMAX phenol red (Thermo Fisher) supplemented with 10% FBS and 1% pen-strep 10,000 U/ml. 3T3-L1 cells were grown in DMEM phenol-red media , supplemented with 10% Bovine calf bovine serum (ATCC) and 1% pen-strep. After 48 h of confluency, DMEM was supplemented with methylisobutylxanthine , dexamethasone , and insulin 10 μg/ml (Sigma) , and serCells were treated for the indicated time with 17 α-E2 (Sigma) or 17 β-E2 (Sigma) at 10 μM concentration. Inflammation was induced by LPS at a concentration of 10 ng/ml (Sigma) either alone or in combination with the respective estrogen.Esr-1 (ERα gene) in the presence and absence of estrogens to determine if ERα mediates estrogens’ effects on inflammation. To do this, 3T3-L1 cells were cultured in 12-well plates and transfected at day 10 of differentiation with siRNA targeting murine Esr-1 . As a control, cells were treated with an unrelated control/scrambled sequence siRNA at the same concentration . Lipofectamine RNAi max was used as the transfection reagent , according to the manufacturer’s instructions. Seventy-two hours after siRNA transfection, cells were treated with estrogens and LPS as indicated, and then lysed for RNA extraction.In this series of experiments, we used siRNA to knockdown Tnf-α); interleukin 6 (Il-6); and Nuclear Factor Kappa B Subunit 1 (Nfκb1), and anti-inflammatory genes interleukin 4 (Il-4), and interleukin 6 receptor alpha (Il-6rα) were quantified following LPS and/or E2s using qPCR. To determine the impact of treatments on ERα, we also quantified Esr-1 gene expression. Cells were washed twice with PBS (Thermo Fisher) and lysed in 500 μl of TRIzol® reagent (Thermo Fisher). RNA from cells was extracted and isolated using the RNeasy kit , according to the manufacturer’s instructions. The concentration and purity of RNA were determined by spectrophotometric analysis , and all samples had a A260/A280 ratio around 2.0 [Gapdh was used as reference gene, and data was normalized and relative expression determined using 2-∆∆ CT method.Relative expression of the pro-inflammatory genes Tumor Necrosis Factor Alpha , supplemented with anti-phosphatase cocktail  and anti-protease cocktail , was used per well. After sample centrifugation, the supernatant was separated and used to quantify protein concentration and prepare samples for gel electrophoresis and Western blotting. For cell fractionation, the protocol adapted from Silva et al. (2005)  was usedProtein concentrations were determined by BCA (Thermo Fisher). Proteins (30–50 μg) were separated by electrophoresis  and electro-transferred to nitrocellulose membrane . Non-specific binding sites were saturated by incubation of membranes for 1 h in TBS (supplemented with 1% Tween 20) and 5% non-fat powdered milk, followed by overnight incubation with primary antibodies . Appropriate secondary antibodies were used , followed by enhanced chemiluminescence assay (Bio-Rad). The optical density of blots was analyzed using ImageLab software (Bio-Rad), and β-actin  or histone  were used for normalization. The results were expressed as arbitrary units in comparison to the control group, which was set as 1.0.In this series, we verified protein expression and location of NFκB p65 and ERα. To do this, cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% saponin. Cells were stained for primary antibodies NFκB p65 or ERα overnight (at the concentration of 1:50), and then incubated with fluorescent-labeled secondary antibody , followed by DAPI incubation. Slides were mounted with Vectashield . Images were captured using Keyence® BZ-9000 microscope and analyzed by Keyence® BZ-9000 software.P < 0.05 was considered statistically significant.Data is presented as mean ± SEM of three independent experiments. For primary cells, two mice were pooled and processed for each experiment, which yielded three biological replicates , 37. StaTnf-α mRNA expression in both male and female