Aryl Hydroxylation Research Articles

Monooxygenase activity in Cunninghamella bainieri: Evidence for a fungal system similar to liver microsomes

The fungus Cunninghamella bainieri effects the oxidative N-demethylation of aminopyrine, O-demethylation of 4-nitroanisole and anisole, the aryl hydroxylation of anisole, aniline, and naphthalene, and the reduction of nitro and azo groups. The hydroxylation of 4-[ 2H]-anisole and 2-[ 2H]-anisole proceeds with migration and retention of isotopic hydrogen (NIH shift). The above reactions and the formation of the trans-dihydrodiol of naphthalene and the incorporation of oxygen-18 from 18O 2 into the trans-dihydrodiol and hydroxylated anisole are characteristic of reactions catalyzed by the cytochrome P 450 monooxygenases of hepatic microsomes. The product ratios in these hydroxylations are very similar to those obtained using liver microsomes providing further evidence that the C. bainieri monooxygenase enzymes are similar to the liver monooxygenases. Furthermore, an epoxide hydrase enzyme similar to that present in hepatic microsomes must also be present in C. bainieri.

Soybean shoot metabolism of isopropyl-3-chlorocarbanilate: Ortho and para aryl hydroxylation

This laboratory reported that isopropyl-3-chlorocarbanilate-phenyl-U- 14C (chlorpropham-phenyl- 14C) was absorbed, translocated, and metabolized by soybean plants. Both polar metabolites and insoluble residues were found in roots, whereas only polar metabolites were found in shoot tissues. In both roots and shoots the polar metabolites were shown to be the O-glucoside of isopropyl-2-hydroxy-5-chlorocarbanilate (2-hydroxy-chlorpropham). In shoot tissue there were other polar metabolites that were not identified. The experiments with soybeans have been repeated, but with new isolation and purification procedures. The plants were root treated with both chlorpropham-phenyl- 14C and isopropyl-3-chlorocarbanilate-2-isopropyl- 14C. The roots and shoots were extracted and separated into the polar, nonpolar, and insoluble metabolic components, using the Bligh-Dyer extraction method. The polar metabolites were separated by gel permeation chromatography. Further purification was accomplished on Amberlite XAD-2. The polar metabolites from the shoot and root tissues were hydrolyzed either by β-glucosidase or hesperidinase. The enzyme liberated aglycones were derivatized and separated by gas-liquid chromatography, and the components were characterized by mass spectrometry or NMR. The results of this study showed that the polar metabolites of soybean shoots were 2-hydroxy-chlorpropham and isopropyl-4-hydroxy-3-chlorocarbanilate (4-hydroxy-chlorpropham). These two hydroxy-chlorpropham metabolites were found in soybean shoots at a ratio of approximately 1:1. The only aglycone found in root tissue was 2-hydroxy-chlorpropham. Using the new procedures, no evidence was obtained for the presence of the unidentified polar metabolites that were previously observed in shoot tissues.

Studies on carcinogenic azo dyes. 3. Retention of tritium during enzymatic hydroxylation of 3'-methyl-4-dimethylaminoazobenzene(4'-3H or 5'-3H) or chemical substitutions of the related compound, and the effect of the repeated administration of this azo dye on the retention of tritium.

This paper is intended to report the retention of tritium during enzymatic hydroxylation at the 4'position of 3'-methyl-4-dimethylaminoazobenzene [4'-3H, or 5'-3H] (3'-Me-DAB) or during nonenzymatic substitutions at the 4 position of 3-methylacetanilide (4-3H, or 5-3H), and the effect of the repeated administration of carcinogenic 3'-Me-DAB upon the aryl hydroxylation activity against 3'-Me-DAB (5'-3H) and retention of tritium in the hydroxylated product. The enzymically formed 3'-methyl-4'-hydroxy-4-dimethylaminoazobenzene from 3'-Me-DAB (4'-3H) retained tritium 94% on an average. Accordingly, it may be proved that the NIH shift also occurs in the enzymatic hydroxylation of 3'-Me-DAB (4'-3H). The chemically produced 4-hydroxy-, 4, 6-dibromo-, and 4-nitro-3-methylacetanilide from 3-methylacetanilide (4-3H) retained tritium approximately 65% in each case. Since no significant effect of feeding 0.06% 3'-Me-DAB for 1-4 weeks on the aryl hydroxylation activity against 3'-Me-DAB (5'-3H) and retention of tritium in the hydroxylated product from 3'-Me-DAB (4'-3H) was observed, it may be suggested that the aryl hydroxylase activity of rat liver against 3'-Me-DAB dose not be qualitatively changed during the early stage of the carcinogenesis by 3'-Me-DAB.

Species Differences in the Biotransformation of a New Antiarrhythmic Agent: Disopyramide Phosphate

The biotransformation of disopyramide phosphate [4-diisopropylamino-2-phenyl-2-(2-pyridyl)butyramide phosphate] was studied in rats and dogs using the 14C-labeled compound and in man using the unlabeled drug. Within 72 hr., 78.7 ± 1.4% of the administered radioactivity was recovered in the urine of dogs after oral administration and 44.1 ± 3.4% was recovered in the urine of rats after intraperitoneal administration. In dogs, 17.3 ± 3.4% of the urinary radioactivity was associated with the unchanged compound, 12.4 ± 1.6% with 4-isopropylamino-2-phenyl-2-(2-pyridyl)-butyramide, and 29.2 ± 2.6% with 3-phenyl-3-(2-pyridyl)-2-pyrrolidone; 18.0 ± 3.3% was present as a water-soluble conjugate which, on acid hydrolysis, gave the pyrrolidone as the major aglycone. In rats, the urinary radioactivity was predominantly (80.9 ± 2.3%) associated with the unchanged disopyramide. In this species the major metabolic pathway was aryl hydroxylation, giving two phenolic compounds, one of which was identified as 4-diisopropylamino-2-(p-hydroxyphenyl)-2-(2-pyridyl)butyramide. These phenolic metabolites were predominantly excreted in the bile as conjugates. In man, 56% of the administered drug was excreted unchanged in the urine while 4% was present as the secondary amine. The structural assignments of the metabolites were based on their detailed spectroscopic analysis and by comparison of their chromatographic properties with authentic samples.

Aryl hydroxylation of isopropyl-3-chlorocarbanilate by soybean plants

Isopropyl-3-chlorocarbanilate-phenyl UL- 14C (CIPC- 14C) is absorbed, translocated and metabolized by soybean plants. Water-soluble metabolites in root and shoot were purified and the root major metabolite characterized. The acetylated aglucones from the β-glucosidase hydrolysis and the esters from the direct acetylation of CIPC- 14C polar metabolites were purified by GLC and analysed by mass spectrometry. The data showed that the phenyl riong of CIPC- 14C was hydroxylated by both root and shoot tissues. Isopropyl-5-chloro-2-hydroxycarbanilate (hydroxy-CIPC) was the predominant aglucone liberated by β-glucosidase from polar metabolites in root and shoot. The o-glucoside of hydroxy-CIPC was shown to be present, by direct acetylation and characterization. In shoot tissue the major metabolites were dechlorinated hydroxy-CIPC and were not hydrolysed by β-glucosidase. These data show that soybean root or shoot tissues hydroxylate the phenyl ring of CIPC- 14C but do not alter either the isopropyl alcohol moiety or the earbamate bond.

Studies on metabolism of 3-deoxysteroids. VII. Migration of deuterium during aryl hydroxylation of 3-deoxyestrone.

In order to examine the occurrence of NIH shiftduring hydroxylation of the aromatic steroid, 2-and 3-deuterio-3-deoxyestrones (IIIb, V) were synthesized as substrate. After oral administration of these labeled steroids to a rabbit, two principal metabolites, 2-hydroxy-3-deoxyestrone (Ib) and 17α-estradiol, were isolated from the collected urine specimen. Inspection of mass and nuclear magnetic resonance spectra revealed that aryl hydroxylation was accompanied by a migration of deuterium from the initially labeled site to an adjacent position. The direction and extent of isotope migration are listed in Table I.

Migration of deuterium during aryl hydroxylation: III. Effect of ortho- and meta- substituents

Cytochrome P450 (P450, CYP) research provides many opportunities for the application of kinetic isotope effect (KIE) strategies. P450s collectively catalyze oxidations of more substrates than any other group of enzymes, and CH bond cleavage is a major feature in a large fraction of these reactions. The presence of a significant primary deuterium KIE is evidence that hydrogen abstraction is at least partially rate-limiting in the reactions, and this appears to be the case in many P450 reactions. The first report of a KIE in (P450-linked) drug metabolism appeared in 1961 (for morphine N-demethylation), and in a number of cases, it has been possible to modulate the in vivo metabolism or toxicity of chemicals by deuterium substitution. A number of efforts are in progress to utilize deuterium substitution to alter the metabolism of drugs in an advantageous manner.

The migration of deuterium during aryl hydroxylation: II.Effect of induction of microsomal hydroxylases with phenobarbital or polycyclic aromatic hydrocarbons

Acetanilide-4- 2H is converted to 4-hydroxyacetanilide-3- 2H during incubation with liver microsomal preparations. The degree of migration and retention of deuterium in this reaction, previously shown to depend on the pH of the media, is also dependent upon the species, strain, and sex of the animal used as the source of microsomal preparations. Pretreatment of animals with phenobarbital causes an increase in the retention of deuterium in 4-hydroxyacetanilide produced from 4-deuteroacetanilide while pretreatment with benzpyrene or 3-methylcholanthrene causes a decrease in the retention. The effect of induction on the migration and retention of deuterium and of tritium is unique to acetanilide-4- 2H, and is not observed during hydroxylation of anisole-4- 2H, chlorobenzene-4- 2H, or 4-fluorobiphenyl-4′- 2H. It is proposed that two “acetanilide hydroxylases” exist in varying amounts in different species, and that one, characterized by exhibiting high retentions of deuterium in the hydroxylation of acetanilide-4- 2H, is induced by phenobarbital, while the other, characterized by exhibiting low retentions of deuterium in the same reaction, is induced by 3-methylcholanthrene and benzpsyrene.

Hydroxylation of alkyl and halogen substituted anilines and acetanilides by microsomal hydroxylases

The hydroxylation of a variety of halo and alkyl substituted anilines and acetanilides with hepatic microsomal preparations was studied. 4 Chloro- and 4-fluoro-acetanilides and anilines were hydroxylated to the corresponding 4-hydroxy derivatives with loss of halogen. The 4-chloro derivatives were extremely poor substrates for the aryl hydroxylases. 3-Methyl-, 3-chloro-, and 3-fluoro-acetanilides were metabolized to the corresponding 4-hydroxy-3-substituted acetanilide. 4-Methylaniline, 4-methyl-acetanilide, and 4-ethylacetanilide were not substrates for aryl hydroxylation, but instead underwent side-chain oxidation to form, respectively, 4-hydroxymethylaniline, 4-hydroxymethylacetanilide, and 4-(1′-hydroxyethyl)acetanilide. The hydroxymethyl compounds were further oxidized to aldehydes.

METABOLITES OF RHO-AMINOBENZOIC ACID. IV. STRUCTURE OF METABOLITE I AND ARYL HYDROXYLATION PRIOR HYDROXYMETHYLATION OF THE BENZENE RING.

METABOLITES OF RHO-AMINOBENZOIC ACID. IV. STRUCTURE OF METABOLITE I AND ARYL HYDROXYLATION PRIOR HYDROXYMETHYLATION OF THE BENZENE RING.

Aryl Hydroxylation of Phenoxyaliphatic Acids by Excised Roots

Aryl Hydroxylation of Phenoxyaliphatic Acids by Excised Roots