12味止血中药对脂多糖诱导小鼠巨噬细胞产生一氧化碳的抑制作用

目的研究12味止血中药对脂多糖(LPS)诱导小鼠巨噬细胞产生一氧化氮(NO)的抑制作用,并探讨其止血机制.方法以1μg·mL-1 LPS刺激小鼠巨噬细胞RAW 264.7,22h后以Griess法测定NO的终产物亚硝酸盐的含量,观察LPS对NO生成的影响.结果12味止血药均可抑制LPS诱导小鼠巨噬细胞RAW 264.7中NO的生成.三七、地榆、仙鹤草、槐花、艾叶、蒲黄、侧柏叶及白茅根的抑制作用显著(P＜0.05).结论本实验为12味止血中药对脂多糖诱导小鼠巨噬细胞产生NO的抑制作用提供了一定的理论依据.     

12味止血中药对脂多糖诱导小鼠巨噬细胞产生一氧化氮的抑制作用

目的:研究12味止血中药对脂多糖(LPS)诱导小鼠巨噬细胞产生一氧化氮(NO)的抑制作用,并探讨其止血机制。方法:以1μg.mL-1LPS刺激小鼠巨噬细胞RAW264.7,22h后以Griess法测定NO的终产物亚硝酸盐的含量,观察LPS对NO生成的影响。结果:12味止血药均可抑制LPS诱导小鼠巨噬细胞RAW264.7中NO的生成。三七、地榆、仙鹤草、槐花、艾叶、蒲黄、侧柏叶及白茅根的抑制作用显著(P<0.05)。结论:本实验为12味止血中药对脂多糖诱导小鼠巨噬细胞产生NO的抑制作用提供了一定的理论依据。     

Shikonin Directly Inhibits Nitric Oxide Synthases: Possible Targets That Affect Thoracic Aorta Relaxation Response and Nitric Oxide Release From RAW 264.7 Macrophages

Recently, an isomeric mixture of herbal anti-inflammatory naphthoquinones shikonin and alkannin, and their derivatives, have been found to impair cellular responses involving nitric oxide (NO) and NO synthesis, like the acetylcholine-induced relaxation response of rat thoracic aorta and NO release from murine RAW 264.7 macrophages. However, the mechanisms of such effects, including whether NO synthase (NOS) activity is affected, remained unclear. We herein investigate possible targets of shikonin in these NOS-related events. Shikonin by itself dose-dependently inhibited the rat thoracic aorta relaxation in response to acetylcholine (pD′₂ value: 6.29). Its optical enantiomer, alkannin, was equally inhibitory in the aorta relaxation–response assay. In RAW 264.7 cells, shikonin inhibited the lipopolysaccharide-induced NO production by 82% at 1 μM. A cell-free assay to verify direct effects on NOS activity showed that shikonin inhibits all isoforms of NOS (IC₅₀ s, 4–7 μM), suggesting NOS as an inhibition target in both the events. Further possible targets of shikonin that might be involved in the inhibitions of the acetylcholine-induced aorta relaxation response and the NO generation by RAW 264.7 cells are also discussed. It is shown for the first time that shikonin inhibits NOS activity.     

Saquinavir‐NO Inhibits IL‐6 Production in Macrophages

Covalent attachment of the nitric oxide (NO) moiety to the HIV protease inhibitor Saquinavir (Saq) produced a new chemical entity, named Saquinavir‐NO, (Saq‐NO) with reduced toxicity and potent immunoregulatory influence on T lymphocytes. In this study, we have compared head‐to‐head the effects of Saq‐NO and Saq on mouse and rat peritoneal macrophage cytokine secretion and NO production upon in vitro, ex vivo and in vivo conditions. The results demonstrate that Saq‐NO, but not Saq, potently decreased interleukin (IL)‐10, IL‐6 and nitrite accumulation and increased the levels of IL‐1β and tumour necrosis factor (TNF) in supernatants of mouse and rat macrophage cultures in vitro. Treatment of mice with Saq‐NO, but not Saq, inhibited ex vivo secretion of IL‐6 from macrophages. Consistent with these findings, Saq‐NO also reduced blood levels of IL‐6 in lipopolysaccharide‐treated mice. The observed inhibitory influence of Saq‐NO on IL‐6 generation in macrophages may be involved in the observed antitumour and immunomodulatory effects of the drug.     

Rac‐ and R‐(+)‐[4,4′,5,5′‐2H4]‐2‐(1′‐[2′′,6′′‐dichlorophenoxy]‐ethyl)‐Δ2‐imidazoline (lofexidine)

The synthesis of the d₄‐forms of rac‐ and R‐lofexidine was accomplished. Two methods are described; one method is a two‐step synthesis of rac‐d₄‐lofexidine from 2‐chloropropionitrile, the second method is a three‐step preparation of R‐d₄‐lofexidine in absolute enantiomeric purity from S‐methyl lactate. The commercial availability of R‐methyl lactate makes this latter enantioselective synthesis applicable also to the synthesis of S‐d₄‐ lofexidine. These procedures also conserve the utilization of the relatively expensive [1,1′,2,2′‐²H₄]ethylene diamine precursor. The availability of S‐ and R‐d₄‐lofexidines will enable pharmacokinetic studies to be carried out to determine if differential in vivo metabolism of the two enantiomers of lofexidine occurs. Copyright © 2009 John Wiley & Sons, Ltd.     

Synthesis of 1,4‐Anthracene‐9,10‐dione Derivatives and Their Regulation of Nitric Oxide,IL‐1β and TNF‐α in Activated RAW264.7 Cells

Mitoxantrone is an anthracenedione antineoplastic and immunosuppressive agent approved for multiple sclerosis treatment. Novel mono‐ and disubstituted anthraquinone derivatives, analogues of mitoxantrone, were synthesized through the addition of lipophilic amino alcohols and evaluated for their effect on IL‐1β, TNF‐α and nitric oxide production by LPS/IFN‐γ‐stimulated RAW264.7 cells. The disubstituted 1,4‐anthracene‐9,10‐dione 10 showed significant inhibition of nitric oxide, TNF‐α and IL‐1β production at the concentration of 5 μg/mL, with a much lower cytotoxicity than mitoxantrone. The monosubstituted 3 , 4 , 11, 12 and 13 also displayed a moderate to good inhibitory capacity on IL‐1β production. However, the methylated compounds 11, 12 and 13 failed to inhibit the TNF‐α production, and compound 13 was the only one to decrease the production of nitric oxide. None of these derivatives was toxic at the tested concentrations. Compounds 10 and 13 had better inhibitory capacity of the inflammatory mediators analyzed, with reliable viability of the cells.     

Synthesis and 11C‐labelling of (E,E)‐1‐(3′,4′‐dihydroxystyryl)‐4‐(3′‐methoxy‐4′‐hydroxystyryl) benzene for PET imaging of amyloid deposits†

Carboxylic acid derivatives of the amyloid‐binding dye Congo red do not enter the brain well and are thus unable to serve as in vivo amyloid‐imaging agents. A neutral amyloid probe, (E,E)‐1‐(3′,4′‐dihydroxystyryl)‐4‐(3′‐methoxy‐4′‐hydroxystyryl)benzene ( 3 ), devoid of any carboxylate groups has been designed and synthesized via a 12‐step reaction sequence with a total yield of 30%. The unsymmetric compound 3 has also been labelled with C‐11 via [¹¹C]methyl iodide ([¹¹C]CH₃I) methylation of a symmetric 4,4′‐dimesyl protected precursor followed by deprotection. Preliminary evaluation indicated that compound 3 selectively stained plaques and neurofibrillary tangles in post‐mortem AD brain, and exhibited good binding affinity (K_i=38±8 nM) for Aβ(1–40) fibrils in vitro. In vivo pharmacokinetic studies indicated that [¹¹C] 3 exhibited higher brain uptake than its carboxylic acid analogs and good clearance from normal control mouse brain. [¹¹C] 3 also exhibited specific in vivo binding to pancreatic amyloid deposits in the NOR‐beta transgenic mouse model. These results justify further investigation of 3 and similar derivatives as surrogate markers for in vivo quantitation of amyloid deposits. Copyright © 2002 John Wiley & Sons, Ltd.     

Synthesis of 1, 1′, 2, 2′, 3, 3′, 4, 4′ ‐ octadeutero‐sulforaphane

Sulforaphane (SFN), a naturally occurring isothiocyanate present in broccoli, shows strong evidence of anti‐carcinogenic activity. The mechanism of action, absorption, distribution, metabolism and excretion of the compound is however still poorly understood and requires a stable isotope labelled version of the compound for further studies. The paper describes an optimized synthesis of octadeutero‐SFN. Copyright © 2004 John Wiley & Sons, Ltd.     

Isotopically labelled tropane alkaloids. The synthesis of (RS)‐[3′, 3′‐2H2]‐ and (RS)‐[1′‐13C, 3′, 3′‐2H2]‐ hyoscyamines for metabolism studies in plants

A synthetic route to isotopically labelled forms of the tropane alkaloid hyoscyamine, including (RS)‐[3′, 3′,‐²H₂]‐ ( 2a ) and (RS)‐[1′‐¹³C, 3′, 3′,‐²H₂]‐ ( 2b ) hyoscyamines, involving the reaction between phenylacetyl tropine and formaldehyde is described. The isotopically labelled products enable the metabolism of hyoscyamine to be studied in plants such as Datura stramonium. Copyright © 2002 John Wiley & Sons, Ltd.     

Synthesis of [1,3, NH2‐15N3] (5′S)‐8,5′‐cyclo‐2′‐deoxyguanosine

To facilitate NMR studies and low‐level detection in biological samples by mass spectrometry, [1,3, NH₂‐¹⁵N₃] (5′S)‐8,5′‐cyclo‐2′‐deoxyguanosine was synthesized from imidazole‐4,5‐dicarboxylic acid in 21 steps. The three ¹⁵N isotopes were introduced during the chemo‐enzymatic preparation of [1,3, NH₂‐¹⁵N₃]‐2′‐deoxyguanosine using an established procedure. The ¹⁵N‐labeled 2′‐deoxyguanosine was converted to a 5′‐phenylthio derivative, which allowed the 8‐5′ covalent bond formation via photochemical homolytic cleavage of the C–SPh bond. SeO₂ oxidation of C‐5′ followed by sodium borohydride reduction and deprotection gave the desired product in good yield. The isotopic purity of the [1,3, NH₂‐¹⁵N₃] (5′S)‐8,5′‐cyclo‐2′‐deoxyguanosine was in excess of 99.94 atom% based on liquid chromatography–mass spectrometry measurements. Copyright © 2013 John Wiley & Sons, Ltd.     

Potent Opioid Peptide Agonists Containing 4′‐[N‐((4′‐phenyl)‐phenethyl)carboxamido]phenylalanine (Bcp) in Place of Tyr

Analogues of the opioid peptides H‐Tyr‐c[d ‐Cys‐Gly‐Phe(pNO₂)‐d ‐Cys]NH₂ (non‐selective), H‐Tyr‐d ‐Arg‐Phe‐Lys‐NH₂ (μ‐selective) and dynorphin A(1‐11)‐NH₂ (κ‐selective) containing 4′‐[N‐((4′‐phenyl)‐phenethyl)carboxamido]phenylanine (Bcp) in place of Tyr¹ were synthesized. All three Bcp¹‐opioid peptides retained high μ opioid receptor binding affinity, but showed very significant differences in the opioid receptor selectivity profiles as compared with the corresponding Tyr¹‐containing parent peptides. The cyclic peptide H‐Bcp‐c[d ‐Cys‐Gly‐Phe(pNO₂)‐d ‐Cys]NH₂ turned out to be an extraordinarily potent, μ‐selective opioid agonist, whereas the Bcp¹‐analogue of dynorphin A(1‐11)‐NH₂ displayed partial agonism at the μ receptor. The obtained results suggest that the large biphenylethyl substituent contained in these compounds may engage in a hydrophobic interaction with a receptor subsite and thereby may play a role in the ligand’s ability to induce a specific receptor conformation or to bind to a distinct receptor conformation in a situation of conformational receptor heterogeneity.     

Synthesis of tritium labelled [2′,6′]‐L‐tyrosine

The synthesis of a specifically ring labelled isotopomer of L ‐tyrosine, (L ‐Tyr), using a combination of chemical and enzymatic methods is reported. The tritium labelled [2′,6′]‐L ‐Tyr has been synthesized via catalytic exchange of phenol with tritiated water in the presence of K₂PtCl₄, reverse acid catalysed removal of tritium from the o‐ and p‐positions of phenol, and subsequent condensation of the resulting [3′,5‐³H₂]‐phenol with S‐methyl‐L ‐cysteine using the enzyme β‐tyrosinase from Citrobacter freundii. Copyright © 2004 John Wiley & Sons, Ltd.