cells; however, following the short-term exposure, only 17 α-E2 was capable of reducing Tnf-α in both sexes  on inflammatory markers in vitro, we directly applied 17 α-E2 and 17 β-E2 to mouse embryonic fibroblast (MEF) cells derived from male and female C57BL/6J mice. To induce inflammation, MEFs were treated with LPS followed by the two different estrogens. First, to determine if estrogens are able to suppress LPS-induced inflammation, cells were pre-treated overnight (12 h) with estrogens, followed by a 2-h LPS treatment. In an additional series of experiments to determine if estrogens are able to suppress LPS-induced inflammation with shorter exposure, cells were pre-treated with E2s for only 1 h, followed by 2 h LPS treatment. Long pre-exposure to 17 α-E2 or 17 β-E2 prevented LPS-induced inflammation as demonstrated by reductions in xes Fig. . Interesale Fig.  and maleale Fig. .Fig. 1EfTnf-α and Il-6 genes both in male  and were tested using a similar paradigm Fig. , 3a, b. ale Fig. –f and feale Fig. –f cells lls Fig. –f. In feion Fig. –f, indicTnf-α and Il-6 when compared to females  is a natural, non-feminizing isomer of 17 β-E2 , 20, andWe and others have reported an important role for ERα in modulating inflammatory responses in various tissues , 42. AddWe have not specifically tested competitive binding of the ERs following exposure to differing E2s; however, reports indicate that 17 α-E2 and 17 β-E2 possess different affinities for ERs. According to Edwards and McGuire (1980) , there a+ cells with a construct containing the IL-6 promoter linked to the luciferase gene NFκB p65 or NFκB p50 with IκBα or control expression vector. The transfection of p50 or p65 vectors alone induced IL-6 promoter activation. However, treatment of cells with estrogens (E2) abolished the IL-6 promoter activity, which was similar to the transfection of p50 and p65 with IκBα construct, which completely abrogated the NFκB-mediated IL-6 promoter activity. When cells were stimulated with phorbol ester (PMA), the levels of cytosolic IκBα protein were hugely decreased, while when co-treated with E2 and PMB, the PMA-induced IκBα protein degradation was inhibited. One possible explanation is that E2 stabilizes IκBα protein by interfering with its phosphorylation or ubiquination, inhibiting its proteolysis. There was no data in this study regarding which ER was transfected to HeLa cells that was mediating E2’s effects.NFkappaB (NFκB) is included within in the inflammatory cascade. Its inactive form resides in the cytoplasm, bound to IKappaB (IκB). When it is activated by a stimulus, IκB is phosphorylated by IKappaK (IκK) and dissociates from NFκB. NFκB then heterodimerizes and translocates to the nucleus, where it binds and modulates expression of target genes such as Tnf-α and Il-6. Estrogen receptors (ERs) can repress NFκB activity through diverse mechanisms. In the study of Quaedackers et al. (2001) , the ostIn the review of Kalaitzidis and Gilmore (2005) , the autThe concentration of E2 is important for its ability to modulate NFκB; according to the literature, E2 concentrations equal to or above 100 pM are necessary to inhibit NFκB activation . AdditioIt is important to note that the objective of the experiments described here was to directly compare the same concentrations of the different E2s in order to discern differences in their ability to modulate inflammatory markers. The concentration used in these studies was equivalent to the pharmacological dose of 17 β-E2 used in vivo , 52. CurTnf-α and Il-6. These findings are important because there is increasing interest in attaining the clinical benefits of estrogen compounds while avoiding adverse side effects. Since 17 α-E2 is a non-feminizing estrogen, its use may not cause the potential deleterious effects of feminizing estrogens. As such, our study suggests that this estrogen may be pharmacologically relevant for treatment of