Inhibitors of sterol synthesis. Chemical syntheses, properties and effects of 4,4-dimethyl-15-oxygenated sterols on sterol synthesis and on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in cultured mammalian cells

The chemical syntheses of a number of 4,4-dimethyl substituted 15-oxygenated sterols have been pursued to permit evaluation of their activity in the inhibition of the biosynthesis of cholesterol and other biological effects. Described herein are the first chemical syntheses of 4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-en-3 beta-ol-15-one, 3 beta,15 alpha-diacetoxy-4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-ene, 3 beta-acetoxy-4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-en-15 beta-ol, 4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta,15 alpha-diol, 4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta,15 beta-diol, 4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-en-15 alpha-ol-3-one, 3 beta-benzoyloxy-4,4-dimethyl-5 alpha-cholest-8(14)-ene-7 alpha,15 alpha-diol, 7 alpha,15 alpha-diacetoxy-3 beta-benzoyloxy-4,4-dimethyl-5 alpha-cholest-8(14)-ene, 4,4-dimethyl-5 alpha-cholest-8(14)-en-3 beta-ol-15-one and 3 beta,7 alpha,15 alpha-tri-o-bromobenzoyloxy-5 alpha-cholest-8(14)-ene. Also prepared for use in the biological experiments were 4,4-dimethyl-5 alpha-cholest-7-ene-3 beta,15 alpha-diol, 4,4-dimethyl-5 alpha-cholest-8-ene-3 beta,15 alpha-diol and 4,4-dimethyl-5 alpha-cholest-8(14)-ene-3 beta,7 alpha,15 alpha-triol. The effects of twelve 4,4-dimethyl substituted 15-oxygenated sterols and of four 4,4-dimethyl substituted 32-oxygenated sterols on sterol synthesis and on the level of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity were evaluated in mouse L cells. With the exception of 4,4-dimethyl-5 alpha-cholest-8(14)-ene-3 beta,7 alpha,15 alpha-triol, all of the 4,4-dimethyl substituted 15-oxygenated sterols caused a 50% inhibition of sterol synthesis at less than 10(-6) M and six of the 4,4-dimethyl substituted 15-oxygenated sterols caused a 50% inhibition of sterol synthesis at less than 10(-7) M. 4,4-Dimethyl-14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta,15 alpha-diol caused a 50% decrease in sterol synthesis at 10(-8) M. The potencies of the 4,4-dimethyl substituted 15-oxygenated and C-32-oxygenated sterols with respect to inhibition of sterol synthesis and suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity have been compared with those of the corresponding sterols lacking the 4,4-dimethyl substitution.     

山玉兰花粉离体萌发和花粉管生长特性的研究

山玉兰(Magnolia delavayi)是木兰科木兰属的常绿乔木或大型灌木,是重要的园林造景、庭院绿化素材,也是重要的育种资源。山玉兰花粉的研究对其杂交育种的成败具有重要影响,但目前尚未见其花粉活力的相关报道。该研究以新鲜的山玉兰花粉为对象,采用悬滴培养法分析了温度、光照以及培养液的蔗糖和硼酸浓度对山玉兰花粉萌发的影响。结果表明:(1)山玉兰花粉萌发时,最适宜的温度为27℃。(2)光暗条件下,山玉兰花粉以浓度为5%的蔗糖培养效果最佳,其萌发率在16%以上;而硼酸浓度则以0.001%的培养效果最佳。(3)蔗糖与硼酸共同作用可有效促进花粉萌发和花粉管生长。其中,在光照条件下,以5.0%蔗糖+0.001%硼酸为最适宜的培养液,花粉萌发率达41.27%,花粉管长达281.49μm;而在黑暗条件下,则以5.0%蔗糖+0.01%硼酸为最适宜的培养液,花粉萌发率达45.71%,花粉管长达254.00μm。该研究结果为进一步开展人工辅助授粉、发掘山玉兰的种质资源工作奠定了基础。     

Identification of the components of the essential oil from Trollius europaeus flowers

The essential oil of Trollius europaeus flowers obtained by hydrodistillation was analyzed by gas chromatography coupled with mass spectrometry (GC–MS). The compounds giving fragrance of essential oils commonly used in perfumery 3,7-dimethyl-1,6-octadien-3-ol, nonanal, 3-methyl-2-pent-2-enyl-cyclopent-2-enone and oxacycloheptadec-8-en-2-one, rare in the Plant Kingdom, were tentatively identified. In the analyzed essential oil, the saturated fatty acids hexadecanoic acid (7.54 %), tetradecanoic acid (4.24 %), dodecanoic acid (3.10 %) and unsaturated fatty acids 9,12,15-octadecatrienoic acid (3.47 %), hydrocarbons, namely eicosane (20.03 %), hexadecane (8.63 %) and 1,2-benzenedicarboxylic acid (2.39 %), were also found.     

Genetic Relationship between a Natural Hybrid Meconopsis× cookei (Papaveraceae) and Its Parents Based on cpDNA trnL- trnF Region Sequence

The Chloroplast DNA trnL- trnF region sequences from a natural hybrid species Meconopsis× cookei and its parents M. punicea and M. quintuplinervia were obtained by using direct sequencing method. The sequence length of trnL- trnF region is 960 bp for M. × cookei, 961 bp for M. punicea, and 957 bp for M. quintuplinervia. The sequences were aligned by the software Clustal X, and then the bases per locus were compared by using the software with manual method. The aligned sequence length is 964 bp, of which trnL intron is 512 bp, the 3′exon of trnL is 50 bp, trnL- trnF intergenic spacer ( IGS) is 361 bp, and the 5′end segment of trnF is 41 bp. Total 25 variable loci were detected from the aligned sequence, of which 21 (84% ) sites are same between M. × cookei and M. punicea, and only one (4%) is same between M. × cookei and M. quintuplinervia , the remaining three loci (12% ) are different among M. × cookei, M. punicea, and M. quintuplinervia. The results show that the cpDNA trnL- trnF region of the natural hybrid species M. ×cookei was inherited from its parent M. punicea. Therefore, according to the plastid inheritance law, our molecular evidences indicate that M. punicea is the mother of hybrid species M. × cookei and M. quintuplinervia is its father.     

绿绒蒿自然杂交种及其亲本cpDNA trnL- trnF 基因的遗传学分析

对自然杂交种Meconopsis× cookei 及其亲本红花绿绒蒿M. punicea 和五脉绿绒蒿M. quintuplinervia 的叶绿体DNA trnL- trnF 区进行了序列测定, 所得序列的长度为957～961 bp , 其中M. × cookei 的序列长度为960bp , 红花绿绒蒿为961 bp , 五脉绿绒蒿为957 bp。利用软件Clustal X 对所得序列进行排序和碱基比较, 排序后的序列长度为964 bp , 其中trnL intron 为512 bp , trnL 3′exon 为50 bp , trnL- trnF intergenic spacer ( IGS) 为361 bp , 还包括41 bp 的trnF 5′端片段。整个trnL- trnF 区序列共有25 个变异位点, 其中杂交种M. × cookei与红花绿绒蒿具有相同碱基的位点有21 个( 占84% ) , M. × cookei 与五脉绿绒蒿具有相同碱基的位点仅有1 个(占4% ) , 余下3 个位点( 占12%) 中, M. × cookei 的碱基与两个亲本均不相同。分析结果表明, 杂交种M. × cookei 的叶绿体基因trnL- trnF 来自红花绿绒蒿, 根据质体细胞质遗传的规律, 从而推测红花绿绒蒿为该杂交种的母本, 五脉绿绒蒿为其父本。     

Photochemical reactions of transition metal organyl complexes with olefins Part 10. Light-induced formal [5+2] cycloadditions at manganese

Tricarbonyl-η⁵-2,4-dimethyl-2,4-pentadien-1-yl-manganese (1) forms upon UV irradiation in THF at 208 K solvent stabilized dicarbonyl-η⁵-2,4-dimethyl-2,4-pentadien-1-yl-tetrahydrofurane-manganese (2). With butynedioic acid dimethyl ester (3) and diphenylacetylene (5) complex 2 yields tricarbonyl-η⁵-1,2-dimethoxycarbonyl-4,6-dimethyl- cyclohepta-2,4-dien-1-yl-manganese (4) and tricarbonyl-η-4,6-dimethyl-1,2-diphenyl-cyclohepta-2,4-dien-1-yl- manganese (6) in a formal [5+2] cycloaddition. Addition of carbon monoxide and a 1,4-H shift completes the reaction. Propynoic acid methyl ester (7) forms the 2:1 adduct dicarbonyl-η^5:2-1,3-dimethyl-6-methoxycarbonyl-6- (E-2′-methoxycarbonylvinyl)-cyclohepta-2,4-dien-1-yl-manganese (8). The crystal and molecular structure of 8 was determined by X-ray structure analysis. The molecular structures of the complexes 4 and 6 were established by IR and NMR spectroscopy. Formation mechanisms of 4, 6 and 8 are discussed. Crystal data for 8: monoclinic space group P2₁/c, a=802.6(3), b=1136.6(1), c=8872.3(3) pm, β=93.14(2)°, V=1.705 nm³, Z=4.     

Ammoniated Forage Poisoning: Concentrations of Alkylimidazoles in Ammoniated Forage and in Milk,Plasma and Urine in Sheep and Cow

4-methylimidazolc (4-MeI) has until now been the only identified toxic compound in ammoniated forage considered to be of possible etiological significance in ammoniated forage poisoning. However, several authors have concluded that 4-MeI alone cannot explain the toxicity observed (Morgan & Edwards 1986, Nielsen et al 1986, Motoi et at. 1997). On this background, we have examined samples of ammoniated forage and of milk, plasma, and urine collected from ewes and lambs during a previous experimental poisoning study (Sivertsen et al. 1993). We have also studied similar samples from a Norwegian Red Cattle dairy cow fed ammoniated hay for the first 5 days after calving. We have, in addition to the previously known ingredients 4-MeI and 2-methylimida-zole (2-MeI), identified 5 new compounds in these samples, all of which were found to be di-alkylsubstituted imidazoles: 1,2-dimcthylimi-dazole (1,2-diMeI), 1,4-dimethyl imidazole (1,4-diMeI), 1,5-dimethyl imidazole (1,5-diMeI), 2,4-dimethylimidazole (2,4-diMeI) and 2-ethyl, 4-methylimidazole (2Et-4MeI) (Müller et al. 1998). In this paper we present quantitative measurements of these di- and monoalky 1 imidazoles in a selection of the collected samples.     

刺山柑化学成分研究

采用硅胶、聚酰胺和Sephadex LH-20等色谱方法从维吾尔药刺山柑中分离得到11个化学成分,通过理化手段及波谱技术分别鉴定为正二十八烷(1)、β-胡萝卜苷(2)、正二十八烷醇(3)、正三十二烷酸(4)、正十六烷酸(5)、琥珀酸(6)、原儿茶酸(7)4、-羟基-5-甲基呋喃-3-羧酸(8)、山萘酚3-O-β-D-芸香糖苷(9)、芦丁(10)和水苏碱(11)。其中化合物13、和4为首次从该属植物中分离得到。     

杉木叶醇提物中石油醚溶解组分的化学成分分析

采用气-质联用法对杉木(Cunningham ia lanceolataHook.)叶醇提物石油醚溶解组分的化学成分进行了研究,经毛细管色谱分析分离出38个峰,共确认出其中36种成分,鉴定出的化学成分质量占总量的98.06%以上。应用色谱峰面积归一法分析各成分的质量分数,含量较高的物质有:十八酸-1,3-甘油二酯(16.929%)、二十七烷(15.178%)、棕榈酸-2-(十八烷氧基)乙酯(11.038%)、油酸(6.531%)、三十七醇(6.877%)等。化合物的类型主要为饱和脂肪酸酯(33.834%)和烃类化合物(22.031%)。     

Design and synthesis of novel and potent amide linked PPARgamma/delta dual agonists

A series of potent amide linked PPARγ/δ dual agonists (1a) has been discovered through rational design. In the ZDF rat model of type 2 diabetes, compound (R)-3-[4-(3-{1-[(5-chloro-1,3-dimethyl-1H-indole-2-carbonyl)-amino]-ethyl}-5-fluoro-phenoxy)-2-ethyl-phenyl]-propionic acid (42) from this series has demonstrated glucose lowering efficacy comparable to the marketed PPARγ agonist rosiglitazone with less weight gain.     

1,3-Dialkyl-substituted tetrahydropyrimido[1,2-f]purine-2,4-diones as multiple target drugs for the potential treatment of neurodegenerative diseases

Adenosine receptors and monoamine oxidases are drug targets for neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease. In the present study we prepared a library of 55 mostly novel tetrahydropyrimido[2,1-f]purinediones with various substituents in the 1- and 3-position (1,3-dimethyl, 1,3-diethyl, 1,3-dipropyl, 1-methyl-3-propargyl) and broad variation in the 9-position. A synthetic strategy to obtain 3-propargyl-substituted tetrahydropyrimido[2,1-f]purinedione derivatives was developed. The new compounds were evaluated for their interaction with all four adenosine receptor subtypes and for their ability to inhibit monoamine oxidases (MAO). Introduction of mono- or di-chloro-substituted phenyl, benzyl or phenethyl residues at N9 of the 1,3-dimethyl series led to the discovery of a novel class of potent MAO-B inhibitors, the most potent compound being 9-(3,4-dichlorobenzyl)-1,3-dimethyl-6,7,8,9-tetrahydropyrimido[1,2-f]purine-2,4(1H,3H)-dione (21g, IC₅₀ human MAO-B: 0.0629 μM), which displayed high selectivity versus the other investigated targets. Potent dually active A₁/A_2A adenosine receptor antagonists were identified, for example, 9-benzyl-1-methyl-3-propargyl-6,7,8,9-tetrahydropyrimido[1,2-f]purine-2,4(1H,3H)dione (19f, K_i, human receptors, A₁: 0.249 μM, A_2A: 0.253 μM). Several compounds showed triple-target inhibition, the best compound being 9-(2-methoxybenzyl)-1-methyl-3-(prop-2-ynyl)-6,7,8,9-tetrahydro pyrimido [1,2-f]purine-2,4(1H,3H)-dione (19g, K_i A₁: 0.605 μM, K_i A_2A: 0.417 μM, IC₅₀ MAO-B: 1.80 μM). Compounds inhibiting several different targets involved in neurodegeneration may exhibit additive or even synergistic effects in vivo.     

Enhancement of Candida rugosa lipase activity in non-aqueous media by co-lyophilization with amphiphiles from aqueous solution

Modified Candida rugosa lipase was co-lyophilized with two gemini-type amphiphiles, l- and d-2-(3-bis-[3-(2,3,4,5,6-pentahydroxy-hexanoylamino)-propyl]-carbamoyl -propionylamino)-pentanedioic acid didodecyl ester or dodecanoic acid 2-[(3-bis-[3-(2,3,4,5,6-pentahydroxy-hexanoylamino)-propyl]-carbamoyl -propionyl)-(2-dodecanoyloxy-ethyl)-amino]-ethyl ester. Enzymatic activities of the modified lipases in the transesterification between racemic 2,2-dimethyl-1,3-dioxolane-4-methanol and vinyl butyrate in cyclohexane were enhanced as much as by 37-78, 1.5–5- and 41–83-fold of magnitude relative to that of native enzyme, respectively. The lack of significant enhancement of the enzymatic activity, only in the case of the d-isomeric amphiphile-modified lipase, was considered from the topological view of the amphiphile.     

Inhibitory constituents of Nardostachys chinensis on nitric oxide production in RAW 264.7 macrophages

The activity-guided fractionation of the MeOH extract of the rhizomes and roots of Nardostachys chinensis led to the isolation of two new sesquiterpenoids, narchinol B (8) and narchinol C (9), along with 10 known compounds, ursolic acid (1), nardosinone (2), pinoresinol (3), desoxo-narchinol A (4), kanshone B (5), epoxyconiferyl alcohol (6), debilon (7), 4α,5-dimethyl-1,3-dioxo-1,2,3,4,4α,5,6,7-octahydronaphthalene (10), p-coumaric acid (11), and isoferulic acid (12). Their structures were determined using spectroscopic techniques, which included 1D- and 2D-NMR. Among the isolates, compounds 2, 4, 5, 8 and 9 showed inhibitory activity against LPS-induced NO production with IC(50) values of 4.6-21.6 μM.     

Two neolignans from Perilla frutescens and their inhibition of nitric oxide synthase and tumor necrosis factor-alpha expression in murine macrophage cell line RAW 264.7

Two neolignans were isolated from leaves of Perilla frutescens (Labiatae) as inhibitors of nitric oxide syntheses (IC50 5.9 microM and 53.5 microM, respectively) and tumor necrosis factor-alpha in lipopolysaccharide-activated RAW 264.7 cells. Their structures were identified as 1beta,2alpha,3beta,4alpha-1,2-dimethyl-3,4-bis-(2,4,5-trimethoxyphenyl)-cyclobutane (magnosalin), and 1alpha,2beta,3beta,4alpha-1,2-dimethyl-3,4-bis-(2,4,5-trimethoxyphenyl)-cyclobutane (andamanicin), and their activities were confirmed as resulted from the suppressed expression of inducible nitric oxide synthase enzyme and from the secretion of tumor necrosis factor-alpha from activated macrophages.     

Delta8(14)-steroids in the bacterium Methylococcus capsulatus.

The 4,4-dimethyl and 4alpha-methyl sterols of the bacterium Methylococcus capsulatus were identified as 4,4-dimethyl- and 4alpha-methyl-5alpha-cholest-8(14)-en-3beta-ol and 4,4-dimethyl- and 4alpha-methyl-5alpha-cholesta-8(14),24-dien-3beta-ol. Sterol biosynthesis is blocked at the level of 4alpha-methyl delta8(14)-sterols.     

Preparation and properties of poly 2''-O-ethylcytidylic acid.

Poly 2'0-ethylcytidylic acid (poly (Ce)) was prepared by polymerization of 2'-0-ethylcytidine-5'-pyrophosphate with Escherichia coli polynucleotide phosphorylase in the presence of Mn++, and its properties compared with those of poly (rC), poly (Cm) and poly (dC). The neutral form of pOLY (Ce) exhibits properties similar to those of poly (rC) and poly (Cm). It also forms an acid twin-stranded helix with a transition pH of 5.9 in 0.1 M NaCl. The neutral form readily forms a double-stranded helical complex with poly (rI). Relative to poly (Cm), replacement of the 2'-0-methyl by 2-0-ethyl leads to increased enhancement of the thermal stabilities of both the acid helical form of poly (Ce) and its complex with poly (rI).     

藏药短管兔耳草挥发性化学成分的研究

采用毛细管气相色谱—质谱联用技术对藏药短管兔耳草挥发性化学成分进行了研究,经毛细管色谱分离出62个峰,共确定了其中41种化学成分,所鉴定的化合物含量占全挥发油的77．32％;用气相色谱面积归一化法测定了各化学成分的相对含量,其主要化学成分为：二苯胺(16．47％)、邻苯二甲酸丁基—8—甲基壬基酯(6．42％)、二十六碳烷(4．76％)、十六烷酸(3．66％)、二十四碳烷(3．40％)、邻苯二甲酸二丁酯(3．38％)、二十二碳烷(3．30％)、二十碳烷(3．26％)、十六烷酸乙酯(2．77％)、十八碳烷(2．76％)、戊酸(2．48％)、3—乙基环辛烯(2．05％)等。     

Pyruvylated glycolipids from Mycobacterium smegmatis. Nature and location of the lipid components

The dipyruvylated glycolipid from Mycobacterium smegmatis (Saadat, S., and Ballou, C.E. (1983) J. Biol. Chem. 258, 1813-1818) has been shown to have the following structure in which FA1 is tetra- or hexadecanoic acid and FA2 is 2,4-dimethyl-2-eicosenoic acid. (formula; see text) The fast atom bombardment mass spectrum showed two major ions [M - H]- at m/z 1511 and 1539 (Mr 1512 and 1540) in a ratio of 1.4:1, suggesting that the glycolipid was a mixture of homologs that differed in fatty acid composition by 2 methylene groups. Analysis revealed C14, C16, and C22 fatty acids in ratios of 0.6:0.4:1.0, indicating that 60% of the molecules contained a C14 and C22 fatty acid whereas 40% contained a C16 and C22 fatty acid. The fragmentation pattern showed that a single glucose unit along with the smaller fatty acid could be lost to yield a tetrasaccharide with attached C22 fatty acid, and a second fragmentation yielded a trisaccharide containing 2 pyruvic acids but without attached fatty acid. The C14 and C16 fatty acids were identified as myristic and palmitic acid, whereas the C22 fatty acid was 2,4-dimethyl-2-eicosenoic acid. Precise localization of the fatty acids came from periodate oxidation and methylation analysis.     

Head Space Constituents of Different Varieties of Osmanthus fragrans

This work deals with the head space constituents of different varietiesof Osmanthus fragrans. A porous crosslinked polystyrene resin (Amberite XAD-4)trap was used to absorbing the head space of fresh flowers and the constituents weredetermined by using GC, GC/MS/DS. Thus, fifty six components have been identifiedand thirty seven of them were unreported: i.e. ethyl acetate, 3-methyl butanone, 3-hydroxy-2-butanone, 5-hexen-2-one, 3,3-dimethyl hexane, undecan-6-one, myrcene, decane,limonene,αand β-ocimene, 6-methyl-3, 5-heptadien-2-one,1,6-diacetoxy-hexane,5-phen-ylmethoxy-l-pentanol,3-eyelohexene-l-methanol, ,7-dimethyl-3,5-octadiene, menthone,menthol, ethyl benzaldehyde, 2-cyclohexen-l-yl-2-methyl-5-(1-methylethenyl) acetate, 4-methyl-2-heptanol, 1,4-benzene dicarboxaldehyde, hexyl butanoate, ethyl benzoate, cin-namic aldehyde, 1,4-dimethyl-3-eyclohexen-l-ol ethanone, carvone, 2-hydroxymethyl be-nzoic acid, benzoic acid, 3,4-dihydro-l-2(H)-naphthalenone, 8,8-dimethyl-4-methylene, 1-oxaspiro-[2, 5]-oct-5-ene, 2,4-dimethyl phenyl ethenone, 1-ethoxyethyl benzene, 4,6,6-tri-methyl-bicyelo-[3, 1, 1]-hept-3-en-2-one, 1,4-phenylene bis ethanone-l,1, diethyl O-phtha-late and dibutyl-O-phthalate.     

Synthesis of bis-(methyl 3,4,6-tri-O-acetyl-D-glucopyranosid-2-yl)-oxamides

The synthesis of a new bis-(D-glucopyranosid-2-yl)oxamides via the key intermediate, N-acetyl N-(methyl 3,4,6-tri-O-acetyl-alpha-D-glucopyranosid-2-yl) oxamic acid chloride (2alpha) is described. Treatment of compound 2alpha with methyl 3,4,6-tri-O-acetyl-2-amino-2-deoxy-beta-D-glucopyranoside afforded N-(methyl 3,4,6-tri-O-acetyl-alpha-D-glucopyranosid-2-yl)-N'-(methyl 3,4,6-tri-O-acetyl-beta-D-glucopyranosid-2-yl)-oxamide. Reaction of 2alpha with 1,2-diaminoethane afforded 1,2-bis-[N,N'-(methyl 3',4',6'-tri-O-acetyl-alpha-D-glucopyranosid-2'-yl)]ethyloxamide as a main product, while 2-N-[N'-(methyl 3',4',6'-tri-O-acetyl-alpha-D-glucopyranosid-2'-yl)oxamide]-ethyl acetamide was formed as a side product. Reaction of 2alpha with 1,3-diamino-2-hydroxypropane gave only 1,3-bis-N,N-[N'-(methyl 3',4',6'-tri-O-acetyl-2'-deoxy-alpha-D-glucopyranosid-2'-yl)-oxamido]-2-propanol.