Recent studies have indicated that Toll-like receptor 4 (TLR4), a pathogen-recognition receptor that triggers inflammatory signals in innate immune cells, is also expressed on sensory neurons, implicating its putative role in sensory signal transmission. However, the possible function of sensory neuron TLR4 has not yet been formally addressed. In this regard, we investigated the role of TLR4 in itch signal transmission.TLR4 was expressed on a subpopulation of dorsal root ganglia (DRG) sensory neurons that express TRPV1. In TLR4-knockout mice, histamine-induced itch responses were compromised while TLR4 activation by LPS did not directly elicit an itch response. Histamine-induced intracellular calcium signals and inward currents were comparably reduced in TLR4-deficient sensory neurons. Reduced histamine sensitivity in the TLR4-deficient neurons was accompanied by a decrease in TRPV1 activity. Heterologous expression experiments in HEK293T cells indicated that TLR4 expression enhanced capsaicin-induced intracellular calcium signals and inward currents.Our data show that TLR4 on sensory neurons enhances histamine-induced itch signal transduction by potentiating TRPV1 activity. The results suggest that TLR4 could be a novel target for the treatment of enhanced itch sensation. Chronic itch is a pathological hallmark of atopic dermatitis and other infectious skin diseases. Recent studies on single nucleotide polymorphisms implicate genes encoding pattern-recognition receptors such as CD14 and toll-like receptors (TLRs) in the development and severity of atopic dermatitis (AD) . TLRs arRecently, some pathogen-recognition receptors including TLR3, 4, and 7 were also found to be expressed on sensory neurons . These fIn addition to TLR3 and 7, TLR4 is reported to be expressed in certain DRG and trigeminal ganglia sensory neurons . This fiper se. We then tested if TLR4 is involved in itch signal transmission triggered by the pruritogens 5-HT, histamine, chloroquine (CQ), imiquimod (R837), and SLIGRL. While scratching responses seen in TLR4 KO mice after 5-HT, SLIGRL, or imiquimod injection were comparable to responses in WT mice, histamine-induced and chloroquine-induced scratching responses were severely compromised in TLR4 KO mice  of DRG neurons of all sizes  in sensory neurons [++]i in 13% of cells, similar to previous results [++]i in the histamine-responsive subpopulation was also reduced by 30% in TLR4-deficient DRG neurons  results . HoweverTo investigate the mechanisms of these results, we tested if TLR4 affected histamine receptor expression in sensory neurons. HRH1 level was not reduced, but was rather increased in TLR4 KO DRG neurons   treatment directly excited sensory neurons and induced a scratch response . HoweverIn our study, TLR4 regulates histamine-induced and chloroquine-induced itch by distinct mechanisms. TLR4 potentiates histamine responsiveness by increasing TRPV1 basal activity. However, TLR4 does not affect TRPA1 activity. Instead, TLR4 expression increased MrgprA3 expression. Crosstalk between TLR4 and TRPV1 signals has been documented . In thisTRPV1 is not only associated with itch signals but also involved in pain signals. In our study, we observed capsaicin-induced paw licking was also slightly reduced in TLR4 KO mice (data not shown). This result suggests that the effect of neuronal TLR4 might not be specific to itch signal, but might be involved in pain transmission as well. Of note, nerve-injury induced mechanical allodynia and hyperalgesia are severely compromised in TLR4 KO mice . In thisItch susceptibility to pruritogens or bacterial/viral infections depends on genetic background. For instance, polymorphisms in genes for the TLR family and associated receptors are implicated in itch-associated diseases such as AD . Our stuIn this study, we have uncovered that TLR4 expression in sensory neurons potentiates histamine-induced itch signal transduction. The histamine-induced scratch response is significantly compromised in TLR4 KO mice. Similarly, histamine-induced intracellular calcium signals and inward currents are reduced TLR4-deficent sensory neurons. As a mechanism for these results, we found that TLR4 expression potentiates histamine-mediated itch by regulating TRPV1 channel activity. Our data suggest that TLR4 could be a novel therapeutic target for treating enhanced itch sensations during inflammatory responses.Eight-week-old C57BL6 mice were purchased from Daehan Biolink . TLR4 KO mice of C57BL6 background were generously provided by Dr. S. Akira . Mice were housed at 23 ± 2°C with a 12-h light–dark cycle and fed food and water ad libitum. All surgical or experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at Seoul National University.Scratching behavioral assays were performed as previously described . BrieflyDRGs collected from 8-week-old mice were incubated in Hank’s balanced salt solution (HBSS)  containing 0.33 mg/ml papain  and 0.65 mg/ml L-cysteine  for 10 minutes at 37°C and then HBSS containing 4 mg/ml collagenase  and 5 mg/ml dispase  for 10 minutes at 37°C. Samples were washed with DMEM/F12 with 10% (v/v) fetal bovine serum (Invitrogen), 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin (Welgene). The cell suspension was filtered through a 70-μm cell strainer and cultured in poly-D-lysine (Sigma-Aldrich) and laminin-coated culture dish or glass coverslips (Sigma-Aldrich).2. HEK293T cells were transfected using Effectene transfection reagent  according to the manufacturer’s instructions.HEK293T cells were maintained in DMEM containing 10% FBS, 100 U/ml ampicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine in a humidified incubator containing 5% COTaq DNA polymerase (Invitrogen). The protocol was 5 min at 95°C followed by 40 cycles of 40 s at 95°C, 40 s at 60°C, and 40 s at 72°C. Reactions were completed with 7 min of final elongation. The second round of amplification was in reaction buffer (20 μl) containing 0.2 mM dNTPs, 0.2 μM inner primers, 5 μl first-round PCR product, and 0.1 μl platinum Taq DNA polymerase following the same amplification procedure as the first round. The following primers were used: TLR4 outer forward 5′- ATC TGA GCT TCA ACC CCT TG-3′; TLR4 outer reverse 5′- AAT TCC CTG AAA GGC TTG GT-3′; TLR4 inner forward 5′- TCA GAA CTT CAG TGG CTG GA-3′; TLR4 inner reverse 5′- TTG ACT TGT GGA TTT TCA CG-3′; TRPV1 outer forward 5′- CAT GCT CAT TGC TCT CAT GG-3′; TRPV1 outer reverse 5′- AAC CAG GGC AAA GTT CTT CC-3′; TRPV1 inner forward 5′- CAT GGG CGA GAC TGT CAA C-3′; TRPV1 inner reverse 5′- CTG GGT CCT CGT TGA TGA TG-3′; MAP2 outer forward 5′- GAA GAG TTC CAA GGC CCA CTT-3′ MAP2 outer reverse 5′- GCC TGA AAT TTG CCT TTT CC-3′; MAP2 inner forward 5′- CCT GTG CAA TTC CAG CTC AGT-3′; MAP2 inner reverse 5′- CCC CCA TGT GGC ATG AAA TAT3′.Single-cell RT-PCR was performed as previously described . BrieflyFor DRG staining, adult mice were anesthetized with urethane and perfused with 4% paraformaldehyde and DRGs were collected. DRGs were incubated in 4% paraformaldehyde, cryoprotected in 30% sucrose, and frozen in OCT. DRG sections (12 μm) were obtained by cryostat and mounted on collagen-coated slides. Sections were stained with mouse anti-histamine receptor H1  or anti-TRPV1 (Santa Cruz) then incubated for 1 h with Cy3-conjugated secondary antibodies , and coverslipped with VectaShield medium .For immunocytochemistry, DRG neuronal cells or transfected HEK293T cells were seeded onto poly-D-lysine (PDL)-coated coverglass. Cells were fixed in 4% PFA in 0.1 M PBS (pH 7.4) for 15 min. After rinsing in 0.1 M PBS, cells were blocked with 0.1 M PBS containing 5% normal goat serum, 5% fetal bovine serum, 2% bovine serum albumin, and 0.1% Triton X-100 for 1 h at RT. Cells were incubated overnight at 4°C with mouse anti-NeuN , rabbit anti-TLR4 , or mouse anti-HA  antibody. Cells were incubated for 1 h at RT with FITC- or Cy3-conjugated secondary antibody and mounted with VectaShield medium.Escherichia coli O55:B5  for 1 h at 37°C. After rinsing in 0.1 M PBS, coverglasses were mounted with VectaShield medium. Images were captured using a confocal laser scanning microscopy .For live cell staining, DRG neurons were seeded on PDL-coated coverglasses and incubated with 5 μg/ml Alexa Fluor 488-conjugated LPS from -∆∆CT method as previously described i.). The net change in [Ca++]i upon drug treatment (Δ ratio (340 nm/38 nm)) was calculated by subtracting basal [Ca++]i from the peak [Ca++]i achieved after exposure to the drug.To determine intracellular calcium levels in HEK293T cell population, cells were detached from plates 24 h after transfection and stained with 2 μM Fura-2 AM in Locke’s solution . Under an upright microscope, whole-cell patch recordings were obtained from acute-isolated DRG neurons in voltage-clamp mode and switched current-clamp configuration for recordings with an Axopatch 700B . Pipette resistance ranged from 3 to 6 MΩ. The internal solution was 140 mM K-gluconate, 10 mM HEPES, 7 mM NaCl, 4 mM Mg-ATP, and 0.3 mM Na3-GTP, pH 7.4. Data were analyzed and plotted with pClamp (Molecular Devices).Cells were plated on PDL-coated cover glasses and incubated in medium for at least 6 h. DRG neuron recordings were performed in HEPES buffer , membranes were incubated with rabbit anti-histamine receptor H1 , or mouse anti-β-actin  antibodies. Proteins were detected with horseradish peroxidase conjugated secondary antibodies using West Save Gold western blot detection kit . Signal was visualized by MicroChemi .t-test. Differences were considered significant when p was less than 0.05.All data are presented as mean value with SEM. Differences between groups were determined by one-way ANOVA with Tukey’s multiple comparison test or Student’s 5-HT: 5-hydrixytryptamineAD: Atopic dermatitisCD: Cluster of differentiationCQ: ChloroquineDRG: Dorsal root gangliaGAPDH: Glyceraldehyde 3- phosphate dehydrogenaseHBSS: Hank’s balanced salt solutionHRH1: Histamine receptor H1HRH4: Histamine receptor H4IL-1β: Interleukine-1βKO: Knock outLPS: LipopolysaccharideMrgprA3: MAS-related G protein-coupled receptor family, member A3R837: ImiquimodTLR4: Toll-like receptor 4TNF-α: Tumor necrosis factor-αTRPA1: Transient receptor potential cation channel, subfamily A, member 1TRPV1: Transient receptor potential cation channel, subfamily V, member 1The authors do not have any financial or non-financial competing interests.HJ carried out behavior study, primary DRG neuron culture, real-time RT-PCR and calcium assay. HL performed single-cell RT-PCR and immunofluorescence staining with primary DRG neuron. HL performed DRG immunofluorescence staining. YHJ contributed to behavior study. SJC and CJL carried out whole cell patch-clamp recording. SJL designed and supervised all the experiments and analyzed data. All authors read and approved the final manuscript.


The molecular structure of the title compound is centrosymmetric. The cyclohexa-2,5-diene moiety is exactly planar and has a bond-length distribution characteristic for one pair of double bonds and two pairs of single bonds. 14H22N2O2Si2, contains one half of the molecule, which is completed by inversion symmetry. The cyclohexa-2,5-diene ring is exactly planar and reflects the bond-length distribution of a pair of located double bonds [1.3224 (14) Å] and two pairs of single bonds [1.5121 (13) and 1.5073 (14) Å]. The tetrahedral angle between the sp3-C atom and the two neighbouring sp2-C atoms in the cyclohexa-2,5-diene ring is enlarged by about 3°.The asymmetric unit of the title compound, C The tetrahedral angles around C1 are likewise distorted due to the ring strain. The angles involving the O atom of the oxy(trimethylsilyl) group and the ring C atoms are enlarged to 110.79 (8)° and 113.26 (8)° while the angle involving the O atom and the C atom of the carbonitrile group is reduced to 104.95 (8)°. The backbone of the 1,1-substituents is nearly perpendicular to the cyclohexa-2,5-diene ring, with a dihedral angle of 86.05 (7).The molecular structure of the title compound is centrosymmetric, leading to a ps Fig. 1. The cycNotable features in terms of non-classical hydrogen bonding interactions are not observed in the crystal structure of the title compound. As a result of the bulky trimethylsilyl groups, π–π stacking interactions between the rings are not possible. The packing of the molecules Fig. 2 seems totrans nitrile groups in the 1,4 positions is listed, namely 3,5-bis(4-(dimethylamino)phenyl)cyclohexa-2,5-diene-1,1,2,4,4-pentacarbonitrile oxy]cyclohexa-2,5-diene-1,4-dicarbonitrile was synthesized by a modified protocol reported by Onaka  al. 1989. The req al. 2000 and actiin vacuo to yield 4.23 g  of a cis/trans (3/1) isomeric mixture of 1,4-bis[(trimethylsilyl)oxy]cyclohexa-2,5-diene-1,4-dicarbonitrile : δ = 6.19 , 0.23  p.p.m.; 13C NMR : δ = 238.3 (s), 129.4 (d), 1.5 (q) p.p.m.1,4-Benzoquinone  was dissolved in 75 ml dichloromethane (0.2 M), purged with argon and cooled to 273 K. Trimethylsilyl cyanide  and Fe-montmorillonite (0.75 g) were added sequentially and the mixture stirred for 1 h at 273 K under an argon atmosphere. The Fe-montmorillonite was filtered off (Por 4 glass filter) and the solvent was evaporated le Fig. 3. CrystalUiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 110.1107/S1600536814014251/su0009sup1.cifCrystal structure: contains datablock(s) general, I. DOI: 10.1107/S1600536814014251/su0009Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536814014251/su0009Isup3.cmlSupporting information file. DOI: 1008752CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


All compounds were in vitro screened for their activity as antioxidants using DPPH , ABTS ) and FRAP (ferric reducing/antioxidant power) methods. The antioxidant activity of the studied compounds against lipid peroxidation was also measured. The highest antioxidant activity was demonstrated by compound 3c. It is obvious that the presence of a hydroxyl group in the structure is essential for the antioxidant properties and should be taken into consideration in further design of structures with potential antioxidant properties.Piperazine nucleus is one of the most important heterocyclic systems exhibiting remarkable pharmacological activities. Thus, in the current study six new aryl/aralkyl substituted piperazine derivatives, containing methylxanthine moiety were synthesized and their structures were confirmed by IR and  The cellular injury caused by oxidative stress and excess of free radicals such as reactive oxygen species has been associated with ageing and great number of clinical disorders including age-associated disorders such as neurodegenerative diseases. For example, Parkinson's disease, which is the second most common neurodegenerative disorder after Alzheimer's disease (AD), has been linked to increased levels of oxidative stress. Also oxiAntioxidant-based drugs and formulations for the prevention and treatment of complex diseases like atherosclerosis, stroke, diabetes, AD, cancer and some psychiatric disorders have appeared during the last decades. This hasPiperazine nucleus is one of the most important heterocycles exhibiting remarkable pharmacological activities. Piperazine is consisting of a six-membered ring containing two opposing nitrogen atoms. Slight change in substitution pattern in piperazine nucleus causes distinguishable difference in their pharmacological activities, such as antipsychotic, anticonvulsant, antiarrhythmic, antimicrobial, antimalarial, citotoxic and antioxidant effects.Thus, in this study some new aryl/aralkyl substituted piperazine derivatives, containing methylxanthine moiety were synthesized and their antioxidant activity was established.254 plates (Merck) and solvent system: ethanol:chloroform:acetone (4:3:3 ). The spots were detected at UV254 nm.Synthetic grade chemicals procured from Acros organics, Belgium, were used for the synthesis of the target compounds, as received. Non-commercially available intermediates required for the synthesis of novel derivatives of arylpiperazine were prepared according to the literature procedures without modifications: 1-(3-iodopropyl)-3,7-dimethylxanthine. The comp2O, sodium acetate and potassium persulphate were purchased from Sigma-Aldrich. All the other chemicals including the solvents were of analytical grade.The reagents: 2,2′-Diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azinobis-(3-ethylbenzo thiazine-6-sulfonic acid) (ABTS), sulfanilamide, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,4,6-tripyridyl-s-triazine (TPTZ), ferric chloride × 6H−1 were recorded on an FT-IR Nicolet iS10 spectrophotometer . The 1H NMR spectra were measured on a Bruker Avance DRX 250 (250 MHz) spectrometer (Germany). 1H NMR spectra were measured for approximately 0.03 mol/L while using CDCl3 and D2O as solvents and chemical shifts, and were expressed as δ values in ppm against TMS as an internal standard. All names were generated by using structure-to-name and name-to-structure algorithms included with ChemBioDraw Ultra 11.0 (CambridgeSoft). Antioxidant assays were carried out using a Shimadzu UV-1203 spectrophotometer (Japan).Melting points were determined on an electrothermal apparatus  in an open capillary tube and are not corrected. The IR spectra 400–4000 cmi-propanol.A mixture of 1-(3-iodopropyl)-3,7-dimethyl-1H-purine-2,6-dione , the appropriate 1-substituted piperazine  and triethylamine (1 mmol) in acetone (10 mL) was refluxed till exhaustion of the starting products. The progress of the reaction was monitored by TLC. Then the acetone was evaporated to dryness. The reaction mixture was cooled overnight if it was necessary. The precipitated crude product was filtered off and purified by recrystallization from 3 and CH2), 1702 (CO xanthine), 1654 (CO xanthine), 1602 (C = N xanthine), 1552 (C = C xanthine), 762 (mono-substituted aromatic ring), 1H–NMR : 7.54 ; 7.36–7.37 ; 7.27 ; 4.09 –2CH); 3.98 ; 3.84 –2CH); 3.56 ; 3.03 ; 2.17 –CH2–2)CH, 1.26, 1.22 , J = 8.43 Hz).Reaction time 10 hours. Yield: 77%, melting point (m.p.) 191–193 °C, Rf = 0.45, IR: 3070 (CH aromatic), 2911–2833 (CH3 and CH2), 1709 (CO xanthine), 1651 (CO xanthine), 1604 (C = N xanthine), 1577 (C = C xanthine), 1143 (C–F), 820 , 1H–NMR : 7.51 ; 6.96 ; 6.85–6.88 ; 4.10 –2CH); 3.99 ; 3.58 ; 3.11 ; 2.63 ; 2.54 , 1.41 –CH2–2)CH.Reaction time 10 hours. Yield: 67%, m.p. 156–158 °C, Rf = 0.64, IR: 3054 (CH aromatic), 2966–2817 (CH3 and CH2), 1701 (CO xanthine), 1657 (CO xanthine), 1609 with shoulder 1595 (C˭N xanthine), 1550 (C˭C xanthine), 1359 (δOH), 1186 (C–OH), 828 , 1H–NMR : 8.39  7.85 ; 6.97–6.85 ; 6.85–6.88 ; 4.04 –2CH); 3.89 ; 3.48 ; 3.34, 3.28 ; 3.13 –CH2–2)CH, 2.06 .Reaction time 10 hours. Yield: 93%, m.p. 263–264 °C with decomposition, Rf = 0.43, IR: 3202 (Aryl-OH), 3075 (CH aromatic), 2926–2815 (CH3 and CH2), 2817 (O–CH3), 1694 (CO xanthine), 1652 (CO xanthine), 1602 (C˭ N xanthine), 1548 (C˭C xanthine), 1454 (δCH3 O–CH3), 1154 (C–O–C), 745 , 1H–NMR : 7.50 ; 7.00–6.97 ; 6.93–6.90 ; 6.86–6.84 ; 4.10 –2CH); 3.99 ; 3.85 ; 3.58 ; 3.06 ; 2.66 ; 2.53 , 1.90 –CH2–2)CH.Reaction time 5 hours. Yield: 75%, m.p. 177–179 °C, Rf = 0.56, IR: 3125 (CH aromatic), 2931–2817 (CH3 and CH2), 2813 (O–CH3), 1698 (CO xanthine), 1651 (CO xanthine), 1604 (C˭N xanthine), 1548 (C˭C xanthine), 1456 (δCH3 O–CH3), 1125 (C–O–C), 844 , 1H–NMR : 7.50 ; 6.90–6.88 ; 6.84–6.82 ; 4.09 –2CH); 3.99 ; 3.76 ; 3.58 ; 3.06 ; 2.62 ; 2.52 , 1.90 –CH2–2)CH.Reaction time 13 hours. Yield: 64%, m.p. 154–156 °C, Rf = 0.58, IR: 3120 (CH aromatic), 2946–2813 (CH3 and CH2), 1703 (CO xanthine), 1655 (CO xanthine), 1602 (C˭N xanthine), 1551 (C˭C xanthine), 1223 (C–F), 826 (p- di-substituted aromatic ring), 1H–NMR : 7.53 ; 7.35–7.27 ; 7.02–6.93 ; 4.36 –CH); 4.08 –2CH); 3.96 ; 3.56 ; 3.14 ; 2.94 ; 2.33 , 1.48 –CH2–2)CH.Reaction time 5 hours. Yield: 60%, m.p. 144–146 °C, Rf = 0.74, IR: 3070 (CH aromatic), 2962–2825  of the sample was determined. Butylated hydroxytoluene (BHT) was used as positive control. All determinations were performed in triplicate (n = 3).ICcontrol is the absorbance of ABTS radical in methanol; Abssample is the absorbance of an ABTS radical solution mixed with sample.For ABTS assay, the procedure followed the method of Arnao et al.  with som50 value (concentration of sample where the absorbance of ABTS decreases 50% with respect to the absorbance of blank) of the sample was determined. BHT was used as positive control. All determinations were performed in triplicate (n = 3).IC2H3NaO2 × 3H2O and 16 mL C2H4O2), pH 3.6, 10 mmol/L TPTZ solution in 40 mmol/L HCl and 20 mmol/L FeCl3 × 6H2O solution. The fresh working solution was prepared by mixing 25 mL acetate buffer, 2.5 mL TPTZ solution and 2.5 mL FeCl3 × 6H2O solution and then warmed at 37 °C before using. 0.15 mL of compound in methanol was allowed to react with 2.8 mL of the FRAP solution for 30 min in the dark condition. Readings of the coloured product (ferrous tripyridyltriazine complex) were then taken at 593 nm. Results are expressed in μg Trolox equivalent (μM TE/mmol/L]). BHT was used as reference. All determinations were performed in triplicate (n = 3).The FRAP assay was done according to the method described by Benzie and Strain  with somn = 3).The antioxidant activity of studied compounds against lipid peroxidation was measured through ammonium thiocyanate assay, as described by Takao et al.,  with somThe target compounds have been synthesized according to the above-presented synthetic procedure. The general synthetic scheme is presented in n-hexane, except 3c which is soluble only in boiling water. The structures of the synthesized compounds were confirmed by IR and 1H NMR. The results were consistent with the assigned structures.All the synthesized compounds were freely soluble in chloroform, dichloromethane, dimethylformamide (DMF) and dimethyl sulfoxide (DMSO), but insoluble in non-polar solvents like −1 exhibit several characteristic bands. Vibrations at 3054–3125 cm−1 belong to stretching vibrations of aromatic C–H bonds. The stretching vibration of phenolic group in the spectrum of 3c appears at 3202 cm−1. The two strong bands at about 1680–1702 cm−1 are ascribable to the stretching vibration of two carbonyl groups in the xanthine ring. The band at about 1550 cm−1 belongs to the stretching vibration of C˭C bonds in xanthine ring.The FT-IR spectra of the studied compounds 3a–f in the region of the 4000–400 cm1H NMR spectra. The 1H NMR spectra of the compounds 3a–f showed the signals of the respective protons of the synthesized compounds, which were verified on the basis of their chemicals shifts, multiplicities and coupling constants. Thus, the strong singlets at 3.48\3.58 and 3.89\3.99 ppm in the spectrum of the studied compounds correspond to N-methyl protons at positions 3 and 7. The signal of the proton at position 8 appears at 7.50\7.54 ppm as strong singlet. The signals of methylene protons from the benzyl side chain at position 1 form strong singlet at 4.80 ppm. The signals of the aromatic protons from substituted benzyl side chain in the spectra of 3b–e correspond to complicated multiplets between 6.82 and 6.97 ppm, while the same protons in unsubstituted benzyl moiety (3a) appear as multiplet 7.27 and 7.37 ppm. The aromatic protons in benzhydryl group in 3f form multiplet at 7.27 and 7.35 ppm. The methylene proton from the benzhydryl residue forms a singlet with weak to medium intensity at 4.36 ppm. The integral curves correspond to the exact number of the protons. The values of the chemical shifts of the protons registered by 1H NMR spectra were compared with simulated values.[1H NMR spectra are in good correlation with experimental ones.More detailed information about the structure of compounds 3a–f was provided by the d values. Due to aIn this paper, we apply four common methods  for the 2) on DPPH  of the samples were determined.The free radicals scavenging activity was measured by using DPPH and ABTS methods with slight modifications. The inhi on DPPH  and ABTS on DPPH  was dete2 solutions of piperazines.The applied FRAP assay was done according to the method described previously. The obtan = 3), and the results are expressed as IC50 μmol L−2 of inhibition in DPPH and ABTS determinations and as mmol/L TE mM−1 in FRAP determination. The obtained values are presented in The corresponding radical scavenging activities against DPPH, ABTS and FRAP of the compounds were compared with those of BHT, used as positive control. All determinations are performed in triplicate (50 371.97 μmol/L), 3c (IC50 189.42 μmol/L) and 3f (IC50 420.57 μmol/L), with the highest antioxidant activity demonstrated by compound 3c. However, the obtained IC50 values of all piperazines were lower than that of BHT (IC50 113.17 μmol/L).Among the analysed structures, DPPH radical scavenging activity was shown by compounds 3a (IC50 3.45 μmol/L) > BHT (IC50 26.29 μmol/L) > 3f (IC50 41.04 μmol/L) > 3a (IC50 55.87 μmol/L) > 3e (IC50 242.48 μmol/L) > 3b (IC50 345.25 μmol/L) > 3d (IC50 1028.99 μmol/L).All studied compounds demonstrated ABTS-radical scavenging decreasing in order: 3c  complex to ferrous form (which has an intense blue colour) at low pH was monitored by measuring the change in the absorption at 593 nm, which is considered to be directly related to the combined or ‘total’ reducing power of the electron donating antioxidants present in the reaction mixture. Among the synthesized compounds only 3b, 3c and 3e (with p-substitution in phenyl ring) manifest some activity in the FRAP method, while compound 3c demonstrated the highest activity (173.99 ± 1.50 μmol/L TE/mmol/L), followed by 3e (83.10 ± 1.84 μmol/L TE/mmol/L) and 3b (27.22 ± 1.35 μmol/L TE/mmol/L). However, piperazines 3b, 3c and 3e exhibited stronger activity compared to BHT (23.26 ± 0.45 μmol/L TE/mmol/L). These effects are probably due to the possibility of the analytes to break up the free radical chain by donating a hydrogen atom.As seen from the presented results for the three discussed methods used for evaluation of the free radicals scavenging activity and FRAP of the newly synthesized structures, the highest antioxidant activity was demonstrated by compound 3c. We believe that this result is due to the presence of hydroxyl group in the structure of this product.in vitro systems.Furthermore, the difference in the chemical structure of the newly obtained compounds will reflect to their relative ability to quench aqueous peroxyl radicals and to reduce the ABTS+, the DPPH free radical and the ferric ion in Moreover, ABTS, DPPH and FRAP assays gave comparable results for the antioxidant activity measured for all compounds, whereas the ABTS technique was simple, rapidly performed and was applicable for the determination of the oxidative properties for all the analysed structures. An additional advantage of the ABTS to DPPH is that the reaction is more rapid, whereas the DPPH reaction takes much longer. Therefore, it would be an appropriate technique for antioxidant evaluation of structural derivatives with piperazine residue.2) against lipid peroxidation was also measured through ammonium thiocyanate assay, as described previously.[In this study, the antioxidant activity of the studied compounds  was synthesized. The structures of the new compounds were confirmed by IR and


The chiral title compounds are closely related hydantoin derivatives with bromo and chloro substituents at the 3-position of the benzene ring of the isopropoxyphenyl subtituent. In the both crystals, hydantoin groups are connected by N—H⋯O hydrogen bonds, forming two-dimensional sheets, made up from  S)-(+)-5-(3-bromo-4-isopropoxyphenyl)-5-methylimidazolidine-2,4-dione, C13H15BrN2O3, (I), the hydantoin groups are connected via intermolecular N—H⋯O hydrogen bonds, forming a terraced sheet structure. In the chloro analogue, (S)-(+)-5-(3-chloro-4-isopropoxyphenyl)-5-methylimidazolidine-2,4-dione, C13H15ClN2O3, (II), the intermolecular N—H⋯O hydrogen-bonding network forms a flat sheet. Comparison of the crystal structures reveals that (II) is more loosely packed than (I).In ( These values are 0.010 (7) and 0.018 (2) for (I)The conformations of the molecules (I)a is shown in Fig. 3z = 0 and z = ½ along a. Groups of four molecules are linked by these N—H⋯O hydrogen bonds, generating The crystal structure of (I)P212121 and the lattice constants are roughly similar for both. However, there are both similarities and significant differences in the packing modes between the two closely related molecules. The crystal structure of (II)a is shown in Fig. 5z = 0 or z = ½, and are connected by N—H⋯O hydrogen bonds (Table 2a.Both (I)s Table 2, formingX bond distances [C13—Br1 = 1.8945 (18) Å in (I)V/Z is larger by ca 4% than that for (I)Comparison of the crystal structures reveals that (II)S-(+)-5-phenyl-5-ethylhydantoin was reported -(−)-5-p-methylphenyl-5-methylhydantoin in a chlathrate compound with permethylated β-cyclodextrin based on the known absolute configuration of the host. Martin et al. (2011S)-(+)-5-phenyl-5-trifluoromethylhydantoin with (+)-α-methylbenzylamine to determine the configuration based on the known absolute configuration of the chiral amine. It is noted that the R and S notation remains unchanged when CH3 at the 5-position of the hydantoin is replaced with CF3, although the priorities of the substituents in the sequence rule are altered. To our knowledge, the present paper is the first to report the absolute configuration of such compounds determined from anomalous-dispersion effects.Structures of 5-phenyl-5-alkylhydantoin derivatives have been investigated to review the relationships between the absolute configuration and optical activity. Knabe & Wunn 1980 determin al. 2006 determin al. 2011 preparedet al., 2016D, of (I)c = 0.98, MeOH, where c is the concentration of units gram per 100 cm−3).Compounds (I)D, of (II)c = 1.0, MeOH).Plate-like crystals of (II)Uiso(H) = 1.2Ueq(C/N) and Uiso(H) = 1.5Ueq(Cmethyl). The thermal displacement ellipsoids of the non-hydrogen atoms of the isopropoxy group in (II)Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989015023014/sj5484sup1.cifCrystal structure: contains datablock(s) I, II, global. DOI: Click here for additional data file.10.1107/S2056989015023014/sj5484Isup4.cdxSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015023014/sj5484Isup6.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015023014/sj5484IIsup5.cdxSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015023014/sj5484IIsup7.cmlSupporting information file. DOI: 1436066, 1439808CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Diffraction data sets were collected to resolutions of 2.40 and 2.66 Å from crystals of l-arabinonate dehydratase and d-xylonate dehydratase, respectively. l-Arabinonate dehydratase (EC 4.2.1.25) and d-xylonate dehydratase (EC 4.2.1.82) are two enzymes that are involved in a nonphosphorylative oxidation pathway of pentose sugars. l-Arabinonate dehydratase converts l-arabinonate into 2-dehydro-3-deoxy-l-arabinonate, and d-xylonate dehydratase catalyzes the dehydration of d-xylonate to 2-dehydro-3-deoxy-d-xylonate. l-Arabinonate and d-xylonate dehydratases belong to the IlvD/EDD family, together with 6-phosphogluconate dehydratases and dihydroxyacid dehydratases. No crystal structure of any l-arabinonate or d-xylonate dehydratase is available in the PDB. In this study, recombinant l-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii (RlArDHT) and d-xylonate dehydratase from Caulobacter crescentus (CcXyDHT) were heterologously expressed in Escherichia coli and purified by the use of affinity chromatography followed by gel-filtration chromatography. The purified proteins were crystallized using the hanging-drop vapour-diffusion method at 293 K. Crystals of RlArDHT that diffracted to 2.40 Å resolution were obtained using sodium formate as a precipitating agent. They belonged to space group P21, with unit-cell parameters a = 106.07, b = 208.61, c = 147.09 Å, β = 90.43°. Eight RlArDHT molecules (two tetramers) in the asymmetric unit give a VM value of 3.2 Å3 Da−1 and a solvent content of 62%. Crystals of CcXyDHT that diffracted to 2.66 Å resolution were obtained using sodium formate and polyethylene glycol 3350. They belonged to space group C2, with unit-cell parameters a = 270.42, b = 236.13, c = 65.17 Å, β = 97.38°. Four CcXyDHT molecules (a tetramer) in the asymmetric unit give a VM value of 4.0 Å3 Da−1 and a solvent content of 69%. Similarly, d-xylonate dehydratase (EC 4.2.1.82) catalyzes the removal of one water molecule from d-xylonate to produce 2-dehydro-3-deoxy-d-xylonate   and it lacks the [Fe–S] cluster. Therefore, structural studies of enzymes belonging to this family are needed in order to understand their structure–function relationship and the role of the [Fe–S] cluster. Here, we describe the overproduction, purification, crystallization and X-ray diffraction analysis of two enzymes from the IlvD/EDD family.2.2.1.araD gene encoding R. leguminosaruml-arabinonate dehydratase (RlArDHT) and the xylD gene encoding C. crescentusd-xylonate dehydratase (CcXyDHT) were purchased as codon-optimized synthetic genes from GeneArt, Germany and a Strep-Tag II (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) was added at the N-terminus . The genes were cloned into a pBAT4 expression vector, as described in Andberg et al.  expression cells were transformed with the recombinant plasmids. Large-scale protein production was performed in 2 l shake flasks containing 500 ml Luria–Bertani (LB) culture medium with 100 µg ml−1 ampicillin. The culture flasks were incubated at 303 K in a shaker incubator at 180 rev min−1. When the OD600 nm reached 0.5–0.6, protein expression was induced by the addition of isopropyl β-d-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM, and cultivation was continued under identical conditions overnight  and 100 µg ml−1 lysozyme . Intracellular proteins were isolated by centrifugation at 18 500 rev min−1, followed by a one-step freeze–thawing of the suspended cells at 193 K and sonication.For protein production, M Tris–HCl pH 8.0, 150 mM NaCl, 5 mM MgCl2). Strep-Tag II-bound proteins were eluted with elution buffer . A final polishing step was conducted by gel-filtration chromatography on a Superdex 200 HR 10/30 column  equilibrated with a buffer consisting of 50 mM Tris–HCl pH 7.5, 5 mM MgCl2. Purified samples were concentrated using an Amicon Ultra-4 centrifugal filter device with a molecular-weight cutoff of 30 kDa . The molecular weights and the purity of the enzymes were checked by SDS–PAGE with silver staining in a PhastSystem . The enzyme activities were checked by the thiobarbituric acid (TBA) assay and the semicarbazide assay  equilibrated with binding buffer . A dog hair was dipped into the seed-stock solution and streaked through pre-equilibrated crystallization drops. Crystallization information is shown in Table 2Crystallization of the purified enzymes was performed by the hanging-drop vapour-diffusion method in a 24-well cell-culture plate  at 293 K. The following commercial crystallization kits were used: Crystal Screen, Crystal Screen 2, PEG/Ion, SaltRx 1, SaltRx 2, Index HT, PEGRx 1 and PEGRx 2 (Hampton Research). Droplets were prepared by mixing protein sample and crystallization reagent in a 1:1 ratio on a siliconized glass cover slide. The cover slides were then turned drop-side down against 500 µl crystallization reservoir reagent and sealed with grease to ensure that they were airtight. The plates were stored at 293 K. A microseeding technique  in England at a wavelength of 0.97949 Å. Data processing was carried out by XDS  for RlArDHT and CcXyDHT, respectively.Purified dehydratase enzymes contain an [Fe–S] cluster, which is necessary for enzyme activity  from Crystal Screen. Crystals appeared within two weeks  and diffracted to 2.4 Å resolution . The space group was P21, with unit-cell parameters a = 106.07, b = 208.61, c = 147.09 Å, β = 97.43°. Assuming eight molecules in the asymmetric unit, the calculated Matthews coefficient (VM) was 3.2 Å3 Da−1, which corresponds to a solvent content of 62%  gave crystals within four weeks  which diffracted to 2.66 Å resolution . The crystals belonged to space group C2, with unit-cell parameters a = 270.42, b = 236.13, c = 65.17 Å, β = 97.38°. Assuming four molecules in the asymmetric unit, the calculated Matthews coefficient (VM) was 4.0 Å3 Da−1, which corresponds to a solvent content of 69%  in the asymmetric unit, with initial R and Rfree values of 37.7 and 46.7%, respectively. The structure of CcXyDHT was subsequently solved by molecular replacement using the coordinates of RlArDHT. A clear solution with a tetramer in the asymmetric unit (Z-score = 9) was found and resulted in initial R and Rfree values of 31.7 and 39.7%, respectively. Three-dimensional structure determination and structure refinement of both RlArDHT and CcXyDHT are ongoing.The structures of 10.1107/S2053230X16010311/nj5256sup1.pdfSupplementary Figure S1.. DOI:


Both metals are bridged by one carbonyl and one chloridoborylene unit. Additionally, one benzene solvent molecule aligns in a staggered position relative to the (pentamethyl)cyclopentadienyl ligand of the FeII centre.The title compound [η 5-(C5(CH3)5)(CO)Fe{(μ-BCl)(μ-CO)}PtCl(P(C6H11)3)]·C6H6, the two metal atoms, iron(II) and platinum(II), are bridged by one carbonyl (μ-CO) and one chloridoborylene ligand (μ-BCl). The PtII atom is additionally bound to a chloride ligand situated trans to the bridging borylene, and a tricyclohexylphosphane ligand (PCy3) trans to the carbonyl ligand, forming a distorted square-planar structural motif at the PtII atom. The FeII atom is bound to a pentamethylcyclopentadienyl ligand [η5-C5(CH3)5] and one carbonyl ligand (CO), forming a piano-stool structure. Additionally, one benzene solvent molecule is incorporated into the crystal structure, positioned staggered relative to the pentamethylcyclopentadienyl ligand at the FeII atom, with a centroid–centroid separation of 3.630 (2) Å.In the molecular structure of the dinuclear title compound [η This compound was isolated upon the reaction of [(η5-C5Me5)(CO)2FeBCl2] with [Pd(PCy3)2] via the loss of one of the tricyclohexylphosphane ligands (CO)Fe(μ-BBr)(μ-CO)PdBr(PCy3)], which was spectroscopically characterized, was reported without structural proof (OC)Fe(μ-CO)Pt(PCy3)(μ-Br)Pt(PCy3)Br(μ3-B)]. Spectroscopic investigations indicated that [(η5-C5Me5)(CO)Fe(μ-BBr)(μ-CO)PtBr(PCy3)] is likely initially formed, and subsequently reacts with a second equivalent of [Pt(PCy3)2] to give the final product. However, once again, no structural proof could be given (CO)Fe{(μ-BCl)(μ-CO)}Pt(PCy3)Cl]·C6H6, (I)3)2] with [(η5-C5Me5)(CO)2FeBCl2] via oxidative addition of a B—Cl bond to the low-valent platinum, resulting in the loss of one phosphane ligand.Herein we report the related heterodinuclear bridging chloridoborylene complex [(CO)2FeBCl2], (II), which has a Fe—B bond length of 1.942 (3) Å, there is an obvious lengthening of this bond in the target molecule. In the structure of (I)5-C5Me5) and one carbonyl ligand (CO), forming an overall piano-stool structure. The 11B NMR resonance in the spectrum of (I)31P NMR spectrum shows a peak at 56.55 p.p.m. with a coupling constant of 1JP–Pt = 4864 Hz, which is typical for a bridging square-planar platinum complex The orientation of the benzene solvent molecule in the crystal structure of (I)5-C5Me5)(CO)2Fe(BCl2)]  was dissolved in 2 ml of benzene and bis(tricyclohexylphosphane)platinum  was added to the solution. After 5 h of stirring, the solvent was removed, by-products were extracted with two portions of 2 ml of hexane, and the bright-yellow residue was redissolved in 2 ml of benzene. Upon slow evaporation, yellow crystals suitable for X-ray diffraction were obtained at room temperature . Elemental analysis calculated (%): C 49.00, H 6.16; found (%): C 50.08, H 6.20. 1H NMR : δ 1.14–2.12 , 1.66 , 2.92 . 11B{1H} NMR : δ 107.4. 13C{1H} NMR : δ 10.1 (C5Me5), 27.0 (P3Cy), 27.8 (P3Cy), 30.6 (P3Cy), 34.4 (P3Cy), 98.1 , 167.6 (μ-CO), 205.9 (CO). 31P{1H} NMR : δ 56.55 (1JP–Pt = 4864 Hz). IR (toluene): 1978, 1913 cm−1.[(η3) or 1.00 Å . Uiso(H) values were fixed at 1.5 (for primary H atoms) and 1.2 times (tertiary H atoms) Ueq of the parent C atoms.Crystal data, data collection and structure refinement details are summarized in Table 110.1107/S1600536814023381/wm5073sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S1600536814023381/wm5073Isup2.hklStructure factors: contains datablock(s) I. DOI: 1030674CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The crystal structures of four indole derivatives with various substituents at the 2-, 3- and 5-positions of the ring system are described. The dominant intermolecular interaction in each case is an N—H⋯O hydrogen bond, which generates either chains or inversion dimers. Weak C—H⋯O, C—H⋯π and π–π interactions occur in these structures but there is no consistent pattern amongst them. Two of these compounds act as modest enhancers of CB1 cannabanoid signalling and two are inactive. H-indol-3-yl)-3-phenylpropanoate, C25H22ClNO2, (I), 2-bromo-3-(2-nitro-1-phenylethyl)-1H-indole, C16H13BrN2O2, (II), 5-methoxy-3-(2-nitro-1-phenylethyl)-2-phenyl-1H-indole, C23H20N2O3, (III), and 5-chloro-3-(2-nitro-1-phenylethyl)-2-phenyl-1H-indole, C22H17ClN2O2, (IV). The dominant intermolecular interaction in each case is an N—H⋯O hydrogen bond, which generates either chains or inversion dimers. Weak C—H⋯O, C—H⋯π and π–π interactions occur in these structures but there is no consistent pattern amongst them. Two of these compounds act as modest enhancers of CB1 cannabanoid signalling and two are inactive.The crystal structures of four indole derivatives with various substituents at the 2-, 3- and 5-positions of the ring system are described, namely, ethyl 3-(5-chloro-2-phenyl-1 All the bond lengths and angles in these compounds lie within their expected ranges and full details are available in the CIF.Each compound crystallizes in a centrosymmetric space group . The C11—O1—C12—C13 torsion angle is −81.27 (19)° and the dihedral angle between the indole ring system and the C14 phenyl ring is 86.55 (4)°.The molecular structure of (I)anti orientation about the C10—C9 bond. The dihedral angle between the indole ring system and the phenyl ring is 81.69 (7)°.The molecular structure of (II)anti [torsion angle = −171.63 (8)°]. The methyl C atom of the methoxy group deviates from the indole plane by −0.1302 (14) Å, i.e. slightly towards the side of the molecule occupied by the C11 phenyl ring.Fig. 3A view of the molecular structure of (IV)i.e., S configuration of C9), with an anticlockwise twist of C9—H9 with respect to the indole ring system.All-in-all, the conformations of (II)As might be expected, the dominant supramolecular motif in all these compounds involve N—H⋯O hydrogen bonds, although the resulting topologies  bond links the molecules into [100] chains with a C(8) chain motif if Fig. 5; adjaceni  hydrogen bonds occur, which generate In (III)ns Fig. 7. The dimi and N1i—H1i⋯O2  hydrogen bonds ds Fig. 8. Just onsp3-hybridized C atom with two further C atoms and one H atom bonded to it at the 3-position (as per C9 in the present structures) yielded 72 hits. An analysis of the dihedral angle in these structures corresponding to C8—C7—C9—H9 in the present structures showed a wide spread of values with no obvious overall pattern.There are over 4000 indole derivatives with different substituents (including H) at the 2, 3 and 5 positions of the ring system reported in the Cambridge Structural Database malonate , [prepared from diethyl benzylidenemalonate and 5-chloro-2-phenylindole in the presence of Cu(OTf)2] in DMSO (10.8 ml) and water (150 ml) was stirred at 443K for 16 h. After cooling to room temperature, water was added until a precipitate formed (25 ml). The mixture was extracted into DCM (3 × 25 ml), washed with saturated NaCl(aq) (15 ml), dried over sodium sulfate, filtered and evaporated to leave a red oil. Flash chromatography  afforded (I)−1) 3391, 2911, 1738, 1629, 1581, 1556, 1445, 1399, 1283, 1271, 1215, 1208, 1145, 1113, 1077, 874, 852,761. HRMS (ESI) for C25H2335ClNO2 [M + H]+ calculated 404.1418, found 404.1416.A mixture of sodium chloride  and diethyl 2-. This was refluxed in ClCl4 (40 ml) with NBS  for 96 h, filtered and the solvent evaporated under reduced pressure to leave a red oily residue. Flash chromatography of the residue  gave (II)−1) 3353, 2987, 2923, 2856, 1548, 1452, 1337, 740 and 701; RMS (ESI) for C16H1379BrN2O2Na [M + Na]+ calculated 367.0058, found 367.0049.A mixture of indole , trans-β-nitrostyrene  and sulfamic acid  were refluxed in EtOH (45 ml) for 24 h. Removal of the solvent and flash chromatography  afforded 3-(2-nitro-1-phenylethyl)-1H-indole , prepared from p-methoxyphenylhydrazine hydrochloride, acetophenone and PPA in EtOH (5 ml) was stirred at 323K for 40 h. The solvent was removed under reduced pressure and the residue was flash chromatographed  to provide (III)−1) 3407, 1629, 1600, 1581, 1534, 1369, 1200 and 1141; HRMS (ESI) for C23H21N2O3 [M + H]+ calculated 373.1553, found 373.1544.A mixture of trans-β-nitrostyrene , sulfamic acid  and 5-methoxy-2-phenyl-1H-indole , trans-β-nitrostyrene  and sulfamic acid  were stirred in EtOH (80 ml) at reflux for 15 h. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography  to give the product as a yellow solid . Rf 0.23 ; m.p. 457–459K; IR  3396, 3034, 1740, 1598, 1510, 1318, 1055 and 839; HRMS (ESI) for C22H18N2O2Cl [M + H]+ calculated 377.1057, found 377.1054.5-Chloro-2-phenyl-1Uiso(H) = 1.2Ueq(carrier) or 1.5Ueq(methyl carrier) was applied in all cases. The methyl H atoms (if any) were allowed to rotate, but not to tip, to best fit the electron density.Crystal data, data collection and structure refinement details are summarized in Table 510.1107/S2056989015008476/lh5763sup1.cifCrystal structure: contains datablock(s) I, II, III, IV, global. DOI: 10.1107/S2056989015008476/lh5763Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989015008476/lh5763IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989015008476/lh5763IIIsup4.hklStructure factors: contains datablock(s) III. DOI: 10.1107/S2056989015008476/lh5763IVsup5.hklStructure factors: contains datablock(s) IV. DOI: Click here for additional data file.10.1107/S2056989015008476/lh5763Isup6.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015008476/lh5763IIsup7.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015008476/lh5763IIIsup8.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015008476/lh5763IVsup9.cmlSupporting information file. DOI: 1062393, 1062392, 1062391, 1062390CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Paullinia pinnata Linn (Sapindaceae) is amedicinal plant, locally used in the West Region of Cameroon for the treatment oftyphoid fever. This work was designed to evaluate the antityphoid and antioxidantactivities of the extracts and compounds of P.pinnata.invivo for its antityphoid potential in a Salmonella typhimurium-induced typhoid fever model in Wistar rats.The methanol extracts of the leaves and stems were tested for antityphoid andantioxidant activities. Compounds were isolated, and their structures elucidated byanalysis of spectroscopic data in conjuction with literature data and tested for thesame activities. The leaf extract was also tested β)-3-O-(2′-Acetamido-2′-deoxy-β-D-glucopyranosyl) oleanolic acid (5), -3-O-(2′-Acetamido-2′-deoxy-β-D-glucopyranosyl) echinocystic acid (6) and -3-O-[β-D-glucopyranosyl-(1″-3′)-2′-acetamido-2′-deoxy-β-D-galactopyranosyl]oleanolicacid (7) were isolated. Compounds 5 and 1 showed the highest antibacterial(MIC = 0.781-1.562 μg/ml) and DPPH radical scavenging (RSa50 = 19.27 ± 4.43 μg/ml)activities respectively. The maximum extract dose (446.00 mg/kg bw) had comparableactivity with ciprofloxacin (7.14 mg/kg bw) and oxytetracycline (5 mg/kg bw). Theextract induced significant dose-dependent increase of WBCs and lymphocytes.Seven known compounds: methylinositol (1), β-sitosterol (2), friedelin (3),3β-(β-D-Glucopyranosyloxy)stigmast-5-ene (4), (3P.pinnata and its isolated Compounds could be useful in thestandardization of antityphoid phytomedicine from it.These results support the ethnomedicinal use of  Salmonella typhi,Salmonella paratyphi A, Salmonellaparatyphi B; which cause typhoid fever, paratyphoid A and B feversrespectively  is a lianaused in the West Region of Cameroon for the treatment of bacterial infections liketyphoid fever, syphilis, gonorrhea, diarrhea and symptoms like stomach-ache andwaist pain. In east Africa, the leaves are reported to be used in the treatment ofgonorrhea, wounds and microbial infections  at room temperature to give crude extracts (233.8 g and 152.17 grespectively) after concentration under reduced pressure. The leaf extract (230 g)was exhaustively and successively partitioned with hexane and acetone to affordthe hexane (45.2 g), acetone (8 g) and methanol residue (156.8 g) fractions whilethe stem extract was partitioned into petroleum ether, ethyl acetate and water toobtain the PE fraction (8.08 g), EtOAc fraction (9.13 g) and aqueous residuefraction (109.89 g).The air-dried leaves (2.04 Kg) and stems (2.02 kg) of 3-MeOH (40:60) to afford methylinositol (28 mg).Fourty grams of the hexane fraction was applied to neutral silica gel 60(0.2-0.5 mm) column (60 × 8 cm) and eluted with mixtures of petroleum ether-ethylacetate of increasing polarity (100:0 → 50:50 with constant polarity increase of5%) to give 40 fractions. These fractions were further grouped on the basis oftheir TLC band pattern similarities into 5 fractions (F1 to F5). Fraction F1(Petroleum ether 100%) was mounted on a silica gel column and eluted with amixture of Hex-AtOAc of increasing polarity (95:5 → 50:50) to yield 30 fractionswhich were equally grouped on the basis of their TLC band pattern similaritiesinto 5 sub fractions (F1.1 to F1.5). Sub fractions F1.1  andF1.4  both yielded white powders which were purified by sephadexgel (LH-20) column chromatography and eluted withCHCl3-MeOH (4:6) to afford β-sitosterol (20 mg). Finally,F2  was mounted on a silica gel column and elutedwith a mixture of Hex-EtOAc of increasing polarity (95:5 → 70:30) to yield 10fractions which were grouped on the basis of their TLC band pattern similaritiesinto 3 sub fractions (F2.1 to F2.3). Sub fractions F2.1  and F2.2 yielded transparent crystals which were purified by sephadex gel(LH-20) column chromatography and eluted with CHCl3-MeOH(4:6) to afford friedelin (18 mg).One hundred and fifty grams of the methanol residue fraction was applied toneutral silica gel 60 (0.2-0.5 mm) column (60 × 8 cm) and eluted with mixtures ofn-hexane/ethyl acetate and ethyl acetate-methanol of increasing polarity(100:0 → 0:100 with constant polarity increase of 5%) to give 60 fractions whichwere further grouped on the basis of their TLC band pattern similarities into 5fractions (F1 to F5). Further column purification of F2 (eluted with EtOAc-MeOH90:10) on silica gel yielded six fractions denoted F2.1 to F2.6. White niddle-likecrystals, formed in F2.3  and F2.4  werecollected and purified on a sephadex gel (LH-20), eluted with an isocratic systemof CHCl2O 50:50 → 100:0) to afford3β-(β-D-Glucopyranosyloxy)stigmast-5-ene (119 mg),3-0-(2′-acetamido-2′-deoxy-β-D-glucopyranosyl)olean-12-en-28-oic acid (170 mg) and8 fractions. Similarly, F4 (3.60 g) was chromatographed on silica gel column andeluted with CHCl3-MeOH (9:1 → 7:3) to give 4 fractions.F4.4 (448 mg) was subjected to sephadex LH-20 gel column chromatography and elutedwith CHCl3-MeOH (1:1) to afford3-0-(2-acetamido-2′deoxy-β-D-glucopyranosyl)-l6α-hydroxyolean-l2-en-28-oic acid(45 mg). The aqueous residue fraction (76.79 g) was mounted on a D101 macroporousresin column and eluted successively with H2O-EtOH  to obtain 5 fractions denoted F1 to F5 respectively. F5(946 mg) was purified on a silica gel column, eluted with a stepwise gradientmixture of CHCl3-MeOH-H2O(8:2:0.5 → 6:4:0.5) to afford3-O-[β-D-glucopyranosyl-(1″-3′)-2′-acetamido-2′-deoxy-β-D-glucopyranosyl]olean-12-en-28-oicacid (40 mg).The EtOAc fraction (7.07 g) was subjected to column chromatography on Rp-18gel  with TMS as the internal standard.ESIMS spectra were recorded on a Bruker HTC/Esquire spectrometer. HREIMS wasrecorded on a Waters AutoSpec Premier P776 spectrometer. Column Chromatography(CC) was performed on silica gel , Rp-18 . Fractions were monitored by TLC , and by heating silica gel platessprayed with 10% HSalmonella species were used in thisstudy, one strain from the American Type Culture Collection  and three clinical isolates from “Centre Pasteur” ofYaounde-Cameroon.Four 8 colony-forming units/ml (CFU/ml)  and 100–0.781 μg/ml (for compounds). The dilution solution,5% Tween 80, did not show inhibitory effects on the growth of the bacteria. Thecultured micro plates were covered and incubated at 37°C for 24 h. Inhibitoryconcentrations of the extracts were detected after addition of 50 μl of 0.20 mg/mlMTT -2,5-diphenyltetrazolium bromide, Sigma-Aldrich,South Africa) and incubation at 37°C for 30 min  were reared in the Animal house of the Department ofBiochemistry, University of Dschang-Cameroon. Only the crude extract was used inthe treatment of infected animals. Prior to the test, animals were housed underthe test conditions for a period of one week.This test was carried out using a The experiments were conducted according to the ethical guidelines ofCommittee for Control and Supervision of Experiments on Animals , Government of India, on the use of animalsfor scientific research.Animals were immunosuppressed two day before infection by the oraladministration of 30 mg/kg bw. of cyclophosphamide as previously described,containing about 108 CFU was orally administered toeach animal  was not infected and received distilledwater during the treatment period. Group two (negative control group) received only distilled water duringthe treatment period.•Group two (negative control group) received only distilled water duringthe treatment period. Groups three and four (positive control groups) received ciprofloxacin(7.14 mg/kg bw) and oxytetracycline (5 mg/kg bw) during the treatmentrespectively.P. pinnata leaf extract at concentrations of55.75, 111.50, 223.00 and 446.00 mg/kg bw, corresponding to MIC, 2MIC, 4MICand 8MIC respectively. Group five, six, seven and eight (test groups) received the P. pinnata leaf extract at concentrations of 55.75,111.50, 223.00 and 446.00 mg/kg bw, corresponding to MIC, 2MIC, 4MIC and 8MICrespectively.• Group five, six, seven and eight (test groups) received the ad libitum. Treatment was done byadministering the extract orally, every morning at the same time. Each day, thefecal matter was collected during the administration process and assessed for thestool bacterial density. The extent to which the animals complied with treatmentwas studied by counting the amount of bacterial colonies in the fecal samplesusing the following protocol.Food and water were given to the animals before and during the treatment 0.10 g of fecal matter was completely dissolved in 5 ml of autoclaveddistilled water. 50 μl of the resulting solution was spread on the surface of solidified0.9% saline SS agar in the 35 mm type Petri dish.• 50 μl of the resulting solution was spread on the surface ofsolidified 0.9% saline SS agar in the 35 mm type Petri dish.Salmonella typhimurium in eachPetri dish was counted and recorded. After incubation for 18 h at 37°C, the number of colonies followinggrowth of  The results were converted into the number of colonies per gram offecal matter per animal.• The results were converted into the number of colonies per gram offecal matter per animal.Each time a group was completely healed, the animals were sacrificed bychloroform anaesthesia and blood was collected, by cardiac puncture afterdissection, into heparinized tubes. Blood parameters including hematocrite, RedBlood Cells (RBCs), White Blood Cells (WBCs), lymphocytes, monocytes, eosinophils,basophils and neutrophils were evaluated using the heparinized blood  free radical  program. The experimentalresults were expressed (where appropriate) as mean ± standard deviation of threereplicates.13C NMRdata were very close to those of L-quebrachitol stigmast-5-ene or daucosterol (4)   and gram-negative bacteria, with MIC value of 300 μg/ml  was formally isolated from invivo on a Salmonellatyphymurium-induced typhoid model in Wistar rats. In infected animals, stools were either soft with mucus,liquid or were moulded and smooth but mucus coated. Sometimes, the presence of bloodand mucus made the stool to appear dark and shinny. These animals were weak and lessactive, with their fur standing at right angles to the body surface instead of thenormal sleeping position. The slender body became more bulky, with some of theanimals even ‘coughing’. All of these characterized the establishment of infectionin the experimental animals, which was clearly revealed by the growth of Salmonellacolonies on Petri dishes after the culturing of fecal matter.The crude extract of the leaves was tested Salmonella typhi orparatyphi on blood cultures  significantly (p < 0.05) droppedwith time in a dose-dependent manner compared to the negative control group 2)during treatment  presented radical scavenging activities against stable DPPH freeradical in a concentration-dependent manner. The other compounds did not displayappreciable radical scavenging potentials -3-O-(2′-Acetamido-2′-deoxy-β-D-glucopyranosyl) oleanolic acid, and 1, methylinositol,respectively as markers.The present findings support the ethno-pharmacological exploitation of


N-methylation of structure (I), no hydrogen-bonding interactions were observed for structure (II).In the crystal structure of (I), the molecules are linked into chains by N—H⋯O hydrogen bonds with  19H17N3O3S (I), was prepared by a [3 + 2]cycloaddition azide condensation reaction using sodium azide and l-proline as a Lewis base catalyst. N-Methylation of compound (I) using CH3I gave compound (II), C20H19N3O3S. The benzothiophene ring systems in (I) and (II) are almost planar, with r.m.s deviations from the mean plane = 0.0205 (14) in (I) and 0.016 (2) Å in (II). In (I) and (II), the triazole rings make dihedral angles of 32.68 (5) and 10.43 (8)°, respectively, with the mean planes of the benzothiophene ring systems. The trimethoxy phenyl rings make dihedral angles with the benzothiophene rings of 38.48 (4) in (I) and 60.43 (5)° in (II). In the crystal of (I), the molecules are linked into chains by N—H⋯O hydrogen bonds with R21(5) ring motifs. After the N-methylation of structure (I), no hydrogen-bonding interactions were observed for structure (II). The crystal structure of (II) has a minor component of disorder that corresponds to a 180° flip of the benzothiophene ring system [occupancy ratio 0.9363 (14):0.0637 (14)].The title compound, C This chemical modification is essential to restrict the tendency toward cis–trans isomerization of the cyano-stilbene moiety in cyano-CA-4 analogs 3I, resulting in compound (II)In continuation of our work on the development of benzothiophene cyano combretastatin A-4 analogs as anti-cancer agents et al., 2007et al., 2014N-methylation, no significant difference is observed for the N1—N2—N3 bond angle [116.2 (1) and 115.9 (1)°, respectively, for (I)Selected geometric parameters are given in Tables 1N⋯O2 and N2—H2N⋯O3), forming ac plane. No significant hydrogen-bonding interactions were found in the structure of (II)Hydrogen bonding and the mode of packing of (I)s Table 3, which pA search of the 2014 release of the Cambridge Structural Database on unit-cell dimensions for (I)et al., 2014The title compounds were prepared according to a previously reported procedure  and 0.95 Å (Csp2H). Coordinates of the N-bound hydrogen were refined freely. Uiso(H) values were set to either 1.2Ueq or 1.5Ueq (RCH3) of the attached atom.Crystal data, data collection and structure refinement details are summarized in Table 4PLATON  were used to fix overlapping fragments. Restraints were used to maintain the integrity of ill-defined or disordered groups (SHELXL commands SAME and RIGU).Refinement progress was checked using In structure (II)10.1107/S1600536814023095/hg5414sup1.cifCrystal structure: contains datablock(s) global, I, II. DOI: 10.1107/S1600536814023095/hg5414Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S1600536814023095/hg5414IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S1600536814023095/hg5414Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S1600536814023095/hg5414IIsup5.cmlSupporting information file. DOI: 1030172, 1030173CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


R)-propane-1,2-diamine (Lpn) are described. One compound is a zigzag cyanide-bridged chain polymer in which the asymmetric unit consists of two independent chiral {[Cu(Lpn)2][Fe(CN)5(NO)]} units and two water molecules, while the second compound is a two-dimensional cyanide-bridged coordination polymer, in which the asymmetric unit consists of two chiral {[Cu(Lpn)2][Cr(CN)6]}− anions bridged by a chiral [Cu(Lpn)2]2+ cation and five water molecules.Two new chiral cyanide-bridged bimetallic coordination polymers involving the ligand ( catena-polycopper(II)]-μ-cyanido-κ2N:C-[tris(cyanido-κC)(nitroso-κN)iron(III)]-μ-cyanido-κ2C:N] monohydrate], {[Cu(Lpn)2][Fe(CN)5(NO)]·H2O}n, (I), and polydichromium(III)tricopper(II)] pentahydrate], {[Cu(Lpn)2]3[Cr(CN)6]2·5H2O}n, (II) , are new chiral cyanide-bridged bimetallic coordination polymers. The asymmetric unit of compound (I) is composed of two independent cation–anion units of {[Cu(Lpn)2][Fe(CN)5)(NO)]} and two water molecules. The FeIII atoms have distorted octahedral geometries, while the CuII atoms can be considered to be pentacoordinate. In the crystal, however, the units align to form zigzag cyanide-bridged chains propagating along [101]. Hence, the CuII atoms have distorted octahedral coordination spheres with extremely long semicoordination Cu—N(cyanido) bridging bonds. The chains are linked by O—H⋯N and N—H⋯N hydrogen bonds, forming two-dimensional networks parallel to (010), and the networks are linked via N—H⋯O and N—H⋯N hydrogen bonds, forming a three-dimensional framework. Compound (II) is a two-dimensional cyanide-bridged coordination polymer. The asymmetric unit is composed of two chiral {[Cu(Lpn)2][Cr(CN)6]}− anions bridged by a chiral [Cu(Lpn)2]2+ cation and five water molecules of crystallization. Both the CrIII atoms and the central CuII atom have distorted octahedral geometries. The coordination spheres of the outer CuII atoms of the asymmetric unit can be considered to be pentacoordinate. In the crystal, these units are bridged by long semicoordination Cu—N(cyanide) bridging bonds forming a two-dimensional network, hence these CuII atoms now have distorted octahedral geometries. The networks, which lie parallel to (10-1), are linked via O—H⋯O, O—H⋯N, N—H⋯O and N—H⋯N hydrogen bonds involving all five non-coordinating water molecules, the cyanide N atoms and the NH2 groups of the Lpn ligands, forming a three-dimensional framework.The title compounds,  Compound (I)2][Fe(CN)5NO]·H2O, synthesized using the racemic form of the same ligand propane-1,2-diamine 5)(NO)]2−·H2O. Atoms Fe1 and Fe2 have distorted octahedral geometries being coordinated by five C atoms from the cyanide ligands  and by one N atom, N2 and N12, respectively, from the nitrosyl group. The average Fe—N distance [1.657 (14) Å] is much shorter than the Fe—C distances, which are between 1.926 (5) and 1.954 (6) Å. These values are in good agreement with those reported for other polymeric structures involving nitroprusside 2][Cr(CN)6]}− anions bridged by a chiral [Cu(Lpn)2]2+ cation. There are also five water molecules of crystallization present. The coordination sphere of the central CuII atom, Cu3, can be described as elongated octahedral, generated by four N atoms of the Lpn ligands and two cyanide N atoms. The outer atoms Cu1 and Cu2 are pentacoordinate; atom Cu1 has a distorted square-pyramidal geometry with a τ value of 0.14 -propane-1,2-diamine –2.081 (15) Å), and the Cr—C≡N bond angles vary over a small range, 174.5 (13)–179.6 (12)°.The asymmetric unit of complex (II)i and Cu2—N3ii, respectively . This axial bonding results in distorted octahedral coordination spheres for the copper(II) atoms. The extremely long semi-coordination Cu—N bonds can be attributed to the co-existence of pseudo-Jahn–Teller elongation and electrostatic interactions in the infinite one dimensional chain. A similar geometry has been found in [CuIIL2][MII(CN)4]·2H2O  s Table 1, formingk Table 1.iii and Cu2—N13iv, are 2.698 (14) and 2.860 (14) Å, respectively . Thus, as for complex (I)6]3− anions binds to three adjacent nickel atoms rk Fig. 6.R)-propane-1,2-diamine, viz. catena-copper(II)nickel(II) hemihydrate clathrate] tetraaquapentadecacyanidohexakishexacopper(II)tetratungsten(V) hydrate] -propane-1,2-diamine, viz. catena-chromiummanganese(II) (S)-1,2-diaminopropane] tetraaquapentadecacyanidohexakishexacopper(II)tetratungsten(V) hydrate] : (R)-propane-1,2-diamine (Lpn) was synthesized according to a reported procedure  in 0.8 ml of water was added under an argon atmosphere. A glass tube  was charged with this solution, and a mixture of methanol and H2O  was gently added as a buffer layer. A solution of Na2[Fe(CN)5NO] (0.07 mmol) in methanol/H2O  was then added carefully as a third layer under an argon atmosphere, and then the tube was sealed. Crystals of complex (I)11H22N10CuFeO2, found: C, 29.86; H, 5.07; N, 31.93%. calc: C, 29.64; H, 4.97 N, 31.42%.Compound (II): Dark-blue block-like crystals of compound (II)3[Cr(CN)6] instead of Na2[Fe(CN)5NO]. Elemental analysis for C30H70N24Cu3Cr2O5, found: C, 30.87; H, 5.80; N, 28.41%. calc: C, 31.56; H, 6.18 N, 29.44%.Uiso(H) = 1.5Ueq(O). The N- and C-bound H atoms were included in calculated positions and treated as riding atoms: N—H = 0.89 Å, C—H = 0.98–1.00 Å with Uiso(H) = 1.5Ueq(C) for methyl H atoms and 1.2Ueq for other H atoms. It was not possible to locate the H atoms of the disordered water molecule, OW5A/OW5B, in compound (II)Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989015005253/hb7374sup1.cifCrystal structure: contains datablock(s) I, II, Global. DOI: 10.1107/S2056989015005253/hb7374Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989015005253/hb7374IIsup3.hklStructure factors: contains datablock(s) II. DOI: 691330, 691331CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The crystal structure features C—H⋯O hydrogen bonds and π–π stacking interactions. Geometric parameters from quantum-chemical calculations are in good agreement with experimental X-ray diffraction results.The title compound, C 11H6Cl2N2O2S, is a Schiff base that incorporates an N-bound 2,4-dichlorophenyl and a C-bound 5-nitrothiophene ring. The molecule is approximately planar, the maximum deviation from the mean plane being 0.233 (4) Å for the C=N N atom. The dihedral angle between the benzene and thiophene rings is 9.7 (2)°. The C=N double bond has an E configuration. The crystal structure features C—H⋯O hydrogen bonds,forming sheets parallel to (10-1), and π–π stacking interactions between symmetry-related thiophene and benzene rings, in which the distance between adjacent ring centroids is 3.707 (4) Å, forming a three-dimensional supramolecular structure. Geometric parameters from quantum-chemical calculations are in good agreement with experimental X-ray diffraction results.The title compound, C The reaction mixture was stirred for 1h under reflux. Crystals suitable for X-ray analysis were obtained from a solution in ethanol by slow evaporation .The compound 2,4-dichloro-−1): 3102.59 (C—H), 1602.71 (C=N), 1503.00 (NO2), 1231.00 , 1192.05 , 1039.60 , 1124.10 , 957.96 , 787.73 IR (KBr/cmUiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016011816/pk2586sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989016011816/pk2586Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989016011816/pk2586Isup3.cmlSupporting information file. DOI: 1494850CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The structures of two substituted coumarin derivatives are reported, one with acetate and the other with pent-4-ynoate substituents. 14H12O6, (1), and C20H16O6, (2), one with acetate and the other with pent-4-ynoate substituents, both the coumarin rings are almost planar. In (1), both acetate substituents are significantly rotated out of the coumarin plane to minimize steric repulsions. One acetate substituent is disordered over two equivalent conformations, with occupancies of 0.755 (17) and 0.245 (17). In (2), there are two pent-4-ynoate substituents, the C≡C group of one being disordered over two positions with occupancies of 0.55 (2) and 0.45 (2). One of the pent-4-ynoate substituents is in an extended conformation, while the other is in a bent conformation. In this derivative, the planar part of both pent-4-ynoate substituents deviate from the coumarin plane. The packing of (1) is dominated by π–π stacking involving the coumarin rings and weak C—H⋯O contacts link the parallel stacks in the [101] direction. In contrast, in (2) the packing is dominated by R22(24) hydrogen bonds, involving the acidic sp H atom and the oxo O atom, which link the molecules into centrosymmetric dimers. The bent conformation of one of the pent-4-ynoate substituents prevents the coumarin rings from engaging in π–π stacking.In the structures of the two title coumarin derivatives, C They are used in the pharmaceutical industry as precursor reagents in the synthesis of a number of synthetic anticoagulant pharmaceuticals   has been shown to inhibit GST activity in a spectroscopic assay   as a chemical reporter of calreticulin’s acyltransferase capabilities , C14H12O6 (1) and C20H16O6 (2), which are to be used as chemical reporters of calreticulin’s acyltransferase capabilities. These two compounds will be first discussed individually and then compared.This paper reports the structures of two derivatives of coumarin   and 0.245 (17). The metrical parameters of both the coumarin ring and acetate substituents are in the normal ranges.In the structure of (1) Fig. 1, the cou2) , C15B≡C16B = 1.169 (11) and C20≡C21 = 1.177 (3) Å].In (2) Fig. 2, the C≡C1) is dominated by π–π stacking involving the coumarin rings . This can be observed in Fig. 3A as well as C6 and O2, C15A and O2  which link the parallel stacks in the [101] direction.The packing of (1), for (2) the packing .In contrast to . 4-Methyl-2-oxo-2H-chromene-7,8-diyl diacetate (DAMC) was synthesized using a previously reported procedure . 0.5 mmol 7,8-dihydroxy-4-methyl coumarin, DHMC , 2.5 equivalents pentynoic anhydride .1H NMR : δ 7.51–7.49 , δ 7.20–7.17 , δ 6.29 , δ 3.01–3.08 , δ 2.89–2.84 , δ 2.61–2.70 , δ 2.44 , δ 2.09–2.11 .Spectroscopic analysis: 1), the H atoms were positioned geometrically and refined as riding: C—H = 0.95–0.98 Å with isoU(H) = 1.5Ueq(C) for methyl H atoms and = 1.2eqU(C) for other H atoms. One acetate substituent is disordered over two equivalent conformations with occupancies of 0.755 (17) and 0.245 (17).Crystal data, data collection and structure refinement details are summarized in Table 32), the H atoms were positioned geometrically and refined as riding: C—H = 0.95–0.99 Å with Uiso(H) = 1.5Ueq(C) for methyl H atoms and = 1.2eqU(C) for other H atoms. The C≡C group of one of the pent-4-ynoate substituents is disordered over two positions with occupancies of 0.55 (2) and 0.45 (2).In the refinement for  1, 2. DOI: 10.1107/S2056989016005892/hg54711sup2.hklStructure factors: contains datablock(s) 1. DOI: 10.1107/S2056989016005892/hg54712sup3.hklStructure factors: contains datablock(s) 2. DOI: Click here for additional data file.10.1107/S2056989016005892/hg54711sup4.cmlSupporting information file. DOI: 1473151, 1473150CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Their structures were established by extensive spectroscopic analysis. The new alkaloids were evaluated for their cytotoxicity in vitro against five human cancer cell lines.Five new hybrid monoterpenoid indole alkaloids bearing an unusual 2,2-dimethyl-4-oxopiperidin-6-yl moiety, namely rauvotetraphyllines F–H (The online version of this article (doi:10.1007/s13659-015-0074-2) contains supplementary material, which is available to authorized users. Rauvolfia genus of the Apocynaceae family, comprising about 60 species, is mainly distributed in America, Africa, Asia, and Oceania [http://www.chemlib.cn] objective to assemble a large-scale natural product library valuable in the discovery of new drug leads from nature, previous chemical study on the ethanolic extract of Rauvolfia tetraphylla had resulted in the isolation of five new indole alkaloids, rauvotetraphyllines A–E , an ethylidene group , and a glucose unit . Comparison of its 13C NMR data with those of rauvotetraphylline B  determined as a 2,2-dimethyl-4-oxopiperidin-6-yl moiety by HMBC correlations , which could also be explained by that the molecule favors the conformation in which larger substituents are in the anti position. This was further supported by ROESY correlations of H-17↔Me-18. The R* configuration of C-17 was implied by ROESY correlations of H-6β↔H-22β and Me-18↔Me-26  in 1 was relatively deshielded compared to that of H-15 (δH 2.79) in its 17-epimer 2 (vide infra). This is attributed to paramagnetic deshielding caused by the proximity of the NH nitrogen atom to H-15 , showing a quasi-molecular ion peak at m/z 568.3031 . The 1H and 13C NMR spectra of 2 , suggesting the molecular formula C25H31N3O. The NMR data . The 13C NMR data (Table δC 54.8 (d), 47.2 (t), 210.4 (s), 55.4 (t), 54.5 (s), 25.2 (q), 32.2 (q)] on the basis of HMBC correlations  (Table 4 caused a marked downfield shift of H-15 (Δ = 0.30 ppm) . The 1H and 13C NMR spectra of 5 (Tables 4 except for the upfield shift of H-15 (Δ = −0.30 ppm) and downfield shifts of Me-18 (Δ = 0.15 ppm) and H-19 (Δ = 0.11 ppm) in the 1H NMR spectrum. This can be rationalized in terms of paramagnetic deshielding experienced by Me-18 and H-19 in 5 and H-15 in 4 .All of the isolated compounds were evaluated for their in vitro growth inhibitory effects against five human tumor cell lines  with cisplatin and taxol serving as positive controls by the MTT method . RegrettOptical rotations were measured on a Jasco P-1020 automatic digital polarimeter. UV data were obtained from online HPLC analysis. IR spectra (KBr) were obtained on a Bruker Tensor-27 infrared spectrophotometer. NMR spectra were acquired with a Bruker DRX-500 or Bruker Avance III 600 instrument  with deuterated solvent signals used as internal standards. ESI-MS (including HR-ESI-MS) were measured on API QSTAR Pulsar i mass spectrometers. Silica gel  and Sephadex LH-20  were used for column chromatography. Medium pressure liquid chromatography (MPLC) was performed on a Büchi Sepacore System equipping with pump manager C-615, pump modules C-605, and fraction collector C-660 , and columns packed with Chromatorex C-18 . Fractions were monitored by TLC  in combination with reversed-phase HPLC .Rauvolfia tetraphylla were collected in Xiaomenglun of Yunnan Province, China, in June 2010 and identified by Mr. Yu Chen of Kunming Institute of Botany, Chinese Academy of Sciences. The voucher specimen (No. BBP0234020RT) was deposited at BioBioPha Co., Ltd.The aerial parts of R. tetraphylla (7.5 kg) were extracted three times with EtOH-H2O  at room temperature, and the solvent was removed under reduced pressure to give crude extract (ca. 400 g), which was then fractionated by silica gel column chromatography (CC) eluted with a gradient solvent system (containing 0.2 % ammonia) of petroleum ether-acetone and then MeOH to yield seven fractions A–G. Fraction D, eluted by acetone, was separated on silica gel CC  to give three subfractions D1–D3. Fraction D1 was purified further by silica gel CC  and then prep. TLC  to afford 4 (9 mg) and 5 (6 mg). Fraction D2 was separated by silica gel CC  and then prep. TLC  to afford 3 (10 mg). Fraction G, eluted by MeOH, was separated further by silica gel CC , repeated MPLC (40 → 45 % MeOH in H2O), and then Sephadex LH-20 (MeOH) to afford 1 (72 mg) and 2 (26 mg).The air-dried and powdered aerial parts of D15 +10.1 ; UV (MeOH) λmax: 225, 281, 290 (sh) nm; IR (KBr) νmax 3404, 2961, 2921, 1700, 1470, 1453, 1384, 1338, 1320, 1302, 1076, 1031, 745 cm−1; 1H and 13C NMR data see Tables m/z 568 [M + H]+; HR-ESI-MS (pos.): m/z 568.3025 .White amorphous powder; [α]D16 +14.9 ; UV (MeOH) λmax: 225, 281, 290 (sh) nm; IR (KBr) νmax 3396, 2962, 2923, 1699, 1626, 1471, 1451, 1384, 1337, 1300, 1075, 1030, 746 cm−1; 1H and 13C NMR data see Tables m/z 568 [M + H]+; HR-ESI-MS (pos.): m/z 568.3031 .White amorphous powder; [α]D14 −22.4 ; UV (MeOH) λmax: 225, 280, 290 (sh) nm; IR (KBr) νmax 3421, 3143, 3057, 2961, 2925, 2855, 1705, 1626, 1473, 1301, 1240, 1169, 741 cm−1; 1H and 13C NMR data see Tables m/z 390 [M + H]+; HR-ESI-MS (pos.): m/z 390.2538 .White amorphous powder; [α]D14 +9.9 ; UV (MeOH) λmax: 220, 262 nm; IR (KBr) νmax 3433, 2965, 2934, 1741, 1707, 1592, 1453, 1380, 1364, 1295, 1033, 773, 753 cm−1; 1H and 13C NMR data see Tables m/z 448 [M + H]+; HR-ESI-MS (pos.): m/z 448.2613 .White amorphous powder; [α]+9.9 c 0.0, CHCl3;D15 +47.3 ; UV (MeOH) λmax: 220, 263 nm; IR (KBr) νmax 3433, 2965, 2936, 1742, 1707, 1592, 1453, 1376, 1268, 1230, 1177, 1032, 774, 753 cm−1; 1H and 13C NMR data see Tables m/z 448 [M + H]+; HR-ESI-MS (pos.): m/z 448.2602 .White amorphous powder; [α]47.3 c 0.0, CHCl3;50 values were calculated by Reed and Muench’s method  method , by use s method .Supplementary material 1 (DOCX 2343 kb)


In the title compound, the 1,3-dioxane ring is in a chair-like conformation, while the fused oxolane ring adopts an envelope form. In the crystal, classical O—H⋯O and N—H⋯O hydrogen bonds link the molecules into a sheet structure. 12H19NO6, the six-membered 1,3-dioxane ring adopts a chair-like conformation. The seat of this chair, containing two O atoms, is essentially planar, with a maximum deviation of 0.0021 (12) Å. The five-membered oxolane ring cis-fused to the 1,3-dioxane ring adopts an envelope form. The bridgehead C atom at the flap, which is bonded to the tetrasubstituted C atom of the oxolane ring, deviates from the mean plane of other ring atoms by 0.539 (4) Å. In the crystal, classical O—H⋯O and N—H⋯O hydrogen bonds link the molecules into a sheet structure enclosing an R44(24) graph-set motif. Weak intermolecular C—H⋯O interactions support the sheet formation.In the title compound, C S,3R,4R,5S,E)-2-amino-3,4,5-trihydroxy-2-methyl-14-oxoicos-6-enoic acid] was isolated from the fermentation broth of Paecilomyces variotii by Horn et al.  Å, θ = 169.6 (3)°, φ = 116.8 (16)°, Q(2) = 0.090 (3) Å and Q(3) = −0.489 (3) Å. The seat of this chair (C1/O2/O4/C5) is essentially planar with a maximum deviation of 0.0021 (12) Å for O4, and atoms C6 and C3, positioned at the headrest and the footrest, respectively, deviate from the mean plane of the seat by 0.524 (4) and −0.646 (3) Å. The equatorially oriented C5—C15 and C3—C17 bonds make angles with the normal of the Cremer & Pople plane being 63.41 (18) and 63.35 (18)°, respectively, while the C1—C9 bond is a little tilted from the ideal equatorial position with an angle of 50.50 (17)° due to the ring-fusion system. The oxolane ring (C1/C6/O7/C8/C9), which is cis-fused to the 1,3-dioxane ring, adopts an envelope form with puckering parameters of Q(2) = 0.345 (3) Å and φ(2) = 254.7 (4)°. The bridgehead atom C1 deviates from the mean plane of the other four ring atoms by 0.539 (4) Å.The molecular structure of the title compound is shown in Fig. 1i; Table 1c axis, with a C(10) graph-set motif  graph-set motif , are registered ; one compound ; and one compound . The conformations of the bicyclic systems in these seven structures are similar to those in the title compound: the 1,3-dioxane rings adopt chair-like forms, and the cis-fused oxolane rings adopt envelope forms with bridgehead C-1 position at the flap.In the Cambridge Structural Database . HRMS (ESI) m/z calculated for C12H19NO6Na+ [M + Na]+: 296.1110; found: 296.1104.The title compound was afforded in the total synthesis of sphingofungin F from a Uiso(H) = 1.2Ueq(C) or 1.5Ueq(methyl C). The hydroxy H atom was placed guided by difference maps, with O—H = 0.84 Å and with Uiso(H) = 1.5Ueq(O). The amide H atom was also placed guided by difference maps, with N—H = 0.88 Å and with Uiso(H) = 1.2Ueq(N).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016006800/is5451sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989016006800/is5451Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989016006800/is5451Isup3.cmlSupporting information file. DOI: 1475848CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Yarrowia lipolytica to biotransform acetophenone and its derivatives has been studied. Thirteen of these strains were derived from a wild-type strain Y. lipolytica A-101; six had the invertase gene (SUC2) from Saccharomyces cerevisiae integrated into their genome, as well as the damaged or undamaged gene encoding orotidine-5′-phosphate decarboxylase (URA3), three had integrated the damaged URA3 gene into their genome and three were UV acetate-negative mutants, not able to growth on acetate as the sole carbon source. The other tested strains included two wild strains, A-101 and PMR-1, and an adenine auxotroph ATCC 32-338A. All strains were capable of reducing acetophenone to the R-alcohol in high enantiomeric excess (80–89 %). In all of the cultures tested, reversibility of the reduction was observed, which led to an increase in the enantiomeric excess. nantioselective reduction of the acetophenone halogen derivatives revealed that the nature and location of the halogen atom had a significant influence on the enantioselectivity of the reduction. In the culture of ATCC 32-338A, after a 3-day biotransformation of 2,4′-dibromoacetophenone the enantiopure R-alcohol was obtained at a rate of 100 % of substrate conversion. In conclusion, using these invertase-containing strains or uracyl auxotrophs provided no additional benefit in terms of biotransformation capacity over the parental strain.The ability of 16 strains of  Yarrowia lipolytica is a dimorphic yeast that forms cream-colored, wrinkled, rough or smooth surfaced colonies . This biomass is a valuable source of vitamins, exogenous amino acids and unsaturated fatty acids for animal feed (fodder yeasts) , considerably high contents of G+C (49 %), the presence of 1,119 introns, chromosomal features of atypical structure (centromers and replication origins), a large number of tRNA genes (510), dispersed 5S rRNA genes (109 copies) and a novel organization of RNA genes  have been obtained by Nicaud et al.  source of carbon, i.e. sucrose. Sucrose undergoes hydrolysis to glucose and fructose in the reaction catalyzed by an invertase, the enzyme encoded by the d et al. , Försterd et al.  and Walcd et al. . Due to Pyrococcus furiosus (PFADH) effectively catalyzes the reduction of various substituted α-chloroacetophenones to form the (R)-enantiomers of the corresponding chlorohydrins with an excellent enantiomeric purity -(−)-formoterol, (R)-(−)-isoproterenol, (R)-(−)-salmeterol, (R)-(−)-denopamine, d-sotalol and (R)-(−)-clorprenalin  and others by non-homologous gene integration (clones Suc+Ura+ and suc−ura−). One of the clones obtained had two copies of the invertase gene , 2,4′-dichloroacetophenone (3), 2,2’,4′-trichloroacetophenone (4), 2-chloro-4′-fluoroacetophenone (5), 2-bromo-4′-chloroacetophenone (6) and 2,4′-dibromoacetophenone (7); all were purchased from Sigma-Aldrich . All racemic alcohols  were prepared by reducing the corresponding ketone with sodium borohydride in methanol.The substrates used in this study are acetophenone  A-101 , (2) A-101-1.31 (acetate-negative UV mutant of A-101), (3) A-101-1.31-K1 (smooth segregant of acetate-negative UV mutant of A-101-1.31), (4) A-101-1.22 (acetate-negative UV mutant of A-101), (5) PMR-1 (isolated from soil contaminated by fuel) and (6) ATCC 32-338A (adenine auxotroph purchased from the American Type Culture Collection). Also tested were transformants of A-101: (7) A18 (Suc+ura−), (8) B54-6 (Suc+ura−), (9) B55-3 (Suc+ura−), (10) B14-6 (Suc+ura−), (11) B57-4 (Suc+ura−), (12) B1-1 (suc−ura−), (13) A50 (suc−ura−), (14) B9-2 (suc−ura−), (15) Klon1 (Suc+Ura+) and (16) B56-5 (Suc+Ura+).The strains of SUC2 invertase gene from S. cerevisaie, flanked by two ura3 sequence fragments, homologous to the native URA3 gene, encoding orotidine-5′-phosphate decarboxylase from Y. lipolytica. Using the transformation procedure described by Walczak and Robak (+ura− phenotype. The second group (#12–14) contained suc−ura− strains, in which the cassette was not fully exchanged and, therefore, these strains did not gain the ability to grow on sucrose and become uracil auxotrophs. The third group  were strains in which the DNA hybridization took place at a location other than the target location, giving the strains the Suc+Ura+ phenotype. Y. lipolytica transformant B56-5 has two invertase encoding genes , peptone (20 g/l) and glucose (20 g/l) and was sterilized in an autoclave at 121 °C for 20 min. Thiamine (0.3 mg/l) and uracil (20 mg/l) were added after sterilization.4)H2PO4 (5 g/l), KH2PO4 (2.5 g/l), MgSO4 ⋅ 7H2O (1 g/l) and 1 ml/l of mineral salts  was transferred into 250-ml Erlenmeyer flasks containing 50 ml of previously sterilized phosphate buffer . After transferring the biomass to the buffer, 10 mg of a substrate dissolved in 1 ml of acetone was added. After 1, 3, 6 or 9 days of incubation at room temperature and continuous shaking (150 rpm at 25 °C), 10-ml samples of the transformation mixture were removed. All procedure were carried out under aseptic conditions in microbiological safety cabinets  or laminar flow clean benches . The samples were extracted with 10 ml of CHCl3, dried over MgSO4, concentrated in vacuo and analyzed by GC. All the experiments were repeated three times.For the analytical tests the cultures were centrifuged under aseptic conditions  and a hexane/acetone mixture  as the eluent.For the preparative-scale biotransformation the substrates (200 mg of each of compounds dissolved in 2 ml of acetone) were added to the cultures cultivated in 2,000-ml flasks (each containing 500 ml of the culture). The biotransformation mixtures were incubated under the same conditions  for 48 h. The mixtures were then extracted three times with 300 ml of CHCl4)2 and 2 % H3[P(Mo3O10)4] in 10 % H2SO4. GC analysis was performed using a Hewlett-Packard 5890A  GC instrument fitted with a flame ionization detector (FID). A chiral capillary column [Chrompack WCOT Fused Silica (CP Chirasil-DEX CB)], 25 m × 0.25 mm × 0.25 μm,  was used to determine the composition of the unreacted substrates and product mixtures. Nuclear magnetic resonance (NMR) spectra were recorded with a DRX 500-MHz Bruker spectrometer  and measured in CDCl3. Optical rotations were measured with an Autopol IV automatic polarimeter .The course of both the biotransformation and oxidation processes were followed using thin layer chromatography (TLC), and the composition of each product mixture was determined by capillary gas chromatography (CGC). Analytical TLC was carried out on silica gel G (Merck) with different developing systems. Compounds were detected by spraying the plates with 1 % Ce(SO2): 90 °C/1 min, 3 °C min−1 to 105 °C, 30 °C min−1 to 200 °C/5 min, Rt (R) 4.97 min, Rt (S) 5.42 min.1-phenylethan-1-ol (8): 142 °C/1 min, 2 °C min−1 to 165 °C, 30 °C min−1 to 200 °C/5 min, Rt (S) 10.05 min, Rt (R) 10.69 min.2,4′-dichloro-1-phenylethan-1-ol (9): 165 °C/1 min, 2 °C min−1 to 178 °C, 20 °C min−1 to 200 °C/5 min, Rt (R) 6.84 min, Rt (S) 7.42 min.2,2′,4′-trichloro-1-phenylethan-1-ol (10): 140 °C/1 min, 2 °C min−1 to 150 °C, 20 °C min−1 to 200 °C/5 min, Rt (R) 4.24 min, Rt (S) 4.64 min.2-chloro-4′-fluoro-1-phenylethan-1-ol (11): 160 °C/1 min, 2 °C min−1 to 178 °C, 20 °C min−1 to 200 °C/5 min, Rt (R) 9.49 min, Rt (S) 9.93 min.2-bromo-4′-chloro-1-phenylethan-1-ol (12): 150 °C/1 min, 2 °C min−1 to 183 °C, 20 °C min−1 to 200 °C/5 min, Rt (R) 14.20 min, Rt (S) 14.73 min.2,4′-dibromo-1-phenylethan-1-ol (The GC conditions were an injector temperature of 200 °C and a detector temperature of 250 °C. The temperature programs for enantiomeric resolution of particular compounds are as follows:1H NMR spectra: R)-1-phenylethan-1-ol (2) [α]D20 = +38.6° (c = 3.0 CHCl3) (79 % ee); {Omori et al. }; 1H NMR (CDCl3) δ 1.47 , 2.79 , 4.79 , and 7.14-7.21 .-2,4’-dichloro-1-phenylethan-1-ol (8) [α]D20 = +27.2° (c = 3.45 CHCl3) (86 % ee); {Wei et al.  δ 2.67 , 3.60 , 3.71 , 4.88 , and 7.26-7.39 .-2,2’,4’-trichloro-1-phenylethan-1-ol (9) [α]D20 = −26.1° (c = 1.0 CHCl3) (68 % ee);  δ 2.82 , 3.52 , 3.87 , 5.26 , 7.31 , 7.38 , and 7.58 .-2-chloro-4’-fluoro-1-phenylethan-1-ol (10) [α]D20 = +34.7° (c = 4.75 CHCl3) (80 % ee); {Nieduzak and Margolin  δ 2.73 , 3.61 , 3.71 , 4.88 , 7.04-7.09 , and 7.32-7.38 .-2-bromo-4’-chloro-1-phenylethan-1-ol (11) [α]D20 = +14.3° (c = 1.2 CHCl3) (42 % ee); {Basavaiah et al.  δ 2.69 , 3.50 , 3.61 , 4.91 , and 7.20-7.28 .-2,4’-dibromo-1-phenylethan-1-ol (12) [α]D20 = −36.1° (c = 1.8 CHCl3) (100 % ee); {Basavaiah et al. α]D20 = +33.8°, 96 % ee}; 1H NMR (CDCl3) δ 2.75 , 3.49 , 3.60 , 4.88 , 7.24-7.28 , and 7.47-7.54 . to reduce acetophenone (1) was assessed. The results showed that genetic modifications only slightly influenced the yield of the reduction and the enantiomeric excess of the obtained alcohol (2) (Table R)-1-phenylethan-1-ol (2) with high enantiomeric excess. Due to the reversibility of the acetophenone reduction reaction in the cultures of the selected strains -alcohol (2) was achieved as many as 9 days after the biotransformation . Our observations revealed that the longer the reaction time, the higher the predominance of this enantiomer, possibly explained by the oxidation of S-alcohol (2) back to the ketone  was noted. However, unlike the other tested strains, ATCC 32-338A produced predominantly (S)-l-phenylethan-1-ol (2), which is in accordance with the Prelog’s rule  is an equilibrium reaction. Therefore, a racemic mixture of 1-phenylethan-1-ol (2) was subjected to biotransformation. We observed a distinctive decrease in (S)-1-phenylethan-1-ol amount (2) and an increase in the R-enantiomer content of almost all tested strains after 6 (data not shown) and 9 days . In the cultures of these strains R-alcohol 2 was obtained with the highest enantiomeric excess (Table The aim of the next experiment was to confirm that the reduction of acetophenone (han-1-ol  was subjhan-1-ol  was subjss Table .Table 2BY. lipolytica A-101 and the strains B56-5 (Suc+Ura+), A18 (Suc+ura−) and A50 (suc−ura−).On the basis of these results we selected the following strains for further experiments: parental strain Y. lipolytica to use sucrose as a carbon source, we cultivation all strains on a minimal growth medium MTM containing sucrose. The biotransformations were performed according to the procedure described in the section Y. lipolytica A18 was considerably inhibited (number of cells 3.2 × 107 cells/ml) and, therefore, the results of the reduction were poorer . The strain Y. lipolytica B56-5 grew better and provided a much higher yield from the reduction (data not shown). The enantiomeric excess was comparable to the results obtained with yeast biomass growing on the rich culture media (YPG).Due to the non-typical characteristic ability of Y. lipolytica A-101 to reduce five halogen derivatives of acetophenone (1): 2,4′-dichloroacetophenone (3), 2,2′,4′-trichloroacetophenone (4), 2-chloro-4′-fluoroacetophenone (5), 2-bromo-4′-chloroacetophenone (6), and 2,4′-dibromoacetophenone (7)  for all of the substrates used in this experiment. As with acetophenone (1), the majority of the substrateswere reduced to the anti-Prelog alcohol. Only for the substrate with the third chlorine atom at C-2′ (4) did we observe the opposite stereospecificity  gave much lower enantiomeric excess   Fig. .Table 3Y. lipolytica demonstrated different abilities to reduce 2,4′-dichloroacetophenone (3). Y. lipolytica B56-5 and A18, which possess the SUC2 gene, showed properties similar to the parental strain and reduced the substrate with an almost 100 % yield and high (up to 70 % obtained after 3 days of transformation) enantiomeric excesses of the resulting S-alcohol (8) (Table Y. lipolytica A-101 A50 (data not shown). In this case, the strain ATCC 32-338A also performed the reduction with the opposite enantiospecificity, producing a poor yied of R-alcohol (8) (not exceeding 30 %) (Table The tested strains of ophenone . Y. lipoophenone . Y. lipo4) explains why this compound is reduced to alcohol (9) with the opposite enantioselectivity, compared to the previous substrates. In this product, the R-enantiomer of alcohol (9) predominated. The results obtained are satisfactory; however, the conversion of the substrate is less efficient than that with the parental strain. For strain ATCC 32-338A, the conversion was much lower than that of the other tested strains, and the enantiomeric excess was also very low (the S enantiomer predominated) . The highest reduction yield, comparable to that of the parental strain, was observed for the clone with the Suc+Ura+ B56-5 phenotype. Strains A18 and A50 showed a lower substrate conversion . The parental strain and all clones gave predominantly the S-enantiomer of alcohol (10). The highest enantiomeric excess was noted for the parental strain (80 %) and clone B56-5 (68 %). A relatively lower enantiomeric excess was observed for strains A18 (54 %) and A50 (48 %). The R-enantiomer of alcohol (10) was obtained in the culture of ATCC 32-338A , but at a low substarte conversion (12 %)  by the mutants of Y. lipolytica A-101. Due to changes in the composition of the reaction mixtures during incubation with the tested microorganisms, large differences among the results were noticed. The yields were high and the enantiomeric excesses were low. The parental strain produced S-alcohol (11) with an ee of 80 %, whereas strain A50, after a 1-day biotransformation, produced the R-enantiomer at an ee of 60 %. For the rest of the tested strains almost equimolar amounts of both enantiomers of (11) were obtained after 1 day of biotransformation. The most effective reduction of substrate (6) was achieved in the culture of strain ATCC 32-338A  in the case of the biotransformation of 2-bromo-4′-chloroacetophenone  with a 100 % conversion rate (after 3 days of biotransformation). Among the strains tested, the highest enantiomeric excess (22 %) of the R-alcohol was observed in the culture of strain Y. lipolytica A50, after 3 days of biotransformation , was used as a model substrate. All of the mutants tested were capable of the reduction of acetophenone (1) to the R-alcohol with high enantiomeric excess. In all of the tested cultures reversibility of the reduction was observed, leading to an increase in enantiomeric excess. The reversibility of the reduction was studied by using racemic phenylethanol as a substrate for biotransformation by the selected strains. The result was a significant decrease in the amount of S-phenylethanol, leading to the R-alcohol with an ee of >50 %.In order to check the ability of the tested strains to perform a reduction, a prochiral mixed aliphatic–aromatic ketone, i.e. acetophenone  in the culture of ATCC 32-338A strain resulted in the production of the enantiopure R-alcohol (12) at a substrate conversion rate of 100 %. Such a good result with respect to the enantiomeric excess was unexpected because the other halogen derivatives were reduced by this strain much less effectively. This is a also notable result because this biotransformation gave no side products at all. In contrast, the production of additional side products have been observed in transformations of this substrate in cultures of other microorganisms (Rocha et al. Enantioselective reduction of acetophenone halogen derivatives was also performed. We observed that the nature and location of the halogen atom exerted a significant influence on the enantioselectivity of the reduction. A 3-day transformation of 2,4′-dibromoacetophenone (Y. lipolytica contain less selective alcohol dehydrogenases than some other biocatalysts catalyzing the enantioselective reduction of acetophenone and its halogen derivatives (Lin et al. R-alcohol. However, the aim of our study, apart from presenting the capability of selected strains to perform the effective reduction, was to show differences in biocatalytic properties of strains within the same species. Moreover, we focused our attention to the reversibility of the reduction which takes place during biotransformation of the tested substrates in the cultures of Yarrowia lipolytica.Despite the fact that the strains of