Eudesmane alcohols from jasonia glutinosa

Three new sesquiterpene alcohols have been isolated from Jasonia glutinosa. Their structures were elucidated by spectroscopic methods and chemical correlations as (?)-[11R]-4α,14-epoxyeudesm-11,12-diol, (?)-[11R]-eudesm-4(14)-en-5β,11,12-triol and (+)-[11R]-eudesm-4(14)-en-5α,11,12-triol and they are called α-epoxy kudtdiol, 5-epi-kudtriol and kudtriol respectively.     

Abietane diterpenoids from the root of Salvia phlomoides

From the root of Salvia phlomoides three new abietane diterpenoids, demethylcryptojaponol, 14-deoxycoleon U and salviphlomone, have been isolated, besides the previously known diterpenes 8,13-abietadiene, 8,11,13-abietatriene, royleanone, 7α-acetoxyroyleanone, taxodione, taxodone, cryptojaponol and sugiol. The structures of demethylcryptojaponol (11,12-dihydroxy-8,11,13-abietatrien-7-one), 14-deoxycoleon U (6,11,12-trihydroxy-5,8,11,13-abietatetraen-7-one) and salviphlomone (6α,7β-dihydroxy-8,13-abietadiene-11,12-dione) were established by chemical and spectroscopic means.     

Taxusecone,a Novel Taxane with an Unprecedented 11,12-Secotaxane Skeleton,from Taxus cuspidata Needles

Taxusecone, 2α,7β,9α,10β-tetraacetoxy-5α,12-dihydroxy-11,12-secotax-4(20)-ene-11,13-dione (1), a novel taxane with an unprecedented skeleton, was isolated from the needles of Taxus cuspidata.     

A revised structure for the diterpene rosmanol

The diterpene rosmanol, previously isolated from Rosmarinus officinalis, has been isolated from the flowers of Salvia canariensis and its structure revised as 7α,11,12-trihydroxyabieta-8,11,13-trien-20-oic acid 20,6-lactone, on the basis of chemical evidence and an X-ray diffraction analysis.     

Diterpenes from the flowers of Salvia canariensis

The new diterpenes, 11,12-dimethoxy-abieta-6,8,11,13-tetraen-20-oic acid methyl ester, rosmanoyl carnosate, 7-oxocarnosic acid, 6-oxo-7β-hydroxycarnosic acid and 6-oxo-7α-hydroxycarnosic acid, together with the already known rosmanol, galdosol, isorol, carnosol and carnosic acid were isolated from the flowers of Salvia canariensis.     

The polar lipid composition of Mesorhizobium ciceri

The extractable lipid composition of Mesorhizobium ciceri strain HAMBI 1750 grown in a phosphate sufficient medium (79CA) is reported. Cardiolipin (CL—27% of total lipids), phosphatidylglycerol (PG—18%), phosphatidylethanolamine (PE—1%), phosphatidylcholine (PC—30%) and two methylated derivatives of PE, i.e. phosphatidyl-N, N-dimethylethanolamine (DMPE—1%) and phosphatidyl-N-monomethylethanolamine (MMPE—1%), were found to make up the phospholipids of the analysed bacteria. Nonphosphorus, ornithine-containing lipid (OL—10%) was also detected. Polar groups of phospholipids were predominantly acylated with cis-11,12-methyleneoctadecanoyl (lactobacillic) residues, whereas the ornithine lipid contained mainly 3-hexadecanoyloxy-11,12-methyleneoctadecanoic acid bound to the α-amino group.     

Degradation of Benzo[a]pyrene by Mycobacterium vanbaalenii PYR-1

Metabolism of the environmental pollutant benzo[a]pyrene in the bacterium Mycobacterium vanbaalenii PYR-1 was examined. This organism initially oxidized benzo[a]pyrene with dioxygenases and monooxygenases at C-4,5, C-9,10, and C-11,12. The metabolites were separated by reversed-phase high-performance liquid chromatography (HPLC) and characterized by UV-visible, mass, nuclear magnetic resonance, and circular dichroism spectral analyses. The major intermediates of benzo[a]pyrene metabolism that had accumulated in the culture media after 96 h of incubation were cis-4,5-dihydro-4,5-dihydroxybenzo[a]pyrene (benzo[a]pyrene cis-4,5-dihydrodiol), cis-11,12-dihydro-11,12-dihydroxybenzo[a]pyrene (benzo[a]pyrene cis-11,12-dihydrodiol), trans-11,12-dihydro-11,12-dihydroxybenzo[a]pyrene (benzo[a]pyrene trans-11,12-dihydrodiol), 10-oxabenzo[def]chrysen-9-one, and hydroxymethoxy and dimethoxy derivatives of benzo[a]pyrene. The ortho-ring fission products 4-formylchrysene-5-carboxylic acid and 4,5-chrysene-dicarboxylic acid and a monocarboxylated chrysene product were formed when replacement culture experiments were conducted with benzo[a]pyrene cis-4,5-dihydrodiol. Chiral stationary-phase HPLC analysis of the dihydrodiols indicated that benzo[a]pyrene cis-4,5-dihydrodiol had 30% 4S,5R and 70% 4R,5S absolute stereochemistry. Benzo[a]pyrene cis-11,12-dihydrodiol adopted an 11S,12R conformation with 100% optical purity. The enantiomeric composition of benzo[a]pyrene trans-11,12-dihydrodiol was an equal mixture of 11S,12S and 11R,12R molecules. The results of this study, in conjunction with those of previously reported studies, extend the pathways proposed for the bacterial metabolism of benzo[a]pyrene. Our study also provides evidence of the stereo- and regioselectivity of the oxygenases that catalyze the metabolism of benzo[a]pyrene in M. vanbaalenii PYR-1.     

Eudesmane sesquiterpenoid lactones and abietane diterpenoids from Ajuga forrestii Diels

Four eudesmane sesquiterpenoid lactones (1–4) and seven abietane diterpenoids (5–11) were isolated from the whole plants of Ajuga forrestii. Among them, 3α-acetoxy-1α,8β-dihydroxyeudesm-7(11)-en-8,12-olide (1), 3α-acetoxy-1α-hydroxyl-eudesm-8,7(11)-dien-8,12-olide (2), 1α-acetoxy-8α-oxyethyl-2-oxo-eudesman-3,7(11)-dien-8,12-olide (3) and 2α,3β,11,12-tetrahydroxy-7β,20-epoxy-8,11,13-abietatriene (11) are novel compounds. The structures of compounds 1–11 were determined by spectroscopic analysis. Compound 2 exhibited weak cytotoxicity on HepG2 and MCF-7 cell lines.     

Synthesis of homoursodeoxycholic acid and [11,12-3H]homoursodeoxycholic acid

Homoursodeoxycholic acid and [11,12-³H]homoursodeoxycholic acid were synthesized from ursodeoxycholic acid and homocholic acid, respectively. Ursodeoxycholic acid (Ia) was converted to 3α,7β-diformoxy-5β-cholan-24-oic acid (Ib) using formic acid. Reaction of the diformoxy derivative (Ib) with thionyl chloride yielded the acid chloride (II) which was treated with diazomethane to produce 3α,7β-diformoxy-25-diazo-25-homo-5β-cholan-24-one (III). Homoursodeoxycholic acid (IV) was formed from the diazoketone (III) by means of the Wolff rearrangement of the Arndt-Eistert synthesis.N-Bromosuccinimide oxidation of homocholic acid (V), which was prepared from cholic acid by the same procedure described above, afforded 3α,12α-dihydroxy-7-oxo-25-homo-5β-cholan-25-oic acid (VI). Reduction of the 7-ketohomodeoxycholic acid (VI) with sodium in 1-propanol gave 3α,7β,12α-trihydroxy-25-homo-5β-cholan-25-oic acid (VII). The methyl ester of 7-epihomocholic acid (VII) was partially acetylated to give methyl 3α,7β-diacetoxy-12α-hydroxy-25-homo-5β-cholan-25-oate (VIII) using a mixture of acetic anhydride, pyridine and benzene. Dehydration of the diacetoxy derivative (VIII) with phosphorus oxychloride yielded methyl 3α,7β-diacetoxy-25-homo-5β-chol-11-en-25-oate (IX). Reduction of the unsaturated ester (IX) with tritium gas in the presence of platinum oxide catalyst followed by alkaline hydrolysis gave [11,12-³H]homoursodeoxycholic acid.     

Microbial transformation of (+)-nootkatone and the antiproliferative activity of its metabolites

Six metabolites were obtained as a result of microbial transformation of (+)-nootkatone (1) by the fungal strains: Botrytis, Didymosphaeria, Aspergillus, Chaetomium and Fusarium. Their structure were established as (+)-(4R,5S,7R,9R)-9α-hydroxynootkatone (2), (+)-(4R,5S,7R)-13-hydroxynootkatone (3) and (+)-(4R,5S,7R,9R,11S)-11,12-epoxy-9α-hydroxynootkatone (4), (+)-(4R,5S,7R,11S)-11,12-epoksynootkatone (5), (+)-(4R,5S,7R)-11,12-dihydroxynootkatone (6) and (+)-(4R,5S,7R)-7,11,12-trihydroxynootkatone (7) on the basis of their spectral data. Two products: (4) and (7) were not previously reported in the literature. The antiproliferative activity of (+)-nootkatone (1) and isolated metabolites (2-7) of its biotransformation has been evaluated.     

A rapid and sensitive liquid chromatographic assay for epoxide hydrase.

A rapid method for the assay of epoxide hydrase activity is described. 3-Methylcholanthrene-11,12-oxide is employed as substrate and high speed liquid chromatography is used to separate and quantitate trans-11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene (product) formation. The determination of product at picomole levels can be obtained.     

Rat mesenteric arterial dilator response to 11,12-epoxyeicosatrienoic acid is mediated by activating heme oxygenase

11,12-Epoxyeicosatrienoic acid (11,12-EET), a potent vasodilator produced by the endothelium, acts on calcium-activated potassium channels and shares biological activities with the heme oxygenase/carbon monoxide (HO/CO) system. We examined whether activation of HO mediates the dilator action of 11,12-EET, and that of the other EETs, on rat mesenteric arteries. Dose-response curves (10(-9) to 10(-6) M) to 5,6-EET, 8,9-EET, 11,12-EET, 14,15-EET, and ACh (10(-9) to 10(-4) M) were evaluated in preconstricted (10(-6) mol/l phenylephrine) mesenteric arteries (<350 microm diameter) in the presence or absence of 1) the cyclooxygenase inhibitor indomethacin (2.8 microM), 2) the HO inhibitor chromium mesoporphyrin (CrMP) (15 microM), 3) the soluble guanylyl cyclase (GC) inhibitor ODQ (10 microM), and 4) the calcium-activated potassium channel inhibitor iberiotoxin (25 nM). The vasodilator response to 11,12-EET was abolished by CrMP and iberiotoxin, whereas indomethacin and ODQ had no effect. In contrast, the effect of ACh was attenuated by ODQ but not by CrMP. The vasodilator effect of 8,9-EET, like that of 11,12-EET, was greatly attenuated by HO inhibition. In contrast, the mesenteric vasodilator response to 5,6-EET was independent of both HO and GC, whereas that to 14,15-EET demonstrated two components, an HO and a GC, of equal magnitude. Incubation of mesenteric microvessels with 11,12-EET caused a 30% increase in CO release, an effect abolished by inhibition of HO. We conclude that the rat mesenteric vasodilator action of 11,12-EET is mediated via an increase in HO activity and an activation of calcium-activated potassium channels.     

11,12,20-Trihydroxy-eicosa-8(Z)-enoic acid: a selective inhibitor of 11,12-EET-induced relaxations of bovine coronary and rat mesenteric arteries

Arachidonic acid is metabolized to four regioisomeric epoxyeicosatrienoic acids (EETs) by cytochrome P-450. 5,6-, 8,9-, 11,12-, and 14,15-EET are equipotent in relaxing bovine coronary arteries (BCAs). Vasorelaxant effects of EETs are nonselectively antagonized by 14,15-epoxyeicosa-5(Z)-enoic acid. The 11,12-EET analogs, 20-hydroxy-11,12-epoxyeicosa-8(Z)-enoic acid (20-H-11,12-EE8ZE) and 11,12,20-trihydroxyeicosa-8(Z)-enoic acid (11,12,20-THE8ZE) were synthesized and tested for antagonist activity against EET-induced relaxations in BCAs. In U-46619-preconstricted arterial rings, 5,6-, 8,9-, 11,12-, and 14,15-EET caused concentration-dependent relaxations with maximal relaxations ranging from 80 to 96%. Preincubation of arteries with 20-H-11,12-EE8ZE (10(-5) M) inhibited relaxations to 14,15- and 11,12-EET, but not 5,6- and 8,9-EET; however, greatest inhibitory effect was against 11,12-EET (maximal relaxation = 80.6 ± 4.6 vs. 26.7 ± 7.4% without and with 20-H-11,12-EE8ZE, respectively). Preincubation with the soluble epoxide hydrolase inhibitor (tAUCB, 10(-6) M) significantly enhanced the antagonist effect of 20-H-11,12-EE8ZE against 14,15-EET-induced relaxations (maximal relaxation = 86.6 ± 4.4 vs. 27.8 ± 3.3%, without and with 20-H-11,12-EE8ZE and tAUCB) without any change in its effect against 11,12-EET-induced relaxations. In contrast to the parent compound, the metabolite, 11,12,20-THE8ZE (10(-5) M), significantly inhibited relaxations to 11,12-EET and was without effect on other EET regioisomers. Mass spectrometric analysis revealed conversion of 20-H-11,12-EE8ZE to 11,12,20-THE8ZE by incubation with BCA. The conversion was blocked by tAUCB. 14,15-Dihydroxy-eicosa-5Z-enoic acid (a 14,15-EET antagonist), but not 11,12,20-THE8ZE (an 11,12-EET antagonist), inhibited BCA relaxations to arachidonic acid and flow-induced dilation in rat mesenteric arteries. These results indicate that 11,12,20-THE8ZE is a selective antagonist of 11,12-EET relaxations and a useful pharmacological tool to elucidate the function of 11,12-EET in the cardiovascular system.     

Novel bioconversion products of andrographolide by Aspergillus ochraceus and their cytotoxic activities against human tumor cell lines

Andrographolide (1), a major labdane diterpenoidal constituent of a famous traditional Chinese of Andrographis paniculata, exhibits a wide spectrum of biological activities including antibacterial, anti-inflammatory, and antitumor properties. Bioconversion of andrographolide (1) by Aspergillus ochraceus (ATCC 1008) was investigated. Five bioconversion products were isolated and identified. Their structures were identified to be 8β-hydroxy-8(17)-dihydroandrographolide (2), 8β-hydroxy-8(17)-dihydro-14-deoxy-11,12-didehydroandrographolide (3), 8β-hydroxy-8(17)-dihydro-14-deoxy-11,12-didehydroandrographolide 19-oic acid (4), 14-deoxy-11,12-didehydroandrographolide (5), and 14-deoxy-11,12-didehydroandrographolide 19-oic acid (6). Metabolites 2–4 were novel compounds. The proposed biosynthetic pathways of andrographolide by A. ochraceus were drawn. Most bioconversion products showed potential cytotoxic activities against human breast cancer (MCF-7), human colon cancer (HCT-116) and leukemia (HL-60) cell lines.     

LC-MS/MS analysis of epoxyalcohols and epoxides of arachidonic acid and their oxygenation by recombinant CYP4F8 and CYP4F22

CYP4F22 and CYP4F8 are expressed in epidermis, and mutations of CYP4F22 are associated with lamellar ichthyosis. Epoxyalcohols (HEETs) and epoxides (EETs) of 20:4n−6 appear to be important for the water permeability barrier of skin. Our aim was to study the MS/MS spectra and fragmentation of these compounds and to determine whether they were oxidized by CYP4F22 or CYP4F8 expressed in yeast. HEETs were prepared from 15-hydroperoxyeicosatetraenoic acid (15-HPETE), 12-HPETE, and their [²H₈]labeled isotopomers, and separated by normal phase-HPLC with MS/MS analysis. CYP4F22 oxygenated 20:4n−6 at C-18, whereas metabolites of HEETs could not be identified. CYP4F8 formed ω3 hydroxy metabolites of HEETs derived from 12R-HPETE with 11,12-epoxy-10-hydroxy configuration, but not HEETs derived from 15S-HPETE. 8,9-EET and 11,12-EET were also subject to ω3 hydroxylation by CYP4F8. We conclude that CYP4F8 and CYP4F22 oxidize 20:4n−6 and that CYP4F8 selectively oxidizes 8,9-EET, 11,12-EET, and 10,11R,12R-HEET at the ω3 position.     

Synthesis and structural identification of four dihydroxy acids and 11,12-leukotriene C4 derived from 11,12-leukotriene A4

Using a partially purified 12-lipoxygenase from porcine leukocytes, (5Z,8Z,10E,14Z)-12-hydroperoxy-5,8,10,14-icosate traenoic acid was synthesized from arachidonic acid with a yield of over 35%. The absolute configuration of C-12 was determined as S by chiral-phase column chromatography. It was chemically converted to at least three epoxides with the conjugated triene structure. Two were identified by proton NMR and mass spectrometry to be (5Z,7E,9E,14Z)-(11S,12S)-11,12-oxido-5,7,9,14-ic osatetraenoic acid (11,12-leukotriene A4) and (5Z,7Z,9E,14Z)-(11S,12S)-11,12-oxido-5,7,9,14-ic osatetraenoic acid (7-cis-11,12-leukotriene A4). 11,12-Leukotriene A4 underwent acid hydrolysis to yield two diastereomers of (6E,8E,10E,14Z)-(12S)-5,12-dihydroxy-6,8,10,14-i cosatetraenoic acid and two isomers of (14Z)-(12S)-11,12-dihydroxy-5,7,9,14-icosatetraenoic acid. Upon incubation with rat liver glutathione S-transferase, 11,12-leukotriene A4 was converted to 11,12-leukotriene C4, a spasmogenic compound.     

A C-formylated azulene from Lactarius deterrimus

1-Formyl-4-methyl-7-isopropyl azulene (11,12-dihydro-lactaroviolin) was characterized from Lactarius deterrimus together with other known compounds.     

Identification of 15-hydroxy-11,12-epoxyeicosatrienoic acid as a vasoactive 15-lipoxygenase metabolite in rabbit aorta

Arachidonic acid (AA) causes endothelium-dependent smooth muscle hyperpolarizations and relaxations that are mediated by a 15-lipoxygenase-I (15-LO-I) metabolite, 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA). We propose that AA is metabolized sequentially by 15-LO-I and hydroperoxide isomerase to an unidentified hydroxyepoxyeicosatrienoic acid (HEETA), which is hydrolyzed by a soluble epoxide hydrolase (sEH) to 11,12,15-THETA. After incubation of aorta with 14C-labeled AA, metabolites were extracted and the HEETAs were resolved by performing HPLC. Mass spectrometric analyses identified 15-Hydroxy-11,12-epoxyeicosatrienoic acid (15-H-11,12-EETA). Incubation of aortic incubates with methanol and acetic acid trapped the acid-sensitive 15-H-11,12-EETA as methoxydihydroxyeicosatrienoic acids (MDHEs) (367 m/z, M-H). Pretreatment of the aortic tissue with the sEH inhibitor 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA; 10(-6) M) increased the formation of 15-H-11,12-EETA, measured as MDHEs. Thus 15-H-11,12-EETA is an acid- and sEH-sensitive precursor of 11,12,15-THETA. Aortic homogenates and endothelial cells contain a 57-kDa protein corresponding to the rabbit sEH. In preconstricted aortic rings, AA (10(-7)-10(-4) M) and acetylcholine (10(-9)-10(-6) M) caused concentration-related relaxations that were enhanced by pretreatment with AUDA. These enhanced relaxations were inhibited by increasing extracellular [K(+)] from 4.8 to 20 mM. AA (3 x 10(-6) M) induced cell membrane hyperpolarization (from -31.0 +/- 1 to -46.8 +/- 2 mV) in aortic strips with an intact endothelium, which was enhanced by AUDA. These results indicate that 15-H-11,12-EETA is produced by the aorta, hydrolyzed by sEH to 11,12,15-THETA, and mediates relaxations by membrane hyperpolarization. 15-H-11,12-EETA represents an endothelium-derived hyperpolarizing factor.     

Effects of autonomic blockers on linear and nonlinear indexes of blood pressure and heart rate in SHR

Endothelium-derived hyperpolarizing factor (EDHF) is released in response to agonists such as ACh and bradykinin and regulates vascular smooth muscle tone. Several studies have indicated that ouabain blocks agonist-induced, endothelium-dependent hyperpolarization of smooth muscle. We have demonstrated that epoxyeicosatrienoic acids (EETs), cytochrome P-450 metabolites of arachidonic acid, function as EDHFs. To further test the hypothesis that EETs represent EDHFs, we have examined the effects of ouabain on the electrical and mechanical effects of 14,15- and 11,12-EET in bovine coronary arteries. These arteries are relaxed in a concentration-dependent manner to 14,15- and 11,12-EET (EC(50) = 6 x 10(-7) M), bradykinin (EC(50) = 1 x 10(-9) M), sodium nitroprusside (SNP; EC(50) = 2 x 10(-7) M), and bimakalim (BMK; EC(50) = 1 x 10(-7) M). 11,12-EET-induced relaxations were identical in vessels with and without an endothelium. Potassium chloride (1-15 x 10(-3) M) inhibited [(3)H]ouabain binding to smooth muscle cells but failed to relax the arteries. Ouabain (10(-5) to 10(-4) M) increased basal tone and inhibited the relaxations to bradykinin, 11,12-EET, and 14,15-EET, but not to SNP or BMK. Barium (3 x 10(-5) M) did not alter EET-induced relaxations and ouabain plus barium was similar to ouabain alone. Resting membrane potential (E(m)) of isolated smooth muscle cells was -50.2 +/- 0.5 mV. Ouabain (3 x 10(-5) and 1 x 10(-4) M) decreased E(m) (-48.4 +/- 0.2 mV), whereas 11,12-EET (10(-7) M) increased E(m) (-59.2 +/- 2.2 mV). Ouabain inhibited the 11,12-EET-induced increase in E(m). In cell-attached patch clamp studies, 11,12-EET significantly increased the open-state probability (NP(o)) of a calcium-activated potassium channel compared with control cells (0.26 +/- 0.06 vs. 0.02 +/- 0.01). Ouabain did not change NP(o) but blocked the 14,15-EET-induced increase in NP(o). These results indicate that: 1) EETs relax coronary arteries in an endothelium-independent manner, 2) unlike EETs, potassium chloride does not relax the coronary artery, and 3) ouabain inhibits bradykinin- and EET-induced relaxations as has been reported for EDHF. These findings provide further evidence that EETs are EDHFs.     

The potential of Cyathus africanus for transformation of terpene substrates

The insecticidal sesquiterpenes cadina-4,10(15)-dien-3-one and aromadendr-1(10)-en-9-one were administered to the fungus Cyathus africanus ATCC 35853. Biotransformation of the former produced (4R)-9α-hydroxycadin-10(15)-en-3-one, while the latter gave 2β-hydroxyaromadendr-1(10)-en-9-one, 2α-hydroxyaromadendr-1(10)-en-9-one and 10α-hydroxy-1β,2β-epoxyaromadendran-9-one. The bioconversion of santonin led to the production of two analogues, 11,13-dihydroxysantonin and the hitherto unreported 8α,13-dihydroxysantonin, while cedrol yielded 3β,8β-dihydroxycedrane and 3α,8β-dihydroxycedrane. Stemod-12-ene, a diterpene, was transformed to 2-oxostemar-13-ene, a hitherto unknown analogue with a rearranged carbon framework. When methyl betulonate, a triterpenoid belonging to the lupane family, was supplied to the fungus 18α-ursane and 18α-oleanane derivatives, namely 19β-hydroxy-3-oxo-18α-oleanan-28-oic acid and 19α-hydroxy-3-oxo-18α-ursan-28-oic acids, were generated. There are no previous reports of fungal transformation of a triterpene in which a skeletal rearrangement occurred. All substrate administration experiments were done in the presence of the terpene cyclase inhibitor chlorocholine chloride (CCC), using the single phase – pulse feed method.