The five-membered chelate ring adopts an envelope conformation, with puckering parameters of Q(2) = 0.446 (6) Å and ϕ(2) = 88.8 (6)°, with the two amine CH3 substituents on opposite sides of the ring. In the crystal, the molecules are linked by C—H⋯Cl interactions into a zigzag chain parallel to [101].In the structure of the title compound, [HgCl II, which facilitates extraordinarily rapid exchange of simple ligands -N1,N2,N2-trimethylethane-1,2-diamine, can be prepared by the reaction of N1,N1,N2-trimethylethane-1,2-diamine and ortho-bromobenzyl bromide. The ligand is moisture sensitive and is difficult to purify by column chromatography. However, it could easily be purified by vacuum distillation. The moisture-sensitive ligand, when treated with n-BuLi in tetrahydrofuran (THF) and mercuric chloride, afforded the title compound, [HgCl2(C12H20N2)], (3)  Fig. 1.II atom is four-coordinated by two tertiary amine N-atom donors, as well as two Cl− anions to give a distorted tetrahedral coordination environment  = 0.446 (6)Å and ϕ(2) = 88.8 (6)° , the Hgnt Fig. 2. The disnt Fig. 2. The fiv3 substituents on opposite sides of the chelate ring, there is no evidence of any π–π interactions.The molecular adducts are linked by C—H⋯Cl interactions Table 1 into a zII with an N2Cl2 coordination sphere gave 96 hits, but the vast majority of these involved aromatic N donors such as pyridine and imidazole. There were only six hits involving aliphatic amine N-atom donors and only two -N1,N2,N2-trimethylethane-1,2-diamine, (1),  in dry THF (15 ml) was treated dropwise with a 1.6 M solution of n-BuLi in hexane  via syringe under N2 at 273 K. On stirring the reaction mixture for 2 h at this temperature, the lithiated product (2) was obtained. Mercuric chloride  was added to the reaction mixture under a brisk flow of N2 gas and stirring was continued for an additional 6 h at room temperature. The reaction mixture was then removed from the N2 line and evaporated to dryness to give a colourless hygroscopic solid. The solid was extracted with dry chloroform. The organic phase was separated, dried over Na2SO4, and filtered. The filtrate was evaporated to dryness to give a colourless crystalline solid of the HgCl2 adduct of N1-benzyl-N1,N2,N2-trimethylethane-1,2-diamine, (3) . The reaction scheme is shown in Fig. 1A stirred solution of Uiso(H) = 1.2Ueq(C), and C—H = 0.98 Å for methyl H atoms, with Uiso(H) = 1.5Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S1600536814017516/wm5031sup1.cifCrystal structure: contains datablock(s) 3. DOI: 10.1107/S1600536814017516/wm50313sup2.hklStructure factors: contains datablock(s) 3. DOI: 1016995CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Background. Precise estimation of the glomerular filtration rate (GFR) and the identification of markers of progression are important. We compared creatinine, cystatin, and combined CKD-EPI equations with 99mTc-DTPA scintigraphy to measure GFR and proteinuria as markers of progression. Methods. Cross-sectional, observational study including 300 subjects. CKD was classified by 99mTc-DTPA scintigraphy. Determinations. Creatinine, 24-hour creatinine clearance, cystatin, Hoek formula, and creatinine, cystatin, and combined CKD-EPI equations. Results. In the global assessment, creatinine CKD-EPI and combined CKD-EPI equations yielded the highest correlations with 99mTc-DTPA: ρ = 0.839, P < 0.0001 and ρ = 0.831, P < 0.0001. Intergroup analysis versus 99mTc-DTPA: control G, creatinine clearance ρ = 0.414, P = 0.013; G3, combined CKD-EPI ρ = 0.5317, P < 0.0001; G4, Hoek ρ = 0.618, P < 0.0001, combined CKD-EPI ρ = 0.4638, P < 0.0001; and G5, creatinine clearance ρ = 0.5414, P < 0.0001, combined CKD-EPI ρ = 0.5288, P < 0.0001. In the global assessment, proteinuria displayed the highest significant correlations with cystatin  and cystatin-based equations . When GFR < 60 mL/min: in stage 3, proteinuria-cystatin ; proteinuria-Hoek ; in stage 4, proteinuria-cystatin ; proteinuria-Hoek . Conclusions. At every stage of GFR < 60 mL/min, cystatin-based equations displayed better correlations with 99mTc-DTPA. Proteinuria and cystatin-based equations showed strong associations and high degrees of correlation. Kidney Disease Outcomes Quality Initiative (K/DOQI) guidelines, widely employed in clinical practice, stratify CKD into 5 stages according to the GFR estimated through the depuration of creatinine . Female or male ≤0.8 133 × (cystatin C/0.8) −1.328  × 0.996age [× 0.932 if female].Female or male >0.8 133 × (cystatin C/0.8) Combined CKD EPI: Female If creatinine ≤ 0.7 or cystatin C ≤ 0.8 age. 130 × (creatinine/0.7) − 0.248 × (cystatin C/0.8) − 0.375 × 0.995 If cystatin C > 0.8 age. 130 × (creatinine/0.7) − 0.248 × (cystatin C/0.8) − 0.711 × 0.995 Female If creatinine > 0.7 or ≤0.8  age. 130 × (creatinine/0.7) − 0.601 × (cystatin C/0.8) − 0.375 × 0.995 If cystatin C > 0.8  age. 130 × (creatinine/0.7) − 0.601 × (cystatin C/0.8) − 0.711 × 0.995 Male  If creatinine ≤ 0.9 or cystatin C ≤ 0.8 age. 135 × (creatinine/0.9) − 0.207 × (cystatin C/0.8) − 0.375 × 0.995  If cystatin C > 0.8 age. 135 × (creatinine/0.9) − 0.207 × (cystatin C/0.8) − 0.711 × 0.995  Male  If creatinine > 0.9 or ≤0.8  age. 135 × (creatinine/0.9) − 0.601 × (cystatin C/0.8) − 0.375 × 0.995  If cystatin C > 0.8  age.135 × (creatinine/0.9) − 0.601 × (cystatin C/0.8) − 0.711 × 0.99599mTc-DTPA gammagraphy was performed in all 300 subjects as the gold standard method to assess GFR and consequently stratify CKD . In . In 6]. mTc-DTPA . HoweverA Tables . These ft Tables –5. ConseBesides, the main purpose of the development of newer equations should be focused on a higher precision of early detection of renal damage. Why is it important to focus on a correct GFR estimation at these early stages of CKD? It is important due to the large quantity of false negative CKD cases that are apparently being reported using the current methods , 14. Thi99mTc-DTPA, particularly at stages 3 and 4 (Tables 99mTc-DTPA scyntigraphy, and higher correlations between proteinuria and cystatin C-related equations when compared to creatinine-based ones, a tempting hypothesis to explain this situation is that 99mTc-DTPA and cystatin C themselves are only filtered by the glomerulus. In this situation, one should assume that in our population proteinuria is composed of albumin, which is exclusively filtered by the glomerulus as well. This phenomenon could also explain the lower correlations encountered between proteinuria and creatinine, as this molecule is excreted by the glomerulus and the proximal tubule. Noteworthy, it has recently been published that in the Asian population, an elevated serum cystatin C level could also be considered as an independent predictor of cardiovascular events in subjects with normal renal function, as it has been demonstrated for age and hypertension [When proteinuria was analyzed with respect to GFR ≤ 60 mL/min, it presented an inverse strong significant correlation with 4 Tables  and 9. W4 Tables . Albeit rtension . In thisrtension . In a rertension . In othertension –41. In o99mTc-DTPA scyntigraphy was used as the gold standard for GFR measurement, but it is not used routinely due to its cost, being time consuming and laborious [With regard to the limitations of the present study, the cohort was not matched according to gender or age, and the different BMI varied significantly among the stages , which caborious . Particu99mTc-D TPA scyntigraphy is employed at GFR < 60 mL/min but shows no advantages at earlier stages of kidney disease. Cystatin C is a more expensive determination, not applicable in many nephrology facilities, and with certain evidence of advantages compared with creatinine, and may be a better surrogate of CKD with respect to cardiovascular risk and proteinuria. However, cystatin C role in nephrology and as a useful tool to measure GFR in CKD has not been established yet, and more clinical data is needed. Finally, we believe that combined CKD-EPI equations require ample validation before being introduced for CKD staging.Kidney function assessment with combined CKD-EPI appears to be superior to other equations when


S,4S,5R,7aR)-2,2,7-trimethyl-3a,4,5,7a- tetrahydro-1,3-benzodioxole-4,5-diol shows that the absolute configuration determined from the synthesis pathway agrees with that determined by X-ray analysis.The crystal structure of enantiopure ° as a result of their cis-fusion. In the crystal, molecules are linked by two pairs of O—H⋯O hydrogen bonds, forming chains along [010]. These chains are further connected by weaker C—H⋯O interactions along [100], creating (001) sheets that interact only by weak van der Waals forces.The absolute configuration of the title compound, C The six-membered ring (C3A–C4–C5–C6–C7–C7A) adopts an envelope conformation with atom C4 as the flap. In this case, the puckering parameters are Q = 0.466 (2) Å, θ = 52.1 (2) and φ = 50.8 (3)°. The fused rings are nearly perpendicular with a dihedral angle of 76.20 (11)° as a result of their cis-fusion.Fig. 2i and O51—H51⋯O41i  hydrogen bonds along [100], forming (001) sheets. Hydrogen bonds of the O—H⋯O type generate In the crystal structure, the molecules are connected in the three crystallographic directions by intermolecular interactions of different strengths Table 1. In the fs Fig. 3. There acis-fused rings). The C7—C7A—C3A—C4 torsion angle is positive if the flap atom is located on the opposite side of the plane (defined by the remaining five atoms of the cyclohexene ring) to O1 and O3 of the 1,3-dioxole ring, as observed in the title compound [33.5 (2)°]. 12 of the 20 mentioned structures, show a positive torsion angle with minimum and maximum values of 17.2 and 36.4°, respectively. From analysis of the above-mentioned torsion angle and the equatorial/axial orientation of the C4 and C5 substituents in the 20 structures, there is no clear trend that allows the relative orientation of the flap to be predicted based only on the size or kind of the substituents.A search of the Cambridge Structural Database  and extracted with dichloromethane (3 x 10 mL). The combined organic layers were washed with saturated NaCl solution (10 mL), dried (Na2SO4) and filtered. Concentration of the filtrate, followed by flash chromatography (hexanes:ethyl acetate 93:7) yielded (2) . FT–IR (KBr): 2983, 2926, 2856, 1672, 1371. 1H-NMR  δ: 6.01 , 4.40 , 3.58 , 3.42 , 1.91 , 1.53 , 1.41 . For the synthesis of diol (3), epoxide (2)  was dissolved in THF (25 mL) and 10% KOH  was added. This mixture was refluxed for 4 h. After completion of the reaction, the mixture was diluted with dichloromethane (20 mL) and the organic phase was washed with 10% HCl until neutralization, washed with saturated NaCl solution (10 mL), dried with (Na2SO4) and filtered. Concentration of the filtrate, followed by flash chromatography (ethyl acetate:hexanes 4:6) yielded (3) . Crystals suitable for X-ray structure analysis were obtained by dissolving (3) in the minimum volume of ethyl acetate, adding hexanes until the solution became slightly turbid and slowly evaporating the solvent at room temperature. (m.p. = 385–386 K). FT–IR (KBr): 3402, 1637, 1371. 1H NMR  δ: 5.45 , 4.48 , 4.33 , 3.59 , 2.52 , 2.30 , 1.79 , 1.38 , 1.35 .The synthesis of the title compound was carried out through the intermediate epoxide (2)  I, New_Global_Publ_Block. DOI: 10.1107/S2056989015014590/gk2644Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989015014590/gk2644Isup3.cmlSupporting information file. DOI: 1416687CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


R)-d-ribit-1-ylsulfonate has an open-chain structure with the potassium cation seven-coordinated in an approximately pentagonal–bipyramidal coordination environment by six different anions through K—O coordinate bonds.The anion of potassium -1,2,3,4,5-pentahydroxypentane-1-sulfonate], formed by reaction of d-ribose with potassium hydrogen sulfite in water, crystallizes as colourless plates. The anion has an open-chain structure in which the S atom and the C atoms of the sugar chain, excepting that of the hydroxymethyl group, form an essentially all-trans chain; the C atom of the hydroxymethyl group lies in a gauche relationship with the three contiguous C atoms. Through complex cation coordination (through seven oxygen atoms of six different anions) and intermolecular O—H⋯O hydrogen bonding, a three-dimensional bonding network exists in the crystal structure.The title compound, K However, conclusive proof for their acyclic structure awaited X-ray studies, initially by Cole et al. , and its solid-state structure.Preparation of aldose adducts requires reaction at high concentrations, with the bisulfite anion produced trans chain -1,2,3,4,5-pentahydroxypentane-1-sulfonate. The torsion angle C2—C3—C4—C5 is indicative of a gauche conformation with C5 pointing out of the all-trans chain. All of the hydroxyl groups form O—H⋯O hydrogen bonds and all, except for the hydrogen bond from O2, have short H⋯O distances with O—H⋯O angles not far from linear  to 3.0085 (11) and are arranged in an approximately pentagonal–bipyramidal form with O4 and O4iv as the apical atoms. This is shown in Fig. 2a axis, indicating the hydrogen-bonding contacts and the K—O coordinate bonds. Potassium ions can show various coordination numbers in related coordination environments: in the d-galactose bisulfite  and d-ribose with displacement of potassium bisulfite; in the aqueous solution used for MS analysis, some decomposition of (1) to afford d-ribose undoubtedly occurs and this is supported by NMR data on the aqueous solution reported below.High-resolution mass spectrometry in negative-ion mode identified the anion at 1H NMR spectrum of (1) in D2O indicates considerable stability of the adduct in aqueous solution, with the species α-furanose, β-furanose, β-pyranose, α-pyranose, and bisulfite adduct, identified by their H-1 resonances, present in the % ratios of 3.6:6.2:10.9:5.1:74.2, which changed only marginally after 18 days. A complete assignment of the spectrum for (1) and consideration of derived coupling constants indicated overall similarity of the conformation in the crystalline state and in solution. Notably, J1,2 was close to zero and assuming Newman projection angles of 120° and using measured torsional angles, a Karplus relationship suggests a value of about 0.3 Hz. The value J2,3 = 8.6 Hz is in accord with an antiperiplanar arrangement of H2 and H3, whereas J3,4 = 4.6 Hz is consistent with the synclinal disposition of H3 and H4, resulting from a gauche arrangement for C2—C3—C4—C5.The 13C NMR spectrum confirmed the presence of the four ring forms of d-ribose as indicated by their C1 signals and the major peak for C1 in the adduct at δC 82.25 was accompanied by a much smaller peak at δC 84.19 which suggests the presence in solution of the diastereoisomer of (1) having the S-configuration at C1.The R-diastereomer was present in the crystal studied.A three-dimensional network exists in the crystal structure through the coordination of each potassium cation  to six different ribose bisulfite residues and through extensive hydrogen bonding between hydroxy hydrogens and oxygen atoms of hydroxyl groups or those on sulfur. Although the addition of the sulfite anion to C1 of the ribose moiety can theoretically afford two isomers, only the d-ribose (0.5 g) in water (0.35 ml), leading to immediate and complete solution of the reaction mixture. Seed crystals were obtained by complete evaporation of a small proportion of the solution, and these were added to the bulk of the solution which was then stored at 277 K, leading to the formation of large, well-separated crystals. The syrupy nature of the mother liquor required its removal with a Pasteur pipette, after which the crystals were dried by pressing between filter papers, to give potassium (1R)-d-ribit-1-ylsulfonate, m.p. 396–400 K (with decomposition); [α]D −6.1 (15 min.) .Water (0.5 ml) was added to potassium metabisulfite (0.37 g), which did not dissolve completely even on warming but which appeared to change its crystalline form as it underwent hydrolysis to yield potassium hydrogen sulfite. To this suspension was added a solution of 1H NMR : δ 5.37 , 5.24 , 4.92 , 4.85 ; signals for acyclic sulfonate: δH 4.67 , 4.18 , 3.94 , 3.82 , 3.77 , 3.69 . 13C NMR : δ 101.55 , 96.89 , 94.43 , 94.15 ; signals for adduct: 82.25 (C1), 73.23, 71.88, 70.61 , 62.56 (C5). A small but significant peak was observed at δC 84.19.1H NMR spectrum, 5 minutes after sample dissolution, indicated the species α-furanose, β-furanose, β-pyranose, α-pyranose, bisulfite adduct were present in the % ratios of 3.6:6.2:10.9:5.1:74.2. Re-measurement after 18 days, gave these % ratios as 1.5:2.6:16.2:8.7:70.9.Integration of the various signals for H-1 in the 2O/MeOH, solution) gave an expected peak at m/z 231.0187 ([C5H11O8S]−), the base peak at 213.0082 ([C5H11O8S−H2O]−) and a significant peak at 299.0987 ([C10H19O10]−). The last peak corresponds to the ion of the product formed by reaction between the bisulfite adduct and d-ribose with displacement of potassium bisulfite.HRESMS  1, New_Global_Publ_Block. DOI: 10.1107/S1600536814022685/sj54241sup2.hklStructure factors: contains datablock(s) 1. DOI: Click here for additional data file.10.1107/S1600536814022685/sj54241sup3.cmlSupporting information file. DOI: 1007337CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The 1:1 co-crystal salt trans-diaquabiszinc chloride 4,5-dimethylbenzene-1,2-diamine exhibits a tetragonally distorted octahedral zinc coordination sphere.The structures of two zinc complexes containing bidentate benzene-1,2-diamine ligands are reported. dichloroidozinc, [ZnCl2(C6H8N2)], (I), displays a distorted tetrahedral coordination sphere for the metal cation. The diamine ligand and the Zn atom reside on a crystallographic mirror plane. In the 1:1 co-crystal salt trans-diaquabiszinc chloride–4,5-dimethylbenzene-1,2-diamine (1/1), [Zn(C8H12N2)2(H2O)2]Cl2·2C8H12N2, (II), the zinc(II) complex cation exhibits a tetragonally distorted octahedral coordination sphere. The Zn atom sits on a crystallographically imposed inversion center and the diamine ligands are tilted 30.63 (6)° with respect to the ZnN4 plane. Both complexes exhibit extensive hydrogen bonding. In (I), a stacked-sheet extended structure parallel to (101) is observed. In (II), the co-crystallized diamine is hydrogen-bonded to the complex cation via O—H⋯N and N—H⋯N linkages. These units are in turn linked into planes along (200) by O—H⋯Cl and N—H⋯Cl hydrogen bonds.The structures of two zinc complexes containing bidentate benzene-1,2-diamine ligands are reported. (Benzene-1,2-diamine-κ For the co-crystallized diamine, N3 and N4 are 0.139 (3) and 0.088 (3) Å out of the plane, respectively.Compound (II)h Table 3 is signiAs seen in Figs. 3Fig. 5κO)zinc zinc (μ3-hydroxido)dizinc] ethanol monosolvate] exhibits alternating octahedral and tetrahedral zinc coordination modes benzene-1,4-diamine]zinc has a tetrahedral coordination sphere with intermolecular N—H⋯Cl hydrogen bonds  of clear, colorless crystals were isolated, which were used for data collection. The diffraction pattern showed signs of degradation as the temperature was lowered to 200 K from 300 K and so data sets were collected at both temperatures.Compound (I) was prepared by mixing a solution of 100. mg (0.734 mmol) zinc chloride dissolved in approximately 5 mL ethanol with a solution of 238 mg (2.20 mmol) benzene-1,2-diamine dissolved in approximately 5 mL ethanol. The mixture became cloudy with a fine white precipitate. After the addition of 4 drops of 6 M HCl, the mixture was gently heated and filtered. The filtrate was divided into three portions and each allowed to slowly evaporate. After several days, a small number of clear, colorless crystals in the shape of hexagonal plates were isolated, one of which was used for data collection.Compound (II)a) and 200 K (Ib). The diffraction pattern showed clear degradation at the lower temperature. Examination of the crystal subjected to the cold stream showed fractures that were not previously present. As seen in Table 7R values and S values are lower for the 300 K data set.Crystal data, data collection and structure refinement details are summarized in Table 7a), the hydrogen atoms bonded to the benzene carbon atoms were refined using a riding model with C—H = 0.93 Å and Uiso(H) = 1.2Ueq(C), whereas these hydrogen atoms were refined with C—H = 0.95 Å and Uiso(H) = 1.2Ueq(C) for (Ib).For both (I) and (II)Uiso(H) = 1.2Ueq(N). The hydrogen atoms of the water ligands were refined freely, including the isotropic displacement parameters. The methyl hydrogen atoms were refined with C—H = 0.98 Å and Uiso(H) = 1.5Ueq(C).For (II)10.1107/S2056989016010033/lh5817sup1.cifCrystal structure: contains datablock(s) global, Ia, II, Ib. DOI: 10.1107/S2056989016010033/lh5817Iasup2.hklStructure factors: contains datablock(s) Ia. DOI: Click here for additional data file.10.1107/S2056989016010033/lh5817Iasup5.molSupporting information file. DOI: 10.1107/S2056989016010033/lh5817IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989016010033/lh5817IIsup7.molSupporting information file. DOI: 10.1107/S2056989016010033/lh5817Ibsup4.hklStructure factors: contains datablock(s) Ib. DOI: Click here for additional data file.10.1107/S2056989016010033/lh5817Ibsup6.molSupporting information file. DOI: 1486732, 1486731, 1486730CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Sixteen derivatives of the homologous series of bis(acrylamido)alkanes have been synthesized with the aim of systematic analysis of their crystal structures and their solid-state [2 + 2] reactivities. Seven out of the 16 found to have reactive alignments for solid-state [2 + 2] reactions yield dimers and polymers. pyversus N—H⋯O=C) and network geometries. In this series, a greater tendency towards the formation of N—H⋯O hydrogen bonds  over N—H⋯N hydrogen bonds was observed. Among all the structures seven structures were found to have the required alignments of double bonds for the [2 + 2] reaction such that the formations of single dimer, double dimer and polymer are facilitated. However, only four structures were found to exhibit such a solid-state [2 + 2] reaction to form a single dimer and polymers. The two-dimensional hydrogen-bonding layer via N—H⋯O hydrogen bonds was found to promote solid-state [2 + 2] photo-polymerization in a single-crystal-to-single-crystal manner. Such two-dimensional layers were encountered only when the spacer between acryl amide moieties is butyl. Only four out of the 16 derivatives were found to form hydrates, two each from 2-pyridyl and 4-pyridyl derivatives. The water molecules in these structures govern the hydrogen-bonding networks by the formation of an octameric water cluster and one-dimensional zigzag water chains. The trends in the melting points and densities were also analyzed.The homologous series of phenyl and pyridyl substituted bis(acrylamido)alkanes have been synthesized with the aim of systematic analysis of their crystal structures and their solid-state [2 + 2] reactivities. The changes in the crystal structures with respect to a small change in the molecular structure, that is by varying alkyl spacers between acrylamides and/or by varying the end groups  on the C-terminal of the amide, were analyzed in terms of hydrogen-bonding interference (N—H⋯N From th Fig. 1a. As theset al., 20131–4 were prepared by changing the spacers between the amide functionalities -layer which promotes the polymerization reaction as anticipated in a single-crystal-to-single-crystal (SCSC) manner to yield crystalline organic polymers  competition between the O atom of the amide and the N atom of pyridine to form hydrogen bonds with the amide N—H group; (2) similarities or differences with previously published bis-amide analogues; (3) formation of the two-dimensional layer versus the β-sheet hydrogen-bond networks -networks can promote solid-state [2 + 2] reactions  pro2.1H NMR (200/600 MHz) spectra were recorded on a Bruker AC 200/600 MHz spectrometer. The MALDI-TOF experiment was carried out using a Bruker ultrafleXtreme MALDI TOF/TOF mass spectrometer.FTIR spectra were recorded with a Perkin–Elmer Instrument Spectrum Rx Serial No. 73713. Powder XRD patterns were recorded with a Bruker AXS-D8-ADVANCE diffractometer (Cu target). 2.1.In a round-bottom flask cinnamic acid  and pyridine (15 ml) were taken and hydrazine hydrate  was added to the reaction mixture and stirred for ∼ 15–20 min. After that triphenyl phosphite  was added to it and the reaction mixture was refluxed for 7–8 h.After cooling to room temperature, the pyridine was distilled out to reduce the volume up to 5 ml. For the work up process, EtOH was added and then the solid product was filtered and washed with EtOH. The white solid product was recrystallized from methanol–DMF.a1. Yield: 78%; m.p.: 280°C. Anal.: calc. for C18H16N2O2: C 74.0, H 5.5, N 9.6; found: C 69.49, H 4.79, N 8.59%.b1–d1, a2–d2 and a3–d3 were prepared by following the above procedure, but by using corresponding diamine and cinnamic acid, (E)-3-(pyridin-2-yl)-acrylic acid and (E)-3-(pyridin-3-yl)-acrylic acid, respectively.Compounds b1. Yield: 84%; m.p.: 250°C. Anal.: calc. for C20H18N2O2: C 75.4, H 5.7, N 8.8; found: C 64.67, H 4.76, N 7.68%.c1. Yield: 72%; m.p.: 268°C. Anal.: calc. for C22H24N2O2: C 75.83, H 6.94, N 8.04; found: C 75.20, H 6.72, N 7.84%.a2. Yield: 56%; m.p.: 248°C. Anal.: calc. for C16H14N4O2: C 68.6, H 7.5, N 20.0; found: C 68.2, H 6.82, N 19.8%.b2. Yield: 42%; m.p.: 210°C. Anal.: calc. for C18H18N4O6: C 56.0, H 4.7, N 14.5; found: C 55.2, H 4.52, N 13.8%.d2. Yield: 60%; m.p.: 182°C. Anal.: calc. for C22H26N4O4: C 64.4, H 6.4, N 13.7; found: C 63.9, H 5.82, N 12.8%.a3. Yield: 57.2%; m.p. 272°C. Anal.: calc. for C16H14N4O2: C 65.68, H 4.42, N 18.76; found: C 65.30, H 4.79, N 19.04%.b3. Yield: 78%; m.p.: 235°C. Anal.: calc. for C18H18N4O2: C 67.1, H 5.6, N 17.4; found: C 66.43, H 5.62, N 16.52%.c3. Yield: 60%; m.p.: 249°C. Anal.: calc. for C20H22N4O2: C 68.55, H 6.33, N 15.99; found: C 67.74, H 6.63, N 14.96%.2.2.E)-3-(pyridine-4-yl) acrylic acid , pentafluorophenol  and DCC  were taken in 20 ml of dry THF solvent and stirred over 24 h at room temperature, then the solvent was distilled out to collect the solid product. The solid product was recrystallized from pet-ether. The recrystallized ester  and hydrazine hydrate  were taken in dry DMF solvent and stirred at room temperature for 24 h. Then the solid product was filtered and recrystallized with MeOH. Yield: 62.8%; m.p. 279°C. Anal.: calc. for C16H14N4O2: C 65.30, H 4.79, N 19.04; found: C 64.94, H 4.53, N 18.76%.In a round-bottom flask (b4–d4 by using the corresponding diamines and pentafluoroester of (E)-3-(pyridine-4-yl) acrylic acid. In these cases, dry THF was used as the solvent.Similar procedures were followed for the synthesis of b4. Yield: 58%; m.p.: 275°C. Anal.: calc. for C18H18N4O2: C 67.1, H 5.6, N 17.4; found: C 66.9, H 5.49, N 16.69%.c4. Yield: 66%; m.p.: 235–238°C. Anal.: calc. for C20H26N4O4: C 62.2, H 6.8, N 14.5; found: C 61.49, H 5.59, N 14.19%.d4. Yield: 58%; m.p.: 175°C. Anal.: calc. for C22H30N4O4: C 63.7, H 7.3, N 13.5; found: C 62.79, H 6.49, N 12.46%.2.3.Kα radiation (λ = 0.71073 Å) at room temperature (293 K) by the hemisphere method. The structures were solved by direct methods and refined by least-squares methods on F2 using SHELX97 -3-(pyridin-2-yl)-acrylic acid and (E)-3-(pyridin-3-yl)-acrylic acid, respectively, in the presence of triphenyl phosphate in pyridine. The same procedure was found to be incompatible for the syntheses of derivatives of 4. Therefore, compounds a4–d4 are synthesized by reacting the pentafluoroester of (E)-3-(pyridin-4-yl)-acrylic acid with corresponding diamines. Single crystals suitable for X-ray diffraction were obtained by the slow evaporation technique from methanol, ethanol or methanol–DMF, methanol–THF solutions of the corresponding compounds. Despite several trails the single crystals suitable for X-ray diffraction analyses could not be obtained for c2 and d3. The comparison of X-ray powder diffraction (XRPD) patterns with the related analogues hinted at their probable crystal structures. The crystal structures of all the derivatives have been analyzed in terms of hydrogen-bonding networks and comparisons were made with respect to the related derivatives of amides and reverse amides. Further, these studies revealed that the series of these 16 structures can be categorized as four types: (1) β-sheet via N—H⋯O hydrogen bonds; (2) β-sheet via N—H⋯N hydrogen bonds; (3) two-dimensional layer via N—H⋯O hydrogen bonds; (4) two-dimensional layer via N—H⋯N hydrogen bonds. In the following sections the results will be described based on the spacers as listed in Fig. 2c1, b2, c2, a3, c3, a4 and c4 as the structures indicated the possibility of such reactions. The pertinent crystallographic details are given in Table 1The compounds 3.1.a1, a2, a3 and a4 all crystallize in monoclinic space groups C2/c, P21/n, P21/c and P21/n, respectively. With the exception of a3, which contains one molecule, all the other three contain half molecules in the asymmetric unit. Interestingly no solvent inclusion was found in all four lattices, making the comparison between these structures more realistic. With the exception of a1, the hydrazine moieties (C—N—N—C: 164° in a2 and a3 and 180° in a4) exhibited near co-planar geometry. In a1 the hydrazine moiety resembles that of a simple hydrazine or H2O2 with C—N—N—C torsion of 115°. The molecules in a1  and a2  assemble via N—H⋯O hydrogen bonds to form β-sheets containing ten-membered rings. Although both form β-sheets the differences in both the structures are apparent given the differences in the geometry of the spacer and molecules: non-coplanar in a1 and nearly planar in a2. In a1 the molecules are aligned in a zigzag manner, while in a2 they are aligned in a plane containing the hydrogen bonds. In a1 the hydrogen bonds  are more linear than those in a2  , while in a2 they have herringbone packing  .Molecules °) Fig. 4. Given ta3 and a4 the pyridyl groups are found to exhibit interference and form N—H⋯N hydrogen bonds. Although both are nearly planar molecules the hydrogen-bonding patterns and packing are found to differ drastically. In a3 the molecules assemble to form a one-dimensional chain that resembles a β-sheet but via N—H⋯N hydrogen bonds and also the molecules are off-set. These one-dimensional chains pack in a parallel fashion which is somewhat similar to that of a1 . The interplanar angles between the amide planes are 0° in b1, b3 and b4, while it is 61° in b2. Further, the plane of C=C—C=O creates the following angles with the central N—C—C—N plane: 19° in b1, 80° and 19° in b2, 77° and 12° in b3 and 72° in b4.The molecules b1 and b3, the molecules assemble via N—H⋯O hydrogen bonds to form the usual β-sheets along the b-axis. The molecules within the sheet are in-plane in b1 with a repeat distance of 4.85 Å, while they are not in-plane in b3 and contain a repeat distance of 4.65 Å. The sheets pack in a parallel fashion in the case of b1, while the sheets exhibit some angularity in the packing in b3  four water molecules involved in four hydrogen bonds each, two O—H⋯O with H2O, one N—H⋯Ow with the amide and one O—H⋯N with the pyridine N-atom; these O atoms exhibit near tetrahedral geometry in terms of hydrogen bonding; (2) two involved in exclusively three Ow—H⋯Ow hydrogen bonds each with water molecules; (3) two involved in one Ow—H⋯Ow and two Ow—H⋯O=C hydrogen bonds each. The second and third categories exhibit nearly planar geometry in terms of hydrogen bonding. In this three-dimensional network, it was found that the double bonds are aligned for a single [2 + 2] reaction  Å, 161°] and contain the required double bond alignment for [2 + 2] polymerization as anticipated. In particular, within the layers the double bonds are aligned with a distance (d1) of 3.812 Å and C=C⋯C=C torsion (τ2) of 0°. The comparison of XRPD patterns of c2 with those of c1 indicates that the crystal structures of c2 could be similar to that of c1  with that of the spacer —C—C—C—C— plane, and the amide planes are parallel to each other. Further, the central butyl amine moiety is found to have non-planar geometry with the N—C—C—C torsion angles of 59° and 62° in c1 and c3, respectively.In this series the single crystals suitable for X-ray diffraction were obtained for 1c Fig. 9. Both 1cc4 is found to be totally different from the above structures as it includes water in the crystal lattice. The crystals exhibit space group P21/c and the asymmetric unit is constituted by half a unit of c4 and one water molecule. The geometry also differs from the above structures as the central plane of the alkyl spacer makes an angle of 64° with that of C=C—C=O. Unlike the above two structures the —N—(CH2)4—N— fragment exhibits all anti geometry with N—C—C—C angles of 180°. The molecules assemble via amide-to-amide N—H⋯O hydrogen bonds to form the β-sheet network along the b axis with a repeat distance of 4.68 Å. These sheets are further connected via Ow—H⋯Npyridine hydrogen bonds to the chain of water molecules leading to the formation of a two-dimensional layer in which each water has three connectivity .In accordance with our previous studies the introduction of a hexyl spacer resulted in the β-sheet structures in a consistent manner. Molecules d Fig. 11. In both3.5.a1, c1 and c3, which form amide-to-amide hydrogen bonds despite having a θ-value below 20°, i.e. 8.45°, 12.83° and 8.42°, respectively (Table S2).In previous studies on amides and reverse amides it was found that the geometry of the molecule and position of the pyridine groups play a key role in the competition of acceptors C=O of amide and the N atom of pyridine to form hydrogen bonds with the N—H group of amides. In particular, the inter-planar angle (θ) between the amide group and the terminal aromatic ring tailors the resultant hydrogen bonds. The θ-value of less than 20° results in the formation of N—H⋯N hydrogen bonds, while above 20° results in the formation of N—H⋯O hydrogen bonds. This phenomenon was also explored using a larger set of compounds from the CSD. In the present series it was found that this hypothesis holds good with the exception of three compounds, namely The probable reasons could be due to their unusual molecular geometries which differ totally from the rest of the structures presented here as they deviate heavily from linearity. Further, it was found earlier that the phenyl and the 3-pyridyl substituted derivatives do not form hydrates, while the 4-pyridyl derivatives do exhibit such a tendency. Similarly, out of four 4-pyridyl derivatives studied here, two form hydrates. Interestingly, the present studies show that the 2-pyridyl groups also have a similar tendency to form such hydrates as two out of three form hydrates.3.6.2 groups, with the caveat that the derivatives with a butyl spacer  deviate from such trends  and hexyl derivatives . A further decrease in the melting point was observed for hydrates compared with their respective analogues. Phenyl and 4-pyrydyl derivatives have been shown to have higher melting points than the 3-pyridyl and 2-pyridyl derivatives. A similar tendency was also observed in the melting points of amides and reverse amides  — groups with the exception of a1. Interestingly, the densities of 4-pyridyl derivatives are found to be higher than those of the other three homologues. In contrast, the homologous series containing a phenyl substituent was found to exhibit lower densities than the other three series .We are in agreement with the general conception that the decrease in melting points was observed with an increase in the number of CH) Fig. 12. Further3.7.b2 reveals that the double bonds are aligned for a single [2 + 2] reaction with a C⋯C distance (d1) between the double-bonded C atoms of 3.84 Å and C=C⋯C=C torsion of (τ2) 0°. The 1H NMR spectra of irradiated b2 in DMSO-d6 shows the appearance of cyclobutane protons at 4.44 and 4.03 p.p.m. with the presence of olefinic protons. From these observations it can be concluded that b2 was converted to a single dimer product in 59% yield after 72 h of irradiation in sunlight -layers as in c1 and c3. Therefore, a polycrystalline material of c2 was irradiated in sunlight for 96 h. The irradiated product was found to be insoluble in common organic solvents similar to those of c1 and c3. However, it was found to be soluble in DMSO or aqueous solution with a drop of HCl, HNO3 or H2SO4.As it was described earlier, the materials 1H NMR spectra of an irradiated sample of c2 in DMSO-d6 and one drop of H2SO4 revealed that the reaction proceeds through the formation of oligomers as the resultant spectra contains some new peaks in addition to the peaks of the monomer and polymer. In the spectra the cyclobutane peaks of a polymer appeared at 4.27 and 4.64 p.p.m. and also the n-butyl protons of the polymer were found to exhibit an up-field shift from monomer to polymer, i.e. 3.22 to 2.56 p.p.m. and 1.51 to 0.77 p.p.m. For oligomers, the cyclobutane peaks appeared at the same p.p.m. as the polymer, however, the corresponding n-butyl peaks appeared at 3.05, 1.22 and 1.08 p.p.m.. From 1H NMR, the yield of the reaction including oligomers was found to be 45%. The result of the polymerization reaction observed here is in line with that of c3 albeit the yield of the polymer is not 100% in the present case. The unreacted c2 and oligomers were removed by repeatedly washing the irradiated material of c2 with hot methanol. The 1H NMR of the separated polymer in DMSO-d6 with one drop of H2SO4 reveals the presence of cyclobutane protons at 4.27 and 4.64 p.p.m. with n-butyl protons at 2.57 and 0.77 p.p.m. and the absence of olefin protons  was determined using MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) with 2,5-dihydroxy benzoic acid (DHB) as a matrix in solution. The highest molecular ion peak was observed at 4415 (m/z), which corresponds to 12-mer of c2  1a, 1b, 1d, 2a, 2b, 2d, 3a, 3b, 4b, 4c, 4d. DOI: 10.1107/S2052252515009987/yc50051asup2.hklStructure factors: contains datablock(s) kb_mg_080. DOI: 10.1107/S2052252515009987/yc50051bsup3.hklStructure factors: contains datablock(s) kb_mg_2. DOI: 10.1107/S2052252515009987/yc50051dsup4.hklStructure factors: contains datablock(s) kbmg109. DOI: 10.1107/S2052252515009987/yc50052asup5.hklStructure factors: contains datablock(s) kbmg181. DOI: 10.1107/S2052252515009987/yc50052bsup6.hklStructure factors: contains datablock(s) kbmg146. DOI: 10.1107/S2052252515009987/yc50052dsup7.hklStructure factors: contains datablock(s) kbmg154. DOI: 10.1107/S2052252515009987/yc50053asup8.hklStructure factors: contains datablock(s) kb_rs_501. DOI: 10.1107/S2052252515009987/yc50053bsup9.hklStructure factors: contains datablock(s) kbmg011. DOI: 10.1107/S2052252515009987/yc50054bsup10.hklStructure factors: contains datablock(s) kbgt001. DOI: 10.1107/S2052252515009987/yc50054csup11.hklStructure factors: contains datablock(s) kbmg176. DOI: 10.1107/S2052252515009987/yc50054dsup12.hklStructure factors: contains datablock(s) kbmg112. DOI: 1055532, 1055533, 1055534, 1055535, 1055536, 1055537, 1055538, 1055539, 1055540, 1055541, 1055542CCDC references:


N-6 inhibits TR3-stimulated transactivation of Gal4-DBD-RXRα-LBD by binding to the ligand binding pocket of RXRα-LBD, suggesting a strategy to regulate TR3 activity indirectly by using small molecules to target its interacting partner RXRα. For its physiological activities, we show that N-6 strongly inhibits tumor necrosis factor α (TNFα)-induced AKT activation and stimulates TNFα-mediated apoptosis in cancer cells in an RXRα/tRXRα dependent manner. The inhibition of TNFα-induced tRXRα/p85α complex formation by N-6 implies that N-6 targets tRXRα to inhibit TNFα-induced AKT activation and to induce cancer cell apoptosis. Together, our data illustrate a new RXRα ligand with a unique RXRα binding mode and the abilities to regulate TR3 activity indirectly and to induce TNFα-mediated cancer cell apoptosis by targeting RXRα/tRXRα.Retinoid X receptor α (RXRα) and its N-terminally truncated version tRXRα play important roles in tumorigenesis, while some RXRα ligands possess potent anti-cancer activities by targeting and modulating the tumorigenic effects of RXRα and tRXRα. Here we describe NSC-640358 (N-6), a thiazolyl-pyrazole derived compound, acts as a selective RXRα ligand to promote TNFα-mediated apoptosis of cancer cell. N-6 binds to RXRα and inhibits the transactivation of RXRα homodimer and RXRα/TR3 heterodimer. Using mutational analysis and computational study, we determine that Arg316 in RXRα, essential for 9- Retinoid X receptor α (RXRα), a unique member of the nuclear receptor superfamily, plays a pivotal role in diverse biological and physiological processes, including cell growth, differentiation, apoptosis, and homeostasis  and LG100754, contain an acidic moiety and a large hydrophobic moiety. The acidic moiety is able to form salt bridges with Arg316 residue in RXRα ligand binding pocket (LBP), which contributes immensely to ligand-receptor interaction  is a selective RXRα antagonist with a unique RXRα binding mode. Through binding to RXRα, N-6 inhibits RXRα transactivation and RXRα-mediated transcriptional activity of TR3. Moreover, N-6 inhibits tumor necrosis factor α (TNFα)-induced AKT activation and enhances TNFα-mediated cancer cell apoptosis in an RXRα/tRXRα dependent manner.cis-RA displaced [3H]-labeled 9-cis-RA for binding to RXRα-LBD protein in a dose-dependent manner -N-ethylidene]droxylamine, N-6), a thiazolyl-pyrazole derived compound, was a potential RXRα ligand r77 Fig. . Thus, Ncis-RA and CD3254 ; CD3254, UVI3003, anti-AKT1 , anti-c-Myc , anti-RXR , anti-RXR  from Santa Cruz Biotechnology ; anti-caspase-8 , anti-phospho-AKT (Ser473)  from Cell Signaling Technology ; anti-poly (ADP-ribose) polymerase  from BD Biosciences ; 9-cis-Retinoic Acid, anti-β-actin antibody from Sigma ; anti-PI3 kinase , polyvinylidene difluoride membranes from Millipore ; TR-FRET Retinoic X Receptor alpha coactivator assay kit (PV4797) from invitrogen ; rhTNF-α  from Promega ; enhanced chemiluminescence reagents from GE Healthcare  and a cocktail of proteinase inhibitors from Roche ; [3H]9-cis-Retinoic Acid from PerkinElmer  were used in this study.NSC-640358 was provided by the NCI/DTP Open Chemical Repository and the DTP website address is MCF-7 breast cancer cells, HCT116 colon cancer cells, HeLa cervix cancer cells, H292 lung cancer cells, A549 lung cancer cells, HEK293T cells, and CV-1 cells were obtained from American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (FBS). All cell lines used were passaged in our lab for fewer than 4 months after resuscitation, and were used at the fifth through tenth passage in culture for this study. Transient transfection was performed using TurboFect according to the instructions of the manufacturer in regular growth medium.A dilution series of the compound N-6 in potassium phosphate buffer  was made on a 96-well polypropylene plate, which was subsequently transferred onto a 96-well, half-area UV-transparent quartz plate in duplicate, 50 μL/well. The stock solution of the protein RXRα ligand binding domain was diluted into potassium phosphate buffer to reach 0.60 μmol/L, which was immediately transferred onto the quartz plate, 10 μL/well. The fluorescent intensities of the compound were acquired on a FlexStation 3 Microplate Reader . In order to minimize the interference from the intrinsic fluorescence of Trp residues, time-resolved fluorescence mode was used. Data analyses were performed with GraphPad Prism 5.0d.MCF-7 cells seeded in 48-well plates were transiently cotransfected with the luciferase reporter plasmid pG5-Gaussia (40 ng), pBind-receptors (40 ng), and β-Gal plasmid (2 ng). Twenty-four hours after transfection, the medium was replaced by medium containing a respective ligand and/or a testing compound. After 18 h, the medium was collected and assayed by using Gaussia-Dura Luciferase Glow Assay Kit (Pierce). Cells were washed, lysed and assayed for β-Gal activity. Transfection efficiency was normalized to β-Gal activity.MCF-7 cells seeded in 48-well plates were transiently cotransfected with the luciferase reporter plasmid pG5-Gaussia (40 ng), pBind-TR3-LBD (40 ng), pACT-RXRα and β-Gal plasmid (2 ng). Twenty-four hours after transfection, the medium was replaced by medium containing a respective ligand and/or a testing compound. After 18 h, the medium was collected and assayed by using Gaussia-Dura Luciferase Glow Assay Kit (Pierce). Cells were washed, lysed, and assayed for β-Gal activity. Transfection efficiency was normalized to β-Gal activity.CV-1 cells seeded in 24-well plates were transiently cotransfected with one of the luciferase reporter plasmids pBLCAT2-TREpal (100 ng)/pBLCAT2-βRARE (100 ng)/pBLCAT2-NurRE (100 ng), one or two NR expressing plasmids pCMV-Myc-RXRα (20 ng)/pCMV-Myc-RARγ (20 ng)/pCMV-Myc-TR3 (20 ng), and pCMV-Myc-β-Gal (5 ng) as internal reference. Twenty-four hours after transfection, the medium was replaced by medium containing a respective ligand and/or a testing compound. After 18 h, cells were harvested and assayed for CAT and β-Gal activity. Transfection efficiency was normalized to β-Gal activity.Invitrogen’s LanthsScreen time-resolved fluorescence resonance energy transfer (TR-FRET) assay was used. Retinoic X Receptor alpha coactivator assay was conducted according to manufacture’s protocol. The TR-FRET ratio was calculated by dividing the emission signal at 520 nm by the emission signal at 495 nm.The binding kinetics between RXRα-LBD and N-6 was analyzed at 25°C on a BIAcore T200 machine with CM5 chips . PBSP was used for all measurements. A blank channel was used as negative control. About 10,000 response units of RXRα-LBD were immobilized on the chip. When the data collection was finished in each cycle, the sensor surface was regenerated with Glycine-HCl 2.5. A series of concentrations of N-6 ranging from 3.125 to 100 μmol/L were applied for experiment. Sensograms were fit globally with BIAcore T200 analysis using 1:1 Langumuir binding mode.siRNAs against RXRα (SASI-HS01-00097639) and control siRNA (SIC001) used were from Sigma. Cells were transfected with 5 μL aliquot of 20 μmol/L siRNA/well in six-well plates using DharmaFECT 1 transfection reagent, according to the manufacturer’s recommendations.Cell lysates were prepared by lysing cells with lysis buffer containing 25 mmol/L Tris, 150 mmol/L NaCl, 1 mmol/L EDTA, 1% NP-40, 5% glycerol, pH 7.4 with a cocktail of proteinase inhibitors on ice for 30 min. Equal amounts of the lysates were electrophoresed on an SDS-PAGE gel (8% or 12%) and transferred onto polyvinylidene difluoride membranes, which were then blocked with 5% nonfat milk in TBST  for 1 h, incubated with various primary antibodies overnight at 4°C and incubated with secondary antibodies for 1 h at room temperature. Immunoreactive products were detected by chemiluminescence with an enhanced chemiluminescence system.For Co-IP assay, cells grown in 10-cm dishes were transfected with various plasmids for 24 h. After transfection and treatments, cells were harvested. Cell lysates were incubated with the appropriate antibody for 2 h, and subsequently incubated with protein A-Sepharose beads for 2 h. The protein-antibody complexes recovered on beads were subjected to Western blotting using appropriate antibodies after separation by SDS-polyacrylamide gel electrophoresis.cis-RA or different concentrations of compounds in 200 μL binding buffer  at 4°C for 1 h. [3H]-9-cis-RA was added to the tubes to a final concentration of 7.5 nmol/L and final volume of 300 μL then incubated overnight at 4°C. The RXRα-LBD was then captured by nickel-coated beads. Bound [3H]-9-cis-RA was quantitated by liquid scintillation counting.The His-tagged human RXRα-LBD (223–462) was incubated in tubes with unlabeled 9-HeLa cells were seeded in 6-well plate (500 cells/well) for 3 days, and were treated with N-6 (5 μmol/L) and TNFα (10 ng/mL) alone or in combination in 1% serum medium for 3 days. Cells were fixed with 4% paraformaldehyde. Colonies were stained with 0.1% crystal violet and counted.2O2 (80 mL methanol + 10 mL ddH2O + 10 mL 30% H2O2) for 10 min. TUNEL reaction mixture was obtained by adding terminal deoxynucleotidyl transferase and Biotin-11-dUTP. Each sample was then incubated with TUNEL reaction mixture in a humidified chamber at 37°C in dark for 60 min, and then labeled with Streptavidin-HRP. DAB staining was used for coloration. Nucleus was stained by hematoxylin.MCF-7 cells were fixed in 4% paraformaldehyde and permeabilized with 1% Triton X-100 in phosphate-buffered saline. Cells were blocked using 3% Hhttp://dtp.nci.nih.gov) was performed using Q-MOL molecular modeling package   of approximate 200,000 compound library of the Developmental Therapeutics Program (DTP) NCI/NIH . Glide was used to study the interaction of ligand-RXRα complex (LG100754 and N-6) in the suite of Schrodinger with default docking parameter settings. The interaction image was produced by the Pymol software.t test. P < 0.05 was considered significant.Data were expressed as mean ± SD. Each assay was repeated in triplicate in three independent experiments. Statistical significance of differences between groups was analyzed by using Student’s


The free base porphyrin is characterized by a significant degree of  42H34N4, the neighbouring N⋯N distances in the center of the ring vary from 2.818 (8) to 2.998 (8) Å and the phenyl rings are tilted from the 24-atom mean plane at angles varying between 62.42 (2)–71.63 (2)°. The NH groups are involved in intramolecular bifurcated N—H⋯ hydrogen bonds. The Ca—Cm—Ca angles vary slightly for the phenyl rings, between 124.19 (18)–126.17 (18)°. The largest deviation from the mean plane of the 24-atom macrocycle is associated with the meso carbon at the substituted tert-butyl position, which is displaced from the mean plane by 0.44 (2) Å. The free base porphyrin is characterized by a significant degree of ruffled (Bu1) distortion with contributions from domed (Au2) and wave [gE(y) and gE(x)] modes. In the crystal, molecules are linked by a number of weak C—H⋯π interactions, forming a three-dimensional framework. The structure was refined as a two-component inversion twin.In the title free base porphyrin, C In the title compound, the Ca—Cm—Ca angles vary slightly with the Ca—Cm(tert-butyl)—Ca angle of 120.55 (18)° at C5 representing the smallest. This is due to the nature of the tert-butyl substitution present. This angle is similar to that observed in the dication, 5,10,15,20-tetrakis(tert-butyl)-22H+,24H+-porphyrindiium ditrifluoroacetate –Ca angle of 119.53° and 5-tert-butylporphyrin published –Ca angle of 119.86°. The Ca—Cm(phenyl)—Ca angle of the title compound at C10 and C20 are quite similar at 126.03 (18) and 126.17 (18)°, respectively. The Ca—Cm(phenyl)—Ca angles in 5,10,15,20-tetraphenylporphyrin, with an average angle of 125.35° —Ca angle at C15 of the title compound is smaller [124.19 (18)°].The molecular structure of the title compound is illustrated in Fig. 1meso-substituents are 67.62 (2)° (C10), 71.63 (2)° (C15) and 62.42 (2)° (C20). These angles are larger than the tilt angles observed in 5,10,15,20-tetraphenylporphyrin, which are ca 60°  distortion with contributions from domed (Au2) and wave [gE(y) and gE(x)] modes  out-of-plane distortions. The title compound exhibits similar contributions from both these modes whereas the free base 5-tert-butylporphyrin shows significantly more contributions in the Bu1 compared to the gE(y) distortions. This can also be seen in the in-plane distortions as both compounds show significant contributions from the Bg2 and smaller contributions from the Ag1 mode, the title compound shows much larger contributions towards the Bg1 in-plane distortions compared to that of the 5-tert-butylporphyrin.The tilt angles of the phenyl gs Fig. 2. A confoes Fig. 3. Contribtert-butyl substitution at C5. Atom C5 deviates from the mean plane by 0.440 (2) Å, whereas atoms C8, C2, C4, C20, N2 and C7 deviate from the mean plane by −0.361 (2), −0.244 (2), 0.232 (2), −0.217 (2), 0.203 (2) and −0.203 (2) Å, respectively. The smallest deviations are for the atoms associated with the pyrrole ring at the N3 position; atoms C11 C12, C13, C14 and N3 deviate from the mean plane by −0.003 (2), −0.027 (2), 0.027 (2), 0.009 (2) and −0.007 (2), respectively. This ring also shows the least tilt in the porphyrin structure.The maximum deviations from the 24-atom mean plane are associated with carbon and nitrogen atoms surrounding the tert-butyl-substituted group. The centroid–centroid distance of the 24-atom mean planes of the porphyrin rings are between 8.762 (2) and 7.758 (2) Å. The rings that stack above each other are separated by 8.762 (2) Å and the rings that are orientated in an edge-on packing are separated by a centroid–centroid distance of 7.758 (2) Å -10,15,20-triphenylporphyrin in which they reported phenyl tilt angles of 59.07–78.15° from the 24-atom mean plane, with the largest deviation on the phenyl group opposite the alternative meso-substituted position. In this structure there was a larger variance of the Ca—Cm(phenyl)—Ca angle of 123.88–125.51° and a Ca—Cm(C5)—Ca larger than the title compound of 127.35°. Senge et al. (1999a—Cm(phenyl)—Ca angle is similar to the title compound with an angle range of 125.46–125.78°. The structure of 5--10,15,20-triphenylporphyrin pyridine clathrate has been reported by Tanaka et al. —Ca angle either side of the unsubstituted meso position being almost equal to each other (123.78–123.95°). The Ca—Cm(phenyl)—Ca opposite the unsubstituted meso position is smaller than the Ca—Cm(H)—Ca angle, 126.20 and 127.93°, respectively. The phenyl tilt angle from the 24-atom mean plane shows a larger tilt angle (73.56–78.16°) associated with the phenyl rings. However, there is a narrower variance in these angle than in the title compound. Ryppa et al. (2005tert-butylporphyrin which presents Ca—Cm(H)—Ca angles of 129.00–129.23° for the C10 and C15 positions and 125.23° for the C15 position which are all larger than in the title compound. The Ca—Cm(tert-butyl)—Ca angle (C5 in both structures) are of similar size at 119.86° (120.28° for the title compound). The overall pyrrole tilt against the mean 24-atom plane shows similar results to that of the title compound. The pyrrole rings (N1 and N2) closest to the tert-butyl meso substitute show significantly higher tilts  compared to the pyrrole rings (N3 and N4) closest to the unsubstituted position at C15 . Yang et al. —Ca angle of 124.36–124.68° similar to the title compound and a Ca—Cm(naphthoate)—Ca angle of 125.25° which is slightly larger compared to the title compound. The tilt angle of the phenyl rings from the 24-atom mean plane is 60.46–83.15° which shows a larger variance than for the title compound.A search of the Cambridge Structural Database  was dissolved in dry CHCl3 (50 ml) and cooled to 273 K. N-Bromosuccinimide  was added and the solution was stirred for 5 h. The resulting solution was quenched with acetone and the crude product was purified via column chromatography on silica gel . The solvent was removed in vacuo yielding 5-tert-butyl-10,15,20-tribromoporphyrin as purple crystals . Rf = 0.44 ; 1H NMR  δ: 9.45 , 9.36 , 9.31 , 9.25 , 2.33 , −1.72 p.p.m. ; HRMS (MALDI): m/z calculated for C24H19N4Br3 600.9238 [M + H]+; found 600.9248.The title compound was prepared previously by Senge  al. 2000 using a tert-butyl-10,15,20-tribromoporphyrin , phenylboronic acid , tetrakis(triphenylphosphine)palladium(0) , cesium carbonate  and dried under vacuum. The mixture was dissolved in anhydrous THF (5 ml) and was degassed via three freeze–pump–thaw cycles and left under argon. The solution was heated to 353 K under an argon atmosphere for 48 h. The solvent was removed under reduced pressure and the residue was dissolved in CH2Cl2 (10 ml). The crude product was washed sequentially with sat. aq. NaHCO3 (20 ml) and deionized H2O (20 ml). The organic phase was dried over Na2SO4 and filtered. The crude product was purified via column chromatography on silica gel . The solvent was removed in vacuo, yielding the title compound as purple crystals . The compound was recrystallized from CH2Cl2 layered with methanol to yield single crystals suitable for X-ray diffraction analysis.A Schlenk tube was charged with 5-Uiso(H) = 1.5Ueq(C-methyl) and 1.2Ueq for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016000025/su5266sup1.cifCrystal structure: contains datablock(s) I, Global. DOI: 10.1107/S2056989016000025/su5266Isup2.hklStructure factors: contains datablock(s) I. DOI: 1444998CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Oligosaccharides of glycoproteins expressed on the cell surface play important roles in cell-cell interactions, particularly sialylated N-glycans having a negative charge, which interact with sialic acid-binding immunoglobulin-like lectins (siglecs). The entire structure of sialylated N-glycans expressed in the mouse brain, particularly the linkage type of sialic acid residues attached to the backbone N-glycans, has not yet been elucidated. An improved method to analyze pyridylaminated sugar chains using high performance liquid chromatography (HPLC) was developed to determine the entire structure of sialylated N-linked sugar chains expressed in the adult and developing mouse cerebral cortices. Three classes of sialylated sugar chains were prevalent: 1) N-glycans containing α(2-3)-sialyl linkages on a type 2 antennary GlcNAc), 2) sialylated N-glycans with α(2-6)-sialyl linkages on a type 2 antennary, and 3) a branched sialylated N-glycan with a [Galβ(1-3){NeuAcα(2-6)}GlcNAc-] structure, which was absent at embryonic day 12 but then increased during development. This branched type sialylated N-glycan structure comprised approximately 2 % of the total N-glycans in the adult brain. Some N-glycans (containing type 2 antennary) were found to change their type of sialic acid linkage from α(2-6)-Gal to α(2-3)-Gal. Thus, the linkages and expression levels of sialylated N-glycans change dramatically during brain development.The online version of this article (doi:10.1007/s10719-014-9566-2) contains supplementary material, which is available to authorized users. Sugar chains envelop the vast majority of the cell surface and are considered to play significant roles in cell-cell and extracellular matrix interactions as mediators and as signal transducers. Increasing evidence suggests that glycosylation is critical to various biological functions and requires the coordinated action of glycosyltransferases , 2. Othein vitro neurite outgrowth at early developmental stages but inhibits neurite outgrowth in adults , which we termed the 6-sialyl Lewis C structure. This branched sialylated structure changed its content dynamically during development in the mouse brain, suggesting that N-glycans with 6-sialyl Lewis C plays important roles during brain development.We have been developing a systematic method to analyze N-glycan expression patterns present in whole tissues without glycoprotein purification after removal of sialic acid residues –25. HereArthrobacter ureafaciens was purchased from Nacalai Tesque . α2,3-sialidase, specific for α(2-3)-NeuAc, was from New England BioLabs . Pyridylaminated (PA)-sugar chains used as a standard were purchased from Takara Bio and Seikagaku Corporation .Anhydrous hydrazine was purchased from Tokyo Chemical Industry , 2-aminopyridine was from Kanto Chemical , and dimethylamine borane was from Wako . Graphite carbon columns  were purchased from GL Science . Cellulose cartridge columns were from Takara Bio  and GL Science (Cat. No. 5010–11130). Microgranular cellulose for packed cellulose columns was from Sigma . Neuraminidase derived from ICR mice were purchased from Japan SLC . All experiments were carried out with permission of the institutional Animal Research Committee of the National Institute for Physiological Sciences.via high-performance liquid chromatography (HPLC) analyses. Excess reagents were removed and PA-N-glycans were purified using cellulose columns. To analyze N-glycans at each stage, the samples were prepared from more than ten mice at each developmental stage and three adult mice for each experiment.N-glycan purification and pyridylamination were performed as described previously –25. BrieTo separate neutral-, mono-, di-, tri-, and tetra-sialyl N-glycans, PA-N-glycans were passed through an anion-exchange column  using HPLC or a Microgranular DE52-packed column . Anion-exchange column HPLC purification was performed at a flow rate of 1.0 ml/min at room temperature. The mobile phase consisted of solvent A (distilled water adjusted to pH 9.0 with aqueous ammonia) and solvent B (0.5 M ammonium acetate titrated to pH 9.0 with aqueous ammonia). Neutral N-glycans were collected in the non-adsorbed fraction. PA-sugar chains were detected at excitation and emission wavelengths of 310 nm and 380 nm, respectively .Sialylated PA-N-glycans were treated with neuraminidase at 37 °C for 14 h in 50 mM ammonium acetate pH 5.0 to cleave sialic acids, followed by heating at 100 °C for 5 min and filtering through a 0.20 μm spin filter .Analyses of neutral PA-N-glycans using HPLC were performed as described previously , 23–26. RP-HPLC for purification of sialylated N-glycans was performed on a Develosil ODS-5 column  at a flow rate of 1.0 ml/min. The column was equilibrated with 100 mM acetic acid titrated to pH 4.0 with triethylamine containing 0.05 % 1-butanol. After injecting each sample, the concentration of 1-butanol was increased linearly to 0.4 % over 90 min. PA-sugar chains were detected at an excitation wavelength of 320 nm and an emission wavelength of 400 nm.PA-N-glycans were quantified and analyzed as described previously , 23–26. The molecular masses of PA-sugar chains and their isobaric monosaccharide compositions were determined by MALDI/TOF-MS. The PA-sugar chains were dissolved in water. One microliter of matrix solution  was applied on the target spot of plate, and 1 microliter of sample solution was added, and then dried by warm air. MALDI/TOF mass spectra of the samples were acquired using a REFREX spectrometer  in the positive and reflector mode at an acceleration voltage of 20 kV and delayed ion extraction. Standard PA-sugar chains were used to achieve a two-point external calibration for mass assignment of ions. The mass spectra shown are the sum of at least 30 laser shots.Xanthomonas manihotis β1,3-galactosidase (New England BioLabs), specific for β(1-3)-Gal, in 50 mM sodium acetate pH 4.5, 100 μg/ml BSA; Diplococcus pneumoniae β-galactosidase , specific for β1,4-Gal, in 50 mM sodium acetate pH 6.0; α1,3/4-L-fucosidase (Takara Bio), specific for α1,3/4-Fuc, in 100 mM sodium phosphate buffer pH 6.0; bovine kidney α1,6-fucosidase , specific for α1-6 > 1-2/3/4-Fuc, in 100 mM sodium phosphate buffer pH 6.0. Neuraminidase, specific for α2,3/6/8-NeuAc, was incubated with PA-N-glycans for 14 h at 37 °C in 50 mM ammonium acetate pH 5.0. α2,3-sialidase, specific for α(2-3)-NeuAc, was incubated with PA-N-glycans for 14 h at 37 °C in 50 mM sodium citrate pH 6.0, 100 mM NaCl, 100 μg/ml BSA. These samples were heated at 100 °C for 5 min. The reaction mixture was centrifuged at 2,300 × g for 10 min, followed by filtering through a 0.20 μm spin filter .Purified PA-N-glycans were digested for 3 h at 37 °C using the following each enzyme: 2, 1 mM CaCl2. Unbound oligosaccharides were washed away with buffer A, and the bound sugar chains were then eluted from the column with 0.1 M lactose in buffer A.SNA agarose beads  were packed into a 50 × 10 mm assist mini-column. One milliliter of the sample was applied to the SNA column after it had been equilibrated with buffer A, which consisted of 20 mM Tris–HCl (pH 7.5) containing 0.15 M NaCl, 1 mM MnClWe have previously identified the structures of neutral and desialylated N-glycans that comprised more than 1 % of the total N-glycan content in the developing mouse brain . The pre2Hex5HexNAc4-PA  were collected separately. The samples were desialylated with neuraminidase and then applied to a DE52 column. The flow-through fraction was subjected to NP-HPLC; however, the peaks were still incompletely separated and could not be identified or quantified at this point Fig. . The elu and S3 w2Hex5HexNAc5-PA , namely A2G1FoF, Lewis X(a + b)-BA-2, Lewis X2-BA-2, Lewis X-H4FB, and Lewis X-H5F, had a Cα2,3/6/8 value of 0, meaning that they did not contain sialic acid residues  and α2,3-sialidase sensitive portion  should be the same. Quantification of the α2,3-sialidase sensitive proportion of the major sialylated N-glycans is presented in Table α2,3 value from the Cα2,3/6/8 value. This difference should indicate the amount of N-glycans containing sialic acid with an α2-6/8-linkage. Therefore, it is highly likely that the mouse brain contains large amounts of N-glycans containing sialic acid with an α(2-6)-linkage. However, this conclusion contrasts with a report by Zamze et al. . Since there was only one major type 1 N-glycan (A2G’2F) in the mouse cortex, we determined the linkage type of sialic acid residues on A2G’2F in the adult mouse cortex.A2G’2F has four potential sialylation sites . After Mono Q HPLC fractionation and neuraminidase treatment, A2G’2F was recovered primarily from the S2 and S3 fractions (data not shown), thus A2G’2F generally contains two or three sialic acid residues. We focused on the S2 fraction for further analysis because the amount of A2G’2F in the S2 fraction was higher than that in the S3 fraction. After α2,3-sialidase treatment of the S2 fraction, A2G’2F was recovered from the S0, S1 and S2 fractions (data not shown), indicating that A2G’2F has two α2,3-sialidase-sensitive sialic acids, one resistant and one sensitive sialic acid residue or two α2,3-sialidase-resistant sialic acids.a). Sugar chains in this fraction were treated with α2,3-sialidase, subjected to Mono Q HPLC and the S2 fraction was collected again . This fraction should contain only sugar chains having two α2,3-sialidase-resistant sialic acid residues. This fraction was separated by RP-HPLC in the presence of triethylamine  form. The PA-sugar chain mixture extracted from 12w mouse brains was subjected to Mono Q HPLC and the S2 fraction was recovered Fig. -a. Sugarain Fig. -b. This ine Fig. . There wine Fig. . This prine Fig. . These rN-acetylhexosaminidase -linked di-sialic acid chains, at the 6th position of GlcNAc, the di-sialylated N-glycan from the peak S2 in Supplementary Fig. S-8-linkedWe examined the expression levels of N-glycans with the branched sialic acid-containing structure [Galβ(1-3){NeuAcα(2-6)}GlcNAc-], termed 6-sialyl Lewis C, in the developing and adult mouse cerebral cortices. Whereas N-glycans with 6-sialyl Lewis C were barely detected at E12, their expression level increased during development, reaching approximately 2 % of the total N-glycan level in the adult as shown in Fig. et al. [It is essential to determine the entire structure of the sialylated sugar chain, including all of the linkages between sugar residues, to understand roles played by siglecs and sialylated sugar chains. It is also important to know their expression levels during development. A previous study by Zamze et al.  reportedvia an α(2-3)-linkage at E12, but in the adult about half of the N-glycan harbors a NeuAcα(2-3)-Gal- moiety  NeuAcα2-3)-Galβ(1-4)-GlcNAc-, 2) NeuAcα(2-6)-Galβ(1-4)-GlcNAc-, 3) Galβ(1-3)-{NeuAcα(2-6)}GlcNAc-. Some of the N-glycans were found to change their type of sialic acid linkage during brain development. A good example is Ga-BA-2, which has only one possible sialic acid attachment site. This sugar chain harbors no sialic acid attached -Galβ. To determine whether there is a great difference in the structures of sialyl Lewis C containing N-glycans between rat and mouse cortices at the type 1 antennary, N-glycan structures in rat cortices were analyzed using the same method applied to analyze mouse cortices. Cortices from both species were found to contain 6-sialyl Lewis C and di-sialyl Lewis C at similar levels (manuscript in preparation). Therefore, we concluded that 6-sialyl Lewis C is also present in the rat brain, where it should play an important role during brain development.We previously reported that the expression level of A2G’2F is increased during brain development . Here, wIt was reported that some glycoproteins in bovine plasma possess N-glycans with 6-sialyl Lewis C –32. HoweESM 1(PDF 605 kb)Below is the link to the electronic supplementary material.


For this reason, the activity of PLA2 was investigated regarding its pharmaceutical properties. This study was conducted to explore the pharmacological properties of a PLA2 from Daboia siamensis (dssPLA2) venom on human skin melanoma cell line (SK-MEL-28).Phospholipase A2 was isolated by ion exchange and gel filtration columns. Various concentrations of dssPLA2 were investigated for cytotoxic activity and inhibition of migration on SK-MEL-28 cells. Cell death analysis, mRNA expression levels of Notch I-III and BRAF V600E genes were also determined.dssPLA2 exhibited cytotoxicity on SK-MEL-28 for 24 and 72 h as compared with untreated cells. However, it had no toxic effects on CCD-1064sk cells under the same conditions. dssPLA2 (0.25 and 0.5 μg/mL) induced 17.16 and 30.60 % of apoptosis, while activated 6.53 and 7.05 % of necrotic cells. dssPLA2 at 0.25, 0.5, 1 and 2 μg/mL could inhibit migration on SK-MEL-28 cells for 24 h by 31.06, 41.66, 50 and 68.75 %, respectively. The action of dssPLA2 significantly reduced the levels of Notch I and BRAF V600E genes expression on SK-MEL-28 cells compared with untreated cells at 72 h.dssPLA2 had potential effects of apoptosis, necrosis, cytotoxicity and inhibition of migration on SK-MEL-28 cells. dssPLA2 could possibly be a selective agent that targets cancer cells without affecting normal cells.This study indicates that dssPLAThe online version of this article (doi:10.1186/s40409-016-0061-z) contains supplementary material, which is available to authorized users. Skin cancer, especially melanoma, is a medical problem with increasing incidence that is generally asymptomatic . The risκB and downregulation of cyclin D1, led to antiproliferation on SK-MEL-28 cells . Z is the negative distance at time 0. Tn is the experiment distance at time 24 h.Wound healing assay was performed for inhibition of cell migration . SK-MEL-2 for 24 and 72 h. Untreated of SK-MEL-28 cells were used as a negative control. One microgram of total RNA was employed to generate cDNA using random hexamer primers. One microliter of 1:10 cDNA dilution was applied to PCR reactions. Sequences of specific primers were Notch1 forward: 5’-CAGCCTGCACAACCAGACAGA-3’; reverse: 5’-TGAGTTGATGAGGTCCTCCAG-3’; Notch2 forward: 5’-AAAAATGGGGCCAACCGAGAC-3’; reverse: 5’-TCATCCAGAAGGCGCACA A-3’; Notch3 forward: 5’-AGATTCTCATCCGAAACCGCTCTA-3’; reverse: 5’-GGGGTCTCCTCCTTGC TATCCTG-3’; BRAF V600E forward: 5’-AGGTGATTTTGGTCTAGCTACAGA-3’; reverse: 5’-TAGTAACTCAGCAGCATCTCAGGG C-3’; β-actin forward: 5’-ACCAACTGGGACGACATG GACAA-3’; reverse: 5’-GTGGTGGTGAAGCTGTAGCC-3’.qRT-PCR was performed by following manufacturers’ recommendations (RBC Sciences). Total RNA was extracted from the SK-MEL-28 cells that were treated at 0.25 μg/mL of dssPLA-(∆∆CT). CT was a threshold cycle.qRT-PCR was performed based on SYBR Green (RBC Biosciences) to quantify the relative expression of BRAF V600E mutation gene and Notch signaling receptors: N1, N2, N3 genes. The mRNA level of the β-actin gene was detected in each cDNA sample to normalize the expression level of gene of interest (GOI). The ratio of GOI and β-actin was compared among samples. The fold change of GOI expression was acquired by setting the values from the untreated cells to one. Calculation of expression level of each gene that was normalized to the expression level of β-actin by Delta Delta Ct equation: ∆∆Ct = ∆Ct (Target gene treated – Reference gene treated) – ∆Ct (Target gene control – Reference gene control). The mRNA expression gene was calculated by 2 t-test (Primer of Biostatistics version 3.02). Differences between the mean values of results were considered statistical significant at p < 0.05.The statistical significance of the results was analyzed using 2 activity that increased 11.67 folds in the final step. The percent yield of dssPLA2 was 27.0 % of crude venom protein. The size of dssPLA2 was confirmed, 16.4 kDa, by using mass spectrometry. The summary of the purified method was displayed in Fig. In the process of purification, Peak 2 from the second step showed the highest PLA2 at concentrations from 0 to 4 μg/mL for 24 and 72 h of incubation. dssPLA2 effects included dose- and time-dependent cytotoxicity on SK-MEL-28 cells. Viability of cells at 0.25, 0.5, 1, 2 and 4 μg/mL were, respectively, 82.26 % ± 6.73, 72.38 % ± 7.66, 49.19 % ± 5.09, 32.52 % ± 3.3 and 17.82 % ± 4.17 for 24 h whereas were 69.15 % ± 5.38, 32.65 % ± 3.42, 15.95 % ± 2.76, 10.23 % ± 3.35 and 5.3 % ± 1.21 for 72 h when compared with untreated cells. Viability of SK-MEL-28 cells at 72-h incubation was decreased approximately three fold at 1, 2 and 4 μg/mL and 1- and 2-fold at 0.25 and 0.5 μg/mL dssPLA2 concentration when compared with 24 h-incubation. However, dssPLA2 was not toxic to CCD-1064sk cells at the same conditions  and 0.5 μg/mL (7.05 %), while untreated SK-MEL-28 cells showed signs of necrosis in 1.92 % of the cells  by MTT assay. These results agree with a previous work that suggested that venom toxins specifically targets cancer cells without affecting normal ones [Snake venom PLAf snakes . In the mal ones . It indimal ones , 28, 29.2 (0.25 and 0.5 μg/mL) on apoptosis (17.6 % and 30.60 %) at 24 h of incubation suggests that dssPLA2 induced moderate apoptosis on SK-MEL-28 cells. Apoptosis pathway contains multiple cascades that may be investigated by various techniques. AnnexinV-FITC is a tool to detect phosphatidylserine (PS) on the cell surface in initiating apoptosis. Optimization of concentration and incubation period of dssPLA2 in order to provoke apoptosis on SK-MEL-28 cells could be improved in further studies. There were a couple of effects of dssPLA2 on SK-MEL-28 cells at 24 h of incubation. First, dssPLA2 induced apoptosis on SK-MEL-28 cells. In that moment, it had also provoked 31.60, 41.66, 50.0 and 68.75 % of migration inhibition at the following concentrations: 0.25, 0.5, 1 and 2 μg/mL. dssPLA2 (0.25 and 0.5 μg/mL) had induced death on SK-MEL-28 cells (23.19 and 37.65 %) at 24 h. The remaining surviving cells (76.81 and 62.35 %) resisted apoptosis and necrosis, and could migrate. Therefore, the cells migration might be inhibited due to other effects of dssPLA2. The minimum concentration of SK-MEL-28 cells (1 × 105 cells/mL) could close a scratch within 24 h without inhibitors .


Here, the expression of F4/80 on the cell surface of murine macrophages was exploited to develop a novel imaging tracer that could visualize macrophages in vivo.50 of anti-F4/80-A3-1, conjugated with diethylenetriaminepentaacetic acid (DTPA) and radiolabelled with 111In, were determined in vitro using murine bone marrow-derived macrophages. In vivo biodistribution studies were performed with 111In-anti-F4/80-A3-1 and isotype-matched control antibody 111In-rat IgG2b at 24 and 72 h post-injection (p.i.) in SCID/Beige mice bearing orthotopic MDA-MB-231 xenografts. In some studies mice were also treated with liposomal clodronate. Macrophage content in tissues was determined immunohistochemically. Micro-single photon emission computed tomography (SPECT)/CT images were also acquired.The immunoreactive fraction and IC111In-anti-F4/80-A3-1 specifically binds F4/80 receptor-positive macrophages. The immunoreactivity of anti-F4/80-A3-1 was 75 % and IC50 was 0.58 nM. In vivo, injection of 10 or 100 μg 111In-anti-F4/80-A3-1 resulted in splenic uptake of 78 %ID/g and 31 %ID/g, respectively, and tumour uptake of 1.38 %ID/g and 4.08 %ID/g, respectively (72 h p.i.). Liposomal clodronate treatment reduced splenic uptake of 10 μg 111In-anti-F4/80-A3-1 from 248 %ID/g to 114 %ID/g and reduced 111In-anti-F4/80-A3-1 uptake in the liver and femur (24 h p.i.). Tracer retention in the blood and tumour uptake increased (24 h p.i.). Tumour uptake of 111In-anti-F4/80-A3-1 was visualized by microSPECT/CT. Macrophage density in the spleen and liver decreased in mice treated with liposomal clodronate. Uptake of 111In-rat IgG2b was lower in the spleen, liver and femur when compared to 111In-anti-F4/80-A3-1.In vitro binding assays showed that Radiolabelled anti-F4/80-A3-1 antibodies specifically localize in tissues infiltrated by macrophages in mice and can be used to visualize tumours. The liver and spleen act as antigen sink organs for macrophage-specific tracers.The online version of this article (doi:10.1007/s00259-015-3084-8) contains supplementary material, which is available to authorized users. Macrophages play an important role in the phagocytosis of microbes and in the regulation of developmental processes and pathophysiological responses, including inflammation and tumour growth . MonocytThe F4/80 receptor is generally considered a murine pan-macrophage marker  and is eThe multiple therapies targeting TAMs could offer new routes in cancer therapy. Being able to image TAMs could aid in the development of novel macrophage-targeted drugs and could be applied to monitor the kinetics of macrophage infiltration in response to antibody-based immunotherapies engaging innate immune cells, such as therapy with anti-epidermal growth factor receptor (EGFR) antibodies . In addi99mTc-labelled anti-mannose receptor nanobodies  in 9 ml of an aqueous solution of 100 mg/ml clodronate disodium salt (Sigma-Aldrich). Subsequently, the 10 ml crude liposome dispersion was sized by multiple extrusion at 60 °C using a medium pressure extruder  equipped with two stacked polycarbonate membrane filters, one with a pore size of 200 nm on top of one with 100 nm pores. Alcohol and free clodronate (not incorporated in liposomes) were removed by repeated cycles of ultrafiltration and replacement of the filtrate with PBS. The resulting formulation consisted of liposomes of approximately 125 nm in diameter as measured by dynamic light scattering, with a polydispersity index of 0.05 and a zeta potential of approximately −30 mV. Content determination was done by extraction using the organic phase for lipid determination (HPLC followed by evaporative light scattering detection) and the aqueous phase to assess the clodronate content . When tumours reached 150–400 mm3 in volume, mice were injected intravenously (i.v.) with purified 10 μg (1 MBq) or 100 μg 111In-anti-F4/80-A3-1 (1.6 MBq) or 111In-rat IgG2b (1.5 MBq) in 200 μl PBS, 0.5 % BSA. Mice were euthanized by CO2/O2 asphyxiation at 24 or 72 h post-injection (p.i.). For biodistribution studies involving liposomes, 300 μl of the liposome mix was injected i.v. per mouse, twice in 4 days . Blood, tumour and the major organs and tissues were dissected, weighed and counted in a gamma counter. Blood samples were also taken at 1 h p.i. of 10 μg 111In-F4/80 or 111In-rat IgG2b in mice treated with or without clodronate or empty liposomes. Tumours, spleens and livers were flash frozen for further immunohistochemical analysis. The percentage injected dose per gram (%ID/g) of each sample was calculated.In biodistribution studies, female SCID/Beige mice were inoculated in the mammary fad pad with 50 μl RPMI-1640 medium containing 2 × 10Tissues were snap-frozen and sections were fixed in acetone, blocked for 30 min with 5 % normal goat serum and incubated with rat anti-mouse anti-CD68 antibody (AbD Serotec) followed by a secondary biotinylated goat anti-rat antibody. Sections were then exposed to an avidin-biotin-enzyme complex  and were incubated with diaminobenzidine for development followed by a haematoxylin counterstain. The number of CD68-positive cells per 1.5 mm × 1.5 mm square (as highlighted in ×10 image) was calculated for the spleen, liver and tumour sections averaged from six to eight squares/areas of tissue from one sample.111In-F4/80 (14 MBq). Mice were euthanized at 24 h p.i. and scanned using the U-SPECT-II/CT  (MILabs) [In microSPECT imaging studies, mice with orthotopic MDA-MB-231 tumour xenografts were injected i.v. with 100 μg purified (MILabs) . A CT sct test. Statistical significance was set at *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001.In in vitro and biodistribution studies, all values are given as mean ± standard deviation. Statistical analysis in biodistribution studies and tabular data Fig.  was perf111In-anti-F4/80-A3-1 and 111In-rat IgG2b were prepared with a maximum specific activity of 12 and 2 MBq/μg, respectively. After purification, a radiochemical purity of ≥95 % was achieved for both tracers (data not shown). A sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gel was run in the evaluation of the antibody integrity, after conjugation, and it showed a band at both 140 and 50 kDa, suggesting the presence of a F(ab) fragment in the preparation.+/F4/80+ macrophages. MDA-MB-231 human breast cancer cells were CD11b−/F4/80− (data not shown).Bone marrow cells isolated from murine femoral bone marrow were differentiated to CD11b111In-anti-F4/80-A3-1 for F4/80 receptors was 75 % . The addition of a blocking dose of ITC-DTPA-anti-F4/80-A3-1 decreased binding of 111In-anti-F4/80-A3-1 to macrophages from 10.9 ± 2.8 to 0.6 ± 0.1 %  in the circulation decreased from 1.27 %ID/g at 24 h p.i. to 0.39 %ID/g at 72 h p.i. Uptake of 111In-anti-F4/80-A3-1 in the tumour, liver and spleen (p ≤ 0.001) also decreased; however, tracer uptake in all three tissues increased relative to blood. At 72 h p.i., the tumour to blood uptake ratio of 111In-anti-F4/80-A3-1 (10 μg) was 4.6 compared to 1.8 at 24 h p.i. The spleen to blood and liver to blood ratios also increased from 195 to 201 and from 22 to 29, at 24 and 72 h p.i., respectively.The results of the biodistribution studies of 10 and 100 μg 111In-anti-F4/80-A3-1 resulted in a lower splenic uptake than injection of 10 μg 111In-anti-F4/80-A3-1: 30.94 %ID/g versus 78.23 %ID/g at 72 h p.i. (Table p ≤ 0.0001). Uptake of 111In-anti-F4/80-A3-1 in the MDA-MB-231 tumour was higher at the higher dose: 4.08 %ID/g versus 1.38 %ID/g.Injection of 100 μg 111In-anti-F4/80-A3-1 at 24 h p.i. was markedly reduced after mice were treated with clodronate liposomes: 114.35 %ID/g compared to 247.88 %ID/g (p ≤ 0.0001), whereas uptake was unaffected in mice treated with empty liposomes  than that of 111In-anti-F4/80-A3-1 (p ≤ 0.0001).Splenic uptake of 111In-anti-F4/80-A3-1 in both the liver (p ≤ 0.0001) and femur (p ≤ 0.01)  compared to the control group . Figure 111In-anti-F4/80-A3-1 did not significantly differ in animals treated with or without clodronate liposomes (p > 0.05). Tumour uptake of 111In-rat IgG2b was significantly higher than 111In-anti-F4/80-A3-1 , yet higher circulation levels of 111In-rat IgG2b meant significantly lower tumour to blood values for 111In-rat IgG2b than for 111In-anti-F4/80-A3-1. Blood levels of 111In-anti-F4/80-A3-1 (10 μg) at 1 h p.i. were clearly higher after clodronate treatment , spleen (p ≤ 0.01) and tumour (p ≤ 0.05) was lowered significantly after treatment with clodronate liposomes, which equalled 363 ± 64, 1,552 ± 274 and 586 ± 260 per square, respectively, when compared to controls . Treatment of mice with empty liposomes did not affect the macrophage content in tissues compared to controls (data not shown).Figure Fused SPECT/CT scans Fig.  show tha111In-anti-F4/80-A3-1 allows visualization of TAMs as well as macrophages in the spleen and liver. MicroSPECT imaging also shows uptake of the tracer in the kidneys.In this study, we demonstrated that 111In-anti-F4/80-A3-1 specifically bound F4/80 receptor-positive macrophages with high affinity. Supplementary Figures 111In-anti-F4/80-A3-1 and that 111In-anti-F4/80-A3-1 mostly localized at F4/80-positive cells . Partial saturation of these sinks, by the injection of a higher antibody dose, led to improved tumour uptake.Initial in vivo experiments showed that 111In-anti-F4/80-A3-1 in the spleen and liver , suggesting that saturated opsonization only partially reduced the tracer uptake and that decreased macrophage density further reduced the hepatic uptake of the tracer.The main exception to this is the liver where treatment with empty liposomes diminished uptake of e tracer , 34. Nev111In-anti-F4/80-A3-1 uptake in the tumour after clodronate liposomes treatment is a result of enhanced levels of the tracer in the blood.Macrophage infiltration of the tumour was also affected by clodronate treatment, yet the increase in 99mTc-labelled anti-macrophage mannose receptor nanobodies [111In-anti-F4/80-A3-1 macrophages could be monitored for up to 72 h p.i. This could be a great advantage when using imaging tracers to monitor response to existing or novel therapies that interfere with macrophage-tumour cell interactions.Apart from fluorescently labelled anti-F4/80-A3-1 antibodies being used to image arthritis in vivo , an intanobodies  and radinobodies . Both tr111In-anti-F4/80-A3-1, or anti-F4/80-A3-1 labelled with a shorter half-life radionuclide to compensate for the shorter than usual circulating time of the antibody, could provide a good alternative to the current armamentarium of macrophage tracers. With an increased interest in developing drug strategies targeting tumour macrophages, a need for imaging these macrophages preclinically has also arisen, which might be met by the tracer presented here, if future studies also prove promising. 111In-anti-F4/80-A3-1, as a tracer targeting murine macrophages, therefore has potential to become an important in vivo tool in the development and characterization of novel macrophage-targeted therapies.Depending on the need of the tracer, the specific kinetics and biodistribution of 111In-anti-F4/80-A3-1 specifically visualized the tumour and macrophages resident in tissues. The spleen and liver act as antigen sink organs, which has implications for the dose of 111In-anti-F4/80-A3-1 that should be administered in order to see high tumour targeting. These results imply that if 111In-anti-F4/80-A3-1 can be used to monitor TAMs in vivo, its rapid uptake in non-tumour tissues should first be prevented and circulation time enhanced to achieve optimal tumour localization of 111In-anti-F4/80-A3-1. Alternatively, imaging TAMs could also be further accomplished by developing a tracer specific to M2 macrophages. This should lead to a comparatively enhanced tumour uptake and diminished non-tumour tissue uptake by circumventing M1 macrophages that were present in the current antigen sinks leading to reduced tumour uptake of 111In-anti-F4/80-A3-1.In conclusion, Supplementary Figure 1(DOCX 35 kb)Supplementary Figure 2(DOCX 1740 kb)Supplementary Figure 3(DOCX 104 kb)Supplementary Figure 4(DOCX 170 kb)