inflammatory diseases in males, as well as in postmenopausal females.In conclusion, we provide new evidence demonstrating an anti-inflammatory role for 17 α-E2 in adipocytes, which appears to require ERα in females, but not in males. Furthermore, our data suggest a potential mechanism by which 17 α-E2 provides an anti-inflammatory effect which is by reducing NFκB p65 subunit transcription and translation, which, in turn, decreases transcription of inflammatory markers such as Additional file 1: Figure S1.A) Cell viability in % after estrogens and LPS treatments. (B) Oil red O staining for lipolysis in all groups. Data were presented in mean ± SEM values. (TIFF 2622 kb)Effects of 17 α-E2 and 17 β-E2 on modulation of viability of cells and lipolysis in 3T3-L1 differentiated cells.  Relative mRNA expression of Esr1. (B) Total protein was extracted for Western blotting detection of ERα after 72 h of siRNA. Data were presented in mean ± SEM values. Symbol (*) indicates P < 0.05 compared to control group (C). (&) indicates P < 0.05 x siRNA group. (n = 3 independent rounds of cells). (TIFF 375 kb)Effects of Additional file 3: Figure S3.Esr2 (A) and Gper1 (B) in primary pre-adipocyte cells derived from male mice. Data were presented in mean ± SEM values. Symbol (&) indicates P < 0.05 compared to WT mice. (n = 3 independent rounds of cells). (TIFF 375 kb)mRNA expression of Additional file 4: Figure S4.Esr2 (A) and Gper1 (B) in Mef cells derived from WT and ERKO male mice. Data were presented in mean ± SEM values. Symbol (&) indicates P < 0.05 compared to WT mice. (n = 3 independent rounds of cells). (TIFF 239 kb)mRNA expression of


As expected, the 4-aminopyridinium moieties are protonated exclusively at their endocyclic nitrogen atom due to a mesomeric stabilization by the imine form which would not be given in the corresponding double-protonated dicationic species. In the crystal, the phosphonyl (–PO3H2) and hydrogen phosphonate (–PO3H) groups of the anions form two-dimensional O—H⋯O hydrogen-bonded networks in the ab plane built from 24-membered hydrogen-bonded ring motifs with the graph-set descriptor R66(24). These networks are pairwise linked by the anions’ alkylene chains. The 4-aminopyridinium cations are stacked in parallel displaced face-to-face arrangements and connect neighboring anionic substructures via medium–strong charge-supported N—H⋯O hydrogen bonds along the c axis. The resulting three-dimensional hydrogen-bonded network shows clearly separated hydrophilic and hydrophobic structural domains.The asymmetric unit of the title molecular salt, [C These networks contain 24-membered rings classified as a third level graph set et al., 1990e.g. Gomathi & Muthiah, 2011et al., 2007c-axis direction, these networks are pairwise linked by the anions’ alkylene chains to form a three-dimensional anionic substructure. The 4-aminopyridinium cations show π–π stacking interactions. The rings are oriented in parallel displaced face-to-face arrangements  salts, see: Ferguson  al. 1998; Fu et a al. 2004; Fuller  al. 2004; Glidewe al. 2000; Kan & M al. 2000; Mahmoud 2000ab,c ▸; vaet al., 199814H28N2O6P2 (382.3): calculated C 44.0, H 7.4, N 7.3; found C 43.6, H 7.9, N 7.1. M. p.: 157 °C. The IR and Raman spectra of the title compound are shown in Fig. 4Equimolar quantities (0.5 mmol) of 4-aminopyridine (47.1 mg) and nonane-1,9-diphosphonic acid (144.1 mg) were dissolved in methanol, separately. The solutions were mixed and stored in an open petri dish. Within several days, colorless platelet-shaped crystals of the title compound were obtained by slow evaporation of the solvent. 4-Aminopyridine was purchased from commercial sources and nonane-1,9-diphosphonic acid was synthesized according to the literature  values. Carbon-bound hydrogen atoms were included using a riding model (AFIX23 option of the SHELX program for the methylene groups and AFIX43 option for the methine groups).