The enzyme systems carrying out this biotransformation are referred to as mixed-function oxidases or monooxygen-ases.24,25 There is a large family that carry out the same basic chemical reactions. Their nomenclature is based on amino acid homology and is summarized in Table 3.2. There are four components to the name. CYP refers to the cytochrome system. This is followed by the Arabic number that specifies the cytochrome family (CYP1, CYP2, CYP3, etc.). Next is a capital letter that represents the subfamily (CYP1A, CYP1B, CYP2A, CYP2B, CYP3A, CYP3B, etc.). Finally, the cytochrome name ends with another Arabic number that specifies the specific enzyme responsible for a particular reaction (CYP1A2, CYP2C9, CYP2C19, CYP3A4, etc.).
The reaction requires both molecular oxygen and the reducing agent NADPH (reduced form of nicotinamide adeno-sine dinucleotide phosphate). During this oxidative process, one atom of molecular oxygen (O2) is introduced into the substrate R-H to form R-OH and the other oxygen atom is incorporated into water. The mixed-function oxidase system26 is actually made up of several components, the most important being the superfamily of CYP enzymes (currently at 57 genes [http://drnelson.utmem.edu/CytochromeP450.html]), which are responsible for transferring an oxygen atom to the substrate R-H. Other important components of this system include the NADPH-dependent CYP reductase and the NADH-linked cytochrome b5. The latter two components, along with the cofactors NADPH and NADH, supply the reducing equivalents (electrons) needed in the overall metabolic oxidation of foreign compounds. The proposed mechanistic scheme by which the CYP monooxygenase system catalyzes the conversion of molecular oxygen to an "activated oxygen" species is elaborated below.
The CYP enzymes are heme proteins.27 The heme portion is an iron-containing porphyrin called protoporphyrin IX, and the protein portion is called the apoprotein. CYP is found in high concentrations in the liver, the major organ involved in the metabolism of xenobiotics. The presence of this enzyme in many other tissues (e.g., lung, kidney, intestine, skin, placenta, adrenal cortex) shows that these tissues have drug-oxidizing capability too. The name cytochrome P450 is derived from the fact that the reduced (Fe2+) form of this enzyme binds with carbon monoxide to form a complex that has a distinguishing spectroscopic absorption maximum at 450 nm.28
One important feature of the hepatic CYP mixed-function oxidase system is its ability to metabolize an almost unlimited number of diverse substrates by various oxidative transformations.29 This versatility is believed to be a result of the substrate nonspecificity of CYP as well as the presence of multiple forms of the enzyme.30 Some of these P450 enzymes are selectively inducible by various chemicals (e.g., phenobarbital, benzo[a]pyrene, 3-methyl-cholanthrene).31 One of these inducible forms of the enzyme (cytochrome P448)32 is of particular interest and is discussed later in this section.
The CYP monooxygenases are located in the endoplas-mic reticulum, a highly organized and complex network of intracellular membranes that is particularly abundant in tissues such as the liver.33 When these tissues are disrupted by homogenization, the endoplasmic reticulum loses its structure and is converted into small vesicular bodies known as microsomes. Mitochondria house many of the cytochrome enzymes that are responsible for the biosynthesis of steroidal hormones and metabolism of certain vitamins.
Microsomes isolated from hepatic tissue appear to retain all of the mixed-function oxidase capabilities of intact hepatocytes; because of this, microsomal preparations (with the necessary cofactors, e.g., NADPH, Mg2+) are used frequently for in vitro drug metabolism studies. Because of its membrane-bound nature, the CYP monooxygenase system appears to be housed in a lipoidal environment. This may explain, in part, why lipophilic xenobiotics are generally good substrates for the monooxygenase system.34
The catalytic role that the CYP monooxygenase system plays in the oxidation of xenobiotics is summarized in the cycle shown in Figure 3.1.35-37 The initial step of this catalytic reaction cycle starts with the binding of the substrate to the oxidized (Fe3+) resting state of CYP to form a P450-substrate complex. The next step involves the transfer of one electron from NADPH-dependent CYP reductase to the P450-substrate complex. This one-electron transfer reduces Fe3+ to Fe2+. It is this reduced (Fe2+) P450-substrate complex that is capable of binding dioxygen (O2). The dioxygen-P450-substrate complex that is formed then undergoes another one-electron reduction (by CYP reductase-NADPH and/or cytochrome b5 reductase-NADH) to yield what is believed to be a peroxide dianion-P450 (Fe3+)-substrate complex. Water (containing one of the oxygen atoms from the original dioxygen molecule) is released from the latter intermediate to form an activated oxygen-P450-substrate complex (Fig. 3.2). The activated oxygen [FeO]3+ in this complex is highly electron deficient and a potent oxidizing agent. The activated oxygen is transferred to the substrate (R-H), and the oxidized substrate product (R-OH) is released from the enzyme complex to regenerate the oxidized form of CYP.
The key sequence of events appears to center around the alteration of a dioxygen-P450-substrate complex to an activated oxygen-P450-substrate complex, which can then effect the critical transfer of oxygen from P450 to the substrate.37,38 In view of the potent oxidizing nature of the activated oxygen being transferred, it is not surprising CYP
Figure 3.2 • Simplified depiction of the proposed activated oxygen-cytochrome P450-substrate complex. Note the simplified apoprotein portion and the heme (protoporphyrin IX) portion or cytochrome P450 and the proximity of the substrate R-H undergoing oxidation.
Figure 3.2 • Simplified depiction of the proposed activated oxygen-cytochrome P450-substrate complex. Note the simplified apoprotein portion and the heme (protoporphyrin IX) portion or cytochrome P450 and the proximity of the substrate R-H undergoing oxidation.
can oxidize numerous substrates. The mechanistic details of oxygen activation and transfer in CYP-catalyzed reactions continue to be an active area of research in drug metabolism.34 The many types of oxidative reaction carried out by CYP are enumerated in the sections below. Many of these oxidative pathways are summarized schematically in Figure 3.3 (see also Table 3.1).37
The versatility of CYP in carrying out various oxidation reactions on a multitude of substrates may be attributed to the multiple forms of the enzyme. Consequently, students must realize that the biotransformation of a parent xenobiotic to several oxidized metabolites is carried out not just by one form of P450 but, more likely, by several different forms. Extensive studies indicate that the apoprotein portions of various CYPs differ from one another in their tertiary structure (because of differences in amino acid sequence or the makeup of the polypeptide chain).27,30,39-41 Because the apoprotein portion is important in substrate binding and catalytic transfer of activated oxygen, these structural differences may account for some substrates being preferentially or more efficiently oxidized by one particular form of CYP. Finally, because of the enormous number of uncommon reactions that are catalyzed by P450, the reader is directed to other articles of interest.42
O OXIDATIVE REACTIONS Oxidation of Aromatic Moieties
Aromatic hydroxylation refers to the mixed-function oxidation of aromatic compounds (arenes) to their corresponding phenolic metabolites (arenols).43 Almost all aromatic hydroxylation reactions are believed to proceed initially through an epoxide intermediate called an "arene oxide," which rearranges rapidly and spontaneously to the arenol product in most instances. The importance of arene oxides in the formation of arenols and in other metabolic and toxicologic
reactions is discussed below.44'45 Our attention now focuses on the aromatic hydroxylation of several drugs and xenobiotics.
Most foreign compounds containing aromatic moieties are susceptible to aromatic oxidation. In humans, aromatic hydroxylation is a major route of metabolism for many drugs containing phenyl groups. Important therapeutic agents such as propranolol,46,47 phenobarbital,48 phenyt-
ethinylestradiol,54,55 and (S)( — )-warfarin,56 among others, undergo extensive aromatic oxidation (Fig. 3.4 shows structure and site of hydroxylation). In most of the drugs just mentioned, hydroxylation occurs at the para posi tion.57 Most phenolic metabolites formed from aromatic oxidation undergo further conversion to polar and water-soluble glucuronide or sulfate conjugates, which are readily excreted in the urine. For example, the major urinary metabolite of phenytoin found in humans is the O-glucuronide conjugate of p-hydroxyphenytoin.49 50 Interestingly, the para-hydroxylated metabolite of phenylbutazone, oxyphenbutazone, is pharmacologically active and has been marketed itself as an anti-inflammatory agent (Tandearil, Oxalid).51,52 Of the two enantiomeric forms of the oral anticoagulant warfarin (Coumadin), only the more active S( —) enantiomer has been shown to undergo substantial aromatic hydroxylation to 7-hydroxywarfarin in humans.56 In contrast, the (R)( + ) enantiomer is metabolized by keto reduction56 (see "Stereochemical Aspects of Drug Metabolism").
Often, the substituents attached to the aromatic ring may influence the ease of hydroxylation.57 As a general rule, microsomal aromatic hydroxylation reactions appear to proceed most readily in activated (electron-rich) rings, whereas deactivated aromatic rings (e.g., those containing electron-withdrawing groups Cl, -N+R3, COOH, SO2NHR) are generally slow or resistant to hydroxylation. The deactivating groups (Cl, -N+H^C) present in the antihypertensive clonidine (Catapres) may explain why this drug undergoes little aromatic hydroxylation in humans.58,59 The uricosuric agent probenecid (Benemid), with its electron-withdrawing carboxy and sulfamido groups, has not been reported to undergo any aromatic hydroxylation.60
In compounds with two aromatic rings, hydroxylation occurs preferentially in the more electron-rich ring. For example, aromatic hydroxylation of diazepam (Valium) occurs primarily in the more activated ring to yield 4'-hydroxydiazepam.61 A similar situation is seen in the 7-hydroxylation of the antipsychotic agent chlorpromazine (Thorazine)62 and in the para-hydroxylation of p-chlorobiphenyl to p-chloro-p'-hydroxybiphenyl.63
Recent environmental pollutants, such as polychlorinated biphenyls (PCBs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), have attracted considerable public concern over their toxicity and health hazards. These compounds appear to be resistant to aromatic oxidation because of the numerous electronegative chlorine atoms in their aromatic rings. The metabolic stability coupled to the lipophilicity of these environmental contaminants probably explains their long persistence in the body once absorbed.64-66
Arene oxide intermediates are formed when a double bond in aromatic moieties is epoxidized. Arene oxides are of significant toxicologic concern because these intermediates are electrophilic and chemically reactive (because of the strained three-membered epoxide ring). Arene oxides are mainly detoxified by spontaneous rearrangement to arenols, but enzymatic hydration to trans-dihydrodiols and enzymatic conjugation with GSH also play very important roles (Fig. 3.5).43,44 If not effectively detoxified by the first three pathways in Figure 3.5, arene oxides will bind cova-lently with nucleophilic groups present on proteins, deoxyribonucleic acid (DNA), and ribonucleic acid (RNA), thereby leading to serious cellular damage.5,43 This, in part, helps explain why benzene can be so toxic to mammalian systems.
Quantitatively, the most important detoxification reaction for arene oxides is the spontaneous rearrangement to corresponding arenols. Often, this rearrangement is accompanied by a novel intramolecular hydride (deuteride) migration called the "NIH shift."67 It was named after the National Institutes of Health (NIH) laboratory in Bethesda, Maryland, where this process was discovered. The general features of the NIH shift are illustrated with the mixed-function aromatic oxidation of 4-deuterioanisole to 3-deuterio-4-hydroxyanisole in Figure 3.6.68
After its metabolic formation, the arene oxide ring opens in the direction that generates the most resonance-stabilized carbocation (positive charge on C-3 carbon is resonance stabilized by the OCH3 group). The zwitterionic species (positive charge on the C-3 carbon atom and negative charge on the oxygen atom) then undergoes a 1,2-deuteride shift (NIH shift) to form the dienone. Final transformation of the dienone to 3-deuterio-4-hydroxyanisole occurs with the preferential loss of a proton because of the weaker bond energy of the C-H bond (compared with the C-D bond). Thus, the deuterium is retained in the molecule by undergoing this intramolecular NIH shift. The experimental observation of an NIH shift for aromatic hydroxylation of a drug or xeno-biotic is taken as indirect evidence for the involvement of an arene oxide.
In addition to the NIH shift, the zwitterionic species may undergo direct loss of D+ to generate 4-hydroxyanisole, in which there is no retention of deuterium (Fig. 3.6). The alternative pathway (direct loss of D+) may be more favorable than the NIH shift in some aromatic oxidation reactions. Therefore, depending on the substituent group on the arene, some aromatic hydroxylation reactions do not display any NIH shift.
Two extremely important enzymatic reactions also aid in neutralizing the reactivity of arene oxides. The first of these involves the hydration (i.e., nucleophilic attack of water on the
epoxide) of arene oxides to yield inactive trans-dihydrodiol metabolites (Fig. 3.5). This reaction is catalyzed by microsomal enzymes called epoxide hydrases.69,70 Often, epoxide hydrase inhibitors, such as cyclohexene oxide and 1,1,1-trichloropropene-2,3-oxide, have been used to demonstrate the detoxification role of these enzymes. Addition of these inhibitors is accompanied frequently by increased toxicity of the arene oxide being tested, because formation of nontoxic dihy-drodiols is blocked. For example, the mutagenicity of benzo[a]pyrene-4,5-oxide, as measured by the Ames Salmonella typhimurium test system, is potentiated when cyclohexene oxide is added.71 Dihydrodiol metabolites have been reported in the metabolism of several aromatic hydrocarbons (e.g., naphthalene, benzo[a]pyrene, and other related poly cyclic aromatic hydrocarbons).43 A few drugs (e.g., phenytoin,72 phenobarbital,73 glutethimide74) also yield dihydrodiol products as minor metabolites in humans. Dihydrodiol products are susceptible to conjugation with glucuronic acid, as well as enzymatic dehydrogenation to the corresponding catechol metabolite, as exemplified by the metabolism of phenytoin.72
A second enzymatic reaction involves nucleophilic ring opening of the arene oxide by the sulfhydryl (SH) group present in GSH to yield the corresponding trans-1,2-dihydro-1-S-glutathionyl-2-hydroxy adduct, or GSH adduct (Fig. 3.5).44 The reaction is catalyzed by various GSH S-transferases.75 Because GSH is found in practically all mammalian tissues, it plays an important role in the detoxification not only of arene oxides but also of other various chemically reactive and potentially toxic intermediates. Initially, GSH adducts formed from arene oxides are modified in a series of reactions to yield "premercap-turic acid" or mercapturic acid metabolites.76 Because it is classified as a phase II pathway, GSH conjugation is covered in greater detail later in this chapter.
Because of their electrophilic and reactive nature, arene oxides also may undergo spontaneous reactions with nucleophilic functionalities present on biomacromol-ecules.44,45 Such reactions lead to modified protein, DNA, and RNA structures and often cause dramatic alterations in how these macromolecules function. Much of the cytotoxicity and irreversible lesions caused by arene oxides are presumed to result from their covalent binding to cellular components. Several well-established examples of reactive arene oxides that cause serious toxicity are presented below.
Administration of bromobenzene to rats causes severe liver necrosis.77 Extensive in vivo and in vitro studies indicate that the liver damage results from the interaction of a chemically reactive metabolite, 4-bromobenzene oxide, with hepatocytes.78 Extensive covalent binding to hepatic
tissue was confirmed by use of radiolabeled bromoben-zene. The severity of necrosis correlated well with the amount of covalent binding to hepatic tissue. Use of diethyl maleate or large doses of bromobenzene in rats showed that the depletion of hepatic GSH led to more severe liver necrosis.
Polycyclic aromatic hydrocarbons are ubiquitous environmental contaminants that are formed from auto emission, refuse burning, industrial processes, cigarette smoke, and other combustion processes. Benzo[a]pyrene, a potent carcinogenic agent, is perhaps the most extensively studied of the polycyclic aromatic hydrocarbons.79 Inspection of its structure reveals that aromatic hydroxylation of benzo[a]pyrene can occur at several positions. The identification of several dihydrodiol metabolites is viewed as indirect evidence for the formation and involvement of arene oxides in the metabolism of benzo[a]pyrene. Although certain arene oxides of benzo[a]pyrene (e.g., 4,5-oxide, 7,8-oxide, 9,10-oxide) appear to display some mutagenic and tumorigenic activity, it does not appear that they represent the ultimate reactive species responsible for benzo[a]pyrene's carcinogenicity. In recent years, extensive studies have led to the characterization of a specific sequence of metabolic reactions (Fig. 3.7) that generate a highly reactive intermediate that covalently binds to DNA. Metabolic activation of benzo[a]pyrene to the ultimate carcinogenic species involves an initial epoxida-tion reaction to form the 7,8-oxide, which is then converted by epoxide hydrase to ( — )-7(R),8(R)-dihydroxy-7,8-dihydrobenzo[a]pyrene.80 The two-step enzymatic formation of this trans--dihydrodiol is stereospecific. Subsequent epoxidation at the 9,10-double bond of the latter metabolite generates predominantly ( + )-7(R),8(S)-dihy-droxy-9(R),10(R)-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene or ( + )7,8-diol-9,10-epoxide. It is this key electrophilic diol epoxide metabolite that readily reacts with DNA to form many covalently bound adducts.81-83 Careful degradation studies have shown that the principal adduct involves attack of the C-2 amino group of deoxyguanosine at C-10 of the diol epoxide. Clearly, these reactions are responsible for genetic code alterations that ultimately lead to the malignant transformations. Covalent binding of the diol epoxide metabolite to deoxyadenosine and to deoxycytidine also has been established.84
Another carcinogenic polycyclic aromatic hydrocarbon, 7,12-dimethylbenz[a]anthracene, also forms covalent adducts with nucleic acids (RNA).85 The ultimate carcinogenic reactive species apparently is the 5,6-oxide that results from epoxidation of the 5,6-double bond in this aromatic hydrocarbon. The arene oxide intermediate binds covalently to guanosine residues of RNA to yield the two adducts.
The metabolic oxidation of olefinic carbon-carbon double bonds leads to the corresponding epoxide (or oxirane). Epoxides derived from olefins generally tend to be somewhat more stable than the arene oxides formed from aromatic compounds. A few epoxides are stable enough to be directly measurable in biological fluids (e.g., plasma, urine). Like their arene oxide counterparts, epoxides are susceptible to enzymatic hydration by epoxide hydrase to form trans-1,2-dihydrodiols (also called 1,2-diols or 1,2-dihydroxy
compounds).69,10 In addition, several epoxides undergo GSH conjugation.86
A well-known example of olefinic epoxidation is the metabolism, in humans, of the anticonvulsant drug carba-mazepine (Tegretol) to carbamazepine-10,11-epoxide.87 The epoxide is reasonably stable and can be measured quantitatively in the plasma of patients receiving the parent drug. The epoxide metabolite may have marked anticonvulsant activity and, therefore, may contribute substantially to the therapeutic effect of the parent drug.88 Subsequent hydration of the epoxide produces 10,11-dihydroxycarba-mazepine, an important urinary metabolite (10%-30%) in humans.87
Epoxidation of the olefinic 10,11-double bond in the antipsychotic agent protriptyline (Vivactil)89 and in the H1-histamine antagonist cyproheptadine (Periactin)90
also occurs. Frequently, the epoxides formed from the biotransformation of an olefinic compound are minor products, because of their further conversion to the corresponding 1,2-diols. For instance, dihydroxyalcofenac is a major human urinary metabolite of the once clinically useful anti-inflammatory agent alclofenac.91 The epoxide metabolite from which it is derived, however, is present in minute amounts. The presence of the dihydroxy metabolite (secodiol) of secobarbital, but not the epoxide product, has been reported in humans.92
Indirect evidence for the formation of epoxides comes also from the isolation of GSH or mercapturic acid metabolites. After administration of styrene to rats, two urinary metabolites were identified as the isomeric mercapturic acid derivatives resulting from nucleophilic attack of GSH on the intermediate epoxide.93 In addition, styrene oxide cova-lently binds to rat liver microsomal proteins and nucleic acids.94 These results indicate that styrene oxide is relatively reactive toward nucleophiles (e.g., GSH and nucleophilic groups on protein and nucleic acids).
There are, apparently, diverse metabolically generated epoxides that display similar chemical reactivity toward nucleophilic functionalities. Accordingly, the toxicity of some olefinic compounds may result from their metabolic conversion to chemically reactive epoxides.95 One example that clearly links metabolic epoxidation as a biotoxification pathway involves aflatoxin B1. This naturally occurring carcinogenic agent contains an olefinic (C2-C3) double bond adjacent to a cyclic ether oxygen. The hepatocarcinogenic-ity of aflatoxin B1 has been clearly linked to its metabolic oxidation to the corresponding 2,3-oxide, which is extremely reactive.96,97 Extensive in vitro and in vivo metabolic studies indicate that this 2,3-oxide binds covalently to DNA, RNA, and proteins. A major DNA adduct has been isolated and characterized as 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1.98,99
Other olefinic compounds, such as vinyl chloride,100 stilbene,101 and the carcinogenic estrogenic agent diethyl-stilbestrol (DES),102,103 undergo metabolic epoxidation. The corresponding epoxide metabolites may be the reactive
Possible covalent binding to proteins and/or nucleic acids species responsible for the cellular toxicity seen with these compounds.
An interesting group of olefin-containing compounds causes the destruction of CYP.104,105 Compounds belonging to this group include allylisopropylacetamide,106,107 secobarbital,108,109 and the volatile anesthetic agent fluroxene.110 It is believed that the olefinic moiety present in these compounds is activated metabolically by CYP to form a very reactive intermediate that covalently binds to the heme portion of CYP.111-113 The abnormal heme derivatives, or "green pigments," that result from this covalent interaction have been characterized as ^-alkylated protoporphyrins in which the N-alkyl moiety is derived directly from the olefin administered.104,105,111-113 Long-term administration of the above-mentioned three agents is expected to lead to inhibition of oxidative drug metabolism, potential drug interactions, and prolonged pharmacological effects.
Carbon atoms attached to aromatic rings (benzylic position) are susceptible to oxidation, thereby forming the corresponding alcohol (or carbinol) metabolite.114,115 Primary alcohol metabolites are often oxidized further to aldehydes and car-boxylic acids (CH2OH ^ CHO ^ COOH), and secondary alcohols are converted to ketones by soluble alcohol and aldehyde dehydrogenases.116 Alternatively, the alcohol may be conjugated directly with glucuronic acid.117 The benzylic carbon atom present in the oral hypoglycemic agent tolbutamide (Orinase) is oxidized extensively to the corresponding alcohol and carboxylic acid. Both metabolites have been isolated from human urine.118 Similarly, the "benzylic" methyl group in the anti-inflammatory agent tolmetin (Tolectin) undergoes oxidation to yield the dicarboxylic acid product as the major metabolite in humans.119,120 The selective cyclooxygenase 2 (COX-2) anti-inflammatory agent celecoxib undergoes ben-zylic oxidation at its C-5 methyl group to give hydroxycele-coxib as a major metabolite.121 Significant benzylic hydroxy lation occurs in the metabolism of the ß-adrenergic blocker metoprolol (Lopressor) to yield a-hydroxymetoprolol.122123 Additional examples of drugs and xenobiotics undergoing benzylic oxidation are shown in Figure 3.8.
Microsomal hydroxylation at allylic carbon atoms is commonly observed in drug metabolism. An illustrative example of allylic oxidation is given by the psychoactive component of marijuana, A ^tetrahydrocannabinol A1-THC. This molecule contains three allylic carbon centers (C-7, C-6, and C-3). Allylic hydroxylation occurs extensively at C-7 to yield 7-hydroxy- A1-THC as the major plasma metabolite in humans.10,11 Pharmacological studies show that this 7-hydroxy metabolite is as active as, or even more active than, A1-THC per se and may contribute significantly to the overall central nervous system (CNS) psychotomimetic effects of the parent compound.124,125 Hydroxylation also occurs to a minor extent at the allylic C-6 position to give both the epimeric 6a-and 6ß-hydroxy metabolites.1011 Metabolism does not occur at C-3, presumably because of steric hindrance.
The antiarrhythmic agent quinidine is metabolized by allylic hydroxylation to 3-hydroxyquinidine, the principal plasma metabolite found in humans.126,127 This metabolite shows significant antiarrhythmic activity in animals and possibly in humans.128
Other examples of allylic oxidation include the sedative-hypnotic hexobarbital (Sombulex) and the analgesic pentazocine (Talwin). The 3'-hydroxylated metabolite formed from hexobarbital is susceptible to glucuronide conjugation as well as further oxidation to the 3'-oxo compound.129,130 Hexobarbital is a chiral barbiturate derivative that exists in two enantiomeric forms. Studies in humans indicate that the pharmacologically less active (R)(—) enan-tiomer is metabolized more rapidly than its (S)(+)-isomer.131 Pentazocine undergoes allylic hydroxylation at the two
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terminal methyl groups of its N-butenyl side chain to yield either the cis or trans alcohol metabolites shown in the diagrams. In humans, more of the trans alcohol is formed.132133 For the hepatocarcinogenic agent safrole, allylic hydrox-ylation is involved in a bioactivation pathway leading to the formation of chemically reactive metabolites.134 This process involves initial hydroxylation at the C-T carbon of safrole, which is both allylic and benzylic. The hydroxyl-ated metabolite then undergoes further conjugation to form a sulfate ester. This chemically reactive ester intermediate presumably undergoes nucleophilic displacement reactions with DNA or RNA in vitro to form covalently bound adducts.135 As shown in the scheme, nucleophilic attack by DNA, RNA, or other nucleophiles is facilitated by a good leaving group (e.g., SO42") at the C-1 position. The leaving group tendency of the alcohol OH group itself is not enough to facilitate displacement reactions. Importantly, allylic hydroxylation generally does not lead to the generation of reactive intermediates. Its involvement in the biotoxification of safrole appears to be an exception.
The mixed-function oxidase system also oxidizes carbon atoms adjacent (i.e., a) to carbonyl and imino (C =N) functionalities. An important class of drugs undergoing this type of oxidation is the benzodiazepines. For example, diazepam (Valium), flurazepam (Dalmane), and nimetazepam are oxidized to their corresponding 3-hydroxy metabolites.136-138 The C-3 carbon atom undergoing hydroxylation is a to both a lactam carbonyl and an imino functionality.
For diazepam, the hydroxylation reaction proceeds with remarkable stereoselectivity to form primarily (90%) 3-hydroxydiazepam (also called N-methyloxazepam), with the (S) absolute configuration at C-3.139 Further N-demethy-lation of the latter metabolite gives rise to the pharmacologically active 3(S)(+)-oxazepam.
Hydroxylation of the carbon atom a to carbonyl functionalities generally occurs only to a limited extent in drug metabolism. An illustrative example involves the hydroxylation of the sedative-hypnotic glutethimide (Doriden) to 4-hydroxyglutethimide.140,141
Alkyl or aliphatic carbon centers are subject to mixed-function oxidation. Metabolic oxidation at the terminal methyl group often is referred to as w-oxidation, and oxidation of the penultimate carbon atom (i.e., next-to-the-last carbon) is called w-1 oxidation.114,115 The initial alcohol metabolites formed from these enzymatic w and w-1 oxidations are susceptible to further oxidation to yield aldehyde, ketones, or carboxylic acids. Alternatively, the alcohol metabolites may undergo glucuronide conjugation.
Aliphatic m and m-1 hydroxylations commonly take place in drug molecules with straight or branched alkyl chains. Thus, the antiepileptic agent valproic acid (Depakene) undergoes both m and m-1 oxidation to the 5-hydroxy and 4-hydroxy metabolites, respectively.142,143 Further oxidation of the 5-hydroxy metabolite yields 2-n-propylglutaric acid.
Numerous barbiturates and oral hypoglycemic sulfonyl-ureas also have aliphatic side chains that are susceptible to oxidation. Note that the sedative-hypnotic amobarbital (Amytal) undergoes extensive m-1 oxidation to the corresponding 3'-hydroxylated metabolite.144 Other barbiturates, such as pentobarbital,145,146 thiamylal,147 and secobarbital,92 reportedly are metabolized by way of m and m-1 oxidation. The n-propyl side chain attached to the oral hypoglycemic agent chlorpropamide (Diabinese) undergoes extensive m-1 hydroxylation to yield the secondary alcohol 2'-hydroxychlorpropamide as a major urinary metabolite in humans.148
Omega and m-1 oxidation of the isobutyl moiety present in the anti-inflammatory agent ibuprofen (Motrin) yields the corresponding carboxylic acid and tertiary alcohol metabolites.149 Additional examples of drugs reported to undergo aliphatic hydroxylation include meprobamate,150 glutethimide,140,141 ethosuximide,151 and phenylbutazone.152
The cyclohexyl group is commonly found in many medicinal agents, and is also susceptible to mixed-function oxidation (alicyclic hydroxylation).114,115 Enzymatic introduction of a hydroxyl group into a monosubstituted cyclo-hexane ring generally occurs at C-3 or C-4 and can lead to cis and trans conformational stereoisomers, as shown in the diagrammed scheme.
An example of this hydroxylation pathway is seen in the metabolism of the oral hypoglycemic agent acetohexamide (Dymelor). In humans, the trans-4-hydroxycyclohexyl product is reportedly a major metabolite.153 Small amounts of the other possible stereoisomers (namely, the cis-4-, cis-3-, and trans-3-hydroxycyclohexyl derivatives) also have been detected. Another related oral hypoglycemic agent, glipizide, is oxidized in humans to the trans-4- and cis-3-hydroxylcyclohexyl metabolites in about a 6:1 ratio.154
Two human urinary metabolites of phencyclidine (PCP) have been identified as the 4-hydroxypiperidyl and 4-hydroxycyclohexyl derivatives of the parent compound.155156 Thus, from these results, it appears that "alicyclic" hydroxy-lation of the six-membered piperidyl moiety may parallel closely the hydroxylation pattern of the cyclohexyl moiety. The stereochemistry of the hydroxylated centers in the two metabolites has not been clearly established. Biotransformation of the antihypertensive agent minoxidil (Loniten) yields the 4'-hydroxypiperidyl metabolite. In dogs, this product is a major urinary metabolite (29%-47%), whereas in humans it is detected in small amounts (~3%).157,158
Nitrogen and oxygen functionalities are commonly found in most drugs and foreign compounds; sulfur functionalities occur only occasionally. Metabolic oxidation of carbon-nitrogen, carbon-oxygen, and carbon-sulfur systems principally involves two basic types of biotransformation processes:
1. Hydroxylation of the a-carbon atom attached directly to the heteroatom (N, O, S). The resulting intermediate is often unstable and decomposes with the cleavage of the carbon-heteroatom bond:
Oxidative N-, O-, and S-dealkylation as well as oxidative deamination reactions fall under this mechanistic pathway.
2. Hydroxylation or oxidation of the heteroatom (N, S only, e.g., N-hydroxylation, N-oxide formation, sulfoxide, and sulfone formation).
Several structural features frequently determine which pathway will predominate, especially in carbon-nitrogen systems. Metabolism of some nitrogen-containing compounds is complicated by the fact that carbon- or nitrogen-
hydroxylated products may undergo secondary reactions to form other, more complex metabolic products (e.g., oxime, nitrone, nitroso, imino). Other oxidative processes that do not fall under these two basic categories are discussed individually in the appropriate carbon-heteroatom section. The metabolism of carbon-nitrogen systems will be discussed first, followed by the metabolism of carbon-oxygen and carbon-sulfur systems.
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