The Biochemistry of Drug Metabolism- an introduction

REVIEW

The Biochemistry of Drug Metabolism – An IntroductionPart 4. Reactions of Conjugation and Their Enzymes

by Bernard Testa*a) and Stefanie D. Kr�merb)

a) Department of Pharmacy, University Hospital Centre (CHUV), Rue du Bugnon, CH-1011 Lausanne(e-mail: [email protected])

b) Department of Chemistry and Applied Biosciences, ETH Zurich, Wolfgang-Pauli-Strasse 10,CH-8093 Zurich

This Part 4 of our biochemical introduction to drug metabolism [1 –4] presentsthe reactions of conjugation and their enzymes. As we shall see, reactions ofconjugation are also a major focus of interest in the metabolism of drugs andother xenobiotics. Books specifically dedicated to conjugation reactions arerare [5], but much recent information can be found in book chapters (e.g.,[6] [7]).

For a reaction of conjugation to occur, a suitable functional group must be presentin the substrate, which will serve as the anchoring site for an endogenous mole-cule or moiety such as CH3, sulfate, glucuronic acid, or glutathione. Conjugationreactions are thus synthetic (i.e., anabolic) reactions whose products are of modestly tomarkedly higher molecular weight than the corresponding substrate. As for theanchoring group, it can either be present in a xenobiotic or be created by afunctionalization reaction. In other words, reactions of conjugation are able to producefirst-generation as well as later-generation metabolites. We, therefore, consider asunfelicitous the term of �phase II reactions� commonly used to designate conjuga-tions.

A first issue when discussing reactions of conjugation will be to offer a cleardefinition. As we shall see, a number of criteria exist, all of which show some degree offuzziness, and only one of which must necessarily be met. This has indeed led to someconfusion with reactions of hydrolysis, which some biochemists have viewed asconjugation. We oppose such a view for reasons previously explained [3]. To repeatwhat we stated, reactions of hydrolysis are not catalyzed by transferases (EC 2) but byhydrolases (EC 3) [8], and water is not an endogenously synthesized molecule ormoiety linked covalently to a cofactor.

Reactions of conjugation, like the reactions of functionalization we saw in Parts 2and 3, act on exogenous substrates (i.e., xenobiotics [1]) as well as endogenoussubstrates (i.e., endobiotics). This dual functionality may create a potential formetabolic interaction between a drug and an endogenous substrate, a frequentlyoverlooked mechanism of toxicity. Thus, there may be competitive affinity for thecatalytic site of an endobiotic-metabolizing enzyme, or there may be competition forthe limited supply of a cofactor. A typical example of the latter case is found with

CHEMISTRY & BIODIVERSITY – Vol. 5 (2008) 2171

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paracetamol, a high-dose drug undergoing extensive glucuronidation whose admin-istration is forbidden to neonates and babies, since it deprives them of the glucuronicacid they need to detoxify bilirubin.

CHEMISTRY & BIODIVERSITY – Vol. 5 (2008)2172

Fig. 4.1. The structure of this Part follows custom as much as logic. First, anintroductory Chapter will present an overview of the reactions of conjugation, theircriteria, and their similarities and differences with functionalization reactions. As forthe major reactions to be discussed in the subsequent Chapters, there is nooverwhelming argument for preferring one order over another. We shall begin withthe rather straightforward case of the reactions of methylation (Chapt. 4.2). Reactionsof sulfonation (Chapt. 4.3) and glucuronidation (Chapt. 4.4) sometimes compete forthe same substrates and will, therefore, be treated in sequence. Together withsulfonation, we will have a few things to say about reactions of phosphorylation, whoserarity should not obscure their significance in the activation of some drugs. Chapt. 4.5and 4.6 center on coenzyme A, but with a difference. Reactions of acetylation(Chapt. 4.5) follow the usual pattern in having the conjugating moiety carried by thecoenzyme, here coenzyme A. In contrast, there is a variety of reactions where thesubstrate (be it a xenobiotic or an endobiotic) is coupled to coenzyme A prior to beingprocessed along vastly different pathways (Chapt. 4.6). Chapt. 4.7 presents glutathioneand its reactions, a topic of marked biocomplexity and great toxicological significance.

A few unclassifiable reactions will be summarized in Chapt. 4.8.



Fig. 4.2. This Figure is a slightly amended form of Fig. 1.12 we saw in Part 1 [1], with afew additional details. The focus here is on the conjugating moiety being transferred tothe substrate as a result of a conjugation reaction. Reference is made to Chapt. 4.2 –4.8to help the readers get a better view of the present Part. We also have here a first glanceat the different pathways which xenobiotic – coenzyme A conjugates can follow. Two ofthese pathways are not conjugations stricto sensu and, for this reason, are written initalics. Nevertheless, they will be discussed here, since a coenzyme A conjugate is theindispensable intermediate. These two pathways are the unidirectional inversion ofconfiguration of profens (and a few other xenobiotics) and the b-oxidation of fatty acidanalogs. As for the reactions in Chapt. 4.8, they represent poorly investigated pathways

nevertheless worthy of some attention.


Fig. 4.3. Conjugation reactions are characterized by a number of criteria which arepresented here [5] [6] [9]. First and above all, they involve an endogenous molecule(called the endogenous conjugating moiety, and sometimes abbreviated as the�endocon�) with which the substrate is coupled. This is the absolute criterion ofconjugation reactions, although, as we shall see, there may be arbitrariness in decidingwhether a conjugating moiety such as CO2 is endogenous. Second, this endogenousmolecule or moiety is generally polar (hydrophilic) or even highly polar, but there areexceptions. Third, the size of the endocon is generally in the range of 100– 300 Da.Fourth, the endogenous conjugating moiety is usually carried by a cofactor, with thechemical bond linking the cofactor and the endocon being a high-energy one such thatthe Gibbs energy released upon its cleavage drives the transfer of the endocon to thesubstrate. Fifth, conjugation reactions are catalyzed by enzymes known as transferases(EC 2) which bind the substrate and the cofactor in such a manner that their closeproximity allows the reaction to proceed. The metaphor of transferases being a �nuptialbed� has not escaped some biochemists. It is important from a biochemical and practicalviewpoint to note that Criteria 2 – 5 considered separately are neither sufficient nornecessary to define conjugation reactions. They are not sufficient, since, in hydro-genation reactions (i.e., typical reactions of oxidoreduction), the hydride is alsotransferred from a cofactor (NADPH or NADH). And they are not necessary, since

they all suffer from some important exceptions (see next Figure).


Fig. 4.4. This Figure summarizes in tabular form the cases of compliance and non-compliance to the five conjugation criteria. As can be seen, Criterion 1 is indeed theonly one that knows no exception, since all conjugating moieties involved are indeedendogenous. Thus, the C2 unit in chain elongation is derived from acetyl-coenzyme A(Chapt. 4.6). Most of the CO2 used in the formation of carbamic acids (Chapt. 4.8) isclearly also produced in vivo. The criterion of polarity of the endocon (Criterion 2)knows only two major exceptions, namely the coupling of xenobiotic carboxylic acids tosterols or to diglycerides (to yield mixed triglycerides), and the C2 chain elongation(Chapt. 4.6). The transfer of a CH3 group is special, since it adds a hydrophobic moietyexcept when forming quaternary ammonium metabolites (Chapt. 4.2). The CH3 group,being small, is also an exception to Criterion 3 as is the acetyl moiety. But we also haverelatively large endocons such as sterols (Chapt. 4.6) and glutathione (Chapt. 4.7). Asfor the conjugating moiety being carried by a coenzyme (Criterion 4), exceptions areglutathione (Chapt. 4.7), carbonyl compounds (Chapt. 4.8), and all reactions inChapt. 4.6, since here and as stated it is the substrate rather than the endocon that isattached to coenzyme A. Finally, catalysis by a transferase (Criterion 5) is almostalways the case, the few exceptions being the coupling of hydrazines with carbonylcompounds (Chapt. 4.8) and some nonenzymatic conjugations with glutathione

(Chapt. 4.7).


Fig. 4.5. This Figure and the next one illustrate the deep analogy between thephysiological reactions of endobiotic conjugation and the conjugation of xenobiotics.Their graphical similarity with Fig. 1.23 in Part 1 [1] is not fortuitous. As shown here, awaste product of physiological metabolism (e.g., bilirubin, a toxic breakdown productof hemoglobin), an endogenous compound (e.g., the neurotransmitter noradrenaline),or a nutrient (e.g., a fatty acid) is captured by a transferase. The latter catalyzes thetransfer of the adequate conjugating moiety from the cofactor to the substrate, yieldinga conjugate. These reactions have evolved to fulfill a variety of functions, as classified inthe Figure. Thus, the toxic bilirubin is detoxified by conjugation with glucuronic acid,the resulting glucuronide being excreted in the bile. The case of noradrenaline isdifferent, being N-methylated to the neurotransmitter adrenaline or O-methylated toan inactive metabolite. The case of metabolic intermediates in anabolism (syntheticmetabolism) and catabolism (breakdown metabolism) is illustrated with fatty acids,whose coenzyme A conjugates can undergo anabolism by C2 chain elongation, or

catabolism by b-oxidation.


Fig. 4.6. Reactions of xenobiotic conjugation have evolved from physiologicalconjugations to fulfill protective functions [10]. Thus, a xenobiotic containing anadequate target group, or a phase I metabolite, is captured and metabolized by atransferase. As we shall see, some transferases recognize endobiotics and xenobioticsalike (e.g., catechol O-methyltransferase), while others have diversified and arespecialized to some extent toward endobiotics or xenobiotics (e.g., UDP-glucuronyl-transferases). As a rule, drug conjugation inactivates the substrate, but there are onlyfew noteworthy exceptions such as the highly active morphine 6-O-glucuronide.Similarly, toxicity is usually greatly decreased by conjugation (e.g., N-methylpyridi-nium), but, as we shall see, there are numerous examples of conjugations leading totoxification. Some conjugates may indeed be reactive (e.g., some acyl glucuronides),whereas others are highly lipophilic and may accumulate in tissues as residues (e.g.,some mixed triglycerides). Such exceptions should not hide the fact that the greatlyincreased hydrophilicity of many conjugates relative to their parent compoundfacilitates their excretion. What is more, a co-evolution of transferases and transportersis believed to have occurred, such that the formation of polar conjugates (e.g.,

glucuronides and glutathione derivatives) is coupled to their active excretion [11].


Fig. 4.7. This Figure opens Chapt. 4.2 dedicated to biomethylation. These reactions arecatalyzed by methyltransferases (EC 2.1.1), and the endogenous conjugating moiety is aCH3 group carried by the cofactor S-adenosyl-l-methionine (4.1; SAM, AdoMet) [5–8] [12] [13]. The CH3 group in SAM is bound to a sulfonium center, giving it a markedelectrophilic character and explaining its reactivity. During the reaction, S-adenosyl-l-methionine loses the CH3 group and the positive charge to become S-adenosyl-l-homocysteine (4.2). As shown in the Figure, three major types of reactions arerecognized, namely the O-methylation of phenolic groups (mainly catechols), the N-methylation of endocyclic or exocyclic amino groups, and the S-methylation of thiols.Arsenic methylation (not shown in this Figure) will also be discussed. An importantaspect made explicit here is the fate of the positive charge carried by the sulfoniumcenter. In most cases, the positive charge is lost in the form of a proton and the N-methylated metabolite is more lipophilic than the parent compound. The exception isthe N-methylation of pyridine-type N-atoms, which forms a quaternary ammoniumgroup retaining the positive charge. This is pharmacokinetically relevant, since suchpositively charged metabolites are markedly more hydrophilic than the parent

compound, resulting in accelerated excretion.


Fig. 4.8. The main enzymes responsible for O-methylation are catechol O-methyl-transferase (COMT) and phenol O-methyltransferase (PMT) [8]. The former is farmore significant as far as xenobiotic metabolism is concerned, and little can be foundabout the latter in the relevant literature. COMT is mainly cytosolic (molecular weightof ca. 25 kDa) but also exists in membrane-bound form [13 – 16]. Both forms areproducts of a single gene, the membrane-bound form including an additional 50 residuesegment at the N-terminus. The cytosolic form is expressed to high levels in the liverand kidneys, and the membrane-bound form predominates in the brain. The expressionof COMT in human red blood cells has greatly facilitated its study. COMT fulfilsimportant physiological functions by methylating (and inactivating) the catecholamineneurotransmitters dopamine, noradrenaline (norepinephrine), and adrenaline (epi-nephrine). As we shall see, it also methylates catecholestrogens. Its genetically reducedactivity in ca. 25% of Caucasians may have therapeutic and physiopathological

significance [13] [17].


Fig. 4.9. O-Methylations are common reactions of compounds containing a catecholmoiety, 4.3, with a usual regioselectivity for the meta-position (i.e., 4.4) over the para-position (i.e., 4.5) [18]. The substrates can be xenobiotics and particularly drugs, asexemplified in the lower part of the Figure which summarizes a few results from anextensive investigation in which the rates of O-methylation of ca. 50 substrates weredetermined in the presence of recombinant human soluble COMT [18a]. In this Figure,selected substrates are arranged according to their rate of methylation by humanrecombinant soluble COMT. Very good substrates were for example 4-nitrocatechol(4.6) and caffeic acid (4.7). Fair substrates were catechol itself (4.8) and the drugdobutamine (4.9), a cardiostimulant b1-adrenoceptor agonist. The last row shows threewell-known substrates whose rate of methylation was comparatively slow in vitro,namely the neurotransmitter dopamine (4.10), the anti-Parkinsonian drug l-DOPA(4.11), and (S)-carbidopa (4.12), a peripheral inhibitor of l-DOPA decarboxylase usedin combination therapy to increase the efficacy of l-DOPA. Structure – metabolismrelationship studies showed that bulky substituents in adjacent positions decreasedmethylation, whereas increased acidity of a OH group favored it. A more refinedanalysis showed that increased ionization enhanced affinity for COMT, while theturnover rate and Vmax were favored by a high electron density on the phenolate O-atom, in other words, by a restricted delocalization of the negative charge. This isconsistent with nucleophilicity of the target group favoring its methylation, inagreement with the electrophilic character of the CH3 group in S-adenosyl-l-

methionine [18].