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016014298/hb7610sup1.cifCrystal structure: contains datablock(s) I, publication_text. DOI: 10.1107/S2056989016014298/hb7610Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989016014298/hb7610Isup3.cmlSupporting information file. DOI: 1503436CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Mycoplasma gallisepticum (MG), which causes chronic respiratory disease in poultry, has resulted in annual epidemics of conjunctivitis in North American house finches since the 1990s. Currently, few immunological tools have been validated for this songbird species. Interleukin-1β (IL-1β) is a prototypic multifunctional cytokine and can affect almost every cell type during Mycoplasma infection. The overall goal of this study was to develop and validate a direct ELISA assay for house finch IL-1β (HfIL-1β) using a cross-reactive chicken antibody.A unique clade of the bacterium A direct ELISA approach was used to develop this system using two different coating methods, carbonate and dehydration. In both methods, antigens (recombinant HfIL-1b or house finch plasma) were serially diluted in carbonate-bicarbonate coating buffer and either incubated at 4 °C overnight or at 60 °C on a heating block for 2 hr. To generate the standard curve, rHfIL-1b protein was serially diluted at 0, 3, 6, 9, 12, 15, 18, 21, and 24 ng/mL. Following blocking and washing, anti-chicken IL-1b polyclonal antibody was added, plates were later incubated with detecting antibodies, and reactions developed with tetramethylbenzidine solution.A commercially available anti-chicken IL-1β (ChIL-1β) polyclonal antibody (pAb) cross-reacted with house finch plasma IL-1β as well as bacterially expressed recombinant house finch IL-1β (rHfIL-1β) in immunoblotting assays. In a direct ELISA system, rHfIL-1β could not be detected by an anti-ChIL-1β pAb when the antigen was coated with carbonate-bicarbonate buffer at 4°C overnight. However, rHfIL-1β was detected by the anti-ChIL-1β pAb when the antigen was coated using a dehydration method by heat (60°C). Using the developed direct ELISA for HfIL-1β with commercial anti-ChIL-1β pAb, we were able to measure plasma IL-1β levels from house finches.Based on high amino acid sequence homology, we hypothesized and demonstrated cross-reactivity of anti-ChIL-1β pAb and HfIL-1β. Then, we developed and validated a direct ELISA system for HfIL-1β using a commercial anti-ChIL-1β pAb by measuring plasma HfIL-1β in house finches. A member of the interleukin (IL)-1 family, IL-1beta (IL-1β) is a pivotal pro-inflammatory cytokine for host-defense responses to infection and injury . It is aSalmonella Enteritidis show increased IL-1β expression  containing 0.05% Tween-20 and 1% BSA) at room temperature (RT) for 45 min.2SO4) then absorbance was quantified spectrophotometrically at 450 nm with a microplate reader. The raw OD values were normalized by subtracting OD value of buffer. The standard curve was drawn and calculated with Excel  and elisaanalysis.com  using a logarithmic equation  + b), which was applied to calculate the concentration of HfIL-1β. The data were analyzed by independent two-sample t-test using program R (https://www.r-project.org/).The primary (rabbit anti-ChIL-1β pAb) and the secondary (goat anti-rabbit) antibodies were prepared by dilution with the blocking buffer at 1:1000 and 1:2000, respectively. Fifty microliters of anti-ChIL-1β pAb were added to each assigned well, followed by incubating on a microplate shaker for 1 h at RT and washing 3 times (washing buffer as PBS [pH 7.4] containing 0.05% Tween-20). The plates were then incubated with 50 μL of the goat anti-rabbit antibody for 1 h at RT with continuous shaking. To develop HRP signal, a 3.3′,5.5′-tetramethylbenzidine  solution was prepared in 1 mL DMSO, 9 mL of 0.05 M phosphate-citrate buffer (pH 5.0) and 2 μL of 30% hydrogen peroxide  per 10 mL total