Ds-erythro-2-amino-4-ethoxy-3-hydroxybutanoic acid from the fruiting bodies of the edible mushroom,Lyophyllum ulmarium

A new β-hydroxy amino acid isolated from the fruiting bodies of Lyophyllum ulmarium was identified as D_s,-erythro-2-amino-4-ethoxy-3-hydroxybutanoic acid by chemical degradation and spectroscopic analyses.     

Oxygenation of 4-Alkoxyl Groups in Alkoxybenzoic Acids by Polyporus dichrous

The degradation of several alkyl ethers of vanillic acid, of 3-ethoxy-4-hydroxybenzoic acid, and of syringic acid, by the lignin-decomposing fungus Polyporus dichrous included (i) 4-dealkylation (e.g., 3-ethoxy-4-isopropoxybenzoic acid was in part dealkylated to 3-ethoxy-4-hydroxybenzoic acid), (ii) hydroxylation of the 4-alkoxyl groups (e.g., 3-ethoxy-4-isopropoxybenzoic acid was oxidized in part to 2-[4-carboxy-2-ethoxyphenoxy]-propane-1-ol), and (iii) reduction of carboxyl groups (older cultures) (e.g., 3-ethoxy-4-isopropoxybenzoic acid was reduced to 3-ethoxy-4-isopropoxybenzaldehyde and 3-ethoxy-4-isopropoxybenzyl alcohol). Some ethers (e.g., tri-O-methyl gallic acid and glycerol-beta-[4-carboxy-2-ethoxyphenyl]-ether) were not affected. The dealkylations and hydroxylations indicate that the fungus has a relatively nonspecific mechanism for oxygenating various 4-alkoxyl groups of alkoxybenzoic acids; no evidence for oxygenation of 3-alkoxyl groups was obtained. Hydroxylation products were generally degraded further, probably via dealkylation. The vanillic acid and 3-ethoxy-4-hydroxybenzoic acid formed by dealkylations were readily metabolized. Although the isopropyl ether of syringic acid was hydroxylated to 2-(4-carboxy-2, 6-dimethoxyphenoxy)-propane-1-ol, neither this compound nor the parent isopropyl ether was dealkylated; syringic acid itself was only slowly and incompletely metabolized. The relationship of these results to lignin degradation is discussed.     

Synthesis and evaluation of (S)-2-ethoxy-3-phenylpropanoic acid derivatives as insulin-sensitizing agents

A series of (S)-2-ethoxy-3-phenylpropanoic acid derivatives were synthesized and their insulin-sensitizing activities were evaluated in 3T3-L1 cells. Compounds 1b and 1d exhibited more potent insulin-sensitizing activity than rosiglitazone.     

Formation of 4-ethoxy-4'-nitrosodiphenylamine in the reaction of the phenacetin metabolite 4-nitrosophenetol with glutathione

Phenacetin, a constituent of several analgesic and antipyretic formulations has been made responsible for a variety of toxic and carcinogenic actions. 4-Nitrosophenetol, the N-oxydation product of intermediate 4-phenetidine, forms methemoglobin and binds covalently to sulfhydryl groups of proteins and glutathione. In the reaction of 4-nitrosophenetol with glutathione and other thiols an intermediate so-called "semimercaptal" is formed from which N-(thiol-S-yl)-4-phenetidine S-oxide, N-(thiol-S-yl)-4-phenetidine and 4-phenetidine derive. Besides thiol adducts, a yellow compound is formed which was isolated as a pure crystalline product (elemental analysis) and identified by FAB-MS, EI-MS, 13C-, 1H-NMR, and UV-VIS spectroscopy as 4-ethoxy-4'-nitrosodiphenylamine. This nitrosoarene is formed by an unknown mechanism from 4-nitrosophenetol and 4-phenetidine under liberation of ethanol. In human erythrocytes this compound is easily reduced to 4-amino-4'-ethoxydiphenylamine (FAB-MS, EI-MS, 13C-NMR). During the reaction of 4-nitrosophenetol with red cells only traces of 4-ethoxy-4'-nitrosodiphenylamine were formed, whereas up to 10% appeared as the reduction product 4-amino-4'-ethoxydiphenylamine. This latter compound is unstable in red cells and is metabolized further to unidentified products.     

Synthesis and evaluation of (S)-2-ethoxy-3-phenylpropanoic acid derivatives as insulin-sensitizing agents

A series of (S)-2-ethoxy-3-phenylpropanoic acid derivatives were synthesized and their insulin-sensitizing activities were evaluated in 3T3-L1 cells. Compounds 1b and 1d exhibited more potent insulin-sensitizing activity than rosiglitazone.     

A facile synthesis of 1-ethoxy-4-cyano-5-ethoxycarbonyl-3H-azuleno[1,2-c]pyran-3-one, a selective 15-lipoxygenase inhibitor

A facile method to synthesize 1-ethoxy-4-cyano-5-ethoxycarbonyl-3H-azuleno[1,2-c]pyran-3-one, in yield of 92%, which showed selective inhibition effect on 15-lipoxygenase(soybean source) at IC(50)=24.2+/-2.7 microM while no inhibition effect was observed at greater than 300 microM on 5-lipoxygenase, lipid peroxidase, phospholipase A(2), protein kinase C, and cyclooxygenase.     

Degradation of the gamma-Carboxyl-Containing Diarylpropane Lignin Model Compound 3-(4'-Ethoxy-3'-Methoxyphenyl)-2-(4'-Methoxyphenyl)Propionic Acid by the Basidiomycete Phanerochaete chrysosporium

The white-rot basidiomycete Phanerochaete chrysosporium metabolized 3-(4'-ethoxy-3'-methoxyphenyl)-2-(4'-methoxyphenyl)propionic acid (V) in low-nitrogen, stationary cultures, conditions under which ligninolytic activity is expressed. The ability of several fungal mutant strains to degrade V reflected their ability to degrade [C]lignin to CO(2). 1-(4'-Ethoxy-3'-methoxyphenyl)-2-(4'-methoxyphenyl)-2- hydroxyethane (VII), anisyl alcohol, and 4-ethoxy-3-methoxybenzyl alcohol were isolated as metabolic products, indicating an initial oxidative decarboxylation of V, followed by alpha, beta cleavage of the intermediate (VII). Exogenously added VII was rapidly converted to anisyl alcohol and 4-ethoxy-3-methoxybenzyl alcohol. When the degradation of V was carried out under O(2), O was incorporated into the beta position of the diarylethane product (VII), indicating that the reaction is oxygenative.     

Degradation of the γ-Carboxyl-Containing Diarylpropane Lignin Model Compound 3-(4′-Ethoxy-3′-Methoxyphenyl)-2-(4″-Methoxyphenyl)Propionic Acid by the Basidiomycete Phanerochaete chrysosporium

The white-rot basidiomycete Phanerochaete chrysosporium metabolized 3-(4′-ethoxy-3′-methoxyphenyl)-2-(4″-methoxyphenyl)propionic acid (V) in low-nitrogen, stationary cultures, conditions under which ligninolytic activity is expressed. The ability of several fungal mutant strains to degrade V reflected their ability to degrade [¹⁴C]lignin to ¹⁴CO₂. 1-(4′-Ethoxy-3′-methoxyphenyl)-2-(4″-methoxyphenyl)-2- hydroxyethane (VII), anisyl alcohol, and 4-ethoxy-3-methoxybenzyl alcohol were isolated as metabolic products, indicating an initial oxidative decarboxylation of V, followed by α, β cleavage of the intermediate (VII). Exogenously added VII was rapidly converted to anisyl alcohol and 4-ethoxy-3-methoxybenzyl alcohol. When the degradation of V was carried out under ¹⁸O₂, ¹⁸O was incorporated into the β position of the diarylethane product (VII), indicating that the reaction is oxygenative.     

Reaction of porcine pancreatic elastase with 7-substituted 3-alkoxy-4-chloroisocoumarins: design of potent inhibitors using the crystal structure of the complex formed with 4-chloro-3-ethoxy-7-guanidinoisocoumarin

The crystal structure of the acyl enzyme formed upon inhibition of porcine pancreatic elastase (PPE) by 4-chloro-3-ethoxy-7-guanidinoisocoumarin has been determined at a 1.85-A effective resolution. The chlorine atom is still present in this acyl enzyme, in contrast to the previously reported structure of the 7-amino-4-chloro-3-methoxyisocoumarin-PPE complex where the chlorine atom has been replaced by an acetoxy group. The guanidino group forms hydrogen bonds with the carbonyl group and side-chain hydroxyl group of Thr-41, and the acyl carbonyl group has been twisted out of the oxyanion hole. Molecular modeling indicates that the orientation of the initial Michaelis enzyme-inhibitor complex is quite different from that of the acyl enzyme since simple reconstruction of the isocoumarin ring would result in unfavorable interactions with Ser-195 and His-57. Molecular models were used to design a series of new 7-(alkylureido)- and 7-(alkylthioureido)-substituted derivatives of 3-alkoxy-7-amino-4-chloroisocoumarin as PPE inhibitors. All the 3-ethoxyisocoumarins were better inhibitors than those in the 3-methoxy series due to better interactions with the S1 pocket of PPE. The best ureido inhibitor also contained a tert-butylureido group at the 7-position of the isocoumarin. Due to a predicted interaction with a small hydrophobic pocket on the surface of PPE, this isocoumarin and a related phenylthioureido derivative are among the best irreversible inhibitors thus far reported for PPE (kobs/[I] = 8100 M-1 s-1 and 12,000 M-1 s-1). Kinetic studies of the stability of enzyme-inhibitor complexes suggest that many isocoumarins are alkylating the active site histidine at pH 7.5 via a quinone imine methide intermediate, while at pH 5.0, the predominant pathway appears to be simple formation of a stable acyl enzyme derivative.     

Preparation of S14161 and its analogues and the discovery of 6-bromo-8-ethoxy-3-nitro-2H-chromene as a more potent antitumor agent in vitro

The small chemical compound 8-ethoxy-2-(4-fluorophenyl)-3-nitro-2H-chromene (S14161) was recently identified as an inhibitor of the phosphoinositide 3-kinase (PI3K). In the present study, we designed a novel synthesis of S14161 and prepared a series of its analogues via the oxa-Michael–Henry reaction in the presence of catalytic amounts of l-proline and triethylamine. Further structural simplification led to the identification of 6-bromo-8-ethoxy-3-nitro-2H-chromene (BENC-511) that exhibited potent antiproliferative activities against a panel of 12 tumor cell lines. Compared with S14161, BENC-511 was more potent in blocking the AKT phosphorylation and inducing cancer cell apoptosis. BENC-511 also displayed more potent effects on human umbilical vein epithelial cells (HUVEC) migration, suggesting its anti-angiogenesis activity.     

Promotion of mesocotyl growth in etiolated rice seedlings by 4-ethoxy-1-(p-tolyl)-s-triazine-2,6(1H,3H)-dione

4-Ethoxy-1-(p-tolyl)-s-triazine-2,6(1H,3H)-dione (TA) promoted mesocotyl growth in dark-grown rice (Oryza sativa L.) seedlings. In cultivars of the japonica type TA alone showed a small promotive effect and TA+gibberellic acid(GA₃) had a marked synergistic effect, while in other cultivars, mostly of the indica type, TA alone showed a great promotive effect and TA+GA₃ had only an additive effect. In cv. Nato, a typical representative of cultivars showing the second type of response, the concentration of TA giving the greatest growth promotion was around 0.1–0.2 mM. In Nato seedlings treated with TA at 0.1 mM, the mesocotyls continued to elongate for 6 days and reached about 75 mm in length, while the mesocotyls of control seedlings grew to a maximum of about 10 mm and growth was limited to the first 3 days after planting. The TA-induced mesocotyl elongation was mainly the consequence of increased cell multiplication in the meristematic area immediately below the coleoptilar node. GA₃, abscisic acid (ABA) and ethylene also stimulated mesocotyl growth in dark-grown Nato seedlings but their effects were much smaller than those of TA. ABA, like GA₃, had an additive effect with TA, but ethylene suppressed the effect of TA and resulted in increased lateral expansion in the upper region of the mesocotyls of TA-treated seedlings.Abbreviations ABA abscisic acid - GA(s) gibberellin(s) - GA₃ gibberellic acid - TA 4-ethoxy-1-(p-tolyl)-s-triazine-2,6(1H,3H)-dione Part 5 in the series Plant Growth-regulating Activities of Isourea Derivatives and Related Compounds; Part 4=Ogawa et al. (1978)     

High-performance liquid chromatographic method for measurement of cytochrome P450-mediated metabolism of 7-ethoxy-4-trifluoromethylcoumarin

An HPLC method for analysis of deethylation of 7-ethoxy-4-trifluoromethylcoumarin (ETFMC), a substrate of various enzymes of the cytochrome P450 superfamily, was developed. ETFMC was incubated at 37°C with human hepatic microsomes or microsomes prepared from a lymphoblastoid cell line that expresses human CYP2B6. Under these conditions, the highly fluorescent metabolite 7-hydroxy-4-trifluoromethylcoumarin (HTFMC) is formed. The metabolite was analyzed by reversed-phase HPLC with fluorescence detection. The limits of detection of the metabolite were 5.0 fmol per injection, a sensitivity at least one order of magnitude greater than the standard method, which does not involve HPLC. This method will be of great utility when quantities of microsomal protein from cell lines expressing human CYP enzymes are limited.     

The C-C bond cleavage of a lignin model compound, 1,2-diarylpropane-1,3-diol, with a heme-enzyme model catalyst tetraphenylporphyrinatoiron(III)chloride in the presence of tert-butylhydroperoxide

The catalytic C-C bond cleavage of a lignin model compound was investigated by use of tetraphenylporphyrinatoiron(III)chloride as a model for enzymic degradation of lignin. The C-C bond of the lignin model compound 1,2-bis(4-ethoxy-3-methoxyphenyl) propane-1,3-diol was oxidatively cleaved by catalysis of iron-porphyrins in the presence of tert-butylhydroperoxide or iodosylbenzene at a room temperature. The products formed after complete oxidation of the substrate were identified as 4-O-ethylvanillin, alpha-hydroxy-4-ethoxy-3-methoxyacetophenone, 4-O-ethylvanillic acid, 4-ethoxy-3-methoxyphenylglycol, 4-ethoxy-3-methoxy-alpha-(4-ethoxy-3-methoxyphenyl)-beta-hydroxypropi ophenone and formaldehyde.     

Efficient introduction of phosphorothioates into RNA oligonucleotides by 3-ethoxy-1,2,4-dithiazoline-5-one (EDITH).

We recently reported on the use of 1,2,4-dithiazolidine-3,5-dione (DtsNH) and 3-ethoxy-1,2,4-dithiazoline-5-one (EDITH) as effective sulfurizing reagents for the preparation of phosphorothioate-containing oligodeoxyribo-nucleotides [Xu et al. (1996) Nucleic Acids Res., 24, 1602-1607]. One challenge in automated solid-phase synthesis of phosphorothioate-containing RNA is to develop sulfurization reagents that are effective in the presence of bulky 2'-OH protecting groups. The present study demonstrates that EDITH is exceedingly effective at low concentrations (0.05 M) and short reaction times (2 min) for the automated synthesis of oligoribonucleotides.     

Discovery of 2-ethoxy-4-(methoxymethyl)benzamide derivatives as potent and selective PTP1B inhibitors

Protein tyrosine phosphatase 1B (PTP1B), a key negative regulator of insulin signaling, is considered as a promising and validated therapeutic target for type 2 diabetes mellitus (T2DM) and obesity. Upon careful study, a series of 2-ethoxy-4-(methoxymethyl)benzamide and 2-ethoxy-5-(methoxymethyl)benzamide analogs designed by the “bioisosteric principle” were discovered, wherein their PTP1B inhibitory potency, type of PTP1B inhibition, selectivity and membrane permeability were evaluated. Among them, compound 10m exhibited high inhibitory activity (IC₅₀ = 0.07 μM), significant selectivity (32-fold) over T-cell PTPase (TCPTP) as well as good membrane permeability (P_app = 2.41 × 10⁻⁶ cm/s). Further studies on cell viability and cellular activity revealed that compound 10m could enhance insulin-stimulated glucose uptake with no significant cytotoxicity.     