Fig. 4.10. In addition to xenobiotic substrates, O-methylation can occur as a late eventin the metabolism of phenolic or aryl groups, after they have been oxidized tocatechols. This is exemplified here with two recently introduced drugs. Thus,traxoprodil (4.13) is a selective N-methyl-S-aspartate receptor antagonist of potentialinterest in neurodegenerative diseases and brain injury. One of the major metabolicpathways of the drug in humans is a CYP2D6-catalyzed oxidation to the catecholmetabolite, followed by meta-O-methylation to 3’-methoxytraxoprodil (4.14) [19].Another informative example is that of duloxetine (4.15). This inhibitor of serotoninand noradrenaline reuptake contains a naphthalen-1-yl moiety which undergoesoxidation in humans at the 4-, 5- or 6-positions, followed by further oxidation [20]. Asignificant metabolite so produced is the catechol derivative 5,6-dihydroxyduloxetine(4.16), which was found to undergo O-methylation at either position with apredominance for the 6-OH group. The last example in this Figure is taken fromphytochemistry, namely the tea polyphenol (�)-epigallocatechin gallate (4.17). Thiscompound shows two trihydroxyphenyl moieties, each of which is a potential target forCOMT. Incubations with human or rodent soluble COMT revealed the fastmethylation of the 4’’-OH group. This reaction was followed in a second step by theformation of the 4’,4’’-dimethylated metabolite [21], the major metabolite of 4.17detected in the urine of humans and rodents. Interestingly, these two metabolites werefound to be strong noncompetitive inhibitors of COMT, possibly potentiating the

activity of endogenous catecholamines [22].


Fig. 4.11. Catechol O-methyltransferase is also of interest in the detoxification ofcatechol estrogens. Indeed, 17b-estradiol (4.18) is oxidized by cytochrome P450 to 2-hydroxy- and 4-hydroxyestradiol (4.19 and 4.20, resp.). The catecholestrogens areoxidized by CYPs or peroxidases (PER) to the catecholestrogen quinones 4.21 and 4.22.These are reactive endogenous metabolites known to react with nucleic acids and toplay a role in estrogen carcinogenesis [23]. In this context, it is important to note thatCOMT-catalyzed O-methylation is a protective pathway which decreases quinoneformation by competing with oxidoreductases for the catechol substrates. The samepathways are known for estrone, the 17-keto analogue of 4.18. Of further interest incatecholestrogen O-methylation is the fact that 2-methoxyestradiol (4.23) attenuatescardiovascular and renal diseases. These effects were first detected after administrationof 2-hydroxyestradiol (4.19). More recent in vivo studies in rats have revealed that the2-O-methylation of 4.19 is so fast that this catecholestrogen is in fact a prodrug of 2-

methoxyestradiol (4.23) [24].


Fig. 4.12. N-Methylation is a common pathway for several neurotransmitters andhormones. Besides these endogenous compounds, a significant number of exogenousamines are N-methylated. Several enzymes catalyze reactions of xenobiotic N-methylation with different substrate specificities, e.g., nicotinamide N-methyltransfer-ase (NNMT), histamine N-methyltransferase (HNMT), phenylethanolamine N-meth-yltransferase, and nonspecific amine N-methyltransferase [8] [12] [13]. This Figurepresents an Enzyme Identity Card of NNMT and HNMT, both of which are cytosolicand are expressed in the liver and in a number of other organs. Histamine NMT has anarrow substrate specificity and plays a limited role in drug metabolism. However, anumber of xenobiotic amines are known to inhibit it, e.g., (�)-(S)-nicotine (seeFig. 4.15). This is a worrying cause of side-effects, all the more so since the enzyme ispolymorphic in humans. Compared to HNMT, nicotinamide NMT is rather promis-cuous and N-methylates a remarkable variety of xenobiotic aromatic azaheterocycles(see Fig. 4.15). As we shall see, the products are quaternary ammonium cations whosepolarity and renal clearance are increased compared to the parent compound. In otherwords, nicotinamide NMT is a useful detoxification enzyme, but its marked inter-individual variability also implies that detoxification of pyridine-type compounds is

reduced in a fraction of the human population.


Fig. 4.13. This Enzyme ID card features two other N-methyltransferases, namelyphenylethanolamine N-methyltransferase (PNMT) and the nonspecific amine N-methyltransferase (NMT) also known as indolethylamine N-methyltransferase (INMT)[8]. Phenylethanolamine NMT is a highly specialized enzyme which at best plays a verylimited role in xenobiotic metabolism due to its restricted location (mainly the adrenalmedulla) and narrow substrate specificity (noradrenaline and analogous phenyl-ethanolamines) [13]. In contrast, amine NMT exists as two or more isozymes withbroad and overlapping substrate specificities which include primary and secondaryaromatic amines as well as aromatic azaheterocycles [25] [26]. Its role in xenobioticmetabolism is further increased by its wide distribution in organs and tissues, especiallyin the lung. However, much remains to be understood about this enzyme, and

particularly the structural variety of substrates and inhibitors it recognizes.


Fig. 4.14. Tetrahydroisoquinolines have been the objects of numerous investigationsdue to their potential toxification ultimately yielding neurotoxic ammonium cationsshowing a close analogy to methyl-4-phenylpyridinium (MPPþ ; see Fig. 2.94 in Part 2[2]) [27]. This is well-illustrated by 1,2,3,4-tetrahydroisoquinoline (4.25) itself, anendogenous and exogenous cyclic secondary amine whose N-methylation (seeminglyby amine NMT) yields the tertiary amine 4.26 [28a]. This metabolite is a precursorwhose oxidation by monoamine oxidase yields the dopaminergic neurotoxic cation N-methylisoquinolinium (4.27). Various endogenous and exogenous close analogues havebeen shown to undergo the same sequence of toxification [28b]. Carcinogenic aromaticprimary amines such as 4-amino-1,1’-biphenyl (4.28) and benzidine (4.31) are alsosubstrates of amine NMT. Their first N-methylation is considered a reaction oftoxification, since the secondary amine (e.g., 4.29) may then be oxidized by flavinmonooxygenase to a hydroxylamine [2]. In contrast, the documented N-methylation of4.29 to the tertiary amine 4.30 [25] is a detoxification, since N-oxygenation here yields anontoxic N-oxide. Another toxicologically significant reaction of N-methylation is thatof theophylline (4.32) to yield caffeine (4.33) [29]. This reaction goes countersense tothe well-known oxidative N-demethylation reactions of methylxanthines [2], and it isnot seen in adult humans. In contrast, it is effective in neonates (5 – 10% of a dose of

theophylline) and may cause unwanted side-effects.


Fig. 4.15. In this Figure, we exemplify the case of aromatic azaheterocycles, which, asmentioned above, can be N-methylated to yield a quaternary ammonium cation. Ourfirst example is quite naturally nicotinamide (4.34), the physiological substrate ofnicotinamide N-methyltransferase [12a]. This enzyme is the only one to utilizenicotinamide as a CH3 acceptor, but it also N-methylates other pyridine compounds.Indeed, amine NMTappears as the major but not only enzyme acting on pyridine (4.35 ;R¼H) and pyridine analogues, 4.35 (R=H) [26] [30]. The N-methylation of pyridineis clearly a reaction of detoxification, and indeed there exists a trend across animalspecies such that the more extensive its N-methylation, the lower its toxicity. A numberof other azaheterocyclic compounds including quinoline (4.36), phthalazine (4.37), andquinoxaline (4.38) are also good substrates. A particularly interesting example isprovided by nicotine [31] [32]. Whereas the unnatural (þ)-(R)-nicotine enantiomer(4.39) has been found to be a good substrate of amine N-methyltransferase from guineapig lung to yield the quaternary ammonium compound 4.40, the natural (�)-(S)-nicotine (4.41) was a potent competitive inhibitor of (R)-nicotine N-methylation. Theresults in the cytosol of human liver cells again showed (R)-nicotine to be a good

substrate, whereas here (S)-nicotine was a weak substrate.


Fig. 4.16. S-Methyltransferases conjugate thiols into thiomethyl sulfides (Fig. 4.7), thetwo major enzymes in humans and animals being thiol methyltransferase (TMT) andthiopurine methyltransferase (TPMT). Whereas no direct comparison between the twoenzymes seems to have been published, it is clear that thiol MT shows a much widersubstrate specificity than thiopurine MT, and that markedly more medicinal substratesof the former are known. In other words, the clinical significance of thiol MT appearsgreater than that of thiopurine MT. Yet, despite these differences and the fact that bothenzymes are polymorphic in humans, studies on the genetics and pharmacogenetics ofTPMT still exceed those on TMT [13] [33]. In particular and at the time of this writing,no gene for TMT has been identified in humans. Besides these two enzymes, thereexists also a thioether methyltransferase (EC 2.1.1.96; TEMT) which is brieflymentioned here. This enzyme methylated thioethers (i.e., R�S�R’) to form sulfoniumcations (i.e., R�Sþ(CH3)�R’), and it also acts on ether-type selenium compounds. Few

if any medicinal examples have been reported.


Fig. 4.17. In Figs. 4.17– 4.19, we survey a number of xenobiotic thiols whose S-methylation has been investigated. In agreement with the caption to Fig. 4.16, most ofthese compounds are substrates of thiol MT. A first and well-known example is that ofcaptopril (4.42), the first marketed inhibitor of angiotensin-converting enzyme (ACE).This drug undergoes marked S-methylation in humans to the inactive metabolite 4.43,although this route is not the main one [34]. A high activity has been characterized inhuman hepatic and renal microsomes. The second example is a more recent one,omapatrilat (4.44), an inhibitor of both ACE and neutral endopeptidase (also known asa vasopeptidase inhibitor) and a potential new drug for the treatment of hypertension[35]. In humans and laboratory animals, this compound undergoes S-methylation toyield the first-generation metabolite 4.45, plus a number of later-generation S-methylated metabolites such as compound 4.46, methyl sulfoxides, and S-methyl-acylglucuronides. As a whole, S-methylated metabolites accounted for a majority of a doseand demonstrated the significance of this pathway in the biotransformation ofomapatrilat (4.44). It is interesting to note that these results do not appear to beisolated observations, since a comparable metabolic pattern was obtained with

gemopatrilat, a closely related compound [36].


Fig. 4.18. Further medicinal examples are shown here, beginning with a-lipoic acid(4.47), an endogenous cofactor as the (R)-enantiomer and an antioxidant drug as theracemate used in the treatment of diabetic polyneuropathy. When administered orallyto humans, dogs, and rats, the drug was found to be extensively metabolized by b-oxidation (C2 chain shortening, see Chapt. 4.6) and reductive opening of the 1,2-dithiolane ring (disulfide reduction) [37]. The latter reaction was followed by mono-and di-S-methylation, yielding compounds 4.48, 4.49, and 4.50 as representative urinarymetabolites in humans. The lower part of the Figure is dedicated to thiopurine MTsubstrates, most notably 6-mercaptopurine (4.52) and 6-thioguanine (4.54). These drugshave been used in the treatment of leukemia and autoimmune disorders, and in organtransplants [33]. Individual differences in response and toxicity have been correlatedwith polymorphism in the TPMT gene and with the resulting differences in the extentof thiol methylation to yield metabolites 4.53 and 4.55, respectively. Another relevantdrug is azathioprine (4.51), which as a prodrug of 4.52 shows similar variability in

response and toxicity.


Fig. 4.19. Returning to thiol methyltransferase, we encounter prasugrel (4.56), a recentanalogue of the well-known platelet anti-aggregant clopidogrel. Like the latter, 4.56 is aprodrug which must undergo a first activation step to a thiolactone (in this casemetabolite 4.57) [38]. However, there is a significant difference between clopidogreland prasugrel, whose thiolactone formation is catalyzed respectively by cytochromeP450 and carboxylesterases (cleavage of the acetate ester, followed by tautomerizationand double-bond rearrangement). The actual activation is the second step, which leadsto a reactive thiol metabolite, 4.58, the active species which irreversibly antagonizesplatelet ADP receptors via a covalent S�S bridge. In the case of prasugrel (4.56), thisstep to the reactive 4.58 is claimed to be catalyzed by CYPs; but given that the overallreaction from 4.57 to 4.58 is a hydrolytic one, all remains to be clarified regarding thepostulated CYP-catalyzed mechanism. Given the reactivity and activity of the thiolmetabolite 4.58, its own metabolism is of pharmacological significance, producing theS-methyl conjugate 4.59 and a cysteinyl disulfide conjugate (not shown). Our secondexample is that of metam (4.60), an agrochemical extensively used as a soil fumigant[39]. This is a toxic dithiocarbamate which is reversibly transformed in animals into thereactive methyl isothiocyanate (4.61) in a reaction involving glutathione (seeChapt. 4.7). The alternative, and irreversible, metabolic reaction is conjugation to S-methyl metam (4.62). This reaction appears as a detoxification although 4.60, 4.62, and

other metabolites are inhibitors of mitochondrial aldehyde dehydrogenase.


Figs. 4.20 and 4.21. Arsenic is present in many regions of the world where it diffusesfrom As-containing ores into water sources. It is thus a natural contaminant to whichhundreds of millions of humans are exposed through drinking water. Arsenic is also anoccupational hazard for workers and populations associated with smelting of copperand other metals. The contamination of the geosphere by arsenic causes severe healtheffects and cancer in exposed individuals. Thus, the inhalation of arsenic trioxideliberated by smelting causes lung cancer. The chemistry of arsenic is a rather complexone, since it exists in three valence states, namely metallic arsenic, trivalent arsenicals,and pentavalent arsenicals. Arsenic also exists as inorganic acids and salts, and asorganoarsenicals produced in the biosphere, most notably methylarsenicals ofrelevance here, and thiol conjugates (Chapt. 4.7). The manifold toxicity of arsenic isdue in particular to its capacity to inhibit numerous functional proteins by bindingavidly to thiol groups. At the level of tissues and organs, arsenic is a carcinogen and mayevoke severe inflammatory responses. For many years, the methylation of arsenic acids/salts in organisms was considered to be a mechanism of detoxification. However, it isnow known that methylated arsenicals contribute significantly to arsenic toxicity andgenotoxicity [40]. Furthermore, some of the most toxic arsenicals are now believed tobe methylated trivalent species [41]. A likely mechanism of DNA damage induced bymethylated trivalent arsenicals involves the formation of reactive oxygen species, a factthat places arsenic redox reactions at the forefront of its toxicity [42].

Turning our attention to the metabolism of arsenic in humans and animals, Fig. 4.21is so arranged that reactions of reduction appear horizontally, whereas reactions ofmethylation appear vertically. This Figure does not pretend to completeness, since nothiol-As metabolite or intermediate is shown [43], no more than dimethylarsine(Me2As�H), the product of reduction of dimethylarsinate (4.67). In humans chroni-


cally exposed to arsenic, major methylated metabolites found in urine are 4.67(cacodylate, dimethylarsinic acid)>dimethylarsinite (4.68 ; dimethylarsinous acid)>methylarsonate (4.65 ; monomethylarsonic acid)>methylarsonite (4.66 ; monomethy-larsonous acid) [44]. Comparable results were obtained in human and rat hepatocytes[45]. The formation and excretion of trimethylated As species appears marginal, andthere are only few reports on the excretion of trimethylarsine oxide (4.69) andtrimethylarsine (4.70) in animals.

An important finding is the fact that methylation occurs almost exclusively fortrivalent arsenic species. Indeed, the major (exclusive?) enzyme in As methylation isspecific for trivalent species and is, therefore, known as arsenic(III) methyltransferase(Fig. 4.20) [8] [46]. In concrete terms, pentavalent arsenicals must be reduced totrivalent arsenicals to allow their methylation [47 – 49]. Thus, arsenate (4.63) is reducedto arsenite (4.64). This step is catalyzed by at least two enzymes, glutaredoxin:arsenateoxidoreductase (EC 1.20.4.1) and arsenate:acceptor oxidoreductase (EC 1.20.99.1) [8].The methylation of the trivalent arsenite (4.64) is catalyzed by AS3MT and yields thepentavalent methylarsonate (4.65). The latter is then reduced to methylarsonite (4.66)by glutathione:methylarsonate oxidoreductase (EC 1.20.4.2), an enzyme with absoluterequirement for glutathione (GSH) and a member of the glutathione-S-transferasessuperfamily [48]. In turn, the trivalent methylarsonite (4.66) is methylated by AS3MTto the pentavalent dimethylarsinate (4.67), the major urinary metabolite of inorganicarsenic. The final steps to 4.68, 4.69, and 4.70 are minor ones whose enzymology is

unclear.

Fig. 4.21.


Fig. 4.22. This Chapter examines the formation of sulfates (R�O�SO�3 ) andsulfamates (R�NR’�SO�

3 ) [5 – 8] [50]. Given the analogies between sulfates andphosphates, the last Section of the Chapter will be dedicated to the (much rarer)reactions of xenobiotic phosphorylation. As shown here, the formation of sulfates andsulfamates consists in a sulfonate group (�SO�3 ) being added to the substrate undercatalysis by a sulfotransferase. This group is carried by the cofactor 3’-phosphoadenylylsulfate (4.71; also known as 3’-phosphoadenosine 5’-phosphosulfate, PAPS). As thegroup is a sulfonate, the reactions of sulfoconjugation are correctly designated assulfonations rather than the common term of sulfations [51]. All criteria of conjugationare met in sulfonation reactions, since they are enzymatic, and the group transferred isof medium molecular weight, ionized, and highly hydrophilic, and is carried by acoenzyme. In PAPS, the sulfuric and phosphoric moieties are linked by an anhydridebond whose cleavage is exothermic and supplies enthalpy to the catalytic reaction.Sulfonation reactions involve the nucleophilic attack on the S-atom by a OH group (inphenols, alcohols, hydroxylamines, and hydroxylamides), or a primary or secondaryamino group. The by-product of the reaction is adenosine 3’,5’-bisphosphate (4.72 ;PAP). As we shall see, some sulfates are unstable under biological conditions and mayundergo heterolytic cleavage to form electrophilic intermediates of considerable

toxicological significance.


Figs. 4.23 and 4.24. Sulfotransferases involved in the metabolism of small endogenousand exogenous molecules are soluble (cytosolic) enzymes. Following major advances inmolecular biology, they are now recognized as being encoded by a gene superfamily ofwhich ca. 50 mammalian genes are known, and whose products are classified intofamilies (>45% residue identity) and subfamilies (>60% residue identity) accordingto their degree of homology [8] [10] [52– 54]. Thus, human sulfotransferases in-clude the SULT1A subfamily which contains the enzymes 1A1, 1A2, and 1A3 (phenol(¼aryl) sulfotransferases, with some correspondence with EC 2.8.2.1); the subfamilySULT1B with the enzyme 1B1 (thyroid hormone sulfotransferase); the subfamilySULT1C with the enzymes 1C1 and 1C2; and the subfamily SULT1E with 1E1(estrogen sulfotransferase, EC 2.8.2.4). All enzymes in the SULT1 family appear tohave a marked preference for phenol substrates.

In contrast, the SULT2 family has a distinct substrate specificity for alcoholsubstrates, and particularly hydroxysteroids. This family includes the subfamilySULT2A with 2A1 (alcohol/hydroxysteroid sulfotransferase, EC 2.8.2.14); thesubfamily SULT2B with the two transcript variants 2B1a and 2B1b (EC 2.8.2.2); alsoincluded are steroid sulfotransferase (EC 2.8.2.15) and cortisol sulfotransferase(glucocorticosteroid sulfotransferase; EC 2.8.2.18). An important family is SULT3with 3A1, which is involved in amine sulfonation and seems to correspond with aminesulfotransferase (EC 2.8.2.3). There is also a family SULT4 (with 4A1) whose substratespecificity is unknown, and which has been described as a brain sulfotransferase-likeprotein.


It is important to note that there are marked and condition-dependent overlaps inthe substrate specificity of all these sulfotransferases, a fact which prevents any strongand one-to-one correspondence between the classification of the NomenclatureCommittee (NC) of the International Union of Biochemistry and Molecular Biology(IUBMB) [8], and the homology-based nomenclatures. Nevertheless, it is well-established that the enzymes of greatest significance in the sulfonation of xenobioticsare the aryl sulfotransferases, the alcohol sulfotransferases, and the amine sulfotrans-

ferases.

Fig. 4.24.


Fig. 4.25. The upper part of this Figure shows a schematic and highly simplifieddepiction of the catalytic mechanism of sulfotransferases [55 – 57]. The numbering isthat of estrogen sulfotransferase, and what the Figure shows is in fact the transition state.The weak bonds (ionic and H-bonds) are represented by red broken lines ofexaggerated length for clearer vision. The sulfonate group in the center is still beingweakly bound to the cofactor (PAPS in the process of becoming adenosine 3’,5’-bisphosphate) and has begun binding to the substrate (here a generic phenol). At thisstage, the transferred group is planar, with excess electronic density on the threeperipheral O-atoms and an electron deficiency on the S-atom. Lys48 plays an importantrole in forming electrostatic bonds with the SO3 group and the phosphate moiety inPAP, thereby stabilizing the former. The nucleophilicity of the target O-atom in thesubstrate is increased by H-bond donation to His108. Other residues (among othersThr45 and Thr51) form H-bonds with the cofactor, the SO3 group, the substrate, orother important residues, thereby acting directly or indirectly to decrease the freeenergy level of the transition state and stabilize it.

The lower part of the Figure states an important generalization about sulfonationreactions, which are of high affinity and low capacity. In concrete terms, they have a fastinitial turnover rate, but their velocity decreases as the concentration of availablecofactor is rapidly depleted [58]. The metaphor of a sprinter is a straightforward one,especially when contrasted with that of a marathon runner (i.e., the low-affinity, high-

capacity reactions of glucuronidation, see Chapt. 4.4).


Fig. 4.26. A more realistic view of the proximity between cofactor and substrate in theactive site of sulfotransferase is presented. The structure of SULT1A1 crystallized withthe cofactor and a phenolic substrate was retrieved from the Protein Data Bank (PDBID: 2D06) [56]. A single monomer was used to build the image. The structure wascompleted by adding the H-atoms and minimized using VEGA; the image wasgenerated by VMD and rendered by POVRay [59]. The optimized protein is coloredusing a color ramp, from blue (N-terminus) to red (C-terminus), and the ligands aredepicted as Van der Waals space-filling models. The substrate is estradiol (4.18 inFig. 4.11; the dark-grey model on the left of the picture), and the cofactor is a PAPanalog (the violet model on the right; courtesy of Dr. Giulio Vistoli, University ofMilan). Secondary structure elements are represented as coils for helices and arrows forb-strands. The Figure is also of general interest as it nicely illustrates the intricate

manner by which the linear peptidic chain folds into a tertiary structure.


Fig. 4.27. Beginning with model and xenobiotic phenols, we encounter two simplecompounds used as probes of the major human hepatic sulfotransferase 1A1. For years,4-nitrophenol (4.73) was used to this end, but recent results dispute its high selectivity,since it is also a substrate of 1B1 and other SULTs. In contrast, 2-aminophenol (4.74)has a high substrate affinity for SULT1A1 and is a poor substrate of other SULT1enzymes [60]. A large number of other phenols were examined for their substratebehavior toward aryl sulfotransferases [61], yielding useful structure –affinity relation-ships. Other phenols stand out for their environmental and toxicological significance,e.g., phenolic metabolites of polyhalogenated 1,1’-biphenyls such as 2’,5’-dichloro-4-hydroxy-1,1’-biphenyl (4.75), 3,5,2’-trichloro-4-hydroxy-1,1’-biphenyl (4.76), and3,5,2’,4’-tetrachloro-4-hydroxy-1,1’-biphenyl (4.77). Phenols 4.76 and 4.77 were foundto be substrates of human hydroxysteroid SULT2A1, making them potentialcompetitors of the sulfonation of endogenous steroids. As for 4.75, it was identifiedas a non-substrate inhibitor of the enzyme [62]. These properties are a cause of concernas they suggest an interference with hormone regulation. Our last examples here aretwo xenobiotics of toxicological concern, namely bisphenol A (4.78 ; R¼H) andtetrabromobisphenol A (4.78 ; R¼Br). The latter is a flame retardant with demon-strated cytotoxicity, whereas the former is an endocrine disruptor and one of thehighest volume chemicals produced worldwide, being used as a monomer and in manyplastic consumer products such as toys and water containers [62]. Their conjugationmay be hypothesized to be a route of detoxification, with formation of the mono-O-glucuronide (4.79 ; see Chapt. 4.4) clearly predominating over formation of the mono-O-sulfate (4.80). Other examples of preferred glucuronidation over sulfonation will be

presented later.


Fig. 4.28. Many natural and endogenous compounds are substrates of sulfotransfer-ases. Dopamine (4.10) is of particular interest, being both an essential neurotransmitterproduced by decarboxylation of the amino acid l-DOPA (4.11; see also Fig. 4.9), andthe active metabolite of the same l-DOPA used as a major anti-Parkinsonian drug.Dopamine of endogenous or exogenous origin is a good substrate of sulfotransferasesand particularly SULT1A3 [63], with sulfonation being its major route of inactivation,and dopamine O-sulfate, 4.81 and 4.82, accounting for ca. 90% of all circulatingdopamine in humans. The high selectivity of dopamine toward SULT1A3 is explainedby the presence in the catalytic site of a carboxylate group forming an ionic bond withthe ammonium group of dopamine and related amines; hence, its name of monoamine-sulfating phenol transferase (see Fig. 4.23). The high regiospecificity of the enzyme forthe meta-OH group (to yield 4.81) is also remarkable. For the sake of a broader vision,the Figure also shows two other routes of inactivation of dopamine, namely COMT-catalyzed O-methylation and MAO-catalyzed oxidative deamination.

Numerous xenobiotics of plant origin are also substrates of sulfotransferases, inparticular, dietary polyphenols [52] [64]. An example is provided here with (E)-resveratrol (4.83), a polyphenol present in grapes and wine, and known to be endowedwith cardioprotective activity. In vivo in rodents and in rat hepatocytes, the majormetabolites were the 3-O-sulfate 4.84 and the 3-O-glucuronide [65]. In humanhepatocytes, the major metabolites were the 3-O-glucuronide and the 4’-O-glucuronide(see Fig. 4.42). In PAPS-fortified human liver cytosol (where no glucuronyltransferasesare present, see Chapt. 4.4), three sulfates were produced, namely the 3-O-sulfate 4.84,4’-O-sulfate 4.85, and 3,4’-O-disulfate. Incubations with recombinant enzymes

uncovered the enzyme selectivities shown.


Fig. 4.29. The previous Figure mentioned the role of monoamine-sulfating phenoltransferase (SULT1A3) in the conjugation of xenobiotic analogues of dopamine. Here,we present three such analogues, namely b2-receptor agonists used in the treatment ofasthma. As a rule, such drugs are quite hydrophilic and undergo substantial presystemicmetabolism mainly by sulfonation. In vitro studies using human intestinal cytosol andrecombinant human SULT1A3 have confirmed the effective sulfonation of several b2-receptor agonists including isoprenaline (4.86 ; isoproterenol), terbutaline (4.87), andformoterol (4.88) [66]. While all drugs examined had comparable Vmax values, theiraffinity proved markedly structure-dependent, with isoprenaline being the bestsubstrate. There was also a modest stereoselectivity such that the inactive (þ)-(S)-enantiomers were somewhat better substrates. Besides drugs, a number of phenolicdrug metabolites undergo sulfoconjugation, often in competition with glucuronidation(Chapt. 4.4). Traxoprodil (4.13 ; see also Fig. 4.10) offers a nice illustration ofsulfonation occurring for both the drug and one of its metabolites [19]. As discussedin Chapt. 4.2, 4.13 undergoes CYP-catalyzed ring oxidation followed by COMT-catalyzed O-methylation to form 3’-methoxytraxoprodil (4.14). Interestingly, both thedrug and this metabolite form the respective sulfate esters 4.89 and 4.90 along with theO-glucuronides. Ring oxidation was the major route in most subjects, but directconjugation by O-glucuronidation and O-sulfonation was the major metabolic pathway

in �poor metabolizers� (i.e., persons with defective CYP2D6 activity; see Part 6).


Fig. 4.30. The sulfonation of alcohols is also of metabolic significance, with the addedfeature that the resulting alkyl or cycloalkyl sulfate esters may be reactive. Like arylsulfates, alkyl sulfates are sensitive to enzymatic and chemical hydrolysis ; in addition,several alkyl sulfates are known to undergo proton-catalyzed heterolytic cleavage asdiscussed below. The enzymes involved in alcohol sulfonation are mainly alcohol/hydroxysteroid sulfotransferases (see Fig. 4.23), but the involvement of other SULTs issometimes documented. The influence of substrate structure on their sulfonation hasled to some interesting generalizations [67]. Thus, the sulfonation of primary alcohols isdistinctly faster than that of secondary alcohols, with that of tertiary alcohols beingminute. This is well illustrated with three isomeric C7 alcohols, namely cyclohexylme-thanol (4.91), trans-4-methylcyclohexanol (4.92), and 1-methylcyclohexanol (4.93).Another important rule is the fact that low-molecular-weight alcohols (less than six C-atoms) are poor substrates. This is fortunate, given that sulfates of low-molecular-weight alcohols such as methyl sulfate and ethyl sulfate are known alkylating agents.However, and as illustrated here and in the next Figure, some higher-molecular-weightsulfates may also be toxic. Thus, the agricultural fungicide N-(3,5-dichlorophenyl)suc-cinimide (4.94 ; NDPS) is a known nephrotoxin. Its oxidation and sulfonation in ratsyielded the sulfate 4.95 which reacted very rapidly with glutathione (GSH; seeChapt. 4.7, hence the blue arrows) either directly or via the carbocation to form theconjugate 4.96 [68]. The latter is believed to be the ultimate nephrotoxin, since it is ableto react with other endogenous nucleophiles in addition to GSH. Interestingly, thepresence of the adjacent �CH2� group allows intramolecular deactivation to the

unsaturated maleimide analogue 4.97.


Fig. 4.31. Reactive electrophiles are formed by the heterolytic C�OSO�3 cleavage

(and N�OSO�3 cleavage, see next Figure) of sulfates [69]. This reaction is already

apparent in the previous Figure, and it is particularly worrying for genotoxic andcarcinogenic polycyclic arylmethanols, as exemplified here [70– 72]. CYP-Catalyzedhydroxylation of polycyclic methylarenes (here 5-methylchrysene (4.98)), followed bysulfonation catalyzed by hydroxysteroid sulfotransferases, produces the sulfate 4.99.This reactive conjugate can be detoxified by a direct substitution catalyzed by GSH-transferases (GST; Chapt. 4.7) to form 4.100 ; it also undergoes a heterolytic C�OSO�

3

cleavage to generate the carbocation. Besides being inactivated by glutathione, thecarbocation forms adducts with DNA, particularly at the nucleophilic NH2 group ofadenine (see 4.101). The molecular features favoring heterolytic cleavage are partlyknown, the stability of the carbocation appearing as a significant determinant [69].Resonance (i.e., delocalization of the positive charge) certainly contributes to thestabilization of the carbocation and hence to an increased likelihood of heterolyticcleavage. Such delocalization can be caused by an adjacent unsaturated system such asa carbonyl group (as in N-(3,5-dichlorophenyl)succinimide (4.94)), an aromatic system(e.g., methylarenes), or an allylbenzene structure (as in safrole, isosafrole, and

analogues [69]).


Fig. 4.32. A number of N-oxygenated compounds are also known substrates ofsulfotransferases, for example, aryl sulfotransferase IV (EC 2.8.2.9) and SULT2B1[70] [72] [73], thereby forming O-sulfates (also known as N,O-sulfates). Most knownsubstrates in this broad class are hydroxylamines and hydroxylamides, as exemplifiedhere with a simple and interesting model compound and industrial chemical, namely 2-nitrotoluene (4.102) [74]. In male rats, three metabolites were found to be substrates ofsulfotransferases, namely 2-nitrobenzyl alcohol (4.103), 2-aminobenzyl alcohol(4.104), and 2-(hydroxyamino)benzyl alcohol (4.107). The sulfonation of the lattertwo was concluded to account for covalent DNA binding in liver. The case of 4.104 isplainly similar to the examples discussed in Figs. 4.30 and 4.31, with the sulfate ester4.105 forming a carbocation and the glutathione conjugate 4.106. The fate of 4.107 is ofrelevance here, since sulfonation of the NHOH group leads to a labile sulfate, 4.108,which undergoes proton-catalyzed heterolytic cleavage to a nitrenium cation stabilized

by resonance as shown.


Fig. 4.33. Hydroxylamides (also known as hydroxamic acids) might be even morecarcinogenic than hydroxylamines following their toxification by sulfonation. Indeed,they are sulfonated faster than the corresponding hydroxylamines by aryl sulfotrans-ferase IV (EC 2.8.2.9), and their sulfate esters are characterized by a great reactivity inaqueous media [75a]. Many conditions are, therefore, fulfilled for aromatic amines andtheir N-acetylated conjugates (see Chapt. 4.5) to form mutagenic, carcinogenic, and/ornecrotic sulfate conjugates. A classical example is that of N-(9H-fluoren-2-yl)-N-hydroxyacetamide (4.109), a metabolite resulting from the CYP-catalyzed N-hydrox-ylation of N-(9H-fluoren-2-yl)acetamide, and a good substrate to rat liver arylsulfotransferase IVand human liver SULT2B1 [75]. Its O-sulfate 4.110 reacts as shownto form a highly electrophilic nitrenium ion (whose detoxification is shown inFig. 4.118). Despite the potential toxicity of sulfates of hydroxylamines and hydrox-ylamides, one should not conclude that O-sulfonation of N-oxygenated compoundsalways implies toxification. This is demonstrated with amidoximes such as benzamid-oxime (4.112). This compound served as a model of amidoximes, some of which are ofinterest as bioreductive prodrugs (see Part 5) of medicinal amidines. The redoxequilibrium between 4.112 and benzamidine (4.111) markedly favors reduction.Nevertheless, there were fears that the conjugation of amidoximes leading to the O-sulfate 4.113 and the predominant O-glucuronide 4.114 (see Chapt. 4.4) might be aroute of toxification. However, the two conjugates proved chemically stable and devoidof mutagenic effects [76]. An intriguing and rare reaction of conjugation occurs forminoxidil (4.115), a hypotensive agent also favoring hair growth. This drug is an N-oxide, and the actual active form responsible for its therapeutic effects is its stable,

zwitterionic N,O-sulfate 4.116 [77].


Fig. 4.34. In contrast to unstable hydroxylamine sulfates, significantly more stableconjugates (i.e., sulfamates) are obtained upon N-sulfoconjugation of amines. Onemedicinally relevant example is that of trovafloxacin (4.117), a quinolone antibacterialagent. Human volunteers administered the drug excreted it partly unchanged andpartly as three major conjugates, one of which was the sulfamate (4.118) whichaccounted for ca. 10% of the dose and was excreted fecally, indicating its stabilityagainst biodegradation by the gut microflora (see also Sect. 4.5.2) [78]. A number ofalicyclic amines, and primary and secondary alkylamines, and arylamines can all yieldsulfamates in the presence of human sulfotransferases, in particular SULT2A1 [79].The involvement of amine sulfotransferase (EC 2.8.2.3) remains to be betterunderstood, especially in humans. Examples of good substrates include the weaklybasic (pKa 4 – 5) aromatic amines naphthalen-2-amine (4.119) and 1,2,3,4-tetrahydro-quinoline (4.120). The basic (pKa 8 – 10) amines 1,2,3,4-tetrahydroisoquinoline (4.121)and octanamine (4.122) are also good substrates, but only when incubated at basic pHwhere they are in their neutral form. This may not necessarily be the rule for lipophilicmedicinal amines such as desipramine, perhaps depending on the enzyme involved[80]. The sulfonation of a non-basic N-atom is also documented with luminol (4.123), aluminescent reagent used in forensic toxicology, and whose fate was investigated in ratsto search for possible toxic metabolites [81]. Besides the parent compound (< 5% of adose), only two inert metabolites were found, namely the sulfamate 4.124 (ca. 30%)

and the N-glucuronide 4.125 (ca. 60%; Chapt. 4.4).


Fig. 4.35. Phosphate conjugates are rare compared to sulfates but are of primarysignificance in the metabolism of anticancer and antiviral agents impacting onendogenous nucleotides. Indeed, phosphorylation is an essential metabolic step in thebioactivation of these agents, and numerous studies document their stepwise phosphor-ylation to mono-, di-, and triphosphates. Such reactions are sometimes, and correctly,labeled as anabolic (i.e., biosynthetic) ones [82]. They are known or postulated to becatalyzed by some among the many phosphotransferases (EC 2.7), for example,adenosine kinase (EC 2.7.1.20), thymidine kinase (2.7.1.21), pyruvate kinase (2.7.1.40),uridine kinase (2.7.1.48), deoxycytidine kinase (EC 2.7.1.74), deoxyadenosine kinase(EC 2.7.1.76), nucleoside phosphotransferase (EC 2.7.1.77), creatine kinase (EC 2.7.3.2),adenylate kinase (EC 2.7.4.3), nucleoside-phosphate kinase (EC 2.7.4.4), nucleoside-diphosphate kinase (2.7.4.6), guanylate kinase (EC 2.7.4.8), (deoxy)nucleoside-phos-phate kinase (EC 2.7.4.13), and pyrimidine nucleoside monophosphate kinase (2.7.4.14)[8]. An example is afforded by the well-known anti-HIV agent zidovudine (4.126; AZT,azidodeoxythymidine). The concentrations of its phosphate anabolites were measured inthe peripheral blood mononuclear cells of AIDS patients treated with the drug [83]. Themonophosphate 4.127 was the predominant compound; the diphosphate 4.128 andtriphosphate 4.129 were present in minor and comparable amounts. The unexpectedactivity of phosphotransferases toward xenobiotic substrates is also seen with FTY720(4.130), a novel immunomodulator used in transplantations and to treat autoimmunediseases [84]. The agent is phosphorylated in rats and humans to the active mono-phosphate 4.131. The reaction is catalyzed by sphingosine kinases and is highly product-enantioselective. Indeed, FTY720 (4.130) itself is prochiral (it bears two enantiotopicCH2OH groups), and its enzymatic phosphorylation yields exclusively the enantiomer of

(S)-configuration (see 4.131), which is also the only active one.


Fig. 4.36. Together with glutathione transferases (Chapt. 4.7), glucuronosyltransfer-ases count as the most significant conjugating enzymes in xenobiotic metabolism. Thisis explained in qualitative terms by the diversity of functional groups to whichglucuronic acid can be coupled, and quantitatively for the vast number and diversity oftheir substrates [5 – 10] [50] [85]. Here, we begin with the biochemistry of glucuroni-dation, leaving the presentation of the enzymes proper (UGTs) for later Figures.Glucuronidation consists in a molecule of glucuronic acid being transferred from thecofactor uridine-5’-diphospho-a-d-glucuronic acid (4.132 ; UDPGA) to the substrate.This cofactor is produced endogenously by the C(6) oxidation of UDP-a-d-glucose,and it is recognized that ca. 5 g are synthesized daily in the adult human body, hence thehigh capacity of this metabolic route. Glucuronosyltransferases are low-affinityenzymes compared to sulfotransferases, which implies that sulfonation is usually fasterthan glucuronidation at low substrate doses or concentrations (see Fig. 4.25).Glucuronic acid in UDPGA (4.132) exists in the (1a)-configuration, but the productsof conjugation are b-glucuronides 4.133. This is due to the mechanism of the reactionbeing a nucleophilic substitution with inversion of configuration. Indeed, all target sitesin substrates are nucleophiles, a common characteristic they share despite their greatchemical variety. An important feature of glucuronides is their acidity. The pKa ofglucuronic acid is 3.0, and that of O-glucuronides in the range 2.9 – 3.1, implying near-

complete ionization in the physiological pH range [86].


