Proteoliposome-based capillary electrophoresis for screening membrane protein inhibitors

A method for screening of monoamine oxidase (MAO) inhibitor was carried out using capillary electrophoresis (CE) based on the interaction of MAO and its substrate kynuramine (Kyn). Bioactive proteoliposome was reconstituted by liposome and MAO and then was applied as the pseudostationary phase (PSP) of CE to mimic the interaction between the enzyme and its substrate. N-prolmrgyl-R-2-heptylamine (R-2-HPA) and rasagiline [N-propargyl-1-(R)-aminoindan], which are two kinds of MAO inhibitors, were added into the running buffers containing proteoliposome. The results showed that the relative migration time ratio (RMTR × 10(-1)) values of Kyn were enhanced from 8.88 to 9.31 with an increase of the concentrations of rasagiline from 10(-6) to 1 mM. However, the RMTR values of Kyn were enhanced from 8.83 to 9.14 with an increase of the concentrations of R-2-HPA from 10(-6) to 1 mM. The RMTR value of Kyn in the presence of rasagiline was larger than that in the presence of R-2-HPA when rasagiline and R-2-HPA were at the same concentration. The results indicated that the interaction between Kyn and MAO was weakened with the increase of the inhibitors. In addition, the results of offline incubation showed that the inhibitions of rasagiline were 100.0, 72.1, 51.8 and 5.4% at the concentration of 1, 10(-2), 10(-4) and 10(-6) mM; moreover, the inhibitions of R-2-HPA were 70.0, 44.9, 4.1 and 0.9% at the concentrations of 1, 10(-2), 10(-4) and 10(-6) mM. The inhibition efficiency of rasagiline was stronger than that of R-2-HPA at the same concentration. Additionally, the interaction between Kyn and liposome was also investigated. This newly developed method might provide a potential tool for screening MAO inhibitor.     

Diels-Alder reactions between dienamides and quinones: stereochemistry of the cycloadditions and cytotoxic activity of the adducts

Tetrahydronaphthoquinones and tetrahydroanthraquinones bearing an amido group have been prepared by Diels-Alder reactions between (E)-1-(N-carbobenzyloxyamino)-1,3-butadiene (2) or (E)-1-(N-benzoyl-N-benzylamino)-1,3-butadiene (5) and benzoquinone or 5-substituted naphthoquinones. The stereochemistry of the cycloadditions was investigated. A high regioselectivity was observed in the reaction of the diene carbamate 2 with 5-methoxy and 5-acetoxy naphthoquinones. This latter gave the unexpected 1,8-regioisomer 3d. The cycloadditions of the dienamide 5 with naphthoquinones 1 (R = OH, OMe, OAc) are regiospecific. Assignment of the structure of the tetrahydroanthraquinone 6b is in good agreement with the known directing effect of the 5-hydroxy group of juglone 1b in analogous Diels-Alder reactions. With 5-methoxy and 5-acetoxy naphthoquinones, the opposite regiochemistry observed is consistent with the electron-donating influence of the methoxy or acetoxy group, making the C-3 carbon atom more electron deficient. Aromatization of the adducts 6b and 7c was accompanied by an unusual elimination of the amido moiety. Thus, 1-hydroxy and 1-methoxy anthraquinones were obtained. Reactions of the dienes 2 and 5 with benzoquinone gave the tetrahydronaphthoquinones 9 and 10 with an endo stereospecificity. Oxidation of 9 by activated manganese dioxide gave the naphthoquinone 11. These compounds were submitted to in vitro cytotoxic assays towards murine L 1210 leukemia cells and clonogenic human tumor cell line MDA-MB 231. The naphthoquinone derivatives 9, 10 and 11 had significant activities with IC50 less than or equal to 0.4 microgram/ml towards these two tumor cell systems.     

Phenolic constituents of licorice. III. Structures of glicoricone and licofuranone, and inhibitory effects of licorice constituents on monoamine oxidase

Two new phenolic compounds, glicoricone (3) and licofuranone (4), were isolated from a species of licorice brought from the northwestern region of China, and their structures were assigned. Among the twelve licorice constituents examined for the inhibition of monoamine oxidase (MAO), six compounds, 3, 4, genistein (6), licopyranocoumarin (7), licocoumarone (14) and glycyrrhisoflavone (15), inhibited the enzyme with the IC50 (concentration required for 50% inhibition of the enzyme activity) values of 6.0 x 10(-5)-1.4 x 10(-4) M. Glycyrrhizin (1) also inhibited MAO with the IC50 value of 1.6 x 10(-4) M.     

High molar mass ethene/1‐olefin copolymers synthesized with acenaphthyl substituted metallocene catalysts

The influence of ligand structure on copolymerization properties of metallocene catalysts was elucidated with three C₁‐symmetric methylalumoxane (MAO) activated zirconocene dichlorides, ethylene(1‐(7, 9)‐diphenylcyclopenta‐[a]‐acenaphthadienyl‐2‐phenyl‐2‐cyclopentadienyl)ZrCl₂ ( 1 ), ethylene(1‐(7, 9)‐diphenylcyclopenta‐[a]‐acenaphthadienyl‐2‐phenyl‐2‐fluorenyl)ZrCl₂ ( 2 ), and ethylene(1‐(9)‐fluorenyl‐(R)1‐phenyl‐2‐(1‐indenyl)ZrCl2 ( 3 ). Polyethenes produced with 1 /MAO had considerable, ca. 10% amount of trans‐vinylene end groups, resulting from the chain end isomerization prior to the chain termination. When ethene was copolymerized with 1‐hexene or 1‐hexadecene using 1 /MAO, molar mass of the copolymers varied from high to moderate (531–116 kg/mol) depending on the comonomer feed. At 50% comonomer feed, ethene/1‐olefin copolymers with high hexene or hexadecene content (around 10%) were achievable. In the series of catalysts, polyethenes with highest molar mass, up to 985 kg/mol, were obtained with sterically most crowded 2 /MAO, but the catalyst was only moderately active to copolymerize higher olefins. Catalyst 3 /MAO produced polyethenes with extremely small amounts of trans‐vinylene end groups and relatively low molar mass 1‐hexene copolymers (from 157 to 38 kg/mol) with similar comonomer content as 1 . These results indicate that the catalyst structure, which favors chain end isomerization, is also capable to produce high molar mass 1‐olefin copolymers with high comonomer content. In addition, an exceptionally strong synergetic effect of the comonomer on the polymerization activity was observed with catalyst 3 /MAO. © 2007 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 373–382, 2008     

Involvement of central noradrenaline, serotonin and dopamine system in the antidepressant activity of fruits of Solanum torvum (Solanaceae)

The methanolic extract (ME) of Solanum torvum seeds and its ethyl acetate fraction (EAF) were investigated for their antidepressant activity using behavioral (forced swim test, FST and tail suspension test, TST) and biochemical (monoamine oxidase, MAO reduced activity) tests. ME (10, 30 and 100?mg?kg(-1)) and EAF (10 and 30?mg?kg(-1)) dose dependently inhibited the immobility period, increased noradrenaline, serotonin and dopamine levels and inhibited the MAO enzymes in FST and TST using mice. Furthermore, we have observed antagonism between the threshold dose of ME (30 and 100?mg?kg(-1)) and EAF (10 and 30?mg?kg(-1)) with antagonists on behaviour mediated by neurotransmitters noradrenaline, serotonin and dopamine. MAO-A inhibition was more prominent as compared to MAO-B inhibition. The study provides evidence for antidepressant actions of S. torvum.     

Natural and Synthetic Xanthones as Monoamine Oxidase Inhibitors: Biological Assay and 3D‐QSAR

Fifty‐nine xanthones (=9H‐xanthen‐9‐ones) of natural or synthetic origin were investigated for their inhibitory activity toward monoamine‐oxidase A (MAO‐A) and MAO‐B. The compounds demonstrated reversible, time‐independent activities, with selectivity toward MAO‐A. The most active inhibitor had an IC₅₀ of 40 nM . Electron absorption spectroscopy revealed the formation of a 1 : 1 charge‐transfer complex between lumiflavine and xanthones. 3D‐QSAR Studies according to the CoMFA/GOLPE procedure provided information on the relationship between steric and electrostatic fields and MAO‐A inhibition. The ALMOND procedure yielded additional topographical information on structural factors favoring activity.     

Mixed Aluminum‐Magnesium‐Rare Earth Allyl Catalysts for Controlled Isoprene Polymerization: Modulation of Stereocontrol

Summary: The performances of readily available Ln(allyl)₂Cl(MgCl₂)₂ · (THF)₄ precursors (Ln = Nd, 1 ; Y, 2 ; La, 3 ), in combination with alkyl aluminum activators [MAO, AlMe₃, AlEt₃, Al(iBu)₃], have been studied in isoprene polymerization. The catalyst combination 1 /MAO (1:30) shows a high activity (average TOFs up to ca. 5 × 10⁴ mol (Ip) · mol (Nd)⁻¹ · h⁻¹ at 20 °C) and produces polyisoprene in a controlled fashion with up to 98.5% cis content, number‐average molecular weights in reasonable agreement with calculated values, and relatively narrow polydispersities index ( = 1.20–1.70). The yttrium precursor 2 affords systems with much lower activity and degree of control, but enables the formation of either 1,4‐cis‐enriched (75%) or 1,4‐trans‐enriched (91%) polyisoprenes, simply replacing the MAO activator by AlEt₃ or Al(iBu)₃, respectively.

Formation of 1,4‐cis‐ or 1,4‐trans‐enriched polyisoprenes upon activation with MAO.     

Monoamine oxidase inhibitors from Cinchonae Cortex

Three strong alkaloidal monoamine oxidase (MAO) inhibitors, quinine (1), cinchonicinol ([ 1S,3'R,4'R]-3-(3-ethenyl-4-piperidinyl)-1-(4-quinolinyl)-1-propanol) (2) and cinchonaminone ([ 3'R,4'S]-2-[2-(3-ethenyl-4-piperidinyl)-acetyl]-1H-indole-3-ethanol) (3), were isolated from Cinchonae Cortex (Cinchona succirubra Pav., Rubiaceae). The structures of 2 and 3 were elucidated on the bases of spectral data and chemical evidence, and 3 is a new alkaloid. The inhibitory effects on MAO of 1, 2, 3 and related alkaloids were assayed. The type of inhibition by 1 with respect to benzylamine as a substrate was competitive.     

非MAO的茂钛均相催化体系催化苯乙烯间规聚合——[Cp~*TiMe_3]/[Ph_3C]~+[B(C_6F_5)_4]~-催化体系

非ＭＡＯ的茂钛均相催化体系催化苯乙烯间规聚合———［ＣｐＴｉＭｅ３］／［Ｐｈ３Ｃ］＋［Ｂ（Ｃ６Ｆ５）４］－催化体系许光学林尚安（中山大学高分子研究所广州５１０２７５）关键词茂钛络合物，茂金属催化剂，苯乙烯，间规聚苯乙烯间规聚苯乙烯（ｓＰＳ）由于具...     

Studies on the syntheses of heterocycles from 3-arylsydnone-4-carbohydroximic acid chlorides with N-arylmaleimides, [1,4]naphthoquinone and aromatic amines

3-Arylsydnone-4-carbohydroximic acid chlorides (1) could react with N-arylmaleimides (3a–b) or 2-methyl-N-phenylmale-imide (3c) to give 3-(3-arylsydnon-4-yl)-5-aryl-3a,6a-dihydro-pyrrolo[3,4-d]isoxazole-4,6-diones (4a–h) or 6a-methyl-3-(3-arylsydnon-4-yl)-5-phenyl-3a,6a-dihydro-pyrrolo[3,4-d]isoxazole-4,6-diones (4i–l), respectively. However, 3-(arylsydnon-4-yl)-naphtho[2,3-d]isoxazole-4,9-diones (6a–d) were obtained in good yield by the reaction of carbohydroximic acid chlorides 1 with [1,4]naphthoquinone. Furthermore, 2-(3-arylsydnon-4-yl)benzoxazoles (9a–d) and 2-(3-arylsydnon-4-yl)benzothiazoles (9e–h) were obtained via the reaction of carbohydroximic acid chlorides 1 with ortho-substituted aromatic amines 7a and b.     

Cyclocopolymerization: a mechanistic probe for dual-site alternating copolymerization of ethylene and alpha-olefins

The copolymerization of ethylene with 1,5-hexadiene or 1-hexene was studied with the series of ansa-metallocenes Me2Si(Cp)(9-Flu)ZrCl2 (1), Me2Si(1-Ind)(9-Flu)ZrCl2 (3), and Me2Si(9-Flu)2ZrCl2 (4). 1,5-Hexadiene, a monomer which requires two insertion events to be cyclopolymerized, when copolymerized with ethylene using 3/MAO, gave a copolymer with a novel architecture. When compared with the copolymerization of 1-hexene with ethylene, the observed striking differences between the two copolymers provided compelling evidence for a dual-site alternating copolymerization mechanism in both cases. The copolymerization results from 1/MAO and 4/MAO further support this.     

Study of enzyme inhibitory activities by dipyridamole

Inhibitory activities of Dipyridamole (DPM, 2,6-bis(diethanolamino)-4,8-dipiperidinopyrimido(5,4-d)py rim idine) against xanthine oxidase (XO), carbonic anhydorase (CA) and monoamine oxidase (MAO) were studied, in vitro. DPM did not inhibit XO and CA, but it strongly inhibited MAO. The type of inhibition by DPM against MAO with respect to benzylamine as a substrate was uncompetitive.     

Olefin terpolymerizations. III. Symmetry,sterics, and monomer structure in ansa-zirconocenium catalysis of EPDM synthesis

Ethylenebis (η⁵-fluorenyl) zirconium dichloride ( 1 ) and rac-dimethylsilylene bis (1-η⁵-in-denyl) zirconium dichloride ( 2 ) were activated with methylaluminoxane (MAO) to catalyze ethylene (E) propylene (P) copolymerizations. The former produces high MW copolymer at 20°C rich in ethylene with reactivity ratio values of r_E = 1.7 and r_P <0.01, whereas the latter produces lower MW random copolymers with r_E = 1.32 and r_p = 0.36. Ethylidene norbornene (ENB) complexes with 1/MAO but does not undergo insertion in the presence of E and P. In contrast, 2/MAO catalyzes terpolymerization incorporating 9-15 mol % of ENB with slightly lower MW and activity than the corresponding copolymerizations. In comparison, 1,4–hexadiene was incorporated by 2/MAO with much lower A and MW . Terpolymerizations were also conducted with vinylcyclohexene using both catalyst systems. The steric and electronic effects in these processes were discussed. © 1995 John Wiley & Sons, Inc.     

Highly active ethylene polymerization and regioselective 1-hexene oligomerization using zirconium and titanium catalysts with tridentate [ONO] ligands

A series of tridentate dianionic ligands [4-(t)Bu-6-R-2-(3-R'-5-(t)Bu-2-OC(6)H(2))N=CH C(6)H(2)O](2-) (L) [R = R' = (t)Bu (L1); R = CMe(2)Ph, R' = (t)Bu (L2); R = adamantyl, R' = (t)Bu (L3); R = R' = CMe(2)Ph (L4); R = SiMe(2)(t)Bu, R' = CMe(2)Ph (L5)] were synthesized. Reactions of TiCl(4) with 1 equiv of ligands L1-L5 in toluene afford five-coordinate titanium complexes with general formula LTiCl(2) [L = L1 (1); L2 (2); L3 (3); L4 (4); L5 (5)]. The addition of tetrahydrofuran (THF) to titanium complex 5 readily gives THF-solvated six-coordinate complex 6, which also was obtained by reaction of TiCl(4) with 1 equiv of ligand L5 in THF. Reactions of ZrCl(4) with 1 or 2 equiv of ligands L1-L5 afford six-coordinate zirconium mono(ligand) complexes LZrCl(2)(THF) [L = L2 (7); L4 (8); L5 (9)], and bis(ligand) complexes L(2)Zr [L = L1 (10); L4 (11)]. The molecular structures of complexes 2, 8, and 11 were established by single-crystal X-ray diffraction studies. Upon activation with methylaluminoxane, complexes 1-9 are active for ethylene polymerization. The activities and half-lifes of the catalyst systems based on zirconium complexes are more than 10(6) g of polyethylene (mol Zr)(-1) h(-1) and 6 h, respectively. Complex 9 is more active and long-lived, with a turnover frequency (TOF) of 2.6 × 10(5) (mol C(2)H(4)) (mol Zr)(-1) h(-1), a half-life of >16 h, and a total turnover number (TON) of more than 10(6) (mol C(2)H(4)) (mol Zr)(-1) at 20 °C and 0.5 MPa pressure. Even at 80 °C, complex 9/MAO catalyst system has a long lifetime (t(1/2) > 2 h), as well as high activity that is comparable with that at 20 °C. When activated with methylaluminoxane (MAO), complex 9 also show moderate catalytic activity and more than 99% 2,1-regioselectivity for 1-hexene oligomerization.     

High-temperature single-site ethylene polymerization behavior of titanate complexes supported by 1,3-bis(3,5-dialkylpyrazol-1-yl)propan-2-olate ligation

Titanate(1-) complexes Na[(THF)(kappa1-O-bdbpzp)TiCl4] (1) and Na[(THF)(kappa1-O-bdmpzp)TiCl4] (2) and titanate(2-) complexes [Na(THF)]2[(kappa1-O-bdbpzp)2TiCl4] (4) and [Na(THF)]2[(kappa1-O-bdmpzp)2TiCl4] (5) were obtained in good yield from reaction of Na[bdbpzp] or Na[bdmpzp] (sodium salt of 1,3-bis(3,5-di-tert-butylpyrazol-1yl)propan-2-ol or 1,3-bis(3,5-dimethylpyrazol-1yl)propan-2-ol) with TiCl4 (in the appropriate molar ratio) at 0-25 degrees C. Protonolysis of TiCl4 with 1 equiv of bdmpzpH furnished related zwitterionic titanate(1-) complex 3 that possessed a kappa2-N,O-coordinated pyrazolyl-alkoxide with pendant pyrazolium group. Methylalumoxane (MAO) activation of 1-5 under high-temperature solution polymerization conditions produced active single-site ethylene polymerization catalysts that exhibit considerably higher thermal stability (especially 2/MAO, 3/MAO, and 5/MAO) than previously reported for Cp2TiCl2/MAO or Ti catalysts supported by related heteroscorpionate or scorpionate ligation.     

Effects of Al(i-Bu)_3 on Propylene Polymerization and Copolymerization by rac-Me_2Si(Ind)_2ZrCl_2/MAO

Propylenepolymerizationbystereorigidansa-Inetallocene/meUlyI-alulhinoxane(MAO)havebeenreportedbymanygroupsinrecentyears."'PolypropylenefromsuchSystemhasthechacteristicsofnarrowmolecularweightandcompositiondistributions,whichcannotbeproducedbytheconditionalheterogeneousZiegler-Nattacatalysts.Inarecentwork,M.C.SacchietaLrepoftedUleeffectsofaddingAl(i-C4Hg)3ahA)totherac-Et(Ind)ZZrCI,/MAOcatalyzedpropylene/1-hexenecopolymerizationSystem.'ItwasfoundthatbyaddingcertainamountsofTBAhlepoly…     

Effects of Different Carbon and Nitrogen Sources on Naphthoquinone Production of Cordyceps unilateralis BCC 1869

The production of six naphthoquinone derivatives, erythrostominone, deoxyerythrostominone, 4-O-methyl erythrostominone, epierythrostominol, deoxyerythrostominol, and 3,5,8-trihydroxy-6-methoxy-2-(5-oxohexa- 1,3-dienyl)-1,4-naphthoquinone, was examined during the growth of Cordyceps unilateralis BCC 1869 on different carbon and nitrogen sources. Erythrostominone production by the fungus accounted for more than 50% of total naphthoquinones, but production of each of the other five derivatives accounted for less than 20% of total naphthoquinones. The highest volumetric production rate of erythrostominone and overall naphthoquinone production rate were obtained on mannose as a sole carbon source and ammonium sulfate as a sole nitrogen source (4922.4 +/- 118.8 mg/[L.d] and 5.03 g/[L.d], respectively). The highest growth rate was obtained on arabinose (0.043 h-1), whereas the maximum overall naphthoquinone concentration was obtained on lactose (2 g/L) at 237 h. These naphthoquinones were produced with no pH control and were first detected at a pH of about 3.0 to 4.0. These results suggest that carbon and nitrogen influenced directly the production of naphthoquinones.     

A high-throughput monoamine oxidase inhibition assay using liquid chromatography with tandem mass spectrometry

A highly efficient method utilizing liquid chromatography with tandem mass spectrometry (LC/MS/MS) was developed and employed for high-throughput screening of compounds for monoamine oxidase (MAO) inhibition. The method used kynuramine as a common substrate for both MAO-A and MAO-B in incubations, and the 4-hydroxyquinoline (4-HQ) resulting from deamination of kynuramine followed by intramolecular condensation was analyzed using LC/MS/MS; formation of 4-HQ was used as the marker of MAO activity to evaluate the effects of test compounds. Isocratic liquid chromatography was applied to reduce the run time to 2 min. Because of the high specificity and sensitivity of detection of 4-HQ by LC/MS/MS, this method was able to use MAO enzymes at very low concentrations and to perform short incubations; as a result, consumable cost was minimized, and sample preparations were completely avoided. The inhibition data are highly reproducible, and the IC(50) values determined by the method are in good agreement with literature values. The results suggest that this method is very robust and can be used as a generic approach to screen for MAO inhibitors in drug discovery.     

Alkannin/shikonin mixture from roots of Onosma echioides (L.) L.: extraction method study and quantification

This work reports the extraction procedures of alkannin/shikonin mixture from roots of six populations of Onosma echioides, by means of three extraction techniques: Soxhlet extraction, maceration and rapid solid-liquid dynamic extraction (RSLDE). Five solvents with different polarity (hexane, petroleum ether, chloroform, ethyl acetate, methanol) were also studied. Analysis of the extracts was performed by an HPLC-DAD (diode array detector) system. The most efficient extraction technique was Soxhlet procedure using ethyl acetate for 6 h. Studied samples of O. echioides showed an alkannin/shikonin content in the range of 0.02-0.24 mg/kg. Other naphthoquinone derivatives (deoxyalkannin/deoxyshikonin and 5,8-dihydroxy-2-(4-methyl-6-oxo-5,6-dihydro-2H-pyran-2-yl)-[1,4]naphthoquinone and arnebin-6) were found for the first time in O. echioides and characterized in the extracts using HPLC-MS apparatus equipped with an ESI ionization source.     

Isofagomine lactams,synthesis and enzyme inhibition

The synthesis of isofagomine lactams (2-oxoisofagomines) corresponding to the biologically important hexoses is presented. The D-glucose/D-mannose analogue (3S,4R,5R)-3,4-dihydroxy-5-hydroxymethylpiperidin-2-one (9) was synthesised in 9 steps from D-arabinose, the D-galactose analogue (3S,4S,5R)-3,4-dihydroxy-5-hydroxymethylpiperidin-2-one (10) was synthesised in 11 steps from D-arabinose and the L-fucose analogue (3R,4R,5R)-3,4-dihydroxy-5-methylpiperidin-2-one (11) was synthesised in 12 steps from L-arabinose. The three lactams 9-11 were found to be glycosidase inhibitors with micro- to nanomolar inhibition constants. The lactam 10 showed slow onset inhibition of beta-galactosidase from A. Oryzae. The rate constants for this process were determined to be k(on) = 2.55 x 10(4) M-1 s-1 and k(off) = 1.7 x 10(-3) s-1. The activation energies and standard thermodynamic functions were also determined.