Dual antioxidant structures with potent anti-inflammatory,hypolipidemic and cytoprotective properties

Novel amide derivatives of trolox, 3,5-di-tert-butyl-4-hydroxybenzoic acid, (E)-3-(3,5-di-tert-butyl-4-hydroxyphenyl)acrylic acid and cinnamic acid with cysteamine and l-cysteine ethyl ester were synthesised. In four cases, the disulfide derivatives were also isolated and tested. All compounds were examined for antioxidant activity, expressed as their ability to inhibit lipid peroxidation and to scavenge free radicals. They were found to demonstrate up to 17-fold better activity than that of the parent antioxidant acids. They could reduce acute inflammation up to 87%. The most active antioxidant compounds were further tested for their in vivo hypolipidemic effect, which ranged from 47% to 73%, and for their ability to protect the liver against oxidative toxicity caused by high paracetamol dose. The disulfide derivatives of 3,5-di-tert-butyl-4-hydroxybenzoic acid and cinnamic acid had no antioxidant activity and presented equal or lower anti-inflammatory effect than their thiol analogues, indicating that their molecular characteristics may not permit biological barrier penetration.     

Synthesis and evaluation of dithiolethiones as novel cyclooxygenase inhibitors

3H-1,2-Dithiole-3-thiones substituted with a 3,5-di-tert-butyl-4-hydroxyphenyl (DTBHP) or a 3,5-di-tert-butyl-4-methoxyphenyl group at the C5 position were prepared and their ability to inhibit the cyclooxygenase isoenzymes, COX-1 and COX-2 was evaluated. Both compounds were potent inhibitors of COX-2 (relative to rofecoxib), and while the phenol was a weak inhibitor of COX-1, the methyl ether gave no measurable inhibition. Docking studies of the two compounds into the COX-1 and -2 active sites showed that the methyl ether could only fit in the COX-2 active site whereas the phenol could be docked into both COX-1 and -2. This study reports a new mode for inhibitor binding to COX-1 and -2 and a novel structural scaffold for the development of COX-2 selective inhibitors.     

Dependence of the cytotoxicity and antioxidant activity of the ammonium derivatives of alkylphenols on their structural characteristics

The influence of seven structurally similar N,N-dimethyl-(4-hydroxyaryl)alkylammonium chlorides on the survival of strains of E. coli cells has been studied. AB1157 and isogenic BH910 defective in the genes of repair enzymes have been analyzed in the presence and in the absence of hydrogen peroxide. Among the studied compounds, only N,N-dimethyl-(3,5-dimethyl-4-hydroxybenzyl)ammonium chloride (C1) did not show cytotoxic properties and increased the survival of the cells of both strains in the presence of H₂O₂ to a larger extent than trolox (water-soluble analogue of α-tocopherol). Analogues of C1, derivatives of 3-methyl-5-di-tert-butyl-4-hydroxybenzyl- and 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propylamines, efficiently protected only mutant BH910 cells against H₂O₂. In a series of structural analogues of C1, the cytotoxicity increased with the replacement of the methyl groups in the aromatic ring by tert-butyl and cyclohexyl groups. Among the seven new compounds, only the C1 derivative is a prospective antioxidant for a subsequent more detailed analysis.     

Reversed-phase chromatography of urinary metabolites of paracetamol using ion suppression and ion pairing

High-performance liquid chromatography (HPLC) has proven particularly useful for the study of paracetamol metabolism. Two alternative methods were developed using reversed-phase C₁₈ columns. A rapid ion suppression technique was used for the analysis of free paracetamol, paracetamol mercapturic acid and cysteine conjugate in urine samples obtained from isolated perfused rat kidney preparations, which has conveniently demonstrated the oxidative metabolic capacity of the kidney towards paracetamol. A somewhat longer, but higher resolution, ion-pair HPLC procedure was developed for the analysis of paracetamol metabolites in urine samples from experimental animals. The ion-pairing solvent was composed of tetrabutylammonium hydroxide, Tris and EDTA buffered to pH 7.2 with phosphoric acid. Gradient programming was further used to enhance resolution. Using this system two new metabolites, the sulphate and glucuronide conjugates of 3-thiomethyl-paracetamol were detected and routinely determined along with other known paracetamol metabolites, viz. free paracetamol, paracetamol sulphate, glucuronide, mercapturic acid, and cysteine conjugates, 3-methoxyparacetamol glucuronide and sulphate, p-aminophenol and its O-glucuronide and O-sulphate conjugates. Phenolic O-substituted glucuronide and sulphate conjugates of N-hydroxyparacetamol were also separated.     

Measurement of synthetic phenolic antioxidants in human tissues by high-performance liquid chromatography with coulometric electrochemical detection

The antioxidants, 2-tert.-butyl-4-methoxyphenol (BHA) and its oxidative peroxidation product 2,2′-dihydroxy-3,3′-di-tert.-butyl-5,5′-dimethoxybiphenyl (di-BHA), 3,5-di-tert.-butyl-4-hydroxytoluene (BHT) and propyl gallate, were measured in plasma and tissue homogenates by HPLC and electrochemical detection, with a sensitivity down to 0.2 (BHA), 0.1 (di-BHA), 0.4 (BHT) and 1 (propyl gallate) ng ml⁻¹ of plasma or tissue homogenate. The data demonstrate that in man, at the current level of exposure to dietary antioxidants, significant amounts of BHA, BHT and propyl gallate are accumulated in the omentum. Furthermore, they provide the first evidence that the peroxidase-catalysed oxidation of BHA is operative in man.     

Antioxidant activities of 5-hydroxyoxindole and its 3-hydroxy-3-phenacyl derivatives: The suppression of lipid peroxidation and intracellular oxidative stress

The antioxidant activities of 5-hydroxyoxindole (1) and newly synthesized 3,5-dihydroxy-3-phenacyl-2-oxindole derivatives against rat liver microsome/tert-butylhydroperoxide system-induced lipid peroxidation and hydrogen peroxide-induced intracellular oxidative stress were investigated. Compound 1 and its derivatives showed significant suppression of lipid peroxidation and an intracellular oxidative stress. The effects of the more lipophilic derivatives tended to be greater than that of the original compound 1. The cytotoxicity of all of the oxindole derivatives on human promyelocytic leukemia HL60 cells was lower than that of 2,6-di(tert-butyl)-4-hydroxytoluene (BHT), a widely used phenolic antioxidant. These results show that compound 1 and its 3-substituted derivatives could be good lead candidates for future novel antioxidant therapeutics.     

Cryoprotective activity of phosphorus-containing phenol

The antioxidant and cryoprotective efficiencies of a 3,5-di-tert-butyl-4-hydroxyphenyl)methylenediphosphonic acid (MDPA) differ significantly for sperm cells of various species of sturgeon fish (Russian sturgeon, beluga and Stellate sturgeon). The ability of phosphorus-containing phenol MDPA to decrease the level of lipid peroxidation of sperm, beneficial effect on the activity indicators of the sperm of native sturgeon and of the defrosted one after deep freezing as well as on the fertility of sperm cells was shown.     

Protective effects of Cornus walteri W. extracts on t-BHP-induced cell damage through antioxidant activity

This study evaluated the antioxidant potential of extracts from Cornus walteri W. (CW) using various assays for natural antioxidants. We determined the polyphenol and flavonoid contents, as well as the antioxidant properties of water and ethanol extracts from the CW compared with those of other natural and synthetic antioxidants. CW extracts had high total phenolic and flavonoid contents in both the water and ethanol extracts. Various radical (DPPH, hydroxyl, and alkyl) scavenging activities of extracts from CW were higher than that of vitamin C. In addition, the antioxidant capacity measured as ferric reducing antioxidant power (FRAP), and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as diammonium salt (ABTS) radical scavenging were higher than that of 2,6-di-tert-butyl-4-methylphenol (BHT), used as a positive control. The cytoprotective effect of CW extracts was also observed in tert-butyl hydroperoxide (t-BHP)-induced oxidative damage in Chang cells. These results showed that CW extracts have antioxidant properties through their ability to enhance cell viability, prevent reactive oxygen species (ROS) generation, and inhibit mitochondrial membrane depolarization. The antioxidant potency of the CW extracts could be exploited for their health promoting potential.     

3,5-Di-t-butyl-4-hydroxytoluene (BHT) and probucol stimulate selectively the reaction of mammalian 15-lipoxygenase with biomembranes

The lipophilic antioxidant 3,5-di-t-butyl-4-hydroxytoluene (BHT) and the structurally-related antiatherogenic drug probucol stimulate the oxygenation of mitochondrial membranes and erythrocyte ghosts by the rabbit 15-lipoxygenase as indicated by an increase in oxygen consumption as well as by an enhanced loss of polyenoic fatty acids and by the formation of specific lipoxygenase products in the membrane phospholipids. The oxygenation of linoleic acid, phospholipids and human low-density lipoproteins was not stimulated. With mitochondrial membranes, BHT causes a quenching of the 1-anilino-8-naphthalene sulfonate fluorescence. Thus, it is suggested that the stimulation of membrane oxygenation may be due to structural changes in the membranes leading to a better susceptibility of the polyenoic fatty acid residues towards lipoxygenase attack. Owing to this unexpected effect of the antioxidants, which is not related to their radical-scavenger capacity, care should be taken in interpreting experimental data on effects of BHT and probucol.     

Mono- and dinuclear (o-thioetherphenolato)-copper(II) complexes. Structural models for galactose oxidase

Three new o-thioetherphenol ligands have been synthesized: 1,2-bis(3,5-di-tert-butyl-2-hydroxyphenylsulfanyl)ethane (H₂bse), 1,2-bis(3,5-di-tert-butyl-2-hydroxyphenylsulfanyl)benzene (H₂bsb), and 4,6-di-tert-butyl-2-phenylsulfanylphenol (Hpsp). Their complexes with copper(II) were prepared and investigated by UV-Vis-, EPR-spectroscopy; their electro- and magnetochemistry have also been studied: [Cu^II(psp)₂] (1), [Cu^II₂(bse)₂] (2), [Cu^II₂(bsb)₂] (3), [Cu^II(bsb)(py)₂] (4). The crystal structures of the ligands H₂bse, H₂bsb, Hpsp and of the complexes 1, 2, 3, 4 have been determined by X-ray crystallography.     

Synthesis of symmetric and asymmetric diamides of citric acid and amino acids

Summary A convenient method for the synthesis of symmetric and asymmetric diamides of amino acids including DOPA and citric acid from 2-tert-butyl-1,3-di(N-hydroxysuccinimidyl)citrate and 1-tert-butyl-2,3-di(N-hydroxysuccinimidyl)citrate is described.Abbreviations AcOtBu tert-butyl acetate - i-Bu iso-butyl - tBu tert-butyl - Bzl benzyl - p-OH-Bzl p-hydroxybenzyl - m,p-(OH)₂-Bzl m,p-dihydroxybenzyl - DCCI dicyclohexylcarbodiimide - Et ethyl - Me methyl - Su succinimidyl - SuOH N-hydroxysuccinimide - Ph phenyl     

Synthesis and antiperoxidant activity of new phenolic O-glycosides

We describe the synthesis of some 3-tert-butyl-4-hydroxyphenyl -glycopyranosides by reaction of tert-butylhydroquinone with β- -pentaacetyl-glucose, β- -pentaacetyl-galactose, 2-acetamido- and 3,4,6-tri-O-acetyl-2-butanamido-2-deoxy-β- -glucopyranosyl chlorides as well as the formation of anomeric 3-tert-butyl-4-hydroxyphenyl 4,6-di-O-acetyl-2,3-dideoxy- -erythro-hex-2-eno-pyranosides by reaction between tert-butylhydroquinone and 3,4,6-tri-O-acetyl- -glucal. All compounds, except 3-tert-butyl-4-hydroxyphenyl α- and β- -glucopyranosides, inhibited lipid peroxidation with a degree of potency comparable to that of tert-butyl hydroxyanisole.     

Catechol oxidase and phenoxazinone synthase activity of a manganese(II) isoindoline complex

The mononuclear [Mn(6′Me₂indH)(H₂O)₂(CH₃CN)](ClO₄)₂ (6′Me₂indH: 1,3-bis(6′-methyl-2′-pyridylimino)isoindoline) complex has been prepared and characterized by various techniques such as elemental analysis, IR, UV-visible and ESR spectroscopy. The title compound was suitable as catalyst for the catalytic oxidation of 3,5-di-tert-butylcatechol (3,5-DTBCH₂) to 3,5-di-tert-butyl-1,2-benzoquinone (3,5-DTBQ) (catecholase activity), and o-aminophenol (OAPH) to 2-aminophenoxazine-3-one (APX) (phenoxazinone synthase activity) with dioxygen at ambient condition in good yields. Kinetic measurements revealed first-order dependence on the catalyst and dioxygen concentration and saturation type behavior with respect to the corresponding substrate. It was also found that the added triethylamine in both systems accelerates the reaction.     

The metabolism of di-(3,5-di-tert.-butyl-4-hydroxybenzyl) ether (Ionox 201) in the rat

1. Up to one-third of a single oral dose of Ionox 201 was absorbed in rats. 2. In rats dosed with [(14)C]Ionox 201 86.8-97.2% of the label is excreted in the faeces in 24 days (much of this is eliminated in the first 4 days after dosage), 5.6% in the urine and not more than 0.8% in the exhaled air; 5.0% of (14)C is present in the carcass and viscera after removal of the gut, and most of this is in the fatty tissues. 3. About 65.0% of (14)C in the faeces is due to unchanged antioxidant, 30.0% to 3,5-di-tert.-butyl-4-hydroxybenzoic acid, 3.5% to unidentified polar constituent(s), 1.4% to 3,5-di-tert.-butyl-4-hydroxybenzaldehyde and 0.1% to 3,3',5,5'-tetra-tert.-butyl-4-,4'-stilbenequinone. A variable proportion of (14)C in the urine is due to 3,5-di-tert.-butyl-4-hydroxybenzoic acid (40-60%) and the remainder (60-40%) to the ester glucuronide, when the animals were treated with different doses of antioxidant. In eight individual animals dosed with 6.78mg. of [(14)C]Ionox 201, one-third of (14)C in the bile is due to the free acid, 45% to the ester glucuronide, 20% to an unidentified constituent and 2% to unchanged antioxidant, and, in two animals dosed with 13.56mg., there is a small proportion of free acid and a larger proportion of ester glucuronide. About 80% of (14)C in the body fat is due to unchanged antioxidant, 19% to the free acid and 1% to 3,5-di-tert.-butyl-4-hydroxybenzaldehyde. 4. At least 36.2% of a single oral dose of Ionox 201 is metabolized: 3,5-di-tert.-butyl-4-hydroxybenzoic acid accounts for 30.2% of a dose, (3,5-di-tert.-butyl-4-hydroxybenzoyl beta-d-glucopyranosid)uronic acid for 1.4%, 3,5-di-tert.-butyl-4-hydroxybenzaldehyde for 1.3%, 3,3',5,5'-tetra-tert.-butyl-4,4'-stilbenequinone for 0.1% and unidentified polar metabolite(s) for 3.2%. 5. The metabolism of Ionox 201 in vivo is closely related to its antioxidant action in vitro.     

3,5-Dialkyl-4-hydroxybenzylidenemalononitrile; A New Group of Fungicidal and Acaricidal Agents

Several 3,5-dialkyl-4-hydroxybenzylidenemalononitriles and related compounds were tested for their fungicidal and acaricidal activities. The influence of structural variation on biological activity was studied by preparing and using a total of 22 compounds of benzylidenemalononitrile analogues.

Amongst the compounds tested 3,5-di-tert-butyl and amyl-4-hydroxybenzylidenemalononitrile were most effective against fungus, Piricularia oryzae Car. and mite, Tetranychus telarius L.     

An inhibitor of free radical processes decreases the rate of protein synthesis in gunshot wound tissues and decelerates the general adaptation syndrome

The dynamics of total protein and procollagen biosynthesis was studied in the muscle tissue in gunshot wound during the natural healing and after treatment with antioxidant (AO) 2,6-di-tert-butyl-4-methylphenol (BHT). AO treatment considerably decreased the rate of both total protein and procollagen synthesis in the muscle on the third day of healing. BHT notably decreased protein biosynthesis and inhibited corticosteroid production as measured from the electron spin resonance (ESR) signal of reduced adrenodoxin. AO treatment also decreased the rate of protein biosynthesis in bone marrow, thymus, and spleen. The decreased functional activity of the endocrine and immune systems suggests that AO considerably decelerates the systemic body response (general adaptation syndrome) to acute wound. Lipid peroxide radicals and the products of lipid hydroperoxide degradation are proposed as the primary mediators of the body response to stress.     

Molecular structure and catechol oxidase activity of a new copper(I) complex with sterically crowded monodentate N-donor ligand

The attempted alkylation of 1,3-bis(2′-pyridylimino)isoindoline (indH) by the use of n-BuLi and subsequent alkyl halides led to quaternization of the pyridine nitrogens and the zwitterionic monodentate N-ligand (Me₂ind)I was formed. By the use of the ligand the copper(I) complex [Cu^I(Me₂ind)I₂] was prepared and its structure determined. It was found to be good catalyst for the oxidation of 3,5-di-tert-butylcatechol (DTBCH₂) to 3,5-di-tert-butyl-1,2-benzoquinone (DTBQ) and H₂O₂ by dioxygen. Detailed kinetic studies revealed first-order dependence on the catalyst and dioxygen concentration and saturation type behavior with respect to the substrate.     

The metabolism of urethane and related compounds

1. Urethane is metabolized in the rat, rabbit and man by a process of N-hydroxylation. This occurs to a smaller extent when methyl, n-propyl and n-butyl carbamates are administered to the rat and rabbit. 2. Other metabolites which have been detected in urine of animals dosed with urethane and N-hydroxyurethane are ethylmercapturic acid, ethylmercapturic acid sulphoxide and N-acetyl-S-carbethoxycysteine. 3. Substances which appear to be S-ethylglutathione and S-ethylglutathione sulphoxide have been detected in the bile of rats dosed with urethane or N-hydroxyurethane. 4. Methyl, ethyl, n-propyl and n-butyl N-hydroxycarbamates are excreted unchanged in the urine of rats dosed with these compounds to extents depending on the dose administered. 5. Animals dosed with methyl, ethyl, n-propyl or n-butyl carbamate or the corresponding N-hydroxycarbamate excrete the corresponding carbamate and N-hydroxycarbamate in the urine. 6. Methyl, n-propyl and n-butyl carbamates and N-hydroxycarbamates are excreted more slowly than are urethane and N-hydroxyurethane. 7. The probable role of N-hydroxyurethane and the processes of alkylation and carbethoxylation, and of hydroxylamine, nitroxyl and hyponitrous acid in carcinogenesis and chemotherapy with urethane, have been discussed.     

Synthesis of the Stereoisomeric Mixture of the Compound Having the Proposed Structure for “Auxin b Lactone”

The composed having the proposed structure for auxin b lactone (XVIII) was synthesized by formic acid hydrolysis of 4-ethoxy-6-(3,5-di-sec-butyl-1-cyclopenten-1-yl)-5,6-dihydro-2-pyrone (XVII) which was, in turn, prepared by the Reformatsky reaction of 3,5-di-sec-butyl-1-cyclopentenealdehyde (XVI) with ethyl γ-bromo-β-ethoxycrotonate.     

On the Acaciin

Synthesis of N-2,2,3,3-tetrafluoropropionyl-amino acids (abbreviated as tetrafluoropropionyl- or Tfp-amino acids) by the reaction of amino acids with tetrafluoropropionyl anhydride was described, Racemization was observed to occur to a considerable extent during the process of the introduction of this group. Optically active Tfp-amino acids were able to be prepared free from racemization by transesterification of methyl tetrafluoropropionate with amino acid tert-butyl esters, followed by treatment of the resulting Tfp-amino acid tert-butyl esters with trifluoroacetic acid. Some Tfp-dipeptide esters were prepared from the corresponding dipeptide tert-butyl esters in this way. This group was cleavable by mild alkaline hydrolysis or by reduction with sodium borohydrde as well as the Tfa-group.