By contrast, TβRII blocked all-trans retinoic acid-induced differentiation through inhibition of TβRII-B. Overall survival was significantly lower in acute myeloid leukemia (AML) patients with high compared to low TβRII expression. Thus, whereas TβRII-B is a potent inducer of cell cycle arrest, apoptosis, and differentiation, higher TβRII expression correlates with poor clinical prognosis in AML.Altered transforming growth factor-beta (TGF-β) signaling has been implicated in the pathogenesis of leukemia. Although TGF-β type II receptor (TβRII) isoforms have been isolated from human leukemia cells, their expression patterns and functions of these variants are unclear. In this study, we determined that two TβRII isoforms (TβRII and TβRII-B) are abnormally expressed in leukemic cells, as compared to normal hematopoietic cells. TβRII-B, but not TβRII, was found to promote cell cycle arrest, apoptosis, and differentiation of leukemic cells. TβRII-B also enhanced TGF-β1 binding and downstream signaling and reduced tumorigenicity  Transforming growth factor-beta 1 (TGF-β1) is a potent inhibitor of hematopoietic cells. Autocrine- and paracrine-mediated binding of TGF-β1 to cell surface receptors regulates hematopoietic cell proliferation, differentiation, and apoptosis . TGF-β1 in vitro and in vivo TGF-β1 were incubated with K562/TβRII, K562/TβRII-B, or control K562 cells. These results indicated that 40–80 pmol/L (1–2 ng/mL) TGF-β1 bound more strongly to K562/TβRII-B cells than to K562/TβRII cells  Table . In addiOur data have revealed that TβRII and TβRII-B mRNA are abnormally expressed in AML cells and normal bone marrow CD34+ cells. TβRII was predominantly expressed in AML cells whereas TβRII-B was predominantly expressed in normal bone marrow CD34+ cells. Higher levels of TβRII and TβRII-B mRNA were also detected in U937, KG-1, HEL, and NB4 cells relative to K562 and HL60 cells. TβRII mRNA was also higher than TβRII-B in U937, KG-1, HEL, and NB4 cells. We transfected TβRII and TβRII-B splice variants into K562 and HL60 cells, which have relatively low TβRII expression, and generated the following cell lines: K562/TβRII, K562/TβRII-B, HL60/TβRII, and HL60/TβRII-B. Our data suggest that K562/TβRII-B and HL60/TβRII-B cells are more sensitive to TGF-β1-induced growth inhibition and apoptosis than K562/TβRII and HL60/TβRII cells.Bcl-2, c-Myc, and hTERT genes, but also activates expression of the cell cycle inhibitor CIP/WAF1p21. We found that treatment with exogenous TGF-β1 reduced c-Myc protein expression and increased p21CIP/WAF1 protein levels to a greater extent in HL60/TβRII-B cells than in HL60/TβRII cells. TGF-β1 is known to regulate target gene expression through phosphorylation of R-Smad. We found that TGF-β1 led to increased R-Smad phosphorylation as well as phosphorylation of downstream targets including PKC, AKT, and SAPK/JNK in HL60/TβRII-B cells compared to HL60/TβRII cells.We previously reported that ectopic expression of TGF-β1 in HL60, which lack endogenous TGF-β1 expression, inhibited cell proliferation and triggered apoptosis through downregulation of Bcl-2, c-Myc, and hTERT . Here, wPrevious studies have suggested that TGF-β1 alone cannot induce significant HL60 cell differentiation. However, TGF-β1 in combination with low-dose ATRA (lower than therapeutic levels) could induce differentiation of HL60 cells . We prevWe performed knock-down and rescue experiments in NB4 cells, which express high levels of endogenous TβRII, to investigate how TβRII expression impacted TβRII-B function. We determined that there was more ATRA-induced differentiation in NB4/TβRII-B cells compared to NB4/TβRII and negative control cells. In contrast, no effects were observed in NB4/TβRII cells compared to the negative controls. These data indicated that ATRA-induced differentiation of NB4/TβRII-B cells was enhanced by TβRII knock-down. Thus, TβRII perturbs ATRA-induced differentiation of NB4 cells through inhibition of TβRII-B.2O3 induced apoptosis in HL60 cells through upregulation of endogenous TGF-β1 and downregulation of Bcl-2. TGF-β1 antisense oligonucleotides inhibited As2O3-induced apoptosis and restored Bcl-2 mRNA and protein to pre-treatment levels, suggesting an important role for endogenous TGF-β1 in As2O3-induced HL60 cell apoptosis through downregulation of Bcl-2 TGF-β1 was obtained from Beijing Atomic Hi-Tech Technology Co., Ltd., reconstituted in 100 mL 4 mM HCl containing 2 mg/mL BSA and diluted with binding buffer to generate a 100 pM stock solution. Aliquots were stored at -80°C until use. The [125I]TGF-β1 binding assays were performed as previously described [125I]TGF-β1 were added to different concentrations of [125I]TGF-β1 (range 5–320 pM). The final volume was 2 mL. The cells were then incubated at 4°C for 3.5 h on a platform that oscillated at 120 rpm. A 50-fold excess of unlabeled TGF-β1 was added immediately before the incubation with [125I]TGF-β1 in order to determine the amount of [125I]TGF-β1 that bound non-specifically to the surface of cells. In parallel, binding buffer without [125I]TGF-β1 was added to six-well plates for cell counting using a Neubauer counting chamber. Based on the work by Kang et al. [125I]TGF-β1 binding to TβRII was calculated:The [escribed . To prepg et al. , the fol7) were subcutaneously inoculated into the right flanks of the mice and tumor growth measured with a caliper. Tumors were allowed to grow until they became palpable (day 22). The mice were then sacrificed and the tumors removed and weighed. The tumor volume (cm3) was calculated according to Bhola et al.32 using the following formula: volume = length/2 × width2.Female BALB/c nude mice (4–6 weeks old) were obtained from the Shanghai Laboratory Animal Breeding Center at the Chinese Academy of Medical Sciences. HL60/TβRII or HL60/TβRII-B cells . Statistical significance was assessed using two-tailed Student's All mice were maintained under pathogen-free conditions at Fujian Medical University, and all experiments were performed in accordance with the protocols of the Institutional Animal Care and Use Committee. Normal and clinical samples were collected from AML patients according to a protocol approved by the Fujian Medical University Ethics Committee. Written informed consent was also obtained.


The molecules are linked into a two-dimensional hydrogen-bonded network parallel to the (100) plane and the Hirshfeld surface analysis indicates that the major contributions for the crystal structure cohesion are the O⋯H (28.5%) and H⋯F (16.4%) interactions. An  8H5FN2O2, whose molecular structure matches the asymmetric unit and is nearly planar with an r.m.s. deviation for the mean plane through all non-H atoms of 0.0363 Å. In the crystal, the molecules are linked by N—H⋯N, N—H⋯O and O—H⋯O hydrogen-bonding interactions into a two-dimensional network along the (100) plane, forming rings with 22R(8) and 12R(5) graph-set motifs. The crystal packing also features weak π–π interactions along the [100] direction [centroid-to-centroid distance 3.9860 (5) Å]. Additionally, the Hirshfeld surface analysis indicates that the major contributions for the crystal structure are the O⋯H (28.50%) and H⋯F (16.40%) interactions. An in silico evaluation of the title compound with the vascular endothelial growth factor receptor-2 (VEGFR-2) was carried out. The title compound and the selected biological target VEGFR-2 show the N—H⋯O(GLU94), (CYS96)N—H⋯O(isatine) and (PHE95)N—H⋯O(isatine) intermolecular interactions, which suggests a solid theoretical structure–activity relationship.The reaction between 5-fluoroisatin and hydroxylamine hydrochloride in acidic ethanol yields the title compound, C In addins Fig. 2 between normd, with transparency and labelled atoms indicates, in magenta colour, the locations of the strongest intermolecular contacts, e.g. H4, H5 and O1, which are important for the intermolecular hydrogen bonding . The Hirshfeld analysis suggests that the major contributions for the crystal packing amount to 25.40% for H⋯O, 16.40% for H⋯F and 16.10% for H⋯H interactions. Other important intermolecular contacts for the cohesion of the structure are : C⋯C = 11.30, H⋯N = 9.80 and H⋯C = 6.40 . Although the crystal packing  for the title compound and the 5-chloroisatin-3-oxime are quite similar, the contributions of the intermolecular interactions to the cohesion of the crystal structures have differences due to the halogen substituents. For example: for 5-chloroisatin-3-oxime, the H⋯O interaction amounts to 23.60% and the H⋯Cl interaction amounts to 18.10%. The contributions to the crystal packing are shown as Hirshfeld surface two-dimensional fingerprint plots with cyan dots. The ed (y axis) and id (x axis) values are the closest external and internal distances (Å) from given points on the Hirshfeld surface contacts  , (CYS96)N—H⋯O(isatine)  and (PHE95)C—H⋯O(isatine)   was carried out. Initially, the semi-empirical equilibrium energy of the small molecule was obtained using the PM6 Hamiltonian, but the experimental bond lengths were conserved. The calculated parameters were: heat of formation = −49.353 kJ mol°] Fig. 7. Another°] Fig. 7.et al., 2016All starting materials are commercially available and were used without further purification. The synthesis of the title compound was adapted from procedures reported previously (Martins Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989017008301/rz5215sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989017008301/rz5215Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989017008301/rz5215sup3.pdfSwissTargetPrediction report (5-flouroisatin-3-oxime). DOI: 10.1107/S2056989017008301/rz5215sup4.pdfNumber of residues in favoured region (VEGFR-2). DOI: 10.1107/S2056989017008301/rz5215sup5.pdfRe-docking of the Lenvatinib (kinase inhibitor and FDA approved drug). DOI: Click here for additional data file.10.1107/S2056989017008301/rz5215Isup6.cmlSupporting information file. DOI: 1554287CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Bcl2 transgenic v-Abl kinase-transformed pro-B cells provide a pseudo-physiological cellular system to study V(D)J recombination. Treatment of v-Abl/Bcl2 pro-B cells with the Abl kinase inhibitor Imatinib leads to G1 cell cycle arrest, the rapid induction of Rag1/2 gene expression and V(D)J recombination. In this system, the Bcl2 transgene alleviates Imatinib-induced apoptosis enabling the analysis of induced V(D)J recombination. Although powerful, the use of mouse models carrying the Bcl2 transgene for the generation of v-Abl pro-B cell lines is time and money consuming. Here, we describe a method for generating v-Abl/Bcl2 pro-B cell lines from wild type mice and for performing gene knock-out using episomal CRISPR/Cas9 targeting vectors. Using this approach, we generated distinct NHEJ-deficient pro-B cell lines and quantified V(D)J recombination levels in these cells. Furthermore, this methodology can be adapted to generate pro-B cell lines deficient for any gene suspected to play a role in V(D)J recombination, and more generally DSB repair.Antigen receptor gene assembly is accomplished in developing lymphocytes by the V(D)J recombination reaction, which can be separated into two steps: DNA cleavage by the recombination-activating gene (RAG) nuclease and joining of DNA double strand breaks (DSBs) by components of the nonhomologous end joining (NHEJ) pathway. Deficiencies for NHEJ factors can result in immunodeficiency and a propensity to accumulate genomic instability, thus highlighting the importance of identifying all players in this process and deciphering their functions.  