The title heterocyclic 1,3,4-oxadiazole derivatives differ from each other in the groups attached to the carbon atoms: a methoxyphenyl ring and a benzonitrile group in (I) and a chlorophenyl ring and an acetamide group in (II). 18H15N3O4 and C17H14ClN3O3, are heterocyclic 1,3,4-oxadiazole derivatives which differ from each other in the groups attached to the carbon atoms: a methoxyphenyl ring and a benzonitrile group in (I) and a chlorophenyl ring and an acetamide group in (II). Short intramolecular C—H⋯O hydrogen bonds occur in both molecules. The crystal structure of (I) features C—H⋯N hydrogen bonds, while in the crystal structure of (II), N—H⋯N, C—H⋯N and C—H⋯O hydrogen bonds are observed.The title compounds, C Compounds containing a heterocyclic ring system are of great importance both medicinally and industrially S(5) ring motifs  and (II)s Figs. 1 and 2 ▸.B⋯O3iii, Table 1A⋯N1i, Table 1C(5) chains propagating along [010]  chains propagating in an anti-parallel manner along [110]. These C—H⋯N hydrogen bonds along with the C—H⋯O dimers form a closed cavity shape arrangement consisting of 26 atoms in the unit cell if Fig. 4. Further0] Fig. 5. There ill Fig. 6. In addiC(10) chains, C—H⋯N hydrogen bonds forming C(8) chains and C—H⋯O interactions forming C(15) chains ns Fig. 7. All thed Table 2. No π–π 2CO3 (3 mmol) in DMF (4 mL), 2-(chloromethyl)-5-(4-methoxyphenyl)-1,3,4-oxadiazole and KI (0.5 mmol). The reaction mixture was stirred at room temperature for about 2 h until the starting material had been consumed (TLC monitoring), and then washed with cold water. The solid product was collected by filtration and dried under vacuum. The pure compound was further recrystallized from ethyl acetate/petroleum ether solution (v:v = 1:1).Compound (I)N-(4-hydroxyphenyl)acetamide (1mmol), K2CO3 (3 mmol) in ACN (5mL), 2-(chloromethyl)-5-(4-chlorophenyl)-1,3,4-oxadiazole and KI (0.5 mmol). The reaction mixture was stirred under reflux condition for about 16 h, until completion of the reaction (TLC monitoring), then it was diluted with ethyl acetate (30 mL) and washed with saturated NaHCO3 and cold water. The organic layer was separated, dried over anhydrous Na2SO4 and concentrated under vacuum. The pure compound was further recrystallized from an ethyl acetate/petroleum ether solution (v:v = 1:1), giving colourless block-like crystals suitable for X-ray diffraction analysis.Compound (II)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 310.1107/S2056989018016754/zq2243sup1.cifCrystal structure: contains datablock(s) I, II, global. DOI: 10.1107/S2056989018016754/zq2243Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989018016754/zq2243IIsup4.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989018016754/zq2243Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989018016754/zq2243IIsup5.cmlSupporting information file. DOI: 1881075, 1881074CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Kα and Mo Kα radiation as part of a continuous crystallization study. In the crystal, chains along the a axis are formed via N—H⋯O hydrogen bonds between acetamide groups, as well as C—H⋯O interactions. These chains arrange themselves into parallel running stacks which display weak C—Cl⋯O=Chalogen bonding as well as weak C—H⋯π interactions.Two independent samples of the title compound were studied using Cu  N-(4-methylphenyl)propanamide, C10H12ClNO, 1, were studied using Cu Kα, 1a, and Mo Kα, 1b, radiation as part of a continuous crystallization study. The molecule crystallizes with disorder in the Cl/terminal methyl positions [occupancies for the major disorder component of 0.783 (2) in 1a and and 0.768 (2) in 1b] and exhibits N—C bond lengths of 1.3448 (19), 1.344 (2) Å, C=O bond lengths of 1.2233 (18) and 1.2245 (19) Å and an acetamide moiety C—N—C—C torsion angle of 179.00 (13), 178.97 (14) ° for 1a and 1b, respectively. In the crystal, chains along the a axis are formed via N—H⋯O hydrogen bonds between acetamide groups, as well as C—H⋯O interactions. These chains arrange themselves into parallel running stacks which display weak C—Cl⋯O=C halogen bonding as well as weak C—H⋯π interactions.Two independent samples of the title compound, alternatively 2-chloro- The disorder occupancy is different in 1a, 1b and in IQOHOL, but to no great extent with the occupancy of the major component being 0.783 (2) for 1a, 0.768 (2) for 1b and for 0.899 IQOHOL.An overlay of the molecular structures of i, see Tables 2et al., 20011.In the extended structure there is, as expected, a strong amide hydrogen bond, between the N—H group and the ketone oxygen  and 2.8632 (6) Å respectively)] The head-to-tail packing and the chelate hydrogen bonding allows an approximately linear arrangement of 1, forming ribbons propagating along the [100] direction, see Fig. 31a and 1.71940 (13)° in 1b).There is also a weaker interaction between the methine group and the ketone  and 1b, 3.1734 (18) Å . A very weak example of a C—H⋯πiii interaction is also present in 1, with the methyl group C12 directed towards the centroid of ring C1–C6  yielded several similar substituted phenylacetamides: CLACTN  in toluene (50 mL) and 40 mL of aqueous NaOH  at 273 K. After the addition was complete, the biphasic suspension was warmed to room temperature and stirred vigorously for 1 h. The organic phase was separated, and the aqueous layer extracted with ethyl acetate (3 × 15 mL). The organic layers were then combined, dried with Na2SO4, filtered and the solvent removed under vacuum. The resulting off-white solid was collected and washed with thoroughly with cold cyclohexane . Single crystals for X-ray analysis were grown by slow evaporation of a toluene solution at room temperature. Spectroscopic data for the obtained product matched that reported in the literature  in toluene (30 mL) was added dropwise (with extreme caution) to a vigorously stirred bi-phasic suspension of 1H NMR : δ 8.21 , 7.42 , 7.15 , 4.54  2.13 , 1.83 . 13C NMR : δ 166.9 134.4, 134.0, 129.1, 119.7, 55.9, 22.4, 20.5. MS (EI) m/z 197 [M]+, [12C10H1235Cl14N16O 197]. HRMS (EI) m/z Found: [M]+ 197.0604, [C10H12ClNO]+ requires 197.0607.1a and 1b, Cl1/Cl1a and C12/C12a were modelled as disordered over two positions using restraints  and constraints . The occupancy was allowed to refine with a population parameter of 1a = 0.783 (2), and 1b = 0.768 (2). The amide N—H H atom was located in a difference-Fourier map and freely refined. H atoms bonded to carbon were placed with idealized geometry and refined using a riding model with C—H = 0.95 Å aromatic, C—H = 0.90 Å methine, with Uiso(H) = 1.2Ueq(C) and C—H = 0.98 Å methyl with Uiso(H) = 1.5Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989018013889/ds2252sup1.cifCrystal structure: contains datablock(s) . DOI: 10.1107/S2056989018013889/ds22521asup2.hklStructure factors: contains datablock(s) 1a. DOI: 10.1107/S2056989018013889/ds22521bsup3.hklStructure factors: contains datablock(s) 1b. DOI: Click here for additional data file.10.1107/S2056989018013889/ds22521asup4.cmlSupporting information file. DOI: 1870782, 1870781CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the crystal structure of the title sulfonamide, intermolecular N—H⋯O hydrogen bonds are present between sulfonamide groups, as well as offset π–π interactions. 10H13NO2S, was synthesized by a nucleophilic substitution reaction between allyl amine and p-toluenesulfonyl chloride. The sulfonate S—O bond lengths are 1.4282 (17) and 1.4353 (17) Å, and the C—N—S—C torsion angle involving the sulfonamide moiety is −61.0 (2)°. In the crystal, centrosymmetric dimers of the title compound are present via intermolecular N—H⋯O hydrogen bonds between sulfonamide groups. These dimers are linked into ribbons along the c-axis direction through offset π–π interactions.The title compound, C Centrosymmetric dimers of compound (I)i distance of 2.900 (3) Å suggests interactions of medium strength with a nearly linear N—H⋯O hydrogen bond of 174 (3)°  Å, with a slippage of 1.320 Å and a plane-to-plane distance between phenyl rings of 3.600 Å .Molecules of the title compound are linked to one another ° Table 1. These ds Figs. 3, 4 ▸. Thet al., 2016p-tolylsulfonamides where there is a –CH2—C=C group bonded to the sulfonamide-N atom. The alkene group in these structures is a part of, for example, furan rings . The mixture was stirred at room temperature for 24 h. Reaction completion was verified by using TLC analysis. The mixture was acidified to pH 2–3 using concentrated HCl. After dilution with 20 ml of CH2Cl2, the organic phase was washed with H2O (3 × 20 ml) and the aqueous layer was back-extracted with CH2Cl2 (20 ml). The combined organic extracts were dried over anhydrous Na2SO4. After solvent evaporation, the residue was obtained as a yellow solid which was recrystallized in cold ethanol to afford pale-yellow crystals .Allylamine  was added in 20 ml of degassed dichloromethane. This was followed by the addition of pyridine . The resulting solution was stirred under an atmosphere of Nsp3—H = 0.95–1.00 Å with Uiso(H) = 1.2Ueq(C) for methine and methylene groups, and Uiso(H) = 1.5Ueq(C) for methyl groups. The hydrogen atom bonded to the nitrogen atom (H1) was located using electron-density difference maps, and the N—H bond length was restrained to 0.84±0.01 Å using the DFIX command as executed in SHELXL  I. DOI: 10.1107/S2056989018010290/wm5455Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989018010290/wm5455Isup3.cmlSupporting information file. DOI: 1856234CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Z′ = 1 is reported.A new monoclinic polymorph of 2,2′-methylenebis with  C2/c) of 2,2′-methylenebis, C17H10N2O4, is reported and compared to the previously reported triclinic polymorph (space group PZ′ = 1). The molecular conformations of the two polymorphs are very similar, as shown by the r.m.s. deviation of 0.368 Å . The intermolecular interactions of both polymorphs are described along with the Hirshfeld surface analysis, and the lattice energies are calculated.In this study, a new monoclinic polymorph (space group  N-heterocycles have been proven to exhibit significant biological and pharmaceutical activities, and have also been used as dyes and heat-resistant polymers in industry  Å, b = 9.5810 (8) Å, c = 10.2780 (6) Å, α = 104.325 (3)°, β = 99.768 (4)°, γ = 96.030 (3)°, Z = 2, Z′ = 1 and V = 712.23 (11) Å3; Cambridge Structural Database  of 2,2′-methylenebis with Z′ = 1 and compare its properties with those of 1α. According to the Online Dictionary of Crystallography, polymorphism is the phenomenon in which the same chemical compound exhibits different crystal structures  derivatives with five-membered α and 1β  and 0.064 (3) Å for 1β. There are two degrees of freedom to characterize the molecular conformations of 1α and 1β: these are the torsion angles C1—N1—C9—N2  and N1—C9—N2—C10 . Generally, the molecule of 1β deviates only slightly from that of 1α, as indicated by a r.m.s. deviation of 0.368 Å  °, which is smaller than that of 88.96 (4)° observed in polymorph 1α. The calculated density and Kitaigorodskii packing index  are slightly higher than those observed for 1α (1.428 Mg m−3 and 69.0%).The asymmetric units of polymorphs 11β Fig. 1 each cons) Fig. 2. The meaα features weak intermolecular C—H⋯O hydrogen bonds and π–π interactions between neighboring phthalimide units. In the crystal structure of 1β  and between N1/C1/C2/C7/C8 and C11–C16 rings  and 1β ], respectively. The percentages of contribution of H⋯C/C⋯H, O⋯C/C⋯O and C⋯C contacts to the Hirshfeld surface are 20.6, 3.3 and 8.9%, respectively, for 1α, and 20.8, 7.9 and 6.7%, respectively, for 1β ]. The C⋯C contacts appear as a unique ‘triangle’ focused at de ≃ di ≃ 1.75 Å [Fig. 4f)]. The intermolecular π–π interactions are illustrated as unique patterns of red and blue ‘triangles’ on the shape-index surface  is slightly larger than for 1β (128.5 kJ mol−1), indicating that 1α is slightly more stable than 1β under ambient conditions.The C—H bond lengths in 1β were obtained from an unsuccessful synthesis of 2-{[(3-iodopyridin-4-yl)amino]methyl}isoindoline-1,3-dione by reacting N-(bromomethyl)phthalimide (1 mmol) and 4-amino-3-iodopyridine (1 mmol) in N,N-dimethylformamide (8 ml) with the presence of a catalytic amount of anhydrous potassium carbonate. The reaction solution was stirred for about 2 h at room temperature. Once the reaction was complete, the resultant mixture was poured into a beaker of ice-cooled water to obtain a precipitate  = 1.2Ueq(C).Crystal data, data collection and structure refinement details of 110.1107/S2056989018017425/jj2205sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989018017425/jj2205Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989018017425/jj2205Isup3.cmlSupporting information file. DOI: 1884044CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The title compound is a salt formed by a hydrazinium (1+) cation and a hexafluoridosilicate anion interconnected by N—H⋯N and N—H⋯F hydrogen bonds. 2H5)2SiF6, the silicon atom at the centre of the slightly distorted SiF6 octahedron [range of Si—F distances = 1.6777 (4)–1.7101 (4) Å] lies on a crystallographic inversion centre. In the crystal, the ions are connected by N—H⋯N and N—H⋯F hydrogen bonds; the former link the cations into [010] chains and the latter (some of which are bifurcated or trifurcated) link the ions into a three-dimensional network. The two-dimensional fingerprint plots show that F⋯H/H⋯F interactions dominate the Hirshfeld surface (75.5%) followed by H⋯H (13.6%) and N⋯H/H⋯N (8.4%) whereas F⋯F (1.9%) and F⋯N/N⋯F (0.6%) have negligible percentages. The title compound is isostructural with its germanium-containing analogue.In the title inorganic molecular salt, (N However, this compound was characterized by chemical analysis, vibrational spectroscopy and X-ray powder photography by Gantar & Rahten  to 1.7101 (4) Å. The minimum and maximum cis F—S—F angles are 89.26 (2) and 90.74 (2)°, respectively. The N—N separation in the cation is 1.4416 (8) Å.Compound (I)6]2− anion interacts with the (N2H5)+ cations through electrostatic attraction and accepts no fewer than ten simple, bifurcated or trifurcated N—H⋯F hydrogen bonds ds Fig. 3. This reon Fig. 4.et al., 2004CrystalExplorer package dnorm . In order to provide quantitative information on the contribution of the intermolecular interactions to the crystal packing, the three-dimensional dnorm surface is resolved into two-dimensional fingerprint plots, generated based on de and di distance scales and illustrated in Fig. 5a)–(f) The F⋯H/H⋯F interactions appear as distinct spikes in the fingerprint plot, and occupy the majority of the total Hirshfeld surface (75.5%) as illustrated in Fig. 5a); the characteristic ‘wingtip’ features indicate the N—H⋯F hydrogen bonds. The H⋯F interaction are represented by a spike  at the bottom left (donor), whereas the F⋯H interactions are represented by a spike  at the bottom right (acceptor) of the fingerprint plot. The H⋯H contacts appear in the middle of the scattered points; these contacts comprise 13.6% of the total Hirshfeld surface [Fig. 5c)]. The N⋯H contacts cover 8.4% of the total surface, as the third important contributor in the crystal packing, Fig. 5d) while the F⋯F and F⋯N/N⋯F contacts make negligible contributions of 1.9% [Fig. 5e)] and 0.6% [Fig. 5f)], respectively.The acceptor atoms in the interactions are shown with negative electrostatic potentials (red regions), and donor atoms are shown with positive electrostatic potentials (blue regions). The N—H⋯F interactions in the structure are apparent from the relatively bright red-spots on the Hirshfeld surface of (I)2H6SiF6, at room temperature, crystallizes in a pseudo-tetragonal orthorhombic space group , with a = 7.605 (1) Å, b = 7.586 (2) Å and c = 8.543 (1) Å  plane.Hydrazinium (2+) hexafluoridosilicate, Net al., 19852H5)2GeF6 crystallizes in the monoclinic system, space group P21/n (Z = 2), with cell parameters a = 6.015 (2) Å, b = 5.249 (1) Å, c = 11.181 (2)Å and β = 100.15 (2)° and is clearly isostructural with (I)Hydrazinium (1+) hexahalogenometallates were studied by Gantar and co-workers (Gantar 2H5)2TiF6 2TiF6  exhibit racemic twinning but are not isostructural with (I)2H5)2TiF6 consists of N2H5+ cations and two types of slightly distorted octahedral (TiF6)2− anions. The N2H5+ cations and (TiF6)2− anions are linked via N—H⋯F and N—H⋯N hydrogen bonds, building a three-dimensional network. Two other isostructural hydrazinium (l+) hexafluorido complexes, (N2H5)2ZrF6 and (N2H5)2HfF6, were prepared and characterized by chemical analysis, vibrational spectroscopy and X-ray powder diffraction .Fluoride complexes of titanium (IV) with ammonium cation derivatives include two hydrazinium hexafluoridotitanates (IV), (N2H5)2SiF6 crystals in the form of colourless blocks were obtained by slow evaporation, at room temperature, of an aqueous solution containing stoichiometric amounts of hydrazine NH2NH2 and H2SiF6. The infrared spectrum was recorded in the range 450–4000 cm−1 with a Vertex 70 FTIR spectrometer.Hydrazinium (1+) hexafluoridosilicate (NUiso(H) = 1.2Ueq(N). The highest peak and the deepest hole in the final Fourier map are at 0.67 Å from F3 and 0.0 Å from Si1.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989019012672/hb7850sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989019012672/hb7850Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989019012672/hb7850sup3.pdfSupporting information file. DOI: Click here for additional data file.10.1107/S2056989019012672/hb7850sup3.rtfSupporting information file. DOI: 1953037CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The syntheses and crystal structures of three cyclotriphosphazenes, all with fluorinated aryloxy side groups that generate different steric characteristics are reported. viz. hexakis(pentafluorophenoxy)cyclotriphosphazene, N3P3(OC6F5)6, 1, hexakis[4-(trifluoromethyl)phenoxy]cyclotriphosphazene, N3P3[OC6H4(CF3)]6, 2 and hexakiscyclotriphosphazene, N3P3[OC6H3(CF3)2]63, are reported. Specifically, each phosphorus atom bears either two pentafluorophenoxy, 4-trifluoromethylphenoxy, or 3,5-trifluoromethylphenoxy groups. The central six-membered phosphazene rings display envelope pucker conformations in each case, albeit to varying degrees. The maximum displacement of the ‘flap atom’ from the plane through the other ring atoms [0.308 (5) Å] is seen in 1, in a molecule that is devoid of hydrogen atoms and which exhibits a ‘wind-swept’ look with all the aromatic rings displaced in the same direction. In 3 an intramolecular C—H(aromatic)⋯F interaction is observed. All the –CF3 groups in 2 and 3 exhibit positional disorder over two rotated orientations in close to statistical ratios. The extended structures of 2 and 3 are consolidated by C—H⋯F interactions of two kinds: (a) linear chains, and (b) cyclic between molecules related by inversion centers. In both 1 and 3, one of the six substituted phenyl rings has a parallel-displaced aromatic π–π stacking interaction with its respective symmetry mate with slippage values of 2.2 Å in 1 and 1.0 Å in 3. None of the structures reported here have solvent voids that could lead to clathrate formation.The syntheses and crystal structures of three cyclotriphosphazenes, all with fluorinated aryloxy side groups that generate different steric characteristics,  Cyclic organophosphazenes have a long history as representatives of inorganic heterocyclic rings, and are also the focus of arguments about reactivity and pseudoaromaticity in inorganic systems 6 (1), hexakis[4-(trifluoromethyl)phenoxy]cyclotriphosphazene N3P3[OC6H4(CF3)]6 (2) and hexakis6 (3) were synthesized by the reactions of hexachlorocyclotriphosphazene with the appropriate sodium fluoro-aryloxides in THF or dioxane solvent. Forcing reaction conditions (boiling dioxane) were required for complete chlorine replacement in the case of the pentafluorophenoxy derivative (1) presumably due to steric hindrance. The three compounds were characterized by NMR spectroscopy in addition to x-ray crystallography.As part of our ongoing work in this area, the three cyclotriphosphazenes hexakis(pentafluorophenoxy)cyclotriphosphazene Npara-fluorophenoxy groups, was reported by other investigators  is 0.308 (5) Å. A similar calculation in 2 shows atom P2 displaced by 0.232 (4) Å, and in 3 atom N3 is displaced the least, only by 0.205 (4) Å. In earlier structure reports: .The molecular structure of ce Fig. 4 with all1, hydrogen bonding is not feasible in that structure ⋯F type hydrogen bonds ⋯F hydrogen bonds  Å, slippage 1.013 Å].The packing diagram of s Table 2, forminget al. 3, CSD refcode KAGKUY: Bullen, 19712)3, VARYES02: Singh et al., 20002)3·THF, GUHPII: Dietrich et al., 2000Earlier, we reported the crystal structures of a number of cyclotriphosphazenes with spirocyclic aryloxy side groups cyclotriphosphazene (1):4, and concentrated to ∼10 ml by rotary evaporation. A small amount of hexanes was added to the concentrated solution and it was chilled to 273 K via an ice bath to yield colorless blocks of 1, which were filtered and rinsed with cold hexanes then dried under vacuum.Sodium pentafluorophenoxide was prepared by the treatment of pentafluorophenol  with a suspension of NaH 60% dispersion in mineral oil  in 50 ml of dioxane. The pentafluorophenoxide was added to a stirred solution of hexachlorocyclotriphosphazene  and the mixture was heated at reflux for 3 d. Dioxane was removed from the mixture by rotary evaporation and the residue was dissolved in 100 ml dichloromethane. The solution was extracted with 3 × 100 ml of deionized water, dried over MgSOSynthesis of hexakis(4-trifluoromethylphenoxy)cyclotriphosphazene (2):1 to yield colorless cubes of 2.The aryloxide was prepared by treatment of 4-trifluoromethylphenol  with a suspension of NaH [60% dispersion in mineral oil ] in 50 ml of THF. To the stirred solution of 4-trifluoromethylphenoxide was added a solution of hexachlorocyclotriphosphazene  in 15 ml of THF and the mixture was stirred at room temperature overnight. The purification steps of this compound were identical to those of compound Synthesis of hexakiscyclotriphosphazene (3):4 and the dichloromethane was removed by rotary evaporation to yield a colorless oil, which crystallized as colorless needles of 3 after standing for several hours. The crystals were rinsed with cold methanol and then dried under vacuum.A stirred suspension of NaH [60% dispersion in mineral oil ] in 25 ml of THF was treated with liquid 3,5-bis-trifluoromethylphenol  by dropwise addition. The resulting aryloxide solution was then added to a stirred solution of hexachlorocyclotriphosphazene  in 25 ml of THF and the reaction mixture was stirred at room temperature overnight. The mixture was concentrated by rotary evaporation and the residue was dissolved in 40 ml dichloromethane. The dichloromethane solution was washed with 40 ml of deionized water, followed by 20 ml of 5% HCl, and finally rinsed with 40 ml of deionized water. The organic layer was dried over MgSO1–3 are as follows: 1: 31P 10.6 1 ppm (in chloroform-d); 19F −153.52 , −157.53 , −161.79 ; 2: 31P 8.64 ppm (in chloroform-d); 1H 747, 706 (d 2H); 19F −62.79 (s); 3: 31P 7.71 ppm (in chloroform-d); 1H 7.74 (s 1H), 7.52 ; 19F −63.85 (s).The NMR data for 2 and 3 were placed geometrically (C—H = 0.93 Å) and refined as riding on their parent atoms with Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details for all three structures are summarized in Table 310.1107/S2056989019012933/hb7853sup1.cifCrystal structure: contains datablock(s) 1, 2, 3. DOI: 10.1107/S2056989019012933/hb78531sup2.hklStructure factors: contains datablock(s) 1. DOI: Click here for additional data file.10.1107/S2056989019012933/hb78531sup5.molSupporting information file. DOI: 10.1107/S2056989019012933/hb78532sup4.hklStructure factors: contains datablock(s) 2. DOI: Click here for additional data file.10.1107/S2056989019012933/hb78532sup6.molSupporting information file. DOI: 10.1107/S2056989019012933/hb78533sup3.hklStructure factors: contains datablock(s) 3. DOI: Click here for additional data file.10.1107/S2056989019012933/hb78533sup7.molSupporting information file. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Enterobacteriaceae family were particularly most sensitive to new pipemidic acid derivatives. Synthesized compounds exhibited very strong activity towards Proteus mirabilis ATCC 12453, Salmonella typhimurium ATCC 14028 and Escherichia coli ATCC 25922. The minimum inhibitory concentrations of new pipemidic acid derivatives which inhibited the growth of these bacteria were 0.98–7.81 µg/ml, 0.98–7.81 µg/ml and 0.98–3.91 µg/ml, respectively. The antibacterial activity of newly synthesized pipemidic acid derivatives in many cases was far better than the activity of substances used as positive controls .This article describes the synthesis and antimicrobial activity evaluation of new pipemidic acid derivatives. New compounds were obtained on the basis of Mannich reaction of 4,5-disubstituted 1,2,4-triazole-3-thiones with pipemidic acid. Antimicrobial tests revealed high antibacterial activity of obtained derivatives. Gram-negative rods belonging to The online version of this article (10.1007/s12272-018-1025-3) contains supplementary material, which is available to authorized users. Microorganisms play a key role in the functioning of the environment but they are also a real threat to human life and health. The widespread diseases and infections caused by different bacteria and fungi have encourage the need for scientific research and advances in medicinal chemistry . The DMSO-d6 was used as solvent and TMS as the internal standard. Chemical shifts in this article are reported in ppm (δ). The coupling constants (J) are presented in Hertz. The purity of obtained compounds and the progress of the reaction were determined by thin-layer chromatography (TLC) with the use of pre-coated aluminum sheet 60 F254 plates (Merck Co. USA), and CHCl3/C2H5OH  solvent system. The spots were identified by the exposure to the UV light at 254 nm. The elemental analysis of obtained compounds was carried out with the use of AMZ 851 CHX analyser . The results of elemental analysis  were within ± 0.4% of the calculated values.The reagents and solvents used in this research were obtained from Merck Co.  and Sigma-Aldrich . Melting points were determined with the use of Fisher-Johns blocks melting point apparatus  and presented without correction. The 1 or 4-tert-butylbenzhydrazide—2, respectively) was dissolved in 10 ml of ethanol (96%). Then 0.0022 mol of appropriate isothiocyanate was added and heated under reflux for 3 h. Subsequently obtained solution was put to the refrigerator for 24 h. After that formed precipitate was filtered off and re-crystallized from ethanol. The procedure of this synthesis was based on our previous article  is presented in Supplementary Materials.Detailed physicochemical data of thiosemicarbazide derivatives (3–18) was dissolved in 5 ml of 2% sodium hydroxide solution and heated under reflux for 2 h. Subsequently, obtained solution was neutralized with diluted hydrochloric acid. Formed precipitate was filtered off and re-crystallized from ethanol.4,5-Disubstituted 1,2,4-triazole-3-thiones were synthesized with the use of the procedure from our earlier research  is presented in Supplementary Materials.Detailed physicochemical data of 4,5-disubstituted 1,2,4-triazole-3-thione derivatives (19–34) was added to the conical flask and dissolved with stirring in 5 ml of ethanol (96%). After that 200 µl of formaldehyde and 0.0033 mol of pipemidic acid was added to the flask. The content of the flask was stirred by magnetic stirrer for 1 h. Subsequently, 15 ml of distilled water was added to the flask. The precipitate which formed was filtered off and re-crystallized from methanol.In order to obtained new pipemidic acid derivatives we applied the Mannich reaction and the procedure reported by our group earlier  is presented in Supplementary Materials.Detailed physicochemical data of new pipemidic acid derivatives  (EUCAST discussion document E. Dis 5.1 The examined compounds 35–50) were obtained with the use of three step reaction scheme (Scheme 3–18) were synthesized on the basis of condensation reaction of appropriate carboxylic acid hydrazides with various isothiocyanates. Subsequently, thiosemicarbazide derivatives (3–18) underwent cyclization reaction with the use of 2% sodium hydroxide solution, which afforded the 4,5-disubstituted 1,2,4-triazole-3-thione derivatives (19–34). Finally, 1,2,4-triazole-3-thione derivatives (19–34) were subjected to Mannich reaction with pipemidic acid to obtain new pipemidic acid derivatives (35–50). Chemical structure of all obtained compounds (3–50) was confirmed on the basis of spectral identification and elemental analysis.New pipemidic acid derivatives (e Scheme . Firstly3–18), 1,2,4-triazoles-3-thiones (19–34) and pipemidic acid derivatives (35–50), were in vitro screened against references strains of bacteria and fungi. The results of antimicrobial assays are presented in the Tables All synthesized compounds, including thiosemicarbazides 3–8, 1,2,4-3–18) defined as MIC  values against Gram-positive bacterial strains ranged from 3.91 µg/ml to 1000 µg/ml , against Gram-negative bacterial strains ranged from 500 µg/ml to 1000 µg/ml (MBC = > 1000 µg/ml). The MIC values against reference fungi were MIC = 250–1000 µg/ml   were within the range of 500–1000 µg/ml against Gram-positive bacterial strains (MBC = 1000 to > 1000 µg/ml), 1000 µg/ml against Gram-negative bacterial strains (MBC = > 1000 µg/ml) and 250–1000 µg/ml for fungal strains (MBC = 1000 to > 1000 µg/ml)  ranged from 3.91 to 1000 µg/ml against Gram-positive bacteria (MBC = 3.91 to > 1000 µg/ml), 0.98–125 µg/ml against Gram-negative bacteria (MBC = 0.98–250 µg/ml) and 1000 µg/ml towards fungi belonging to Candida spp. (MBC = > 1000 µg/ml) , 4,5-disubstituted 1,2,4-triazoles-3-thiones (19–34) and pipemidic acid derivatives (35–50) in this research was confirmed on the basis of spectral (1H NMR and 13C NMR) and elemental analysis.The pathway for the synthesis of new pipemidic acid derivatives Scheme  was desi3–18) showed three typical singlet signals at δ 7.99–10.52 ppm, on the 1H NMR spectra, which correspond to three NH groups. Whereas on the 13C NMR spectra of this group of compounds (3–18), signals for carbonyl group (C=O) and thiocarbonyl group (C=S) were found at δ 155.1–159.5 ppm and around δ 166.0 ppm, respectively.Thiosemicarbazide derivatives (19–34), on the 1H NMR spectra, singlet signal for NH group was noticed in the range of δ 13.75–14.15 ppm. On the 13C NMR spectra the signal for thiocarbonyl group (C=S) of 1,2,4-triazole-3-thiones (19–34) was found at δ 166.5–169.3 ppm.In the case of 4,5-disubstituted 1,2,4-triazole-3-thione derivatives (1H NMR spectra of new pipemidic acid derivatives (35–50) showed characteristic singlet signal for CH2 group in the range of δ 5.18–5.36 ppm, what confirmed common aminomethylation reaction of 1,2,4-triazole-3-thiones. Signals for other aliphatic and aromatic fragments of synthesized compounds on 1H NMR and 13C NMR were shown at expected shift range.The 35–50) and six compounds among 4,5-disubstituted 1,2,4-triazole derivatives (19–34) are new in the literature and their synthesis, physicochemical data and biological activity have not been reported so far.It is worth to mention that all sixteen synthesized pipemidic acid derivatives (3–18), especially compounds: 4–10, 12, 13, 15, 18, had no activity against all reference microorganisms  values, was 32 times better against M. luteus ATCC 10240 (MIC = 3.91 µg/ml) and two times better against B. cereus ATCC 10876 (MIC = 7.81 µg/ml) in comparison with the activity of pipemidic acid , used as positive control  and moderate or mild effect against other reference staphylococci and some Gram-negative rods (MIC = 250–1000 µg/ml and MBC > 1000 µg/ml). Moreover, the compounds 3, 11, 14 and 16 exhibited mild activity (MIC = 1000 µg/ml and MBC > 1000 µg/ml) towards some Gram-positive bacteria. The substances 3 and 11 showed additional effect against Bordetella bronchiseptica ATCC 4617 (MIC = 500–1000 µg/ml and MBC > 1000 µg/ml)  , only a few, namely 19, 20, 21 and 34 exhibited moderate or mild antibacterial activity against some of reference microorganisms  . The remaining substances 22–33 were inactive towards bacteria and fungi from ATCC  were highly active against all reference bacteria (Table Enterobacteriaceae family were particularly most sensitive to these compounds. All substances (35–50) showed bactericidal effect against them. These compounds exhibited very strong activity towards Proteus mirabilis ATCC 12453, Salmonella typhimurium ATCC 14028 and Escherichia coli ATCC 25922. The minimum concentrations of 35–50 compounds, which inhibited the growth of these bacteria were 0.98–7.81 µg/ml, 0.98–7.81 µg/ml and 0.98–3.91 µg/ml, respectively. Klebsiella pneumoniae ATCC 13883 was slightly less susceptible to these substances. The compounds 36, 37, 38, 44, 46 and 50 indicated very strong activity (MIC = MBC = 3.91–7.81 µg/ml), compounds 35, 39 and 45—strong , while remaining compounds 40, 41, 42, 43, 47, 48, 49—good activity (MIC = MBC = 31.25–62.5 µg/ml) towards reference K. pneumoniae ATCC 13883. In addition, all pipemidic acid derivatives (35–50) exhibited a similar good effect against Bordetella bronchiseptica ATCC 4617 and Pseudomonas aeruginosa ATCC 9027   towards B. bronchiseptica ATCC 4617 were two times better in case of compounds 36, 37, 45, 46 and 50 (MBC = 31.25 µg/ml). Against K. pneumoniae ATCC 13883, the compound 36 showed four times better activity (MIC = 3.91 µg/ml), compounds 37, 38, 44, 46, and 50 showed two times better activity (MIC = 7.81 µg/ml) than pipemidic acid (MIC = 15.62 µg/ml). The compounds 35 and 45 showed two times lower MBC values (MBC = 15.62 µg/ml) than pipemidic acid (MBC = 31.25 µg/ml) against this bacterium. In the case of the activity against P. mirabilis ATCC 12453 the MIC values for the compound 36 were two times lower (MIC = 0.98 µg/ml) and the MBC values for the compounds 35, 38, 46 were two times lower (MBC = 1.95 µg/ml) than such values for pipemidic acid . The activity of synthesized derivatives was also better than pipemidic acid towards Salmonella typhimurium ATCC 14028. The compounds 37 and 38 showed two times better activity on the basis of MIC values (MIC = 0.98 µg/ml) than pipemidic acid (MIC = 1.95 µg/ml) against this bacterium. The MBC values for compounds 35, 36, 45 and 46 (MBC = 1.95 µg/ml) were two times lower than for pipemidic acid used as positive control (MBC = 3.91 µg/ml). In addition to this, the MBC values for compounds 36, 37, 38, and 45 against E. coli ATCC 25922 (MBC = 0.98 µg/ml) were two times lower than for pipemidic acid (MBC = 1.95 µg/ml)  values of pipemidic acid derivatives 5–50 towa35-50 indicated also high activity towards Gram-positive bacteria but slightly weaker compared to Gram-negative microorganisms. Most of the substances showed very strong bactericidal or bacteriostatic effect against Bacillus subtilis ATCC 6633 with MIC = 3.91–7.81 µg/ml, MBC = 3.91–250 µg/ml and MBC/MIC = 1–32. The other compounds 43 and 49 indicated good (MIC = MBC = 31.25 µg/ml) or strong (MIC = MBC = 15.62 µg/ml) bactericidal activity towards this bacterium. Bacillus cereus ATCC 10876 was slightly less sensitive to these substances. Among them the compound 37 showed very strong activity  against this bacterium. In turn, substances 35, 36, 38, 39, 41, 45, 46 and 50 had a strong activity , while compounds 40, 42, 43, 44, 47, 48 and 49—good activity  (Table The compounds 35–50) showed also good activity with bactericidal or bacteriostatic effect against microorganisms belonging to reference staphylococci: S. aureus ATCC 25923, S. aureus ATCC 6538 and S. epidermidis ATCC 12228 . Among them, compounds 36 and 50 showed strong bactericidal activity towards S. aureus ATCC 6538 , while compound 50—very strong  and compounds 36 and 46—strong activity  towards S. epidermidis ATCC 12228 (Table Pipemidic acid derivatives 5–50 show35–50) showed only moderate activity towards Micrococcus luteus ATCC 10240 (MIC = 250–500 µg/ml and MBC > 1000 µg/ml) and most of them except 39 and 47 against Staphylococcus aureus ATCC 43300 (MIC = 250–500 µg/ml and MBC = 500 to > 1000 µg/ml). The compounds 39 and 47 indicated good (MIC = 125 µg/ml and MBC > 1000 µg/ml) or mild (MIC = 1000 µg/ml and MBC > 1000 µg/ml) activity, respectively against S. aureus ATCC 43300  was four times better and for the compounds 35, 37, 38, 44, 45, and 46 (MIC = 31.25 µg/ml) was two times better against S. aureus ATCC 6538 on the basis of MIC values. The compound 50 showed two times lower MIC values (MIC = 7.81 µg/ml) with bactericidal effect and the compounds 36 and 46 showed two times lower MBC values (MBC = 31.25 µg/ml) with bactericidal effect against S. epidermidis ATCC 12228 than pipemidic acid . The MBC values for the derivative 36 towards B. subtilis ATCC 6633 (MBC = 3.91 µg/ml) were two times lower than for pipemidic acid (MBC = 7.81 µg/ml). In the case of the activity against B. cereus ATCC 10876 the MIC values for the substance 37 were two times lower (MIC = 7.81 µg/ml) than for pipemidic acid (MIC = 15.62 µg/ml) (Table 35–50) against B. cereus ATCC 10876 is especially important due to the fact that this bacterium is responsible for an increasing number of foodborne diseases in industrial countries as well as postoperative and posttraumatic wound infections , the activity of the compounds 35–50) indicated also moderate or mild activity towards reference fungi belonging to yeasts. Among them the compound 42 inhibited the growth of all Candida spp. (MIC = 500–1000 µg/ml and MFC > 1000 µg/ml). In turn, substances 38, 41, 43 and 49 showed mild activity towards some of them (MIC = 1000 µg/ml and MFC > 1000 µg/ml). The remaining compounds were inactive against reference Candida spp. Below is the link to the electronic supplementary material.


C2v symmetry and crystallizes in the chiral space group P21.The planar achiral title compound has  10H10O4, was synthesized from tetramethyl-1,4-benzoquinone. In the crystal, the almost planar molecule (r.m.s. deviation = 0.024 Å) forms intramolecular hydrogen bonds between the aldehyde and hydroxy groups and exhibits C2v symmetry. This achiral molecule crystallizes in the chiral space group P21 with intermolecular O—H⋯O and C—H⋯O hydrogen bonding and C—H⋯π and C=O⋯π interactions stabilizing the crystal packing.The title compound, C Furthermore, inhibition of nucleic acid biosynthesis and of the activities of coenzyme Q mediated enzyme systems are also known for related compounds composed of 3,6-dihydroxy-4,5-dimethylbenzene-1,2-dicarbaldehyde . The C—C bond lengths of the methyl substituents are 1.511 (2) and 1.508 (2) Å, the C—O bond lengths of the hydroxy substituents are 1.354 (2) and 1.350 (2) Å, and the C—C bond lengths of the aldehyde substituents are 1.464 (2) and 1.462 (2) Å. Two intramolecular O—H⋯H hydrogen bonds between the hydroxy and aldehyde functions are observed  Å (Cg1 is the centroid of ring C1–C6).The C8=O2 carbonyl group is stacked on top of the aromatic ring, with the O2⋯B⋯Cg1 (3.131 Å) is also found  and 50% acetic acid (35 ml) was added dropwise at 353 K. After 10 min, the reaction mixture was poured onto crushed ice (100 g). The solution was filtered by vacuum filtration and a crude compound was obtained. The crude compound was dissolved in toluene and purified by silica column chromatography to afford 0.567 g (yield 23.5%) of the title compound as a yellow solid . IR : 1633 (s), 3436 (m).A mixture of tetramethyl-1,4-benzoquinone  and concentrated piperidine  was stirred at room temperature for 35 h. The mixture was evaporated and a white intermediate product was obtained. To a solution of the obtained intermediate product dissolved in acetic acid (18 ml), a mixture of CrOUiso(H) = 1.5Ueq(C) for methyl H atoms, and C—H = 0.95 Å and Uiso(H) = 1.2Ueq(C) for the aldehyde H atoms]. O-bound H atoms were constrained using a riding model [O—H = 0.84 Å and Uiso(H) = 1.5Ueq(O) for hydroxy H atoms].Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989018012495/vm2211sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989018012495/vm2211Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989018012495/vm2211Isup3.cmlSupporting information file. DOI: 1865811CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The first of these has an unusually short N⋯N separation of 2.616 (2) Å: refinement of different models against the present data set could not distinguish between a symmetrical hydrogen bond (H atom lying on the twofold axis and equidistant from the N atoms) or static or dynamic disorder models (i.e. N—H⋯N + N⋯H—N).In the crystal, two AcAP molecules related by a crystallographic twofold axis link to H R*, 8S)-2-acetamidooctahydropyrrolizin-4-ium chloride–N-[-hexahydro-1H-pyrrolizin-2-yl)acetamide (1/1)], 2(C9H16N2O)·HCl or C9H17N2O+·Cl−·C9H16N2O, arose as an unexpected product when 1-exo-acetamidopyrrolizidine  was dissolved in CHCl3. Within the AcAP pyrrolizidine group, the unsubstituted five-membered ring is disordered over two orientations in a 0.897 (5):0.103 (5) ratio. Two AcAP molecules related by a crystallographic twofold axis link to H+ and Cl− ions lying on the rotation axis, thereby forming N—H⋯N and N—H⋯Cl⋯H—N hydrogen bonds. The first of these has an unusually short N⋯N separation of 2.616 (2) Å: refinement of different models against the present data set could not distinguish between a symmetrical hydrogen bond (H atom lying on the twofold axis and equidistant from the N atoms) or static or dynamic disorder models (i.e. N—H⋯N + N⋯H—N). Computational studies suggest that the disorder model is slightly more stable, but the energy difference is very small.The title compound [systematic name: (1 The ‘flap’ atoms, C2 and C6, for the substituted (C1–C3/N4/C8) and unsubstituted (C5–C8/N4) rings, respectively, are displaced from the mean plane of the four remaining atoms (C1/C3/N4/C8 and C7/C5/N4/C8) by 0.622(1) and 0.633(2) Å, respectively. The dihedral angle between these mean planes is 56.07 (9)°. The acetamide group is characteristically almost planar (r.m.s. deviation = 0.0181 Å), but twisted from the mean plane of C1/C8/N4/C3 by a dihedral angle of 70.56 (7)°. Aside from an unusually short N—H⋯N hydrogen bond, which will be discussed in subsequent sections, all geometrical parameters are within their expected ranges  −x + 1, y, −z + dN⋯N = 2.616 (2) Å . Similarly, since the refined H+ position lies on the twofold axis and is equidistant from N4 and N4i, the refined N⋯H interatomic distance appears unusually long at 1.3080 (12) Å. The whole hydrogen-bonded ensemble makes an N⋯N4i hydrogen bond, a difference-Fourier map , statically disordered  or dynamically disordered . Alternative strategies for H4N inclusion in the model refined equally well , so we settled on the simplest approach, following the recommendations of Fábry . In addition to the strong N—H⋯N hydrogen bond, weaker N2—H2—Cl1—H2i—N2i interactions link the twofold-related acetamide groups to the Cl− anion [N⋯Cl = 3.2263 (11) Å]. The twist of the twofold-related hydrogen-bonded pyrrolizidine moieties relative to each other, as defined by the torsion angle C8—N4⋯N4i—C8i is −69.82 (16)°. The almost planar acetamide group forms a dihedral angle with its twofold-related counterpart of 20.18 (3)°. The only other intramolecular interactions are van der Waals contacts. Estimates of the relative fractions of intermolecular contacts between individual atom types derived from a Hirshfeld-surface analysis using CrystalExplorer . The Cl− anion and the acetamide O atom each reside in pockets surrounded by hydrogen atoms, giving H⋯Cl/Cl⋯H (16.2%) and H⋯O/O⋯H (12.4%), with the remainder being N⋯H/H⋯N and C⋯H/H⋯C contacts.The primary structural motif within crystals of 2AcAP·HCl consists of a pair of homochiral AcAP molecules hydrogen bonded to HÅ Table 1, it is eap Fig. 4 clearly ábry 2018. Nonetheet al., 2016et al., 2017A search of the Cambridge Structure Database -H-N(X)-C′, where ‘X′ denotes ‘any group’, gave 45 hits. Rejection of cases where apparent close N⋯N distances were due to disorder (four entries), and those in which the N-bound H atom was missing from the model (two entries), left 39 structures, of which three were duplicates. In the remaining 36 structures the N—H⋯N hydrogen bonds are intramolecular in 22 and intermolecular in 14. The closest N⋯N separations occur in the intramolecular N—H⋯N hydrogen-bonded structures, the shortest being 2.419 Å in EBOKOV  Å, although the difference is not significant, and well within the quoted precision estimate of BECHOG and the accuracy limits imposed by the spherical-atom scattering-factor approximation , and one unconstrained (B). The volume difference between these theoretical models  was negligible (Avol = 1918.85 Å3versus Bvol = 1919.63 Å3). In the symmetric model, the N—H⋯N hydrogen atom  is equidistant between the two nitrogen atoms (N—H = 1.290 Å), whereas in model B the N—H distances differ (N—H = 1.194 and 1.406 Å). This is in agreement with the computed charge-density line profile of N—H⋯N in structure B, as shown in Fig. 5B is calculated to be slightly more stable, but the energy difference (4.7 meV per unit cell) is vanishingly small , both with and without symmetry constraints on charge density. In the case where symmetry constraints were absent, a small displacement (0.06 Å) was applied to the hydrogen atom in the N—H⋯N hydrogen bond to break symmetry in the initial geometry. The relaxations led to two structures, one with constrained twofold symmetry  with Perdew–Burke–Ernzerhof (PBE) exchange-correlation functional  calculations were carried out using the Vienna et al., 20143 in a 10 ml round-bottom flask and allowing the solution to stand in a refrigerator for about a month.AcAP was synthesized and purified according to the published procedure (Pan RCH3), 0.99 Å (R2CH2) and 1.00 Å (R3CH). Following the advice of Fábry (2018N) was placed into difference-Fourier electron density and refined, albeit constrained to the twofold axis. An alternative model in which this H atom was allowed to ride at 50% occupancy on both N4 and N4i [symmetry code: (i) −x + 1, y, −z + N) was refined freely. Uiso(H) parameters for nitrogen-bound hydrogen atoms were refined, while for carbon-bound H atoms, Uiso(H) were set to values of either 1.2Ueq  or 1.5Ueq (RCH3) of the attached atom.Crystal data, data collection, and structure refinement details are given in Table 2ábry 2018, the hyd− anion  were close (0.37 and 0.47 Å respectively) to Cl1. Refinement of the anion as mixed Cl and Br gave an occupancy ratio of 0.934 (2):0.066 (2), a lower R-value (3.02%), and a flatter difference map (Δρ = 0.29/-0.19 e Å−3). However, the reaction included no known source of Br−, so the mixed anion model was not retained.The refined displacement parameters for the ClSHELXL commands EXYZ and EADP) were used to fix overlapping fragments. Restraints were used to ensure the integrity of ill-defined or disordered groups . An alternative model using space group Cc (50:50 inversion twinned) was considered but rejected as it required hefty restraints and did not resolve the H4N atom ambiguity.To ensure satisfactory refinement for disordered groups in the structure, a combination of constraints and restraints were employed. The constraints  I. DOI: 10.1107/S2056989019016517/hb7870Isup2.hklStructure factors: contains datablock(s) I. DOI: 1970639CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


S(6) graph-set motifs, while C—H⋯O and C—H⋯N interactions form S(5) graph-set motifs.The title compound is a novel halogen-substituted hydrazine derivative. Intramolecular N—H⋯O interactions form  16H14BrN3O5, is a novel halogen (Br) substituted hydrazine derivative. The hydrazine derivatives were the group of compounds with the general structure, R1R2C=NNH2  results in an extended molecular conformation. The dihedral angle between the 5-bromo-2-methoxyphenyl ring and the nitrophenyl ring is 4.4 (3)°. Intramolecular N—H⋯O interactions form S(6) graph-set motifs, while C—H⋯O and C—H⋯N interactions form S(5) graph-set motifs. Symmetry-related molecules are linked by C—H⋯O intermolecular interactions forming an R21(10) graph-set motif. There are nearly face-to-face directional specific π–π stacking interactions between the centroids of the nitrophenyl ring and the benzene ring of the 5-bromo-2-methoxy group [centroid–centroid distance = 3.6121 (5) Å and slippage = 1.115 Å], which also contributes to the molecular packing. The Hirshfeld surface analysis was performed in order to visualize, explore and quantify the intermolecular interactions in the crystal lattice of the title compound.The title compound, C The central bridging moiety R2C=NNHR1 adopts an all-trans conformation about the C10—C9, C9—N3, N3—N2 and N2—C5 bonds, with torsion angles of 176.0 (6), −178.1 (5), −177.0 (6) and 173.6 (6)°, leading to an extended molecular conformation, thereby causing the terminal bromomethoxyphenyl ring and nitrophenylring to occupy almost the same plane; the dihedral angle between the rings is 4.4 (3)°.Fig. 1x, y, z) and the benzene ring of the 5-bromo-2-methoxy group  [centroid–centroid distance = 3.6121 (5) Å and slippage = 1.115 Å], which also contributes to the molecular packing. The Br atom does not take part in any interactions. The nearest Br⋯C7, which are summarized in Table 2et al., 2013et al., 2011Hirshfeld surface analysis serves as a powerful tool for gaining additional insight into intermolecular interactions of molecular crystals. The Hirshfeld surfaces are mapped with 2D fingerprint plots presented using et al., 2016et al., 2011et al. 2010et al. 2010et al., 2015et al., 1985et al., 2003A and B), 9.30 (6)°, and 13.01 (10) and 14.05 (10)° (in molecules A and B), respectively, compared to 4.4 (3)° in the title compound. The crystal packing of the two compounds is significantly different. In AYSOD, N—H groups do not form hydrogen bonds, in DUSBID, the molecules are linked by N—H⋯π interactions, and in DUSNUB, both molecules form inversion dimers linked by pairs of N—H⋯O hydrogen bonds, thereby generating et al., 1995et al., 2013While searching for 2-phenylhydrazine in the Cambridge Structural Database  with a slight excess of 5-bromo-2-methoxybenzaldehyde (0.215 mg) in an acetic acid solution (10 ml). The reaction mixture was refluxed for 8 h. The solid product formed during reflux was filtered off, washed and dried over anhydrous calcium chloride in a vacuum desiccator (yield 75%). The final product was soluble in acetone, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), methanol, ethanol and ethyl acetate, Uiso(H) = xUeq(C), where x = 1.5 for methyl and x = 1.2 for all other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989018011325/dx2007sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989018011325/dx2007Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989018011325/dx2007Isup3.cmlSupporting information file. DOI: 1860856CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


S),18(R)‐EpETE and 17(R),18(S)‐EpETE. In this study, we demonstrated stereoselective differences of 17(S),18(R)‐EpETE and 17(R),18(S)‐EpETE in amelioration of skin contact hypersensitivity and found that anti‐inflammatory activity was detected in 17(S),18(R)‐EpETE, but not in 17(R),18(S)‐EpETE. In addition, we found that cytochrome P450 BM‐3 derived from Bacillus megaterium stereoselectively converts EPA into 17(S),18(R)‐EpETE, which effectively inhibited the development of skin contact hypersensitivity by inhibiting neutrophil migration in a G protein‐coupled receptor 40‐dependent manner. These results suggest the new availability of a bacterial enzyme to produce a beneficial lipid mediator, 17(S),18(R)‐EpETE, in a stereoselective manner. Our findings highlight that bacterial enzymatic conversion of fatty acid is a promising strategy for mass production of bioactive lipid metabolites.Dietary intake of ω3 polyunsaturated fatty acids such as eicosapentaenoic acid and docosahexaenoic acid is beneficial for health control. We recently identified 17,18‐epoxyeicosatetraenoic acid  as a lipid metabolite endogenously generated from eicosapentaenoic acid that exhibits potent anti‐allergic and anti‐inflammatory properties. However, chemically synthesized 17,18‐EpETE is enantiomeric due to its epoxy group—17( One enantiomer may be responsible for the therapeutic effects, whereas the other enantiomer can be inactive or exert undesired effects. For example, 2(S)‐hydroxy oleic acid induces greater reduction in the tumor volume of lung cancer than does 2(R)‐hydroxy oleic acid.R),18(S)‐EpETE—but not 17(S),18(R)‐EpETE—has vasodilatory effects on arteries, again suggesting that chirality is important for determination of biological activity.The stereochemistry of oxidized fatty acids is a critical determinant of their biological activity. Indeed, 17,18‐EpETE is enantiomeric due to its epoxy groups—17(R)‐HEPE which plays important roles as a SPM and a precursor of resolvin E1 (RvE1) was shown to be generated by hydroxylation of EPA using Bacillus megaterium homogenates.B. megaterium, catalyzed the epoxidation of unsaturated fatty acids and converted EPA into 17(S),18(R)‐EpETE in a stereoselective manner,Although 17,18‐EpETE is endogenously generated from EPA by cytochrome P450 (CYP), microorganisms also harbor various types of CYPs which catalyze a wide range of reactions including epoxidation and hydroxylation.Contact hypersensitivity is a commonly used mouse model of human allergic contact dermatitis, which includes sensitization and elicitation phases. In the sensitization phase, dendritic cells (DCs) migrate to the draining lymph nodes and activate T cells for the induction of memory‐type T cells. In the elicitation phase, on exposure to the same contact allergen as experienced during the sensitization phase, neutrophils and memory‐type T cells infiltrate the inflamed skin, where they produce pro‐inflammatory cytokines that lead to the development of skin swelling, rashes, and edema.S),18(R)‐EpETE produced by BM‐3 (BM‐3 17(S),18(R)‐EpETE) ameliorated CHS by inhibiting neutrophil migration into inflamed skin in a GPR40‐dependent manner. These findings reveal the activity of the 17(S),18(R)‐EpETE enantiomer and the availability of a bacterial enzyme that produces this bioactive lipid metabolite in a stereoselective manner.In this study, we found that 17(22.1Wild‐type (WT) C57BL/6J female mice  were purchased from SLC  and kept in a specific pathogen‐free animal facility at the National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN) for at least 1 week before use in experiments. GPR40‐deficient mice have been described previously2.2S),18(R)‐EpETE and 17(R),18(S)‐EpETE ((±)17,18‐EpETE), a commercially available Cayman (±)17,18‐EpETE . In some experiments, mice were treated with stereoselective 17(S),18(R)‐EpETE (>99% enantiomeric excess) or 17(R),18(S)‐EpETE (>99% enantiomeric excess), which were purified from synthesized (±)17,18‐EpETE, or BM‐3 17(S),18(R)‐EpETE. These lipids were injected intraperitoneally into mice by 100 ng/animal at 30 minutes before DNFB treatment. In some experiments, BM‐3 17(S),18(R)‐EpETE were injected intraperitoneally into mice by 1 µg, 100 ng, or 10 ng/animal in order to evaluate dose response. We used 0.5% (vol/vol) ethanol dissolved in PBS  as a vehicle control.Contact hypersensitivity was induced as described previously.2.3The (±)17,18‐EpETE was prepared from EPA as described previously with some modifications.For purification of stereoselective 17,18‐EpETE, HPLC was performed using a CHIRALCEL OJ‐RH packed column . The isocratic mobile phase was a mixture of methanol (Wako) and water containing 0.1% (vol/vol) formic acid , which was pumped at a flow rate of 1.2 mL/min. The column was maintained at 30°C, and the eluent was monitored at a wavelength of 205 mm. As a control, commercially available Cayman (±)17,18‐EpETE was used for HPLC analysis.2.4E. coli DH5α (Nippon Gene), and transformed by heat shock at 42°C for 30 seconds. After the transformation, E. coli DH5α was inoculated to modified LB agar medium  containing 25 μg/mL kanamycin sulfate (Wako) and cultured at 37°C for 18 hours. Modified LB agar medium was comprised of 1% (wt/vol) Difco select soytone , 0.5% (wt/vol) Bacto yeast extract , 1% (wt/vol) sodium chloride (Wako) and 1.5% (wt/vol) agar powder (Wako). The resultant colonies were picked and inoculated to modified LB liquid culture medium  containing 25 μg/mL kanamycin sulfate and cultured at 37°C for 18 hours. Modified LB liquid culture medium was comprised of 1% (wt/vol) Difco select soytone, 0.5% (wt/vol) Bacto yeast extract and 1% (wt/vol) sodium chloride. The plasmid DNA was extracted using QIAprep Spin Miniprep Kit (Qiagen) according to the manufacturer's instructions. In this way, pFusionF87V‐Km plasmid was constructed.BM3‐encoding pFusionF87V plasmid was ligated into pET‐24d (+) (Merck) at KNC Laboratories .E. coli DH5α and transformed by heat shock at 42°C for 30 seconds. The recombinant E. coli was inoculated to modified LB agar medium  containing 25 μg/mL kanamycin sulfate and cultured at 37°C for 18 hours. The resultant colonies were inoculated to modified LB liquid culture medium  containing 25 µg/mL kanamycin sulfate and cultured at 37°C for 18 hours. The plasmid DNA was extracted using QIAprep Spin Miniprep Kit. In this way, pFusionBM3‐WT plasmid which has no mutation was constructed.We next used pFusionF87V‐Km plasmid as a template for construction of pFusionBM3‐WT plasmid to give rise to large amount of 17,18‐EpETE. The primers for inverse PCR were as follows: primer 1, 5'‐TTTACAAGCTGGACGCATGA‐3' and primer 2, 5'‐TAACCCGTCTCCTGCAAAATCAC‐3'. The fragments were self‐ligated using Ligation‐Convenience kit after phosphorylation of 5' ends by T4 polynucleotide kinase (Takara Bio). The resultant solution was suspended with ECOS Competent E. coli BL21 (DE3) which is expression host strain, the pFusionBM3‐WT was suspended with ECOS Competent E. coli BL21 (DE3) (Nippon Gene) and transformed by heat shock at 42°C for 30 seconds. After the transformation, E. coli BL21 (DE3) was inoculated to modified LB agar medium  containing 25 μg/mL kanamycin sulfate and cultured at 37°C for 18 hours. The resultant colonies were inoculated to modified LB liquid culture medium  containing 25 µg/mL kanamycin sulfate and cultured at 28°C for 18 hours. The glycerol stock of pFusionBM3‐WT/BL21 (DE3) was made by mixing cultured solution and 50% (vol/vol) glycerol (Wako) in 2:1 (vol/vol) and stored at −20°C.In order to carry pFusionBM3‐WT into 2.5Cultivation was performed at KNC Laboratories. For preculture, 100 µL of pFusionBM3‐WT/BL21 (DE3) glycerol stock was inoculated in 500 mL of modified LB liquid culture medium  containing 25 µg/mL kanamycin sulfate and cultured at 25°C for 22 hours with shaking at 120 rpm.D‐thiogalactopyranoside  and cultured at 20°C for 47 hours with ventilation rate of 75 L/min. 2 × YT medium was comprised of 1.6% (wt/vol) Difco select soytone, 1% (wt/vol) Bacto yeast extract and 0.5% (wt/vol) sodium chloride. pH in culture was maintained at pH 7.0 ± 0.1 using 25% (vol/vol) ammonia solution (Wako) and 2 mol/L phosphoric acid (Wako), and dissolved oxygen was maintained at DO 1.5 ± 0.5 ppm by stirring.1 L of precultured liquid was added to 150 L of modified 2 × YT medium  containing 25 µg/mL kanamycin sulfate, 80 µg/mL 5‐aminolevulinic acid (Wako), 100 µM ammonium iron (II) sulfate hexahydrate (Wako), 250 µmol/L isopropyl β‐2.6Bioconversion of EPA into 17,18‐EpETE was performed at KNC Laboratories. 1.5 L of 1 mol/L EPA  was added to cultured medium and incubated at 20°C for 71.5 hours with ventilation rate of 20 L/min in order to convert EPA into 17,18‐EpETE. pH was maintained at pH 7.0 ± 0.1 using 25% (vol/vol) ammonia solution and 2 mol/L phosphoric acid, and dissolved oxygen was maintained at DO 1.5 ± 0.5 ppm by stirring.In order to stop reaction and kill bacteria, 35 L of ethanol (Wako) was added to reaction mixture and cultured at 20°C for 46 hours with ventilation rate of 20 L/min. pH was maintained at pH 7.0 ± 0.1 using 25% (vol/vol) ammonia solution and 2 mol/L phosphoric acid, and dissolved oxygen was maintained at DO 1.5 ± 0.5 ppm by stirring. In order to confirm whether the bacteria are dead, the reaction liquid was inoculated to modified LB agar medium  and cultured at 37°C for 18 hours, then we confirmed that the colonies were not formed.2.7S),18(R)‐EpETE was performed at KNC Laboratories. Fifteen kilograms of Diaion HP20  were added to 150 L of EPA reaction solution (20% (vol/vol) ethanol aqueous solution) and stirred for 1 hour to adsorb 17,18‐EpETE, which was confirmed by the analysis of the supernatant with HPLC Prominence System (Shimadzu). The condition of HPLC analysis is as follows: flow rate; 1.0 mL/min, column temperature; 40°C, UV wavelength; 205 nm, injection volume; 10 µL, column; C18 column , mobile phase; (A) 0.05% (vol/vol) formic acid aqueous solution and (B) acetonitrile. The eluent gradients were 45%‐55% (vol/vol) B for 0‐12 minutes, 55%‐75% (vol/vol) B for 12‐19 minutes, 75%‐100% (vol/vol) B for 19‐20 minutes, 100% (vol/vol) B for 20‐28 minutes, 100%‐45% (vol/vol) B for 28‐29 minutes, and 45% (vol/vol) B for 29‐40 minutes. After the absorption of 17,18‐EpETE, HP20 was removed from the EPA reaction solution, and washed four times with purified water. Then, HP20 was soaked in 20% (vol/vol) ethanol aqueous solution and stored at 4°C.Purification of BM‐3 17  ethanol aqueous solution containing 0.05% (vol/vol) acetic acid). This solution was charged to Inertsil ODS‐3 column  that had been equilibrated with 20% (vol/vol) ethanol aqueous solution containing 0.05% (vol/vol) acetic acid at 50 mL/min flow rate. After charged, the column was washed with 20% (vol/vol) ethanol aqueous solution containing 0.05% (vol/vol) acetic acid, and eluted with 70% (vol/vol) ethanol aqueous solution containing 0.05% (vol/vol) acetic acid at 45 mL/min flow rate. The elution sample containing 17,18‐EpETE was stored at −80°C as a low purity sample .In order to obtain high purity sample, the low purity sample was charged to Inertsil ODS‐3 column  that had been equilibrated with 20% (vol/vol) ethanol aqueous solution containing 0.05% (vol/vol) acetic acid at 15 mL/min flow rate. After charged, the column was washed with 20% (vol/vol) ethanol aqueous solution containing 0.05% (vol/vol) acetic acid, and eluted with 55% (vol/vol) ethanol aqueous solution containing 0.05% (vol/vol) acetic acid at 10 mL/min flow rate. The elution sample containing 17,18‐EpETE was stored at −80°C as a high purity sample .S),18(R)‐EpETE for biological assay.In order to replace the solvent to ethanol, the elution sample containing 17,18‐EpETE was diluted with Milli‐Q water containing acetic acid  ethanol aqueous solution containing 0.05% (vol/vol) acetic acid). The resultant solution was charged to Inertsil ODS‐3 column  that had been equilibrated with 20% (vol/vol) ethanol aqueous solution containing 0.05% (vol/vol) acetic acid at 15 mL/min flow rate. After charged, the column was washed with 20% (vol/vol) ethanol aqueous solution containing 0.05% (vol/vol) acetic acid, followed by washed with 20% (vol/vol) ethanol aqueous solution, and eluted with 100% (vol/vol) ethanol at 15 mL/min flow rate. The elution sample containing 17,18‐EpETE was stored at −80°C as a very high purity sample , and used as BM‐3 17 staining, frozen tissue sections were stained in hematoxylin solution (Wako) for 10 minutes and washed with water for 30 minutes. Then, the sections were stained in 1% eosin Y solution (Wako) for 1 minute and dehydrated through increasing concentrations of ethanol . Finally, stained tissue sections were dehydrated in xylene  for 3 minutes and mounted in Permount .For immunohistologic analysis, frozen tissue sections were washed with PBS for 10 minutes and then blocked in 2% (vol/vol) newborn calf serum (Equitech‐Bio) in PBS for 30 minutes at room temperature in an incubation chamber (Cosmo Bio). Tissue sections were then incubated in an incubation chamber overnight at 4°C with fluorescein isothiocyanate–anti‐Ly6G monoclonal antibody  in 2% (vol/vol) newborn calf serum in PBS. Then, samples were washed once for 5 minutes in 0.1% (vol/vol) Tween‐20  in PBS and then in PBS only for 5 minutes. To visualize nuclei, tissue sections were stained with 4ʹ,6‐diamidino‐2‐phenylindole  for 10 minutes at room temperature in the incubation chamber. Finally, tissue sections were washed twice with PBS for 5 minutes each, and mounted in Fluoromount (Diagnostic BioSystems) and examined under a fluorescence microscope .2.9The isolation of cells from ear tissue and their flow cytometric analysis were performed as described previously.Cell suspensions were filtered using a cell strainer  and cells counted. Cells were stained using an anti‐CD16/32 monoclonal antibody  to avoid nonspecific staining, and dead cells were stained with 7‐aminoactinomycin D . The cells were further stained with the following antibodies: fluorescein isothiocyanate–anti‐Ly6G , allophycocyanin–Cy7–anti‐CD11b , and BV421–anti‐CD45 . Samples were analyzed using MACSQuant . Data were analyzed using FlowJo 9.9 software (TreeStar).2.105 cells) were suspended in HBSS  containing 0.2% bovine serum albumin (Sigma Aldrich) and allowed to adhere to fibronectin‐coated coverslips (Neuvitro) for 15 minutes at 37°C in a 5% CO2 incubator. Neutrophils were treated with either 1000, 100, 10, or 1 nmol/L of commercially available Cayman (±)17,18‐EpETE, BM‐3 17(S),18(R)‐EpETE, 18‐HEPE , RvE1 , or 0.03% (vol/vol) ethanol (vehicle control) for 15 minutes and then stimulated with 1 μmol/L N‐formyl‐methionyl‐phenylalanine  or 100 nmol/L leukotriene B4  for 2 minutes at 37°C in a 5% CO2 incubator. Neutrophils were fixed in 4% paraformaldehyde , permeabilized using 0.5% (vol/vol) Triton X‐100  in PBS, and stained with 100 nmol/L Acti‐stain 488–phalloidin (Cytoskeleton) for 30 minutes at room temperature. Finally, cell nuclei were stained by incubating neutrophils with 4ʹ,6‐diamidino‐2‐phenylindole for 30 seconds at room temperature. Images were obtained with Leica TCS SP8 confocal microscopy (Leica Microsystems).Neutrophils were purified from bone marrow as described previously.2.11Actinβ sense, 5'‐aaggccaaccgtgaaaagat‐3'; Actinβ antisense, 5'‐gtggtacgaccagaggcatac‐3'; interferon‐γ (Ifn‐γ) sense, 5'‐atctggaggaactggcaaaa‐3'; Ifn‐γ antisense, 5'‐ttcaagacttcaaagagtctgaggta‐3'; interleukin (Il)‐17 sense, 5'‐cagggagagcttcatctgtgt‐3'; Il‐17 antisense, 5'‐gctgagctttgagggatgat‐3'; Cxcl1 sense, 5'‐gactccagccacactccaac‐3'; Cxcl1 antisense, 5'‐tgacagcgcagctcattg‐3'; Cxcl2 sense, 5'‐aaaatcatccaaaagatactgaacaa‐3'; Cxcl2 antisense, 5'‐ctttggttcttccgttgagg‐3'; Cxcl9 sense, 5'‐cttttcctcttgggcatcat‐3'; Cxcl9 antisense, 5'‐gcatcgtgcattccttatca‐3'; Cxcl10 sense, 5'‐gctgccgtcattttctgc‐3'; Cxcl10 antisense, 5'‐tctcactggcccgtcatc‐3'.Reverse transcription and quantitative PCR analysis were performed as described previously.2.12P < 0.05 was considered significant.Statistical significance was evaluated through one‐way ANOVA using Prism 3.03 software (GraphPad Software). A 33.1S),18(R)‐EpETE from 17(R),18(S)‐EpETE. In this issue, we first chemically synthesized a large amount of (±)17,18‐EpETE from EPA and found that, like commercially available Cayman (±)17,18‐EpETE, synthesized (±)17,18‐EpETE showed 2 peaks using HPLC system with chiral column. The bacterial enzyme BM‐3 has been reported to stereoselectively convert EPA into 17(S),18(R)‐EpETE.S),18(R)‐EpETE was identical with the second peak of (±)17,18‐EpETE ,18(S)‐EpETE.We previously reported that commercially available Cayman (±)17,18‐EpETE showed potent anti‐allergic and anti‐inflammatory properties.3.2S),18(R)‐EpETE showed anti‐inflammatory activity in inhibiting ear swelling but 17(R),18(S)‐EpETE had little effect 17,18‐EpETE, 17,18(R)‐EpETE exerted anti‐inflammatory activity, we next examined whether 17(S),18(R)‐EpETE stereoselectively produced by BM‐3 suppresses CHS. We found that BM‐3 17(S),18(R)‐EpETE suppressed ear swelling ,18(R)‐EpETE decreased inflammation in the skin ,18(R)‐EpETE decreased the number of neutrophils in inflamed ears ,18(R)‐EpETE ,18(R)‐EpETE in CHS by evaluating ear swelling and neutrophil numbers. Both ear swelling and neutrophil numbers were decreased at the dose of 1 µg and 100 ng/animal, but the anti‐inflammatory effects were hardly observed at the dose of 10 ng/animal ,18(R)‐EpETE was shown to be effective at the dose more than 100 ng/animal.Given that 17,18(R)‐EpETE on pseudopod formation in neutrophils isolated from bone barrow. We found that fMLP‐induced pseudopod formation was inhibited by treatment with BM‐3 17(S),18(R)‐EpETE ,18(R)‐EpETE was absent when the neutrophils were prepared from GPR40‐deficient mice. We also found that BM‐3 17(S),18(R)‐EpETE inhibited LTB4‐induced pseudopod formation in a GPR40‐dependent manner ,18(R)‐EpETE treatment ,18(R)‐EpETE did not inhibit the expression of Ifn‐γ ,18(R)‐EpETE acts on neutrophil selectively.We next evaluated the inhibitory effect of BM‐3 17,18(R)‐EpETE with EPA‐derived fatty acid metabolites, RvE1 and 18‐HEPE, for inhibition of pseudopod formation ,18(R)‐EpETE showed inhibitory effects at the dose of 10 nmol/L, while RvE1 and 18‐HEPE lost their activities. BM‐3 17(S),18(R)‐EpETE also lost its activity at the dose of 1 nmol/L.We compared the effectiveness of BM‐3 17,18(R)‐EpETE at the dose of 10 nmol/L, while RvE1 and 18‐HEPE showed inhibitory effects. Both RvE1 and 18‐HEPE lost its activity at the dose of 1 nmol/L. These results indicated that EPA‐derived bioactive lipid mediators of BM‐3 17(S),18(R)‐EpETE, RvE1, and 18‐HEPE all possess anti‐inflammatory activity by inhibiting neutrophil pseudopod formation.In the case of LTB44, which is secreted by neutrophils, promotes secondary neutrophil migration.S),18(R)‐EpETE inhibited both fMLP‐ and LTB4‐induced pseudopod formation, and RvE1 and 18‐HEPE also inhibited both fMLP‐ and LTB4‐induced pseudopod formation. Consistent with these findings, a previous report showed that RvE1 and 18‐HEPE reduced neutrophil transmigration.4 receptor, BLT1.4‐induced pseudopod formation through BLT1 antagonistic activity in DC.S),18(R)‐EpETE inhibited neutrophil pseudopod formation by GPR40‐mediated pathway, while 18‐HEPE and RvE1 used ChemR23 and/or BLT1 as functional receptors.Actin polymerization and neutrophil migration are induced by different kinds of chemoattractants. The fMLP promotes primary neutrophil migration in response to bacterial infection, while LTBS)‐ and (R)‐thalidomide was introduced as a sedative medicine in the late 1950s, but it was withdrawn due to teratogenicity of (S)‐thalidomide, indicating the importance of stereoselective production of candidate medicines.S)‐ and (R)‐enantiomers of salbutamol exert different effects: the (R)‐enantiomer of salbutamol binds the B2‐adrenergic receptor with greater affinity than the (S)‐enantiomer and is responsible for salbutamol's bronchodilation activity.The use of single‐enantiomer medicines can potentially lead to simpler and more selective pharmacologic profiles, because the enantiomers of a chiral compound may differ significantly in their bioavailability.S),18(R)‐EpETE stereoselectively showed anti‐inflammatory activity in CHS. Conversely, it is reported that 17(R),18(S)‐EpETE—but not 17(S),18(R)‐EpETE—is a potent vasodilator and stimulates calcium‐activated potassium channels, which lead to the relaxation of rat cerebral artery vascular smooth muscle cells.S),18(R)‐EpETE–GPR40 axis suppresses CHS, whereas the 17(R),18(S)‐EpETE–calcium‐activated potassium‐channel axis achieves arterial relaxation.S),18(R)‐EpETE as a single‐enantiomer therapy might decrease the risk of side effects due to vasodilatory activity, such as a rapid decrease in blood pressure and increased skin redness.In this study, we found that BM‐3 17(R),18(S)‐EpETE, whereas Cyp4f18 in mice and CYP2D6 in humans—like BM‐3—selectively generate 17(S),18(R)‐EpETE.R),18(S)‐EpETE and 17(S),18(R)‐EpETE.We previously found that dietary linseed oil, which contains large amounts of α‐linolenic acid, a precursor of EPA and DHA, increases the amount of ω3 PUFA‐derived metabolites in the body.S),18(R)‐EpETE might reflect differences in CYP activity or expression level. For example, the expression level of Cyp1a2 is upregulated by the ligand‐activated transcription factor aromatic hydrocarbon receptor (AhR).R),18(S)‐EpETE might be modulated through diet.S),18(R)‐EpETE and 17(R),18(S)‐EpETE.CYPs harbor gene polymorphisms, which cause different enzymatic activities among individuals.Bacillus bacteria, including B. megaterium, are used for the production of fermented foods, such as the soybean products natto and miso. Therefore, the production level of lipid metabolites likely is affected not only by the enzymes in the body but also by enzymes derived from microorganisms in fermented food, suggesting that the production levels of 17(S),18(R)‐EpETE could be increased by eating fermented foods containing Bacillus bacteria.B. megaterium homogenate reportedly yields 17,18‐EpETE and 18‐HEPE, whereas BM‐3 primarily catalyzes epoxidation and produces 17,18‐EpETE from EPA, thus suggesting that not only BM‐3, various types of CYPs in B. megaterium contribute to the production of 17,18‐EpETE and 18‐HEPE.Because microorganisms metabolize fatty acids, microorganisms can affect the lipid profile. In particular, microorganisms are used for the production of fermented foods, which may often contain abundant amounts of lipid metabolites. For example, S),18(R)‐EpETE can reduce CHS. The 17(S),18(R)‐EpETE–GPR40 axis played a key role in the amelioration of CHS by inhibiting neutrophil migration. These results suggest that bacterial fermentation with BM‐3 activity is a promising tool for the stereoselective mass‐production of 17(S),18(R)‐EpETE.In conclusion, the present study showed enzymatically produced 17(There are no conflict of interest to declare. AS, TN, and JK designed the research and wrote the paper. AS, TN, SK, SP, NM, MS, SM, EN, and JO performed experiments, analyzed data, and discussed the results. TH, PT, SH, KH, and KK provided technical help and discussed the results. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Recently oxidative stress induced maladies have amplified owing to sedentary lifestyle and monotonous diet. Introduction of plant based biomolecules may be a suitable strategy to cope with the lipid peroxidation. In this context, black tea polyphenols (theaflavin & thearubigins) are in fame among the scientific community as cost effective therapeutic agents owing to their safety, economics, structural diversity and ability to modulate various lipid peroxidation responses by halting the expression of different metabolic targets.0 (control diet without supplementation), T1 , T2  & T3  for the period of 56 days. Alongside, a control study was also carried out for comparison by involving normal rats fed on arginine free diet. The body weight, lipid profile, glycemic responses, Renal function test, liver function test, antioxidant indices and hematological parameters were estimated at the termination of study.The mandate of present investigation was to first time check the synergism among the isolated theaflavins & thearubigins against lipid peroxidative indicators both in vitro and in vivo. Purposely, theaflavins and thearubigins were isolated from black tea through solvent partition methods by using different solvents  and time intervals  and subjected to in vitro characterization through different antioxidant indices to access the in vitro lipid peroxidation shooting effect of these bioactive moieties. Moreover, individual theaflavins contents also estimate through HPLC. For evaluation of in vivo antioxidant effect, renal malfunction was induced through arginine and forty rats were divided in four groups  and 04 types of diets were given i.e. TThe results indicated that theaflavins and thearubigins isolation was significantly affected by time of extraction and solvent. In this context, aqueous ethanol at 60 min extraction interval caused maximum extraction. Likewise, theaflavins isolate exhibited more antioxidant activity as compared to thearubigins. Moreover, the theaflavins and thearubigins based experimental diets imparted significant reduction in Lipid profile, glucose content, renal function tests and TBARS with enhancement in insulin, HDL and hematological parameters. In this context, theaflavin based diet caused maximum reduction in lipid profile and TBARS better as compared to thearubigins and theaflavins + thearubigins based. However, theaflavin+ thearubigins based diet caused highest glucose, urea & creatinine decline and maximum insulin increase & antioxidant indices as compared to other nutraceuticals.It was deduced that theaflavins & thearubigins have strong antioxidative potential both in in vitro as well as in vivo to tackle the menace associated with lipid peroxidation. Likewise, preventive role of theaflavin in lipid peroxidation is mainly attributed to its ability to cease the chain reaction. Apart from free radical scavenging and metal chelating abilities, theaflavin has potential to activate certain antioxidant enzymes like glutathione-S-transferase (GST), glutathione peroxidase (GPX), superoxide dismutase (SOD) and catalase (CAT) thereby reduces lipid peroxidation. Likewise, thearubigins or polymeric black tea polyphenols (PBPs) are the oxidative products of phenolics and their production accelerated after 75% conversion of catechins into flavan-3-ol molecules. They have ability to activate phase II enzymes by inducing transcriptional upregulation in lung and liver [Globally, therapeutic worth of tea has been established against numerous maladies. The health escalating perspectives of tea has been attributed to his distinguished polyphenols. Accordingly, theaflavins and thearubigins are the promising polyphenols belong to subclass flavanolos. They produced when fresh tea leaves undergo enzymatic fermentation triggered by polyphenol oxidase and catalase resulted in structural variations in catechins thus formed theaflavin and thearubigins –3. Strucnd liver .Chronic renal failure (CRF) is an irreversible loss to functioning nephrons resulting numerous disorders of blood vessels, glomeruli, tubules and renal interstitium . VariousThe mandate of current investigation to isolate the theafavins and thearubigins from black tea Qi-men variety by using different solvents and time intervals, in vitro characterization and preparation of nutraceutical intervention against renal malfunctioning in arginine induced liver damage rats.Black tea variety (Qi-Men) was procured from the National Tea Research Institute (NTRI), Shinkiari, Mansehra. The reagents  and standards were purchased from Merck  and Sigma-Aldrich . For efficacy trial, Male Sprague Dawley rats were housed in the Animal Room of Physiology department of GCUF. For biological assay, diagnostic kits were purchased from Sigma-Aldrich, Bioassay  and Cayman Chemicals .Theaflavin and thearubigins were extracted by using water, methanol and ethanol at 30, 60 and 90 min intervals and isolated by solvent partition method . Initial18 column , 10 μL sample through auto sampler (WISP Model 710) and column temperature 40 °C. The composition of mobile phase was acetonitrile, ethylacetate and 0.05% phosphoric acid in ratio of 21:3:76 with flow rate of 1 mL/min and using on UV/vis detector (model 481) and measurement wavelength was 278 nm.The isolated samples of theaflavins were characterized for their fractions through HPLC . The conditions for HPLC were CAntioxidant capacity of isolated theaflavin and thearubigins fractions were determined through antioxidant indices including total antioxidant activity, free radical scavenging activity and ferric reducing antioxidant power as following methods. For this purpose, the isolated fractions both from theaflavin and thearubigins were mixed in water, methanol and ethanol (1 mg/mL) to be further utilized in antioxidant indices estimation.V/vis Spectrophotometer (CECIL CE7200) against control and expressed results as mg gallic acid/100 g.Total phenolics of resultant isolates were estimated spectrophotometricaly using Folin-Ciocalteau method . Brieflyβ-carotene and linoleic acid assay was applied to measure the total antioxidant capacity . PurposeDPPH radical scavenging activity was measured according the procedure of . InitialThe FRAP test was performed according to the guidelines of . 0.5 mL Effect of isolated theaflavin and thearubigins fraction on glucose diffusion was assessed using glucose oxidase kit and 1.65 mM D-glucose solution .For biological assay, rats were divided in to four homogeneous groups fed on experimental diet. The common experimental diet was formulated using corn oil (10%), protein (10%), corn starch (64%), cellulose (10%), mineral (3%) vitamin mixture (1%) alongside arginine @ 2% for the induction of renal malfunctioning. For comparison a control study was also conducted by providing the normal diet (same composition except for arginine). However, common experimental diet from both studies further divided into four groups on the bases of addition of active ingredients, Diet 1 , Diet 2, (theaflavins supplementation @ 1 g), Diet 3, (Thearubigins supplementation @ 1 g) and Diet 4 . Afterwards All the ingredients were mixed then oven baked for 10 min. The dose was selected by carried out a 21 days safety trial (Data not included).n = 6 rats/dose). The treatments were given in both acute (single dose followed by a 48-h observation period) and sub-acute . The hematological analysis as well as general physical examination was carried out [In safety trial, rats were provided all the active ingredients orally @ of 250, 500, 1000, 1500, 2000 and 3000 mg/kg-bw  and relative humidity (55 ± 5%) along with 12 h light-dark period. At the initiation of study, some rats  were sacrificed to establish the baseline trend. For the induction of renal malfunctioning initially high arginine diet @ 2% was administrated for a period of 7 days. During that tenure urea and creatinine levels were observed to estimate the onset of renal malfunctioning. Afterwards when values of both test deviate 25% from normal then the original study was started. The study comprised of four groups of rats ten in each . Accordingly, four types of experimental diets were given i.e. T0 , T1, T2  and T3  by protocols of , high deGlucose concentration was estimated by GOD-PAP method by following the protocol of , howeverThe serum samples were also analyzed for urea by GLDH-method and creatinine by Jaffe-method using commercial kits  to assesGlutathione contents were assessed by adapting the guidelines as mentioned by . The reaLiver function tests including aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were assessed. Levels of AST and ALT were measured by the dinitrophenylhydrazene (DNPH) method using Sigma Kits 59–50 and 58–50, respectively and ALP by Alkaline Phosphates–DGKC method .Red blood cells indices including total red blood cells (TRBCs), hemoglobin (Hb), hematocrit (Hct) and mean corpuscular volume (MCV) were estimated. Likewise, white blood cell indices including monocytes, lymphocytes and neutrophils were measured by using Automatic Blood Analyzer  .Statistical program SAS  was utilized to analyze the data collected from this study. Two-way analysis of variance (ANOVA) was conducted to evaluate the effect of extraction time and solvent on polyphenol isolation. Moreover, One-way ANOVA were applied in efficacy trial and significance among the treatments were determined by applying LSD.p ≥ 0.001) and time (p ≥ 0.002), highest theaflavin (3.42 ± 0.27 g/100 g) were detected in ethanolic extract followed by methanol (2.77 ± 0.25 g/100 g) whilst water exhibited the lowest yield (2.32 ± 0.13 g/100 g). Similarly, solvent (p ≥ 0.001) & time (p ≥ 0.001) also imparted pronounced impact for thearubigins, highest in ethanol and minimum in water. Extraction efficiency was also influenced by time and maximum yield for theaflavins and thearubigins was obtained at 60 min 3.41 ± 0.17 g/100 g and 13.17 ± 0.59 g/100 g, respectively (Table Means for extraction yield illuminated the significant effect of solvents p ≥ 0.00 and timep ≥ 0.001) and extraction time (p ≥ 0.0031) whilst their interactive effect showed non-significant trend (p ≥ 0.521). Means for TF1 indicated highest value (2.18 ± 0.06 mg/g) in ethanolic extract followed by methanol (1.96 ± 0.03 mg/g) and water (0.751 ± 0.001 mg/g). However, effect of time revealed maximum output (1.79 ± 0.006 mg/g) at 60 min and minimum (1.48 ± 0.001 mg/g) at 30 min. Likewise trend was observed for TF2A, TF2B & TF3, among the solvents highest ratio in ethanolic fractions and minimum in water and among time intervals maximum in 60 min and minimum in 30 min , ß-carotene (p ≥ 0.004) and FRAP (p ≥ 0.000) whilst non-significant differences (p ≥ 0.185) were observed for glucose diffusion. Means for DPPH in theaflavin and thearubigins indicated highest activity in ethanolic extract 83.27 ± 1.23 & 68.72 ± 3.61%, respectively followed by methanol and water. For the time factor, the highest DPPH activity of theaflavin was 82.69 ± 5.10% at 60 min whilst 78.03 ± 4.31 & 75.89 ± 3.92% at 90 and 30 min, respectively. Similarly, thearubigins also exhibited highest DPPH activity at 60 min  followed by the methanolic (89.96 ± 2.31%) and water extract (87.02 ± 3.12%). Likewise, in thearubigins the values for this parameter in respective extracts were 87.71 ± 6.12, 84.92 ± 6.42 and 83.02 ± 7.12%. Considering time factor, highest glucose diffusion was reported at 60 min for the both parameters (Table p ≥ 0.001) of experimental diets on weight in both control and renal malfunctional rats and recorded values for weight in renal malfunctional rats at the termination of study were 213.10 ± 6.11 & 213.73 ± 8.12, 217.74 ± 5.61 & 220.52 ± 9.26 and 214.17 ± 4.25 & 215.73 ± 6.23 g/rat for T1 T2 and T3, respectively  effect on serum cholesterol level of rats, highest cholesterol level was observed in T0 (86.31 ± 4.63 mg/dL) that significantly reduced in T1 (81.14 ± 2.12 mg/dL) trailed by T3 (82.29 ± 4.25 mg/dL) and T2 (82.99 ± 5.63 mg/dL). In contrary, the tested diets imparted non-significant (p ≥ 0.091) enhancement in HDL levels of study animals. In this context, T0 drink consuming group exhibited least HDL value as 38.54 ± 2.36 mg/dL, respectively that elevated non-significantly to 39.70 ± 2.32, 38.92 ± 1.09 and 39.64 ± 1.56 mg/dL in T1, T2 and T3 groups, respectively. As for as LDL levels were concern, the tested diets caused momentous (p ≥ 0.000) decline on this trait. The maximum LDL was noticed in T0 that reduced substantially to in T1, T2 and T3, correspondingly. Triglycerides values for T0, T1, T2 and T3 differed non-momentously (p ≥ 0.128) i.e. 67.15 ± 4.20, 64.96 ± 5.15, 65.20 ± 4.12 and 65.13 ± 2.12 mg/dL (Table The prepared diets caused significant ( 86.31 ± .63 mg/dLp ≥ 0.000) impact of experimental diets on blood glucose levels of tested groups. In renal malfunctional rats, the highest glucose level 89.18 ± 6.14 in T0 group however, black tea polyphenols supplemented diets lowered the glucose value to 84.70 ± 6.13, 85.60 ± 3.20 and 83.98 ± 7.14 mg/dL in T1, T2 and T3 groups, correspondingly. Likewise, insulin also varied significantly (p ≥ 0.004) minimum insulin values 7.76 ± 0.22 μU/mL were observed in T0 whilst highest level 8.11 ± 0.12 μU/mL in T3 followed by 8.06 ± 0.41 μU/mL in T1 and 7.96 ± 0.52 μU/mL in T2  impact on these traits however, the diet supplemented with both theaflavin and thearubigins caused maximum effect in comparison with others. The highest urea level 37.83 ± 2.46 was recorded in T0 that suppressed to 33.44 ± 1.45, 34.09 ± 2.56 and 34.95 ± 2.91 mg/dL in T3, T1 and T2 groups. Likewise, highest creatinine level 1.15 ± 0.03 mg/dL was recorded in T0 group (control drink) that significantly suppressed to 1.04 ± 0.05 mg/dL in T3 (drink containing theaflavin+thearubigins), 1.08 ± 0.01 mg/dL in T1 (drink containing theaflavin) and 1.09 ± 0.02 mg/dL in T2 (drink containing thearubigins).The means in Table 0 group showed decreased glutathione content 43.12 ± 3.01 mg/L that momentously (p ≥ 0.001) enhanced to 46.19 ± 2.56, 44.91 ± 4.14 and 47.87 ± 4.12 mg/L in T1, T2 and T3 groups. Similar significant (p ≥ 0.000) effect of experimental diets was observed for MDA, the recorded values for this trait in T0 was 8.01 ± 0.21that differed significantly in T1, T2 and T3 by 6.78 ± 0.24, 7.55 ± 0.01 and 6.95 ± 0.4 mmol/L (Table T0 (49.06 ± 3.01 IU/L) was significantly ((p ≥ 0.001)) varied in T1 T2 and T3 groups with mean values 48.50 ± 2.42, 49.12 ± 2.13 and 46.38 ± 2.45 IU/L. Moreover, T0 group showed maximum AST level 105.27 ± 5.14 IU/Lthat reduced substantially ((p ≥ 0.002) in T1 (101.68 ± 9.12 IU/L), T2 (103.00 ± 9.02 IU/L) and T3 (109.01 ± 9.45 IU/L). Likewise, ALP level in T0 (196.89 ± 17.23 IU/L) was significantly ((p ≥ 0.001)) higher than that of T1 (177.89 ± 15.20 IU/L), T2 (180.90 ± 13.25 IU/L) and T3 (167.68 ± 9.62 IU/L).Mean values in Table 0, T1, T2 and T3, respectively. Mean Hb level in T0, T1, T2 and T3 were 10.88 ± 0.71, 11.15 ± 0.61, 10.98 ± 0.81 and 11.20 ± 0.71 g/L, respectively. Moreover, the recorded hematocrit value for T0 (36.79 ± 1.52%) was improved non-significantly in T1, T2 and T3 groups as 37.25 ± 2.71, 36.99 ± 2.12 and 37.03 ± 2.81%, respectively. Likewise, mean MCV values for T0, T1, T2 and T3 were 49.96 ± 2.48, 51.03 ± 3.82, 50.92 ± 3.97 and 51.65 ± 3.30 fl, respectively. Similarly, the mean WBCs in T0 were 17.29 ± 0.58 cells/nL that non-significantly decreased to 16.99 ± 0.49, 17.26 ± 0.65 and 16.85 ± 0.35 cells/nL, respectively in tested groups. Likewise, means for Neutrophils in T0, T1, T2, and T3 were 62.25 ± 1.28, 63.85 ± 2.82, 62.45 ± 3.25 and 64.63 ± 2.63%, respectively. Mean monocytes values for T0, T1, T2 and T3 groups were 5.29 ± 0.21, 5.35 ± 0.22, 5.41 ± 0.25 and 5.65 ± 0.63%, respectively. In study IV, values for Lymphocytes were 33.92 ± 1.21, 35.25 ± 1.82, 34.91 ± 1.25 and 35.29 ± 1.63% in T0, T1, T2 and T3 group, respectively  determination to enhance the authenticity of the results. Likewise, human efficacy trial for insulin sensitivity/resistance by different test like HOMA-insulin resistance, QUIKI, and Matsuda should be conducted to unveil the mechanistic concerns.


In the crystal, the disaccharide and water molecules form layers parallel to the  13H24O9·H2O, a structural model for part of bacterial O-antigen polysaccharides from Shigella flexneri and Escherichia coli, crystallizes with four independent disaccharide molecules and four water molecules in the asymmetric unit. The conformation at the glycosidic linkage joining the two rhamnosyl residues is described by the torsion angles φH of 39, 30, 37 and 37°, and ψH of −32, −35, −31 and −32°, which are the major conformation region known to be populated in an aqueous solution. The hexopyranose rings have the 1C4 chair conformation. In the crystal, the disaccharide and water molecules are associated through O—H⋯O hydrogen bonds, forming a layer parallel to the bc plane. The layers stack along the a axis via hydrophobic interactions between the methyl groups.The title compound, C Shigella flexneri , which are defined, respectively, by H1A—C1A—O2B—C2B, C1A—O2B—C2B—H2B and H1B—C1B—O7B—C7B. These torsion angles are (I)H =39°, ψH = −32° and φH(C7) = 49°, (II) φH = 30°, ψH −35° and φH(C7) = 52°, (III) φH = 36°, ψH = −31° and φH(C7) = 51°, and (IV) φH = 37°, ψH = −32° and φH(C7) = 51°, where (I)–(IV) correspond to the four independent disaccharide molecules 1–4, respectively, in Fig. 2H, ψH and φH(C7) angles are 35 (4), −33 (2) and 51 (1)°, respectively. The φH torsion angle is governed by the exo-anomeric effect and should be approximately 40° for an α-l-sugar, which is also the case in the title rhamnose-containing disaccharide  mixture solution at ambient temperature.The title compound was synthesized according to the published procedures  and treated as riding with Uiso(H) = 1.2–1.5Ueq. The O—H bond and H⋯H distances in the water molecules were restrained to 0.85 (1) and 1.34 (1) Å, respectively. The orientation of each water molecule was adjusted and restrained with additional DFIX commands using parameters derived from a solid state DFT optimization of the crystal structure.Crystal data, data collection and structural refinement details are summarized in Table 210.1107/S2056989019006935/is5512sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989019006935/is5512Isup2.hklStructure factors: contains datablock(s) I. DOI: 1915954CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: