Although the understanding of the genetic and molecular basis of cancer has advanced significantly over the past several decades, imaging and treatment options for glioblastoma patients have been more limited . This is in part due to difficulties in diagnosing this disease early, combined with its diffuse, infiltrative growth. This study was aimed at the development of a novel diagnostic tool for glioblastoma through the synthesis of a small molecule based on radioiodinated poly(ADP-ribose)polymerase 1 (PARP1) targeted tracers. This PARP1 is a biomarker that is overexpressed in glioblastoma tissue, but has only low expression levels in the healthy brain .131I and 124I. Biodistribution as well as imaging experiments were performed in orthotopic and subcutaneous mouse models of glioblastoma.A library of PARP1 inhibitors (iodo-PARPis) was synthesized. Based on their pharmacokinetic properties and nuclear PARP1 binding, the most successful inhibitor was radiolabeled with 50 values in the nanomolar range (9 ± 2–107 ± 4 nM) and were able to inhibit the uptake of a fluorescent PARP1 inhibitor analog (PARPi-FL). I2-PARPi was able to reduce the uptake of PARPi-FL by 78 ± 4 % in vivo. In mouse models of glioblastoma, we show that the radioiodinated inhibitor analog has high uptake in tumor tissue . PET and SPECT imaging performed in orthotopic glioblastoma models with [124I]- and [131I]-I2-PARPi showed selective accumulation in the tumor tissue. These results were also verified using autoradiography of tumor sections, which displayed focal selective uptake of the tracer in the tumor regions as confirmed by histology. The uptake could be blocked through pre-injection of excess unlabeled PARP1 inhibitor (Olaparib).One member of our iodo-poly(ADP-ribose)polymerase 1 (PARP1) inhibitor library, I2-PARPi, shows promising biophysical properties for in vivo application. All synthesized tracers have ICWe have successfully synthesized and radioiodinated the PARP1 selective tracer I2-PARPi. The novel tracer shows selective binding to tumor tissue, both in vitro and in models of glioblastoma, and has the potential to serve as a selective PET imaging agent for brain tumors.The online version of this article (doi:10.1186/s13550-015-0123-1) contains supplementary material, which is available to authorized users. State-of-the-art cancer care is stricken by our inability to efficiently treat, inhibit, and ultimately cure tumor growth for the vast majority of primary cancers. And while for most types of cancer, the diagnosis and detection of lesions has seen significant improvements over the past decades , some ar131I and 124I . Specifically, we show the design of small molecular targeted agents and their validation in biochemical assays, both in cells and in vivo. We further use one tracer for 131I/124I SPECT and PET imaging of PARP1 in subcutaneous and orthotopic mouse models of cancer, thereby illustrating that the expression of PARP1 is highly upregulated and that our labeled tracer accumulates inside the nuclei of glioblastoma cells, where PARP1 is expressed.The goal of this project was therefore to develop a non-invasive PET-based imaging platform for PARP1. Based on our successful optical PARP1 imaging agents, we chose a 2H-phthalazin-1-one scaffold  previousN-succinimidyl-4-(tributylstannyl) benzoate was purchased from Synthonix , 3-(3-iodophenyl)propionic acid and 3-(4-iodophenyl)propionic acid from Matrix Scientific , and 4-iodophenyl acetic acid from Alfa Aesar . Olaparib (AZD2281) was purchased from LC Laboratories . 4-(4-Fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one was synthesized as described previously +. HRMS-ESI [M-H+]− m/z calculated for [C27H22FIN4O3]− 595.0642, found 595.0660.To a solution of 4-(4-Fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one , triethylamine  and HBTU  in dimethyl formamide  were added to 3-iodobenzoic acid . The mixture was stirred at room temperature for 20 h. The crude product was then purified by preparative HPLC and dried under vacuum, yielding a white solid . 1H-NMR (CDCl3) δ = 10.48 , 8.40–8.39 , 7.74–7.66 , 7.27–7.26 , 7.09–7.07 , 4.22 , 3.73–3.14 . LC-ESI-MS (+) m/z = 597.1 [M+H+]+. HRMS-ESI [M-H+]− m/z calculated for [C27H22FIN4O3]− 595.0642, found 595.0640.A solution of 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one , HBTU , triethylamine , and 4-iodobenzoic acid  in DMF (500 μL) was stirred overnight at room temperature. The crude product was purified by preparative HPLC and dried under vacuum, yielding a white solid . N-hydroxy succinimide (NHS), and 600 μL DMF was stirred for 30 min at room temperature. Then, 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one  was added to the solution, and the mixture was stirred at room temperature overnight. The reaction was washed with 500 μL of H2O and extracted with 500 μL dichloromethane (DCM). The resulting organic solution was purified on silica gel, using a gradient elution from neat DCM to DCM/hexane 5:1 to obtain the desired product as a white solid . 1H-NMR (CDCl3) δ = 9,82 , 8.40–8.38 , 7.71–7.69 , 7.55–7.53 , 7.51–7.50 , 7.25–7.24 , 7.09–6.90 , 4.20 , 3.64–3.31 , 2.84 . LC-ESI-MS (+) m/z = 633.1 [M+Na+]+. HRMS-ESI [M+H+]+ m/z calculated for [C28H24FIN4O3]+ 611.0955, found 611.0948.A solution of 3-iodophenyl acetic acid , 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) , 2O (500 μL) was added, the mixture was extracted with DCM (2 × 500 μL), and the combined extracts were dried under vacuum. The crude mixture was purified by silica column chromatography (100 % DCM), and the product obtained was a white solid . 1H-NMR (CDCl3) δ = 9.82 , 8.40–8.38 , 7.83–7.81 , 7.77–7.75 , 7.70–7.69 , 7.63–7.56 , 7.00–6.89 , 4.20 , 3.63–3.11 , 2.84 . LC-ESI-MS (+) m/z = 632.9 [M+Na+]+. HRMS-ESI [M+H+]+ m/z calculated for [C28H24FIN4O3]+ 611.0955, found 611.0971.A solution of 4-iodophenyl acetic acid , EDC , NHS, and 600 μL DMF was stirred for 30 min at room temperature. After this time, the 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one  was added to the solution and the mixture was stirred at room temperature overnight. H1H-NMR (CDCl3) δ = 10.33 , 8.41–8.39 , 7.71–7.63 , 7.51–7.45 , 7.27–7.25 , 7.12–6.92 , 4.22 , 3.65–3.12 , 2.88–2.83 , 2.59–2.48 . LC-ESI-MS (+) m/z = 647.1 [M+Na+]+. HRMS-ESI [M+H+]+ m/z calculated for [C29H26FIN4O3]+ 625.1112, found 625.1111.A solution of 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one , HBTU , triethylamine , and 3-(3-iodophenyl)propionic acid  in 400 μL of AcN was stirred overnight at room temperature. The crude product was then purified by preparative HPLC and the isolated product dried at vacuum to obtain a white solid . 1H-NMR (CDCl3) δ = 9.71 , 8.40–8.38 , 7.70–7.69 , 7.64–7.63 , 7.55–7.52 , 7.27–7.25 , 7.00–6.97 , 6.91–6.87 , 4.20 , 3.64–3.11 , 2.87–2.85 , 2.63–2.47 . LC-ESI-MS (+) m/z = 647.1 [M+Na+]+. HRMS-ESI [M+Na+]+ m/z calculated for [C29H26FIN4O3Na]+ 647.0931, found 647.0941.4-(4-Fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one  was mixed with HBTU , triethylamine , and 4-iodo-3-phenyl propionic acid  in 400 μL of AcN, and the solution was stirred overnight at room temperature. The crude product was then purified by preparative HPLC and the isolated product dried at vacuum to obtain a white solid . 131I]-NaI was purchased at Nordion  in NaOH solution (0.1 M) with a concentration of 0.99–2.5 mCi/μL. [124I]-NaI was produced at Memorial Sloan-Kettering Cancer Center  in NaOH solution (0.5 M) with a concentration of 0.20–0.40 mCi/μL.-NaI in NaOH 0.1 M (1–2.5 mCi). After 5 min at room temperature, the reaction was purified by HLPC on a C18 Waters Atlantis T3 column , using water (solvent A) and AcN (solvent B) as mobile phase, with an elution gradient from 5 to 100 % for solvent B over 15 min and then 100 % of solvent B from 15 to 25 min. The retention time of [131I]-NHS benzoate was 14.3 min, and its identity was established by co-elution with the reference cold compound. The radiochemical yield was 67 ± 6 % (n = 12), and the radiochemical purity was >98 %. The collected fraction containing [131I]-NHS benzoate was concentrated to dryness under vacuum.Precursor 124I]-NHS benzoate. In this case, the radiochemical yield was 32 ± 5 % (n = 5) and the purity was >95 %.The same procedure was followed for the synthesis of [131I]-NHS-benzoate precursor was dissolved in 200 μL of AcN, and an excess of HBTU  and 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one  was added and allowed to react for 3 h at 32 °C. The final product was purified by HPLC, using water (solvent A) and AcN (solvent B) as solvents with a gradient elution from 5 to 100 % of solvent B over 15 min and then 100 % of B from 15 to 25 min. The retention time of [131I]-I2-PARPi was 13.1 min, and its identity was established by co-elution with the reference cold compound. The radiochemical yield was 72 ± 8 % (n = 12) and the radiochemical purity >95 %. The collected fraction containing [131I]-I2-PARPi was concentrated to dryness under reduced pressure.The dried radiolabeled [124I]-I2-PARPi. In this case, the radiochemical yield was 68 ± 5 % (n = 5) and the purity >95 %.The same procedure was followed for the synthesis of -I2-PARPi in 200 μL of solution PBS/PEG300 (10:1). The blood was sampled from the saphenous vein at 5, 15, 30, 60, 120, and 240 min post injection. The blood was weighed and radioactivity was measured on a Wizard 2470 Automatic Gamma Counter . Measurements in counts per minute were calculated as the mean %ID/g. The blood half-life was calculated using Prism 6.0c .Blood half-life was determined by measuring the activity in serial blood samplings. Specifically, healthy female nude mice , or injected with saline alone. One hour post injection, the mice were sacrificed and the tumors were resected and imaged with the IVIS spectrum fluorescence imaging system  using Living Image 4.4 software. The tumors were also imaged microscopically with the 5Live fluorescent confocal microscope using the 488 nm laser for PARPi-FL excitation.To verify the specificity of tumor uptake of I2-PARPi in vivo, the level of blocking of the fluorescent PARP1 inhibitor on a macroscopic and microscopic scale was determined. Nude mice bearing subcutaneous U87 MG tumors were injected with either the fluorescent PARP1 inhibitor PARPi-FL alone , PARPi-FL 30 min after a pre-injection of a 50-fold excess of I2-PARPi  and then vigorously vortexed for 30 s to precipitate out serum protein. The sample was centrifuged at 3000 RCF for 3 min at 4 °C, and the supernatant was collected. This procedure was repeated three times, and the combined supernatants were analyzed by HPLC equipped with radioactive detector , collecting samples every 30 s. Radioactivity of each fraction was measured on a Wizard 2470 Automatic Gamma Counter , and the blood stability was analyzed using Prism 6.0c .The in vitro stability was assessed by incubating 6 μCi [n = 21) bearing U87 MG or U251 MG subcutaneous xenografts. The radiolabeled small molecule preparation (30–20 μCi of [131I]-I2-PARPi in 200 μL of a solution 90 % PBS 10 % PEG300) was administrated via the lateral tail vein. To determine the optimal specific activity to achieve the highest tumor to organ ratio, various specific activities were tested  in mice bearing U87 MG tumors. The compound was allowed to circulate for 2 h post injection at which time the mice were sacrificed and organs were harvested (n = 3). After determining the optimal specific activity, the optimal time for imaging was determined by testing the drug distribution in nude mice bearing U87 MG tumors at different time points. The drug was allowed to circulate for various times , after which the mice were sacrificed (n = 3). The radioactive content in the tissue of interest  was measured on a Wizard 2470 Automatic Gamma Counter and the tissue-associated activity was calculated as the mean %ID/g.Biodistribution experiments were conducted on female nude mice  were orthotopically implanted in athymic nude mice, using a stereotaxic device, and the tumors were allowed to grow for approximately 4 weeks. Once tumors reached the sufficient size, the orthotopic U251 MG tumor-bearing mice were injected intravenously with 500 μCi [131I]-PARPi  alone or with a pre-injection of 15 μmol Olaparib  30 min prior to the injection of [131I]-PARPi. Additionally, healthy mice were also injected with 500 μCi [131I]-PARPi. After 2 h of circulation time after the [131I]-PARPi injection, the mice were sacrificed. Liver, tumor, muscle, and brain tissues were excised and embedded in O.C.T. compound  and frozen at −20 °C, and a series of 8 μm frozen sections was cut and mounted on microscope slides. To determine radiotracer distribution, digital autoradiography was performed by placing tissue sections in a film cassette against a phosphor image plate  for 48 h at −20 °C. Phosphor imaging plates were read at a pixel resolution of 25 μm with a Typhoon 7000IP plate reader . After autoradiographic exposure, the same frozen sections were then used for immunohistochemical staining. Areas of brain slides containing tumor tissue were identified using the H&E staining and then overlaid with the autoradiographic data. Intensity of tumor areas and non-tumor areas were then quantified using ImageJ 1.47u.U251 MG glioblastoma cells (5 × 10131I]-I2-PARPi in 200 μL PBS solution (10 % PEG300) via the lateral tail vein and then anesthetized with isoflurane mixed with medical air (2 % for induction and maintenance). Animals were placed in prone position, and scans were then performed 90 min after injection for 60 min using a SPECT/CT small animal imaging system . SPECT Images were reconstructed using HiSPECT software, and in vivo Scope software was used for CT image reconstruction.SPECT/CT was acquired in athymic nude mice (6–10 weeks old). Before administration of the radioiodinated tracer, in terms to block the thyroid, the animals were treated with an intraperitoneal injection of NaI  60 min previous to the injection of 450–600 μCi (145–210 mCi/μmol) [124I]-I2-PARPi in 200 μL PBS solution (10 % PEG300) via the lateral tail vein under isoflurane anesthesia (2 % for induction and 1.5 % for maintenance). Mice were also treated with an intraperitoneal injection of NaI , 60 min previous to the administration of the radioiodinated tracer. Animals were immediately placed in prone position under isoflurane anesthesia, and scans were then performed 90 min after injection for 30 min using the Inveon PET/CT imaging system . PET and CT Images were reconstructed using Inveon research workplace software.In the case of PET imaging, images were acquired after the injection of 200–250 μCi (110–170 mCi/μmol) -NaI and chloramine T in acetic acid . The resulting radioactive N-succinimidyl-4-(131I-iodo) benzoate was then conjugated to a PARP1 targeting 2H-phthalazin-1-one in the presence of HBTU and AcN at room temperature  and a radiochemical purity >95 %. HPLC chromatograms and mass spectrometry data are shown in Fig. n = 5) and a radiochemical purity >95 %.The preparation of the radiotracer 131I]-I2-PARPi was realized in two synthetic steps. First, the precursor 31I-I2-PAure Fig. . The cru131I]-I2-PARPi. We did not observe other major peaks, which would indicate small molecule metabolites, which confirmed the stability of the drug over the course of 120 min  showed only one main peak at 15 min, corresponding to the pure compound [131I]-I2-PARPi in healthy athymic nude mice  = 17.1 min  in U87 MG tumor-bearing mice . Similar to SPECT/CT, orthotopic U251 MG xenografts were clearly visualized non-invasively, whereas healthy mice showed negligible uptake of the tracer , which is close to the original Olaparib -I2-PARPi and [124I]-I2-PARPi, respectively  is characterized by aggressive malignant infiltrative growth and is associated with a dismal prognosis . CurrentOlaparib  and loweOlaparib , 27. Theely Fig. . The trasue Fig. . At the sue Fig.  showing sue Fig.  and 9, w124/131I]-I2-PARPi, was tested as a PET and SPECT tracer. Our data demonstrates the specific binding of our I2-PARPi tracer to PARP1 and illustrates the potential of this tracer for glioblastoma detection.In summary, we have designed, radiolabeled, and tested a library of iodinated PARP1 inhibitors, based on the high affinity of PARP1 to the 2H-phthalazin-1-one scaffold. The high tolerability of PARP1 for small molecules appended to 2H-phthalazin-1-one resulted in a number of high affinity binders in our small molecule library, the best representative of which, [


Notably, both the primary amino group and the cyano groups are involved in hydrogen-bonding interactions with DMF ligands to direct the assembly and stabilize the crystal packing.The title compound, [Cd(C 5H2N5)2(C3H7NO)2]n or [Cd(adci)2(DMF)2]n, the Cd2+ ion is located on a twofold rotation axis and is six-coordinated in a CdN4O2 manner by four imidazole N atoms of four symmetry-related 2-amino-4,5-dicyanoimidazolate (adci) anions in the equatorial plane and by two O atoms of symmetry-related N,N-dimethylformamide (DMF) ligands in axial positions. The adci− anions bridge adjacent Cd2+ ions [shortest Cd⋯Cd separation = 6.733 (3) Å] into a layered coordination polymer extending parallel to (001). The primary amino group and the non-coordinating cyano groups of adci− anions are involved in hydrogen-bonding interactions with DMF ligands to stabilize the crystal structure.In the title structure, [Cd(C The asyA⋯O1 hydrogen bonds between the primary amine group of adci− and the O atoms of an DMF ligand as well as C7—H7C⋯N5 interactions between the methyl C atoms of DMF and the non-coordinating N atoms of the cyano group of an adci− anion play a crucial role in directing and stabilizing the assembly of the supramolecular structure . The lengths of these three hydrogen bonds fall in or approach the range (3.2–4.0 Å) of weak hydrogen-bonding interactions up Fig. 3b. The let al., 19993 topology has been reported 6]Cl]}n 4(H2O)3]·3H2O3}n and [Co(IMDN)2(H2O)2]n  with chain structures. However, the coordination modes of the imidazoles in these complexes are different.The cyano groups of the dci ligands exhibit a strong electron-withdrawing effect. Consequently, the formation of anionic species is relatively straightforward  were mixed in 2 ml DMF. After stirring for 0.5 h, Cd(NO3)2·4H2O  in 6 ml methanol was added dropwise. The mixture was further stirred for another hour and then filtrated. The filtrate was kept at ambient temperature. After about three weeks, yellow block-shaped crystals of (I)−1): 3436, 3346, 2930, 2217, 1658, 1525, 1486, 1444, 1385, 1328, 1305, 1115, 675.Compound (I)d(C—H) = 0.93 Å for sp2-bound H atoms and Uiso(H) = 1.2Ueq(C), and d(C—H) = 0.96 Å for methyl H atoms and Uiso(H) = 1.5Ueq(C). H atoms of the amino group were located from a difference map and were refined with d(N—H) = 0.86 Å and Uiso(H) = 1.2Ueq(N).Crystal data, data collection and refinement details are summarized in Table 210.1107/S2056989015014516/wm5187sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989015014516/wm5187Isup2.hklStructure factors: contains datablock(s) I. DOI: 1416545CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


ALL1-fused from chromosome 1q (AF1q), originally considered as an oncogenic factor, has been implicated in neuronal development; however, its upstream regulatory mechanisms in neural system remained elusive.AF1q. The promoter assay identified a neuron-restrictive silencer element at −383 to −363 bp of human AF1q promoter. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (CHIP) confirmed the binding of REST to the NRSE in AF1q gene promoter. Additionally, the negative correlation between the expression levels of Af1q and Rest in mice neurodevelopment supported the negative regulation of AF1q by REST and the potential functions of AF1q in neurodevelopment.Our study showed that REST (RE1 silencing transcription factor), a key transcription factor in neurodevelopment, could down-regulate the gene expression of AF1q gene transcription through directly binding to a NRSE at −383 to −363 bp of AF1q promoter.These results demonstrate that REST regulates  AF1q gene was initially identified as a mixed lineage leukaemia (MLL) fusion partner from an infant acute myelomonocytic leukemia patient carrying the t translocation [AF1q mRNA levels had been shown in leukemic and immature hematopoietic cells [Af1q, homologue of human AF1q, was found to be significantly up-regulated during the neuronal production from neural stem/progenitor cells [The  t1;11)q1;q23 tra;11q1;q23ic cells –8, thougic cells . In addiic cells . The mouor cells , but theREST (RE1 silencing transcription factor) regulates embryonic and neural stem cells by affecting their derivatives –14 and pAF1q transcription could be down-regulated by REST. We also identified AF1q promoter sequence and demonstrated that REST was a key transcriptional factor participating in AF1q down-regulation via a NRSE site at −383 to −363 bp of human AF1q promoter. Furthermore, our study suggested that the expression of REST and AF1q were negatively correlated during neurodevelopment, implying that AF1Q may be involved in neurodevelopment.In the present study, we demonstrated that AF1q gene transcription was regulated by REST, the endogenous AF1q mRNA levels were determined by RT-PCR in cells transfected with REST expression plasmid pREST or its pSuper based silencing vector psiREST. The knock-down efficiency of psiREST was detected by real time PCR using SYBR Green gene expression assay. Silencing plasmid pSuper was performed as control. Real time PCR confirmed that REST expression was reduced by psiREST to 37.57 ± 3.81 % of controls , P7, P14 and adult. The real-time RT-PCR results showed that mRNA levels of Af1q and Rest were coordinately expressed with significant negative correlation during neurodevelopment  was obtained by PCR of genomic DNA isolated from BAC-human-rp11 using a pair of primers (5′-CGGCTAGCAGGTCTCCACCCTGTCCCTGC-3′ and 5′-CCCTCGAGTTCCCTCCACCCAGCTCTGGTC-3′). The first base of the first exon is referred as bp +1. Then they were cloned into pGL3basic vector  containing firefly luciferase reporter gene. Primers used to generate a series of promoter deletion plasmids were as follows: forward primers, 5′-CGGCTAGCGTCAGGAGTTCCAGACCAGC-3′ (pAF1qAluc), 5′-CGGCTAGCTGTAATCCCAGCTACTTGGG-3′ (pAF1qBluc), and reverse primers: 5′-CCCTCGAGCAGAAATGGCCTTGTTCTCT-3′. The pAF1qNRSEmut was constructed from pAF1QAluc in which the NRSE site 5′-TTAGCTGGGCGTGGTGGCGGA-3′ was mutated to 5′-TTAGCTGGaaGTctgGaaGGA-3′. All the constructs were verified by sequencing and restriction enzyme digestions. Human REST siRNA was generated using pSuper vector. The target sequence for human REST siRNA is GCTACAATACTAATCGATA. The two strands of RESTsuper sense (5′-gatccccGCTACAATACTAATCGATAttcaagagaTATCGATTAGTATTGTAGCttttta) and RESTsuper antisense (5′-agcttaaaaaGCTACAATACTAATCGATAtctcttgaaTATCGATTAGTATTGTAGCggg) were annealed and inserted into pSuper plasmid to generate the pSuper-REST construct. The vectors pREST were generated as previously described [The 5′-upstream region of human escribed .2. All transfections were carried out with lipofectamine™2000 transfection reagent (Invitrogen) according to the manufacturer’s instructions. Luciferase activity was determined as previously described [HEK293 cells were cultured as previously described [escribed .REST and AF1q was quantified by TOYOBO R SYBR Green gene Expression Analysis kit . Primers for real-time quantitative and semi-quantitative PCR were as follows: human AF1q (270 bp), forward, 5′-CCGCTCGAGGCCACCATGAGGGACCCTGTGAG-3′, and reverse, 5′-GGGGTACCGAGCAAGTCCAGTTCGAAG-3′; human REST (137 bp), forward, 5′-ACTCATCACGGAGAACGCCC-3′, and reverse, 5′-GAGGCCACATAACTGCACTG-3′; mouse Rest (244 bp), forward, 5′-CGAGTCTCAGGAAATTGATGA-3′, and reverse, 5′-GCCGTTACCCACTCACTAATAC-3′; human and mouse β-actin (141 bp), forward, 5′-GACAGGATGCAGAAGGAGAT-3′, and reverse, 5′-TGATCCACATCTGCTGGAAGGT-3′. All animal protocols were approved by Shandong University Institutional Animal Care and Use Committee and by the Institutional Ethics Committee on Animal Research of Qilu Hospital.Total RNA was isolated from mouse brains or cells using TRIzol reagent (Invitrogen). The mRNA of The sense sequences of AF1qNRSE, consensus NRSE and mutant AF1qNRSE were 5′-AAAGATTAGCTGGGCGTGGTGGCGGATGCCTGTA, 5′-TTCAGCACCACGGACAGCGCC, 5′-AAAGATTAGCTGGAAGTCTGGAAGGATGCCTGTA. EMSA and ChIP were performed as previously described . Chip-PCWestern blot was performed as previously described . Anti-REt test analysis.All experiments were repeated three to five times. In figures one representative picture was shown; quantifications were from three or five independent experiments. The values represent the mean ± SEM. The data were evaluated for statistical significance with Student’s


Several findings from cell culture and mouse experiments suggest intercellular α-syn transfer.in vitro. A single exposure to amyloid fibrils of human α-syn was sufficient to induce aggregation of endogenous α-syn in human neuroblastoma SH-SY5Y cells. Remarkably, endogenous wild-type α-syn was sufficient for the formation of these aggregates, and overexpression of the protein was not required.Through a methodology used to obtain synthetic mammalian prions, we tested whether recombinant human α-syn amyloids can promote prion-like accumulation in neuronal cell lines Our results provide compelling evidence that endogenous α-syn can accumulate in cell culture after a single exposure to exogenous α-syn short amyloid fibrils. Importantly, using α-syn short amyloid fibrils as seed, endogenous α-syn aggregates and accumulates over several passages in cell culture, providing an excellent tool for potential therapeutic screening of pathogenic α-syn aggregates. Each sample analysis was performed in fifteen replicates. Each well contained one 3-mm glass bead (Sigma). The plate was covered with sealing tape (Fisher Scientific), incubated at 37°C under continuous shaking, and read on SpectraMax M5 fluorescence plate reader (Molecular Devices) by top fluorescence reading every 5 min at excitation of 444 nm and emission of 485 nm.http://www.imagemet.com) software.AFM analysis was performed in accordance with the method previously described . Three tThe α-syn amyloid solution was transferred from the 96-well plate into a 1.5 mL Eppendorf tube and collected under sterile conditions. The solution was ultracentrifuged at 100,000 g for 30 min at 4°C (Beckman Coulter). Pellets were resuspended in 1X PBS and then sonicated (Branson 2510) for 5 min prior to adding to the cultured cell plate.Three hundred μg of α-syn amyloids was added to GT1 and SH-SY5Y cell plates (10 cm-plate) and exposed in the cell culture media for 7 days before the next splitting and media change. Cells were split and maintained for six additional passages. Cell lysates were collected at each passage for Western blotting and immunofluorescence studies. For immunofluorescence of amyloids internalization, cells were cultured in 12-well plates with coverslips; 30 μg of α-syn amyloids was added to the cell culture media and incubated for 7 days.The total protein content of samples was measured by bicinchoninic acid assay (BCA) (Pierce). Fifty μg of cell lysates protein was used and 5X loading buffer was added in a 1:5 ratio. The samples were boiled for 5 min at 100°C, loaded onto a 10% Tris-Glycine SDS-PAGE gel, and transferred overnight onto Immobilon P PVDF membranes (Millipore). Membranes were blocked by 5% nonfat milk, incubated with 0.4 μg/mL rabbit polyclonal anti-α-syn antibody (Santa Cruz) followed by incubation with goat anti-rabbit IgG F(ab)2 fragment conjugated with horseradish peroxidase. Blots were developed with the enhanced chemiluminescent system  and visualized on Hyperfilm (Amersham Biosciences). To analyze α-syn aggregation in cell samples, cells were scraped in TBS buffer containing 1% Triton X100, protease cocktail inhibitors and phosphatase inhibitor. After sonication (Sonicrep 150) with 10 amplitude microns for 3 times (30 seconds of sonication and 30 seconds intermediate stop) cells were centrifuged at 2000 RPM for 5 min before the protein was quantified. Fifty μg of sample was centrifuged at 100,000 g for 30 min. Supernatant (in term S-TX) was collected and the pellets were resuspended in 2% SDS. Samples in 2% SDS were centrifuged, supernatant (S) and pellet (P) were collected. All the fractions were added to loading buffer 5X in ratio 1:5 and prepared similarly for Western blotting.Total RNA was extracted from cultured cells using the Trizol® Reagent (Life Technologies) extraction method. From 1 μg of total RNA, cDNA was synthesized using 200 units MoMuLV-reverse transcriptase , oligo (dT) primers and 1 μl 10 mM dNTP Mix in a final volume of 13 μl. Ten microliters of the 25-fold diluted solution was subjected to quantitative PCR analysis. The following primer pairs were used for α-syn: Hu_Syn_FW 5′-ATGGATGTATTCATGAAA-3′, Hu_Syn REV 5′-TTAGGCTTCAGGTTCGTA-3′. PCR amplification was conducted using the Phusion® High-Fidelity DNA Polymerase protocol, under the following conditions: initial denaturation at 98°C for 30s, 10s at 98°C, 30s at 45°C and 30s at 72°C for 25 cycles, followed by 10 min at 72°C. After amplification, 10 μl aliquots were electrophoresed in 1.5% agarose gel, followed by photographic recording of the gel stained with ethidium bromide.Cells on coverslips were washed with PBS and fixed with 4% paraformaldehyde, then washed twice, 15 min/time with PBS and blocked in blocking buffer [5% normal goat serum (NGS) in PBS + 0.3% Triton] for 1 hour. For Thioflavin S (ThS) staining, fixed cells were incubated with 0.025% of the fluorophore (Sigma) for 8 min and washed three times with 80% ethanol for 5 min each time, prior to the antibody incubations. Fluorescence immunocytochemistry was performed using the primary and secondary antibodies listed in Additional file All dopaminergic neuroblastoma SH-SY5Y cells were grown on ibidi dishes  for microscopy. After washing in PBS, cells were fixed using 4% (w/v) paraformaldehyde (Sigma) in PBS for 15 min and permeabilized with 0.01% Triton X-100 (Sigma) in PBS for 3 min, washed and blocked in 2% Bovine serum albumin (Sigma) in PBS for 20 minutes. Cells were immunostained using anti-α-syn  and anti-phospho S129 α-syn (Abcam) followed by secondary antibody coupled to Alexa 488 (Invitrogen). Cells were mounted in Aqua Poly/Mount (Polysciences) and pictures were acquired using white field Axiovision microscope (Zeiss) with a 63x objective. Quantification experiments were carried out independently at least three times; more than 150 cells were counted for each condition. Individual differences were assessed using individual student’s t-tests in GraphPad Prism software . Data are shown as mean ± standard error of the mean (SEM).α-syn: α-synuclein; LBs: Lewy bodies; PD: Parkinson disease; AD: Alzheimer disease; DLB: Dementia with lewy bodies; MSA: Multiple system atrophy.The authors declare that they have ho competing interests.SA and TTNL contributed to design the study, performed most experiments, analyzed the data, performed the statistical analysis, and helped draft the manuscript. SC performed the AFM experiments and analyzed the data. SAb performed the P-S129 immunofluorescence experiment. LC and CZ contributed to the design and coordination of the study. FM and FT helped conceive the study, participated in its design and coordination and helped draft the manuscript. SG provided the human cell lines used in this work. GL conceived, designed and coordinated the study, analyzed the data, and drafted the manuscript. All authors read and approved the final manuscript.Plasmids used for expressing human α-syn and FLAG-human α-syn in E. coli BL21(DE)3. (A) Homology comparison between mouse α-syn and human α-syn, (B) highlighting amino acid substitutions. (C) Typical chromatogram obtained from human α-syn protein purification by anion-exchange chromatography with HiTrap Q Sepharose Fast Flow column. The protein was eluted with a 0–0.5 M NaCl gradient in 20 mM Tris pH8.0. Fractions B7-B2 correspond to purified protein. (D) Expression of recombinant human α-syn protein, 15% SDS-PAGE. Lane 1 and 5, molecular mass marker; lane 2, whole cell extract before IPTG induction; lanes 3 and 4, cell extract after IPTG induction: 5 hours (lane 3), overnight induction (lane 4); lane 6, purified α-syn protein.Click here for fileSummary of dimensions of the three α-syn amyloid preparations.Click here for fileInfection of the transfected SH-SY5Y cells with α-syn short amyloid fibrils and sub-passing over time. The SH-SY5Y cells that stably overexpress human wild-type α-syn (SH-OE) were infected with recombinant human FLAG-α-syn short fibrils. Cells were cultured on coverslips for each passage (P0 to P6). The deposition of exogenous short amyloid fibrils FLAG-α-syn (red) was detected by anti-FLAG antibody. Human endogenous α-syn detected by anti-human α-syn (C-20)-R antibody (green). The nuclei were stained with DAPI (blue). Bar, 12 μm. Corrected total cell fluorescence (CTCF) from immunofluorescence imaging shows the induction of endogenous α-syn in SH (neuroblastoma SH-SY5Y cell line) infected with human α-syn short amyloid fibrils during the passages (bottom panel). The analysis was performed on at least 150 cells, n = 3, ***p < 0.005. Values are mean ± SD.Click here for fileThioflavin-S (ThS) positive staining of endogenous α-syn aggregates in stably transfected SH-SY5Y cell line. The SH-SY5Y cells that overexpress wild-type human α-syn (SH-OE) were infected with recombinant human α-syn short amyloid fibrils (SF: short amyloid fibrils of α-syn) and sub-passaged at sixth passage p6). The deposition and level of α-syn (red) in α-syn-infected cell lines after six passages were detected by anti-α-syn antibody  induces the same behavior. (A) Immunofluorescence images show the induction of endogenous mouse α-syn in GT1 cells after infection with human α-syn short amyloid fibrils over four passages.The detection of exogenously added short amyloid fibrils was performed using an anti human α-syn antibody [LB 509] (red). Mouse endogenous α-syn was detected by anti mouse α-syn antibody D37A6 (green). The nuclei were stained with DAPI (blue). Bars of Ctrl, p0 and p1 are 12 μm; bars of p2, p3, p4 are 24 μm. The graph shows the analysis of the relative corrected total cell fluorescence (CTCF) of endogenous α-syn in GT1 cells infected with human α-syn short amyloid fibrils during the passages. The analysis was performed on at least 150 cells, n = 3, ***p < 0.005. Values are mean ± SD. (B) ThS staining of endogenous aggregated in GT1 cells. (C) Western blotting of cell lysates exposed to anti-α-syn antibody; SFp5: human α-syn short amyloid infected cells at fifth passage.Click here for fileAntibodies used in this study. Click here for file


The cyclohexenone ring in the title compound adopts an envelope conformation and in the crystal, molecules are linked by N—H⋯O and C—H⋯N hydrogen bonds, forming ribbons of edge-fused rings propagating along [010]. 19H17Cl3N2O3, has been prepared in a cyclocondensation reaction between 2,3,5-trichlorobenzaldehye and 4-acetyl-2-methyl-1H-imidazole. The cyclohexenone ring adopts an envelope conformation with the C atom substituted by the trichlorophenyl ring as the flap. The mutually trans ester and aryl substituents both occupy equatorial sites. In the crystal, a combination of N—H⋯O and C—H⋯N hydrogen bonds links the molecules into ribbons of edge-fused centrosymmetric rings, which enclose R22(14) and R44(16) alternate ring motifs, propagating along the b-axis direction.The title compound, C The maximum deviation from the mean plane through atoms (C1–C5) is 0.023 (2) Å for atom C4, with an r.m.s. deviation of 0.0144 Å, and with the flap atom C6 displaced by 0.684 (3) Å.The central cyclohexenone ring (C1–C6), has puckering parameters of trans to one another and both occupy equatorial sites  chains, and inversion-related pairs of such chains are linked by C—H⋯N hydrogen bonds, forming ribbons or molecular ladders of edge-fused centrosymmetric rings, in which n + 1/2, 1/2, 1/2) alternate with n, 1/2, 1/2); where n represents an integer in each case se Fig. 3. There aet al., 2008et al., 2008et al., 2011abcet al., 2012et al., 2015R,6S) and  forms, with mutually trans substituents at the sites corresponding to atoms C1 and C6 in compound (I)trans disposition of the substituents at sites C1 and C6. Of particular interest is the structure of methyl -4-(4-chlorophenyl)-6-[4-(propan-2-yl)phenyl]-2-oxocyclohex-3-ene-1-carboxylate, which exhibits enantiomeric disorder where the reference site contains both  and  forms with occupancies of 0.923 (3) and 0.077 (3), respectively H-imidazol-4-yl)-3-prop-2-en-1-one (A), aqueous sodium hydroxide solution  was added to a mixture of 2,3,5- trichlorobenzaldehyde (0.02 mol) and 4-acetyl-2-methyl-1H-imidazole (0.02 mol), and the mixture was stirred at 275 K for 3 h. The resulting solid product was collected by filtration and recrystallized from ethanol. For the synthesis of the title compound, (I)A  and ethyl 3-oxobutanoate  in methanol (30 cm3) containing aqueous sodium hydroxide  was heated under reflux for 10 h. The reaction mixture was then cooled to ambient temperature and the resulting solid product (I)The synthesis of the title compound is illustrated in Fig. 1Uiso(H) = 1.2Ueq(N), giving an N—H distance of 0.79 (3) Å. The C-bound H atoms were subsequently treated as riding atoms in geometrically idealized positions: C—H distances 0.93–98 Å with Uiso(H) = 1.5Ueq(Cmethyl) and 1.2Ueq(C) for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989015023245/su5251sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989015023245/su5251Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989015023245/su5251Isup3.cmlSupporting information file. DOI: 1440150CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


E)-1-(5-chlorothiophen-2-yl)-3-(2-methylphenyl)-prop-2-en-1-one (I) are linked into simple hydrogen-bonded C(5) chains, while three distinct conformers of (2E)-1-(anthracen-9-yl)-3-[4-(propan-2-yl)phenyl]prop-2-en-1-one (II) co-exist in the crystal but each conformer forms hydrogen-bonded C(8) chains, albeit of two different types.Molecules of  chains. Compound (II), C26H22O, crystallizes with Z′ = 2 in space group P-1; one of the molecules is fully ordered but the other is disordered over two sets of atomic sites having occupancies 0.644 (3) and 0.356 (3). The two disordered components differ from one another in the orientation of the isopropyl substituents, and both differ from the ordered molecules in the arrangement of the central propenone spacer unit, so that the crystal of (II) contains three distinct conformers. The ordered and disordered conformers each form a C(8) chain built from a single type of C—H⋯O hydrogen bond but those formed by the disordered conformers differ from that formed by the ordered form.In the crystal of compound (I), C R1—C(=O)—CH=CH—R2, are versatile intermediates in synthesis -1-(5-chlorothiophen-2-yl)-3-(2-methylphenyl)-prop-2-en-1-one (I)E)-1-(anthracen-9-yl)-3-[4-(propan-2-yl)phenyl]prop-2-en-1-one (II)Chalcones, et al., 2005et al., 2005et al., 2014In the molecule of compound (I)Z′ = 2 in space group PCompound (II)11 Fig. 2 is fully11 Fig. 2 has occu11 Fig. 2 has occun19—Cn1—Cn2—Cn3 are 11 (3)° and 12 (5)° for n = 2 and 3 respectively, corresponding to a rotation of approximately 180° about the bond Cn1—Cn2 in conformers 2 and 3 as compared with conformer 1. In addition, in conformers 2 and 3 the torsional angles Cn33—Cn34—Cn37—Hn37 are −14° and −170° for n = 2 and 3, respectively, so that the orientation of the CHMe2 group in these two forms differs by a rotation of approximately 180° about the bond Cn34—Cn37; the corresponding value in conformer 1 is ca 162°. Hence three different conformations of compound (II)cf. Figs. 2In the fully ordered molecule containing atom O11 the torsional angle C119—C11—C12—C13 is 177.72 (16)° whereas in the two disordered components containing atoms O21 and O31 the values of the corresponding torsional angles Cn19,Cn1,Cn2,Cn3,Cn31 is effectively planar with r.m.s. deviations from the mean planes of 0.011, 0.036 and 0.043 Å for n = 1–3, respectively. This spacer unit makes dihedral angles with the central ring of the anthracene unit of 67.70 (11), 65.7 (5) and 71.7 (10)° for n = 1–3, respectively, and the corresponding dihedral angles with the adjacent aryl rings Cn31—Cn36 are 6.26 (18), 1.5 (11) and 7(2)°, respectively. These values confirm that the principal difference between conformer 1 and conformers 2 and 3 is simply a rotation about the bond Cn1—Cn2.In each conformer, the central spacer unit encompassing atoms Cn11—Cn12, Cn13—Cn14, Cn15—Cn16 and Cn17—Cn18 are very much shorter than the other C—C bonds in these units, while the C—C distances in the central rings show rather little variation. These observations are consistent with an electronic structure for the anthracene units where a central ring displaying aromatic delocalization is flanked by two isolated diene units  chain running parallel to the [001] direction  chains running parallel to the [100] direction d Table 1 to form on Fig. 5. In the on Fig. 5, but theon Fig. 6. In addion Fig. 6.et al., 2015et al., 2015E)-1-(5-chlorothiophen-2-yl)-3-(4-ethylphenyl)prop-2-en-1-one and (2E)-1-(5-bromothiophen-2-yl)-3-(4-ethylphenyl)prop-2-en-1-one, but in the isostructural compounds (2E)-1-(5-chlorothiophen-2-yl)-3-(4-ethoxyphenyl)prop-2-en-1-one and (2E)-1-(5-bromothiophen-2-yl)-3-(4-ethoxyphenyl)prop-2-en-1-one the molecules are linked by C—H⋯O hydrogen bonds to form simple C(7) chains, while C(5) chains are present in the structure of (2E)-1-(5-bromothiophen-2-yl)-3-(3-methoxyphenyl)prop-2-en-1-one. In the structure of -3, 3′--bis(1-(anthracene-9-yl)prop-2-en-1-one), which is related to compound (II)et al., 2015E)-3--1-(2-methoxyphenyl)prop-2-en-1-one w/v, 5 ml). The reaction mixture was then stirred at 301 K for 4 h, when the resulting solid product was collected by filtration, washed with cold water and dried. Crystals suitable for single-crystal X-ray diffraction were grown by slow evaporation, at ambient temperature, of a solution in acetone-dimethylformamide : m. p. 387–389 K. For the synthesis of compound (II)w/v, 15 ml) was added to a mixture of 4-isopropylbenzaldehyde  and 9-acetylanthracene  in ethanol (50 ml), and the resulting mixture was stirred at 278 K for 3 h. The resulting solid product was collected by filtration and recrystallized from ethanol solution: m.p. 369–371 K. Crystals suitable for single-crystal X-ray diffraction were grown by slow evaporation, at ambient temperature, of a solution in dimethylformamide.For the synthesis of compound (I)Uiso(H) = kUeq(C) where k = 1.5 for the methyl groups, which were permitted to rotate but not to tilt, and 1.2 for all other H atoms. The H atoms in the minor-disorder component were included in the refinement in calculated positions under exactly the same conditions. For compound (II)K = mean (Fo2)/mean (Fc2) for the group of 287 very weak reflections having Fc/Fc(max) in the range 0.000 < Fc/Fc(max) < 0.006. For compound (II)K, 10.808, for the group of 733 very weak reflections having Fc/Fc(max) in the range 0.000 < Fc/Fc(max) < 0.005.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989016011592/su5313sup1.cifCrystal structure: contains datablock(s) global, I, II. DOI: 10.1107/S2056989016011592/su5313Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989016011592/su5313IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989016011592/su5313Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989016011592/su5313IIsup5.cmlSupporting information file. DOI: 1494027, 1494026CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The equatorial positions are occupied by the N atoms of two 2,5-bis(pyridin-2-yl)-1,3,4-thiadiazole ligands, whereas the axial positions are occupied by N atoms of the azide anions. The thiadiazole and pyridine rings linked to the metal are almost coplanar, with a maximum deviation from the mean plane of 0.0273 (16) Å. The cohesion of the crystal is ensured by weak C—H⋯N hydrogen bonds and by π–π interactions between pyridine rings [intercentroid distance = 3.6356 (11) Å], forming a layered arrangement parallel to (001). The structure of the title compound is isotypic with that of the analogous nickel(II) complex .In the mononuclear title complex, [Co(N al. 2013. Acta Cr An interesting feature of the metal–ligand chemistry of these compounds is that the resulting complexes can be mononuclear 2(H2O)2]·2BF4, in which the water molecules are substituted by azide ions which at the same time neutralize the positive charge of Co2+  Å] and by weak C—H⋯N hydrogen bonds Table 1, formingL) was synthesized as described previously by Lebrini et al. (2005L2(N3)2] was synthesized in bulk quantity by dropwise addition with constant stirring at room temperature of an aqueous solution of NaN3  to an ethanol/water solution (1:1 v/v) of L  and CoCl2·6H2O . The red-coloured solid precipitated was filtered and washed with cold ethanol. Single crystals of the title compound suitable for X-ray data collection were obtained by slow interdiffusion of a solution of CoCl2·6H2O and L in acetonitrile into NaN3 dissolved in water. Red block-shaped single crystals appeared after one month. The crystals were washed with water and dried under vacuum (yield 60%). Analysis calculated for C24H16N14CoS2: C, 46.23; H, 2.59; N, 31.45 S, 10.28. Found: C, 46.42; H, 2.63; N, 31.35; S, 10.51.The ligand 2,5-bis(pyridin-2-yl)-1,3,4-thiadiazole  = 1.2 Ueq(C). Two outliers  I. DOI: 10.1107/S2056989015006544/rz5153Isup2.hklStructure factors: contains datablock(s) I. DOI: 1057234CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


H-1,2,4-triazol-1-yl)methyl]pyridine, (I), and the salt 1,1′-. The overall packing of structure (II) can be described as two-dimensional with the layers stacking parallel to the (001) plane. In the crystal, the predominant intermolecular interactions in (I) and (II) involve the acidic hydrogen atom in the third position of the triazole ring, with either the triazole N-atom acceptor in weak C—H⋯N hydrogen bonds in (I), or with halide counter-ions through C—H⋯I interactions, in (II).In the structures of the 2,6-bismethyl-substituted pyridine compound, C Herein, we report the structures of 2,6-bispyridine with methyl groups at the fourth nitrogen positions of the triazole rings, with mixed triiodide/iodide anions. This compound crystallizes in the space group C2/m with half of the dication, half of a triiodide (I1—I2—I3) and half of one iodide (I4) in the asymmetric unit it Fig. 2. The tria) the nitrogen atoms involved are in the fourth position of the triazole ring (C1—H1⋯N7 and C11—H11⋯N7), and b) the nitrogen atom is in the second position of the ring (C2—H2⋯N6). Pyridine nitrogen atoms, on the other hand, are involved as acceptors in hydrogen bonds arising from the methylene hydrogen atoms, forming a stack of one molecule on top of the other  (Table 1In compound (I)er Fig. 4, althoug) Table 1. The ovec-axis, the triiodide anion lies on the mirror plane in the middle of the dication-iodide units, filling up a pore-like groove within the structure  the meta-hydrogen atoms of the pyridine ring (C6—H6⋯I1), and b) the methylene hydrogen atoms (C4—H4B⋯I2) re Fig. 5. There are Fig. 5,  Fig. 7. The minH-Imidazol-1-yl){6-[(1H-imidazol-1-yl)methyl]-2-pyridyl}methane methyl]-1H-pyrrole methyl]-2-pyridyl}methyl)-1H-imidazole bromide methyl]pyridine and 1,2,4-triazole  were dissolved in 10–12 mL of acetonitrile by stirring. Once these had completely dissolved, K2CO3  was added and briefly stirred to deprotonate the triazole. 2,6-Bis(bromomethyl)pyridine  was then dissolved separately in 5 mL of acetonitrile. The two solutions were then combined in a 10–20 mL microwave vessel and placed in the microwave reactor for 15 min at 403 K, after which the acetonitrile was removed in vacuo. Compound (I)1H NMR  δ 8.23 , 7.98 , 7.69 , 7.12 , 5.44 . 13C NMR  δ 152.5, 144.0, 138.6, 121.8, 54.8.For the synthesis of compounds (I)H-1,2,4-triazol-4-ium) iodide and iodomethane  was added to a 10 mL acetonitrile solution of 2,6-bis[methyl]pyridine  in a microwave vial. The mixture was placed in the microwave reactor for 10 min at 413 K, after which the acetonitrile was removed in vacuo. Compound (II)1H NMR  δ 10.15 , 9.17 , 7.99 , 7.51 , 5.76 , 3.95 . 13C NMR  δ 152.88 (2C), 146.02 (2C), 144.24 (1C), 139.19 (2C), 122.97 (2C), 55.75 (2C), 34.55 (2C), 25.75 (iPrOH).For (II)Uiso(H) = 1.2Ueq(C). In compound (II)Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989014027881/zs2322sup1.cifCrystal structure: contains datablock(s) General, I, II. DOI: 10.1107/S2056989014027881/zs2322Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989014027881/zs2322IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989014027881/zs2322Isup4.cmlSupporting information file. DOI: 1040607, 1040608CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


AbstractSpinaethorax, whose proposal is based on specimens of Megalothoraxspinotricosus Palacios-Vargas & Sánchez, 1999, is given a new name combination and a redescription. The type species comes from two caves in Campeche State, México. A new combination is also suggested for Megalothoraxtonoius Palacios-Vargas & Sánchez, 1999. The new genus is similar to Megalothorax Willem, 1900 and Neelus Folsom, 1896, but it clearly differs from all genera within family Neelidae by a peculiar combination of characters and the presence of some new features, e.g. globular sensillum on Ant. III, sword-like macrosetae on oral fold. A comparative table and an identification key for all Neelidae genera as well as some summary tables of antennae chaetotaxy and legs setation for type species are provided.The new genus  Neelidae comprises five genera and 41 species in the world. However, only 9 species in 3 genera are known from Mexico , they have nor eyes or scales, however, their antennae are shorter than their heads. The main differences among the genera of this family are in the structure of forehead setation, antennae, sensory fields and furcula.The family m Mexico . This faAcanthoneelidus, with only one species from Europe, and Zelandothorax from New Zeland plus three cosmopolitan genera. Megalothorax is the most diversified with 28 species, Neelus with 6 species Neelides with 5, both widely spread, too. The new genus described herein has two species distributed in Mexican caves.Members of this family are cosmopolitan. Therefore, they have been found in various localities mainly associated with soil and litter at different altitudes from sea shore up to 3,000 m a.s.l. . They haNeelidae. The catalog by The most remarkable contribution on this family from Mexico is the work by Megalothoraxspinotricosus Palacios-Vargas & Sánchez, 1999, is based on original slides deposited in Facultad de Ciencias, UNAM. Specimens were obtained from samples of bat guano and soil that were processed by Berlese-Tullgren funnels and preserved in 75% ethylalcohol. Slides were mounted using Hoyer´s solution.The present redescription of Ti. I, II, III width (middle part)” that can be used as additional character, was also included.Body length was measured on slides excluding antennae and furcula. Lengths of unguis and unguiculus were measured between the most basal  point and the tip on their inner margins. Besides the common measurements, the ratio “unguis I, II, III (inner margin): Neelus revision  is applied according to We followed nomenclature used in last revision  for labr—antennal segmentAnt.; —thoracic segmentTh.; —abdominal segmentAbd.; —tibiotarsusTi.; —subcoxaescx; —sensory fields.f.; —free wax rod generating cryptwrc; —swollen sensillas1, s2, s3, s3´; —proximal part of densdp; —distal part of densdd; —Universidad Nacional Autónoma de MéxicoUNAM.Abbreviations: PageBreakTaxon classificationAnimaliaCollembolaNeelidaehttp://zoobank.org/9E27A3C3-9464-4A6A-94B8-5BB470502D2CNeelidae Folsom, 1896 with the following diagnostic characters:A genus of the Neelidae. Small size, about 0.6 mm. Color white. Tegumentary grain fine and uniform. Apex of head with sword-like spines, body with several such spines, mainly around sensorial fields. Ant. III and IV fused and Ant. III with small globular sensillum in proximal position. Anterior labral setae R1 and R2 thick, curved and smooth. Oral fold with 1+1 sword-like macrosetae. Basomedian field of labium furnished with 6+6 setae. Presence of 3 setae around abdominal sensory fields, no E3 spine/setae on dd. Mid abdomen with swollen sensilla s3 and s3´. Base of Abd. IV sternite with 1+1 neosminthuroid setae, smooth and with pointed tip.Habitus of Spinaethoraxspinotricosus , comb. n.Figs UNAM.Holotype: female mounted on slide. Original label: 23/00/1991, Mexico, Campeche, Cueva Xtancumbilxunaan (cave), 29.xii.1996, A. Ruíz and S. Aguilar col., ext. soil. Paratypes: 1 female on slide, 23.viii. 1991, J. G. Palacios col., direct collection and 2 juveniles on slides, the same data as the holotype. Type material deposited at Facultad de Ciencias, UNAM.Mexico, Campeche, Cueva Actún Guachapil (cave), 1 male, 1 female and 2 juveniles on slides. Original label: 14/iv/2012, 22.iii.1997, A. Ruiz and S. Aguilar col., ext. guano; 1 female on slide, 29.v. 1997, J. G. Palacios col., ext. guano. Material deposited at Facultad de Ciencias, 1 and R2 thick, curved and smooth. Oral fold with 1+1 sword-like macrosetae. Basomedian field of labium with 6+6 setae. Ant. III with small globular sensillum. Manubrium with 4+4 posterior setae. Mucro with both lamellae serrated and with rounded tip.Unpaired seta a0 between antennal basis. Labral chaetae RNeelidae. No pigmentation, cuticle finely granulated, linea ventralis without crossing with integumentary channels on ventral head back. Mid and hind Abd. with numerous spine-like microsetae, stouter spines around all sensory fields on body and apex of head.For lengths of different character see Table s.f. ordinary or slightly spine-like setae (20 µm); posterior part with 3+3 stouter spines IL1–IL3 of different lengths (28–42 µm) and 1+1 smaller axial spines IL4 (17–21 µm), others similar to smaller setae (10–14 µm). Labrum with 5,5,4 setae, 4 prelabrals. Pattern of labral setae . Anterior R1 and R2 slightly thick, smooth and curved, R2 (11 µm) longer than R1 (9 µm). Medial setae (m-row) equal (11 µm), smooth median setae in one line with others. Maxillary palp simple, with 1 enlarged terminal seta (18 µm), 1 basal seta (14 µm) and 1 sublobal hair  than others (10 µm); basolateral field with 1+1 setae (10 µm), oral fold with 1+1 basal setae (8 µm) and 1+1 terminal sword-like macrosetae (25–30 µm). Head with 3+3 smooth postmedian setae ventrally , 6+6 wax rods  with straight setae (3–4 µm) and 2+2 (wrc 3 and 5) with thicker and longer straight setae (6–7 µm). All wrc placed in small cuticular depressions. Trichobothria or their sockets not observed. Th. II with 3+3 ordinary setae (12–14 µm) and 6+6 stouter spines (25–36 µm) around thoracic sensory fields, axial spines smaller (25 µm); sensory fields at leg II base with 2+2 ordinary setae (20 µm), 3+3 stouter spines, anterior ones longer (45 µm) than posterior (26–28 µm), 1+1 lateral sensillum s1 (8 µm) broadened at tip and 1+1 swollen transparent rod with bifid tip and no base (8 µm) above s.f. of leg II. Th. III with 5+5 ordinary setae (6–8 µm), 4+4 wrc (wrc 1–4) and several small spine-like microsetae, whose overall number is not seen clearly; at leg III base with 1+1 ordinary seta (16 µm), 3+3 stouter spines, medial ones longer (42 µm) than lateral (22 µm) and 2+2 wrc . Anterior Abd. medially with 1+1 wrc 7 and 2+2 swollen sensilla s3 and s3´; hind Abd. with abdominal sensory fields, which are surrounded by 2+2 ordinary setae (12 and 18 μm), 1+1 stouter spines (30 µm), 1+1 swollen sensilla s2 and 1+1 wrc 8 above Abd. s.f. Dorso and dorso-lateral anterior and hind abdomen covered with numerous spine-like microsetae (4–6 μm) arranged as in Figs Abd. tergum VI with 3+3 setae (17 µm) and 1 unpaired axial seta (14 µm). Anal complex with three anal valves, each with on seta (7 µm). Abd. VI sternum with 4+4 setae (18–20 µm) and 2+2 very small globular structures (1–2 µm) next to anal valve setae. Female genital plate , one seta more laterally and 1+1 distal setae (8 µm). Lateral part of Abd. IV sternum with 1+1 short and pointed neosminthuroid setae (6 µm)  Fig. .Ant. III and IV not separated . Ant. II with 1 medial seta and 5 apical setae arranged in a whorl. Ant. III organ consists of 2 transparent rods (7 µm), 1 leaf-like transparent sensillum Sg (12 µm) and spine-like seta (7 µm). Proximal part of Ant. III bears 1 globular sensillum Sg (4 µm). Ant. IV with 13 curved macrosensilla S finely blunt at tip (24–32 µm); subapically with 1 long and thick subapical sensillum Sy (16–18 µm) and with 1 thick shorter apical sensillum Sx broadened at tip (12–14 µm); Ant. IV organ like a tiny, hardly visible spine (5 µm); apically with curved setae a and sa apical in position (10 µm). Complete chaetotaxy of antennae provided in Table scx I: 1, 1, 3 (one spine); scx II: PageBreakPageBreak1, 1, 1; coxae: 1, 1, 2; trochantera: 4, 3, 3; femora: 8, 8, 8 and tibiotarsi: 13, 14, 13. Thin meso- or microsetae as in following numbers on leg I: femur with 2; on leg II: femur with 1; on leg III: trochanter with 1, femur with 1. For complete setation of legs see Table PageBreakPageBreakrespectively. Length ratio unguis I, II, III / ti. I, II, III width  = 1.8, 1.7, 1.5. Unguis I and III with three auxiliary lamellae la, lp, Bp, unguis II with only lp and Bp  and mucro: 56, 31, 71 and 74 µm, respectively. Manubrium with 4+4 setae posteriorly, lateral ones shorter (10–15 µm) than axial (16–20 µm). Dens in proximal part (dp) with 2+2 posterior setae, lateral ones (14 µm) shorter than those axial (22 µm); distal part (dd) apically with 2+2 broad, blunt lateral spines (9 µm) and 1 medial sharp spine (7 µm) on anterior side; with 2 external (E1–E2) posteriorly and 2 internal (J1–J2) spines , and 1 medial, subapical seta (18 µm). Mucro with serrated lamellae and rounded tip. Base of mucro furnished with small scale without base. Middle mucro width 7 µm.Appendages. ted Fig. . Length t tip 12– µm; Ant.ent rods  µm, 1 lert setae –10 µm. As.f. placed in depressions each with secretory rod (10–12 µm), i.e. blunt seta with basal part inserted on cuticle and placed in upper margin of the field. Following arrangement: (a) anterior and posterior field on head  each with secretory rod and 1 seta on margin (14–18 µm); (b) thoracic field  with secretory rod, 3 internal spines PageBreak(4 µm) arranged in triangle, 2 external marginal setae (12–14 µm) and 6 sword-like spines of different lengths (25–36 µm), 3 spines are in anterior position and 3 spines above s.f. in axial position; (c) fields at base of legs II and III  each with secretory rod, 2 internal spines (4 µm) in s.f. 4 and 1 internal spine (4 µm) in s.f. 5. S.f 4 with 2 marginal external setae (20 µm), 3 stouter sword-like spines, medial ones longer (45 µm) than lateral (26–28 µm), 1 lateral sensillum s1 broadened at tip (8 µm) and 1 swollen rod with split tip and without base (8 µm) above s.f. 4. S.f. 5 with 1 marginal external seta (16 µm), 3 stouter sword-like spines, medial ones longer (42 µm) than lateral (22 µm); (d) abdominal field  with secretory rod, 1 internal spine (4 µm), 2 marginal ordinary setae (12 and 18 μm), 1 sword-like spine above s.f. (30 µm) and 1 swollen sensillum s2 on the margin of s.f. (6 μm). Wrc 8 is located above Abd. s.f.Sensory fields Figs  and 15. Both sexes known.The genus is named after the spine-like setae on thorax and abdomen.Spinaethoraxspinotricosus is currently known from two caves and is putatively spread in the cave systems of Yucatán Peninsula, mainly in places with accumulation of bat guano or other type of rich organic material.The young have only one proximal seta on dens.Taxon classificationAnimaliaCollembolaNeelidaecomb. n.Ant. III., stouter spines on Th., fusion of Ant. III and IV, 3+3 setae around Abd. s.f., 2+2 setae on proximal part of dens and absence of E3 spine on distal part of dens. The main differences consist in setation of hind Abd. (numerous thickened macrosetae in Spinaethoraxtonoius comb. n. vs. numerous spine-like microsetae in Spinaethoraxspinotricosus comb. n.), in chaetotaxy of the apex of head (only one spine IL 1 in Spinaethoraxtonoius comb. n. vs. three spines IL 1–IL 3 in Spinaethoraxspinotricosus comb. n.), in the structure of tenent hairs on Ti. (more developed in Spinaethoraxtonoius comb. n. vs. shorter in Spinaethoraxspinotricosus comb. n.) and in setation around Abds.f. . Species description is based only on one adult specimen (holotype) and does not allow us to describe it in an appropriate way, along with drawings and measurements.This species, described from a cave in State of Guerrero, shares with new type species of the genus similar generic characters like presence of sword-like macrosetae on oral fold, globular sensillum on Neelidae was for a long time an overlooked group of Collembola, mainly due to its small size and lack of diagnostic characters. In spite of the foregoing facts recent years molecular phylogenetic analysis have revealed unexpected diversity within this family . Soon after additional new taxonomical characters in genus Megalothorax were defined , because some features  indicated that those specimens belonged to this genus. On the other hand, some characters link those specimens to Neelus . Megalothoraxspinotricosus clearly differs from the other Megalothorax by the presence of great number of microsetae on Abd. and pointed out that Megalothoraxspinotricosus deserved its own genus. On the basis of recent diagnostic features, material of these two species was re-examined arriving to the same conclusion as Neelidae. Spinaethorax gen. n. differs from other genera by striking morphological features and combinations, which clearly separate them, e.g. sword-like macrosetae on oral fold, six setae on basomedian field of labium, Ant. III and IV fused, presence of small globular sensillum Sg on Ant. III, three marginal setae around abdominal sensory field, absence of spine E3 on dd and dp with two seate. For comparison with other genera see Table  The family s family . Recentl revised  as well  defined . SpinaetThe identification key is based on that of


In the crystal, molecules are linked by C—H⋯O hydrogen bonds and C—H⋯π interactions, forming double-chains parallel to the  5H5)(C34H28N3O)], the four-fused-rings system of the 11H-indenoquinoxaline unit is approximately planar [maximum deviation = 0.167 (4) Å] and forms a dihedral angle of 37.25 (6)° with the plane of the benzene ring of the methylbenzoyl group. Both pyrrolidine rings adopt a twist conformation. An intramolecular C—H⋯O hydrogen bond is observed. In the crystal, molecules are linked by C—H⋯O hydrogen bonds and weak C—H⋯π interactions, forming double chains extending parallel to the c axis.In the title compound, [Fe(C In the fused pyrrolidine system, both five-membered rings adopt a twist conformation, as indicated by the puckering parameters . Single crystals suitable for the X-ray diffraction analysis were obtained by slow evaporation of the solvent at room temperature.Ninhydrin (1 mmol) and 1,2-phenylenediamine (1 mmol) were mixed and stirred with methanol (10 ml) for 10 min. To this mixture, proline (1 mmol) and 1-ferrocenyl-3-(4-methylbenzoyl)prop-2-ene dipolarophile (1 mmol) were added and refluxed up to the end of the reaction as observed by thin-layer chromatography. The solvent was removed from the mixture under reduced pressure and the crude product was obtained using column chromatography. The crude extract was purified by petroleum ether and ethyl acetate (4:1 Uiso(H) = 1.5Ueq(C) for methyl groups or 1.2Ueq(C) otherwise. DELU restraints were applied to atoms C24 and C25.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S1600536814017644/rz5131sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S1600536814017644/rz5131Isup2.hklStructure factors: contains datablock(s) I. DOI: 1017369CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the cations, a Co/P/N/P four-membered slightly bent metallacycle is the key structural element. The pendant NH group is not coordinated to the CoI atom, which displays a distorted trigonal–bipyramidal coordination geometry. Weak interionic hydrogen bonds are observed between the NH groups and a carbonyl group of the tetrahedral [Co(CO)4]− anions.The asymmetric unit of the title compound, [Co(C N-(diphenylphosphanyl)-N,N′-diisopropyl-diaminophenylphosphine, Ph2PN(i-Pr)P(Ph)N(H)i-Pr, with the cobalt precursor Co2(CO)8 was performed to prepare a noble-metal-free catalyst for light-driven water reduction to produce hydrogen. These compounds are attractive in terms of environmental acceptability as well as for economic reasons. Several very active intermolecular water-reduction systems using 3d metal complexes as catalytically active centres are known, examples include work on iron 6(PNPNH) 6(PNPNH) and therefore the IR and NMR spectra were measured only from freshly prepared samples.Reaction of the PNPNH ligand P21/n with eight cations, eight anions as well as two molecules of toluene in the unit cell. The toluene solvent molecules are found to be disordered about inversion centres. The asymmetric unit is shown in Fig. 1I atom is fivefold coordinated by three carbonyl ligands and the PNPNH ligand, which is bound via both P atoms  and 14.44 (9)°]. The terminal secondary amine is not involved in complexation with the CoI atom and acts as a spectator group. In the cyclic units the following bond lengths are observed: Co1—P1 2.1948 (7), P1—N2 1.698 (2), N2—P2 1.695 (2), P2—Co1 2.1800 (7), Co2—P3 2.1884 (7), P3—N4 1.695 (2), N4—P4 1.702 (2), P4—Co2 2.1971 (7) Å. A similar coordination mode was previously found for this ligand in a variety of transition metal complexes –1.770 (3) Å and are comparable to those observed for a range of complexes displaying tetracarbonylcobaltate anions (vide supra), including ionic salts of tetracarbonylcobaltate with alkali 4]  anions Table 1. Other tet al. -N,N′-diisopropyl-diaminophenylphosphine was synthesized by a literature method 8 was purchased from Strem and used without further purification. Toluene was dried over CaH2 and distilled prior to use. Synthesis: A solution of Co2(CO)8  in toluene (10 ml) was added to N-(diphenylphosphanyl)-N,N′-diisopropyl-diaminophenylphosphine, Ph2PN(i-Pr)P(Ph)N(H)i-Pr  in a glove box. After gas evolution subsided, the 50 ml Schlenk flask was closed and heated to 383 K for 35 min without stirring to preserve the two-phase system. After crystallization from toluene at room temperature for three days, two crystal fractions were separated from the solvent and washed with n-hexane (2 × 5 ml). The fraction of cubic brown crystals showed space-group and lattice parameters identical to X-ray diffraction data published previously : δ (p.p.m) 61.8 , 59.9 ; IR : ν−1 [cm−1] 3335 (w), 3191 (w), 3058 (w), 2975 (m), 2869 (m), 2081 , 2021 , 1979 (w), 1872 , 1586 (w), 1462 (w), 1436 (m), 1390 (w), 1369 (w), 1311(w), 1165 (w), 1125 (m), 1097 (m), 1064 (m), 999 (w), 896 (m), 869 (m), 747 (m), 716 (w), 694 (m), 631 (w), 612 (w), 550 (s), 501 (m), 426 (m).General: d(C—H) = 0.95–1.00 (CH) and 0.98 Å (CH3) and refined using a riding model with Uiso(H) fixed at 1.2 Ueq(C) for CH and 1.5 Ueq(C) for CH3. The ring of the half-occupied toluene molecule was constrained to resemble an ideal hexagon with C—C distances of 1.39 Å. SADI instructions were used to improve the geometry of one phenyl ring  and one i-propyl group .Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S1600536814024908/rz5139sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S1600536814024908/rz5139Isup2.hklStructure factors: contains datablock(s) I. DOI: 1033917CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


X—H⋯N hydrogen bonds links the ions in (I) into a ribbon of alternating centrosymmetric In each of the title compounds, the anion shows evidence of extensive electronic delocalization. A combination of N—H⋯N and  10H9N2+·C9H5N4O−, (I), the ethyl group in the anion is disordered over two sets of atomic sites with occupancies 0.634 (9) and 0.366 (9), and the dihedral angle between the ring planes in the cation is 2.11 (7)°. The two independent C(CN)2 groups in the anion make dihedral angles of 10.60 (6) and 12.44 (4)° with the central propenide unit, and the bond distances in the anion provide evidence for extensive electronic delocalization. In bis 1,1,3,3-tetracyano-2-(dicyanomethylene)propane-1,3-diide [alternative name bis tris(dicyanomethylene)methanediide], 2C10H9N2+·C10N62− (II), the dihedral angles between the ring planes in the two independent cations are 7.7 (2) and 10.92 (17)°. The anion exhibits approximate C3 symmetry, consistent with extensive electronic delocalization, and the three independent C(CN)2 groups make dihedral angles of 23.8 (2), 27.0 (3) and 27.4 (2)° with the central plane. The ions in (I) are linked by an N—H⋯N hydrogen bond and the resulting ion pairs are linked by two independent C—H⋯N hydrogen bonds, forming a ribbon containing alternating R44(18) and R44(26) rings, where both ring types are centrosymmetric. The ions in (II) are linked by two independent N—H⋯N hydrogen bonds and the resulting ion triplets are linked by a C—H⋯N hydrogen bond, forming a C21(7) chain containing anions and only one type of cation, with the other cation linked to the chain by a further C—H⋯N hydrogen bond.In 2,2′-bipyridin-1-ium 1,1,3,3-tetracyano-2-ethoxyprop-2-en-1-ide, C Thus such anions readily form binary complexes with transition-metal and ternary complexes in which a transition-metal centre is also coordinated by other bridging or chelating ligands, and such materials exhibit interesting magnetic properties  phenomenon (Dupouy Compound (I)cf. Wood et al., 2009In none of the cations are the two rings exactly parallel: the dihedral angle between the mean planes of the two rings in the cation of compound (I)et al., 1987A)–(F) (see scheme below) all playing a role in the overall electronic structure. Accordingly, the N—H⋯N hydrogen bond linking the two ions within the selected asymmetric unit of (I)et al., 19942 units are twisted out of the plane of the central C3O core in a conrotatory fashion, and the dihedral angles between the planes of the C(CN)2 units and that of the central core are 10.60 (6)° and 12.44 (4)° respectively for the two units containing atoms C31 and C33.In the anion of compound (I)2 units is rotated out of the plane of the central four-atom core, with dihedral angles between the planes of these three units and that of the central core of 23.8 (3), 27.0 (3) and 27.4 (2)°, respectively, for the C(CN)2 units containing atoms C51, C52 and C53. These rotations are in a concerted sense, giving approximate molecular, but not crystallographic, symmetry of D3 (32) type for the anion. Although the bond distances involving the cyano substituents show some variations s Table 4 the appret al., 1995n − ½, n, n − ½) alternate with n, n + ½, n), where n represents an integer in both cases  make a dihedral angle of 2.11 (7)°: the ring-centroid separation is 3.7395 (8) Å and the shortest perpendicular distance from the centroid of one ring to the plane of the other is 3.3413 (5) Å, corresponding to a ring-centroid offset of ca 1.65 Å, so that there is only a very modest overlap of the two rings in question if Fig. 3. The comes Fig. 3. A singlon Fig. 4. If thison Fig. 4 into a sc-glide plane at y = 0.75 are linked by a C—H⋯N hydrogen bond (Table 2Despite the presence of three independent ions in the structure of compound (II)d Table 2, formingon Fig. 5. This chH-imidazole)copper(II) cation Pbcn, so that while two of the twofold rotation axes are only approximate, the third is a crystallographic axis. As in compound (II)2O)6]2+ 6]2+  were prepared using published methods . For the synthesis of salt (I)v/v, 15 cm3) and then held in the autoclave at 393 K for 3 d. After slowly cooling to room temperature, pale-orange crystals of (I)2tcpd  instead of K(tcnoet), giving yellow crystals suitable for single-crystal X-ray diffraction (yield 40%).The salts K(tcnoet) and KUiso(H) = 1.2Ueq(C): for H atoms bonded to N atoms, the atomic coordinates were refined with Uiso(H) = 1.2Ueq(N), giving the N—H distances shown in Tables 1Uiso(H) = 1.5Ueq(C) for the methyl groups, which were permitted to rotate but not to tilt, and C—H distances of 0.99 Å with Uiso(H) = 1.2Ueq(C) for the CH2 groups.Crystal data, data collection and structure refinement details are summarized in Table 510.1107/S2056989015007306/hb7404sup1.cifCrystal structure: contains datablock(s) global, I, II. DOI: 10.1107/S2056989015007306/hb7404Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989015007306/hb7404IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989015007306/hb7404Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015007306/hb7404IIsup5.cmlSupporting information file. DOI: 1059034, 1059033CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The central pyran rings (B) of compounds (I) and (III) adopt half-chair conformations, whereas that of compound (II) adopts a sofa conformation. The pyran rings (A) of the chromene ring systems of compounds (II) and (III) adopt half-chair conformations, while that of compound (I) adopts a sofa conformation. The mean plane of the central pyran rings (B) make dihedral angles of 70.02 (6), 61.52 (6) and 69.12 (7)°, respectively, with the mean planes of the chromene moieties (C+A) of compounds (I), (II) and (III). The bicyclic coumarin ring system (C+A+B+E) in compound (I) is almost planar (r.m.s. deviation = 0.042 Å). The carbonitrile side chain in compound (III) is very nearly linear, with the C—C≡N angle being 176.6 (2)°. The cyclohexene ring (E), fused with the central pyran ring (B) in compound (II) adopts a sofa conformation. In the molecular structures of compounds (II) and (III), there are C—H⋯O short contacts, which generate S(7) ring motifs. In the crystal structures of the title compounds, molecules are linked by C—H⋯O hydrogen bonds, which generate molecular sheets parallel to the ab plane, with R43(28) loops in (I), inversion dimers with R22(10) loops in (II) and chains along [010] with R22(12) ring motifs in (III). In the crystal structures of (I) and (III), there are also C—H⋯π interactions present, leading to the formation of a three-dimensional framework in (II) and to sheets parallel to (101) in (III).The title compounds, C A number of drugs containing chromene are used in the treatment of ailments such as hypertension, asthma, ischemia and urinary incontinence. Chromene derivatives are known to possess antitumor, antivascular  fused with a chromene ring system (C+A). The central pyran ring (B) is fused with a second chromene ring system (E+F) in (I)E) in (II)E) in (III)D) are attached to the central pyran ring (B), in adjacent positions, whereas in (III)B).The molecular structures of compounds, (I)B) adopt half-chair conformations with puckering amplitudes Q = 0.5166 (15) Å, θ = 51.22 (17), φ = 259.4 (2)° and Q = 0.486 (2) Å, θ = 128.3 (2), φ = 74.5 (3)°, respectively. In compound (II)B) adopts a sofa conformation . The cyclohexene ring (E) fused to the central pyran ring (B) in compound (II)Q = 0.497 (2) Å, θ = 109.8 (2), φ = 5.9 (2)°]. The pyran ring (A) of the chromene moiety adopts a half-chair conformation in compounds (II)Q = 0.4850 (14) Å, θ = 53.26 (17), φ = 271.70 (19)° and Q = 0.507 (2) Å, θ = 128.9 (2), φ = 92.7 (3)°, respectively] and a sofa conformation in compound (I)Q = 0.5130 (16) Å, θ = 57.83 (18), φ = 234.6 (2)°].In compounds (I)A – sofa conformation) of the chromene moiety is 14.95 (8)°, whereas in (II)A rings here have half-chair conformations). The decrease in the value of the dihedral angle in compounds (II)S(7) ring motifs. The second coumarin ring system (E+F) is almost planar with the dihedral angle between the pyran and benzene rings being 3.73 (7)°. Atom O4 deviates from the mean plane of this coumarin ring system by 0.111 (1) Å. The phenyl ring (D) is inclined to the mean plane of the central pyran ring (B), by 60.48 (8)°.In compound (I)B) makes dihedral angles of 22.63 (8) and 56.99 (9)° with the mean plane of the six-membered carbocylic ring (E) and the phenyl ring (D), respectively. Atom O3 deviates from the mean plane of ring (E) by 0.199 (1) Å.In compound (II)B) makes dihedral angles of 7.36 (9) and 58.24 (10)° with the pyrimidine (E) and ethyl-substituted benzene (D) rings, respectively. Atom O3 and the methyl group C atom, C16, deviate significantly from the mean plane of the pyrimidine ring (E) by 0.106 (1) and −0.107 (2) Å, respectively.In compound (III)ab plane ne Fig. 4. The shene Fig. 4.In compound (II)) Table 2.b-axis direction, enclosing In compound (III)fs Fig. 6. The chafs Fig. 6.c]chromene yielded 14 hits. The bond distances and bond angles in compounds (I)–(III) are in agreement with those in the reported structures. For example: compounds (I)et al., 2013B), OLEZIP : A mixture of (E)-methyl 2-[(2-formylphenoxy)methyl]-3-phenylacrylate  and 4-hydroxy-2H -chromen-2-one  was placed in a round bottom flask and heated at 453 K for 1 h. After completion of the reaction as indicated by TLC, the crude product was washed with 5 ml of ethylacetate and hexane mixture (1:49 ratio) which successfully provided compound (I)Compound (II): A mixture of (E)-methyl 2-[(2-formylphenoxy)methyl]-3-phenylacrylate  and cyclohexane-1,3-dione  was placed in a round bottom flask and heated at 453 K for 1 h. After completion of the reaction as indicated by TLC, the crude product was washed with 5 ml of ethylacetate and hexane mixture (1:49 ratio) which successfully provided the crude product of compound (II)Compound (III): A mixture of (E)-2-[(2-formylphenoxy)methyl]-3-(4-ethylphenyl)acrylonitrile  and 1,3-dimethylpyrimidine-2,4,6-trione  was placed in a round-bottom flask and heated at 453 K for 1 h. After completion of the reaction as indicated by TLC, the crude product was washed with 5 ml of ethylacetate and hexane mixture (1:49 ratio) which successfully provided pure compound (III)d(C—H) = 0.93, 0.96, 0.97 and 0.98 Å for aryl, methyl, methylene and methine H atoms, respectively, and with Uiso(H)= 1.5Ueq(C) for methyl H atoms and 1.2Ueq(C) for other H atoms.Crystal data, data collection and structure refinement details for compounds (I)10.1107/S2056989015012967/su5160sup1.cifCrystal structure: contains datablock(s) I, II, III, global. DOI: 10.1107/S2056989015012967/su5160Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989015012967/su5160IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989015012967/su5160IIIsup4.hklStructure factors: contains datablock(s) III. DOI: Click here for additional data file.10.1107/S2056989015012967/su5160Isup5.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015012967/su5160IIsup6.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015012967/su5160IIIsup7.cmlSupporting information file. DOI: 1410607, 1410606, 1410605CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


III luminophores, Eu(hfa)x(CPO)yand Eu(hfa)x(TCPO)y , were synthesizedby the complexation of EuIII ions with hfa moieties and CPO or TCPOligands. The thermal and luminescent stabilities of the luminophores are extremelyhigh. The decomposition temperature of Eu(hfa)x(CPO)y andEu(hfa)x(TCPO)y were determined as 200 and450 °C, respectively. The luminescence ofEu(hfa)x(TCPO)y under UV light irradiation was observedeven at a high temperature, 400 °C. The luminescentproperties of Eu(hfa)x(CPO)y andEu(hfa)x(TCPO)y were estimated from emission spectra,quantum yields and lifetime measurements. The energy transfer efficiency from hfamoieties to EuIII ions in Eu(hfa)x(TCPO)y was59%. The photosensitized luminescence of hyper-stableEu(hfa)x(TCPO)y at 400 °C isdemonstrated for future photonic applications.Novel organo-Eu There has been significant interest in the development of luminescent lanthanidematerials for use in devices such as fluorescent lamps1245678910101112III = Nd, Sm, Eu, Gd) andtris-(4-carboxylphenyl)phosphine oxide has a high decomposition temperature(500 °C)IIIluminophores (η < 1%). Thermostablelanthanide luminophores with effective photosensitization are expected to open up a newfield of luminescent material science. We have attempted to prepare an organo lanthanidematerial with high thermostability and effective photosensitized luminescence. In thisstudy, novel organo-EuIII luminophores with hfa moieties (hfa:hexafluoroacethylacetonato) and carboxy phosphine oxide are reported, the structures of which are shown in III luminophoresand play an important role in the suppression of non-radiative transition viavibrational relaxation due to their lower vibrational frequenciesIII complex, Eu(hfa)3(TPPO)2 (TPPO:triphenylphosohine oxide) was prepared as a standard reference. The thermostability ofthe organo-EuIII luminophores was evaluated using thermogravimetricanalysis (TGA). The luminescent properties were estimated from emission spectra, quantumyields and lifetime measurements. The bright luminescence ofEu(hfa)x(TCPO)y at 400 °C wassuccessfully observed and the energy transfer efficiency ofEu(hfa)x(TCPO)y was calculated to be 47%. Thus, thermostableand effective photosensitized organo-EuIII luminophores were demonstratedfor the first time.There are currently various types of organo lanthanide luminophores based oncharacteristic ligand design that have been developed as strongly luminescentmaterials141516171819202122232425262728293031323334III luminophore with carboxy phosphine oxidehave been reportedx(TCPO)y andEu(hfa)x(CPO)y were synthesized by the complexation ofthe carboxy phosphine oxide (CPO or TCPO) withEu(hfa)3(H2O)2 in methanol under reflux.The phosphine oxide parts (P = O) and the carboxy groups(COO−) in CPO and TCPO ligands effectivelypromote the formation of polymeric structures. The significant vibrational bandsat C=O and P=O groups of Eu(hfa)x(CPO)y were shifted toshorten wavenumbers (1658 and 1143 cm−1)(CPO ligand: 1702 and 1151 cm−1). The IRbands of Eu(hfa)x(TCPO)y were also observed at 1622 and1102 cm−1, which are shorter thanthose of the ligand (TCPO ligand: 1692 and1115 cm−1) (see x(TCPO)y without basecondition. This chelate reaction is a new method for preparation ofEu(hfa)x(TCPO)y. On the other hand,Eu(hfa)x(CPO)y is prepared under base-condition(addition of triethyl amine). The reaction difference is might be due to moietyof the joint ligands, CPO and TCPO. The x and y in formulas inEu(hfa)x(CPO)y andEu(hfa)x(TCPO)y are defined0 < x < 1 and0 < y < 3. Weestimated x = 0.38, y = 2.12 inEu(hfa)x(CPO)y and x = 0.03,y = 1.92 in Eu(hfa)x(TCPO)y usingEDX data (see x(TCPO)y, we tried to measure by single-crystalX-ray structure analysis. The structure was determined to be eight-coordinatedstructure with two water molecules and five TCPO ligands. The two TCPO ligandsshow bidentate bridged connection between two EuIII ions . TheEu(hfa)x(TCPO)y crystal provides three dimensionalnetwork structure. This single crystal is including four methanol molecules inone unit (x(CPO)y at around 20° and28° (x(CPO)y has an amorphous structure at roomtemperature. In contrast, the as-prepared white powder ofEu(hfa)x(TCPO)y has noticeable peaks at11.29°, 12.41°, 13.45°, 14.76°,18.88°, 19.47°, 22.62°, 23.44°,24.23°, 25.41°, 28.48°, and30.11° (x(TCPO)y after heat treatment alsohas noticeable peak at 11.32°, 11.99°,14.05°, 15.21°, 18.22°, 18.73°,20.21°, 20.37°, 22.29°, 22.88°,23.23°, and 29.07° (x(TCPO)y compared with that ofEu((CH3)2NCHO)x(TCPO)y inprevious workx(TCPO)y is much different from that ofEu((CH3)2NCHO)x(TCPO)y.Identification of the polymeric structure was performed using fast atombombardment-mass spectrometry (FAB-MS) and energy dispersive X-ray spectroscopy(EDX) measurements. The fragment peaks of Eu(hfa)x(CPO)yand Eu(hfa)x(TCPO)y in the FAB-MS spectra agree with thosecalculated for[Eu2(hfa)3(CPO)2]+ and[Eu(hfa)2(TCPO)·5H2O]+fragments, respectively (see x(CPO)y andEu(hfa)x(TCPO)y for EDX measurements calibrated withEu(hfa)3(TPPO)2 as a standard. The EDX measurementsindicated the percentage of hfa moieties in Eu(hfa)x(CPO)yand Eu(hfa)x(TCPO)y were 10.8% and 0.89%, respectively. Wepropose that the small amount of hfa molecules attached on the crystal surface.The hfa molecules on the surface were successfully detected by ionized-fragmentinformation using FAB-MS spectrum (2TCPO·5H2O). In contrast, the EDXsignals of the XRF measurement gave the average information about total elementratio of Eu(hfa)x(TCPO)y.In previous work, Eum−1) see . Wesuccdata see . In ordeone unit .These s and28° . TheEuy andEu(hfa)x(TCPO)y polymeric structures were evaluatedusing TGA and the results are shown in III complex,Eu(hfa)3(TPPO)2, was also measured as a standardreference. The decomposition temperature ofEu(hfa)3(TPPO)2 was 200 °C.The weight of Eu(hfa)x(CPO)y gradually decreases from200 °C, which may be due to the loose packing structurein amorphous Eu(hfa)x(CPO)y to promote partial eliminationof the hfa moieties. The decomposition temperature ofEu(hfa)x(TCPO)y was 450 °C. Wecannot observed the elimination of solvent from the material. This resultindicates that Eu(hfa)x(TCPO)y have no solvent in thestructure after heat treatment. Therefore, XRD measurements ofEu(hfa)x(TCPO)y were kept under450 °C (see x(TCPO)y is the highest among theorgano-EuIII luminophores with photosensitized hfa moieties.Thus, a luminescent organo-EuIII luminophore with extra-highthermostability was successfully synthesized.The thermo-stabilities of the Eu(hfa)0 °C see . The dec3(TPPO)2,Eu(hfa)x(CPO)y, andEu(hfa)x(TCPO)y heated on a hot plate under UV lightirradiation (λ = 365 nm) areshown in 3(TPPO)2exhibits red luminescence at 50 °C but does not emitphotons at 250 °C due to their thermal decomposition.The red luminescence of Eu(hfa)x(CPO)y faded out at around250 °C. In contrast, bright red luminescence wassuccessfully observed from Eu(hfa)x(TCPO)y under400 °C. (See emission spectra under control oftemperature in x(TCPO)y exhibits botheffective photosensitized luminescence and thermostability.The luminescence images for Eu(hfa)3(TPPO)2,Eu(hfa)x(CPO)y andEu(hfa)x(TCPO)y in the solid state detected at613.5 nm and excited at 365 nm are shown in x(CPO)y at around 300 nm isassigned to π–π* transitionof hfa moietiesx(TCPO)y crystals.The emission bands were observed at around 578, 591, 613, 651, and699 nm, which are attributed to the 4f-4f transitionsof EuIII. Thespectra were normalized with respect to the magnetic dipole transitionintensities at 591 nm (EuIII:5D0—7F1),which is known to be insensitive to the surrounding environment of thelanthanide ions. The normalized emission intensity ofEu(hfa)x(TCPO)y at 613 nm is larger thanthat of Eu(hfa)x(CPO)y. These spectral shapes ofEu(hfa)x(TCPO)y andEu(hfa)x(CPO)y are different from that of crystallineEu(hfa)3(TPPO)2.Excitation and emission spectra for Eu(hfa)x(CPO)y andEu(hfa)x(TCPO)y are shown in x(CPO)y indicates single-exponential decays ofmillisecond scale. We estimated the emission lifetime ofEu(hfa)x(TCPO)y using single-exponential decay. Thelifetime and R2 under single-exponential analysis were found tobe 0.61 ms and 0.996, respectively. The lifetime is similar to thatof Eu(hfa)x(CPO)y. In this paper, we used thesingle-exponential analysis for estimation of lifetime ofEu(hfa)x(TCPO)y. We consider that the luminescence ofEu(hfa)x(TCPO)y comes from one-dominantEuIII species with hfa moieties on the crystal surface. Theemission lifetimes of Eu(hfa)3(TPPO)2,Eu(hfa)x(CPO)y andEu(hfa)x(TCPO)y were determined to be 0.80, 0.60 and0.61 ms, respectively.Time-resolved emission profiles of Eu(hfa)f − 4f emission quantumyields (Φf−4f4) and theradiative (kr) and non-radiative (knr) rateconstants of these EuIII compounds were calculated using thefollowing equations.The 4τrad) is defined as anideal emission lifetime without non-radiative processes. The radiative lifetimeis expressed by AMD,0is the spontaneous emission probability for the5D0—7F1transition in vacuo (14.65 s−1),n is the refractive index of the medium , and(Itot/IMD) is the ratio of the totalarea of the corrected EuIII emission spectrum to the area of the5D0—7F1band. The emission quantum yields, and the radiative and non-radiative rateconstants are summarized in The radiative lifetime (x(TCPO)y excited at355 nm (π − π*transition band of hfa moieties) was also measured to calculate the energytransfer efficiency (η), which was determined as 34%. Theenergy transfer efficiency of Eu(hfa)x(TCPO)y is larger than that ofthe previously reported thermostable organo-EuIII luminophore,[Eu(hfa)3(dpbp)]n biphnyl, decompositiontemperature = 308 °C,η = 40%)The emission quantum yield of Eu(hfa)III luminophore, Eu(hfa)x(TCPO)y,with effective thermostability and photosensitized luminescent property wassuccessfully synthesized. Thermostable Eu(hfa)x(TCPO)yexhibits bright red luminescence at 400 °C under UV lightirradiation. The luminescence of Eu(hfa)x(TCPO)y is due tophotosensitized energy transfer from hfa moieties to EuIII ions.Thermostable organo-lanthanide luminophores are expected to open up the frontierfields of photophysical science, material chemistry and industrial applications.A organo-Eun-hydrate (99.9%), diphenyl(p-tolyl)phosphine andtri-p-tolylphosphine were purchased from Wako Pure ChemicalIndustries Ltd. Hexafluoroacetylacetone and triphenylphosphine oxide (TPPO) wereobtained from Tokyo Kasei Organic Chemicals Co., Inc. Dimethylsulfoxide-d6  was obtained from KantoChemical Co., Inc. All other chemicals and solvents were reagent grade and wereused without further purification.Europium acetate 1H NMR (400 MHz) spectra were recorded on a JEOLECS400. Chemical shifts were reported in δ ppm, referenced to aninternal tetramethylsilane standard for 1H NMR spectroscopy.Infrared spectra were measured using a Thermo Nicolet AVATAR 320 FT-IRspectrometer. FAB-MS spectra were recorded on a JEOL JMS-700TZ. Elementalanalyses were performed on a J-Science Lab Micro Corder JM 10 and an ExeterAnalytical CE440. In addition, the ratio of F to Eu was measured using EnergyDispersive X-ray Fluorescence Spectrometer EDX-8000 with 3(TPPO)2). XRD patterns were characterized by aRIGAKU SmartLab X-ray diffractometer with Cu Kα radiation, a D/teXUltra detector, and a temperature control unit .Thermogravimetric Analysis was performed on a Seiko Instruments Inc. EXSTAR 6000(TG-DTA 6300) at first heating rate of 10 °Cmin−1 up to 100 °C,cooling rate of 10 °C min−1up to 40 °C, and second heating rate of1 °C min−1 up to500 °C.p-tolyl)phosphine withpotassium permanganate, according to the procedure described in theliterature1H NMR: δ8.10—8.06 , 7.78—7.72 ,7.67—7.61 , 7.60—7.54  ppm;IR (ATR): 1658, 1592, 1540, 1498, 1411, 1254, 1144,1118 cm−1; Elemental analysis calcd(%) for C19H15O3P: C 70.81, H 4.69; found: C70.15, H 4.49.CPO was synthesized by the oxidation of diphenyl: δ8.12—8.08 , 7.88—7.75 ppm; IR (ATR): 1692, 1395, 1246, 1162, 1102,1016 cm−1; Elemental analysis calcd(%) for[C21H15O7P + H2O]:C 58.89, H 4.00; found: C 58.67, H 4.08.TCPO was synthesized by the oxidation of tri-n-hydrate  wasdissolved in distilled water (20 mL). A solution ofhexafluoroacetylacetone was added dropwise to the solution. The reaction mixtureproduced a precipitation of white yellow powder after stirring for3 h at room temperature. The reaction mixture was filtered, and theresulting powder was used without further purification for next step. Yield:95%; IR (KBr): 1650,1258–1145 cm−1; Elementalanalysis calcd (%) for C15H7EuF18O8:C 22.27, H 0.87; found: C 22.12, H 1.0142Europium acetate 3(H2O)2  and TPPO  wasrefluxed under stirring for 12 h. The reaction mixture wasconcentrated using a rotary evaporator. Reprecipitation by addition of excesshexane solution produced crude crystal, which were washed in toluene severaltimes. Recrystallization from hot toluene/cyclohexane gave white needlecrystals. Yield: 74%; 1H-NMR :δ = 5.42 ,7.58—7.71 , 7.76—7.86 ,8.67  ppm; IR (KBr): 1650, 1250—1150,1125 cm−1; Elemental analysis calcd(%) for C51H33EuF18O8P2:C 46.07, H 2.50; found: C 45.94, H 2.57Methanol (100 mL) containingEu(hfa)3(H2O)2  were dispersed in methanol (30 mL), andtriethylamine was added to neutralize. The dispersion was stirred for5 h at 60 °C. The precipitate was washedwith methanol several times, and dried in vacuo. Yield:45.3 mg; IR (ATR) 1658, 1592, 1540, 1498, 1411, 1254, 1144,1118 cm−1; FAB-MS (m/z):[Eu2(hfa)3(CPO)2]+ calcd forC53H31Eu2F18O12P2,1566.9; found 1566.7; EDX found (%): CPO, 60.0; Eu, 28.3; hfa, 10.7.CPO  andEu(hfa)3(H2O)2  were dispersed in methanol (30 mL). Thedispersion was stirred for 9 h at 60 °C. Thewhite precipitate was washed with methanol several times, and dried invacuo oven at 90 °C :[Eu(hfa)2(TCPO)·5H2O]+calcd for C31H27EuF12O16P, 1067.01;found 1067.3; EDX found (%): TCPO, 64.9; Eu, 33.8; hfa, 0.9.TCPO  andEu(hfa)0 °C see . Yield:τobs) were measured using the third harmonics(355 nm) of a Q-switched Nd:YAG laser  and aphotomultiplier . The Nd:YAG laser response was monitored with adigital oscilloscope  synchronizedto the single-pulse excitation. The emission quantum yield excited at355 nm .