volume. One hundred microliters of the prepared TMB solution were added to each well, followed by incubation for 30–45 min at RT. Colorimetric development was stopped by adding 100 μL of 2 N sulfuric acid . To improve coating the antigen on the plate, the dehydration method was adopted. The ELISA results showed increased optical density (OD450) in a dose-dependent fashion  is the most common coating buffer, and typical coating condition involves 50–100 μL of coating buffer containing 1–10 μg/mL of either antigen or antibody incubated overnight at 4°C or for 1–3 h at 37°C. However, optimal coating conditions should be tested during the development of a new ELISA system. In this study, the typical coating condition of incubation at 4°C overnight did not work in detecting HfIL-1β in plasma samples. To increase adsorption to the plate, we applied a dehydration method for antigen coating, although we were concerned with the background level of spectrometric readings. To eliminate such background, we used the coating buffer itself as a negative control. We also incubated rHfIL-1β and random plasma samples with goat anti-rabbit antibody alone. In both cases, the absorbance at 450 nm was in the 0.034–0.044 range, which was significantly low compared to the 2 ng/mL rHfIL-1β, the minimum concentration that the established ELISA system can detect. It is currently unclear why the dehydration method rather than a typical coating method is superior for detecting HfIL-1β in house finch plasma. One potential explanation is the denaturation of HfIL-1β during the heat-based dehydration, resulting in the increased binding affinity between HfIL-1β and rabbit anti-ChIL-1β, as well as adsorption to the plate.Unlike commercial systems, which mostly use a sandwich ELISA with capture and detection antibodies, we developed a direct ELISA by coating the antigens on the plate using dehydration. Therefore, the sensitivity of the developed HfIL-1β ELISA system is low, and the assay can only detect a limited concentration range (2–20 ng/mL). In our trials with random plasma samples and non-infected and MG-infected plasma samples, a 1:20 dilution with PBS fell within the measurable standard curve range. However, it is necessary to serially dilute plasma samples for every ELISA assay to obtain the best fit within the standard curve range. For the standard curve with rHfIL-1β, first we attempted to use a simple logarithmic equation (i.e. Y = a*ln (x) + c); however, the adjusted R-square value was 0.89, resulting in high variation among samples. Thus, we modified the logarithmic equation to “Y = a – b*ln (x + c)”, which yielded an adjusted R-square value of 0.94–0.95.t-test showed no statistical significance between non-infected and MG-infected birds (P = 0.28), likely due to the low sample size tested. However, the MG-infected birds showed average plasma HfIL-1β levels almost 5-fold higher than those of non-infected birds. Further studies should use a larger number of birds to examine how plasma levels of HfIL-1β change during MG infection.The ELISA results showed the non-infected house finches had an average of 2.89 ng/mL of plasma HfIL-1β, while the MG-infected birds had on average 13.93 ng/mL of plasma HfIL-1β. Two-sample We validated the cross-reactivity of anti-ChIL-1β pAb with HfIL-1β using immunoblotting. Then, a direct ELISA system was developed using anti-ChIL-1β antibody to quantify HfIL-1β plasma levels using the dehydration coating method. The developed ELISA system can quantify HfIL-1β in plasma of test birds including non-infected and MG-infected house finches. This developed direct ELISA system using commercially available chicken antibody will provide valuable research and diagnostic tools for house finch research.