HR requNHEJ is essential for V(D)J recombination as illustrated by the severe combined immunodeficiency observed in some human patients and mouse models with NHEJ defects . V(D)J rv-Abl pro-B cell lines that enable the manipulation and study of V(D)J recombination ex vivo are a powerful tool for gaining a fuller understanding of DSB repair.Altogether, as highlighted by the data outlined above, the V(D)J recombination reaction is cell cycle stage specific (i.e. G1-arrested progenitor lymphocytes), generates unique structures of DNA ends  and relies on NHEJ, and, to a lesser extent, the ATM-dependent DNA damage response machinery. It thus provides a unique physiological system to discover novel DSB repair factors and elucidate their precise functions. Accordingly, Abl oncogene of Abelson murine leukemia virus induces transformation of early B cells (v-Abl transformed B cell lines (v-Abl progenitor B cell transformants (v-Abl pro-B cells) typically display cell surface markers characteristic of a pro- or early pre-B stage of development (B220+ CD43+ CD25− IgM−). They are also characterized by a rapid proliferative state, low expression of Rag1 and Rag2 and infrequent immunoglobulin light chain gene rearrangement. Treatment of v-Abl pro-B cell lines with the Abl kinase inhibitor Imatinib  leads to G1 arrest, rapid induction of Rag1 and Rag2 expression and RAG-mediated DNA breakage at immunoglobulin light chain genes J recombination at endogenous RAG target loci as well as any chromosomally integrated reporter substrate - B cells . Fundamell lines . v-Abl pin genes . STI-571in genes . This liransgene . In thisubstrate . Althougv-Abl/Bcl2 pro-B cell lines from wild type mice and for performing gene knock-out using episomal CRISPR /Cas9 targeting vectors. In this system, the anti-apoptotic Bcl2 gene is retrovirally introduced into v-Abl transformed pro-B cells thus bypassing the need of Bcl2 transgenic animals. Additionally, CRISPR/Cas9-mediated gene knock-out can be achieved in approximately 4 weeks, facilitating the interrogation of candidate V(D)J recombination genes. Using this approach, we generated distinct NHEJ-deficient pro-B cell lines and quantified V(D)J recombination levels in these cells  were used for generating wild type 2.2v-Abl plasmid encoding for Abl  twice during the transformation period. 1 ml of culture media containing 2 × 106v-Abl transformed pro-B cells were subsequently transduced with 1 ml of Bcl2/puro retroviral supernatant (see v-Abl/Bcl2 (hereafter named v-Abl) pro-B cells were further cultured in RPMI 1640 supplemented with Glutamine, 10% FBS, penicillin (100 U/ml)/streptomycin (100 μg/ml) and 50 μM 2-mercaptoethanol and frozen in 90% FBS/10% DMSO.One mouse femur was isolated, washed five times in Dulbeccos's phosphate-buffered saline (PBS) (Gibco) with penicillin (100 U/ml)/streptomycin (100 μg/ml) (Gibco) and cut at both ends near the joints . The bontant see  was thentant see  Fig. 1)v-Abl ret2.42.4.1http://crispr.mit.edu/). Two gRNA-related oligonucleotides (A and B) containing the 18 to 20 bp gRNA sequence and the linker sequences (5′-ACACCG-gRNA-G-3′ for the oligonucleotide A and 5′-AAAAC-gRNA′-CG-3′ for the oligonucleotide B) were used for cloning into the BsmBI cloning site of MLM3636. Briefly, annealing of gRNA-related oligonucleotides A and B (100 μM stock) was performed by mixing 10 μl of each oligonucleotide  with 80 μl of buffer containing 10 mM Tris pH 7,5, 1 mM EDTA and 50 mM NaCl and incubating at 95 °C for 15 min and then at room temperature for at least 1 h. MLM3636 plasmid was digested by BsmBI enzyme (NEB). Linearized MLM3636 plasmid was run on a 1% gel, extracted and purified . Ligation was performed by incubating 100 ng of linearized MLM3636 plasmid with 0.05 μM annealed gRNA-related oligonucleotides, 2 μl 10 × Ligase buffer (NEB) and 1 μl T4 Ligase (NEB) in a 20 μl volume reaction for 1 h at room temperature. DH5α bacteria (Invitrogen) were transformed with the ligation product and amplified. Purified plasmids were Sanger sequenced using LKO.1 5′ primer (5′-GACTATCATATGCTTACCGT-3′) to verify correct gRNA cloning.Cas9 was expressed from plasmid pCas9-GFP (Addgene plasmid #44719) and guide RNAs (gRNAs) were expressed from plasmid MLM3636 (Addgene plasmid # 43860) . Two gRN2.4.22.4.2.16 cells/ml and left to recover for 24 to 36 h. The next day, single cells expressing GFP were sorted using a BD FACSAria™ cell sorter into round bottom 96-well plates containing 200 μl of culture media. The percentage of GFP positive cells was typically low (2 to 5%) but sufficient to sort up to 384 cells into four 96-well plates (6 cells/ml), 1 ml was transferred into a 2 ml Eppendorf tube. The tube was spun at 10,000 rpm for 2 min  and genomic DNA was prepared as described in 15 million pro-B cells were nucleofected using the Cell Line Nucleofector® Kit V from Lonza  and 6 μg of plasmid mix  . Electrol plates . Plates 2.4.2.26 cells) was resuspended in 1 ml of lysis buffer containing 0.2 M NaCl, 0.4% SDS, 0.1 M Tris pH 7.5, 0.01 M EDTA pH 8 and 0.4 mg/ml proteinase K  and incubated at 55 °C for a minimum of 1 h. The tube was then left to cool down to room temperature. 1 ml of isopropanol was added to the lysis buffer and the tube was mixed thoroughly until genomic DNA precipitate formed. The tube was centrifuged at 14,000 rpm for 2 min  and the supernatant was removed. The DNA pellet was washed once with 70% Ethanol  and air dried for approximately 15 min. Genomic DNA preparation was suspended in 400 μl 0.1 × TE solution. For PCR screening, 5 μl of genomic DNA was mixed with 5 μl 10 × Taq buffer , 1 μl 50 mM MgCl2 , 5 μl 750 μM dNTP, 1 μl 10 μM primer 1, 1 μl 10 μM primer 2, 0.5 μl Taq polymerase  and 30 μl H2O . After a few days in culture, purified pMX-INV v-Abl pro-B cells (106/ml) were treated with 3 μM of the Abl kinase inhibitor STI-571 (Novartis) or 0.3 μM of the STI-571 analogous Abl kinase inhibitor PD180970 (Sigma) for 72 h and assayed for recombination levels by FACS analysis of GFP/hCD4 expression   pro-B cell lines from wild type C57BL/6J mice  that enables GFP expression upon successful RAG-dependent chromosomal inversional recombination (v-Abl pro-B cell lines with the Abl kinase inhibitor Imatinib (STI-571) for 3 days led to an almost complete G1 arrest (< 2% of large sized cycling pro-B cells) without significant cell death (< 6% cell death) J recombination in G1-arrested pro-B cells.We first generated /6J mice   C. In addays 53%, C, D  from wild type v-Abl pro-B cells, generating Paxx−/−v-Abl pro-B cell clones and Xlf (Δexon1) from wild type and Paxx−/− cells to generate Xlf−/− and Paxx−/−Xlf−/−v-Abl pro-B cell clones, respectively  and Xlf−/− (26%) cells (Paxx−/−Xlf−/− (0.42%) cells as compared with the WT, Paxx−/− and Xlf−/−v-Abl pro-B cells (Paxx−/−Xlf−/− cells was similar to that of Xrcc4−/− (0.04%) and Lig4−/− (0.02%) cells  cells D  cells . Of note%) cells . In shar-B cells . Notably%) cells D, indicaphocytes . These s4v-Abl pro-B cell lines from wild type mice in approximately 6 weeks. When combined with CRISPR/Cas9 technology, v-Abl pro-B cells can be effectively used to test the role of candidate genes in V(D)J recombination J recombination in these settings. V(D)J recombination reporter assays revealed severely impaired inversional rearrangement in Xrcc4−/− and Lig4−/−v-Abl pro-B cells J recombination.Here, we selectively knocked-out four NHEJ genes: -B cells D, consis-B cells . Conversing NHEJ D (Lescaling NHEJ . Notably fertile . In cont−/− mice . Thus, Cv-Abl pro-B cell lines also provide a valuable experimental system to test more general DSB response and repair activities in cycling or G1-arrested cells. For instance, over the past years, this cell system has been used to assay cell sensitivity to irradiation and genotoxic drugs, nuclear DNA damage foci formation, DNA breakage sites, translocation formation and genetic and epigenetic modifications that occur upon DSB formation and repair (d repair . With th


Oncorhynchus mykiss), concentrating on tumor necrosis factor and interleukin-1 ligand/receptor systems and acute phase response genes. The dataset includes expression values for drpt, il11a, il1b1, il1b2, il1b3, il1r-like-1(e3-5), il1r-like-1(e9-11), il1r1-like-a, il1r1-like-b, il1r2, saa, tnfa1, tnfa2, tnfa3, tnfrsf1a, tnfrsf1a-like-a, tnfrsf1a-like-b, tnfrsf5, and tnfrsf9. Gene expression was measured at four time-points post-challenge in both a resistant line (ARS-Fp-R) and a susceptible line (ARS-Fp-S) of rainbow trout. In addition, fish body weight, spleen index and the Flavobacterium psychrophilum load are reported. These data are an extension of information presented and discussed in “Proinflammatory cytokine and cytokine receptor gene expression kinetics following challenge with Flavobacterium psychrophilum in resistant and susceptible lines of rainbow trout (Oncorhynchus mykiss)”  A GeXP multiplex, RT-PCR assay was developed and optimized that simultaneously measures expression of a suite of immune-relevant genes in rainbow trout ( Specification TableValue of the data•Data are provided describing the optimization and validation of a new GeXP assay for measuring rainbow trout proinflammatory cytokines.•The data include PCR product relative migration and identification that are of use to researchers when performing this assay.•The gene expression values from the rainbow trout resistant line, ARS-Fp-R, and the susceptible line, ARS-Fp-S, can be used in future studies for meta-analyses comparing the response to other pathogens or environmental conditions.1Oncorhynchus mykiss) following challenge with Flavobacterium psychrophilumil11a  was used to quantify transcript expression. Primers were designed as described 2.2tnfrsf5 and il11a, with gapdh as a reference gene. Primers sequences were identical to those used in the GeXP multiplex assay. The real time PCR was performed with an ABI7900 Sequence Detection System (Applied Biosystems) using a 2-step PCR procedure with the following protocol. Temperature was held at 50 °C for 2 min followed by 95 °C for 10 min. Forty PCR cycles of 95 °C (15 s) and 60 °C (1 min) were completed, followed by melt curve analysis to confirm a single PCR product per reaction. PCR efficiencies were initially determined using a 6–10-step two-fold serial cDNA dilution. PCR efficiencies were 2.05, 2.08, and 2.19 for tnfrsf5, il11a, and gapdh, respectively.One microgram of DNase treated RNA was used in a 40 μL reverse transcription reaction random primers  and MMLV  per manufacturer׳s protocol. Four μL of cDNA was used in a 15 µL PCR reaction with 7.5 µL 2X SYBR Green Master Mix , 833 nM forward primer and 833 nM reverse primer. Real-time PCR reactions were completed in duplicate for


Herein, we demonstrate that significant amounts of DXA3 are rapidly attached to phosphatidylethanolamine (PE) forming four esterified eicosanoids, 16:0p, 18:0p, 18:1p and 18:0a/DXA3-PEs that can activate neutrophil integrin expression. These lipids comprise the majority of DXA3 generated by platelets, are formed in ng amounts (24.3±6.1 ng/2×108) and remain membrane bound. Pharmacological studies revealed DXA3-PE formation involves cyclooxygenase-1 (COX), protease-activated receptors (PAR) 1 and 4, cytosolic phospholipase A2 (cPLA2), phospholipase C and intracellular calcium. They are generated primarily via esterification of newly formed DXA3, but can also be formed in vitro via co-oxidation of PE during COX-1 co-oxidation of arachidonate. All four DXA3-PEs were detected in human clots. Purified platelet DXA3-PE activated neutrophil Mac-1 expression, independently of its hydrolysis to the free eicosanoid. This study demonstrates the structures and cellular synthetic pathway for a family of leukocyte-activating platelet phospholipids generated on acute activation, adding to the growing evidence that enzymatic PE oxidation is a physiological event in innate immune cells.Activated platelets generate an eicosanoid proposed to be 8-hydroxy-9,10-dioxolane A3 (DXA  •3-PE in platelets.Four enzymatically oxidized phospholipids are described, termed DXA•3-PE forms by COX-1 oxidation of arachidonate, then esterification into PE.DXA•3-PE formation requires calcium, phospholipase C and phospholipase A2.DXA•3-PEs purified from platelets activates neutrophil Mac-1 expression.DXA We recently described a new platelet eicosanoid, proposed to be 8-hydroxy-9,11-dioxolane eicosatetraenoic acid (DXA3) 3 both primes and activates human neutrophils at nM-μM concentrations Platelets generate eicosanoids including thromboxane Asn2 position sn1. We recently showed that thrombin-activated platelets generate over 100 diverse eoxPL on acute activation of platelets, including n3 and n6 fatty acids, and both single and multiply oxygenated forms at sn2in vitro including enhancing clotting factor activities, modulating monocyte cytokine generation and enhancing neutrophil antibacterial actions Platelets also form enzymatically-oxidized phospholipids (eoxPL) that contain PGs or HETEs at the 3. A co-ordinated series of enzymes and signaling mediators are required, including the fast esterification of newly formed DXA3 free acid. A quantitative assay showed the majority of platelet DXA3 generated on thrombin activation is PE-esterified. DXA3-PEs remain cell associated, are detected in human clots and activate neutrophil integrin expression, independently of their hydrolysis to the free acid analog. In summary, DXA3-PEs are platelet-derived lipids that can activate neutrophils adding to the growing evidence for enzymatic phospholipid oxidation as a physiological process of importance during early innate immunity.Herein, we describe the detailed cellular and enzymatic biosynthesis pathways for four PE-esterified forms of DXA22.12-d4: 9-oxo-11α,15S-dihydroxy-prosta-5Z,13E-dien-1-oic-3,3,4,4-d4 acid, ≥99% deuterated forms. HPLC grade solvents were from Thermo Fisher Scientific . PAR-1 and −4 agonists and U73112, U73343 were from Tocris Biosciences . COX-1 inhibitor (Sc-560) was from Cayman Chemical. Platelet signaling inhibitors (oleyloxyethylphosphocholine (OOEPC), bromoenol lactone (BEL), cytosolic phospholipase A2α (cPLA2α) inhibitor −4-(Biphenyl-2-ylmethyl-isobutyl-amino)-1-[2--benzoyl]-pyrrolidin-2-ylmethyl}-3-[4--phenyl]acrylamide, HCl), Gö 6850 and wortmannin were from Calbiochem (UK). All other reagents were from Sigma-Aldrich unless otherwise stated. Ovine COX-1 was from Cayman Chemical or purified as described Lipids and lipid standards were purchased from Avanti Polar Lipids  or Cayman Chemical . Deuterated standards are as follows: PGE2.23-PE was analyzed using reverse phase LC-MS/MS as described below.Apo-COX-1 was stored in 80 mM Tris, pH 7.8, at −80 °C. For heme reconstitution, Apo-COX-1 (35 µg) was preincubated on ice for 20 min with 2 M equivalents of hematin in phosphate buffer . Then, 3.5 μg holo-enzyme was added to 1 ml phosphate buffer with 500 µmol/L phenol and incubated for 3 min at 37 °C in the presence of 150 µM arachidonate (AA), 1-stearoly-2-arachidonyl-PE (SAPE) or both. The reaction was stopped using ice-cold lipid extraction solvent, and immediate extraction of lipids, after addition of 5 ng DMPE internal standard. In some experiments, 10 μM diethylenetriaminepentaacetic acid (DTPA) was added just before holoCOX-1. 18:0a/DXA2.3g for 10 min at room temperature. Platelet-rich plasma was collected and centrifuged at 900g for 10 min, and the pellet resuspended in Tyrode's buffer  containing ACD . Platelets were centrifuged at 800g for 10 min then resuspended in Tyrode's buffer at 2×108 ml−1. Platelets were activated at 37 °C in the presence of 1 mmol/L CaCl2 for varying times, with 0.2 U ml−1 thrombin, 10 μg/ml collagen, 10 μmol/L A23187, 20 μmol/L TFLLR-NH2, or 150 μmol/L AY-NH2 before lipid extraction as below. Experiments involving signaling inhibitors included a 10 min preincubation at room temperature. In some experiments, calcium was omitted from buffers. For separation of cells from microparticles, platelets were centrifuged at 970g for 5 min, then supernatants re-spun at 16,060g for 5 min. For aspirin supplementation, blood samples were first obtained following a 14-day NSAID-free period for baseline determinations of eicosanoids. Subjects were administered 75 mg/day aspirin for 7 days, then provided a second blood sample. Platelets were isolated and activated in vitro using 0.2 U/ml thrombin, as described above, then lipids extracted as described below.Human blood donations were approved by the Cardiff University School of Medicine Ethics Committee and were with informed consent , and according to the Declaration of Helsinki. Exclusion criteria was a known sensitivity to aspirin. For studies on isolated platelets, whole blood was collected from healthy volunteers free from non-steroidal anti-inflammatory drugs for at least 14 days into acid-citrate-dextrose   and centrifuged at 2502.4g for 10 min. Clot samples were placed in a pre-frozen mortar and pestle on dry ice and further cooled with liquid nitrogen. 250 mg clot was ground up in 3 ml PBS pH 7.4 containing 10 mM DTPA. Lipids were extracted from 1 ml samples using the method below.Blood was allowed to clot for 1 h at 24 °C, and spun at 17302.52-d4, and di-14:0-phosphatidylethanolamine (DMPE) were added to samples before extraction, as internal standards. After vortexing and centrifugation, lipids were recovered in the upper hexane layer. The samples were then re-extracted by addition of an equal volume of hexane. The combined hexane layers were dried and analyzed for free or esterified PGs using LC-MS/MS as below.Lipids were extracted by adding a solvent mixture ) to the sample at a ratio of 2.5–1 ml sample, vortexing, and then adding 2.5 ml of hexane 2.5.118 Luna, 3 µm, 150 mm×2 mm column (Phenomenex), using a gradient of 50–100% B over 10 min followed by 30 min at 100% B  with a flow rate of 200 μl/min. Esterified DXA3 was monitored on a 4000 Q-Trap (Sciex) as precursors of m/z 770.6, 796.6, 798.6 and 814.7 fragmenting to product ions at m/z 351.2. For high resolution analysis, a reversed-phase UPLC Fourier Transform MS method was used (Thermo Scientific Orbitrap Elite). Analysis was performed using heated electrospray ionization (h-ESI) in negative ion mode at sheet, auxiliary and sweep gas flows of 70, 20, and 0, and capillary and source heater temperatures at 300 and 350 °C, respectively. Data dependent MS3 of m/z 351 from esterified DXA3 was carried out in negative FTMS mode with a resolving power of 15,000. Lipid extracts were separated on a C18 Hypersil Gold, 1.9 µm, 100×2.1 mm column using a gradient  with flow rate 200 μl min –1, starting at 50% B and maintaining for 10 min. The gradient increased to 100% B over 15 min and returning to initial conditions for 5 min. Orbitrap analysis was performed using heated electrospray ionization (h-ESI) in negative ion mode at sheath, auxiliary, and sweep gas flows of 30, 10, and 0, respectively. Capillary and source heater temperatures were 275 and 250 °C, respectively, at 30,000 resolution, in full scan mode. LC/MS of precursor ions were monitored using accurate mass in FTMS mode. Negative MS/MS spectra were acquired using higher energy collision-induced-dissociation (HCD). Data dependent MS3 of m/z 351 was carried out in ITMS mode on the LTQ Ion Trap. Where fold changes are presented, data are shown as A/IS: analyte divided by internal standard.For MRM analysis, lipids were separated on a C2.5.2−13s was performed by injecting 20 μl of each fraction under flow (1 ml min−1) in methanol into the electrospray source, with specific MRM transitions monitored using m/z 351 as the product ion, on a 4000 Q-trap. PE fractions were dried using N2, then resuspended in 1 ml buffer , pH 8.9). 200 μg snake venom phospholipase A2 (PLA2) from Sigma-Aldrich was added, and incubated for 60 min at 37 °C. Lipids were re-extracted as above, using hexane:isopropanol:acetic acid. As a purified standard is not yet available, we synthesized and purified a biogenic standard using COX-1 . Quantitation was achieved using PGE-d4 as internal standard. For analysis of free DXA3, the following HPLC conditions were used: C18 Spherisorb ODS2, 5 µm particle size, 150×4.6 mm  on a Sciex 4000 Q-Trap. The solvent system was 75% water, 25% acetonitrile, 1% glacial acetic acid Solvent A, 60% methanol, 40% acetonitrile, 1% glacial acetic acid at 1 ml min−1 Solvent B. Solvent B was increased from 50–90% over 20 min and at 25 min returned to 50% PE was purified from thrombin-activated platelet lipid extracts using normal phase HPLC as follows: extracts resuspended in normal phase solvents ) were separated on a Spherisorb S5W 4.6×150-mm column  using a gradient of 50–100% B over 25 min at a flow rate of 1.5 ml min2.62 followed by lipid extraction. DXA3-PEs were isolated using a Discovery C18 HPLC column ) at 1 ml min−1, and 50–100% mobile phase B  over 15 min, held at 100% B for 20 min. Fractions were collected at 1 min intervals then analyzed using LC-MS/MS. DXA3-PE containing fractions were combined and evaporated to dryness using a Rapidvap N2/48 evaporation system (Labconco Corporation), resuspended in methanol, then stored at −80 °C until use.Washed human platelets were activated using A23187  with 1 mM CaCl2.7g for 20 min at 4 °C. The pellet was resuspended in ice-cold PBS and 0.4% sodium tricitrate (wt/vol) and centrifuged at 400g for 5 min at 4 °C. Contaminating erythrocytes were removed using up to three cycles of hypotonic lysis. Finally, cells were resuspended in a small volume of Krebs buffer , counted and kept on ice. Neutrophils were diluted to 2×106 cells/ml and incubated with or without HPLC purified DXA3-PE , vehicle (methanol or DMSO), 1 μM 1-stearoyl-2-arachidonyl-PE (SAPE), or 1 μM fMLP for 20 min at 37 °C. In some experiments, neutrophils were pre-treated with 1.60 µM U-75302  or 1 µM LY255283 (Sigma-Aldrich) for 10 min at room temperature. Cells were blocked using 5% mouse serum in PBS  for 1 h on ice and centrifuged at 320g for 5 min at 4 °C. Anti-human CD11b-Alexa Fluor 647  or isotype control were added and incubated for 30 min on ice. Neutrophils were washed twice with ice-cold PBS  and analyzed on a Cyan ADP flow cytometer (Beckman) and identified by forward and side scatter and Alexa Fluor 647.Human neutrophils were isolated from 20 ml citrate anticoagulated whole blood, and resuspended in Krebs buffer. Briefly, blood was mixed 1:3 with 2% trisodium citrate (wt/vol) and HetaSep (Stemcell technologies) and allowed to sediment for 45 min at 20 °C. The upper plasma layer was recovered and under laid with ice-cold Lymphoprep (2:1 for plasma/Lymphoprep) and centrifuged at 8002.8t-test. Where the difference between more than two sets of data was analyzed, one-way ANOVA was used followed by Bonferroni multiple comparisons test, as indicated on legends. p<0.05 was considered statistically significant.Data on platelets are representative of at least three separate donors, with samples run in triplicate for each experiment. Data are expressed as mean±SEM, of three separate determinations. Statistical significance was assessed using an unpaired, two-tailed Students 33.1m/z 351.2 in lipid extracts from thrombin-activated platelets, in order to find esterified prostaglandins. This demonstrated several series of ions consistent with four phosphatidylethanolamine (PE) species, namely 16:0p, 18:1p, 18:0p and 18:0a-PE, where p refers to plasmalogen and a to acyl, for the sn1 fatty acid 2/D2, but others remained uncharacterised. Herein, a later eluting series are identified as DXA3-PEs using MS/MS and MS3 both for the esterified forms and following cPLA2 hydrolysis of purified platelet PE fractions  mode, two lipids were detected for each PE parent ion . This indicates that the majority of DXA3 is esterified, with structural data confirming the four lipids as 16:0p/DXA3-PE, 18:1p/DXA3-PE, 18:0p/DXA3-PE and 18:0a/DXA3-PE.MS/MS of the third lipid in each series  or in vitro inhibition  inhibition  elevated free DXA3, while suppressing DXA3-PE formation , inhibited formation of DXA3-PE, but also partially blocked free DXA3 formation , neutrophil Mac-1 expression increased significantly . This would be a new concept for eicosanoid signaling, since traditionally these were only thought of as soluble signaling agents. Previous, eicosanoids were not considered to be re-esterified into phospholipids in significant amounts. This study showing DXA3 esterification strengthens previous reports of formation of PGE2- and HETE-PE formation as a regulated biological process Herein, DXAree DXA3 D-H. This3-PE might activate LTB4 receptors, either BLT1 or BLT2, since both are present in neutrophils and known to upregulate Mac-1 expression 3-PE likely activates other pathways to upregulate Mac-1 expression and further studies are required to delineate this. Indeed whether the free and esterified forms of this lipid can also stimulate additional neutrophil activities including calcium mobilization, superoxide generation and degranulation, also will be determined.We suspected that DXA2 could metabolise DXA3-PEs from activated platelets generating the free acid analog and thus further supporting innate immune responses to acute injury via soluble diffusion, with this lipid able to signal either in free or esterified forms. In summary, DXA3-PEs represent new platelet lipids generated through receptor-regulated cell signaling pathways. The full structural characterization of DXA3 and generation of synthetic standards will enable further study of its functions in innate immunity.Neutrophil phospholipids that have incorporated exogenous eicosanoids can be subsequently hydrolyzed by secondary cell stimulation, for example releasing free acid 15-HETE CH, MA, HP, MH, VH, SC, DS conducted experiments. CH, MA, VOD, RCM, PWC designed experiments. LJM, CP and SGO provided reagents/clinical samples. MA and VOD wrote the paper. All authors edited the paper.The authors declare no competing financial interests.


