Supramolecular chains arise in the crystal as a result of charge-assisted O—H⋯O and N—H⋯O hydrogen-bonding.The mefloquinium cation in the title salt is  17H17F6N2O+·C2Cl3O2−, comprises a single ion-pair. The hydroxy-O and ammonium-N atoms lie to the same side of the cation, a disposition maintained by a charge-assisted ammonium-N—H⋯O(hydroxy) hydrogen bond [the Oh—Cm—Cm–Na  torsion angle is 58.90 (19)°]. The piperidin-1-ium group is approximately perpendicular to the quinolinyl residue [Cq—Cm—Cm–Na (q = quinolinyl) is −178.90 (15)°] so that the cation, to a first approximation, has the shape of the letter L. The most prominent feature of the supramolecular association in the crystal is the formation of chains along the a-axis direction, being stabilized by charge-assisted hydrogen-bonds. Thus, ammonium-N+—H⋯O−(carboxylate) hydrogen bonds are formed whereby two ammonium cations bridge a pair of carboxylate-O atoms, leading to eight-membered {⋯O⋯HNH}2 synthons. The resulting four-ion aggregates are linked into the supramolecular chain via charge-assisted hydroxyl-O—H⋯O−(carboxylate) hydrogen bonds. The connections between the chains, leading to a three-dimensional architecture, are of the type C—X⋯π, for X = Cl and F. The analysis of the calculated Hirshfeld surface points to the importance of X⋯H contacts to the surface  along with a significant contribution from O⋯H hydrogen-bonds (10.2%). Conversely, H⋯H contacts, at 12.4%, make a relatively small contribution to the surface.The asymmetric unit of the centrosymmetric title salt, C Z′ would usually be greater than 1 (unless the molecule lies on a rotation axis) and in which enantiomeric molecules, when present, are related by a non-crystallographic symmetry, e.g. a non-crystallographic centre of inversion. Reviews of this phenomenon have appeared for organic compounds -erythro-mefloquinium] isomer. That protonation from the carboxylic acid to the base occurred during co-crystallization is readily seen in the equivalence of the C18 O2, O3 bond lengths, i.e. 1.238 (3) and 1.245 (3) Å, respectively. The formation of the piperidin-1-ium cation is supported by the pattern of hydrogen bonding involving the ammonium-N—H H atoms. Indeed, an intramolecular ammonium-N+—H⋯O(hydroxy) hydrogen bond is formed ensuring the hydroxyl-O1 and ammonium-N2 atoms are orientated to the same side of the cation with the O1—C12—C13—N2 torsion angle of 58.90 (19)° angle indicating a + syn-clinal relationship. The r.m.s. deviation for the 10 atoms comprising the quinolinyl residue is 0.0147 Å, with the hydroxy-O1 [−0.299 (3) Å] and ammonium-N2 [1.490 (4) Å] atoms lying to either side of the plane. The dihedral angle of 74.00 (5)° formed between the fused ring system and the best plane through the piperidin-1-ium ring indicates that, overall, the molecule has the shape of the letter L. This is confirmed by the +syn-clinal C3—C12—C13—C17 torsion angle of 60.1 (2)°.The two ions comprising the asymmetric unit of salt (I)2O2 atoms is 0.0131 Å with the Cl3 atom lying to one side of the plane [deviation = 1.7153 (3) Å] whereas the Cl1 [−0.9341 (3) Å] and Cl2 [−0.6170 (4) Å] atoms lie to the other side.In the anion, the r.m.s. deviation through the Ca-axis direction in the crystal of (I)a). The most prominent feature of the packing is the formation of centrosymmetric, eight-membered {⋯O⋯HNH}2 synthons, which arise as a result of ammonium-N+—H⋯O−(carboxylate) hydrogen bonds whereby two ammonium cations bridge, via both hydrogen atoms, a pair of carboxylate-O2 atoms. The four-ion aggregates are linked into the chain via charge-assisted hydroxyl-O—H⋯O−(carboxylate) hydrogen bonds. These lead to larger centrosymmetric agglomerates, i.e. 18-membered {⋯OCO⋯HOC2NH}2 synthons. The connections between the chains are of the type C—X⋯π, for X = Cl and F. Such interactions are inherently weak, providing energies of stabilization less than 4 kcal mol−1, with those for interactions involving chloride atoms being greater than those with fluoride  interactions are formed whereby the C—Cl bond is approximately parallel to the C6 ring. Each of the fluoride atoms bound to the C10 atom participates in a C—F⋯π contact as these CF3 groups lie in regions flanked by quinolinyl residues. Two of the contacts are as for the chloride atom, i.e. side on, whereas the other is best described as an end-on C—F⋯π contact as the angle subtended at the F1 atom is 170.95 (14)°. The aforementioned interactions combine to form a three-dimensional architecture. A view of the unit-cell contents is shown in Fig. 2b).The presence of charge-assisted hydrogen bonds between the constituent ions lead to linear, supramolecular chains along the et al., 2018N atom and carboxylate-O2 and O3 atoms of the anion on the Hirshfeld surfaces mapped over dnorm in Fig. 3b). The presence of bright- and broad-red spots near the ammonium-H1N and H2N, hydroxyl-H1O, carboxylate-O2 and O3 atoms on the dnorm-mapped Hirshfeld surfaces indicate the influence of the charge-assisted N—H⋯O and O—H⋯O hydrogen bonds, as indicated in Fig. 4a) and (b). The donors and acceptors of intermolecular interactions in the crystals of (I)3 atoms as well as the other atoms of the cation, Fig. 4a) and (c), and the Cl1 atom, Fig. 3b), indicate the involvement of these atoms in short interatomic contacts, Table 2X⋯π interactions , Table 1The Hirshfeld surface calculations for the title salt (I)et al., 2007i.e. 12.4%, from H⋯H contacts to the Hirshfeld surfaces of (I)The overall two-dimensional fingerprint plot for (I)de + di ∼1.7 Å in the delineated fingerprint plot. The largest percentage contribution to the Hirshfeld surface are from F⋯H/H⋯F contacts, i.e. 25.4%. This is due to the presence of a number of short interatomic H⋯F contacts, Table 2de + di ∼ 2.5 Å in the corresponding delineated fingerprint plot. An arrow-like tip at de + di ∼ 2.8 Å in the fingerprint delineated into F⋯F contacts is due to the effect of the short interatomic F⋯F contact summarized in Table 2a), is viewed as the spear-shaped distribution of points with the pair of adjoining tips at de + di ∼ 3.5 Å in the fingerprint plot delineated into C⋯Cl/Cl⋯C contacts. Although the interatomic Cl⋯H/H⋯Cl and Cl⋯Cl contacts make significant contributions to the Hirshfeld surface of (I)The intermolecular N—H⋯O and O—H⋯O hydrogen-bonds in the packing of (I)Chemical context, there are two mefloquine derivatives that exhibit kryptoracemic behaviour with both examples being isolated after attempts at chiral resolution of racemic mefloquine with different carboxylic acids. In one example, two mefloquinium cations are related across a pseudo centre of inversion, and the charge balance is provided by two crystallographically independent 3,3,3-trifluoro-2-methoxy-2-phenylpropanoate anions, i.e. (+)-PhC(CF3)(OMe)CO2−  δ: 1.20–1.35, 1.55–1.75, 3.04 , 3.53 , 5.90 , 6.94 , 8.01 , 8.13 , 8.42 , 8.72 , 9.48 ; resonances due to OH and NH were not observed. 13C NMR (DMSO-d6) δ: 21.43 (2×), 21.59, 44.51, 58.90, 67.85, 135.50. 121.17 , 121.21 , 123.64 , 126.37, 127.93 , 128.32, 128.68. 129.9 , 142.78, 146.73 , 150.97, 159.82 . 19F NMR (DMSO-d6) δ: −58.65, −58.84, −66.68.A solution of mefloquinium chloride (1 mmol) and sodium difluorochoroacetate (1 mmol) in EtOH (10 ml) was refluxed for 20 min. The reaction mixture was left at room temperature and after two days, colourless slabs of (I)Uiso(H) set to 1.2Ueq(C). The O- and N-bound H atoms were refined with distance restraints 0.84±0.01 and 0.88±0.01 Å, respectively, and refined with Uiso(H) = 1.5Ueq(O) and 1.2Ueq(N), respectively. Owing to poor agreement, most likely due to interference from the beam-stop, two reflections, i.e. (100) and (101), were omitted from the final cycles of refinement.Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989018016389/hb7786sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989018016389/hb7786Isup2.hklStructure factors: contains datablock(s) I. DOI: 1879700CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The cyclopent-3-ene ring has an envelope conformation with the tertiary C atom as the flap atom. tert-butyl 1-(2-iodobenzoyl)-cyclopent-3-ene-1-carboxylate, C17H19IO3, was synthesized and structurally characterized. The 2-iodobenzoyl group is attached to the tertiary C atom of the cyclopent-3-ene ring. The dihedral angle between the benzene ring and the mean plane of the envelope-type cyclopent-3-ene ring is 26.0 (3)°. In the crystal, pairs of C-H⋯O hydrogen bonds link the molecules to form inversion dimers.1-(2-Iodobenzoyl)-cyclopent-3-ene-1-carboxylates are novel substrates to construct bicyclo[3.2.1]octanes with antibacterial and antithrombotic activities. In this context,  The deviation of C8 from the mean plane defined by atoms C9–C12 is 0.097 (4) Å. The dihedral angle between the benzene ring and the alkene plane (C9–C12) of the cyclopent-3-ene ring is 26.51 (19)°.The molecular structure of the title compound is shown in Fig. 1b axis and form layers parallel to the bc plane. There are no other significant intermolecular interactions present in the crystal.In the crystal, molecules are linked by a pair of C—H⋯O hydrogen bonds forming inversion dimers Table 1. They stet al., 2016viz. methyl 4-[(1-methylcyclopent-3-en-1-yl)carbonyl]benzoate in the space group Pnma : δ 8.0 , 7.4 , 7.3 , 7.1 , 5.6 , 3.1 , 1.2 . HRMS (ESI) calcd for [C17H19IO3+Na]+ 421.0271, found 421.0272. Crystallization from a 5:1 mixture (v/v) of dichloromethane and n-hexane by slow evaporation at room temperature for about 7 d gave block-shaped crystals of the title compound.The title compound was prepared according to a general literature protocol (Yuan Uiso(H) = 1.5Ueq(C-methyl) and 1.2Ueq(C) for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989019011514/wm5513sup1.cifCrystal structure: contains datablock(s) global, I. DOI: Click here for additional data file.10.1107/S2056989019011514/wm5513Isup2.cmlSupporting information file. DOI: 10.1107/S2056989019011514/wm5513Isup3.hklStructure factors: contains datablock(s) 20180530A_0m_a. DOI: 1899475CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Lysosomal storage disorders (LSDs) are characterized by the accumulation of lipids, glycolipids, oligosaccharides, mucopolysaccharides, and other biological substances because of the pathogenic deficiency of lysosomal enzymes. Such diseases are rare; thus, a multiplex assay for these disorders is effective for the identification of affected individuals during the presymptomatic period. Previous studies have demonstrated that such assays can be performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring (MRM) detection. An assay procedure to quantify the activity of 11 enzymes associated with LSDs was provided. First, a validation study was performed using dried blood spot (DBS) samples with 100% and 5% enzyme activity for quality control (QC). Under the assay condition, the analytical range, defined as the ratio of the peak area of the enzyme reaction products from the DBS for QC with 100% enzyme activity to that from the filter paper blank sample, was between 14 for GALN and 4561 for GLA. Based on these results, the distribution of the enzyme activity for the 11 LSD enzymes was further examined. Consistent with the previous data, the enzyme activity exhibited a bell-shaped distribution with a single peak. The averaged enzyme activity for the healthy neonates was as follows: GLA, 3.80 ± 1.6; GAA, 10.6 ± 4.8; IDUA, 6.4 ± 2.3; ABG, 8.6 ± 3.1; ASM, 3.3 ± 1.1; GALC, 2.8 ± 1.3; ID2S, 16.7 ± 6.1; GALN, 1.2 ± 0.5; ARSB, 17.0 ± 8.7; NAGLU, 4.6 ± 1.5; and GUSB, 46.6 ± 19.0 μmol/h/L . In contrast, the enzyme activity in disease-affected individuals was lower than the minimum enzyme activity in healthy neonates. The results demonstrate that the population of disease-affected individuals was distinguished from that of healthy individuals by the use of LC-MS/MS. The aMass spectrometry (MS)-based LSD assays were first reported by Dr. M.H. Gelb at the University of Washington ,7. The pN-acetylgalactosamine-6-sulfatase (ARSB) for MPS IVA, N-acetylgalactosamine-4-sulfatase (GALN) for MPS VI, N-acetyl-α-D-glucosaminidase (NAGLU) for MPS IIIB, and β-glucuronidase (GUSB) for MPS VII [Because the number of new therapies has been emerging based on new technologies, the number of assay reagents for LSD enzyme activity has also been growing. Assay reagents for iduronate-2-sulfatase (ID2S) for MPS II,  MPS VII , as well MPS VII ,16 were  MPS VII . Subsequ MPS VII . Previou MPS VII ,17. One  MPS VII ,17. For  MPS VII ,16,17. A MPS VII . The cur22.1N-Acetylglucosamine thiazoline (NAG-thiazoline) was purchased from Toronto Research Chemicals . The acetonitrile was purchased from Thermo Fischer Scientific . The deionized water was obtained from a Milli-Q water system . The formic acid was purchased from Kanto Chemical . A set of DBS samples with high (100%) and low (5%) activity was provided by PerkinElmer for QC. The other reagents used in this study were of the highest commercially available grade.The reagents required for the 6-plex LSD enzyme assay for the GAA, GLA, IDUA, ABG, ASM, and GALC were purchased from PerkinElmer . The reagents for the ID2S, GALN, and ARSB were purchased from PerkinElmer under a custom manufacturing agreement. The reagents for the NAGLU and GUSB were provided by Prof. Michael H. Gelb . The 2.2This study was approved by the Research Ethics Board of the National Center for Child Health and Development.2.3The DBS samples were stored at −20 °C according to previous procedures . One unt2.4g for 5 min at room temperature using a plate centrifuge , an aliquot of the organic layer (200 μL) was transferred to a fresh 96-well plate.The assay cocktail contained the known concentrations of substrate for the GAA for Pompe disease (0.35 mM), the GLA for Fabry disease (1.2 mM), the IDUA for MPS I (0.25 mM), the ABG for Gaucher disease (0.5 mM), the ASM for Niemann-Pick disease type A/B (0.75 mM), and the GALC for Krabbe disease (0.85 mM) and of IS for the GAA (24 μM), GLA (24 μM), IDUA (15 μM), ABG 20 μM), ASM (15 μM), and GALC (10 μM)  μM, ASM . All of 2.5Z)-Pugnac. All of the assays were carried out with a 3-mm punch in 30 μL of the assay cocktail in a polypropylene 96-well plate and incubated at 37 °C for 20 h. To terminate this enzyme reaction, a mixture of methanol/ ethyl acetate  was added. Next, to extract the enzyme reaction products, ethyl acetate (400 μL) and 0.5 M sodium chloride (200 μL) were added and mixed vigorously using a pipette. After the centrifugation of these plates at 700 ×g for 5 min at room temperature using a plate centrifuge, an aliquot of the organic layer (200 μL) was transferred to a fresh 96-well plate.The assay cocktail contained the known concentrations of substrate for the ID2S for MPS II (0.5 mM), the GALN for MPS IVA (1 mM), and the ARSB for MPS VI (1 mM) and of IS for the ID2S (5 μM), GALN (5 μM), and ARSB (5 μM) . The enz2.6g for 5 min at room temperature using a plate centrifuge, an aliquot of the organic layer (200 μL) was transferred to a fresh 96-well plate.The assay cocktail contained the known concentrations of substrate for the NAGLU for MPS IIIB (0.5 mM) and the GUSB for MPS VII (0.5 mM) and of IS for the NAGLU (10 μM) and GUSB (10 μM) . The enz2.7For the 11-plex assay, an aliquot of the ethyl acetate layer from Plates A (200 μL), B (200 μL), and C (200 μL) were combined in a deep-well plate. Next, the solvent of the extracts was removed under a nitrogen stream. Finally, the enzyme reaction products were reconstituted with a reconstitution solvent . In this study, it was estimated that a 3-mm DBS punch contained 3.1 μL of whole blood. Enzyme activity was calculated in μmol/h/L blood as was previously reported .2.8An aliquot (5 μL) was injected into a Quattro Premier XE tandem mass spectrometer equipped with an Acquity UPLC chromatograph (Waters). An aliquot of the samples was injected by autosampler into an analytical column ACQUITY CSH C18  equilibrated with 80% mobile phase A (0.2% formic acid in 5% acetonitrile/95% water) and 20% mobile phase B (0.2% formic acid in acetonitrile) at a flow rate of 0.6 mL/min at 40 °C. The enzyme reaction product and IS were eluted with 20% mobile phase B for 0–0.1 min, 20–100% for 0.1–1.0 min, 100% for 1.0–1.5 min, and 20% for 1.51–2.0 min. The data were collected using MassLynx software V4.1 (Waters). The other analytical conditions are described in Supplementary Tables 1–3.33.1Z)-Pugnac, an inhibitor for O-linked N-acetylglucosamine, was included in this batch of ID2S, ARSB, and GALN reagents at the time of production . This implies that the suppression for NAGLU observed above in the preliminary 11-plex assay could be associated with (Z)-Pugnac in assay reagent for ID2S, ARSB, and GALN. Thus, a decision was made to examine a 11-plex assay using a combination of three 96-well plates (6/3/2-plex assay using Plate A/B/C). For comparison, a 6-plex assay for the GAA, GLA, IDUA, ABG, ASM, GALC; a 3-plex assay for the ID2S, ARSB, and GALN; and a 2-plex for the NAGLU and GUSB were performed . As was previously demonstrated, the accumulation of enzyme reaction products was proportional to the percentage of enzyme activity in the DBS . As was demonstrated, the CV values for the QC DBS with high enzyme activity for intraday and interday precision were within 12% in the 11-plex assay . The ana3.2The distribution of the enzyme activity in the healthy controls for the 11 LSD enzymes was examined. There was one peak in the distribution of the activity in the 11 LSD enzymes . Note that enzyme activity of neonates is usually higher than that of adults due to higher leukocyte counts: our results were comparable to previous studies because the enzyme activity for neonatal controls were examined . Next, tThe activity of the tested LSD enzymes in the healthy controls was as follows : GLA enzyme activity, 3.8 ± 1.6 ; GAA enzyme activity, 10.6 ± 4.8 ; IDUA enzyme activity, 6.4 ± 2.3 ; ABG enzyme activity, 8.6 ± 3.1 ; ASM enzyme activity, 3.3 ± 1.1 ; GALC enzyme activity, 2.8 ± 1.3 ; ID2S enzyme activity, 16.7 ± 6.1 ; ARSB enzyme activity, 17.0 ± 8.7 ; GALN enzyme activity, 1.2 ± 0.5 ; NAGLU enzyme activity, 4.6 ± 1.5 ; and GUSB enzyme activity, 46.6 ± 19.0 .In addition, the enzyme activity in the disease-affected individuals was assessed. The IDUA enzyme activity found for one MPS I-affected individual was 0.39 μmol/h/L, the mean ID2S enzyme activity for four MPS II-affected individuals was 0.18 ± 0.10 μmol/h/L , the NAGLU enzyme activity for one MPS IIIB-affected individual was 0.05 μmol/h/L, the mean GALN enzyme activity for two MPS IVA-affected individuals was 0.08 μmol/h/L , and the mean ARSB enzyme activity for two MPS VI-affected individuals was 0.43 μmol/h/L .4This study has demonstrated a quantification procedure for 11-plex LSD enzyme activity using LC-MS/MS. First, the assay performed 3 separate reactions at 37 °C for 20 h. The reaction products were then combined in a single plate, and the enzyme activity was subsequently determined through each accumulating enzyme reaction product by LC-MS/MS . The enzGLA is frequently found in Taiwan; the phenotype of this mutation seems to be associated with cardiac damage with the adult onset of the disease [GLA gene in other areas [NBS for LSDs using an MS/MS-based technique has been implemented in several states in the U.S. and Taiwan ,12. Curr disease . In facter areas ,30.The analytical range is defined as the ratio of the peak area of enzyme reaction products in controls to that in the filter paper blank samples . This vaIn sum, an 11-plex LSD enzyme assay was examined by LC-MS/MS. The maximum interday CV% values were 10% when the activity of 11 enzymes in the rehydrated QC DBS with 100% enzyme activity was examined. The distribution of the 11 LSD enzyme activity exhibited a bell-shaped single peak. Although the number of samples was limited, the DBS for the disease-affected individuals was properly identified. Thus, future studies will be able to examine the putative cut-off values using this assay procedure. When cut-off value is to be determined, age-matched controls are to be used because leukocyte count of neonatal blood is 2–10-times higher than that of older population. The typical run-time for this assay is 2 min. This suggests that NBS for these LSD enzymes can be performed using this assay procedure.


In the title quinoline derivative, the two benzyl rings are inclined to the quinoline ring mean plane by 74.09 (8) and 89.43 (7)°. 24H19NO3, the two benzyl rings are inclined to the quinoline ring mean plane by 74.09 (8) and 89.43 (7)°, and to each other by 63.97 (10)°. The carboxylate group is twisted from the quinoline ring mean plane by 32.2 (2)°. There is a short intramolecular C—H⋯O contact forming an S(6) ring motif. In the crystal, molecules are linked by bifurcated C—H,H⋯O hydrogen bonds, forming layers parallel to the ac plane. The layers are linked by C—H⋯π interactions, forming a supramolecular three-dimensional structure.In the title quinoline derivative, C The FMOs play an important role in the optical and electric properties, as well as in quantum chemistry and UV–Vis spectra. The frontier orbital gap helps characterize the chemical reactivity and the kinetic stability of the molecule. A molecule with a small frontier orbital gap is generally associated with a high chemical reactivity, low kinetic stability and is also termed a soft molecule. DFT quantum-chemical calculations for the title compound were performed at the B3LYP/6–311 G level -one skeleton gave ten hits. The dihedral angle between the benzyl and quinoline rings varies from ca 71.0 to 89.6°, compared to 89.43 (7)° in the title compound. Only two of these compounds have a carboxylate group in position 4 on the quinoline ring, viz. ethyl 1-benzyl-3-hydroxy-2-oxo-1,2-dihydroquinoline-4-carboxylate , benzyl chloride  and tetra n-butylammonium bromide as catalyst in DMF (50 ml) was stirred at room temperature for 48 h. The solution was filtered by suction and the solvent was removed under reduced pressure. The residue was chromatographed on a silica-gel column using hexane and ethyl acetate  as eluents to afford the title compound. Colourless prismatic crystals of the title compound were obtained by slow evaporation of a solution in ethanol (yield 53%).A mixture of 2-oxo-1,2-dihydroquinoline-4-carboxylic acid , KUiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989019007989/su5500sup1.cifCrystal structure: contains datablock(s) I, Global. DOI: 10.1107/S2056989019007989/su5500Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989019007989/su5500Isup3.cmlSupporting information file. DOI: 1920509CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the crystal, C—H⋯N(nitrile) short contacts and stacking interactions combine to link the anions into layers parallel to the 2 dinitrile moieties are slightly twisted from the polymethine main chain to which they are attached [C(CN)2/C5 dihedral angles = 6.1 (2) and 7.1 (1)°]. The C—C bond distances along the heptadienyl chain vary in the narrow range 1.382 (2)–1.394 (2) Å, thus indicating the significant degree of conjugation. In the crystal, the anions are linked into zigzag chains along the [10The title compound, C In the linear anion, the bond lengths vary in the narrow range 1.382 (2)–1.394 (2) Å, thus indicating a significant degree of conjugation along the hydrocarbon chain. Such a structural and electronic configuration in which the difference in bond lengths along the conjugated backbone approaches zero is known as a cyanine-like structure  and 7.1 (1)°. The bond lengths and angles and the overall conformation of the anion are close to those reported for the same anion in N-(7-(dimethylamino)hepta-2,4,6-trienylidene)-N,N-dimethylammonium 1,1,7,7-tetracyanohepta-2,4,6-trienide hepta-2,4,6-trienylidene)-N,N-dimethylammonium 1,1,7,7-tetracyanohepta-2,4,6-trienide pyridinium chloride. Pyridine  was added to a solution of 2,4-dinitrochlorobenzene  in dry acetone (4 mL). The resulting mixture was brought to reflux for 1 h before being cooled to room temperature. The crude product was collected by filtration and recrystallized from ethanol to give N-pyridinium chloride , m.p. 462–463 K. 1H NMR  δ 9.38 , 9.12 , 8.99 , 8.95 , 8.44 .Tetramethylammonium -1,1,7,7-tetracyanohepta-2,4,6-trien-1-ide. Malononitrile  was added to a refluxing solution of fresh sodium ethoxide, prepared by adding sodium metal  to absolute ethanol (5 mL). To this solution, was added a solution of N-pyridinium chloride  in ethanol (2 mL), and the reaction mixture was stirred at reflux for 1 h before being cooled to room temperature and stirred for a further hour. A solution of tetramethylammonium bromide  in water (10 mL) was added to the reaction. After about an hour, the deep-red mixture was extracted with dichloromethane (3 × 30 mL), dried over magnesium sulfate, and the solvent was removed in vacuo. The deep-violet residue was purified by column chromatography . 1H NMR  δ 7.06 , 6.95 , 6.06 , 3.68 .Crystallization. Crystals of the title compound were grown over a period of 2–4 weeks by the vapour-diffusion method using dichloromethane as the solvent and hexane as the non-solvent.Uiso(H) set to 1.2Ueq(C) for aromatic and 1.5Ueq(C-methyl). To obtain an idealized geometry of the cation, 1,2 and 1,3 restraints for C—N mean bond distances and C—N—C bond angles were used. In the tetramethyl ammonium cation, three methyl groups are each disordered over two positions about the N5—C12 axis and were refined with partial occupancies of 0.66 (1) and 0.34 (1). The positions of all disordered atoms were refined in an isotropic approximation.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989019011411/nr2075sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989019011411/nr2075Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989019011411/nr2075Isup3.cmlSupporting information file. DOI: 1947007CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


CAFs derived from hepatocellular carcinoma (HCC) express high αSMA and low LXRα levels, whereas hepatocarcinoma cells exhibit an inverse expression pattern. All hepatoma cells analyzed responded to the LXRα agonist T0901317 by inducing fatty acid synthase (FASN) expression. On the other hand, T0901317 antagonized TGFβ-induced fibroblastic marker responses, such as fibronectin and calponin, in a subset of hepatoma cells and all CAFs analyzed. Mechanistically, LXRα antagonized TGFβ signaling at the transcriptional level. Smad3 and LXRα were recruited to adjacent DNA motifs of the ACTA2 promoter. Upon cloning the human ACTA2 promoter, we confirmed its transcriptional induction by TGFβ stimulation, and LXRα overexpression repressed the promoter activity. Hepatosphere formation by HCC cells was enhanced upon co-culturing with CAFs. T0901317 suppressed the positive effects exerted on hepatosphere growth by CAFs. Taken together, the data suggest that LXRα agonists limit TGFβ-dependent CAF differentiation, potentially limiting primary HCC growth.Transforming growth factor β (TGFβ) is deposited in the extracellular space of diverse tissues. Resident fibroblasts respond to TGFβ and undergo myofibroblastic differentiation during tissue wound healing and cancer progression. Cancer-associated fibroblasts (CAFs) communicate with tumor cells during cancer progression, under the guidance of TGFβ signaling. We report that agonist-activated liver X receptors (LXR) limit the expression of key components of myofibroblast differentiation, including the  TGFβ halts cell proliferation and accelerates cell death, as it occurs in immune cells or during liver homeostasis3. Moreover, TGFβ promotes epithelial-mesenchymal transition (EMT) and stimulates fibroblasts to secrete cytokines, chemokines and extracellular matrix (ECM) molecules3. Cellular responses initiate when TGFβ binds to the type II (TβRII) and type I (TβRI) kinase receptor complex4. Within this complex, TβRII trans-phosphorylates TβRI, activating TβRI to phosphorylate Smad2 and Smad3, which, in complex with Smad4, regulate gene transcription. The TGFβ receptor also activates mitogen activated protein kinase (MAPK), the tyrosine kinase Src and phosphatidylinositol-3´-kinase pathways. Upon ligand-induced cleavage, the TβRI intracellular domain regulates transcription5.Misregulation of transforming growth factor β (TGFβ) signaling occurs in infectious, cardiovascular diseases and cancer7. Transcriptional programs become activated in epithelial cells by TGFβ, mediated for example by Snail and Slug, leading to repression of cell-cell contact genes, induction of cytokine genes as well as ECM genes, such as fibronectin8. EMT generates mesenchymal-like cells that partially resemble fibroblasts and contribute to chronic tissue fibrosis and cancer progression9. Cancer-associated fibroblasts (CAFs) secrete TGFβ and other cytokines to assist tumor cell invasion and suppress anti-tumor immune responses11. Myofibroblast differentiation is a key response to TGFβ signaling, mediated by Smad complexes with β-catenin and parallel MAPK signaling12. The contractility of CAFs depends on microfilament networks built by α smooth muscle actin (αSMA) and associated myosins, tethered via membrane receptors to specialized collagen fibers, all induced by TGFβ signaling12.In adult tissues, TGFβ regulates EMT and fibroblast maturation14. TGFβ suppresses HCC by arresting hepatocyte proliferation at the early G1 phase of the cell cycle, and inducing apoptotic responses14. TGFβ promotes EMT, survival and CAF differentiation, contributing to HCC invasion and metastasis16. Similar to TGFβ, the nuclear liver X receptors (LXRα/NR1H3 and LXRβ/NR1H2), either suppress or promote cancer, by inhibiting cell proliferation or helping tumor cells to escape from immune detection, respectively17. The LXRs are expressed in hepatocytes among other tissues; agonistic LXR ligands include the oxysterols, metabolic derivatives of cholesterol, inducing the transcriptional activity of LXRs18.Among several malignancies, TGFβ regulates the progression of hepatocellular carcinoma (HCC), a cancer with high-mortality rate worldwide19, and, in the context of HCC, we described a role for the transcription factor Snail as a target of the crosstalk between TGFβ and LXRα20. Due to the established role of TGFβ on CAF biology, we here analyzed signaling crosstalk between TGFβ and LXRα in human HCC fibroblastsWe previously demonstrated that LXRα suppresses TGFβ-induced differentiation21. Using E-cadherin  and vimentin  as marker genes, we found good concordance of mRNA and protein expression profiles with the classification  and as mesenchymal-like cells with a late signature ion Fig. , but alsion Fig. . Snail mlls Fig. .Fig. 1LXFASN) mRNA, a direct readout of LXRα transcriptional activity17, had a rather similar to the LXRα mRNA expression profile , SM22α and calponin mRNA expression were induced by TGFβ; in AKH38 cells, T0901317 suppressed the TGFβ effects, whereas suppression of αSMA was measurable but not significant in AKH12 cells, where calponin expression was even enhanced after co-stimulation The mRNA expression analysis was corroborated by analysis of the corresponding proteins Fig. . InductiMicroscopic analysis of actin-based cytoskeletal organization confirmed abundant accumulation of stress fibers containing β-actin and αSMA and formation of actin-supported membrane ruffles in the three CAFs Fig. . CAFs stαSMA/ACTA2 gene. Since previous studies of ACTA2 gene regulation focused on mouse or rat cells, we cloned a fragment of the human ACTA2 gene spanning the transcriptional start site, extending from −1400 to +50 bp relative to the start site  gene promoter, a well-characterized target of TGFβ signaling, was analyzed under the same conditions , and LXR-responsive elements Fig. . ChIP exifs Fig. . As a poons Fig. . ChIP asACTA2 promoter, we analyzed direct binding of Smad3 and LXRα to a synthetic human ACTA2 gene promoter DNA fragment, which encompasses a cluster of CAGA and LXRα motifs  and examined the role of CAFs and LXRα signaling. Hep3b cells assembled typical hepatospheres, while SNU449 formed multicellular aggregations  Minimum Essential Medium  with 0.1 mM non-essential amino acids ; SNU449, SNU398 and SNU423 in RPMI-1400 medium ; PLC/PRF5, Huh7, HLF, HepG2, HaCaT and 293-T in Dulbecco’s Modified Earle Medium . All media contained 10% fetal bovine serum , 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mM glutamine .Hep3b, HepG2, PLC/PRF5, Huh7, SNU449, SNU398, SNU423, HLF, HEK-293T (abbreviated as 293-T), HaCaT and AG1523 cells were previously reported22. Patient consent and ethical approval were based on regulations of the Comprehensive Cancer Center Vienna. AKH12 were isolated from a patient with low-to-medium differentiated HCC, cirrhosis and vascular invasion (stage 3 by Edmondson and Steiner); AKH14 from a stage 2 HCC with focal fibrosis but no cirrhosis; AKH38 from a multifocal, fibrolamellar stage 2 HCC. None of the CAFs were infected by hepatitis-A, B, C or human immunodeficiency viruses22. CAFs were maintained in RPMI-1400, 5% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 10 mM Hepes, 0.5 mM sodium pyruvate, 2 mg/ml bovine serum albumin (BSA), 10 μg/ml apotransferrin, 10 μM phosphoethanolamine, 10 μM ethanolamine, 25 nM sodium selenite, 50 nM hydrocortisone, 100 pM 3,3´,-5-triiodothyronine, 100 μg/ml insulin , and 1 ng/ml epidermal growth factor . Cells were free of mycoplasma (tested every 2 months) and all established cell lines were authenticated using PCR-single-locus-technology .HCC CAFs were isolated at the University of Vienna HospitalCells were serum-deprived for 16 h; 5 ng/ml TGFβ1  in the absence or presence of 5 μM T0901317 or GW3965  were added at 80% confluency for 48 or 72 h, as specified. CAFs were stimulated with ligands without prior serum deprivation. Dimethylsulfoxide (DMSO), vehicle to T0901317 and GW3965, was added as control.3 per well) were cultured alone or co-cultured with AKH12 CAFs , for 96 h in 10% FBS/MEM (Hep3b) or RPMI-1400 (SNU449), respectively. For hepatosphere formation, the 96-well plate GravityPLUSTM Hanging Drop System  was used according to the manufacturer´s instructions. Where indicated, 5 μM T0901317 was added separately on SNU449 and AKH12 cells for 24 h prior to sphere formation, in order to assess compound effects on a single population. LY2157299  was administered during sphere formation. Hepatospheres were photographed in the InSPHERO plates using a Zeiss Axioplan-2 microscope with 10 × objective lens, at ambient temperature and without immersion oil, via a Hamamatsu C4742-95 CCD digital camera and acquisition software Volocity® . Hepatospheres were assessed as the ratio of wells containing uniform spheres larger than 50 µm in diameter , relative to the total number of wells. Sphere size was determined via Image J analysis . Triplicate (nb = 3) biological experiments were performed in 10 technical replicates (nt = 10) per condition.Hep3b and SNU449 cells (1.5 × 104 cells per 12-well dish), Hep3b and SNU423 (5 × 105 cells per 60 mm dish) and SNU398, SNU449, HLF, Huh7 and PLC/PRF5 (4 × 105 cells per 60 mm dish), after the specified treatments, were washed in ice-cold phosphate buffer saline (PBS), pH 7.4, lysed and analyzed by immunoblot as described20, with antibodies at the following dilutions: fibronectin, 1:10,000 ; fatty acid synthase (FASN), 1:1,000 (ab22759); calponin, 1:2,000 ; LXRα, 1:1,000 (ab41902); Smad3, 1:1,500 (ab40854), all from Abcam, Cambridge, United Kingdom; α-smooth muscle actin (αSMA), 1:500 ; GAPDH, 1:50,000 . Horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies  were used at 1:20,000 dilution. Triplicate (nb = 3) biological experiments were performed in 2 technical replicates (nt = 2) per condition. Densitometric quantification of protein bands was performed using the Fujifilm Intelligent Dark Box II program of a Fuji Aida digital scanner .CAFs (2 × 104 cells per 8-chamber well) were treated at 50% confluency with TGFβ1 (5 ng/ml) and/or T0901317 (5 μM) for 48 h prior to F-actin and αSMA detection, as described20. The antibodies used were: anti-αSMA  in 1% BSA/PBS overnight (16 h) at 4 °C; anti-mouse Alexa Fluor-488 secondary antibody  for 1 h in the dark. Tetramethylrhodamine-isothiocyanate-phalloidin  staining lasted for 30 min at 24°C, and incubation with 4’,6-diamidino-2-phenylindole  for 5 min was followed by three rinses in PBS. Triplicate (nb = 3) biological experiments were performed in 2 technical replicates (nt = 2) per condition.CAFs .The human 4 cells per 24-well) were transfected with luciferase-encoding together with pCMV-β-galactosidase plasmids (100 ng), the latter as reference, using Lipofectamine-3000  for 48 h, and assayed as described20. The plasmids were: synthetic Smad-binding promoter CAGA12-luc24, −1400/+50 bp hACTA2 promoter-luciferase, pCDNA3-Flag-Smad3/pCDNA3-Flag-Smad430, and pCMX-LXRα20. Serum-starved cells were stimulated with TGFβ1 (5 ng/ml) for 16 h in the absence or presence of T0901317 (5 μM), GW3965 (5 μM) or their combination. Quadruplicate (nb = 4) biological experiments were performed in 3 technical replicates (nt = 3) per condition.HepG2 or AG1523 cells (1.8 × 105 per 6-well) and HepG2 (4 × 105 per 60-mm dish) cells were transfected with pCMX-LXRα and/or pCDNA3-Flag-Smad3 plasmids (200 ng) for 48 h, using calcium phosphate (293-T) or lipofectamine-3000 . Intact or transfected cells were lysed in 20 mM Tris, pH 7.5, 100 mM NaCl, 0.5% NP-40, 0.5 mM EDTA and protease inhibitor cocktail . Lysates were pre-cleared with protein-A beads , incubated with 0.9 μg biotinylated DNA probe  and 15 μg salmon sperm DNA (in-house) for 90 min (293-T) or 3 h (HepG2), and with magnetic streptavidin-sepharose beads  for 45 min; three washes were performed with lysis buffer prior to sequential immunoblot analysis on the same membrane using LXRα and Smad3 antibodies (see immunoblotting). The biotinylated double-stranded hACTA2 promoter probe sequence was: 5’-CAAGGAGGTTAGTGGGCAGAGAGGAGGGCTACAGAGGC-3’. Quadruplicate (nb = 4) biological experiments were performed in 2 technical replicates (nt = 2) per condition.293-T , forward, 5′-GCAGGACATCCGGGAGAGA-3′; reverse, 5′-CCAATAGCCTTGGCCTGAGA-3′; human ACTA2 , forward, 5′-CAGCTGGTCATGGCTGTAAAATAAAG-3′; reverse, 5′-CTCATAAAGAAATATTTTTGTGGGTACTG-3′; human ACTA2 (−805/−685 bp), forward, 5′-CATATCACCTGGTCTCTCTACTTC-3′; reverse 5′-CCCAG CAGCCTGTCAAAGA-3′; human ACTA2 (−161/−71 bp), forward, 5′-CCGCCTCCCGTT TCATG-3′; reverse, 5′-GACCTCAGCACAAAACTCTC-3′. Triplicate (nb = 3) biological experiments were performed in four technical replicates (nt = 4) per condition.Cells were fixed in 2% formaldehyde for 10 min at 37 °C, washed in ice-cold PBS twice, scraped in PBS and centrifuged at 4000 rpm for 5 min. Cells were lysed in 1% SDS, 10 mM EDTA, 50 mM Tris, pH 8.1, with protease inhibitors, for 20 min on ice, and sonicated (250 bp average DNA size). Lysates were diluted 10 times in 0.01% SDS, 1.0% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl, with protease inhibitors, and immunoprecipitated using anti-Smad2/3  or control rabbit antiserum, overnight at 4 °C, followed by protein-A dynabead incubation for 2 h at 4°C, washing once in 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl, once in 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl, once in 0.25 M LiCl, 1% IGEPAL, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1, and twice in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA. Beads and input samples were re-suspended in 1% SDS, 1 mM NaHCO4 per 12-well), Hep3b and SNU423 (5 × 105 per 60-mm dish), and SNU398, SNU449, HLF, Huh7 and PLC/PRF5 cells (4 × 105 per 60-mm dish), were cultured for total RNA isolation using the NucleoSpin RNA II kit , according to the manufacturer´s instructions. Reverse transcriptase real-time PCR was performed using primers . At least three biological (nb = 3) experiments, each in technical triplicate (nt = 3) are described.CAFs (2 × 10nb), most of which included at minimum technical triplicates (nt = 3), as indicated in the figure legends. Sample size depended on the assay type: for sphere quantification assays a minimum of 25 spheres was measured per experiment to reach statistical power for discrimination between conditions. For all groups that are statistically compared, the variance within each group was very similar, close to identical. Representative results are reported as averages minus-plus standard deviation, s.d. Statistical analysis was performed with GraphPadPrism  using two-sided t-test, and statistical significance was assigned with *p-value < 0.05; **p < 0.01; ***p < 0.001.Experiments were performed in biological replicates (Supplemental FigureFigure S1


Their structures were elucidated on the basis of extensive spectroscopic analysis  and acidic hydrolysis. The possible transformation pathway of these compounds were also speculated from ginsenoside Rg1. Compound 1, with a unique α,β-unsaturated ketene in its side chain, showed significant inhibitory effects against NO production on Murine macrophage cells (IC50 = 4.12 ± 0.20 μM) and comparable cytotoxicities against five human cancer cell lines  to positive control, cisplatin (DDP).Four new protopanaxatriol-type triterpenes (The online version of this article (10.1007/s13659-019-0211-4) contains supplementary material, which is available to authorized users. Panax notoginseng (Burk.) Chen , a well-known member called Sanqi or Tianqi in Ginseng family for the treatment of cardiovascular diseases, has been domesticated and cultivated for more than 400 years in the southwest of China +), corresponding to six degrees of unsaturation. The IR spectrum indicated the presence of hydroxy groups (3391 cm−1), keto (1738 cm−1), double bond (1674 cm−1) and methyls (2954 cm−1). The UV spectrum presented a weak absorption at 254 nm (K-band) indicating the existence of a conjugated α,β-unsaturated ketene in this structure. The 13C NMR spectrum (Table δC 124.9), seven methines with three oxygen-bearing ones , and seven quaternary carbons, including one oxygenated carbon (δC 72.9), one olefinic carbon (δC 145.2) and one ketone (δC 203.2). The 1H NMR spectrum , two olefinic protons at δH 6.06 and 5.66 , three oxymethines at δH 4.43 , 3.54 , and 3.95 , and four active hydrogen protons at δH 7.39, 7.02, 5.79, 5.32 . The two of six unsaturated degrees were from the carbonyl and double bond. Above analysis displayed 1 to be a tetracyclic triterpene. The NMR data of 1 were similar to those of 20(S)-protopanaxatriol (PPT)  +). The 13C NMR spectrum of 2 showed an olefinic , two carbonyl groups , while the 1H NMR indicated the existence of three active hydrogens at δH 5.48, δH 5.87, δH 6.46, two oxymethines at δH 4.42 , δH 4.27, and two vinyl protons at δH 5.25 and δH 4.97. An oxygen-bearing methine at δH 4.42 (H-24) was correlated with δC 31.4 (C-23)/δC 150.4 (C-25)/δC 18.4 (C-27) in HMBC spectrum ,24(S)-trihydroxydammar-3,12-dione-25-ene (5) +). The 13C NMR and DEPT spectra showed six more carbon resonances than those of 1, suggesting the existence of a hexosyl unit . So far, all hexosyl units in ginsenosides isolated from Panax species is only glucosyl (Glc) +). An β-d-glucopyranosyl unit  could be recognized by 13C NMR data and the anomeric proton at δH 5.04 , and confirmed by GC analysis after acidic hydrolysis. Furthermore, the HMBC correlations from H-5/H-7/H-1′ to C-6 (δC 79.8) indicated the glucosyl group was substituted at C-6. The 1H and 13C NMR data of 4 were in good agreement with those of 20(S)-ginsenoside-Rh1  with that in 3-oxo-20(R)-ginsenoside-Rh1  +, .White amorphous powder; mentclass2pt{minimc 0.08, MeOH); IR (ATR) νmax: 3478, 3452, 3405, 2956, 2916, 2880, 1690, 1111; UV (MeOH) λmax (log ε): 195 (0.35) nm; 13C and 1H NMR data: see Tables m/z 511.3399 [M+Na]+, .White amorphous powder; mentclass2pt{minimc 0.12, MeOH); IR (ATR) νmax: 3356, 2956, 2934, 2880, 1692, 1425, 1380, 1076, 1035; UV (MeOH) λmax (log ε): 195 (0.37) nm; 13C and 1H NMR data: see Tables m/z 691.4024 [M+Na]+, .White amorphous powder; mentclass2pt{minimc 0.13, MeOH); IR (KBr) νmax: 3422, 2964, 2931, 2877, 1691, 1637, 1458, 1383, 1077, 1022; UV (MeOH) λmax (log ε): 196 (0.52), 254 (0.07) nm; 13C and 1H NMR data: see Tables m/z 659.4126 [M+Na]+, .White amorphous powder; mentclass2pt{minim3 and 4 (each 2.0 mg) were hydrolyzed in 2 M HCl (3 mL) at 65 °C for 4 h, respectively. The reaction mixture was extracted with CH2Cl2, three times (3 × 3 mL). The aqueous layer was neutralized with 2 M NaOH and dried to produce a monosaccharide mixture. Next, l-cysteine methyl ester hydrochloride (1.0 mg) was added to a no aqueous pyridine solution of the sugar mixture (1.0 mL) and kept at 60 °C for 1 h. After this, trimethylsilylimidazole (1.0 mL) was added to the reaction mixture and kept at 60 °C for 30 min, followed the extraction with n-hexane (1 × 2 mL). The n-hexane layer was subjected to GC analysis, run on a Agilent Technologies HP5890 gas chromatograph, equipped with a HP-5 quartz capillary column (30 mm × 0.32 mm × 0.25 mm) and a H2 flame ionization detector with the following conditions: column temperature, 180–280 °C; programmed increase, 3 °C/min; carrier gas, N2 (1.5 mL/min); injector and detector temperature, 250 °C; injection volume, 2.0 μL; and split ratio 1/50. The configuration of the sugar moiety was determined by comparing the retention time with the derivatives of the authentic samples. The retention times of d-/l-glucose were 20.418/20.825 min, and the configuration of the respective sugar moiety from compounds 3 and 4 was determined as d-glucose.Compounds 5 cells/well) and treated with serial dilutions of the compounds with a maximum concentration of 50 μM in triplicate, followed by stimulation with 1 μg/mL LPS  for 18 h. Nitric oxide production in the supernatant was assessed by Griess reagents . The absorbance at 570 nm was measured with a microplate reader . NG-Methyl-l-arginine acetate salt , a well-known nitric oxide synthase (NOS) inhibitor, was used as a positive control. All the compounds were prepared as stock solutions in DMSO. The viability of RAW264.7 cells was evaluated by the MTS assay simultaneously to exclude the interference of the cytotoxicity of the test compounds. Concentration of a compound inhibiting 50% of cell growth (IC50) was calculated by the Reed and Muench method.As reported previously , 32, mur50) was calculated by the Reed and Muench method.Five human cancer cell lines, myeloid leukemia HL-60, lung cancer A-549 cells, hepatocellular carcinoma SMMC7721, breast cancer MCF-7, and colon cancer SW480, were used in the cytotoxic assay. All the cells were cultured in RPMI-1640 or DMEM medium , supplemented with 10% fetal bovine serum . The cytotoxicity assay was performed according to the MTS [3--5(3-carboxymethoxy-phenyl)-2-(4-sulfopheny)-2H-tetrazolium] method in 96-well microplates . Briefly1–2, 5, 8) and saponins  were isolated from the rot roots of P. notoginseng for the first time. Among them, 1–4 are new compounds. Their structures were determined on the basis of the extensive spectroscopic analyses and acidic hydrolysis. Compound 1 with a novel terminal ketene at C-24 to C-26 showed significant cytotoxic and anti-inflammatory activities.In summary, eight PPT-type triterpenes . (DOCX 5775 kb)Supplementary material 1D and 2D NMR, HRESIMS, IR, UV, OR, and CD spectra of compounds Below is the link to the electronic supplementary material.


We report new findings on multilayer silicene grown on Si(111)√3 × √3 R30°–Ag template, after the recent first compelling experimental evidence of its synthesis. Low-energy electron diffraction, reflection high-energy electron diffraction, and energy-dispersive grazing incidence X-ray diffraction measurements were performed to show up the fingerprints of √3 × √3 multilayer silicene. Angle-resolved photoemission spectroscopy displayed new features in the second surface Brillouin zone, attributed to the multilayer silicene on Si(111)√3 × √3 R30°–Ag. Band-structure dispersion theoretical calculations performed on a model of three honeycomb stacked layers, silicene grown on Si(111)√3 × √3 R30°-Ag surface confirm the experimental results. The first synthesis of multilayer silicene on Si111)√3 × √3 R30°–Ag interface used as a template was recently reported ), giving better real-space structural resolution, which is crucial in our experiment. Indeed, the inverse relation between X-ray energy and dhkl implies that when X-rays of high energy are available , then the diffraction from small d values can be recorded, resulting in a larger spanned q range.Among the advantages of this technique, the ED method gains data over a larger q range (q = (4π.sin ϑ)/λ = 2π/dImportantly, for the experiments presented in this paper, the fixed geometry of the ED experimental setup allowed a set of cradles to be allocated in the optical center of the diffractometer, enabling in-plane and out-of-plane rotations to be performed.In this way, since ED mode permits a multiple acquisition, the data collection, simultaneously as a function of the scattering angle ϑ, energy dispersive grazing wide-angle X-ray scattering (GWAXS), and grazing small-angle X-ray scattering (GSAXS) measurements was performed, tilting the sample along the in-plane direction (± α) and out-of-plane direction (Ψ), as shown in At first, a rocking curve (RC) analysis was performed upon the 10 MLs √3 × √3 Si film grown on reconstructed Si(111)√3–Ag, moving symmetrically the sample along the ±α directions, thus keeping the whole scattering-angle unchanged see , to miniz Si(111) = 2.003 (5) Å−1, corresponding to dz Si(111) = 3.136 Å, matching literature [ICDD card Nr. 00-04-0783], and as the RC analysis was performed, interestingly, the presence of multilayer silicene diffraction peaks located at qz 10 MLs = 2.032 (5) Å−1 became evident (red and blue dots) dz 10 MLs = 3.092 (5) Å, in good agreement with reference [Si(111) = 0.023 Å−1; FWHM 10 MLs = 0.011 Å−1 at α = 0.50°; FWHM Si(111) = 0.033 Å−1; FWHM 10 MLs = 0.019 Å−1 at α =1.50°.The Si111) diffraction peak (black dots) was observed at q11 diffraeference . In Figuz) were observed, but the in-plane Si(111)√3–Ag reconstruction and 10 MLs √3 × √3 Si film in-plane reflections were detected.Subsequently, to deduce in-plane structural information, the cradle was tilted along the Ψ direction, thus partially projecting the X-ray momentum transfer on the film plane. In this way, tilting both α and Ψ, simultaneous acquisition of in-plane and out-of-plane structural features was gained, and the substrate contribution was minimised. Indeed, as visible in xy 10 MLs = 1.947 (5) Å−1 was now visible, as well as the Si(111)√3-Ag reconstruction and 10 MLs √3 × √3 Si film, corresponding to the in-plane qxy Si(111)√3-Ag = 0.947 (5) Å−1 and qxy 10 MLs = 0.969 (5) Å−1 and plotted in the right inset. Furthermore, the left inset shows the in-plane multiple order reflection qxy 10 MLs = 0.482 (5) Å−1 (FWHMxy 10 MLs = 0.015 Å−1) from 10 MLs √3 × √3 Si film unit cell.Multilayer silicene in-plane second order reflection at qThe Si(111)√3-Ag template only was also characterized in order to observe out-of-plane and in-plane reflections of both Si and √3-Ag reconstruction, as an internal reference. As reported in Subsequently, to minimize the strong silicon contribution, rocking curve analysis was performed and ED-GIXRD pattern was collected, fixing α ~ 0.020° and ψ ~ 0.060°, respectively.xy Ag-Ag Triangles = 2.212 (5) Å−1, corresponding to dxy Ag-Ag Triangles = (2.840 ± 0.007) Å, and xy Si-Si Trimers = (2.910 ± 0.005) Å−1, dxy Si-Si Trimers = (2.159 ± 0.005) Å, related to the honeycomb-chained triangle (HTC) model of Si(111)√3–Ag [xy Si(111) = 2.91 Å−1, d220 = (1.920 ± 0.003) Å, producing a lattice parameter of Si(111) = 3.842 Å, in agreement with the results reported in the reference [11)√3–Ag ,33,34 anxy (in-plane)/qz (out-of-plane) and the corresponding lattice parameters/vertical separations d from the Si(111) substrate, Si(111)√3–Ag template, and 10 MLs multilayer film, grown at ∼ 200 °C on the Si(111)√3–Ag template.In These results are in close agreement with the atomic distances between Ag triangles and Si trimers of the well-known HCT model for the √3-Ag reconstruction on Si111) at room temperature ,32,33,3411 at rooP = 18.6 keV) electron beam along the [11The presence of √3 × √3 multilayer silicene reconstruction was also confirmed by the RHEED pattern, collected at RT √3–Ag, extensively explored in the past [1 state, with a downward convex dispersion, the less dispersive S2, and the upward convex largely dispersing S3 were attributed, by first-principle calculation and scanning tunneling spectroscopy measurements, to surface states derived mainly from Ag 5px and 5py orbitals (S1), and states consisting mainly of Ag 5s orbitals with some Ag 5p contribution (S2 and S3) [The bands dispersion at RT, reported in the past ,40,41,42 and S3) ,44,45. A1, S2, and S3 were observed in the dispersion of 1 surface state was slightly down-shifted in energy, most likely due to an extra doping from Ag atoms [These characteristic surface states SAg atoms ,46,47. T1, albeit with slightly different dispersion, could also be obtained for certain multilayer silicene structures without on-top Ag atoms. However, a band similar to S1 and S3 structures disappeared, giving place to two new structures S and S*, located at ~−1.15 and ~−0.6 eV below the Fermi level, set at 0 eV, (separated by 0.55 eV) with a gap of ~0.6 eV at 2 band was replaced by a well-evident flat band (FB), even located at ~−1.0 eV. To better distinguish these bands, we have reported in 1 and S3 (in red) from panel (c) and traced the new dispersion of features S and S * with a dotted line (in white). It can be immediately noticed that both their shape and energy position are different from S1 and S3 bands, suggesting that they belong to different origins. In addition, we fit the S1 state in 1, Ag-derived state and S*, Si-derived state, we superimpose this parabola to S*. The FB is also labeled.Let us now examine the dispersion of the panel shown in We hypothesize that these extremely localized bands around  Ag(111) ,48.1 and disperses down until the 1, pinned to the Fermi level with Si 3pz character and the dispersive band crossing the EF due to Ag antibonding d orbitals √3–Ag interface. This model predicts a layers stacking with internal honeycomb structure, showing a silicene √3 × √3 supercell on top of unperturbed Ag atoms. Clearly, band S* with similar intensity variations can be observed in tals see f. Since 11√3–Ag i−1 in the second BZ along the [11We reported new fingerprints of multilayer silicene grown on Si(111)√3 × √3 R30°–Ag at ~200 °C, by performing the energy-dispersive out-of-plane rocking curves and grazing incidence XRD patterns that clearly show the stacked distance between the silicene layers of 3.092 (5) Å and the first and multiple XRD orders of the √3 × √3 silicene unit cell size of 6.484 (5), both values different from tetragonal Si. Experimental angle-resolved photoemission spectroscopy on multilayer silicene evidences new electronic structures S, S*, and a flat band, FB, at This work corroborates the results already presented in Ref. , through


In each crystal of the title compounds the packing is driven by C—H⋯F intertactions, along with a variety of C—F⋯π, C—H⋯π, C—Br⋯N, C—H⋯N, and C—Br⋯π contacts. Hirshfeld surface analysis was conducted to aid in the visualization of these various influences on the packing. viz. 2,3,5,6-tetrafluoro-4-phenoxypyridine, C11H5F4NO (I), 4-(4-bromophenoxy)-2,3,5,6-tetrafluoropyridine, C11H4BrF4NO (II), 2,3,5,6-tetrafluoro-4-[oxy]pyridine, C15H7F4NO (III), 4-[oxy]-2,3,5,6-tetrafluoropyridine, C15H6BrF4NO (IV), and 2,2′-bis[(perfluoropyridin-4-yl)oxy]-1,1′-biphenyl, C22H8F8N2O2 (V). The dihedral angles between the aromatic ring systems in I–IV are 78.74 (8), 56.35 (8), 74.30 (7), and 64.34 (19)°, respectively. The complete molecule of V is generated by a crystallographic twofold axis: the dihedral angle between the pyridine ring and adjacent phenyl ring is 80.89 (5)° and the equivalent angle between the biphenyl rings is 27.30 (5)°. In each crystal, the packing is driven by C—H⋯F interactions, along with a variety of C—F⋯π, C—H⋯π, C—Br⋯N, C—H⋯N, and C—Br⋯π contacts. Hirshfeld surface analysis was conducted to aid in the visualization of these various influences on the packing.Five new crystal structures of perfluoropyridine substituted in the 4-position with phenoxy, 4-bromophenoxy, naphthalen-2-yloxy, 6-bromonaphthalen-2-yloxy, and 4,4′-biphenoxy are reported,  It is commercially available and its chemistry is well understood. As a result of the presence of five fluorine atoms, in addition to the nitrogen atom of the pyridine ring, these systems are highly electrophilic and undergo substitution reactions readily in a predictable pattern  and 80.89 (5)°. In V, the rings of the biphenyl system are rotated by 27.30 (5)° from each other.Compounds ly Fig. 1. With thII, the packing is aided by C—H⋯π and C—Br⋯N interactions, the latter of which lead to the formation of chains in the pyridine (III) To a stirred solution of potassium carbonate , naphthalen-2-ol , pentafluoropyridine , and DMF (100 ml) were added. The resulting solution was allowed to stir at room temperature for 24 h. Dichloromethane (50 ml) and saturated aqueous ammonium chloride (100 ml) were added and the biphasic solution stirred vigorously for an additional 24 h. The organic layer was separated, washed with water (5 × 200 ml), dried over MgSO4, and solvent removed via rotary evaporation. The resulting pale brown solid was dissolved in refluxing EtOH (50 ml) and cooled to 298 K for 12 h. Vacuum filtration, washing with cold EtOH (20 ml) and vacuum drying afforded the target compound as a white, crystalline solid . Colourless rectangular prisms were obtained from a saturated EtOH solution by cooling to 298 K. 1H NMR : 7.90-7.86 , 7.76 , 7.54-7.47 , 7.35–7.31 . 19F NMR : −88.3, −154.0.2,3,5,6-Tetrafluoro-4-[oxy]pyridine (IV) To a stirred solution of potassium carbonate , 6-bromo-2-naphthol , pentafluoropyridine , and DMF (60 ml) were added. The resulting solution was allowed to stir at room temperature for 24 h. Diethyl ether (50 ml) and saturated aqueous ammonium chloride (100 ml) were added and the biphasic solution stirred vigorously for an additional 2 h. The organic layer was separated, washed with water (5 × 200 ml), dried over MgSO4, and solvent removed via rotary evaporation. The resulting off-white solid was dissolved in refluxing EtOH (40 ml) and cooled to 298 K for 12 h. Vacuum filtration, washing with cold EtOH (20 ml) and vacuum drying afforded the target compound as a white, crystalline solid . Colourless plates were obtained from a saturated EtOH solution by cooling to 298 K. 1H NMR : 8.00 , 7.81–7.75 , 7.63–7.55 , 7.35–7.27 . 19F NMR : −87.9, −153.8.2,2′-Bis[oxy]-1,1′-biphenyl (V) To a stirred solution of potassium carbonate , 2,2′-biphenol , pentafluoropyridine , and DMF (30 ml) were added. The resulting solution was allowed to stir at room temperature for 24 h. Diethyl ether (50 ml) and saturated aqueous ammonium chloride (100 ml) were added and the biphasic solution stirred vigorously for an additional 2 h. The organic layer was separated, washed with water (5 × 200 ml), dried over MgSO4, and solvent removed via rotary evaporation. The resulting off-white solid was dissolved in refluxing EtOH (10 ml) and cooled to 298 K for 12 h. Vacuum filtration, washing with cold EtOH (20 ml) and vacuum drying afforded the target compound as a white solid . Colourless rectangular prisms were obtained from a saturated EtOH solution by cooling to 298 K. 1H NMR : 7.45–7.24 , 6.99 . 19F NMR : −88.9, −155.0.Uiso(H) = 1.2Ueq(C). The absolute structures of I and III were intermediate in the present refinement. Compound IV was refined as an inversion twin.Crystal data, data collection and structure refinement details are summarized in Table 1110.1107/S2056989019009344/hb7833sup1.cifCrystal structure: contains datablock(s) global, I, II, III, IV, V. DOI: 10.1107/S2056989019009344/hb7833Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989019009344/hb7833Isup7.cmlSupporting information file. DOI: 10.1107/S2056989019009344/hb7833IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989019009344/hb7833IIsup8.cmlSupporting information file. DOI: 10.1107/S2056989019009344/hb7833IIIsup4.hklStructure factors: contains datablock(s) III. DOI: Click here for additional data file.10.1107/S2056989019009344/hb7833IIIsup9.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989019009344/hb7833IVsup10.cmlSupporting information file. DOI: 10.1107/S2056989019009344/hb7833IVsup5.hklStructure factors: contains datablock(s) IV. DOI: Click here for additional data file.10.1107/S2056989019009344/hb7833Vsup11.cmlSupporting information file. DOI: 10.1107/S2056989019009344/hb7833Vsup6.hklStructure factors: contains datablock(s) V. DOI: 1937619, 1937620, 1937621, 1937622, 1937623CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In summary, we present a system for the creation of inducibly αSyn-overexpressing cell lines holding high potential for the screening for modulators of αSyn aggregation and αSyn-mediated toxicity.Parkinson’s disease (PD) represents an increasing problem in society. The oligomerization of alpha-synuclein (αSyn) is a suggested key event in its pathogenesis, yet the pathological modes of action remain to be fully elucidated. To identify potential disease-modifying therapeutics and to study αSyn-mediated toxic mechanisms, we established cell lines with inducible overexpression of different αSyn constructs: αSyn, αSyn coupled to the fluorescence protein Venus (αSyn-Venus), and αSyn coupled to the N-terminal or C-terminal part of Venus  for a bimolecular fluorescence complementation assay (BiFC). Inducibility was achieved by applying modified GAL4-UAS or Cre-loxP systems and addition of tebufenozide or 4-OH-tamoxifen, respectively. Expression constructs were stably integrated into the host genome of H4 neuroglioma cells by lentiviral transduction. We here demonstrate a detailed investigation of the expression characteristics of inducible H4 cells showing low background expression and high inducibility. We observed increased protein load and aggregation of αSyn upon incubation with DMSO and FeCl The loss of dopaminergic neurons in the substantia nigra (SN) and Lewy bodies or Lewy neurites in some of the remaining neurons – with αSyn occurring as highly ordered amyloid-type fibril as principal component – represent the histopathological hallmarks of PDH-pyrazole shows beneficial effects on pathology in in vivo models of synucleinopathies5, yet – to date – the distinct toxic αSyn species and the pathological mechanisms are not completely understood6. It has also been suggested and discussed critically that αSyn may exist mainly as physiological tetramer8 and that the disruption of tetrameric αSyn to unfolded monomers represents the starting point for pathological αSyn aggregation9. Interestingly, the overexpression of αSyn appears to be sufficient to induce pathological effects, since duplication or triplication of the SNCA gene cause parkinsonian symptoms with the age of onset and severity of symptoms correlating with the gene copy number12. Additionally, modulating SNCA transcription by targeting the β2-adrenoreceptor (β2AR) using salbutamol is associated with reduced risk of developing PD13. In several epidemiological studies environmental risk factors for the development of PD have been shown to influence αSyn aggregation14, including the exposure to heavy metals and iron15.To date, several lines of evidence point towards small αSyn oligomers as driver of toxicity, and the small molecule modulator of αSyn oligomerization 3--5-(3-bromophenyl)-116 or viral transduction17 or in cell models with stable insertion and constitutive overexpression19. Both strategies hold several drawbacks: (a) when using transient transfection or viral transduction, the fraction of transgene expressing cells and the strength of overexpression are subject to great inter-experimental variation. Moreover, individual experiments are rather time-consuming and expensive. Additionally, the initiation of expression cannot be defined accurately, and for several cell lines transient transfection is very inefficient. (b) Constitutive overexpression of αSyn on the other hand enables the investigation of αSyn oligomers only in steady state but not the investigation of de novo oligomer formation. This hinders the identification of compounds which prevent αSyn aggregation but are not capable of degrading or dissociating preformed αSyn aggregates leading to false-negative results in drug screening. Moreover, the constitutive overexpression of αSyn and the resulting αSyn-mediated toxicity may result in selection for cells that are resistant to αSyn-mediated toxicity. This might interfere with the investigation of potential toxic effects.So far, formation, modulation, and toxicity of αSyn oligomers have mainly been studied in cell models in which the overexpression of αSyn is induced by transient transfection20, αSyn-140 coupled to Venus (SV)  Fig.  which isSV) Fig. , the N-tSV) Fig. 19,21.FigT2-loxP (CET2) system23  showed higher transgene induction than the H4 cell lines relying on the CET2 system (H4_CET2 cells), the expression characteristics of the tebufenozide-dependent H4_GE cells were analysed in detail. Using this cell model we further found that incubation with DMSO and ferric iron led to an increase in fluorescence intensity in the BiFC assay indicating αSyn oligomer formation which was confirmed by sucrose gradient centrifugation. Increased aggregation was accompanied by an increase in cytotoxicity as demonstrated by quantification of condensed nuclei in both H4_GE cells expressing V1S + SV2 and untagged αSyn, further validating the value of our model for studying risk factors of PD development, analysing clearance mechanisms, or screening for therapeutic compounds.Overexpression relied on the Cre_ERm23 Fig.  or the Gtem Fig. . Both syAfter production and purification of the required lentiviruses to prepare stable cell lines we established an optimized transduction protocol to obtain as many lentivirally transduced H4 cells as possible , H4_mC_GE-S  resulting in red fluorescence in the nucleus of positively transduced cells and inducible overexpression of αSyn), H4_GE-V (inducible overexpression of Venus), H4_GE-SV (inducible overexpression of αSyn coupled to Venus), and H4_GE-V1S + SV2 (inducible overexpression of V1S and SV2).T2 system were created: H4_CET2-S (inducible overexpression of αSyn), H4_CET2-V (inducible overexpression of Venus), H4_CET2-SV (inducible overexpression of αSyn coupled to Venus), and H4_CET2-V1S + SV2 (inducible overexpression of V1S and SV2)  with different tebufenozide concentrations ranging from 100 pM to 100 µM or DMSO as control, respectively.No increase in mean cellular Venus fluorescence was observed in H4, H4_GE-S, or H4_mC_GE-S cells Fig. . In lineOn the contrary, the fluorescence signal increased in a time- and tebufenozide concentration-dependent manner from day 0 on in H4_GE-V, H4_GE-SV, and H4_GE-V1S + SV2 cells Fig. .In H4_GE-V cells, maximum fluorescence was obtained upon incubation with 1 µM tebufenozide on DPI 5 Fig.  with an For H4_GE-SV cells, maximum fluorescence was obtained upon incubation with 10 µM tebufenozide on DPI 6 Fig.  with an In H4_GE-V1S + SV2 cells fluorescence intensity increased more slowly than in H4_GE-V and H4_GE-SV cells and reached its maximum on DPI 6 upon incubation with 10 µM tebufenozide Fig.  with an For H4_GE-V, H4_GE-SV, and H4_GE-V1S + SV2 cells, fluorescence intensity increased with increasing tebufenozide concentrations in the range of 100 pM to 10 µM and decreased when cells were incubated with 100 µM tebufenozide Fig. .For all cell lines the number of cells increased until DPI 4 or 5. No obvious effect on cell number was observed for all tebufenozide concentrations except for treatment with 100 µM tebufenozide where the number of cells was decreased in all cell lines.In order to further characterize the cell lines regarding their transgene expression properties in dependence of the tebufenozide concentration, the cells were incubated for 4 days with different tebufenozide concentrations ranging from 10 nM to 10 µM or with DMSO as control, respectively. After 4 days cells were harvested, lysed and subjected to Western blot analyses.As expected, we observed no transgene expression in H4 cells Fig. . For allCell lines H4_GE-S, H4_mC_GE-S, H4_GE-V, and H4_GE-SV showed a maximum in transgene expression after incubation with 10 µM tebufenozide, whereas maximum transgene expression in H4_GE-V1S + SV2 was observed upon incubation with 1 µM tebufenozide. According to the quantification of Western blot signal intensity we obtained a maximum increase in transgene expression compared to DMSO by a factor of 16.8 in H4_GE-S, 11.5 in H4_mC_GE-S, 5.6 in H4_GE-V, 7.2 in H4_GE-SV, and 11.9 in H4_GE-V1S + SV2 for SV2 expression. Concerning the expression of V1S in H4_GE-V1S + SV2 the factorial increase in transgene expression could not be evaluated since no background signal for treatment with DMSO was detected.In order to further characterize the cell lines regarding their expression kinetics according to protein levels, the cells were incubated for up to 5 days with 10 µM tebufenozide since this reached the highest transgene expression in most cell lines see Figs , 4. CellAs expected, no transgene expression was observed in H4 cells Fig. . For allin vitro15. In order to validate our model, we investigated if this effect of DMSO and ferric iron on αSyn aggregation could be reproduced in the H4_GE cell model. For this, H4_GE-V1S + SV2 and H4_GE-V cells were incubated with different combinations of DMSO (ranging from 0.1% to 1.0%) and FeCl3 concentrations (ranging from 0 µM to 1 mM) for 3 days. Transgene expression was induced with 10 µM tebufenozide in H4_GE-V1S + SV2 cells and with 10 nM tebufenozide in H4_GE-V cells for three days in order to reach comparable fluorescence intensities. After 3 days, cellular fluorescence intensity was determined using the Opera® setup.DMSO and ferric iron have been shown to increase aggregation of αSyn 3 concentrations in H4_GE-V1S + SV2 cells  compared to H4_GE-V cells overexpressing Venus. Since we observed many dead and rounded cells upon incubation with 1% DMSO and iron precipitation upon incubation with 1 mM FeCllls Fig. . An incr3 for H4_GE-S and H4_GE-V1S + SV2 cells compared to cells treated with 0.1% DMSO. In contrast, we found no difference between both treatments in H4_GE-V cells  when cells were treated with 0.75% DMSO and 100 µM FeCllls Fig. .3.Moreover, we developed an evaluation script for the quantification of cell death relying on the analysis of nuclear morphology. We here observed a strong increase in the fraction of condensed nuclei in H4 cell lines overexpressing αSyn compared to Venus-overexpressing cells upon incubation with 0.75% DMSO and 100 µM FeClin vitro findings from Kostka et al.15, these findings suggest increased aggregation of αSyn in H4_GE cells upon incubation with high concentrations of DMSO and FeCl3 which goes along with increased cytotoxicity. The findings from the BiFC-assay in H4_GE-V1S + SV2 cells were confirmed in cell lines expressing untagged αSyn, further validating the significance of our model system.In line with the T2 cells were created using lentiviral transduction resulting in long-term stable gene expression , the YFP variant Venus (V), αSyn coupled to Venus (SV), and constructs for a BiFC assay V1S and SV2). Inducible H4_GE and H4_CES and SV2T2 system we performed a detailed characterization of its expression characteristics using the Opera® system and Western blot analyses and obtained almost 100% of transgene expressing cells for all H4_GE cell lines that express fluorescence proteins . Thus, E-S Fig. . This coE-S Fig. . Recentlion Figs , 5. A reet al. observed αSyn immunoreactivity in H4 BiFC cells which did not display Venus fluorescence suggesting the presence of monomeric αSyn27.Interestingly, the fluorescence intensity and the fraction of positive cells increased more slowly in the H4_GE-V1S + SV2 cells compared to H4_GE-V or H4_GE-SV cells Fig. , while p24. The decreased fluorescence intensity observed when incubating cells with 100 µM tebufenozide can most likely be explained by precipitation of tebufenozide. Background expression in the absence of tebufenozide was very low or not detectable at all for all H4_GE cell lines . S was amplified from plasmid #3 using primers MB8_SV_Hind3_f and MB11_Syn_Not1_r, V1S from plasmid #1 (primers MB24_V1_HindIIIf and MB6_V1S_Not1_r), SV2 from plasmid #2 (primers MB3_SV2_Hind3_f and MB25_V2_NotI_r), SV from plasmid #3 (primers MB8_SV_Hind3_f and MB9_SV_PspOMI_r), V from plasmid #3 (primers MB10_V_Hind3_f and MB9_SV_PspOMI_r).® Technology47. Vector #92 was created by amplifying mCherry-NLS from plasmid #8748 (pmCherry-NLS was a gift from Martin Offterdinger (Addgene plasmid # 39319)) using primers 42MB_SalI-mCherry_f and 43MB_NsiI_mCherry_r and subcloning between the XhoI and NsiI sites of plasmid #45.For lentiviral expression plasmids, constructs were inserted between the HindIII and NotI site of pCR8/GW/TOPO + pCS2-MCS and then transferred to the destination vectors 636, 597, #44, and #92 . Both constructs were subcloned between the HindIII and NotI sites of P12-HA-TGFa-FLAG  were maintained in DMEM  supplemented with 1% glutamine  and 10% fetal bovine serum . H4 human neuroglioma cells  were maintained in Opti-MEM  supplemented with 10% FBS . Cells were grown at 37 °C in a humidified 95% air/5% CO® 2000 according to the manufacturer’s instructions.Packaging plasmids #97 and #98, and corresponding expression plasmids ), sterile filtered) overnight, then centrifuged . Supernatant was frozen in 20 µl aliquots at −80 °C.Virus-containing medium was collected after 48 h and 72 h and centrifuged . Afterwards, supernatant was filtered using a 0.45-µm filter and centrifuged , Sorvall Discovery 90SE ultracentrifuge). Pellets were incubated in TBS-5 buffer (50 mM Tris-HCl (pH 7.8), 130 mM NaCl, 10 mM KCl, 5 mM MgClH-pyrazole or anle138c (synthesized as described previously5) with a final DMSO concentration of 0.1%. After 24 h, cells were washed with DPBS and fresh normal growth medium was added. Afterwards, cells were transiently transfected with plasmids #71 and #75 using X-tremeGENETM HP DNA Transfection Reagent according to the manufacturer’s instructions . After 2 h, cells were washed with DPBS and fresh normal growth medium containing the corresponding compound or DMSO was added for 48 h.100,000 cells/ml were incubated with 0.1% DMSO as a control or 10 µM 3--5-(3-bromophenyl)-1ImageJ software.To optimize the transduction protocol we determined the fraction of fluorescing cells for different protocols manually using the Protocol A: V99 and V41 were diluted 1:200 in 2 ml of cell-containing medium .Protocol B: V99 and V41 were diluted 1:50 in 2 ml cell-containing medium .Protocol C: V99 and V41 were diluted 1:12 in 200 µl cell-containing medium .Protocol D: V41 was diluted 1:3.5 in 50 µl cell-containing medium. Cells were expanded and maintained as described above. After two weeks, the obtained H4_41 cells were transduced with V99.For all protocols, cell nuclei were stained with 0.5 µg/ml Hoechst33342  72 h after transduction.H4_100-41 (H4_GE-SV), H4_100-422 (H4_GE-S), H4_100-43 (H4_GE-V), H4_100-120 (H4_mC_GE-S) and H4_100-392-462 (H4_GE-V1S + SV2). For the CET2 system, cells were transduced with V102, V32, V33, V35, V36, or V37, respectively (see Supplementary Table H4_102-32-33 (H4_ CET2-V1S + SV2), H4_102-35 (H4_CET2-SV), H4_102-36 (H4_ CET2-S), and H4_102-37 (H4_ CET2-V).H4 cells were transduced according to protocol D (described above). For the GE system, cells were transduced with V100, V39 (twice), V41, V42 (twice), V43, V46 (twice) and V120, respectively, resulting in the following cell lines: Cells were expanded and stored in the gas phase of liquid nitrogen. For each experiment a new aliquot was unfrozen to obtain reproducible cell populations.Tebufenozide  and (z)-4-OH-tamoxifen  were diluted in DMSO to a final concentration of 100 mM. To induce transgene expression, cells were incubated with 100 pM to 100 µM and a final DMSO concentration of 0.1%.50, 4B12 ), anti-GFP , anti-GAPDH , anti-tubulin , anti-actin ) diluted in 5% milk in TBS-T supplemented with 0.02% NaN3. After washing, the membranes were incubated with a secondary antibody (rabbit-anti-rat IgG (H&L) -conjugated) , goat-anti-rabbit IgG (AP-conjugated) , goat-anti-mouse IgG (AP-conjugated) ), goat-anti-rabbit (horseradish peroxidase (HRP)-conjugated) , horse-anti-mouse (HRP-conjugated) ) diluted in 5% milk in TBS-T for 1 h. After washing, the membranes were prepared for signal detection using CDP-Star  for AP coupled antibodies or using the ClarityTM Western ECL Substrate  for HRP coupled antibodies, respectively. Chemiluminescence signals were detected with the ChemiLux camera system and the ChemoStar software (Intas).Cell pellets were resuspended in lysis buffer ), incubated at 4 °C and 1,000 rpm on a Thermomixer Comfort (Eppendorf) for 30 min and centrifuged . Protein concentration was adjusted to 2.08 mg/ml. After blotting, the membranes were incubated overnight with primary antibodies (anti-αSyn (15G7Cell pellets were resuspended in 40 µl SC lysis buffer (50 mM Tris (pH 7.4), 175 mM NaCl, 0.1% NP-40, 1x protease inhibitor) and incubated on ice for 15 min twice before centrifugation . Concentration of the supernatant adjusted to 50–200 µg of total protein in 200 µl in 50 mM Tris (pH 7.4), 175 mM NaCl, and 0.1% NP-40. Centrifuge tubes were filled with buffer A (50 mM Tris-HCl (pH 7.5), 0.1% NP-40, 0–60% Sucrose) in layers in the given order: 200 µl 60% sucrose, 400 µl each of 50–10% sucrose, 200 µl sample without sucrose. The samples were ultracentrifuged in a Sorvall WX Ultra 90 (Thermo Scientific) , 70 min, 4 °C) and 12 fractions of 200 µl each were collected, mixed with 800 µl of 12.5% trichloroacetic acid (TCA) and incubated at 20 °C overnight. After centrifugation at 4 °C and 20,000 rcf for 15 min, pellets were washed with 1 ml precooled acetone (−20 °C) and centrifuged at 4 °C and 20,000 rcf for 15 min. Pellet was allowed to dry at RT for 5 min. 30 µl of 5x Lämmli sample buffer were added and samples were shaken at 30 °C and 1,400 rpm for 10 min followed by incubated at 96 °C for 5 min.51 using an InsightTM Reader (Evotec-Technologies). The total brightness (Itot) was adapted to 50 to 100 kHz by dilution in RIPA buffer . Laser power of the argon-ion laser (488 nm) was adjusted to 200 µW. Measurement was performed for 10–15 seconds at RT, samples were scanned in a length of 100 µm with a frequency of 50 Hz of the beam scanner and a displacement of 2,000 µm of the sample carrier. Data were analysed by fluorescence intensity distribution analysis52 (FIDA) using the FCSPP evaluation software version 2.0 (Evotec-Technologies).Cell lysis and single particle spectroscopy was performed as described previously® high-throughput confocal imaging platform . Nuclei were stained by with 5 µM Draq5 . All measurements were performed at RT using the 20x air objective. A detailed description of the setup and data analysis can be found in the supplementary Methods  in order to quantify cellular Venus fluorescence and the fraction of condensed nuclei (Supplementary Fig. For data analysis, an automated image analysis tool was developed using the AcapellaThe datasets generated during the current study are available from the corresponding author on reasonable request.Supplementary Information


In the crystal of the title salt, cations and anions are linked by N–H⋯Br hydrogen bonds forming inversion-related dimers. The dimers are connected by weak C–H⋯Br hydrogen bonds into chains. 16H15FN3S+·Br−, the phenyl ring is disordered over two sets of sites with a refined occupancy ratio of 0.503 (4):0.497 (4). The mean plane of the thiazolidine ring makes dihedral angles of 13.51 (14), 48.6 (3) and 76.5 (3)° with the fluorophenyl ring and the major- and minor-disorder components of the phenyl ring, respectively. The central thiazolidine ring adopts an envelope conformation. In the crystal, centrosymmetrically related cations and anions are linked into dimeric units via N—H⋯Br hydrogen bonds, which are further connected by weak C—H⋯Br hydrogen bonds into chains parallel to [110]. Hirshfeld surface analysis and two-dimensional fingerprint plots indicate that the most important contributions to the crystal packing are from H⋯H (44.3%), Br⋯H/H⋯Br (16.8%), C⋯H/H⋯C (13.9%), F⋯H/H⋯F (10.3%) and S⋯H/H⋯S (3.8%) interactions.In the cation of the title salt, C The mean plane of the thiazolidine ring makes dihedral angles of 13.51 (14), 48.6 (3) and 76.5 (3)° with the fluorophenyl ring (C5–C10) and the major- and minor-disorder components (C11–C16 and C11′–C16′) of the phenyl ring, respectively. The N2—N1—C4—C5 bridge that links the thiazolidine and 4-fluorophenyl rings has a torsion angle of −177.36 (19)°.The thiazolidine ring (S1/N2/C1–C3) in the cation of the title salt Fig. 1 adopts avia pairs of N—H⋯Br hydrogen bonds  to 1.2166 a.u. (blue) is shown in Fig. 3Hirshfeld surface analysis was used to investigate the presence of hydrogen bonds and intermolecular interactions in the crystal structure. The Hirshfeld surface analysis  shows the 2D fingerprint of the sum of the contacts contributing to the Hirshfeld surface represented in normal mode. These represent both the overall 2D fingerprint plots and those that represent H⋯H (44.3%), Br⋯H/H⋯Br (16.8%), C⋯H/H⋯C (13.9%), F⋯H/H⋯F (10.3%) and S⋯H/H⋯S (3.8%) contacts, respectively . The most significant intermolecular interactions are the H⋯H interactions (44.3%) . All the contributions to the Hirshfeld surface are given in Table 2Fig. 5y Figs. 5b–f. Theet al., 2016viz. UDELUN  contacts.In the crystal of UDELUN :0.179 (3). The system of hydrogen bonds connecting two cations and two anions into 12-membered rings is identical in the racemic and in the optically active crystals. YITCEJ  : 4.56 , 4,87 , 5.60 , 7.32–8.16 , 8.45 , 10.37 . 13C NMR : 45.39, 55.97, 116.05, 127.81, 128.91, 129.13, 129.60, 131.05, 131.17, 137.55, 150.00, 167.89. MS (ESI), m/z: 300.36 [C16H15FN3S]+ and 79.88 Br−.To a solution of 3-amino-5-phenylthiazolidin-2-iminium bromide (1 mmol) in ethanol (20 ml) was added 4-fluorobenzaldehyde (1 mmol). The mixture was refluxed for 2 h and then cooled. The reaction product precipitated from the reaction mixture as colourless single crystals, was collected by filtration and washed with cold acetone . Analysis calculated (%) for CUiso(H) = 1.2Ueq. The phenyl ring in the cation is disordered over two sets of sites with an occupancy ratio of 0.503 (4):0.497 (4). Seven outliers  I. DOI: 10.1107/S2056989019004973/rz5254Isup2.hklStructure factors: contains datablock(s) I. DOI: 1909594CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: