Short-chain 3-ketoceramides, strong apoptosis inducers against human leukemia HL-60 cells

Ceramides act as a second messenger of the apoptotic signaling process. The allylic alcohol portion comprising the C-3, C-4, and C-5 carbons is essential for this function. The suggestion has been made that this alcohol moiety is oxidized in mitochondria to a carbonyl moiety, with the generation of reactive oxygen species. However, there is no established precedent for the apoptotic performance of 3-ketoceramides thus presumed. In this work, we have synthesized three different types of short-chain 3-ketoceramides, that is, (2S,4E)-2-acetylamino-3-oxo-4-octadecen-1-ol (A), (2S,4E,6E)-2-acetylamino-3-oxo-4,6-octadecadien-1-ol (B), and (2S,4E)-2-acetylamino-1-methoxy-3-oxo-4-octadecene (C), and demonstrated that these 3-ketoceramides are capable of inducing effective apoptosis in human leukemia HL-60 cells. In particular, the two monoenoic compounds, A and C, are far more powerful than the corresponding alcoholic analogue, N-acetyl-D-erythro-sphingosine. Observations of DNA fragmentation, caspase-3 activation, and cytochrome c release from mitochondria provide substantiated evidence for mitochondrial apoptosis and the effects of exogenous glutathione on these phenomena are also discussed.     

2S,4R-carboxy-2-acetylamino-4-piperidine dans les feuilles de Calliandra haematocephala

A new naturally occurring pipecolic acid derivative has been isolated from leaves of the legume Calliandra haematocephala. Its structure was shown to be 2S,4R-carboxy-2-acetylamino-4-piperidine by chemical and spectrosopic methods.     

Benzoic acid and pyridine derivatives as inhibitors of Trypanosoma cruzi trans-sialidase

Benzoic acid and pyridine derivatives inhibit recombinant trans-sialidase from Trypanosoma cruzi with I50 values between 0.4 and 1mM. The best compounds, 4-acetylamino-3-hydroxymethylbenzoic acid and 5-acetylamino-6-aminopyridine-2-carboxylic acid, provide new leads to inhibitors not containing the synthetically complex sialic acid structure. The weak inhibition by such compounds contrasts with their much stronger inhibition of neuraminidase from Influenza virus.     

Effects of glutathione reductase inhibition on cellular thiol redox state and related systems

Although inhibition of glutathione reductase (GR) has been demonstrated to cause a decrease in reduced glutathione (GSH) and increase in glutathione disulfide (GSSG), a systematic study of the effects of GR inhibition on thiol redox state and related systems has not been noted. By employing a monkey kidney cell line as the cell model and 2-acetylamino-3-[4-(2-acetylamino-2-carboxy-ethylsulfanylthio carbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA) as a GR inhibitor, an investigation of the effects of GR inhibition on cellular thiol redox state and related systems was conducted. Our study demonstrated that, in addition to a decrease in GSH and increase in GSSG, 2-AAPA increased the ratios of NADH/NAD⁺ and NADPH/NADP⁺. Significant protein glutathionylation was observed. However, the inhibition did not affect the formation of reactive oxygen species or expression of antioxidant defense enzyme systems [GR, glutathione peroxidase, catalase, and superoxide dismutase] and enzymes involved in GSH biosynthesis [γ-glutamylcysteine synthetase and glutathione synthetase].     

A Method for the Synthesis of Aldehydes

Aldehydes were synthesized via α-acetoxy sulfldes from alkyl aryl sulfoxides by the Pummerer reaction with acetic anhydride in the presence of base. By this method, isobutyraldehyde, pentanal, benzaldehyde, and 2-acetylamino-5-methy]benzaldehyde were obtained from isobutyl phenyl sulfoxide, pentyl p-tolyl sulfoxide, benzyl phenyl sulfoxide, and 2-acetylamino-5-methylbenzyl phenyl sulfoxide, respectively. The starting sulfoxides are easily synthesized from olefins, alkyl halides, or alkyl aryl sulfldes.     

Inhibition of Bovine Carbonic Anhydrase by New Sulfonamide Compounds

Inhibitory effects of three new derivatives of 2-acetylamino-1,3,4-thiadiazole-5-sulfonamide on bovine carbonic anhydrase have been investigated. The new compounds are 2-(3-chloropropionylamino)-1,3,4-thiadiazole-5-sulfonamide, 2-(2,2-dichloroacetylamino)-1,3,4-thiadiazole-5-sulfonamide, and 2-(3-phenylpropionylamino)-1,3,4-thiadiazole-5-sulfonamide. The new compounds inhibit the esterase activity of carbonic anhydrase noncompetitively and have inhibition constants and I ₅₀ values very similar to those for 2-acetylamino-1,3,4-thiadiazole-5-sulfonamide, the latter being clinically used in the treatment of glaucoma.     

Reaction products of the carcinogen N-hydroxy-4-acetylamino-4'-fluorobiphenyl with DNA in liver and kidney of the rat

The binding of AABP4'F and ABP4'F residues to rat liver and kidney DNA in vivo was studied at different periods of time after administration of N-[G-3H]hydroxy-AABP4'F at dose levels of 5 and 25 mg/kg body weight. DNA preparations from both organs were hydrolyzed enzymatically at pH 8--9 with mixtures of DNAase, snake venom phosphodiesterase and alkaline phosphatase from Escherichia coli. The enzymatic digests were analysed by Sephadex LH-20 chromatography using synthetic N-([G-14C] deoxyguanosin-8-yl)-AABP4'F as marker. Elution with 30% ethanol gave three major peaks of tritium activity. The first peak consisted largely of N-(deoxyguanosin-8-yl)-ABP4'F decomposition products, which were not further characterized. The second product has similar chromatographical and chemical properties as 3-(deoxyguanosin-N2-yl)-AAF; and was also persistent in liver as well as in kidneys. The third peak of tritium activity co-chromatographed with the marker compound N-([G-14C] deoxyguanosin-8-yl)-AABP4'F. Kinetic studies revealed that the latter product was removed rapidly from liver and kidney DNA at equal rates (t1/2 = 2 days). Approximately 80% of the total radioactivity bound to DNA consisted of deacetylated material, which was removed at a much slower rate (t1/2 = 10 days) in both organs. An initial rapid removal of all products in kidney during the first 7 days (t1/2 = 3.3 days) at dose levels of 25 mg/kg is probably due to toxic effects on the kidneys, because this phenomenon was not observed at dose levels of 5 mg/kg. The synthetic ester N-OSO3K-AABP4'F was at least twice as reactive towards L-methionine and guanosine as compared to the corresponding AABP derivative, but had 40% of the reactivity of N-acetoxy-AAF under similar conditions. The new compounds 3-methylmercapto-4-acetylamino-4'-fluorobiphenyl and N-(deoxyguanosin-8-yl)-4-acetylamino-4'-fluorobiphenyl have been characterized by means of their NMR and mass spectra. Attempts to devise an unambiguous synthesis for 3-(deoxyguanosin-N2-yl)arylamides have been unsuccessful.     

Evaluation of a dithiocarbamate derivative as an inhibitor of human glutaredoxin-1

Context: Glutaredoxins (GRX) are involved in the regulation of thiol redox state. GRX-1 is a cytosolic enzyme responsible for the catalysis of deglutathionylation of proteins. To date, very few inhibitors of GRX-1 have been reported.

Objective: The objective of this paper is to report 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethyl-sulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA) as an inhibitor of human GRX-1.

Materials and methods: The mechanism of inhibition of GRX-1 was investigated using dialysis, substrate protection, and mass spectrometry.

Results: 2-AAPA inhibits GRX-1 in a time and concentration dependent manner. The activity did not return following dialysis indicating that inhibition is irreversible. Results of substrate protection and mass spectrometry indicate that the inhibition is occurring at the active site. The compound also produced GRX inhibition in human ovarian cancer cells.

Discussion: 2-AAPA is an irreversible GRX-1 inhibitor with similar or greater potency compared to previously reported inhibitors.

Conclusion: The inhibition of GRX-1 by 2-AAPA could be used as a tool to study thiol redox state.     

O(3)-(2-Acetylamino-2-deoxy-β-d-glucopyranosyl)-oleanolic acid,a novel triterpenoid glycoside from two Pithecellobium species

A new triterpenoid glycoside containing an amino sugar moiety has been isolated from Pithecellobium cubense and P. arboreum and identified as O(3)- (2-acetylamino-2-deoxy-β-d-glucopyranosyl)oleanolic acid. β-d-Glucopyranosyl-α-spinasterol was also obtained from P. cubense.     

Antioxidant and anti-inflammatory activities of N-acetyldopamine dimers from Periostracum Cicadae

A known N-acetyldopamine dimer, (2R,3S)-2-(3',4'-dihydroxyphenyl)-3-acetylamino-7-(N-acetyl-2'-aminoethyl)-1,4-benzodioxane (1) and a new N-acetyldopamine dimer, (2R,3S)-2-(3',4'-dihydroxyphenyl)-3-acetylamino-7-(N-acetyl-2'-aminoethylene)-1,4-benzodioxane (2) were isolated from the methanolic extracts of Periostracum Cicadae. Compounds 1 and 2 inhibited the Cu2+ -mediated, 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH)-mediated, and 3-morpholinosydnonimine (SIN)-1-mediated LDL oxidation in the thiobarbituric acid-reactive substances (TBARS) assay. The antioxidant activities of 1 and 2 were tested with respect to other parameters, such as lag time of conjugated diene formation, relative electrophoretic mobility (REM), and apoB-100 fragmentation on copper-mediated LDL-oxidation. Compounds 1 and 2 also showed 1,1-diphenyl-2-picrylhydrasyl (DPPH) radical scavenging activity. Compound 2 was more efficient than compound 1 at inhibiting the reactive oxygen species (ROS) generation, nitric oxide (NO) production, and nuclear factor-kappaB (NF-kappaB) activity as well as the expression of pro-inflammatory molecules such as inducible nitric oxide synthase (iNOS), interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, and cyclooxygenase (COX)-2 in LPS-induced RAW264.7 cells.     

The Synthesis of Ribosides of Asymmetrically-Substituted Aminohalogenobenzimidazoles

Abstract

A series of ribosides of asymmetrically-substituted amino-halogenobenzimidazoles has been prepared by reaction of a suitably protected ribose with 6(5)-fluoro-5(6)-formylaminobenzimidazole (1), 5-amino-4-chloro-6-fluorobenzimidazole (2), 5-acetylamino-4,7-dichloro-benzimidazole (13), 5(6)-amino-6(5)-fluoro-2-methylbenzimidazole (20) and 6(5)-fluoro-5(6)-formylamino-2-methylbenzimidazole (21).     

Formation of a diazoquinone-type mutagen from acetaminophen treated with nitrite under acidic conditions

Acetaminophen (paracetamol) showed mutagenicity to Salmonella typhimurium TA100 and TA98 either with or without S9 mix when treated with excess nitrite in acidic solution. The mutagen was isolated and identified as 4-acetylamino-6-diazo-2,4-cyclohexadienone. Mutation tests and product analysis by high-performance liquid chromatography, however, suggested that this type of mutagen was hardly formed in the digestive tract of normal subjects.     

Extractionless method for the determination of urinary caffeine metabolites using high-performance liquid chromatography coupled with tandem mass spectrometry

Caffeine is metabolised in humans primarily by cytochromes P450 1A2 and 2A6, xanthine dehydrogenase/oxidase, and N-acetyltransferase 2. The activities of these enzymes show a large variation due to genetic polymorphisms and/or induction by xenobiotics. Ratios of different caffeine metabolites in urine or other body fluids are frequently used to characterise the individual/actual activity of these enzymes. The common analytical method involves extensive sample preparation, followed by HPLC-UV. The presence of numerous other UV-absorbing chemicals in body fluids affects the sensitivity and selectivity of this method. We have developed an HPLC-electrospray-MS-MS method for the determination of 11 caffeine metabolites and two internal standards after a simple, extractionless preparation. Blank urine, obtained after 5 days on a methylxanthine-free diet, contained small amounts of some caffeine metabolites, but no other components producing any confounding signals. Eleven metabolites and internal standards were recovered at 90 to 110% after addition to the blank urine (0.1 to 2.5 micro M in the final sample involving a 20-fold dilution of urine) in the 0.1-2.5 micro M concentration range. Other metabolites, 5-acetylamino-6-amino-3-methyluracil (AAMU) and 5-acetylamino-6-formylamino-3-methyluracil (AFMU), were detected with similar recovery and precision, but required higher concentrations (3 to 30 micro M). AFMU was completely converted into AAMU by a short alkalisation of urine. The method was explored in six healthy individuals after consuming coffee (4 mg caffeine per kg body mass). These experiments demonstrated the simplicity, high sensitivity and selectivity of the method under conditions used for phenotyping.     

Antigenic polysaccharides of Shigella bacteria. Structure of the polysaccharide chain of the lipopolysaccharide from Shigella boydii, type 11]

On mild acid degradation of the Shigella boydii, type 11 lipopolysaccharide, the corresponding O-specific polysaccharide composed of D-glucuronic acid, 2-acetylamino-2-deoxy-D-glucose, D-ribose and L-rhamnose residues in the ratio 1:1:1:3 was obtained. Methylation, partial acid hydrolysis and 13C-NMR spectral data for the polysaccharide led to the structure of the oligosaccharide repeating unit as a branched hexasaccharide: [formula: see text]. Numerous O-acetyl groups attached non-stoichiometrically to the residues of D-glucuronic acid, L-rhamnose and 2-acetylamino-2-deoxy-D-glucose were located with the use of 13C-NMR spectroscopy.     

Isolation and identification of non-chlorinated phenylbenzotriazole (non-ClPBTA)-type mutagens in the Ho River in Shizuoka Prefecture, Japan

We previously identified 2-[2-(acetylamino)-4-amino-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA) congeners as major mutagens in water concentrates from several rivers that flow in three different areas, i.e. Kyoto, Aichi, and Fukui Prefectures, in Japan. In synthesis studies, these PBTAs were shown to be formed from corresponding dinitrophenylazo dyes via non-chlorinated derivatives (non-ClPBTAs). However, only non-ClPBTA-1, i.e. 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-6-amino-4-bromo-2H-benzotriazole, had been detected as a minor contaminant in the Nishitakase River in Kyoto. In this study, analysis of mutagens in water concentrate from the Ho River, which flows through an area with a textile dyeing industry in Shizuoka Prefecture, Japan, allowed the isolation of four compounds (I, II, III, and IV). These four mutagens were identified as 2-[2-(acetylamino)-4-[N-(2-cyanoethyl)ethylamino]-5-methoxyphenyl]-6-amino-4-bromo-2H-benzotriazole (non-ClPBTA-2), 2-[2-(acetylamino)-4-[(2-hydroxyethyl)amino]-5-methoxyphenyl]-6-amino-4-bromo-2H-benzotriazole (non-ClPBTA-3), 2-(2-acetylamino-4-amino-5-methoxyphenyl)-6-amino-4-bromo-2H-benzotriazole (non-ClPBTA-4), and 2-[2-(acetylamino)-4-(diethylamino)-5-methoxyphenyl]-6-amino-4-bromo-2H-benzotriazole (non-ClPBTA-7) by spectral data and co-chromatography using synthesized standards. Non-ClPBTA-3 and -7 were highly mutagenic in Salmonella typhimurium YG1024, inducing 159,000 and 178,000 revertants/microg, respectively, in the presence of S9 mix. Like PBTAs, non-ClPBTAs might have been produced from azo dyes during industrial processes in dyeing factories and released into rivers.     

Evaluation of a dithiocarbamate derivative as a model of thiol oxidative stress in H9c2 rat cardiomyocytes

Thiol redox state (TRS) refers to the balance between reduced thiols and their corresponding disulfides and is mainly reflected by the ratio of reduced and oxidized glutathione (GSH/GSSG). A decrease in GSH/GSSG, which reflects a state of thiol oxidative stress, as well as thiol modifications such as S-glutathionylation, has been shown to have important implications in a variety of cardiovascular diseases. Therefore, research models for inducing thiol oxidative stress are important tools for studying the pathophysiology of these disease states as well as examining the impact of pharmacological interventions on thiol pathways. The purpose of this study was to evaluate the use of a dithiocarbamate derivative, 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA), as a pharmacological model of thiol oxidative stress by examining the extent of thiol modifications induced in H9c2 rat cardiomyocytes and its impact on cellular functions. The extent of thiol oxidative stress produced by 2-AAPA was also compared to other models of oxidative stress including hydrogen peroxide (H₂O₂), diamide, buthionine sulfoximine, and N,N׳-bis(2-chloroethyl)-N-nitroso-urea. Results indicated that 2-AAPA effectively inhibited glutathione reductase and thioredoxin reductase activities and decreased the GSH/GSSG ratio by causing a significant accumulation of GSSG. 2-AAPA also increased the formation of protein disulfides as well as S-glutathionylation. The alteration in TRS led to a loss of mitochondrial membrane potential, release of cytochrome c, and increase in reactive oxygen species production. Compared to other models, 2-AAPA is more potent at creating a state of thiol oxidative stress with lower cytotoxicity, higher specificity, and more pharmacological relevance, and could be utilized as a research tool to study TRS-related normal and abnormal biochemical processes in cardiovascular diseases.     

Synthesis and pharmacological evaluation of 4H-1,4-benzothiazine-2-carbonitrile 1,1-dioxide and N-(2-cyanomethylsulfonylphenyl)acylamide derivatives as potential activators of ATP sensitive potassium channels

1,2,4-Thiadiazine derivatives, like 3-methyl-7-chlorobenzo-4H-1,2,4-thiadiazine 1,1-dioxide, diazoxide and 7-chloro-3-isopropylamino-4H-benzo-1,2,4-thiadiazine 1,1-dioxide, BPDZ 73, are potent openers of Kir6.2/SUR1 K(ATP) channels. To explore the structure-activity relationship of this series of K(ATP) openers, 4H-1,4-benzothiazine-2-carbonitrile 1,1-dioxide and N-(2-cyanomethylsulfonylphenyl)acylamide derivatives were synthesized from 2-acetylamino-5-chloro-benzenesulfonic acid pyridinium salt or 2-aminobenzenethiols. The 4H-1,4-benzothiazine-2-carbonitrile 1,1-dioxide derivatives (e.g., 7-chloro-3-isopropylamino-4H-1,4-benzothiazine-2-carbonitrile 1,1-dioxide, 3f) were found to activate K(ATP) channels as indicated by their ability to hyperpolarize beta cell membrane potential, to inhibit glucose-stimulated insulin release in vitro and to increase ion currents through Kir6.2/SUR1 channel as measured by patch clamp. The potency and efficacy of, for example, 3f is however significantly reduced compared to the corresponding 4H-1,2,4-benzothiadiazine 1,1-dioxide derivatives. Opening of the 4H-1,2,4-thiadiazine ring to get (e.g., 2-cyanomethylsulfonyl-4-fluorophenyl) carbamic acid isopropyl ester (4c) gives rise to compounds, which are able to open K(ATP) channels but with considerable reduced potency compared to, for example, diazoxide. Compound 3a, 7-chloro-3-methyl-4H-1,4-benzothiazine-2-carbonitrile 1,1-dioxide, which inhibits insulin release in vitro from beta cells and rat islets, reduces plasma insulin levels and blood pressure in anaesthetized rats upon intravenous administration.     

Emeriamine, an antidiabetic beta-aminobetaine derived from a novel fungal metabolite

Emeriamine [(R)-3-amino-4-trimethylaminobutyric acid], derived from a novel fungal metabolite "emericedin" [(R)-3-acetylamino-4-trimethylaminobutyric acid], was proved to be a strong and specific inhibitor of carnitine-dependent oxidation of long chain fatty acid (IC50; 3.2 X 10(-6)M) and its main inhibition site was shown to be carnitine palmitoyltransferase I located on the outer-surface of the mitochondrial inner membrane. Emeriamine also showed hypoglycemic and antiketogenic activities in a dose-dependent manner (1 - 10 mg/kg) when administered orally to fasted normal and diabetic animals.     

Oxidative transformation of 2-acetylaminofluorene by a chemical model for cytochrome P450: A water-insoluble porphyrin and tert-butyl hydroperoxide

Oxidation of 2-acetylaminofluorene (AAF), a carcinogen, by a chemical model for cytochrome P450 was investigated to identify an active mutagen and elucidate the oxidation pathway. The oxidation system consisted of a water-insoluble tetrakis(pentafluorophenyl)porphyrinatoiron(III) chloride and tert-butyl hydroperoxide. The mutagen derived from AAF by the chemical model was 2-nitro-9-fluorenone (NO(2)=FO), which was mutagenic in Salmonella typhimurium TA1538. AAF was oxidized initially at position 9 of the fluorene carbon by the chemical model forming 2-acetylamino-9-fluorenol (AAF-OH), and then oxidized further to 2-acetylamino-9-fluorenone (AAF=O) as a major product. Initial oxidation of the nitrogen formed 2-nitrofluorene (NO(2)F), and further oxidation yielded 2-nitro-9-fluorenol (NO(2)F-OH) as a minor product. These products, AAF-OH, AAF=O, NO(2)F, and NO(2)F-OH, and their presumable common intermediate, N-hydroxy-2-acetylaminofluorene, were oxidized by the chemical model, and the formation of NO(2)F=O was determined. These results showed that NO(2)F=O was the mutagen derived from AAF in the presence of the chemical model and was formed via oxidation of N-OH-AAF, NO(2)F, and NO(2)F-OH. These results may lead to a new metabolic pathway of AAF.     

Synthesis of imidazol-2-yl amino acids by using cells from alkane-oxidizing bacteria

Sixty-one strains of alkane-oxidizing bacteria were tested for their ability to oxidize N-(2-hexylamino-4-phenylimidazol-1-yl)-acetamide to imidazol-2-yl amino acids applicable for pharmaceutical purposes. After growth with n-alkane, 15 strains formed different imidazol-2-yl amino acids identified by chemical structure analysis (mass and nuclear magnetic resonance spectrometry). High yields of imidazol-2-yl amino acids were produced by the strains Gordonia rubropertincta SBUG 105, Gordonia terrae SBUG 253, Nocardia asteroides SBUG 175, Rhodococcus erythropolis SBUG 251, and Rhodococcus erythropolis SBUG 254. Biotransformation occurred via oxidation of the alkyl side chain and produced 1-acetylamino-4-phenylimidazol-2-yl-6-aminohexanoic acid and the butanoic acid derivative. In addition, the acetylamino group of these products and of the substrate was transformed to an amino group. The product pattern as well as the transformation pathway of N-(2-hexylamino-4-phenylimidazol-1-yl)-acetamide differed in the various strains used.