The Schiff base molecule is transformed into a substituted dibenzoiminodioxocin compound featuring a folded butterfly-like conformation with a dihedral angle of 84.72 (7)° between the benzene rings. 17H17NO4, lacks crystallographic symmetry with one molecule per asymmetric unit. The molecule exists in a folded butterfly-like conformation; the benzene rings form a dihedral angle of 84.72 (7)°. The central eight-membered imino-bridged dioxocin ring adopts a twisted-boat conformation. In the crystal, inversion-related molecules are linked by pairs of weak C—H⋯O hydrogen bonds, forming double-stranded chains parallel to the a axis.The title compound, C The resulting Schiff base apparently underwent self-condensation to form the substituted dibenzoiminodioxocin, 4,10-dimethoxy-13-methyl-6H,12H-6,12-epiminodibenzodioxocine, (I)N-methyliminomethyl)phenol, a liquid product of a similar condensation of salicylaldehyde and methylamine, after a few months storage in mild conditions  dibenzo(3.3.1)nonane  and two six-membered heterocyclic rings (O11/C11/C12/C121/N1/C221 and O21/C21/C22/C221/N1/C121) Fig. 1. The orga-axis direction 2·2H2O  dissolved in 5 ml dimethylformamide was added to the yellow solution of the Schiff base formed in situ, and the resulting deep-yellow solution was stirred at room temperature for an hour. The addition of N(Et)3 (1 ml) produced a light precipitate which was filtered off. The solution, which was kept cold (283–285 K), changed colour from yellow to brown. It was diluted twice with methanol (4 ml) since it was thickening. Brown plate-like crystals of the title compound formed over two months after successive addition of PriOH (4 ml) in two portions. They were collected by filter-suction, washed with dry PriOH and finally dried in air (yield: 23%). Analysis calculated for C17H17NO4 (299.31): C, 68.21; H, 5.72; N, 4.68%. Found: C, 68.55; H, 5.49; N, 4.87%. 1H NMR : δ (ppm) 6.89–6.79, m ; 5.69, s ; 3.71, s ; 2.51, s . The IR spectrum of powdered (I)−1 shows all characteristic functional groups peaks: ν(CH) due to aromatic =C—H and alkyl –C—H stretching above and below 3000, respectively, the aromatic rings vibrations in the 1600–1400 region, ν(CO) and ν(CN) at 1300–1000 and aromatic CH bending in the 900–600 cm−1 region (see Supporting information).2-Hydroxy-3-methoxy-benzaldehyde  and methylamine hydrochloride  were added to methanol (5 ml) and stirred magnetically for 10 min. Zn(CHUiso(H) = 1.2UeqC for CH, C—H = 0.98 Å, Uiso(H) = 1.5UeqC for CH3). Anisotropic displacement parameters were employed for the non-hydrogen atoms.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989017002328/hg5482sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989017002328/hg5482Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989017002328/hg5482sup3.pdfIR spectrum in KBr. DOI: Click here for additional data file.10.1107/S2056989017002328/hg5482Isup4.cmlSupporting information file. DOI: 1532218CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the molecule of the complex, the Re atom is coordinated to two N atoms of the two 4-pyOH ligands, three carbonyl C atoms, in a facial configuration, and the Cl atom. The resulting geometry is slightly distorted octahedral. In the crystal structure, both fragments are associated by hydrogen bonds; two 4-HpyO molecules bridge between two molecules of the complex using the O=C group as acceptor for two different HO– groups of coordinated 4-pyOH from two neighbouring metal complexes. The resulting square arrangements are extented into infinite chains by hydrogen bonds involving the N—H groups of the 4-HpyO molecule and the chloride ligands. The chains are further stabilized by π-stacking interactions.The asymmetric unit of the title compound, [ReCl(C In the present work, we present the structure of a rhenium complex, where the square architecture is achieved by a coordinative Re—L link (where L is 4-hydroxypyridine) and by hydrogen-bonding interactions involving a 4-pyridone molecule (a tautomer of 4-hydroxypyridine L).The structural stability of the fac-[ReCl(4-pyOH)2(CO)3] (where 4-pyOH represents 4-hydroxypyridine) and pyridin-4(1H)-one (4-HpyO) in a 1:1 ratio 2(CO)3] is similar to other tricarbonylrhenium(I) complexes with two pyridine-based ligands 2(CO)3] using the ketone O=C group as the hydrogen-bonding acceptor to two different HO– groups, forming g Wyckoff site . Although the centroid-to-centroid distance between the pyridone and hydroxypyridone is rather long (3.791 Å), some distances between the atoms and centroids of the rings  was obtained by refluxing for 90 min a mixture of 4-hydroxypyridine  and [ReCl(CH3CN)2(CO)3] in chloroform–methanol . The solution was concentrated , diethyl ether was added and the mixture cooled to 277 K. Finally, the solid was filtered off and vacuum dried on CaCl2 . Analysis, calculated for C13H10ClN2O5Re: C 31.5, H 2.0, N 5.6%; found: C 31.9, H 1.9, N 5.5%. MS–ESI [m/z (%)]: 461 (100) [M – Cl]+. IR : 2016 (m), 1865 , ν(CO).The complex fac-[ReCl(CO)3(4-pyOH)2] in CHCl3:CH2Cl2:ether (1:1:1) stored at 253 K .Single crystals of the title compound  were obtained from solutions of via difference Fourier analyses and refined with Uiso(H) = 1.5Ueq(O) and 1.2Ueq(N). Other H atoms were included at calculated sites and allowed to ride on their carrier atoms, with Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989017013512/zl2716sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989017013512/zl2716Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989017013512/zl2716Isup3.cdxSupporting information file. DOI: 1575682CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Streptomyces sp. KIB-H0495 was detected to be active. Subsequent large scale fermentation and isolation led to the isolation of four α-pyrone derivatives including two new compounds, violapyrones J (2) and K (3), and two known analogues, violapyrones B (1) and I (4). Further bioactivity assays indicated that only 1 and 3 exerted potent resensitization effects on MCF-7/TamR cells at a concentration of 1 μM. Owing to the simple structures of 1 and 3, these two compounds might have potential for further investigation as novel tamoxifen resensitization agent in breast cancer chemotherapy.Tamoxifen resistance (TamR) is the underlying cause of treatment failure in many breast cancer patients receiving tamoxifen. In order to look for noncytotoxic natural products with the ability to reverse TamR, an extract from strain The online version of this article (doi:10.1007/s13659-017-0136-8) contains supplementary material, which is available to authorized users. Resistance to chemotherapeutic drugs is a severe clinical impediment to the successful treatment in cancer chemotherapy . For theEndophytic strains isolated from the medicinal plants or their rhizosphere are prolific sources of novel natural products –9. It haStreptomyces sp. KIB-H0495 significantly inhibited resistant strains MCF-7/TamR. Further fermentation and isolation towards this strain led to the identification of four α-pyrone derivatives including two new ones, violapyrones J (2) and K (3), and two known analogues, violapyrones B (1) + peak at m/z 197.1176  in the HRESIMS, indicating four degrees of unsaturation. Comparing with the 1H and 13C NMR data of violapyrone B (1)  CC, yielding five fractions A-E. Fraction B (1.93 g) was separated on MCI CC eluting with gradient CH3OH/H2O . From 50% CH3OH/H2O part, compounds 1 (2.2 mg), 2 (1.0 mg), 3 (1.1 mg), and 4 (1.3 mg) were obtained after a preparation by semipreparative HPLC eluting with 68% CH3OH/H2O at the flow rate of 3.0 mL/min.The 2): Yellowish amorphous solid; UV (MeOH) λmax (log ε) 204 (4.3), 289 (3.9) nm; 1H and 13C NMR see Table m/z 197 [M+H]+; HRESIMS 197.1176 .Violapyrone J (3): Yellowish amorphous solid; UV (MeOH) λmax (log ε) 204 (4.2), 289 (3.9) nm; 1H and 13C NMR see Table m/z 211 [M+H]+; HRESIMS 211.1328 .Violapyrone K ; A549 was cultured in DMEM medium; Breast cancer cells MDA-MB-231 were cultured in DMEM/F12 medium with 1.2 g/L NaHCO3. All the above mentioned medium were also supplemented with 10% Fetal bovine serum (FBS)  and 1% penicillin–streptomycin . Normal breast epithelial cells MCF-10A were cultured in DMEM/F12 medium with 1.2 g/L NaHCO3, 5% horse serum , 10 μg/mL insulin , 20 ng/mL EGF2 , 100 ng/mL cholera toxin α subunit  and 0.5 μg/mL hydrocortisone .Parental breast cancer cell line MCF-7 and its derived tamoxifen resistant lines MCF-7/TamR were kfindly provided by Dr. Tao Zhu  and cultured as described before . Breast α-pyrone derivatives were tested alone against breast epithelial cells , tamoxifen resistant cells (MCF-7/TamR), breast cancer cells , gastric cancer cells (BGC-823) and lung cancer cells (A549). The cytotoxicity of MCF-7/TamR was detected by inputting different concentrations of tamoxifen under separately quantitative compounds 1–4 and DMSO.Cytotoxicity of KIB-H0495 extract was investigated using resistant cell line MCF-7/TamR. Four The SRB assay was modified according to the method described by Pauwels et al. . BrieflySupplementary material 1 (PDF 658 kb)Below is the link to the electronic supplementary material.