IV alkoxide dimer, supported by a novel monoanionic [NNN] pincer-type ligand. The bis[2--4-methylphenyl]amine ligand is the first reported ligand that bears hydrogen atoms on its ketimine side arms.The title compound is an LiBr-bridged Ti 3[NNN] ligand, namely di-μ-bromido-μ-{2--N-[2--4-methylphenyl]-4-methylaniline}-bis[(diethyl ether)lithium], [Li2Br2(C24H33N3)(C4H10O)2] (1) and a dinuclear metal complex, namely di-μ-bromido-2:3κ4Br:Br-bis{2--N-[2--4-methylphenyl]-4-methylaniline}-1κ3N,N′,N′′;4κ3N,N′,N′′-tetra-μ-isopropanolato-1:2κ4O:O;3:4κ4O:O-diisopropanolato-1κO,4κO-2,3-dilithium-1,4-dititanium, [Li2Ti2Br2(C24H32N3)2(C3H7O)6] or {[NHNNH]Ti(OiPr)3(LiBr)2}2 (2). Complex 1, which sits on a twofold rotation axis, is a rare example of a pincer-type ligand which bears ketimine side arms. A unique feature of complex 1 is that the ketimine N atoms have an LiBr(Et2O) fragment bonded to them, with the Li atom adopting a distorted tetrahedral geometry. This particular fragment creates an LiBr bridge between the two ketimine sidearms, which leads to a cage-type appearance of the ligand. Complex 2 consists of the previously described ligand and a TiIV metal atom in an octahedral environment, and is located on an inversion center. Complex 2 crystallizes as a dinuclear species with the metal atoms being bridged by an LiBr entity [the Br atoms are disordered and refined in two positions with their site occupation factors refining to 0.674 (12)/0.372 (12)], and the Li cation being bonded to the isopropoxide O atoms . The organic ligand of compound 2 exhibits disorder in its periphery groups; isopropyl and tert-butyl groups (occupation factors fixed at 0.6/0.4). The novel [NNN]H3 pincer-type ligand was characterized by multinuclear and multidimensional NMR, HRMS and X-ray crystallography. The dinuclear metal complex 2 was characterized by X-ray crystallography. Although each structure exhibits donor N—H groups, no hydrogen bonding is found in either one, perhaps due to the bulky groups around them. One of the ethyl groups of the ether ligand of 1 is disordered and refined in two parts with site-occupation factors of 0.812 (8) and 0.188 (8). One and a half toluene solvent molecules are also present in the asymmetric unit of 2. The toluene molecules were significantly disordered and could not be modeled properly, thus SQUEEZE  was used to remove their contributions to the overall intensity data.This report describes a synthetic protocols and the crystal structures involving a novel pincer-type HSpek 2015. Acta Cr The ligand moiety of 1  Å and a N3—Li1 bond length of 2.065 (7) Å. The two lithium ions are both bridged by two bromides with an Li1—Br1 bond length of 2.504 (6) Å and an Li—Br1 Å. The short C=N bond length of 1.277 (4) Å is comparable to reported C=N bond lengths. For instance the C=N bond length in furazan is 1.29 Å  exhibits a singlet at 2.26 ppm attributable to the methyl groups on the aryl backbone of the ligand framework. Another characteristic singlet that appears at 1.20 ppm has three times the intensity of the backbone CH3 and is attributable to the tert-butyl CH3 protons residing on each ligand arm. Furthermore, the 1H NMR spectrum exhibits a quartet at 3.48 ppm and a triplet at 1.20 ppm, both signals can be assigned to the –C2H and –C3H groups of bound Et2O. The central backbone N—H resonates as a singlet at 5.32 ppm, and the ketimine N—H protons resonate at 9.42 ppm. 1H–15N gHMBC indirect detection demonstrates that the central nitrogen resonates at 77.00 ppm. In contrast, the chemical shifts of the ketimine nitrogen atoms are not observable. Furthermore, in a NOESY1D experiment the tert-butyl CH3 groups show an nOe with the Et2O –CH3 group when irradiated selectively at 1.28 ppm. From the occurrence of this nOe, it can be concluded that one Et2O molecule is bonded to every lithium atom.Similar to its solid-state structure, 2 is located on an inversion center  Å confirms that the central pincer nitrogen atom is deprotonated. The slightly elongated N1—Ti and N3—Ti bonds of 2.136 (3) and 2.130 (3) Å are indicative of an L-type bonding of the ket-mine nitrogen atoms. The bond lengths and the fact that the titanium metal atom is coordinated by three isopropoxide ligands supports the claim that the [NNN] ligand within 2 must be monoanionic with both ketimine N—H protons still present. The Ti—O1, Ti—O2 and Ti—O3 bond lengths are 1.805 (2), 1.901 (2) and 1.934 (2) Å, respectively. The increase in bond length between Ti—O2 and Ti—O3 in comparison to Ti—O1 is attributed to the coordination of Li to O2 and O3. While the O3—Ti—O1 bond angle of 173.58 (9)° deviates slightly from the optimal angle of 180°, the angle N1—Ti—N3 is 160.94 (11)°. This distortion is due to the short bond length that can be found in a C=N bond. The dinuclear complex also exhibits four disordered regions. The isopropyl groups on C25, C28, C31 and the tert-butyl group on C21 are all disordered and were refined in two parts. The bridging Br ligands are also disordered and were refined in two parts; namely Br1 and Br2.In the solid state, complex r Figs. 2 and 3 ▸ 2O) were dried using a GlassContours drying column. Chlorofrom-d1 (Cambridge Isotopes) was dried over anhydrous CaCl2; vacuum transferred, passed over a plug of basic alumina, and stored over 4 Å molecular sieves. Di-p-tolylamine, nBuLi (2.5 M in hexanes), titanium(IV)isopropoxide, and HCl (1 M in Et2O) were purchased from Sigma Aldrich and used as received. Trimethylacetonitrile was vacuum distilled and freeze pump thawed prior to use. Bis(2-bromo-4-methylphenyl)amine was prepared by literature methods   was added dropwise to a stirring solution of bis(2-bromo-4-methylphenyl)amine. The reaction mixture color changed from colorless to yellow. After stirring for 120 min, pivalonitrile was added dropwise, resulting in a color change from yellow to orange. After an additional 180 min of stirring, excess HCl (1 M in Et2O) was added dropwise, resulting in a color change from orange to yellow and the formation of a white microcrystalline powder. The pale-yellow solution was filtered through Celite™. The volatiles in the resulting filtrate were removed in vacuo and the oily residue was triturated three times (3 × 2 mL) with pentane. Single crystals were obtained by cooling a concentrated toluene solution of 1 to 238 K. Yield: 0.091 g . 1H NMR : δ = 1.20 2), 1.28 2), 2.26 , 3.48 , 6.79 , 6.96  ppm. 1H–13C gHMBC NMR : δ(ppm) = 15.3 , 20.2 , 28.1 , 40.9 , 65.8 , 119.8 , 127.6 , 129.5 , 129.7 , 132.3 , 137.3  and 190.6 ; HRMS calculated (found) for C24H33N3 (M+) 364.2747 (364.2755).In a nitrogen-filled glove-box, a glass vial was charged with bis(2-bromo-4-methylphenyl)amine , 3.0 mL of Et3(LiBr)2 (1) , and 3 mL of Et2O. 1.1 eq. Ti(OiPr)4  was added dropwise to a stirring solution of 1. The reaction mixture changed color instantaneously from yellow to dark red. After stirring for 120 min, the dark-red solution was filtered through Celite™. The volatiles in the resulting filtrate were removed in vacuo and the resulting oily residue was washed three times (3 × 2 mL) with pentane. Single crystals were obtained by preparing a concentrated solution of the oily complex 2 in toluene and cooling it for two weeks at 238 K. Yield: 0.107 g .In a nitrogen-filled glove-box, a glass vial was charged with [NNN]HUiso(H)= 1.2/1.5Ueq(C), except for the –N—H hydrogen atoms which were obtained from a difference Fourier map and refined freely. The dimer complex is located on a twofold rotation axis of symmetry and thus only a half is contained in the asymmetric unit. One ethyl group of the Li-coordinating ether ligand is disordered and was refined in two parts (C15–C16/C15′–C16′). Their site-occupation factors dependently refined to 0.812 (8) and 0.188 (8), for the major and minor parts, respectively.Crystal data, data collection and structure refinement details are summarized in Table 1Uiso(H)= 1.2/1.5Ueq(C), except for the –N—H hydrogen atoms which were obtained from a difference-Fourier map and refined freely. The Ti dimer is located on an inversion center and thus a half dimer is present in the asymmetric unit. One and a half toluene solvent molecules are also present in the asymmetric unit. The half toluene molecule is disordered around inversion symmetry while the one in a general position is disordered in two parts. The toluene molecules were significantly disordered and could not be modeled properly, thus SQUEEZE  and 0.326 (12), respectively. The –N—H hydrogen atoms were obtained from a difference-Fourier map and refined freely.The non-H atoms were refined with anisotropic displacement parameters and all of the H atoms were calculated in idealized positions (C—H = 0.93/1.00 Å) and refined riding on their parent atoms with 10.1107/S2056989016019964/lh5823sup1.cifCrystal structure: contains datablock(s) 1, 2. DOI: 10.1107/S2056989016019964/lh58231sup2.hklStructure factors: contains datablock(s) 1. DOI: 10.1107/S2056989016019964/lh58232sup3.hklStructure factors: contains datablock(s) 2. DOI: 10.1107/S2056989016019964/lh5823sup4.pdfSupporting information file. DOI: 1523135, 1523134CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The synthesis and structure of a rare example of a trigonal–bipyramidal titanium coordination complex with three chlorides and two oxygen donor ligands is reported. 24H33O2)Cl3(C4H8O)], is a rare example of a trigonal–bipyramidal titanium coordination complex with three chloride and two oxygen donor ligands. The asymmetric unit contains two independent molecules having essentially the same conformation. The molecules feature the titanium(IV) metal cation complexed with three chloride ligands, a tetrahydrofuran molecule, and one oxygen atom from the resolved ligand precursor (R)-(+)-5,5′,6,6′-tetramethyl-3,3′-di-t-butyl-1,1′-biphenyl-2,2′-diol, where the remaining phenolic hydrogen atom engages in intermolecular O—H⋯Cl hydrogen bonding. In one molecule, the THF ligand is disordered over two orientations with refined site occupancies of 0.50 (3).The title compound, [Ti(C The complex, [BIPHEN-κ1O]TiCl3(THF), is a rare example of a trigonal–bipyramidal coordination geometry for titanium(IV), with a Chemical Bonding Classification (CBC) designation of TiLX4 , contains two independent molecules  and Hooft y = 0.03 (2)  x, y + 1, z] with H⋯Cl distances of 2.62 (4) and 2.59 (4) Å, respectively 2 comprises TiCl3 moieties additionally coordinated by each phenoxide O atom of the ligand , also has a bulky phenoxide ligand in an equatorial position and THF in an axial position on the trigonal–bipyramid  units coordinated by phenoxides derived from the diol 2,2′-bis[phenol]  derived from a fluorenediol also contains titanium in a trigonal–bipyramidal coordination environment; however, all three chlorides are in the equatorial plane and the ketone and phenoxide are axial -(+)-5,5′,6,6′-tetramethyl-3,3′-di-t-butyl-1,1′-biphenyl-2,2′-diol  in C6H6 (2.5 ml) and the benzene was allowed to slowly evaporate yielding red plate crystals within seven days. The synthesis could be scaled up and the material collected by filtration, yielding a dark-red crystalline powder; however, the material quickly powders into a pink amorphous solid upon loss of coordinating THF, as observed by 1H NMR of the decomposition product.Under a nitrogen atmosphere, tetrachloridobis(tetrahydrofuran)titanium(IV)  was added to (Uiso(H) = 1.2, 1.5 and 1.2Ueq(C) of the aryl, methyl and methylene C atoms, respectively. The position of the phenolic hydrogen atoms were found in the difference map and the atom refined semi-freely using a distance restraint d(O—H) = 0.84 Å, and with Uiso(H) = 1.2Ueq(O).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016020156/rz5202sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989016020156/rz5202Isup2.hklStructure factors: contains datablock(s) I. DOI: 1523643CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information: