Aromatic Amines and Heterocyclic Nitrogen Compounds. The biotransformation of aromatic amines parallels the carbon and nitrogen oxidation reactions seen for aliphatic amines.221-223 For tertiary aromatic amines, such as N,N-dimethylaniline, oxidative N-dealkylation as well as N-oxide formation take place.224 Secondary aromatic amines may undergo N-dealkylation or N-hydroxylation to give the corresponding N-hydroxylamines. Further oxidation of the N-hydroxylamine leads to nitrone products, which in turn may be hydrolyzed to primary hydrox-ylamines.225 Tertiary and secondary aromatic amines are encountered rarely in medicinal agents. In contrast, primary aromatic amines are found in many drugs and are often generated from enzymatic reduction of aromatic nitro compounds, reductive cleavage of azo compounds, and hydrolysis of aromatic amides.
N-oxidation of primary aromatic amines generates the N-hydroxylamine metabolite. One such case is aniline, which is metabolized to the corresponding N-hydroxy prod-uct.223 Oxidation of the hydroxylamine derivative to the nitroso derivative also can occur. When one considers primary aromatic amine drugs or metabolites, N-oxidation constitutes only a minor pathway in comparison with other biotransformation pathways, such as N-acetylation and aromatic hydroxylation, in humans. Some N-oxygenated metabolites have been reported, however. For example, the antileprotic agent dapsone and its N-acetylated metabolite are metabolized significantly to their corresponding N-hydroxylamine derivatives.226 The N-hydroxy metabolites are further conjugated with glucuronic acid.
Methemoglobinemia toxicity is caused by several aromatic amines, including aniline and dapsone, and is a result of the bioconversion of the aromatic amine to its N-hydroxy derivative. Apparently, the N-hydroxylamine oxidizes the Fe2+ form of hemoglobin to its Fe3+ form. This oxidized (Fe3+) state of hemoglobin (called methemoglobin or ferrihemoglo-bin) can no longer transport oxygen, which leads to serious hypoxia or anemia, a unique type of chemical suffocation.227
Diverse aromatic amines (especially azoamino dyes) are known to be carcinogenic. N-oxidation plays an important role in bioactivating these aromatic amines to potentially reactive electrophilic species that covalently bind to cellular protein, DNA, or RNA. A well-studied example is the carcinogenic agent N-methyl-4-aminoazobenzene.228'229 N-oxidation of this compound leads to the corresponding hydroxylamine, which undergoes sulfate conjugation. Because of the good leaving-group ability of the sulfate (SO42_) anion, this conjugate can ionize spontaneously to form a highly reactive, resonance-stabilized nitrenium species. Covalent adducts between this species and DNA, RNA, and proteins have been characterized.230,231 The sulfate ester is believed to be the ultimate carcinogenic species. Thus, the example indicates that certain aromatic amines can be bioactivated to reactive intermediates by N-hydroxylation and O-sulfate conjugation. Whether primary hydroxylamines can be bioactivated similarly is unclear. In addition, it is not known if this biotoxification pathway plays any substantial role in the toxicity of aromatic amine drugs.
N-oxidation of the nitrogen atoms present in aromatic heterocyclic moieties of many drugs occurs to a minor extent. Clearly, in humans, N-oxidation of the folic acid antagonist trimethoprim (Proloprim, Trimpex) has yielded approximately equal amounts of the isomeric 1-N-oxide and 3-N-oxide as minor metabolites.232 The pyridinyl nitrogen atom present in nicotinine (the major metabolite of nicotine) undergoes oxidation to yield the corresponding N-oxide metabolite.233 Another therapeutic agent that has been observed to undergo formation of an N-oxide metabolite is metronidazole.234
Amides. Amide functionalities are susceptible to oxidative carbon-nitrogen bond cleavage (via a-carbon hydroxylation) and N-hydroxylation reactions. Oxidative dealkylation of many N-substituted amide drugs and xenobiotics has been reported. Mechanistically, oxidative dealkylation proceeds via an initially formed car-binolamide, which is unstable and fragments to form the N-dealkylated product. For example, diazepam undergoes extensive N-demethylation to the pharmacologically active metabolite desmethyldiazepam.235
Various other N-alkyl substituents present in benzodiazepines (e.g., flurazepam)136-138 and in barbiturates (e.g., hexobarbital and mephobarbital)128 are similarly oxidatively N-dealkylated. Alkyl groups attached to the amide moiety of some sulfonylureas, such as the oral hypoglycemic chlorpropamide,236 also are subject to dealkylation to a minor extent.
In the cyclic amides or lactams, hydroxylation of the ali-cyclic carbon a to the nitrogen atom also leads to carbino-lamides. An example of this pathway is the conversion of cotinine to 5-hydroxycotinine. Interestingly, the latter carbinolamide intermediate is in tautomeric equilibrium with the ring-opened metabolite y-(3-pyridyl)-y-oxo-N-methylbutyramide.237
Metabolism of the important cancer chemotherapeutic agent cyclophosphamide (Cytoxan) follows a hydroxylation pathway similar to that just described for cyclic amides. This
drug is a cyclic phosphoramide derivative and, for the most part, is the phosphorous counterpart of a cyclic amide. Because cyclophosphamide itself is pharmacologically inactive,238 metabolic bioactivation is required for the drug to mediate its antitumorigenic or cytotoxic effects. The key biotransformation pathway leading to the active metabolite involves an initial carbon hydroxylation reaction at C-4 to form the carbinolamide 4-hydroxycyclophosphamide.239,240
4-Hydroxycyclophosphamide is in equilibrium with the ring-opened dealkylated metabolite aldophosphamide. Although it has potent cytotoxic properties, aldophos-phamide undergoes a further elimination reaction (reverse Michael reaction) to generate acrolein and the phospho-ramide mustard N,N-bis(2-chloro-ethyl)phosphorodiamidic acid. The latter is the principal species responsible for cyclophosphamide's antitumorigenic properties and
Cotinine 5-Hydroxycotinine y-(3-Pyridyl)-y-oxo-A/-
Cotinine 5-Hydroxycotinine y-(3-Pyridyl)-y-oxo-A/-
methylbutyramide chemotherapeutic effect. Enzymatic oxidation of 4-hydroxy-cyclophosphamide and aldophosphamide leads to the relatively nontoxic metabolites 4-ketocyclophosphamide and carboxycyclophosphamide, respectively.
N-hydroxylation of aromatic amides, which occurs to a minor extent, is of some toxicological interest, because this biotransformation pathway may lead to the formation of chemically reactive intermediates. Several examples of cytotoxicity or carcinogenicity associated with metabolic N-hydroxylation of the parent aromatic amide have been reported. For example, the well-known hepatocarcinogenic 2-acetylaminofluorene (AAF) undergoes an N-hydroxyl-ation reaction catalyzed by CYP to form the corresponding N-hydroxy metabolite (also called a hydroxamic acid).241 Further conjugation of this hydroxamic acid produces the corresponding O-sulfate ester, which ionizes to generate the electrophilic nitrenium species. Covalent binding of this reactive intermediate to DNA is known to occur and is likely to be the initial event that ultimately leads to malignant tumor formation.242 Sulfate conjugation plays an important role in this biotoxification pathway (see "Sulfate Conjugation," for further discussion).
Acetaminophen is a relatively safe and nontoxic analgesic agent if used at therapeutic doses. Its metabolism illustrates the fact that a xenobiotic commonly produces more than one metabolite. Its metabolism also illustrates the effect of age, because infants and young children carry out sulfation rather than glucuronidation (see discussion at the end of this chapter). New pharmacists must realize that at one time acetanilide and phenacetin were more widely used than acetaminophen, even though both are considered more toxic because they pro duce aniline derivatives. Besides producing toxic aniline and p-phenetidin, these two analgesics also produce acetaminophen. When large doses of the latter drug are ingested, extensive liver necrosis is produced in humans and animals.243,244 Considerable evidence argues that this hepatotoxicity depends on the formation of a metabolically generated reactive intermediate.245 Until recently,246,247 the accepted bioactiva-tion pathway was believed to involve an initial N-hydroxyla-tion reaction to form N-hydroxyacetaminophen.248
Spontaneous dehydration of this N-hydroxyamide produces N-acetylimidoquinone, the proposed reactive metabolite. Usually, the GSH present in the liver combines with this reactive metabolite to form the corresponding GSH conjugate. If GSH levels are sufficiently depleted by large doses of acetaminophen, covalent binding of the reactive intermediate occurs with macromolecules present in the liver, thereby leading to cellular necrosis. Studies indicate, however, that the reactive N-acetylimidoquinone intermediate is not formed from N-hydroxyacetaminophen.245-247 It probably arises through some other oxidative process. Therefore, the mechanistic formation of the reactive metabolite of acetaminophen remains unclear.
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