The crystal structures of three 9,9-disubstituted-3,7-diazabicyclo[3.3.1]nonane-2,4,6,8-tetraones and their supramolecular features were studied by X-ray diffraction. 9H10N2O4, (II), C11H12N2O4, and (III), C14H12N2O4·C3H7NO represent 9,9-disubstituted-3,7-diazabicyclo[3.3.1]nonane-2,4,6,8-tetraone derivatives with very similar molecular geometries for the bicyclic framework: the dihedral angle between the planes of the imide groups is 74.87 (6), 73.86 (3) and 74.83 (6)° in (I)–(III), respectively. The dimethyl derivative (I) is positioned on a crystallographic twofold axis and its overall geometry deviates only slightly from idealized C2v symmetry. The spiro-cyclopentane derivative (II) and the phenyl/methyl analog (III) retain only internal Cs symmetry, which in the case of (II) coincides with crystallographic mirror symmetry. The cyclopentane moiety in (II) adopts an envelope conformation, with the spiro C atom deviating from the mean plane of the rest of the ring by 0.548 (2) Å. In compound (III), an N—H⋯O hydrogen bond is formed with the dimethylformamide solvent molecule. In the crystal, both (I) and (II) form similar zigzag hydrogen-bonded ribbons through double intermolecular N—H⋯O hydrogen bonds. However, whereas in (I) the ribbons are formed by two trans-arranged O=C—N—H amide fragments, the amide fragments are cis-positioned in (II). The formation of ribbons in (III) is apparently disrupted by participation of one of its N—H groups in hydrogen bonding with the solvent molecule. As a result, the molecules of (III) form zigzag chains rather than the ribbons through intermolecular N—H⋯O hydrogen bonds. The crystal of (I) was a pseudo-merohedral twin.Compounds (I), C The molecule of (I)C2 (2)], and its geometry deviates only slightly from the perfectly symmetrical C2v. As a result of the presence of spiro-9-cyclopentane [in the case of (II)] and 9-phenyl and 9-methyl [in the case of (III)] substituents, the overall symmetry of these molecules decreases to Cs (m). However, in the crystal, the intrinsic Cs symmetry remains only for the molecule of (II)Compounds (I)s Figs. 1–3 ▸ ▸. The two imide fragments in the molecules of (I)–(III) are almost planar . The dihedral angles between the imide planes are 74.87 (6), 73.86 (3) and 74.83 (6)° for (I)–(III), respectively. Moreover, the four carbonyl carbon atoms in (I)–(III) are each coplanar with r.m.s. deviations of 0.018, 0.000, and 0.031 Å, respectively; the bridged carbon atom lies by 1.854 (3), 1.846 (1), and 1.858 (2) Å, respectively, above this plane in (I)–(III). The cyclopentane substituent in (II)Importantly, in (III)et al., 2008et al., 1981et al., 2007et al., 2014et al., 2014et al., 2007et al., 2005In general, any compound of type (I)–(III) could form up to six intermolecular hydrogen bonds utilizing two hydrogen-bond donor NH groups and four hydrogen-bond acceptor carbonyl oxygen atoms. In the literature, even the unsubstituted analogue cis disposed. As one of the two NH groups in (III)Despite the geometrical similarity of compounds (I)-(III), they form different supramolecular structures in the solid state. Thus, in the crystals of (I)et al., 1998The title compounds (I)–(III) were synthesized Fig. 7 accordinDinitrile subproducts were obtained by adding 2-cyanoacetamide to the corresponding ketone . The other hydrogen atoms were placed in calculated positions with C—H = 0.95–1.00 Å and refined in the riding/rotating model with fixed isotropic displacement parameters [Uiso(H) = 1.5Ueq(C) for the CH3-groups and 1.2Ueq(C) for the other groups]. The crystal of (I)Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989017009458/ld2140sup1.cifCrystal structure: contains datablock(s) global, I, II, III. DOI: 10.1107/S2056989017009458/ld2140Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989017009458/ld2140IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989017009458/ld2140IIIsup4.hklStructure factors: contains datablock(s) III. DOI: Click here for additional data file.10.1107/S2056989017009458/ld2140Isup5.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989017009458/ld2140IIsup6.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989017009458/ld2140IIIsup7.cmlSupporting information file. DOI: 1558317, 1558316, 1558315CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


II atom displays a distorted square-planar coordination.The synthesis and crystal structures of two platinum(II) complexes containing one or two Cl atoms, an eugenol derivative and 2-aminopyridine as ligand are described. The central Pt trans-(2-aminopyridine-κN)dichlorido{4-ethoxycarbonylmethoxy-3-methoxy-1-[-prop-2-en-1-yl]benzene}platinum(II), [PtCl2(C5H6N2)(C14H18O4)], (I), and (2-aminopyridine-κN)chlorido{5-ethoxycarbonylmethoxy-4-methoxy-1-[-prop-2-en-1-yl]phenyl-κC1}platinum(II), [Pt(C14H17O4)Cl(C5H6N2)], (II), the central PtII metal atom displays a distorted square-planar coordination, with the PtII atom coordinated by the pyridine N atom, the C=C double bond of the eugenol ligand and two Cl atoms for (I) or one Cl atom and a C atom of the phenyl ring for (II). The allyl fragment in (I) is disordered, with population parameters 0.614 (14) and 0.386 (14) for the two positions of the central C atom. The least-squares planes through the two aromatic ring systems make a dihedral angle of 51.10 (13)° for (I) and 78.5 (2)° for (II). Intramolecular N—H⋯O and N—H⋯π interactions occur in (I). In (I), inversion dimers formed by C—H⋯Cl interactions are further linked into chains parallel to the b axis by C—H⋯O hydrogen bonds. Both aromatic rings are involved in π–π interactions, with centroid-to-centroid distances of 3.508 (3) and 3.791 (3) Å. In (II), inversion dimers form chains parallel to the b axis by C—H⋯O interactions.In the title complexes,  Both aromatic rings show π–π stacking, with Cg1⋯Cg1iii = 3.791 (3) Å for the phenyl ring and Cg2⋯Cg2iv = 3.508 (3) Å for the pyridine ring , can be rationalized by the replacement of the Cl atom in the trans position with respect to the C=C bond. Verification of the Pt—Cl distances in the dinuclear complex di-μ-chlorido-bis[carbonyl]methoxy}phenyl-κC1)platinum(II)], [Pt2(IsoPreug-1H)2Cl2]  Å] is cleaved, leading to a cis position of 2-aminopyridine with respect to the C=C bond.The synthesis of (II)3(Eteug)] and the dinuclear chelate ring complex [Pt2Cl2(Eteug-1H)2] were synthesized following the protocol of Da and co-workers ] (0.2 mmol) in acetone (15 ml). After 2 h, a white precipitate of KCl was separated out. After stirring for 3 h at room temperature, ethanol (2 ml) was added to the obtained solution. Slow evaporation of the solvent at room temperature afforded the desired product as bright orange–yellow crystals. The yield was 80%. The product is soluble in acetone and chloroform, but only slightly soluble in ethanol and insoluble in water. Single crystals suitable for X-ray diffraction were obtained from an acetone/ethanol (3:1 v/v) solution via slow evaporation of the solvents at 277–278 K.While stirring, a solution of 2-aminopyridine (0.22 mmol) in acetone (2 ml) was added slowly to a solution of K (0.1 mmol) and acetone/ethanol . After stirring for 2 h at room temperature, a yellow solution was obtained. A white precipitate was formed by slow evaporation of the solvent at 277–278 K. The precipitate was collected by filtration and washed with ethanol. The product is soluble in acetone and chloroform, but only slightly soluble in ethanol and insoluble in water. The yield was 75%. Single crystals suitable for X-ray diffraction were obtained from a acetone/ethanol (1:1 v/v) solution via slow evaporation of the solvents at 277–278 K.A solution of 2-aminopyridine (0.22 mmol) in acetone (2 ml) was added slowly to a mixture of  and was refined with constraints for bond lengths and anisotropic displacement parameters present in this fragment.In (I)10.1107/S2056989017004285/rz5208sup1.cifCrystal structure: contains datablock(s) I, II. DOI: 10.1107/S2056989017004285/rz5208Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989017004285/rz5208IIsup3.hklStructure factors: contains datablock(s) II. DOI: 1538567, 1538566CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


H-chromen-4-one, C17H11F3O4, are reported. This isoflavone is used as a starting material in the preparation an array of potent and competitive FPR antagonists.The synthesis and crystal structure of 7-hydroxy-3-(2-methoxyphenyl)-2-trifluoromethyl-4 H-chromen-4-one, C17H11F3O4, are reported. This isoflavone is used as a starting material in the preparation an array of potent and competitive FPR antagonists. The pyran ring significantly deviates from planarity and the dihedral angle between the benzopyran mean plane and that of the exocyclic benzene ring is 88.18 (4)°. In the crystal, O—H⋯O hydrogen bonds connect the molecules into C(8) chains propagating in the [010] direction.Herein, the synthesis and crystal structure of 7-hydroxy-3-(2-methoxyphenyl)-2-trifluoromethyl-4 The fused aromatic benzopyran ring system shows a slight distortion from planarity as a result of the puckering of the pyran ring. The dihedral angle between the pyran ring and the exocyclic benzene ring is 89.26 (6)°. The mean plane of the ten atoms of the benzopyran ring system was used to evaluate the degree of twisting of the 2-methoxyphenyl ring in relation to the chromone as depicted in Fig. 1sp3 character, with a bond length of 1.4983 (17) Å. This conformation is to be expected and probably results from minimization of the steric hindrance between the 2-methoxy substituent of the exocyclic benzene ring with the voluminous –CF3 group and/or the oxo oxygen atom of the chromone ring.The pyran ring is not planar as the weighted average absolute torsion angle is 6.77 (7)°; for planarity this should be below 5.00°  Å and −0.1398 (10) Å, respectively], showing again that the benzopyran ring itself does not show the typical planarity observed for similar chromone and coumarin structures  link forms a C(8) chain, which runs parallel to the b axis. This is formed by the action of the c-glide plane at y = Details of the hydrogen-bonding interactions are given in Table 2x, 1 − y, 1 − z) interaction forms an x, 1 − y, 1 − z) interaction forms an a axis, Fig. 3The molecules are linked into alternating pairs of dimers to form a ladder. The C36—H36⋯O7 was calculated with TONTO  plots °. KUZJIA, 7-hydroxy-3-phenyl-4H-chromen-4-one, dihedral angle = 55.23 (8)°. KUZJIA01, 7-hydroxy-3-phenyl-4H-chromen-4-one, dihedral angle = 56.83 (7)°(molecule A), 48.27 (6)° (molecule B). KUZNIE, 7-hydroxy-3-phenyl-4H-chromen-4-one; dimethyl sulfoxide solvate, dihedral angle = 45.91 (7)°. KUZJUM, 7-hydroxy-3-phenyl-4H-chromen-4-one N,N-dimethylformamide solvate, dihedral angle = 41.70 (7)°. KUZKAT, 7-hydroxy-3-phenyl-4H-chromen-4-one propan-1-ol, solvate, dihedral angle = 45.18 (9)°. KUZKEX, 7-hydroxy-3-phenyl-4H-chromen-4-one butan-1-ol solvate, dihedral angle = 45.11 (11)°. In all solvated structures, the 7-OH hydroxyl group is involved in hydrogen bonding with the solvent. In the two KUZJIA(01) structures, –OH⋯O chains are formed as in 1.A search made in the Cambridge Structural Database,  and heated at 358 K, for 90 min. Then the mixture was poured into water and stirred until the formation of a solid and extracted with ethyl acetate. The combined organic phases were washed with water, dried over anhydrous sodium sulfate, filtered and evaporated. The product was recrystallized from ethyl acetate solution and used in the subsequent reaction to obtain the isoflavone.The title compound was obtained by a two-step synthesis -2-(2-methoxyphenyl)ethan-1-one (1 mmol), trifluoroacetic anhydride (3 mmol) and trimethylamine (2 ml) were refluxed for 1 h. After cooling, water (15 ml) was added. The solution was acidified (pH 5) with 2 1H NMR (DMSO-d6): 3.69 , 6.95 , 7.00 , 7.02 , 7.09 , 7.15 , 7.42 , 7.92 , 11.13 .Uiso = 1.5Ueq(C-methyl) and 1.2Ueq(C) for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989017009896/hb7688sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989017009896/hb7688Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989017009896/hb7688Isup3.cmlSupporting information file. DOI: 1453086CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The coordination geometry for each molecule is based on an octahedron with the lone-pair of electrons occupying a position opposite to the n-butyl group.Two chemically similar molecules comprise the asymmetric unit and these are connected  12H17ClOTe. The molecules, with the same chirality at the methine C atom, are connected into a loosely associated dimer by Te⋯O interactions, leading to a {⋯Te—O}2 core. The resultant C2ClO2 donor set approximates a square pyramid with the lone pair of electrons projected to occupy a position trans to the n-butyl substituent. Interestingly, the TeIV atoms exhibit opposite chirality. The major difference between the independent molecules relates to the conformation of the five-membered chelate rings, which is an envelope with the O atom being the flap, in one molecule and is twisted about the O—C(methine) bond in the other. No directional intermolecular interactions are noted in the molecular packing beyond the aforementioned Te⋯O secondary bonding. The analysis of the Hirshfeld surface reveals the dominance of H⋯H contacts, i.e. contributing about 70% to the overall surface, and clearly differentiates the immediate crystalline environments of the two independent molecules in terms of both H⋯H and H⋯Cl/Cl⋯H contacts.Two independent molecules comprise the asymmetric unit in the title benzoxatellurole compound, C While the bridging-O2 atom forms a significantly longer Te⋯O2 bond than the Te—O1 bond, Table 1trans to the n-butyl group. The coordination geometry for the Te2-containing molecule is essentially the same.The asymmetric unit of (I)IV atoms in the independent molecules are similar, Table 1i.e. 0.8°, seen in the Cl—Te—Olong angle. A distinguishing feature of the independent molecules is noted in the conformation of the five-membered, chelate rings. Thus, in the Te1-molecule, the chelate ring has the form of an envelope with the flap atom being the O1 atom . For the Te2-molecule, the chelate ring is twisted about the O2—C13 bond, as seen in the Te2—O2—C13—C15 torsion angle of 12.1 (7)°.The bond lengths about the Te2 core of the dimeric aggregate, Fig. 1n-butyl groups lie to either side of the dimeric aggregate, there is a suggestion that the independent molecules are related across a pseudo centre of inversion. However, the configuration of the chiral-C2 and C13 atoms in the Te1- and Te-molecules, respectively, is R. This is highlighted in the overlay diagram shown in Fig. 2n-butyl) bond, the chirality about the Te1 atom is S and that about Te2, R.The central {⋯Te—O}PLATON i.e. ca 15%. Others interactions each contribute less than 5% to the Hirshfeld surface. It should be noted that the C—H⋯O contacts, Te⋯O secondary interactions and most of the C—H⋯Te contacts are formed between the two independent molecules, thus they are overlapped and do not contribute to surface area of the asymmetric unit.Referring to Fig. 5a and H⋯Cl/Cl⋯H contacts, Fig. 6b, highlight the distinctive features of the interactions for the two molecules. For example, short H⋯H interactions for the Te2-molecule, Fig. 6a, occur at shorter distances that those of the Te1-molecules. With regard to the H⋯Cl/Cl⋯H contacts, there is a wider spread at lower de + di for the Te1- cf. the Te2-molecule.The main differences between the surface areas of the independent molecules are in the interactions of the type H⋯H and C—H⋯Cl. Referring to Fig. 6et al., 20163 donor set as in (I)H-2,1-benzoxatellurol  compounds studied earlier R)-MeCH(OH)]C6H4Te(nBu) , prepared as in the literature . 1H  δ 8.20 , 7.6–7.5 , 7.31 , 5.59 , 3.31 , 1.90 , 1.59 , 1.46 , 0.93 . 13C  δ 148.1, 131.6, 131.2, 128.7, 127.8, 125.4, 75.5 (Br), 45.4, 28.4, 24.6, 23.7, 13.0. 125Te  δ 847.2 (minor), 801.1 (major). 125Te  δ 1201.5 (minor), 1189.1 (major).The compound was prepared following a literature procedure  set to 1.2–1.5Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989017003887/hg5483sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989017003887/hg5483Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989017003887/hg5483Isup3.cmlSupporting information file. DOI: 1537011CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In vitro, both C60 derivatives had dose-dependent suppressive effects on T cell receptor-mediated activation of T cells and antibody production by B cells under anti-CD40/IL-4 stimulation, similar to the actions of the antioxidant N-acetylcysteine. In addition, C60-P suppressed ovalbumin-specific antibody production and ovalbumin-specific T cell responses in vivo, although T cell-independent antibodies responses were not affected by C60-P. Together, our data suggest that fullerene derivatives can suppress acquired immune responses that require T cells.The therapeutic effects of fullerene derivatives on many models of inflammatory disease have been demonstrated. The anti-inflammatory mechanisms of these nanoparticles remain to be elucidated, though their beneficial roles in allergy and autoimmune diseases suggest their suppressive potential in acquired immunity. Here, we evaluated the effects of C Fullerenes have a highly unsaturated structure and excellent electron-receptor properties, and for these reasons, these molecules—particularly water-soluble fullerene derivatives in which hydrophilic group moieties are added to the carbon cage for biological uses—have been investigated extensively as radical scavengers [Fullerenes Cavengers . Fullereavengers , reductiavengers , 4, exteavengers , and UV-avengers  and enzyavengers .60 derivatives, namely C60 pyrrolidine tris-acid (C60-P) and polyhydroxylated fullerene (C60(OH)36). Our data suggest that fullerene derivatives can suppress acquired immune responses that require T cells and support their potential for use in the treatment of inflammatory diseases.Many studies have shown the anti-inflammatory effects of fullerene derivatives, which are expected to be candidates for use as anti-inflammatories –12. Howe60 pyrrolidine tris-acid (C66O6NH7) (C60-P) was purchased from FLOX . Polyhydroxylated fullerene (C60(OH)36·8H2O; C60(OH)36) was synthesized as previously described [60-P by using liquid chromatography–mass spectrometry. C60(OH)36 is a mixture of isomers; we prepared and purified it by using a method described in the literature [60 powders were stored in the dark at room temperature. Immediately before use, the powder was dispersed in dimethyl sulfoxide , sonicated at 400 W for 5 min at 25 °C, and then vortexed for 1 min. This suspension was then diluted with cell culture medium or saline, further sonicated at 400 W for 5 min at 25 °C, and then vortexed for 1 min.Cescribed . We confterature . The C60N-acetylcysteine (NAC), and lipopolysaccharide  were purchased from Sigma–Aldrich . NP49-AECM-Ficoll  and NP30-BSA were obtained from Biosearch Technologies .Mitomycin C was purchased from Wako. Anti-mouse CD3ε (145-2C11), CD28 (37.51), and CD40 (HM40-3) were obtained from Biolegend . Recombinant mouse IL-4 was purchased from R&D Systems . Ovalbumin (OVA), Female C57BL/6 and BALB/c mice were purchased from SLC  and used at 6 to 8 weeks of age.6 cells/well). Stimulator splenocytes from C57BL/6 mice were treated with 250 μg/mL mitomycin C at 37 °C for 30 min, followed by three extensive washes with complete RPMI1640 supplemented with 10 % fetal bovine serum, 10 mL/L of a 100× nonessential amino acid solution , 50 μM 2-mercaptoethanol (Gibco), and 1 % antibiotic cocktail . They were then added at 3 × 106 cells/well to responder splenocytes. C60-P or C60(OH)36 was added to responder splenocytes 30 min before addition of the stimulator splenocytes. After incubation of the cells for 4 days at 37 °C , the amount of interleukin 2 (IL-2) released into an aliquot of culture supernatant was measured with a murine IL-2 enzyme-linked immunosorbent assay (ELISA) kit  in accordance with the manufacturer’s instructions.Responder splenocytes from BALB/c mice were seeded onto 96-well flat-bottomed plates  (1 × 1060-P or C60(OH)36 was added to the well 30 min before the addition of OVA (100 μg/mL). After incubation of the cells for 3 days at 37 °C , the amount of interleukin-4 (IL-4) released into an aliquot of culture supernatant was measured with an IL-4 ELISA kit (eBioscience) in accordance with the manufacturer’s instructions.C57BL/6 mice were intraperitoneally immunized with OVA (10 μg/mouse) and Imject Alum adjuvant (1 mg/mouse)  weekly. One week after the third injection, the mice were euthanized, and single-cell suspensions of splenocytes were prepared. C+ T cells, we coated 96-well flat-bottomed plates (Nunc) with anti-CD3ε (2.5 μg/mL) by overnight incubation at 4 °C. CD4+ T cells were negatively isolated with a CD4+ T cell isolation kit  in accordance with the manufacturer’s instructions. CD4+ T cell purity was determined by flow cytometry . Immediately after cell preparation, CD4+ T cells were added to the coating plate (1 × 105 cells/well). C60-P, C60(OH)36, or NAC was added to the well 30 min before the addition of anti-CD28 (1 μg/mL). After incubation of the cells for 3 days at 37 °C , the amount of IL-2 released into an aliquot of culture supernatant was measured with an IL-2 ELISA kit (eBioscience) in accordance with the manufacturer’s instructions.To stimulate CD4+ cells >90 % purification). Immediately after cell preparation, B cells were seeded into 96-well flat-bottom plates (Nunc) (1 × 106 cells/well). B cells were incubated in complete RPMI1640 with 10 ng/mL IL-4 and 3 μg/mL anti-CD40 for 10 days. C60-P, C60(OH)36, or NAC was added to the wells 30 min before the addition of IL-4 and anti-CD40. After incubation of the cells for 10 days at 37 °C , the amount of total IgE released into an aliquot of culture supernatant was measured with a total IgE ELISA kit (BD Biosciences) in accordance with the manufacturer’s instructions.B cells were purified from splenocytes by positive selection with CD19 MicroBeads (Miltenyi Biotec) in accordance with the manufacturer’s instructions. B cell purity was determined by flow cytometry (B22049-Ficoll (50 μg/mouse), or with a mixture of one of these plus C60-P (62.5 to 250 μg/kg), by intraperitoneal injection. Seven days after the third (in the case of the OVA experiment) or second (in the case of the LPS and NP-Ficoll experiments) treatments, the blood or blood and spleens, were collected to evaluate antibody responses and effector cytokine responses. Splenocytes (1 × 106 cells/well) were re-stimulated with OVA (100 μg mL). After incubation of the cells for 72 h at 37 °C, the levels of IL-4 released into an aliquot of culture supernatant were measured by ELISA (eBioscience) in accordance with the manufacturer’s instructions.C57BL/6 mice were treated weekly with OVA (10 μg/mouse), LPS (30 μg/mouse), or NP30-BSA (5 μg/mL). The coated plates were incubated with 2 % Block Ace (for OVA-coated plates)  or 1 % BSA (for LPS or NP30-BSA-coated plates) for 2 h at room temperature. Plasma dilutions were added to the antigen-coated plates. After incubation with the plasma for 2 h at room temperature, the coated plates were incubated with a horseradish peroxidase-conjugated goat anti-mouse IgG1, IgM, or IgG3 solution  for 2 h at room temperature. After the incubation, the color reaction was developed with tetramethylbenzidine , stopped with 2 N H2SO4, and measured at OD450–620 on a microplate reader. To detect OVA-specific IgE, we coated ELISA plates with purified rat anti-mouse IgE (2 μg/mL) and detected OVA-specific IgE with biotin-conjugated OVA (5 μg/mL) followed by horseradish peroxidase-coupled streptavidin .Plasma levels of antigen-specific antibodies were determined by ELISA. To detect OVA-specific IgG1, LPS, or NP-specific IgM or IgG3, we coated ELISA plates  with OVA (10 μg/mL), LPS (25 μg/mL), or NP60-P-added groups were determined by using the Williams test; a P value less than 0.05 was considered significant.Statistical analyses were performed with Ekuseru-Toukei 2012 software . Significant differences between control groups and C60 derivatives, namely C60-P and C60(OH)36, which had shown the strongest anti-inflammatory effects among more than 20 C60 derivatives that we had screened by using IL-1β-stimulated Caco-2 cells  (paper in preparation). In a preliminary experiment, to evaluate the immune suppressive effects of C60-P and C60(OH)36, we evaluated the cytotoxicity of the C60 derivatives on murine splenocytes by using a lactate dehydrogenase assay. After 3 days’ co-incubation with each C60 derivative, no cytotoxicity was induced by either one, at least at the maximum dose that we used, 100 μM (data not shown). We therefore used 100 μM of each C60 derivative as the maximum dose in the following in vitro assays.We used two hydrophilized C60 derivatives on T cells by using MLR and measured IL-2 as a mediator of T cell expansion. C60-P and C60(OH)36 dose-dependently suppressed IL-2 production by responder cells 36. To confirm the effect of C60-P on T cells, CD4+ T cells were purified from splenocytes and then stimulated with anti-CD3 and anti-CD28. To some groups of CD4+ T cells, we added each C60 derivative or the antioxidant NAC at various concentrations 30 min before the addition of anti-CD28 antibodies. IL-2 production induced by anti-CD3 and anti-CD28 stimulation was decreased by both C60 derivatives 36. Our results suggested that the C60 derivatives had some suppressive effects on B cells. In addition, NAC treatment significantly decreased IgE levels, suggesting that the decrease in IgE production caused by the C60 derivatives is associated with their antioxidant properties.We evaluated the effects of the Cion Fig. ; the eff60 derivatives revealed that both had the potential to suppress T and B cells; overall, C60-P seemed to have more potent effects than C60(OH)36  than that in mice treated with OVA alone  are important messengers in TCR signaling , 18; suplls Fig. . B cell 60-P successfully suppressed OVA-specific immune responses, which require T cells . Nanopa18.8 nm; ; thus, d60 and C60-P have been suggested to promote antigen presentation by DCs by another group [60(OH)36, which might have weaker suppressive effects than C60-P on T and B cells, failed to suppress OVA-specific IL-4 production by OVA-immunized splenocytes 36 on T cells and DCs might have canceled each other out. Thus, it is possible that C60 derivatives inconsistently affect each aspect of the acquired immune response in vivo. However, considering the ultimate phenotype of the OVA-specific immune response induced by C60-P in vivo appeared to be suppressive . Cer group . C60(OH)tes Fig. . In thatOur data suggest that fullerene derivatives can suppress acquired immune responses that require T cells. Some groups have attempted to use fullerenes as tumor-inhibitory reagents by activating anti-tumor immunity , 23. Wit


The result is a two-dimensional coordination polymer extending parallel to the ab plane with square channels along the b axis. The CdII atom geometry is based on a distorted cis-N2S4 octahedron. The 3-methylpyridine molecules reside in the channels aligned along the b axis, being held in place by methylene-C—H⋯N(3-methylpyridine) and (3-methylpyridine)-C—H⋯π(pyridyl) interactions. Pyridyl-C—H⋯S and dithiocarbamate-methyl-C—H⋯π(pyridyl) interactions provide connections between layers along the c axis.The title compound, {[Cd(C This arises owing to the presence of equal numbers of chelating ligands and tridentate ligands, with the latter chelating one CdII atom while bridging a second. The coordination geometry defined by the resulting S5 donor set is invariably highly distorted and intermediate between trigonal-bipyramidal and square-pyramidal 2 have μ3-bridging ligands exclusively and six-coordinate, S6, geometries. Systematic crystallization studies indicate these transform to the binuclear motif with the egress of time Me2]2}2(pyridine) Ph]2(pyridine)2 2}n CH2CH2OH]2}23], isolated as its tetra acetonitrile solvate, both bidentate bridging (× 1) and monodentate (× 2) modes of coordination are found for 1,2-bis(pyridin-4-yl)ethylene, resulting in a cis-N2S4 coordination geometry CH2CH2OH]22} leading to a NS4 donor set for cadmium 2, see above, was retained. Thus, the structure of {Cd[S2CN(iPr)CH2CH2OH]2}2, isolated as its hydrate CH2Py]2 CH2(CH2py)]2}2 CH2py]2.3-methylpyridine}2, containing a pyridyl-functionalized dithiocarbamate ligand, is described.The varied and interesting structures notwithstanding, it is obvious that Cd2CN(Et)CH2py]2, Fig. 1II atom as just described and simultaneously bridging another via the pyridyl-N atom so that the coordination geometry about the CdII atom is cis-N2S4, distorted octahedral, Table 1a axis being formed via S3/S4–N4 bridges and those along the b axis being sustained by S1/S2–N2 bridges. The result is a two-dimensional architecture in the ab plane, Fig. 2b-axis direction and these are occupied by the solvent 3-methylpyridine molecules, Fig. 2a and b. The slats along the a axis are defined by the pyridyl residues and these block access along this direction, Fig. 2c.The asymmetric unit of (I)c axis are of the type pyridyl-C—H⋯S and dithiocarbamate-methyl-C—H⋯π(pyridyl). Two illustrations of the molecular packing are given in Fig. 3A summary of specific intermolecular interactions contributing to the molecular packing of (I)−[S2CN(Et)CH2py], found in (I)et al., 2012M[S2CN(R)CH2py]2, a notable example being one with R = CH2py, namely, {Hg[S2CN(CH2Py)2]2]}n   CH2Py]2}n  CH2py]2 precursor  was dissolved in an excess of 3-methylpyridine (ca 10 ml). The solution was filtered, transferred to a 50 ml test tube and layered with hexanes (ca 60 ml). Colourless crystals of (I)−1): 2973(w), 2923(w), 1608(s), 1473(s), 1408(s), 1283(m), 1249(m), 1219(s), 1167(s), 1107(m), 1071(m), 991(s), 946(s). NMR 1H: δ (ppm) 8.56 , 8.43 , 8.38 , 7.61 , 7.30 , 5.19 , 3.88 , 2.30 , 1.22 . M.p. 531 – 533 K (uncorrected). TGA: two steps, the first corresponding to loss of 3-methylpyridine , the second step corresponds to the decomposition to CdS . Total theoretical mass loss 77.1%, observed mass loss 74.9%.The Cd[SUiso(H) set to 1.2–1.5Ueq(C). Owing to interference from the beam-stop, the (100) reflection was removed from the final cycles of refinement.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989017003516/hb7664sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989017003516/hb7664Isup2.hklStructure factors: contains datablock(s) I. DOI: 1535967CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


SK2YWS-L. Their structures were elucidated on the interpretation of spectroscopic data. 1 and 4 were further constructed by analysis of X-ray diffraction. Antioxidant properties based on 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl radical scavenging activities and the ferric reducing ability power (FRAP) of the new compounds were assayed. All of them exhibited significant effects, of which 1 showed more potent activity than ascorbic acid in scavenging DPPH radical with IC50 value of 18.1 μM.Three novel 2,3-diaryl indone derivatives, ascomindones A−C ( Overproduction of reactive oxygen species (ROS), such as hydroxyl radical and superoxide radical, playing an important role in chronic metabolic and degenerative diseases, have been proven to be specific signaling molecular under both physiological and pathophysiological conditions1Kandelia candel, used as a folk medicine to cure inflammationKanelia candel such as the sesterterpenoids aspterpenacids A and BKanelia candel endophytic fungus SK2YWS−L was carried out and five new polyketones, ascomindone A−C (1−3), ascomfuran A (4) and B (5), together with four known compounds (6−9) were obtained  to afford the five new compounds (1−5) together with the three known compounds methyl 2--3-hydroxy-5-methoxybenzoate (6), 2-(2-carboxy-3-hydroxy-5-methylphenoxy)-3-hydroxy-5-methoxybenzoic acid (7)8) and emodin (9)The EtOAc extract of the fermentation broth was fractionated by repeated silica gel chromatography, Sephadex LH-20 column chromatography and reversed-phase C1) was obtained as brown crystals. The molecular formula was determined as C32H26O11 based on the quasi-molecular ion peak at m/z 585.1401  from HRESIMS, indicating 20 degrees of unsaturation. The IR spectrum exhibited the absorption bands of hydroxyl groups (3425 and 3219 cm−1) and carbonyl groups (1636 and 1670 cm−1). The 1H NMR (Nuclear Magnetic Resonance) spectrum , eight aromatic protons at δH 5.92 , δH 6.09 , δH 6.24 , δH 6.34 , δH 6.34 , δH 6.38  and δH 6.51 , two methoxyl groups at δH 3.54  and 3.71 , two methyls at δH 2.09  and 2.12 . 13C NMR and DEPT spectra  and the degrees of unsaturation suggested there should be an 2,3-disubstituded indone moiety in compound 1, which was further evidenced by the correlation from H-7 to C-1 in HMBC spectrum. The remaining two tetra-substituted benzene rings should be incorporated into a diphenyl ether moiety based on their corresponding 13C chemical shifts. Additionally, the placements of the hydroxyl, methyl and methoxyl groups were unambiguously elucidated by the chemical shifts and the HMBC correlations  was an axially chiral biaryl natural product, which should have had the specific optical rotation and Cotton effects (CEs) in Circular Dichroism (CD) spectrum. However, the lack of any specific optical rotation or significant CEs indicated that 1 was a racemic mixture of M- and P-helicity enantiomers with the ratio of 1:1. Further analysis of the molecular packing in crystals presented the mixture of (±)-1 -1 .2), isolated as brown powder, was deduced to possess the molecular formula of C33H28O11 based on the HRESIMS at m/z 599.1558 . Analysis of the 1D NMR spectra suggested that compound 2 was structurally similar to 1 except for the presence of an additional methoxyl group. The HMBC correlation from OMe-7′ (δH 3.64) to the carbonyl carbon (C-7′) indicated that the methoxyl was linked to C-7′. The gross structure was elucidated based on 2D NMR data (Ascomindone B (NMR data .3) was obtained as brown powder and showed the molecular formula of C32H26O11 based on the HRESIMS at m/z 567.1298 , suggesting that 3 was the dehydration product of 1. Comparison of 1D and 2D NMR with those of 1 indicated that 3 constructed the same 2,3-disubstituded indanone and the symmetric tetra-substituted benzene ring moieties. The major difference between them was that the diphenyl ether moiety in 1 was transformed to a depside in 3 through intramolecular esterification, which was confirmed by the upfield shift (∆δC = 7.8 ppm) of the carbonyl carbon (C-7′) in 3. The complete structure of 3 was deduced by the HMBC spectra (Ascomindone C ( spectra  as shown2 and 3) were both inferred to be racemates since the specific optical rotation were zero and there were no distinct CEs in CD data.Ascomindones B and C (4) was isolated as yellow crystals. HRESIMS analysis afforded an [M − H]− ion peak at m/z 287.0927, indicating the molecular formula as C16H16O5. The IR data exhibited absorption of hydroxyl (3395 cm−1) functionality. The 1H NMR spectrum  and δH 6.20 , a pair of chemically equivalent aromatic protons at δH 6.16 , a couple of oxygenated methylene in AB spin system protons at δH 5.33  and δH 5.00 , a methoxyl signal at δH 3.73  and a methyl signal at δH 2.16 . 13C NMR and DEPT spectra exhibited 16 carbon resonances, containing eight quaternary carbons, five methines, one methylene and two methyls. A comprehensive analysis of the 1D NMR data suggested that there should be two tetra-substituted benzene rings including a symmetric one.Ascomfuran A . In addition, the cross-peaks of H3-15 to C-5 and H3-14 to C-10(C-12)/C-11 were detected, which revealed the attachments of the methoxyl and the methyl group at C-5 and C-11, respectively. Thus, the planar structure of 4 was constructed as shown in The HMBC correlations  from H2-4 was deduced to be racemic mixtures at C-8. A further X-ray diffraction experiment was carried out and the enantiomers were performed in molecular packing of crystal , isolated as yellowish amorphous power, was assigned a molecular formula of C16H14O6 based on the HREI-MS m/z 302.0790 . The IR spectrum exhibited hydroxyl (3212 cm−1) and additional carbonyl (1702 cm−1) functional groups. 1H NMR data were similar to those of ascomfuran A (4), except for the absence of the methylene signals. In the 13C NMR spectrum, the disappearance of the corresponding methylene signal and the presence of an additional carbonyl carbon signal at δC 173.5 suggested that the methylene in 4 was oxidized to a carbonyl group in 5, which was confirmed by the HMBC correlations  gave the benzophenone intermediate (structure i), which could further transform into diphenyl ether derivatives (7 and 8) via grisendience15i and 8ii could afford compound 1−3 through an aldol condensation as well as the additional esterification. In addition, 4 and 5 were transformed from intermediate iii by esterification and reduction, which was a reductive product of intermediate i.The hypothetical biosynthesis pathways of 1−3), represent the first examples of 2,3-diarylindone derivatives constructing diphenyl ether or depside moiety. It is challenging to elucidate such a class of structures with low H/C ratio using NMR spectroscopic methods based on the Crews’s rule1−3 were deduced unambiguously. In addition, ascomfurans A (4) and B (5), belong to the derivatives of 1-aryl isobenzofuran, were naturally obtained racemic mixture like isopestacin and pestacin since the enantiomers of 1S and 1R could transform to each other through a stable cationic intermediate19To the best of our knowledge, 2,3-diarylindone derivatives are quite rare in natural productsin vitro antioxidative activities based on 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl radical scavenging capacities and ferric reducing ability power (FRAP) assays. Compared to the positive control ascorbic acid (Vc), ascomindone A (1) exhibited more potent capacity in scavenging DPPH radical with a IC50 value of 18.1 μM while compounds 2−4 also showed significant effect (1−3) exhibited strong activity with the IC50 values in the range from 80 to 100 μM  are given in ppm with reference to TMS, and coupling constants (J) are given in Hz. Column chromatography (CC) was carried out on silica gel  and sephadex LH-20 . Solvents were distilled prior to use. Semipreparative HPLC was performed on a Waters Breeze HPLC system using a Phenomenex Luna  C18 column , flow rate, 2.0 mL/min.Melting points were determined on a Fisher-Johns hot-stage apparatus and were uncorrected. UV data were measured on a UV-240 spectrophotometer . HRMS (ESI) were determined with a Q-TOF high-resolution mass spectrometer (Waters). HREIMS data were measured on a MAT95XP high-resolution mass spectrometer (Thermo). IR spectrum was recorded using Bruker Vector spectrophotometer 22. Optical rotation was recorded using a an MCP300 . CD data were recorded with a J-810 spectropolarimeter . The NMR data were recorded on a Bruker Avance 600 spectrometer  at 600 MHz for Kandelia candel, which were collected in April 2012 from Shankou Mangrove Nature Reserve in Guangxi Province, China. It was obtained using the standard protocol for the isolation. Fungal identification was carried out using a molecular biological protocol by DNA amplification and sequencing of the ITS region. The sequence data obtained from the fungal strain have been deposited at GenBank with accession no. KX389270. A BLAST search result showed that the sequence was the most similar (100%) to the sequence of Ascomycota sp. (compared to KC857277.1 HQ647349.1). A voucher strain was deposited in School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou, China.The fungus used in this study was isolated from the healthy leaf of the marine mangrove 4•7H2O (0.3 g/L), KH2PO4 (0.5 g/L), yeast extract (3.0 g/L), corn steep liquor (1.0 g/L)) in 80 Erlenmeyer flasks for 45 days at room temperature under static condition. After fermentation, the former was extracted with EtOAc and concentrated under reduced pressure to yield residual gum in 5.4 g. The residue was subjected to silica gel CC using gradient elution with petroleum ether-EtOAc from 90:10 to 0:100 (v/v) to give twelve fractions (Frs.1–10). Fr. 5 (207 mg) was further purified by silica gel CC using CHCl3/MeOH (99:1) to obtain 4 (3.5 mg), 5 (3.3 mg), 6 (9.2 mg), 7 (9.8 mg) and 8 (11.9 mg) Fr. 8 (117 mg) was further purified by silica gel CC using CHCl3/MeOH (97:3) to afford six subfractions (Frs.8.1-8.6). Fr. 8.5 (20.1 mg) was applied to Sephedx LH-20 CC, eluted with MeOH to obtain 1 (10.8 mg) and 9 (8.3 mg). After purification by RP-HPLC , Fr. 8.2 (30.2 mg) afforded 2  and 3 .The fungus was grown on liquid cultured medium , mannitol (20.0 g/L), glucose (10.0 g/L), monosodium glutamate (10.0 g/L), MgSOm.p. 301 ~ 302 °C; [α]D = 0 (0.1 M in methanol); UV (MeOH): λmax: 275, 382 nm. IR (KBr): 3425, 3219, 1670, 1636, 1430, 1278, 1199, 1055 cm−1; HRESIMS m/z 585.1401 [M − H]− ; 1H and 13C NMR data: see brown crystal; m.p. 277 ~ 279 °C; [α]D = 0 (0.1 M in methanol); UV (MeOH) λmax: 275, 358 nm. IR (KBr): 3391, 1695, 1619, 1430, 1303, 1148, 1063 cm−1; HRESIMS m/z 599.1558 [M − H]− ; 1H and 13C NMR data: see yellowish powder; m.p. 279 ~ 281 °C; [α]D = 0 (0.1 M in methanol); UV (MeOH) λmax: 275, 360 nm. IR (KBr): 3408,1687, 1610, 1455, 1190, 1131, 1055 cm−1; HRESIMS m/z 567.1298 [M − H]− ; 1H and 13C NMR data: see yellowish powder; m.p. 218 ~ 220 °C; [α]D = 0 (0.1 M in methanol); UV (MeOH) λmax: 245 nm. IR (KBr): 3395, 2922, 2850, 1459, 1346, 1202, 1149 cm−1; HRESIMS m/z 287.0927 [M − H]− ; 1H and 13C NMR data: see yellowish crystal; m.p. 232 ~ 233 °C. [α]D = 0 (0.1 M in methanol). UV (MeOH) λmax: 252, 284 nm. IR (KBr): 3411, 3212, 2968, 1702, 1416, 1303, 1209, 1154, 1055 cm−1. HREIMS m/z 302.0790 [M]+ ; 1H and 13C NMR data: see yellowish powder; 1) were obtained from MeOH containing a small amount of H2O at room temperature. Data were collected on Agilent Xcalibur Nova single-crystal diffractometer using Cu Kα radiation. The crystal structure was refined by full-matrix least-squares calculation with the SHELXL-97. Crystallographic data for the structure of 1 have been deposited in the Cambridge Crystallographic Data Centre (deposition number: CCDC 1483330). Crystal data of 1: C32H26O11∙2CH3OH∙H2O (M = 668.63); block crystal (0.4 × 0.4 × 0.38); space group P21/c; unit cell dimensions a = 17.5917(5) Å, b = 25.5009(10) Å, c = 14.2645(5) Å, α = 90°, β = 93.021(3)°, γ = 90°, V = 6390.2(4) Å3, Z = 8; T = 150(2) K; ρcald = 1.390 mg/m3; absorption coefficient 0.918 mm−1; F(000) = 2816, a total of 11658 reflections were collected in the range 3.05° < θ < 68.67°, independent reflections 9974 [R(int) = 0.0334]; the number of data/parameters/restraints were 11658/896/0; goodness-offit on F2 = 1.024; final R indices [I > 2σ(I)] R1 = 0.0438, ωR2 = 0.1156; R indices  R1 = 0.0528, ωR2 = 0.1230.Brown crystals of ascomindone A (4) were obtained from MeOH containing a small amount of H2O at room temperature. Data were collected on Agilent Xcalibur Nova single-crystal diffractometer using Mo Kα radiation. The crystal structure was refined by full-matrix least-squares calculation with the SHELXL-97. Crystallographic data for the structure of 1 have been deposited in the Cambridge Crystallographic Data Centre (deposition number: CCDC 1483331). Crystal data of 1: C16H16O5 (M = 288.29); block crystal (0.4 × 0.4 × 0.35); space group P21/c; unit cell dimensions a = 9.6005(6) Å, b = 18.5783(9) Å, c = 8.3575(5) Å, α = 90°, β = 101.567(6)°, γ = 90°, V = 1460.38(14) Å3, Z = 4; T = 293(2) K; ρcald = 1.311 mg/m3; absorption coefficient 0.098 mm−1; F(000) = 608, a total of 3253 reflections were collected in the range 3.67° < θ < 27.40°, independent reflections 2532 [R(int) = 0.0467]; the number of data/parameters/restraints were 3253/195/0; goodness-offit on F2 = 1.097; final R indices [I > 2σ(I)] R1 = 0.0607, ωR2 = 0.1296; R indices  R1 = 0.0826, ωR2 = 0.1428.Yellow crystals of ascomfuran A  of the tested compounds 1−5 was added to 150 μL (0.16 mmol/L) DPPH solution in MeOH in each well. Absorbance at 517 nm was recorded after 45 min and the percentage of inhibition was calculated. Vitamin C was used as a positive control.The DPPH radical scavenging test was based on the previous reported methodμL) was mixed with 25 μL of FeSO4 solution (3 mM) and 25 μL of 1,10-phenanthroline . Furthermore, 0.01% (v/v) H2O2 peroxide (25 μL) was added into the mixture. After incubated at 37 °C for 1 h, the absorbance was measured at 536 nm.The hydroxyl radical-scavenging assay was determined based on the described in previous reports3COOH buffer (pH 3.6), 0.1 M 2,4,6-Tris(2-pyridyl)-s-triazine and 0.2 M FeCl3 at 10:1:1 volume ratio in 0.4 M HCl. 180 μL FRAP reagent and 20 μL tested compound (100 μM) were added in 96-well microplates. After incubated at 37 °C for 20 min, the absorbance of the mixture was meansured at 595 nm. Vitamin C was used as a positive control. The FRAP value was expressed in Trolox  equivalents using the linear slope of the compounds tested versus that of Trolox.The FRAP assay performed was slight modified according to the previous reported literature2526How to cite this article: Tan, C. et al. Antioxidative Polyketones from the Mangrove-Derived Fungus Ascomycota sp. SK2YWS-L. Sci. Rep.6, 36609; doi: 10.1038/srep36609 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The title compound was synthesized using a novel one-pot method under mild conditions and fully characterized using NMR, ESI–MS and SXRD. The supramolecular structure of the title compound is defined by a combination of C—H⋯N and π–π interactions. 26H25N, was achieved by the condensation of an ethanolic mixture of benzaldehyde, cyclohexanone and ammonium acetate in a 2:1:1 molar ratio. There are two crystallographically independent molecules in the asymmetric unit. The two cyclohexyl rings adopt an anti-envelope conformation with the benzyl moiety adopting a cis conformation with respect to the nitrogen atom of the phenanthridine segment. In the crystal, molecules are linked through C—H⋯N interactions into hydrogen-bonded chains that are further arranged into distinct layers by weak offset π–π interactions.The preparation of the title compound, C Full refinement details are given in Section 5. In both of the crystallographically independent molecules, the phenyl and benzylidene groups are rotated out-of-plane with respect to the octahydrophenanthrine moieties: in molecule A the angle between the mean planes of the phenyl and pyridine rings is 46.92 (5)° with the equivalent angle in molecule B of 53.43 (5)°. The angle between the mean planes of the benzylidine and pyridine rings in molecule A is 48.53 (5)° and the corresponding angle in molecule B is 41.37 (5)°.The asymmetric unit contains two crystallographically independent molecules, A to the N1 position in a B molecule , with an equivalent weak contact formed from the C109 position in molecule B to the N2 position of a neighbouring molecule A . Geometric parameters for these contacts are given in Table 2D⋯A distances for weak hydrogen bonds of 3.2–4.0 Å, the D—H⋯A angles being slightly more linear than the expected values of 90–150°  with a centroid–centroid distance of 3.9853 (14) Å and shift distance of 2.285 (3) Å.The crystal structure features a combination of weak hydrogen bonds and weak offset π–π interactions. A weak C—H⋯N contact is formed from the octahydrophenanthridine C6 position in molecule et al., 2016Version 5.38 of the Cambridge Structural Database . The direction of these contacts runs parallel to the axis of the C—H⋯N contacts between molecules on neighbouring hydrogen-bonded chains and appears to result from the intercalation of these chains. As these contacts are not associated with either of the major attractive interactions (A–B C—H⋯N hydrogen bonds or B–B π–π stacking), it is probable that this contact arises solely from the packing arrangement required to maximize the number and strength of these favourable interactions.Fingerprint analysis of the intermolecular interactions by the generation of Hirschfeld surfaces using The title compound was isolated from the reaction mixture using a flash column chromatography and as follows: A solution of benzaldehyde , ammonium acetate  and cyclohexanone  in ethanol (20 mL) was heated to reflux for 2 h. The obtained residue was purified from the crude product by flash chromatography with an eluent mixture of 33% ethyl acetate in hexane, m.p. = 467–469 K, yield: 42%. Colourless crystals suitable for X-ray single crystal analysis were obtained by slow evaporation of a methanol solution of the compound.−1: 1600 ν (C=N), 1508 ν (C=C)aromatic ring. NMR data (ppm) : 7.81 , 7.52–7.35 , 7.28–7.21 , 2.79 , 2.71 , 2.66 , 2.61 , 1.80 , 1.62 . 13C NMR, δc : 155.15 (C-1), 147.72 (C-5), 144.86 (C-3), 140.98 (C-9) and 137.45 (C-2), 136.07 (C-15), 129.61 (C-4), 129.44 (C-21), 129.14, 129.03, 128.76, 128.33, 128.00 and 127.76 and 126.70 (C-Ar), 124.60 (C-14), 27.83 C-8), 26.90 (C-6), 25.80 (C-10), 24.91 (C-13), 22.18 , 21.96 (C-7). The electrospray (+) mass spectrum showed the parent ion peak at m/z = 352.2068 (M + H)+ for C26H26N; requires =352.2065. Elemental analysis: calculated for C26H25N: C 88.85%, H 7.17%, N 3.99%; found: C 88.76%, H 7.20%, N 3.88%. cmiUso(H)= 1.2Ueq(C). Disorder at C90/C91/C92/C93 was modelled by splitting the component atoms across two positions and refining the occupancy using FVAR to 82% for C90A–C93A and 12% for C90B–C93B. 1,2 distances were restrained using SADI and ADPs for C90A/C90B and C93A/C93B constrained using EADP commands.Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989017009537/ff2149sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989017009537/ff2149Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989017009537/ff2149Isup3.cmlSupporting information file. DOI: 1506784CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: