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.     

The first synthesis of 4-phenylbutenone derivative bromophenols including natural products and their inhibition profiles for carbonic anhydrase,acetylcholinesterase and butyrylcholinesterase enzymes

The first synthesis of (E)-4-(3-bromo-4,5-dihydroxyphenyl)but-3-en-2-one (1), (E)-4-(2-bromo-4,5-dihydroxyphenyl)but-3-en-2-one (2), and (E)-4-(2,3-dibromo-4,5-dihydroxyphenyl)but-3-en-2-one (3) was realized as natural bromophenols. Derivatives with mono OMe of 2 and 3 were obtained from the reactions of their derivatives with di OMe with AlCl₃. These novel 4-phenylbutenone derivatives were effective inhibitors of the cytosolic carbonic anhydrase I and II isoenzymes (hCA I and II), acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with K_i values in the range of 158.07–404.16 pM for hCA I, 107.63–237.40 pM for hCA II, 14.81–33.99 pM for AChE and 5.64–19.30 pM for BChE. The inhibitory effects of the synthesized novel 4-phenylbutenone derivatives were compared to acetazolamide as a clinical hCA I and II isoenzymes inhibitor and tacrine as a clinical AChE and BChE enzymes inhibitor.     

Synthesis of halogenated 4-quinolones and evaluation of their antiplasmodial activity

Treatment of 4-hydroxyquinolines with (2-methyl)allyl bromide in the presence of K₂CO₃ resulted in the formation of novel N-[(2-methyl)allyl]-4-quinolones through selective N-alkylation. Further reaction of N-(2-methylallyl)-4-quinolones with bromine or N-bromosuccinimide yielded the corresponding 3-bromo-1-(2,3-dibromo-2-methylpropyl)-4-quinolones and 3-bromo-1-(2-methylallyl)-4-quinolones, respectively. Furthermore, a copper-catalyzed C–N coupling of the latter 3-bromo-4-quinolones with (5-chloro)indole afforded novel 3-[(5-chloro)indol-1-yl]-4-quinolone hybrids. Antifungal and antiplasmodial assays of all new 4-quinolones were performed and revealed no antifungal properties but moderate antiplasmodial activities. All 15 compounds displayed micromolar activities against a chloroquine-sensitive strain of Plasmodium falciparum, and the five most potent compounds also showed micromolar activities against a chloroquine-resistant strain of P. falciparum with IC₅₀-values ranging between 4 and 70 μM.     

Unusual 4-hydroxybenzaldehyde synthase activity from tissue cultures of the vanilla orchid Vanilla planifolia

Tissue cultures of the vanilla orchid, Vanilla planifolia, produce the flavor compound vanillin (4-hydroxy-3-methoxybenzaldehyde) and vanillin precursors such as 4-hydroxybenzaldehyde. A constitutively expressed enzyme activity catalyzing chain shortening of a hydroxycinnamic acid, believed to be the first reaction specific for formation of vanilla flavor compounds, was identified in these cultures. The enzyme converts 4-coumaric acid non-oxidatively to 4-hydroxybenzaldehyde in the presence of a thiol reagent but with no co-factor requirement. Several forms of this 4-hydroxybenzaldehyde synthase (4HBS) were resolved and partially purified by a combination of hydrophobic interaction, ion exchange and gel filtration chromatography. These forms appear to be interconvertible. The unusual properties of the 4HBS, and its appearance in different protein fractions, raise questions as to its physiological role in vanillin biosynthesis in vivo.     

Brominated anisoles and cresols in the red alga Polysiphonia sphaerocarpa

The red marine alga Polysiphonia sphaerocarpa was extracted by a simultaneous steam distillation-solvent extraction technique and several brominated compounds were identified by gas chromatography-mass spectrometry. The compounds detected were 2,4-dibromoanisole, 2,4,6-tribromoanisole, 3-bromocresol, 3,5-dibromocresol, 3-bromo-4-hydroxybenzaldehyde, 3,5-dibromo-4-hydroxybenzaldehyde, 2-bromophenol, 4-bromophenol, 2,4-dibromophenol, 2,6-dibromophenol and 2,4,6-tribromophenol. This is the first time brominated anisoles and cresols have been detected in marine algae.     

Biosynthesis of carnitine and 4-N-trimethylaminobutyrate from lysine

The conversion of l-[U-(14)C]lysine into carnitine was demonstrated in normal, choline-deficient and lysine-deficient rats. In other experiments in vivo radioactivity from l-[4,5-(3)H]lysine and dl-[6-(14)C]lysine was incorporated into carnitine; however, radioactivity from dl-[1-(14)C]lysine and dl-[2-(14)C]lysine was not incorporated. Administered l-[Me-(14)C]methionine labelled only the 4-N-methyl groups whereas lysine did not label these groups. Therefore lysine must be incorporated into the main carbon chain of carnitine. The methylation of lysine by a methionine source to form 6-N-trimethyl-lysine is postulated as an intermediate step in the biosynthesis of carnitine. Radioactive 4-N-trimethylaminobutyrate (butyrobetaine) was recovered from the urine of lysine-deficient rats injected with [U-(14)C]lysine. This lysine-derived label was incorporated only into the butyrate carbon chain. The specific radioactivity of the trimethylaminobutyrate was 12 times that of carnitine isolated from the urine or carcasses of the same animals. These data further support the idea that the last step in the formation of carnitine from lysine was the hydroxylation of trimethylaminobutyric acid, and are consistent with the following sequence: lysine+methionine --> 6-N-trimethyl-lysine --> --> 4-N-trimethylaminobutyrate --> carnitine.     

4-Bromo-2-octenoic acid specifically inactivates 3-ketoacyl-CoA thiolase and thereby fatty acid oxidation in rat liver mitochondria

In an attempt to develop a compound which would specifically inhibit 3-ketoacyl-CoA thiolase (EC 2.3.1.16) in whole mitochondria, 4-bromo-2-octenoic acid was synthesized and studied. After rat liver mitochondria were preincubated with 4-bromo-2-octenoic acid for 3 min, respiration supported by either palmitoylcarnitine or pyruvate was completely abolished, whereas no inhibition was observed with rat heart mitochondria. Addition of carnitine stimulated respiration supported by pyruvate without relieving inhibition of palmitoylcarnitine-dependent respiration. Hence, this compound seems to be a specific inhibitor of beta-oxidation. When the enzymes of beta-oxidation were assayed in a soluble extract prepared from mitochondria preincubated with 4-bromo-2-octenoic acid, only 3-ketoacyl-CoA thiolase was found to be inactivated. 4-Bromo-2-octenoic acid is metabolized by mitochondrial beta-oxidation enzymes to 3-keto-4-bromooctanoyl-CoA which effectively and irreversibly inhibits 3-ketoacyl-CoA thiolase but not acetoacetyl-CoA thiolase (EC 2.3.1.9). Even though 3-keto-4-bromooctanoyl-CoA inhibits the latter enzyme reversibly, 4-bromo-2-octenoic acid does not inhibit ketogenesis in rat liver mitochondria with acetylcarnitine as a substrate. It is concluded that 4-bromo-2-octenoic acid specifically inhibits mitochondrial fatty acid oxidation by inactivating 3-ketoacyl-CoA thiolase in rat liver mitochondria.     

Metabolism of 4-hydroxybenzaldehyde, 3-bromo-4-hydroxybenzaldehyde and bromide by cell-free fractions of the marine red alga Odonthalia floccosa

Cell-free fractions from Odonthalia floccosa incubated with 4-hydroxybenzaldehyde-[U-¹⁴C], 3-bromo-4-hydroxybenzaldehyde-[U-¹⁴C] and ⁸²Br^? formed the dibromo-dihydroxybenzaldehyde derivatives of the bromophenols (brominated benzylalcohols) which were also identified as naturally occurring products.     

The addition of hypobromous acid to 3β,17β-diacetoxy-4-estrene and an equilibration study involving neighboring group participation by the a-ring acetoxy group

The reaction of 3β,17β-diacetoxy-4-estrene with N-bromoacetamide in a two phase ether/water solvent mixture gave 5-bromo-4β,17β-diacetoxy-5α-estran-3β-ol as the major product (75%). Four minor products were also isolated and identified. These were: 4α-bromo-3β, 17β-diacetoxy-5α-estran-5-ol (5%), 5-bromo-3g,17β-diacetoxy-5α-estran-4β-ol (6%), 5-bromo-4α,17β-diacetoxy-5αt-estran-3β-ol (3%), and 4β-bromo-3β,17β-diacetoxy-5α-estran-5-ol (4%). The 5-bromo-4β, 17β-diacetoxy-5α-estran-3β-ol was equilibrated by heating with oxalic acid in refluxing benzene for $\text{ca}$. 16h to give a mixture of it and 5-broino-3β,17β-diacetoxy-5α-estran-4β-ol in the ratio of 16:84 respectively. A similar equilibration mixture (14:86) was obtained under identical conditions when 5-bromo-3β,17β-diacetoxy-5α-estran-4β-ol was the starting material.     

Identification of urinary 3-ethoxy-4-hydroxybenzoic and 3-ethoxy-4-hydroxymandelic acids after dietary intake of ethyl vanillin

It has been discovered recently that several patients undergoing urinary organic acid profiling excrete high concentrations of 3-ethoxy-4-hydroxybenzoic acid and traces of 3-ethoxy-4-hydroxymandelic acid. These are believed to be the consequences of feeding the patients with synthetic diets flavoured with 3-ethoxy-4-hydroxybenzaldehyde, a vanilla-like artificial flavouring added to improve patient acceptance and taste. The syntheses, mass spectra and gas chromatographic behaviour of these and related compounds are presented.     

Inactivation of GABA transaminase by 3-chloro-1-(4-hydroxyphenyl)propan-1-one

Previously it was found that 4-hydroxybenzaldehyde is a competitive inhibitor of GABA transaminase. Here 3-chloro-1-(4-hydroxyphenyl)propan-1-one (9), a 4-hydroxybenzaldehyde analogue, was found to inactivate potently the enzyme in a time-dependent manner. α-Ketoglutarate prevented the enzyme from inactivation, suggesting that the inactivation occurs in its active site. Several experiments indicated that the inactivation is irreversible. This study provides a novel strategy for the design of more effective inhibitors.     

Synthesis and stability assay of 4-methylumbelliferyl (1-->3)-beta-D-pentaglucoside

The synthesis and stability of 4-methylumbelliferyl (1 --> 3)-beta-D-pentaglucoside 3 are described. The (1 --> 3)-beta-D-glucan isolated from the cell walls of Saccharomyces cerevisiae was recovered from the aqueous medium as water-insoluble particles by the spray drying (GS) method. The acid-solubilized (1 --> 3)-beta-D-oligoglucosides were prepared by partial acid hydrolysis of glucan. The peracetylated (1 --> 3)-beta-D-pentaglucoside 1 was obtained by isolation of peracetylated (1 --> 3)-beta-D-oligoglucoside mixture. The peracetylated 4-methylumbelliferyl (1 --> 3)-beta-D-pentaglucoside 2 was synthesized by treating compound 1 with the 4-methylumbelliferone and a Lewis acid (SnCl4) catalyst. NaOMe in dry methanol was used for the deacetylation of the blocked derivative, to give the target compound 3 in an overall yield of 35%. Activity assays with beta-glucosidase indicated that compound 3 was much more stable than the corresponding pentasaccharide.     

Indium-promoted Barbier-type allylations in aqueous media: a convenient approach to 4-C-branched monosaccharides and (1-->4)-C-disaccharides

Starting from methyl 6-bromo-4,6-dideoxy-alpha-D-threo-4-enopyranoside, 4-C-branched sugars have been prepared through indium-promoted Barbier-type allylation of various aldehydes in aqueous media followed by hydroboration of the resulting double bond. The intermediate unsaturated monosaccharides were shown to rearrange in acidic media to give 4-C-acetyl-5-C-alkyl pyranose compounds. From beta-1-formyl sugars the corresponding beta-(1-->4)-C-disaccharides were obtained.     

Biotransformation of 4-Hydroxybenzen Derivatives by Hairy Root Cultures of Polygonum multiflorum Thunb.

The biotransformation of four 4-hydroxybenzen derivatives （1,4-benzenediol （compound 1）, 4-hydroxybenzaldehyde （compound 2）, 4-hydroxybenzyl alcohol （compound 3） and 4-hydroxybenzoic acid （compound 4）） by the hairy root cultures of Polygonum multiflorum Thunb. as a new biocatalyst was investigated. It was found that the substrates were transformed to their corresponding glucosides, 4-hydroxyphenyl β-D-glucopyranoside （arbutin, compound la）, 4-hydroxymethylphenyl β-D-glucopyranoside （gastrodin, compounds 2a, 3a） and 4-carboxyphenyl α-D- glucopyranoside （compound 4a）, respectively. In the meantime, the hairy roots of P. multiflorum were able to stereoselectively and regioselectively glucosylate phenolic hydroxyl groups of compounds 1-4, but the cultures could not glucosylate the aldehyde group of compound 2 or the benzyllc hydroxyl group of compound 3, and no glucosyl esterification of carboxyl groups of compound 4 was detected. On the other hand, the result also showed that the hairy roots of P. multiflorum were able to reduce the 4-hydroxybenzaldehyde to its corresponding alcohol. This is the first report that substrate 4 has been converted into its α-D-glucopyranoside by a plant biotransformα- tion system.     

The pentose phosphate pathway as a source of NADPH for lignin synthesis

The aim of this work was to investigate whether the pentose phosphate pathway provides reducing power for lignin synthesis. Explants of the stem of Coleus blumei and the storage tissue of Helianthus tuberosus were cultured for 4 days on media which caused extensive lignification. [3-³H]-glucose and either [3-¹⁴C]- or [U-¹⁴C]-glucose were supplied to such 4-day-cultured explants, and also to the roots of 5-day-old seedlings of Pisum sativum. Significant amounts of ³H and ¹⁴C were recovered in syringaldehyde, vanillin, p-hydroxybenzaldehyde, and ligothio-glycollic acid from the explants of Coleus and Helianthus; and in vanillin, p-hydroxybenzaldehyde, and milled-wood lignin from pea roots. The ³H/¹⁴C ratios in these derivatives and preparations of lignin are held to indicate that much of the reducing power for lignin synthesis comes from the pentose phosphate pathway.     

Milligram-scale preparation and purification of oligosaccharides of defined length possessing the structure of chondroitin from defructosylated capsular polysaccharide K4

Escherichia coli K4 bacterium synthesizes a nonsulfated capsule polysaccharide (K4) composed of a repeating disaccharide subunit of D-glucuronic acid (beta1-->3) and N-acetyl-D-galactosamine (beta1-->4) to which beta-fructofuranose units are linked to C-3 of D-glucuronic acid residues. The K4 polyanion is easily defructosylated under acid conditions with no fragmentation of the polymer to produce a polysaccharide having a repeated disaccharide unit of chondroitin consisting of D-glucuronic acid (beta1-->3) and N-acetyl-D-galactosamine (beta1-->4) (K4d). K4 and K4d were depolymerized by partial digestion with testicular hyaluronidase and separated into uniform-size oligosaccharides from 4-mers to 16-mers by preparative anion-exchange chromatography after removal of the hyaluronidase. The purity and size of each oligosaccharide was confirmed by using anion-exchange HPLC, HPSEC analysis, and FACE. Mg-scale K4d oligosaccharides were obtained from 50 mg K4d starting material. Under the conditions used to degrade the K4 polysaccharide by testicular hyaluronidase, fructose is slowly liberated forming the defructosylated K4. As a consequence, a mixture of uniform- size K4 and K4d oligosaccharide species, from approximately 4- to 20-mers, are generated and size-separated by anion-exchange chromatography. These pure, uniform-size, and large ranges of K4d oligosaccharides having the structure of a chondroitin, -->4)-GlcUA-beta(1-->3)GalNAc-beta(1-->, will be available for investigating important biological functions of this polymer.     

Synthesis and CYP26A1 inhibitory activity of novel methyl 3-[4-(arylamino)phenyl]-3-(azole)-2,2-dimethylpropanoates

The role of all-trans-retinoic acid (ATRA) in the development and maintenance of many epithelial and neural tissues has raised great interest in the potential of ATRA and related compounds (retinoids) as pharmacological agents, particularly for the treatment of cancer, skin, neurodegenerative and autoimmune diseases. The use of ATRA or prodrugs as pharmacological agents is limited by a short half-life in vivo resulting from the activity of specific ATRA hydroxylases, CYP26 enzymes, induced by ATRA in liver and target tissues. For this reason retinoic acid metabolism blocking agents (RAMBAs) have been developed for treating cancer and a wide range of other diseases.The synthesis, CYP26A1 inhibitory activity and molecular modeling studies of novel methyl 3-[4-(arylamino)phenyl]-3-(azole)-2,2-dimethylpropanoates are presented. From this series of compounds clear SAR can be derived for 4-substitution of the phenyl ring with electron-donating groups more favourable for inhibitory activity. Both the methylenedioxyphenyl imidazole (17, IC₅₀ = 8 nM) and triazole (18, IC₅₀ = 6.7 nM) derivatives were potent inhibitors with additional binding interactions between the methylenedioxy moiety and the CYP26 active site likely to be the main factor. The 6-bromo-3-pyridine imidazole 15 (IC₅₀ = 5.7 nM) was the most active from this series compared with the standards liarozole (IC₅₀ = 540 nM) and R116010 (IC₅₀ = 10 nM).     

ApoE secretion modulating bromotyrosine derivative from the Australian marine sponge Callyspongia sp.

High throughput screening of a pre-fractionated natural product library identified 11 active fractions showing ApoE modulation activity. Mass-directed fractionation of one active crude extract from the Australian marine sponge Callyspongia sp. resulted in the isolation of 13 metabolites, including three new bromotyrosine derivatives, callyspongic acid (1), 3,5-dibromo-4-methoxyphenylpyruvic acid (2), N-acetyl-3-bromo-4-hydroxylphenylethamine (3), and ten known compounds (4–13). The structure elucidation of compounds 1–3 was based on their 1D and 2D NMR and MS spectroscopic data. 3,5-Dibromo-4-methoxyphenylpyruvic acid (2) showed weak activity in increasing the apolipoprotein E secretion from human CCF-STTG1 cells at the concentration of 40 μM.     

Structure of the O-specific polysaccharide of Proteus vulgaris O4 containing a new component of bacterial polysaccharides, 4,6-dideoxy-4

A high-molecular-mass O-specific polysaccharide was obtained by mild acid degradation of Proteus vulgaris O4 lipopolysaccharide followed by GPC. The polysaccharide was studied by chemical methods along with 1H and 13C NMR spectroscopy, including two-dimensional COSY, TOCSY, NOESY, H-detected 1H,13C HMQC, and 1H,13C HMBC experiments. Solvolysis of the polysaccharide with trifluoromethanesulfonic (triflic) acid resulted in a GlcpA-(1 --> 3)-GlcNAc disaccharide and a novel amino sugar derivative, 4,6-dideoxy-4-[N-[(R)-3-hydroxybutyryl]-L-alanyl]amino-D-glucose [Qui4N(HbAla)]. On the basis of the data obtained, the following structure of the tetrasaccharide repeating unit of the O-specific polysaccharide was established: --> 4)-beta-D-GlcpA-(1 --> 3)-beta-D-GlcpNAc-(1 --> 2)-beta-D-Quip4N(HbAla)-(1 --> 3)-alpha-D-Galp-(1 -->. This structure is unique among the O-specific polysaccharides, which is in accordance with classification of the strain studied in a separate Proteus serogroup.     

马尾伸筋草化学成分及抗骨质疏松活性研究

为研究马尾伸筋草(Lycopodium fargesii Hert.)的化学成分及其抗骨质疏松活性,采用硅胶、MCI、RP-18、Sephadex LH-20柱色谱及半制备高效液相色谱对马尾伸筋草的乙醇提取物进行系统分离,从乙酸乙酯部位中得到18个化合物,综合利用NMR、HR-ESI-MS等技术,分析鉴定化合物的结构,分别鉴定为α-芒柄蜡素(1)、26-去甲-8-氧化-α-芒柄蜡素(2)、serrat-14-en-3β,21α,24-triol(3)、serrat-14-en-3β,21β,24-triol(4)、1,20-eicosanediol(5)、二十三烷(6)、glycerolmonolinoleate(7)、glyceryl linolentate(8)、3-(4′-formylphenoxy)-4-methoxybenzaldehyde(9)、芹菜素(10)、对羟基苯甲醛(11)、香草酸(12)、3-羟基-4-甲氧基苯甲酸(13)、2-羟基-1-(4-羟基-3-甲氧基-苯基)-丙基-1-酮(14)、3-甲氧基-4-羟基桂皮醛(15)、8-acetoxy-5-hydroxyumbelliprenin(16)、trans-4-hydroxy-2-nonenoic acid(17)和正十烷硫醇(18)。化合物1~18均为首次从马尾伸筋草中分离得到,其中化合物16对破骨细胞活性具有显著的抑制作用,其IC₅₀为1.98μM。