Some oxidation products of ethoxyquin including those found in autoxidising systems

2,4-Dimethyl-6-ethoxyquinoline (2), 1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline nitroxide (3), 2,6-dihydro-2,2,4-trimethyl-6-quinone imine N-oxide (4), 2,6-dihydro-2,2,4-trimethyl-6-quinone imine (5), 1,8′-di(1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline) (6) and 1,2-dihydro-6-hydroxy-2,2,4-trimethylquinoline (7) have been prepared from 1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline (1) (ethoxyquin) and their spectroscopic properties (UV, IR, mass and NMR) examined.     

Champanones, yellow pigments from the seeds of champa (Campomanesia lineatifolia)

Chemical investigation of the methanol extract of the seeds of Campomanesia lineatifolia Ruiz and Pav. (Myrtaceae) led to the isolation of two new beta-triketone type compounds, named champanones A (1) and B (2), together with the known 2,3-dihydro-5-hydroxy-6,8,8-trimethyl-2-phenyl-4H-1-benzopyran-4,7(8H)-dione (champanone C) (3). The structures of 1 and 2 were determined to be 2,2,4,4-tetramethyl-6-(1-oxo-3-phenylprop-2-enyl) cyclohexane-1,3,5-trione (occurs as an enol form) and 2,2,4-trimethyl-6-(1-oxo-3-phenylprop-2-enyl)cyclohexane-1,3,5-trione (occurs as an enol form), respectively, by means of spectroscopic analysis. The three compounds showed mild antimicrobial activity.     

Initial water content and lipase-mediated ester formation in hexane

Lipase biocatalysis was investigated as a tool for the production of esters by two model reactions, esterification of 1-butanol with 2-methyl-1-pentanoic acid and irreversible transesterification between 2-methyl-1-pentanol and vinyl acetate. The reactions were carried out in hexane using lipases from Candida cylindracea and porcine pancreas. The initial water content influenced both the yield of the ester and the enantioselectivity of the reaction (esterifica-tions) or the ester formation only (transesterifications).     

Identification of opdA, a gene involved in biodegradation of the endocrine disrupter octylphenol

Octylphenol (OP) is an estrogenic detergent breakdown product. Structurally similar nonylphenols are transformed via type II ispo substitution, resulting in the production of hydroquinone and removal of the branched side chain. Nothing is known, however, about the gene(s) encoding this activity. We report here on our efforts to clone the gene(s) encoding OP degradation activity from Sphingomonas sp. strain PWE1, which we isolated for its ability to grow on OP. A fosmid library of PWE1 DNA yielded a single clone, aew4H12, which accumulated a brown polymerization product in the presence of OP. Sequence analysis of loss-of-function transposon mutants of aew4H12 revealed a single open reading frame, opdA, that conferred OP degradation activity. Escherichia coli subclones expressing opdA caused OP disappearance, with the concomitant production of hydroquinone and 2,4,4-trimethyl-1-pentene as well as small amounts of 2,4,4-trimethyl-2-pentanol. These metabolites are consistent with a type II ipso substitution reaction, the same mechanism described for nonylphenol biodegradation in other sphingomonads. Based on opdA's sequence homology to a unique group of putative flavin monooxygenases and the recovery of hydroxylated OP intermediates from E. coli expressing opdA, we conclude that this gene encodes the observed type II ipso substitution activity responsible for the initial step in OP biodegradation.     

Chromenes from Peperomia serpens (Sw.) Loudon (Piperaceae)

Chromatographic separation of the CH2Cl2 extract from leaves of Peperomia serpens yielded two chromenes [5-hydroxy-8-(3',7'-dimethylocta-2',6'-dienyl)-2,2,7-trimethyl-2H-1-chromene (1) and 5-hydroxy-8-(3'-methyl-2'-butenyl)-2,2,7-trimethyl-2H-1-chromene-6-carboxylic acid (2)], besides the known chromene [methyl 5-hydroxy-2,2,7-trimethyl-2H-1-chromene-6-carboxylate (3)] and the flavonoid, dihydrooroxylin (4). Their structural elucidation were achieved by spectroscopic analyses. The antifungal activities of the CH2Cl2 extract and the isolated chromenes were measured bioautographically against Cladosporium cladosporioides and C. sphaerospermum, when it was found that the crude extract showed higher activity as compared to the pure compounds.     

In vitro toxicity of solubilized 2,3,4-trimethylpentane I. Cytotoxicity and metabolism of TMP using primary hepatocytes

Summary Primary rat hepatocyte suspension cultures (∼2×10⁶ cells) exposed to solubilized 2,3,4-trimethylpentane at concentrations ranging from 7.9 to 31.5 mM under two different culture conditions resulted in a linear dose response, as determined by lactate dehydrogenase leakage and viability data. A significant increase in the 2,3,4-trimethylpentane effective concentration 50 for primary hepatocytes occurred when exposures were implemented in medium containing 0.05% albumin. The effective concentration 50 for hepatocytes exposed to 2,3,4-trimethylpentane in medium lacking and containing albumin were 17.1 and 20.7 mM, respectively. Metabolite analysis by gas chromatography-mass spectrometry of supernatant (lacking or containing albumin) and cell extracts from hepatocyte cultures exposed to 2,3,4-trimethylpentane for 4 h indicated the presence of three metabolites: 2,3,4-trimethyl-1-pentanol, 2,3,4-trimethyl-2-pentanol, and 2,3,4-trimethyl-1-pentanoic acid. Electron microscopic examination of 2,3,4-trimethylpentane-exposed primary hepatocytes indicated ultrastructural changes which included abnormal condensed chromatin association with the nuclear membrane, swollen mitochondria, increased amounts of cytoplasmic lipid, significant los of microvilli from the cell surface, increased vacuolation, and increased numbers of peroxisomes. Although these changes were observed under both culture conditions, they were more severe in cultures lacking albumin. This study indicates that primary hepatocyte suspension cultures provide a useful system for rapidly identifying liver metabolites of selected test compounds of interest. Animals used in this study were handled in accordance with the principles stated in the Guide for the Care and Use of Laboratory Animals, prepared by the Committee on Care and Usage of Laboratory Animals of the Institute of Laboratory Animals Resources, National Research Council, DHHS, National Institute of Health publication 85–23, 1985, and the Animal Welfare Act of 1966, as amended. This material has been funded wholly or in part by the United States Air Force under contract F33615-85-C-0532 to NSI Technology Services Corporation. It has been subject to review by the United States Air Force and it has been approved for publication as a customer document. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.     

New Bitter C27 and C30 Terpenoids from the Fungus Ganoderma lucidum (Reishi)

Four new bitter terpenoids, lucidenic acids A (1), B (2), C (3) and ganoderic acid C (5), were isolated from the fruiting bodies of Ganoderma lucidum, together with the known bitter ganoderic acid B (4). On the basis of spectroscopic data and chemical conversion, their structures were determined to be 7β-hydroxy-4,4,14α-trimethyl-3,11,15-trioxo-5α-chol-8-en-24-oic acid, 7β,12β-dihydroxy-4,4,14α-trimethyl-3,11,15-trioxo-5α-chol-8-en-24-oic acid, 3β,7β,12β-trihydroxy-4,4,14α-trimethyl-11,15-dioxo-5α-chol-8-en-24-oic acid and 7β-hydroxy-3,11,15,23-tetraoxo-5α-lanost- 8-en-26-oic acid, respectively.     

5(Z)-benzylidene-1,2-dihydro-9-hydroxy-10-methoxy-2,2,4-trimethyl-5H-1-aza-6-oxa-chrysenes as non-steroidal glucocorticoid receptor modulators

A series of 5-benzylidene-1,2-dihydro-2,2,4-trimethyl-5H-1-aza-6-oxa-chrysenes was synthesized and profiled for their ability to act as selective glucocorticoid receptor modulators (SGRMs). The synthesis and structure-activity relationships for this series of compounds are presented.     

Identification of opdA, a Gene Involved in Biodegradation of the Endocrine Disrupter Octylphenol

Octylphenol (OP) is an estrogenic detergent breakdown product. Structurally similar nonylphenols are transformed via type II ispo substitution, resulting in the production of hydroquinone and removal of the branched side chain. Nothing is known, however, about the gene(s) encoding this activity. We report here on our efforts to clone the gene(s) encoding OP degradation activity from Sphingomonas sp. strain PWE1, which we isolated for its ability to grow on OP. A fosmid library of PWE1 DNA yielded a single clone, aew4H12, which accumulated a brown polymerization product in the presence of OP. Sequence analysis of loss-of-function transposon mutants of aew4H12 revealed a single open reading frame, opdA, that conferred OP degradation activity. Escherichia coli subclones expressing opdA caused OP disappearance, with the concomitant production of hydroquinone and 2,4,4-trimethyl-1-pentene as well as small amounts of 2,4,4-trimethyl-2-pentanol. These metabolites are consistent with a type II ipso substitution reaction, the same mechanism described for nonylphenol biodegradation in other sphingomonads. Based on opdA's sequence homology to a unique group of putative flavin monooxygenases and the recovery of hydroxylated OP intermediates from E. coli expressing opdA, we conclude that this gene encodes the observed type II ipso substitution activity responsible for the initial step in OP biodegradation.     

Biotransformation of alpha-isomethylionone to 1-(2,6,6-trimethyl-2-cyclohexen-1-yl)propan-2-one

The main biodegradation product of (+/-)-alpha-isomethylionone (2) with standard activated sludge was characterized as (+/-)-1-(2,6,6-trimethyl-2-cyclohexen-1-yl)propan-2-one (1) by its analysis and synthesis. Both enantiomers (1a and 1b) of 1 were synthesized by starting from (R)- and (S)-2,4,4-trimethyl-2-cyclohexen-1-ol (3a and 3b), respectively.     

Modulation of insulin secretion by lipoxygenase products of arachidonic acid. Relation to lipoxygenase activity of pancreatic islets

Glucose (16.7 mM)-induced insulin secretion from isolated pancreatic islets of rats was inhibited by nordihydroguaiaretic acid (NDGA), 1-phenyl-3-pyrazolidinone (phenidone), 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline (BW755C), 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone (AA861), and 2,6-di-tert-butyl-4-methylphenol (BHT). Indomethacin and aspirin, however, failed to inhibit the glucose-induced insulin secretion but rather tended to enhance it. The glucose-induced insulin secretion was inhibited by 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) (50 microM), 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) (100 microM), and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) (100 microM), but not by 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) (100 microM). Exogenous 5-HETE (10 microM) induced significant insulin secretion in a low glucose (3.3 mM) medium. Racemic 5-HETE also showed insulinotropic effect in a concentration-dependent manner with the concentrations 20 microM or above, whereas 12-HETE, 15-HETE, 15-HPETE, 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid, 5-hydroxy-6-glutathionyl-7,9,11,14-eicosatetraenoic acid, 5-hydroxy-6-cysteinylglycinyl-7,9,11,14-eicosatetraenoic acid, prostaglandin E2, and prostaglandin F2 alpha failed to induce insulin secretion. Although significant insulin release was observed with arachidonic acid (greater than or equal to 100 microM), reduce cell viability was evident at 200 microM. When the 10,000 X g supernatant of isolated pancreatic islet homogenate was incubated with [3H]arachidonic acid at 37 degrees C in the presence of GSH and Ca2+, and the labeled metabolites then extracted with ethyl acetate and subjected to reverse phase high pressure liquid chromatography, several radioactive peaks, coeluted with authentic 15-, 12-, and 5-HETE, were observed. The radioactive peaks were completely suppressed by the addition of either NDGA, BW755C, or phenidone into the medium. The results support our contention i.e. the involvement of lipoxygenase product(s) in the secretory mechanism of insulin, and further suggest that 5-lipoxygenase system may play a role.     

Apotrisporin-E: A new sesquiterpenoid isolated fromPhycomyces blakesleeanus andBlakeslea trispora

A new sesquiterpenoid has been identified in two species of Mucorales. It is apotrisporin-E: 2,4,4-trimethyl-3-[5′-hydroxyl-3′-methylpenta-(1′E,3′E)-dienyl]-(5S)-hydroxylcyclohex-2-enone. Nuclear Overhauser enhancement studies indicated that thecis-trans isomeric assignments at carbon atom 3′ based upon the NMR signals for the hydrogens on carbon atoms 1′ and 2′ were valid. The existence of apotrisporin-E implies that trisporin-D and trisporin-E, two putative pheromones, are made by the Mucorales.     

Formation of the metabolic intermediate 2,4,4-trimethyl-2-pentanol during incubation of a shape Sphingomonas sp. strain with the xeno-estrogenic octylphenol

Degradation of branched octylphenol was studied in abacterial culture of a Sphingomonas sp. strain.Octylphenol is considered to be the most stabledegradation intermediate formed from the correspondingnonionic octylphenol polyethoxylates surfactantsduring biological wastewater treatment. Sinceoctylphenol can exert estrogenic effects in wildlife,a detailed study of its biodegradation is warranted.The aerobic microbiological transformation ofoctylphenol was examined with and without the additionof the easily assimilable sodium acetate. In bothcases the formation of the metabolite2,4,4-trimethyl-2-pentanol, representing the intactalkyl chain as a tertiary alcohol, was observed. Sincethe octylphenol degradation rate was not affected bythe presence of acetate, this strain did not show anydiauxic metabolic behaviour when incubated withoctylphenol and sodium acetate as the sources ofcarbon and energy. As a result of thebiotransformation of octylphenol, its estrogenicpotency was removed because it is the phenolic moietythat interacts with the estrogen receptors. Thisfeature opens perspectives for the use of this strainin the framework of an adequate treatment ofwastewater with high levels of alkylphenolpolyethoxylates.     

Chemical Studies on Components of Dried Bonito, “Katsuobushi”

The flavor concentrate obtained by the extraction of “Katsuobushi” of bonito (Katsuwonus pelamis) with 80% ethanol and by the subsequent steam distillation of the extract was fractionated by the usual method and the resulting neutral, non-carbonyl oxygenated fraction was investigated by gas chromatography. The following components were tentatively identified: 2-pentanol and 2-methyl-1-heptanol as free alcohols, and 4 alcohols of n- and isobutanol, n-pentanol and n-dodecanol and 9 carboxylic acids of propanoic, n-butanoic, n-pentanoic, n-octanoic, n-nonanoic n-decanoic, n-dodecanoic, n-tetradecanoic and n-hexadecanoic acid as the constituents of esters. A constituent alcohol existing in the largest amount was isolated by gas chromatography and identified as 2-methyl-1-heptanol by elemental analysis, NMR, IR, and MS. A constituent acid existing in large amount was also isolated and investigated similarly, and the structure was partially estimated. 2-Methyl-1-heptanol holds a fresh woody aroma and seems to have a major effect on “Katsuobushi” flavor.     

Antiparasitic activity of prenylated benzoic acid derivatives from Piper species

Fractionation of dichloromethane extracts from the leaves of Piper heterophyllum and P. aduncum afforded three prenylated hydroxybenzoic acids, 3-[(2E,6E,10E)-11-carboxy-3,7,15-trimethyl-2,6,10,14-hexadecatetraenyl)-4,5-dihydroxybenzoic acid, 3-[(2E,6E,10E)-11-carboxy-13-hydroxy-3,7,15-trimethyl-2,6,10,14-hexadecatetraenyl]-4,5-dihydroxybenzoic acid and 3-[(2E,6E,10E)-11-carboxy-14-hydroxy-3,7,15-trimethyl-2,6,10,15-hexadecatetraenyl]-4,5-dihydroxybenzoic acid, along with the known compounds, 4,5-dihydroxy-3-(E,E,E-11-formyl-3,7,15-trimethyl-hexadeca-2,6,10,14-tetraenyl)benzoic acid (arieianal), 3,4-dihydroxy-5-(E,E,E-3,7,11,15-tetramethyl-hexadeca-2,6,10,14-tetraenyl)benzoic acid, 4-hydroxy-3-(E,E,E-3,7,11,15-tetramethyl-hexadeca-2,6,10,14-tetraenyl)benzoic acid, 3-(3,7-dimethyl-2,6-octadienyl)-4-methoxy-benzoic acid, 4-hydroxy-3-(3,7-dimethyl-2,6-octadienyl)benzoic acid and 4-hydroxy-3-(3-methyl-1-oxo-2-butenyl)-5-(3-methyl-2-butenyl)benzoic acid. Their structures were elucidated on the basis of spectroscopic data, including homo- and heteronuclear correlation NMR experiments (COSY, HSQC and HMBC) and comparison with data reported in the literature. Riguera ester reactions and optical rotation measurements established the compounds as racemates. The antiparasitic activity of the compounds were tested against three strains of Leishmania spp., Trypanosoma cruzi and Plasmodium falciparum. The results showed that 3-(3,7-dimethyl-2,6-octadienyl)-4-methoxy-benzoic acid exhibited potent and selective activity against L. braziliensis (IC₅₀ 6.5 μg/ml), higher that pentamidine used as control. Moreover, 3-[(2E,6E,10E)-11-carboxy-3,7,15-trimethyl- 2,6,10,14-hexadecatetraenyl)-4,5-dihydroxybenzoic acid and 4-hydroxy-3-(3-methyl-1-oxo-2-butenyl)-5-(3-methyl-2-butenyl)benzoic acid showed moderate antiplasmodial (IC₅₀ 3.2 μg/ml) and trypanocidal (16.5 μg/ml) activities, respectively.     

Interference of 7,8-dihydroneopterin with peroxynitrite-mediated reactions

By in vitro studies 7,8-dihydroneopterin, which is secreted by macrophages stimulated by interferon-gamma, was reported to be a radical scavenger as well as a prooxidative agent depending on the experimental settings. In this study, we investigated the interference of 7,8-dihydroneopterin with peroxynitrite mediated reactions by different analytical procedures. Luminol chemiluminescence and oxidation of the spin probe 1-hydroxy-2,2,6,6-tetramethyl-4-oxo-piperidine induced by peroxynitrite were inhibited by 7,8-dihydroneopterin. On the other hand, we found that 7,8-dihydroneopterin very efficiently inhibits nitration of tyrosine by peroxynitrite. Hydroxylation, however, was rather enhanced than inhibited, suggesting that 7,8-dihydroneopterin reacts in quite different manner with the intermediates generated from peroxynitrite. We provide the first evidence that a pterin radical is formed from a dihydropterin using EPR spectroscopy and 2,2,4-trimethyl-2H-imidazole-1-oxide as a spin trap. We conclude that 7,8-dihydroneopterin while being a weak scavenger of superoxide acts as a very efficient inhibitor of tyrosine nitration induced by peroxynitrite.     

Three isoflavanones with cannabinoid-like moieties from Desmodium canum

Three further derivatives of 5,7,2',4'-tetrahydroxy-6-methyl isoflavanone have been isolated from the root extract of Desmodium canum and assigned the structures 2,3-dihydro-5,7-dihydroxy-6-methyl-3-(1a,2,3,3a,8b,8c-hexahydro-6-hydroxy-1,1,3a-trimethyl-1H-4-oxabenzo[f]cyclobut[c,d]inden-7-yl)-4H-1-benzopyran-4-one (1) 2,3-dihydro-5,7-dihydroxy-6-methyl-3-(6a,7,8,10a-tetrahydro-3-hydroxy-6,6,9-trimethyl-6H-dibenzo[b,d]pyran-2-yl)-4H-1-benzopyran-4-one (2) 2,3-dihydro-5,7-dihydroxy-6-methyl-3-(3-hydroxy-6,6,9-trimethyl-6H-dibenzo[b,d]pyran-2-yl) 4H-1-benzopyran-4-one (3). The three compounds and the previously isolated chromene 4 all derive from the geranylated precursor 5 by a series of cannabinoid-like oxidative rearrangements.     

Insulinotropic effect of the tumor promoter teleocidin in isolated pancreatic islets

A tumor promoter teleocidin induced insulin secretion from isolated pancreatic islets in a concentration-dependent manner. The teleocidin-induced secretion was inhibited by p-bromophenacyl bromide, nordihydroguaiaretic acid, 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline and 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone, but not by indomethacin. Insulinotropic concentrations of teleocidin stimulated 6-keto-prostaglandin F1 alpha release from pancreatic islets. These results suggest that phospholipase A2 activation and lipoxygenase product(s) are involved in the mechanism of teleocidin-induced insulin secretion.     

Unusual naphthoquinones, catechin and triterpene from Byrsonima microphylla

The new triterpene Delta1-lupenone (1), together with lupeol, beta-amyrin and betulin were isolated from the wood of Byrsonima microphylla (Malpighiaceae). The new compounds 3-hydroxy-2-methoxy-8,8,10-trimethyl-8H-antracen-1,4,5-trione (2), 3,7-dihydroxy-2-methoxy-8,8,10-trimethyl-7,8-dihydro-6H-antracen-1,4,5-trione (3), (2S*,10aR*)-2,8-dihydroxy-6-methoxy-1,1,7-trimethyl-2,3,10, 10a-tetrahydro-1H-fenantren-9-one (4) and (2S,3S)-3'-hydroxy-4',5,7-trimethoxy-flavan-3-ol (5) were also isolated through monitored TLC using the antioxidant beta-carotene reagent. The antioxidant potential of the compounds 2-5 was measured and none of them showed activity. The structures of these compounds were elucidated by chemical and spectroscopic analysis based on NMR techniques (1H, 13C NMR, COSY, nOe difference, HMQC and HMBC), UV and MS.     

Effects of Cyclohexanonic Long-Chain Fatty Alcohol, tCFA15 on Amino Acids in Diabetic Rat Brain: A Preliminary Study

The aim of this study was to evaluate the effects of streptozotocin-induced type 1diabetes and a subchronic treatment with cyclohexanonic long-chain fatty alcohol, 3-(15-hydroxypentadecyl)-2,4,4-trimethyl-2-cyclohexen 1-one (tCFA15) on contents of amino acids including aspartate, glutamate, glutamine, GABA, glycine, taurine, alanine, serine, threonine, and arginine in the prefrontal cortex, hippocampus and striatum. Levels of glutamate, threonine, taurine, alanine, arginine, and the ratio of glutamate/glutamine were altered region-differently in the brain of diabetic rats. However, tCFA15 region-specifically antagonized the changes in taurine and arginine levels and the ratio of glutamate/glutamine. The alteration in glutamate/glutamine ratio may indicate that experimental models of type 1 diabetes have abnormalities of neuron-gria interaction in brain.