Arylglycerol-γ-Formyl Ester as an Aromatic Ring Cleavage Product of Nonphenolic β-O-4 Lignin Substructure Model Compounds Degraded by Coriolus versicolor

4-Ethoxy-3-methoxyphenylglycerol-γ-formyl ester (compound IV) was identified as a degradation product of both 4-ethoxy-3-methoxyphenylglycerol-β-syringaldehyde ether (compound I) and 4-ethoxy-3-methoxyphenylglycerol-β-2,6-dimethoxyphenyl ether (compound II) by a ligninolytic culture of Coriolus versicolor. An isotopic experiment with a ¹³C-labeled compound (compound II′) indicated that the formyl group of compound IV was derived from the β-phenoxyl group of β-O-4 dimer as an aromatic ring cleavage fragment. However, compound IV was not formed from 4-ethoxy-3-methoxyphenylglycerol-β-guaiacyl ether (compound III). γ-Formyl arylglycerol (compound IV) could be a precursor of 4-ethoxy-3-methoxyphenylglycerol (compound VI), because 3-(4-ethoxy-3-methoxyphenyl)-1-formyloxy propane (compound VII) was cleaved to give 3-(4-ethoxy-3-methoxyphenyl)-1-propanol (compound VIII) by C. versicolor. 4-Ethoxy-3-methoxyphenylglycerol-β,γ-cyclic carbonate (compound V), previously found as a degradation product of compound III by Phanerochaete chrysosporium (T. Umezawa, and T. Higuchi, FEBS Lett., 25:123-126, 1985), was also identified from the cultures with compound I, II, and III and degraded to give the arylglycerol (compound VI). An isotopic experiment with ¹³C-labeled compounds II′ and III′ indicated that the carbonate carbon of compound V was derived from the β-phenoxyl groups of β-O-4 substructure.     

Arylglycerol-gamma-Formyl Ester as an Aromatic Ring Cleavage Product of Nonphenolic beta-O-4 Lignin Substructure Model Compounds Degraded by Coriolus versicolor

4-Ethoxy-3-methoxyphenylglycerol-gamma-formyl ester (compound IV) was identified as a degradation product of both 4-ethoxy-3-methoxyphenylglycerol-beta-syringaldehyde ether (compound I) and 4-ethoxy-3-methoxyphenylglycerol-beta-2,6-dimethoxyphenyl ether (compound II) by a ligninolytic culture of Coriolus versicolor. An isotopic experiment with a C-labeled compound (compound II') indicated that the formyl group of compound IV was derived from the beta-phenoxyl group of beta-O-4 dimer as an aromatic ring cleavage fragment. However, compound IV was not formed from 4-ethoxy-3-methoxyphenylglycerol-beta-guaiacyl ether (compound III). gamma-Formyl arylglycerol (compound IV) could be a precursor of 4-ethoxy-3-methoxyphenylglycerol (compound VI), because 3-(4-ethoxy-3-methoxyphenyl)-1-formyloxy propane (compound VII) was cleaved to give 3-(4-ethoxy-3-methoxyphenyl)-1-propanol (compound VIII) by C. versicolor. 4-Ethoxy-3-methoxyphenylglycerol-beta,gamma-cyclic carbonate (compound V), previously found as a degradation product of compound III by Phanerochaete chrysosporium (T. Umezawa, and T. Higuchi, FEBS Lett., 25:123-126, 1985), was also identified from the cultures with compound I, II, and III and degraded to give the arylglycerol (compound VI). An isotopic experiment with C-labeled compounds II' and III' indicated that the carbonate carbon of compound V was derived from the beta-phenoxyl groups of beta-O-4 substructure.     

Epoxidation and reduction of cholesterol, 1,4,6-cholestatrien-3-one and 4,6-cholestadien-3beta-ol

Many naturally occurring polyhydroxylated sterols and oxysterols exhibit potent biologic activities. This paper describes reagent and position selectivity of epoxidation and reduction of cholesterol derivatives. Cholesterol was reacted with m-chloroperoxybenzoic acid (m-CPBA) to form 5alpha,6alpha-epoxycholestan-3beta-ol, but in reaction with 30% H(2)O(2), it did not reacted. 1,4,6-cholestatrien-3-one was obtained from cholesterol and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone in dioxane. 1,4,6-cholestatrien-3-one was reacted with 30% H(2)O(2) and 5% NaOH in methanol to give 1alpha,2alpha-epoxy-4,6-cholestadien-3-one, which was stereoselectively reduced with NaBH(4) to form 1alpha,2alpha-epoxy-4,6-cholestadien-3beta-ol and reduced with Li metal in absolute ethanol to give 2-ethoxy-1,4,6-cholestatrien-3-one. And 1,4,6-cholestatrien-3-one was epoxidized with m-CPBA in dichloromethane to afford 6alpha,7alpha-epoxy-1,4-cholestadien-3-one, which was reacted with NaBH(4) to synthesize 6alpha-hydroxy-4-cholesten-3-one and reduced Li metal in absolute ethanol to form 2-ethoxy-1,4,6-cholestatrien-3-one, respectively. 1,4,6-cholestatrien-3-one was reduced with NaBH(4) in absolute ethanol to form 4,6-cholestadien-3beta-ol, which was reacted with 30% H(2)O(2) to leave original compound, but was reacted with m-CPBA to give 4beta,5beta-epoxy-6-cholesten-3beta-ol as the major product and 4beta,5beta-epoxy-6alpha,7alpha-epoxycholestan-3beta-ol as the minor product.     

Aromatic ring cleavage of a non-phenolic beta-O-4 lignin model dimer by laccase of Trametes versicolor in the presence of 1-hydroxybenzotriazole

The novel cleavage products, 2,3-dihydroxy-1-(4-ethoxy-3-methoxyphenyl)-1-formyloxypropane (II) and 1-(4-ethoxy-3-methoxyphenyl)-1,2,3-trihydroxypropane-2,3-cyclic carbonate (III) were identified as products of a non-phenolic beta-O-4 lignin model dimer, 1,3-dihydroxy-2-(2,6-dimethoxylphenoxy)-1-(4-ethoxy-3-methoxypheny l)propane (I), by a Trametes versicolor laccase in the presence of 1-hydroxybenzotriazole (1-HBT). An isotopic experiment with a 13C-labeled lignin model dimer, 1,3-dihydroxy-2-(2,6-[U-ring-13C] dimethoxyphenoxy)-1-(4-ethoxy-3-methoxyphenyl)propane (I-13C) indicated that the formyl and carbonate carbons of products II and III were derived from the beta-phenoxy group of beta-O-4 lignin model dimer I as aromatic ring cleavage fragments. These results show that the laccase-1-HBT couple could catalyze the aromatic ring cleavage of non-phenolic beta-O-4 lignin model dimer in addition to the beta-ether cleavage, Calpha-Cbeta cleavage, and Calpha-oxidation.     

Use of 1,2,4-dithiazolidine-3,5-dione (DtsNH) and 3-ethoxy-1,2,4-dithiazoline-5-one (EDITH) for synthesis of phosphorothioate-containing oligodeoxyribonucleotides.

Previous methods for the preparation of phosphorothioate-containing oligodeoxyribonucleotides rely on the reaction of phosphite triesters with sulfurizing reagents such as tetraethylthiuram disulfide (TETD) and 3H-1,2-benzodithiol-3-one 1,1-dioxide (Beaucage reagent). However, these and other sulfurizing reagents suffer from several disadvantages, and there is great impetus for the development of improved methods for sulfur transfer that are fully compatible with standard automated DNA synthesis. The present report describes the use of 1,2,4-dithiazolidine-3,5-dione (DtsNH) and 3-ethoxy-1,2,4-dithiazoline-5-one (EDITH) as effective sulfurizing reagents that meet these needs. Both reagents are easily prepared, and are stable upon prolonged room temperature storage in acetonitrile solution. The reagents are used at low concentrations (0.05 M) and for short reaction times (30 s). The methodology has been proven for the automated synthesis on 0.2-1.0 micromol scales of oligodeoxyribonucleotides, of length 6-20 bases, containing the phosphorothioate substitution at either a single site or at all positions.