Fig. 4.37. The previous Figure contains a summary of the functional groups beingpotential substrates for UDP-glucuronyltransferases. This overview is presented herein greater detail and shows the electron-rich target sites of glucuronidation. First, thereare the OH groups in phenols, alcohols, hydroxylamines, and hydroxylamides, whichform O-glucuronides. An important class of O-glucuronides are also the acylglucuronides formed from carboxylic acids (shown here) and carbamic acids(RR’N�COOH; see Chapt. 4.8). The N-glucuronides are generated from primaryand secondary aliphatic or aromatic amines, saturated secondary heterocyclic amines,amides and sulfonamides (all of which are represented as RR’NH in the Figure).Another group of N-glucuronides are formed from tertiary amines, be they aliphatic,saturated heterocyclic, or aromatic (i.e., of the pyridine type). These N-glucuronidesare special in the sense that they contain a permanent positive charge in addition to thenegative charge of the carboxylate; they are thus zwitterions [87]. Thiols and thioacidscan lead to S-glucuronides. A few strongly acidic enolic acids are known to form C-glucuronides. In summary, the products of glucuronidation are grouped into four mainclasses, namely O-, N-, S-, and C-glucuronides, depending on the site of attachment of

the C(1)-atom in glucuronic acid.


Figs. 4.38 – 4.40. The enzymes catalyzing the highly diverse reactions of glucuronida-tion are known as UDP-glucuronosyltransferases (UDP-glucuronyltransferases;UGTs) and consist in a number of proteins coded by genes of the UGT superfamily[6– 8] [88– 94]. These enzymes are part of the glycosyltransferases, and we shall see inSect. 4.4.9 that UGTs are also able to catalyze conjugations with glucose and a few otherhexoses [95]. The human UGTs known to metabolize xenobiotics are the products of(to date) four gene families (UGT1, UGT2, UGT3, and UGT8). These are membrane-bound enzymes found in the endoplasmic reticulum. Their tissular distribution is quitebroad, with noteworthy organs being the liver and bile ducts, kidneys, gastrointestinaltract (esophagus, stomach, small intestine, and colon), reproductive organs (themammary glands, testes, and prostate), and the skin. Endogenous substrates include avariety of androgens (testosterone, androsterone, epiandrosterone), estrogens (b-estradiol, estriol) and gestagens (17a-hydroxyprogesterone), biliary acids (lithocholicacid, deoxycholic acid, chenodeoxycholic acid), and bilirubin (a waste product ofhemoglobin) whose detoxification by UGTs is of major significance in humans [92]. Anumber of polymorphisms have been reported in the UGT1 and -2 families. Thus,mutations in 1A1 cause hereditary unconjugated hyperbilirubinemia (i.e., Crigler–Najar and Gilbert syndromes). Other polymorphisms are known in, e.g., UGT1A3 to1A10, 2B4, 2B7, 2B10, 2B15, and 2B17.

A list of human UGTs is shown in Fig. 4.39 together with the major classes of theirsubstrates [96] [97]. This is complemented in Fig. 4.40 with the evolutionary relation-

ship of human UGTs in families 1 and 2 [88] [89].


Fig. 4.39.

Fig. 4.40.


Fig. 4.41. The Figure shows the ribbon structure of UGT2B7 complexed with UDP-glucuronic acid and morphine. The ribbon is colored using a ramp of color from bluefor N-terminus to red for C-terminus. The ligands are represented as Van der Waalsspheres (green for morphine and cyan for the cofactor UDPGA). The structure of theenzyme was generated combining the C-terminal domain (Ala285 – Ser446) whosestructure was experimentally resolved (PDB 2O6L [98]) with the model of N-terminus(Gly24 – Pro284, the putative signal peptide from Met1 to Cys23 was discarded) whichwas generated by homology techniques. In detail, the N-terminal domain was modeledby Fugue exploiting its homology with the macrolide glycosyltransferase fromStreptomyces antibioticus (PDB 2IYF). The predicted backbone was completed byadding the side chains and the H-atoms using VEGA and was joined to theexperimental C-terminus by superimposing the common residues. With the apoproteinstructure minimized, the tertiary complex was generated by docking first UDPGA,followed by morphine. Specifically, the ligands were docked manually optimizing theirknown interactions with pivotal residues of the UGT2B7. The resulting structure wasfinally minimized to optimize the relative position of the bound ligands (courtesy of Dr.

Giulio Vistoli, University of Milan; see also Fig. 4.26).


Fig. 4.42. Our overview of UGTs� substrates begins with phenols, and quite logicallywith plant phenols. Indeed, herbivores and omnivores have been exposed to such non-nutritious compounds throughout their evolutionary history, which explains theevolution of detoxifying enzymes to facilitate their elimination, SULTs and UGTsamong them [99]. Thus, (E)-resveratrol (4.83) is found in a variety of plant sources,most notably grapes, and is known for its antioxidant, lipid-lowering, cardioprotectiveand chemopreventive activities. Both sulfonation (see Fig. 4.28) and glucuronidationare significant reactions in humans, their relative importance depending on dose (i.e.,low doses allow for higher degrees of sulfonation, see Fig. 4.36). The two O-glucuronides of (E)-resveratrol (4.83) are shown here together with the human UGTsthat catalyze their formation. Human liver microsomes produced more 3-O-glucur-onide, 4.135, than 4’-O-glucuronide, 4.136, a product regioselectivity reversed in humanintestine microsomes. Overall, the latter were markedly more active than the former[100]. Two further examples of product regioselective glucuronidation are seen withbioflavonoids such as baicalin and quercetin. Baicalin (4.137) bears OH groups on itsB-ring only; in the presence of human or rat liver or intestinal microsomes, the rates ofO-glucuronidation were higher at 7-OH than at 6-OH, with no reaction detected at the5-OH group. 7-O-Glucuronidation was catalyzed by the human enzymes 1A9>1A8>1A3>2B15>1A7. UGT1A1 was the most efficient, but only at very low substrateconcentrations [101]. Quercetin (4.138) is more complex as it contains four phenolicand one phenol-like OH group. Its regioselective glucuronidation is also markedlydependent on enzyme and tissue, with the 3- and 3’-positions seemingly emerging as

preferred over the 7- and 4’-positions [102].


Fig. 4.43. An apt transition between natural products and medicinal compounds isprovided by morphine (4.139). This major medicine contains a phenol function (the 3-OH group) and a secondary alcohol function (the 6-OH group). Both groups areglucuroconjugated, yielding morphine 3-O-glucuronide (4.140) and morphine 6-O-glucuronide (4.141), respectively. In most laboratory species and in humans, the formeris produced in higher amounts than the latter. In humans, these reactions are catalyzedmainly by UGT2B7. Lower amounts of two other conjugates have also been found in anumber of species, namely the 3,6-O-diglucuronide and morphine 3-O-sulfate (notshown here) [103]. Of great clinical significance is the fact that morphine 6-O-glucuronide is a highly active agonist at the opiate m-receptor, and that both the 3-O-and the 6-O-glucuronides accumulate in the serum of renally impaired patientschronically treated with morphine. The presence of UGT2B7 in human central nervoussystem provides further evidence for the therapeutic contribution of its 6-O-glucuronide to the analgesic activity of morphine. Another important example is thatof paracetamol (4.142), a nonprescription drug widely used for its mild analgesic andantipyretic properties. At therapeutic doses, its main metabolite is the O-glucuronide4.143 (ca. 50– 60% of total urinary metabolites). A number of UGTs can glucuronidateparacetamol, the most active ones being UGT1A1, 1A6, and 1A9. A further butsomewhat less important conjugate is the O-sulfate 4.144 (ca. 30 – 40% of total urinarymetabolites). CYP-Catalyzed oxidation (a route of toxification) represents less than5% of a dose [104] (see also Parts 2 and 3). At hepatotoxic doses, the sulfonationcapacity is exceeded and glucuronidation predominates (2/3 to 3/4 of metabolites),

with oxidation also increasing (7 – 15%).


Fig. 4.44. Another medicinal example of glucuronidation is ezetimibe (4.145), aninhibitor of the intestinal absorption of exogenous cholesterol used in the treatment ofprimary hypercholesterolemia, alone or in combination with cholesterol-loweringdrugs acting by another mechanism (e.g., statins). Ezetimibe (4.145) is interesting as asubstrate of UGTs in that it contains two target sites, namely a phenol and an alcoholgroup. The phenolic glucuronide 4.146 (formed by UGT1A1, 1A3, and 2B15) isproduced in much larger proportions than the benzylic metabolite 4.147 (formed byUGT2B7) [105]. In fact, the phenolic metabolite is the main circulating metabolite inhuman plasma; it is also and by far the major in vitro conjugate in human livermicrosomes. In contrast, human jejunum microsomes produced the two O-glucur-onides in similar amounts. Both conjugates are excreted mainly in bile and undergoenterohepatic cycling. This phenomenon is clinically relevant, given that the pharma-cological target of 4.145 is an intestinal cholesterol transporter, and that itsglucuronides are also active. A number of other drugs have their therapeutic effectsprolonged by enterohepatic cycling, as nicely illustrated in a pharmacokinetic study ofestradiol in postmenopausal women administered a single oral dose of 1.5 mg [106]. Inthis Figure, we simply show the structure of estradiol (4.18) in redox equilibrium with

its metabolite estrone (4.148).


Fig. 4.45. This Figure continues and complements the previous one by illustratingsome of the pharmacokinetic results obtained in the clinical study under discussion[106]. Both estradiol (4.18) and its metabolite estrone (4.148) were extensively O-glucuronidated and underwent enterohepatic cycling. An experimental proof of thisphenomenon can be seen in the time profile of the serum concentrations of bothhormones. The time profile of estradiol shows a first phase of absorption– eliminationwith an approximate half-life of 2 h. However, a second absorption phase rapidlykicked in and resulted in sustained levels of estradiol for 24 h and more. The serumconcentration curve of the metabolically produced estrone showed enterohepaticabsorption phases after ca. 24 and 50 h, extending its half-life from 4 h after the firstabsorption phase to 11 h. Estrone levels were markedly higher than estradiol levels(cmax�130 vs. 30 ng/l). It is known from numerous literature data that large hydrophilicanions are actively secreted in the bile ducts as substrates of organic anion transporterswhose primary substrates are biliary acids produced in the liver. By virtue of theirnegative charge, the glucuronides of sufficiently large aglycones fulfill the physico-chemical conditions for biliary excretion. As a rule of thumb, the biliary excretionoccurs above a threshold of ca. 450– 500 Da in humans and 350– 400 Da in rats.However, biliary excretion is a necessary but not sufficient condition for enterohepaticcycling, since the glucuronide must also be hydrolyzable by bacterial and/or intestinalglucuronidases [6] [107] [108]. And the aglycone liberated by hydrolysis must obviously

be absorbable by the intestinal wall.


Fig. 4.46. Alcohols can be glucuronidated with relative ease, producing O-glucuro-nides whose chemical stability contrasts with the reactivity of O-sulfates (seeSect. 4.3.3). To follow the same sequence as Chapt. 4.3, we devote a Figure to naturalalcohols, beginning with steroidal hormones [109]. These endogenous compounds andtheir synthetic analogues are well known substrates of UGTs in humans and animals.Interestingly, recent studies using powerful new technologies have identified a novelclass of glucuronides resulting from the sequential glucuronidation of a single OHgroup. This is illustrated here with 5a-dihydrotestosterone (4.149), the activemetabolite of testosterone. Its glucuronidation at the 17b-OH group to yield themonoglucuronide 4.150 was catalyzed by human UGT2B17, 2B15, 1A8, and 1A4 indecreasing order. Incubation of the monoglucuronide resulted in a low but measurableproduction of the diglucuronide 4.151 having the two glucuronyl moieties linked at the1’’!2’ position. This reaction was catalyzed by UGT1A8 and, to a lesser extent, by1A1 and 1A9. However, UGT1A8 was the only human UGT capable of producing thediglucuronide from 5a-dihydrotestosterone. Alcohols of plant origin are numerousand, like natural phenols, have been consumed by herbivores for hundreds of millionsof years, explaining for a good part the evolution of the UGT gene superfamily. Asexamples, (� )-borneol and (� )-linalool are two terpenoid alcohols known to form therespective glucuronides 4.152 and 4.153 [110]. Note that borneol is a secondary alcohol,

whereas linalool is a tertiary one, a point we shall take up in the next Figure.


Fig. 4.47. This Figure addresses the issue of chemoselectivity and regioselectivity inreactions of glucuronidation (see also Part 1 [1] [4]). Chemoselectivity implies thediscrimination between chemically diverse target groups, here between primary,secondary and tertiary alcoholic groups, independently of their presence in the same ordifferent compounds. When chemically diverse alcohol groups are present in the samemolecule, chemoselectivity overlaps partly with regioselectivity, as seen with theantibiotic chloramphenicol (4.154). Human microsomes produced both the 3-O-glucuronide (at the primary OH group) and the 1-O-glucuronide (at the secondary OHgroup), with a ca. tenfold preference for the former [111]. However, such a resultcannot be generalized given its dependence on species and isozymes. Thus, theantithrombotic agent AZ11939714 (4.155) was glucuronidated at the secondary OHgroup at C(2) in dog microsomes, at the primary 4-CH2OH group in humanmicrosomes, and mainly at the secondary OH group at C(3) (with some glucuroni-dation at the 4-CH2OH group) in rat microsomes [112a]. The results in humans areconsistent with the high O-glucuronidation of AZT (4.126 ; Fig. 4.35) at its correspond-ing CH2OH position, a reaction catalyzed by human UGT2B7 [112b]. The lower partof the Figure shows relevant metabolites of 1,1-dimethylpropyl methyl ether (4.156) inrats [113], which affords a further example of the glucuronidation of a tertiary alcohol.Indeed, CYP-catalyzed oxidation of 4.156 led to 1,1-dimethylpropan-1-ol (4.157), aminor urinary metabolite. Major urinary metabolites were 2-methylbutane-2,3-diol(4.158) resulting from further oxidation, the glucuronide (4.159) of the tertiary alcohol4.157, and the 3-O-glucuronide 4.160 of 2-methylbutane-2,3-diol (4.158 ; i.e., at itssecondary alcoholic group). Comparable urinary ratios were seen in a single humanvolunteer, except for 4.157 which was a major metabolite, and the glucuronide 4.160

which was a minor.


Fig. 4.48. Another type of selectivity is seen in some glucuronidation reactions as wellas in various other phase I and phase II metabolic reactions, namely the preference ofsome UGTs for one enantiomer over the other, and more generally for onestereoisomer over the other(s). Because we prefer to use the term �selectivity� forreactions and �specificity� for enzymes [1] [4], what is presented here are examples ofthe enantiospecificity of UGTs. The b-blocker propranolol (4.161) is a chiral drugexisting as two stable forms, the active (S)-enantiomer (i.e., the eutomer) and theinactive (R)-enantiomer (i.e., the distomer). Because d-glucuronic acid is itself achiral, enantiomerically pure compound, the glucuronidation of propranolol (4.161)yields two diastereoisomeric glucuronides. In humans administered racemic propra-nolol, plasma and urinary levels of (S)-propranolol glucuronide are higher than thoseof (R)-propranolol glucuronide. In other words, the overall reaction of propranololglucuronidation is substrate-enantioselective. The origin of this selectivity has nowbeen ascribed (at least in part) to three UGTs, namely UGT1A9 and 1A10, which showa marked enantiospecificity for (S)- and (R)-propranolol, respectively, and UGT2B7,whose preference for (R)-propranolol is minute [114]. The case of oxazepam (4.162) isboth similar to and different from that of propranolol. The stereogenic center in thiscompound is a highly unstable one, such that the drug racemizes with an estimated half-life of 1 – 4 min under physiological conditions of pH and temperature [115]. Yet,UGTs acting on this substrate do show enantiospecificity, since UGT1A9 and 2B7glucuronidate (R)-oxazepam, whereas UGT2B15 is specific for (S)-oxazepam [116].


Fig. 4.49. Hydroxylamines and hydroxylamides may also form O-glucuronides. Thus, afew drugs and a number of aromatic amines are known to be N-hydroxylated and thenO-glucuronidated. An example is found in the metabolism of the candidatehypoglycemic agent 9-[(1S,2R)-2-fluoro-1-methylpropyl]-2-methoxy-6-(piperazin-1-yl)purine (4.163) [117]. When administered to monkeys, more than half of a dosewas recovered as a compound found to be the O-glucuronide, 4.165, of the N-hydroxylated metabolite 4.164. The possibility of the metabolite being an N-glucuronide was conclusively excluded. It is also interesting to note that all resultsreported in this study are consistent with a good chemical stability of the O-glucuronide4.165, in contrast to some O-sulfates of hydroxylamines and hydroxylamides asdiscussed in Sect. 4.3.4. Zileuton (4.166), an inhibitor of 5-lipoxygenase, has value in thetreatment of asthma and other inflammatory pathologies. It is an unusual drug in that itcontains a hydroxylamido group which proved to be a major target of human UGT[118]. Indeed, ca. 75% of a dose are recovered as the O-glucuronide 4.167 in humanurine. Interestingly, human and monkey liver microsomes catalyze the formation of thismetabolite, but rat liver microsomes do not. Substrate enantioselectivity is alsodocumented, since the glucuronidation rate of (S)-zileuton in human liver microsomesis ca. four times higher than that of its (R)-enantiomer, explaining the faster clearance

of the (S)-isomer in humans.


Figs. 4.50 – 4.52. An important reaction catalyzed by UGTs is the formation of acylglucuronides, 4.168, whose significance is now recognized due to their potentialtoxification [119]. These metabolites are quite reactive due to the combination of anester and an acetal function. Intermolecular reactions with nucleophilic compoundsinclude hydrolysis [107] [108], transacylation with glutathione (Chapt. 4.7) [120], anddirect transacylation of proteins [121], leading to potentially immunogenic andantigenic proteins. These reactions are in competition with intramolecular nucleophilicrearrangements, particularly internal migration of the acyl moiety to the 2-O, 3-O, and4-O positions [122]. In the resulting positional isomers 4.169, 4.171, and 4.173,respectively, the 1-hydroxy hemiacetal is free and able to undergo the well-knownreversible ring opening of reducing sugars, yielding the acyclic isomers 4.170, 4.172, and4.174, respectively. These are reactive hydroxy aldehydes which can bind covalently tonucleophilic groups in biomacromolecules, particularly to NH2 groups in blood andtissue protein. The resulting imine 4.175 (a Schiff base) isomerizes to a more stableform known as an Amadori compound, 4.176 [123] [124]. Covalent binding of reducingsugars to proteins (i.e., glycation) is a phenomenon of toxicological concern. Indeed,the protein adducts (and to a minor extent the acyl glucuronides in their acyclic form4.172) can undergo autoxidation catalyzed by transition metal ions. The enaminol 4.177and enediol 4.179 tautomers appear to be intermediates, while the products ofautoxidation are reactive oxygen species (ROSs), reactive carbonyl species (RCSs; e.g.,4.178 and 4.180), and advanced glycation endproducts (AGEs) [125]. These processesof glycation and autoxidation are known as glycoxidation and may in some cases lead toa variety of pathologies [126] in a few sensitive individuals, and in case of abuse of acyl

glucuronide-forming drugs.


Fig. 4.51.

Fig. 4.52.


Fig. 4.53. A specific reaction of intramolecular nucleophilic elimination is shown by anumber of statins, i.e., cholesterol-lowering drugs that act by inhibition of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Two of the marketed statins areused in the lactone, prodrug form, namely lovastatin and simvastatin (4.181), whilemost others are used as the active hydroxy acids (e.g., atorvastatin, fluvastatin, andpravastatin) [107]. The metabolism of statins is a complex one. There is anonenzymatic lactone/hydroxy acid equilibrium which is comparatively fast undergastric conditions of acidity (t1/2 of ca. 1 h with an equilibrium constant close to one) butmuch slower at neutral pH [127]. Enzymatic lactone-ring opening can also occur, inparticular, by serum paraoxonase [3] [4]. The COO group in the hydroxy acid form ofstatins is an active target of phase-II metabolism involving conjugates as intermediatesand/or metabolites. Of relevance here is the fact that glucuronidation of the COOgroup leads to an acyl glucuronide, 4.183, which was detected in the in vitro metabolismof simvastatin acid (4.182 ; and atorvastatin, among others). These acyl glucuronideswere characterized both as metabolites and as intermediates, since they spontaneouslyundergo cyclization – elimination to form the d-lactone [128]. Other examples of acyl

glucuronides undergoing cyclization – elimination begin to be reported [129].


Fig. 4.54. Figs. 4.50 – 4.52 might leave the reader with the conclusion that acylglucuronides are consistently dangerous metabolites. Fortunately, this is not the case,and numerous drugs metabolized to acyl glucuronides have been cleared for safety,with the usual proviso that dosage guidelines must be respected and exaggerated useavoided. In addition, a minute proportion of patients may be sensitive to theimmunogenic and antigenic potential of proteins adducts [130]. The endogenous UGTsubstrate bilirubin (4.184) is of high interest in this context. This breakdown product ofhemoglobin is markedly toxic beyond the physiological threshold, causing jaundice andneurological disorders. Its route of detoxification and elimination is via glucuroconju-gation, followed by biliary excretion. Bilirubin contains two COOH groups, and indeedthe three possible acyl glucuronides (namely the two monoglucuronide isomers 4.185and 4.186, and the diglucuronide 4.187) have been found in rat bile [131]. Theirincubation with human serum albumin under physiological conditions of pH andtemperature resulted in a marked formation of acyl glucuronide –albumin adducts.Covalent binding occurred by the Schiff-base mechanism, predominantly to the freeamino group of Lys190. We note here that the formation of albumin adducts does notprevent bilirubin glucuronidation from being a reaction of detoxification which evolved

to protect animals from the intrinsic toxicity of this breakdown product.


Fig. 4.55. Our overview of acyl substrates of UGTs continues with benzoic acids [132].A significant finding connected with potential toxicity of acyl glucuronides is a linearrelationship between their rate of degradation (hydrolysis plus acyl migration) andtheir propensity to bind covalently to serum albumin [123c]. Thus, the diureticfurosemide (4.188) forms a comparatively stable glucuronide and few adducts. Incontrast, the uricosuric agent probenecid (4.189) forms a labile glucuronide and ismarkedly reactive toward plasma proteins [133]. In contrast, the nonsteroidal anti-inflammatory drug (NSAID) mefenamic acid (4.190) forms a comparatively stable acylglucuronide [121a] [134]. Electronic and steric factors are postulated to determine therelative reactivities of these glucuronides. The NSAID diflunisal (4.191) is interestingdue to the presence of two target sites for UGTs. While the phenolic glucuronide isstable and readily excreted, the acyl glucuronide is reactive and undergoes in parthydrolysis or isomerization, followed by covalent binding. In rats dosed with the drug,protein adducts were found in the serum as well as in tissues such as the liver, kidneys,and lungs [124a] [135]. Our last two examples illustrate the fact that many UGTs areable to form acyl glucuronides. In the case of salicylic acid (4.192), twelve humanexpressed UGTs were examined (eight in subfamily 1A and four in subfamily 2B), allof which, except 1A4, 2B15, and 2B17, were found to be active toward this substrate[136]. The ratio of phenolic over acyl glucuronide formed varied markedly amongthese enzymes. As for the anti-allergic drug tranilast (4.193), its main metabolicpathways in humans are O-demethylation and glucuronidation [137]. The latterreaction is catalyzed mainly by UGT1A1, leading in some individuals to hyper-

bilirubinemia due to competition with bilirubin (4.184) glucuronidation.


Fig. 4.56. The medicinal UGT substrates presented here were selected for theadditional information they offer. Zomepirac (4.194) and tolmetin (4.195) are twoarylacetate NSAIDs known for their relatively high reactivity and covalent binding toproteins [122] [123c]. Looking at chiral drugs [138], (S)-etodolac (4.196) is the activeenantiomer of etodolac. This drug is extensively conjugated in humans to a ratherreactive acyl glucuronide, the reaction in human liver microsomes being ca. fourfoldfaster for the active (S)-etodolac than for its enantiomer [139]. Human UGT1A9 wasthe major enzyme involved in (S)-etodolac glucuronidation, with low contributionsfrom 1A10 and 2B7. With the exception of 2B7, individual UGTs showed very lowactivity toward (R)-etodolac. Ketoprofen is a NSAID representative of the chiral 2-arylpropanoates known as profens. In contrast to the previous examples, its acylglucuronide is of relatively low reactivity. Interestingly, the overall rate of degradationand the 1b- to 2b-acyl migration of the glucuronide of (R)-ketoprofen (4.197) weretwofold faster than those of its epimer formed from (S)-ketoprofen [122] [138].Naproxen (used as the (S)-enantiomer 4.198) is another member of the profen family.Its glucuronidation by rat UGT1A1 was markedly enantioselective for the (R)-form,whereas no substrate enantioselectivity was seen with the human 1A1 orthologue[140a]. At pH 7.4 and 378, naproxen glucuronide hydrolyzed and isomerized at similarrates [121] [140]. Also, covalent binding occurred by both transacylation and glycation.Our last examples are the anti-HIV candidate 3-O-(3,3-dimethylsuccinyl)betulinic acid(4.199) [141] and hypolipidemic agent gemfibrozil (4.200) [124b], both of whichcontain hindered COOH groups. While 4.199 formed relatively stable acyl glucur-onides, gemfibrozil-glucuronide was rather unstable and reactive. This illustrates the

difficulty of making qualitative reactivity previsions based on chemical structure.


Fig. 4.57. Second in importance after O-glucuronidations are the N-glucuronidations,due to the variety of target groups and substrates. One characteristic of many N-glucuronides compared to O-glucuronides is their greater chemical and enzymaticstability, excepting their easier hydrolysis in the acidic pH range. Also noteworthy arethe species differences in the glucuronidation of tertiary amines to form quaternary N-glucuronides [142]. Among carboxamides and sulfonamides, an important example isthe antiepileptic drug carbamazepine (4.201) whose primary carboxamido group isconjugated to form the N-glucuronide 4.202 [143]. The reaction is catalyzed byUGT2B7 in microsomes from human liver, kidney, and intestine. Lactam glucuroni-dation is illustrated with MaxiPost (4.203 ; GMS-204352), a potent and specific maxi-Kchannel opener [144]. Its major metabolism in humans was the N-glucuronide, whichproved stable in buffers and in the presence of glucuronidases; only 2B7 catalyzed itsformation. Examples of primary and secondary sulfonamides are provided byvaldecoxib and sulfonamides, respectively. Valdecoxib (4.204) was extensively metab-olized in humans by oxidation and further conjugations. In addition, a majormetabolite was its N-glucuronide resulting from direct glucuronidation at thesulfonamido group [145]. The case of antibacterial sulfonamides is of toxicologicalsignificance. The metabolism of these drugs occurs mainly by conjugation and involvesa competition between N-acetylation of the primary N(4)H2 group (see Chapt. 4.5) andN-glucuronidation at the secondary N(1)HSO2 group. In a number of first-generationsulfonamides, N-acetylation produced a poorly soluble metabolite which maycrystallize in the kidneys and was nephrotoxic. In contrast, N(1)-glucuronidationproduces a soluble and nontoxic metabolite, as seen with sulfadimethoxine (4.205)

whose N(1)-glucuronide accounted for 40– 45% of a dose in humans [142] [146].


Fig. 4.58. Among primary and secondary amines that are substrates of UGTs, we finddrugs as well as a number of nonmedicinal xenobiotics, in particular, some industrialaromatic amines known to undergo toxification to mutagenic and carcinogenicmetabolites [142] [147]. Interestingly, their N-glucuronidation may, in some cases, beinvolved in such toxification, witness the example of benzidine (4.206) shown here[148]. This xenobiotic can cause bladder cancer in humans, an organ-selective toxicityto be contrasted with the liver cancer selectivity prevailing in rats. The hepaticconjugation of benzidine by N-acetylation (see Chapt. 4.5) leads to N-acetyl- and N,N’-diacetylbenzidine (4.207 and 4.208, resp.). In humans, N,N’-diacetylbenzidine (4.208) isa minor metabolite due to extensive deacetylation by hydrolases [3] [4] [107], whereasN-glucuronidation is an effective reaction yielding appreciable levels of benzidine N-glucuronide (4.209) and mainly of N-acetylbenzidine N’-glucuronide (4.210). These twoN-glucuronides are easily excreted via the kidneys, but their lability under even theslightly acidic conditions prevailing in the urinary tract results in their hydrolysis tobenzidine (4.206) and N-acetylbenzidine (4.207), which will then be able to undergooxidative toxification, in particular, by prostaglandin G/H synthase present atrelatively high levels in bladder epithelium [2] [4] [149] [150]. The metabolic situationis different in rats, where a) N,N’-diacetylbenzidine is a major metabolite which can be

oxidized in the liver to reactive intermediates, and b) N-glucuronidation is minor.


Fig. 4.59. The upper part of this Figure completes the previous Figure by drawing thereader�s attention to the role played by the N-hydroxy metabolites of carcinogenicaromatic amines. Indeed and as illustrated with naphthalen-1-amine (4.211), itsmetabolite N-hydroxynaphthalen-1-amine (4.212) [2] [4] [149] [150] is also a substrateof UGTs. Its glucuronidation proceeds mainly if not exclusively by reaction at the N-atom rather than at the O-atom [142]. Like other N-glucuronides, the resultingconjugate 4.213 is excreted by the kidney and undergoes acid hydrolysis in urine,followed by toxification to the ultimate electrophilic carcinogen [2] [4] [151]. The otherexamples in the Figure are of medicinal interest. Norverapamil (4.214) is the N-demethylated metabolite of the cardiovascular drug verapamil [152]. Like a number ofsimilar N-dealkylated metabolites of medicinal tertiary amines [147], it is N-glucuronidated in humans and rats. The case of the antiepileptic agent D-23129(4.215) is particularly interesting, since the compound contains both a primary and asecondary amino group. Its oxidative metabolism was minimal in human livermicrosomes and liver slices, in contrast to N-glucuronidation which produced the two

regioisomeric glucuronides 4.216 and 4.217 [153].


Fig. 4.60. As shown in Fig. 4.37, a number of tertiary arylalkylamines can beglucuronidated to form N-quaternary glucuronides (also known as Nþ-glucuronides).This permanent positive charge together with the high acidity of the carboxylate group(pKa ~ 3; see Fig. 4.36) result in such conjugates being zwitterions. Many substrates ofNþ-glucuronidation are medicinal N,N-dimethylated arylalkylamines such as antihist-amines and neuroleptics [142] [147] [154]. This is illustrated here with the nonsteroidalantiestrogen tamoxifen (4.218). This compound and its active 4-OH metabolite (notshown) both form an Nþ-glucuronide (4.219 in the case of tamoxifen). For bothsubstrates, the reaction in human liver microsomes was catalyzed exclusively byUGT1A4 among all UGTs tested [155]. In contrast, all tested UGTs, except 1A3 and1A4, catalyzed the O-glucuronidation of 4-hydroxytamoxifen, showing that even amodest structural change may alter the substrate specificity of transferases. The secondimportant group of arylalkylamines forming Nþ-glucuronides are N-methylpiperidinesand -piperazines, as exemplified here with the antipsychotic drug clozapine (4.220).Interestingly, this compound yields two direct N-glucuronides in humans, namely thequaternary N-glucuronide (4.221) and the N(5)-glucuronide (4.222) [156]. Both wereexcreted by patients administered the drug, and both were formed by incubation withhuman liver microsomes. The stability of the two N-glucuronides differed markedly;whereas 4.221 was hydrolyzed by glucuronidases and resistant to acid hydrolysis, 4.222

was resistant toward enzymatic hydrolysis but was labile under acidic conditions.


Fig. 4.61. Another important group of substrates of Nþ-glucuronidation are pyridines,i.e., compounds containing a pyridine ring. A typical substrate is (S)-nicotine (4.41)whose N-methylation was discussed in Fig. 4.15. Indeed, the natural (S)-nicotine (andits lactam metabolite (S)-cotinine resulting from 5’-oxidation; not shown) are Nþ-glucuronidated by human UGT1A4 and to some extent also by UGT1A9 [32] [157].Nicotine Nþ-glucuronide (4.223) and cotinine Nþ-glucuronide are found in the urine ofsmokers, with large quantitative differences depending on the activity of the manyenzymes involved in the metabolism of nicotine. The second example in the Figure isthat of indinavir (4.224), a well-known HIV protease inhibitor. This drug features apyridine ring which undergoes N-oxygenation and Nþ-glucuronidation [158]; both aremajor routes of metabolism in human liver slices and in vivo. The Nþ-glucuronideshowed a marked species dependence in that it was formed in monkeys but not in dogsor rats. Our last example here is that of a xenobiotic, namely the tobacco smokecomponent 4-(methylnitrosamino)-1-(pyridin-3-yl)butan-1-one (4.225 ; NNK). In mosttissues, this compound undergoes carbonyl reduction to 4-(methylnitrosamino)-1-(pyridin-3-yl)butan-1-ol (4.226 ; NNAL). Both compounds are potent lung carcinogensfollowing the oxidative toxification of their nitrosamino group. Alternative metabolicroutes may thus lead to detoxification, as is the case with the glucuronidation of NNAL.Interestingly, both Nþ- and O-glucuronidation occur in humans, with the twoglucuronides, 4.227 and 4.228, respectively, each accounting for ca. 1/4 to 1/3 of thetotal NNAL excreted in the urine of smokers [159]. Nþ-Glucuronidation is catalyzed inhumans by UGT1A4; rats form the O-glucuronide only, a reaction catalyzed by

UGT2B1.


Fig. 4.62. Besides the pyridine derivatives, a marked number of aromatic diaza- andpolyazaheterocyclic compounds are known to undergo N-glucuronidation. Theirinterest lies in the fact that both N- and Nþ-glucuronides may be formed whentautomerism is possible. Tautomerism is structurally excluded in the imidazoleantifungal drug tioconazole (4.229), and indeed only the quaternary N-glucuronide(4.230) is formed, being a major urinary metabolite in humans [160]. The same reactionhas been seen in a variety of N-aryl- or N-arylalkyl-substituted imidazoles, withUGT1A4 catalyzing their glucuronidation [161]. Polyazaheterocycles having anendocyclic NH (i.e., unsubstituted) moiety yield tertiary N-glucuronides, as illustratedwith the methyl-1,1’-biphenyl derivatives 4.231, 4.233, and 4.236 used as modelcompounds [162]. Compounds 4.231, 4.233, and 4.236 contain an 1H-imidazole, a 1,2,3-triazole, and a tetrazole ring, respectively. The tertiary N-glucuronides 4.232, 4.234,4.235, and 4.237 were formed in human, monkey, dog, and rat liver microsomes at an N-atom distant from the substituted C-atom, presumably due to steric hindrance. The1,2,3-triazole ring (in 4.233) and the tetrazole ring (in 4.236) have two such distal N-atoms; they are distinct in 4.233 and identical in 4.236, explaining why the former yieldstwo N-glucuronides (i.e., 4.234 and 4.235), while the latter yields one (i.e., 4.237). Notethat the tetrazole ring is of particular interest in medicinal chemistry, being an acidicgroup (pKa ~5) used as isostere of a carboxylic group in, e.g., angiotensin II receptorantagonists (sartans). The N-glucuronide 4.232 was resistant to enzymatic and chemicalhydrolysis, whereas 4.234, 4.235, and 4.237 were labile. It can be hypothesized that N-glucuronide lability increases with the acidity of the target N-atom in the substrate.


Fig. 4.63. Here, we look in more detail into the problem of tautomerism. JNJ-10198409(4.238) is a novel candidate under development with promises as an antitumor agentacting by a combination of anti-angiogenetic and antiproliferative properties [163]. Asshown, the compound exists as two major tautomers at the pyrazole ring, namely the1H- and the 2H-tautomers. The exocyclic N-atom may also be involved in a minortautomeric equilibrium, but this is not considered here. Both tautomers at the pyrazolering appear to be substrates of UGTs, as deduced from the formation of the tertiaryN(1)-glucuronide, 4.239, in human, monkey and rat liver microsomes, and of thetertiary N(2)-glucuronide, 4.240, in rat liver microsomes (but not in human and monkeyliver microsomes). Needless to say, these two N-glucuronides are different chemicalentities that do not interconvert. A third glucuronide was formed in human, monkey,and rat liver microsomes, namely the N-glucuronide 4.241 which, as shown, has its twopyrazole N-atoms free to undergo tautomerism. The three N-glucuronides differed alsoin their chemical stability, since 4.240 and 4.241 were sensitive to enzymatic (b-glucuronidase) and chemical hydrolysis, whereas 4.239 was stable. Perhaps differences

in basicity can explain this contrast.


Fig. 4.64. A case simultaneously comparable and different from the previous one isfound with the anticonvulsant drug lamotrigine (4.242) [164]. This compound containsfive N-atoms arranged in a diaminotriazine system, two of which are targets for N-glucuronidation. This pathway is the major route of metabolism of lamotrigine (4.242)in humans, who excrete in their urine ca. 80% of a dose as the quaternary N(2)-glucuronide, 4.243. The remainder of the dose is excreted as the secondary N(5)-glucuronide, 4.244, the quaternary N(2)-methyl derivative, the N(2)-oxide, and theunchanged drug plus unidentified metabolites. Inspection of the structure of 4.242reveals three possible amino > imino tautomeric equilibria, one of which is shown.None of the resulting imino tautomers appears favored over the aromatic diamino-triazine structure, yet their possible role during the catalytic phase cannot bediscounted. A further factor with 4.242 is the possibility for its quaternary N(2)-glucuronide, 4.243, to deprotonate to a tertiary N-glucuronide as shown. We are notaware of any experimental evidence revealing the basicity of the N(2)-glucuronide orsupporting the presence of its deprotonated form, but such information might be

informative in terms of catalytic mechanism and excretion.


Fig. 4.65. Some examples of S-atom glucuronidation are reported in the literature, inline with the occurrence of S-glycosides as natural products. An early example of the S-glucuronidation of a xenobiotic is that of the hepatotrophic agent malotilate (4.245).This compound is first transformed to the dithiol metabolite 4.246 which is then S-glucuronidated. The resulting S-glucuronide 4.247 was excreted as a major biliarymetabolite in rats [165]. A recent example is that of the nonsteroidal progesteronereceptor agonist tanaproget (4.248). Its S-glucuronide 4.249 has been characterizedunambiguously and was a major metabolite (>10%) in human and rat livermicrosomes [166]. The formation of this metabolite implies a tautomeric equilibriumbetween the oxazine-2-thione moiety and its iminothiol species.

Xenobiotic C-glucuronidation is an even rarer reaction than S-glucuronidation andinvolves very few acidic C�H moieties. The best known example is that of theuricosuric drug sulfinpyrazone (4.250), which features an acidic C(4)�H group(pKa ~ 2.5) in the pyrazolidine-3,5-dione ring. The glucuronidation of 4.250 is a majormetabolic reaction in humans, yielding conjugate 4.251 in which the C(4)-atom islinked to b-d-glucuronic acid via a C�C bond [167]. The same reaction was seen in

analogues (e.g., phenylbutazone) and at an activated acetylenic group.


Fig. 4.66. The catalytic spectrum of UDP-glucuronosyltransferases is not limited toUDP-glucuronic acid as cofactor, since several among these enzymes are able to useUDP-glucose and transfer d-glucose to the substrate, thereby forming b-d-glucosides[95]. There are also reports of UDP-galacturonic acid being a cofactor of UGTs [168].Furthermore, glycosylations may involve enzymes others than UGTs, as suggested bythe formation of xenobiotic a-d-glucosides by rat liver a-glucosidases [169]. A fewanecdotical reactions have also been reported, such as lactose conjugates ofantibacterial sulfonamides in the milk of lactating dairy cows [170]. Fast developmentin analytical and structural techniques, coupled with the multidisciplinary scienceknown as metabonomics, allow ever smaller traces of metabolites to be characterizedand is expected to reveal yet other unexpected conjugates [171]. Mycophenolic acid(4.253) offers a nice example of glucosidation reactions [172]. This antineoplastic andimmunosuppressant agent is the active metabolite of the prodrug mycophenolatemofetil (4.252). Mycophenolic acid (4.253) contains two target sites for UGTs, namelyits phenol and COOH groups. Its phenol O-glucuronide 4.254 was the primarymetabolite in the plasma of patients treated with the prodrug. The acyl glucuronide4.255 and the phenol O-glucoside 4.256 were also characterized. Mycophenolic acid O-glucuronidation was catalyzed by all recombinant UGTs tested, and the O-glucur-onides 4.254 and 4.255 were formed in microsomes from various human tissues. Thesame was true for the phenol O-glucoside 4.256. In addition, human kidney

microsomes produced the acyl glucoside 4.257.


Fig. 4.67. Two examples of xenobiotic O-glucosidation are presented here. Dogsadministered cannabidiol (4.258 ; CBD) produced a range of oxidized metabolites,notably 4’’-hydroxy-CBD, 5’’-hydroxy-CBD, and 6-oxo-CBD [173]. These metaboliteswere the O-glucosidated (e.g., 4’’-hydroxy-CBD O-glucoside; 4.259) and excreted asmajor urinary metabolites. Our second example is that of bis(2-ethylhexyl) phthalate(4.260) [174]. Plasticizers in the chemical class of phthalates are suspected of beingendocrine disruptors, hence the interest in their biotransformation. When administeredto mice, bis(2-ethylhexyl) phthalate (4.260) was oxidized on one of its 2-ethylhexyl sidechains to a secondary alcohol, a ketone and a carboxylic acid, while the other side chainwas cleaved by hydrolysis. The carboxy group so liberated became available forconjugation, yielding three acyl glucosides which were characterized in the animals�

urine. One of these three acyl glucosides was the conjugate 4.261.


Fig. 4.68. Reactions of N-glucosidation seem to be somewhat better documented thanreactions of O-glucosidation, with a marked preference for acidic N�H groups [175].Typical substrates are barbiturates, which were the subject of a number of informativestudies published several years ago [176] [177]. Thus, phenobarbital (4.262) isconjugated to its N-glucoside 4.263 as a minor urinary metabolite in humans, whereno N-glucuronidation of barbiturates occurs. Interestingly, C(5) in N-unsubstitutedbarbiturates is a prochirality center, to be transformed into a stereogenic center by areaction of N-glucuronidation or N-glucosidation. Given that sugars are chiral,barbiturate N-glucuronides and N-glucosides will exist as two diastereoisomers [178],as illustrated here. Product stereoselectivity is documented in this reaction ofconjugation, with (5S)-phenobarbital N-glucoside being the preferred product ofhuman UGTs (probably UGTs in the 2B subfamily). Among mammals, only themouse showed a capacity to N-glucuronidate barbiturates comparable to that ofhumans. In the mouse, phenobarbital N-glucuronide was excreted in several-foldhigher amount than phenobarbital N-glucoside, and product stereoselectivity in N-glucosidation was the opposite of that in humans. Our second example is ranirestat(4.264), a new aldose reductase inhibitor [179]. This compound has a succinimide ringanalogous to the barbiturate ring, and it undergoes both N-glucosidation and N-glucuronidation in humans. However, human UGTs of the 2B subfamily catalyzed onlythe former reaction. Note that, like the barbiturate ring, the succinimide ring inranirestat (4.264) is labile to chemical hydrolysis followed by decarboxylation, andindeed its sugar conjugates were characterized as the ring-opened metabolites 4.265.


Fig. 4.69. The case of the new xanthine oxidoreductase inhibitor FYX-051 (4.266)illustrates again the interplay of tautomerism and N-glycosylation [180]. Thecompounds exists as the 1H- and 2H-tautomers as shown, and both N�H groups aretarget of UGTs, with a consistent preference for conjugation at the N(1)-atom. Boththe N-glucuronides, 4.267 and 4.268, and the N-glucosides, 4.269 and 4.270, wereformed, but large species differences were seen. Little N-glucuronidation and N-glucosidation occurred in rats. N-Glucuronidation (but no N-glucosidation) was seen inhumans and monkeys, while N-glucosidation was predominant in dogs (see Part 6).Besides weakly acidic N�H groups, there are very few reported cases of weakly basicamino groups being N-glucosidated. One example is 5-aminosalicylic acid (4.271), adrug used to treat inflammatory bowel disease [181]. Its major metabolite in humans isthe N-acetyl conjugate (see Chapt. 4.5), but traces of the N-glucoside 4.272 were alsoseen. This conjugate was unstable in solution, and its formation was partly or totally

nonenzymatic.


Fig. 4.70. To conclude this important Chapter, we summarize some of the properties ofglucuronidation and glucuronides, keeping in mind that the statements made here arenot rules but mere generalizations having numerous exceptions. From a pharmaco-logical and pharmacokinetic viewpoint, the really significant reactions in this Chapterare the glucuronidations of hydroxy, carboxy, and amino groups. This neat classificationdoes not correspond to well-defined families or subfamilies of UDP-glucuronosyl-transferases, and yet major species differences are apparent (see Part 6). Therespective products of these reactions, namely O-glucuronides, acyl glucuronides,and N-glucuronides, differ greatly in their stability. Thus, O-glucuronides areconsistently good substrates of b-glucuronidases and may undergo enterohepaticcycling when of sufficient molecular weight to be excreted in the bile. As a rule, acylglucuronides are reactive molecules undergoing rearrangement and forming adductswith blood and tissue proteins. As for the N-glucuronides, they are usually stabletoward b-glucuronidases and labile under acidic conditions, but a number of exceptionshave been mentioned in this Chapter. A subclass of N-glucuronides are the quaternaryN-glucuronides ; their permanent positive charge renders them zwitterionic and may be

hypothesized to affect their recognition by anion transporters.


Fig. 4.71. In this Chapter, we focus on reactions of acetylation. Compared tosulfonations and glucuronidations, they are modest in terms of the number and varietyof substrates, but remain significant in a toxicological perspective [5 –7] [182]. As far asxenobiotic metabolism is concerned, three reactions of acetylation are known, namelyN-acetylations, O-acetylations, and N,O-acetylations. The former two types ofreactions use acetyl-coenzyme A (abbreviated acetyl-CoA or AcCoA; 4.273) as acetyldonor and are shown here. N-Acetylations involve mainly primary aromatic amines,hydrazines, and hydrazides, in other words, weakly basic or non-basic NH2 groups.However, some basic arylalkylamines are also N-acetylated. The xenobiotic substratesof O-acetylation are aromatic hydroxylamines of toxicological significance. Reactionsof N,O-acetylations are not shown here and will be discussed separately, being enzyme-catalyzed reactions of trans-acetylation whose acetyl donor is a xenobiotic arylacet-amide.

Coenzyme A (4.274) is an essential cofactor in the metabolism of lipids; we willencounter it again in Chapt. 4.6 where it will be shown to play a critical role insignificant yet lesser known reactions of conjugation. As shown here, coenzyme Aresults from the conjugation of adenosine 3’-phosphate 5’-diphosphate with (R)-pantothenic acid (in the larger oval) and cysteamine (in the smaller oval). Thecatalytically essential group in coenzyme A is the thiol, which forms a reactive thioester

group with acyl moieties such as acetyl (i.e., in 4.273).


Fig. 4.72. The acetylation of xenobiotics is mainly a reaction of N-acetylation. Themajor enzyme here is arylamine N-acetyltransferase (NAT), which targets primaryaromatic amines and hydrazines [8] [10] [182– 185]. Two genes exist in humans, whoseproducts show different yet partly overlapping substrate specificities. Both the NAT1and NAT2 enzymes are polymorphic, the latter distinctly more than the former. Somelarge species differences have been reported in substrate specificities. Also noteworthyis the fact that the mouse has three Nat genes and three functional enzymes (Nat1,Nat2, and Nat3), with the mouse Nat1 being functionally analogous to the humanNAT2 [186]. The second enzyme in this Figure is arylalkylamine N-acetyltransferase,the product of the AANAT gene. Its distribution is markedly restricted compared toNAT, and its preferred substrates are arylethylamines. Several of these substrates arephysiological compounds such as neurotransmitters, hence the name serotonin N-acetyltransferase. A number of xenobiotic basic amines have also been reported, as we

shall see.


Fig. 4.73. A number of acetyltransferases catalyze the O-acetylation of alcoholicgroups in endogenous compounds, the best known of which is choline O-acetyltrans-ferase (EC 2.3.1.6; human gene CHAT) which synthesizes the neurotransmitteracetylcholine [8]. Another O-acetyltransferase of physiological significance iscarnitine O-acetyltransferase (EC 2.3.1.7; human gene CRAT). However, and to thebest of our knowledge, no O-acetylation of xenobiotics has been documented inmammals except of N-arylhydroxylamines and N-aryl-N-hydroxyacetamides (alsoknown as N-arylhydroxamic acids). The two enzymes presented here are the onescatalyzing the O-acetylation of such xenobiotics, but they represent enzymatic activitiesrather than distinct proteins. Indeed, much evidence points to arylamine N-acetyltransferases (EC 2.3.1.5) as the enzymes catalyzing these reactions. The literatureappears rather consistent in assigning acetyl-coenzyme A-dependent O-acetylations ofN-arylhydroxylamines and N-arylhydroxylamides to mainly EC 2.3.1.118, and N-aryl-

N-hydroxyacetamide-dependent N,O-transacetylations to EC 3.2.1.56 [8].


Fig. 4.74. Several primary arylamines of medicinal interest are known to be goodsubstrates of NAT in humans. This is the case of para-aminobenzoic acid (NAT1) and 5-aminosalicylic acid (4.271; see Sect. 4.4.9 above) (NAT1�NAT2) [181] [182]. Otherdrugs include the antileprosy agent dapsone (Fig. 2.67 in Part 2 [2]), and variousantibacterial sulfonamides. Thus, sulfamonomethoxine (4.275) was extensively N(4)-acetylated in humans to metabolite 4.276 (ca. 35– 38% of a dose), whereas N(1)-glucuronidation was low (ca. 11– 13%). Unchanged drug accounted for ca. 13– 14%,leaving ca. 35– 40% of a dose unaccounted for [187]. There was no difference betweenslow and fast acetylators (NAT2 phenotypes, see Part 6), suggesting NAT1 to be theenzyme involved in the reaction. Its low glucuronidation-to-acetylation ratio distin-guishes sulfamonomethoxine (4.275) from sulfadimethoxine (4.205 ; see Fig. 4.57). Oursecond example deals with a nonsteroidal androgen receptor modulator bearing an N-Ac substituent, namely (S)-3-[4-(acetylamino)phenoxy]-2-hydroxy-2-methyl-N-[4-ni-tro-3-(trifluoromethyl)phenyl]propanamide (4.277) [188]. This agent is deacetylated inhumans to the primary arylamine 4.278, an active metabolite back-acetylated to theparent compound 4.277. N-Acetylation of 4.278 was indeed fast in human liver cytosol(NAT2>NAT1) and in rats (in vivo and in vitro), but did not occur in dogs. An evenmore complex case is seen with levosimendan (4.279) developed for the treatment ofcongestive heart failure [189]. Two plasma metabolites have been characterized inhumans, the arylamine 4.280 produced by a reductive reaction catalyzed by intestinalbacteria, and the N-acetylated metabolite 4.281 likely produced by NAT2. Interest-ingly, the latter metabolite has a pharmacological activity similar to that of the parent

drug.


Fig. 4.75. Hydrazines and hydrazides are good and highly selective substrates ofhuman NAT2 [182]. Few such compounds are used as drugs, two of which are shownhere. Besides undergoing oxidation reaction, the antihypertensive agent hydralazine(4.282) is extensively conjugated in humans (NAT2) and other species to its N-acetylmetabolite [190]. Earlier studies yielded contradictory results regarding the nature ofthis metabolite, until further studies proved its structure to be 3-methyl[1,2,4]triazo-lo[3,4-a]phthalazine (4.283). The latter product derives from the N-Ac intermediate,which is unstable and spontaneously cyclizes to 4.283 presumably via its enol tautomer.The antituberculosis drug isoniazid (4.284) is another good substrate of human NAT2,and one whose N-acetylation to the inactive conjugate 4.285 shows significantquantitative difference between fast and slow acetylators [191] [192]. The problem iscomplicated by the fact that 4.284 also undergoes hydrolase-catalyzed hydrolysis tohydrazine (4.286), a toxic xenobiotic used in particular as a rocket propellant. Itappears that at least part of the hepatotoxicity and neurotoxicity which threatens slowacetylators more than fast acetylators is due to free radicals derived from hydrazine(4.286) and its monoacetyl conjugate 4.287. In contrast, the subsequent formation of

1,2-diacetylhydrazine (4.288) is a reaction of detoxification.


Fig. 4.76. In Sect. 4.3.5, we encountered the N-sulfonation of trovafloxacin (4.117) tothe sulfamate 4.118. This antibacterial agent of the quinolone class is of further interest,since its metabolism is almost exclusively by conjugation in humans and rats, with noPhase-I metabolite being reported. This is presumably due to the steric and electronicshielding of CYP target groups in the molecule. Formation of the N-acetyl metabolite4.289 and acyl glucuronide 4.290 were the two additional pathways in these two species,with acyl glucuronidation being the major one [78]. The situation in dogs was morecomplex, with two minor Phase I metabolites being seen, and the N-Ac conjugate 4.289seemingly being produced by the intestinal microflora. Independently of its formationin the liver or intestine, this N-Ac conjugate 4.289 is an interesting metabolite, since it isone among a rather limited number of known examples of the N-acetylation of a basicamino group. While we are not aware of secondary arylamines being acetylated, rare

cases of acetylation of secondary basic amines are known.


Fig. 4.77. Another example of the N-acetylation of a primary basic amine is found inthe metabolism of the b-adrenoceptor blocker propranolol (4.161; see also Sect. 4.4.2).In this case, however, conjugation involves a metabolite (i.e., the primary amine 4.291)rather than the parent drug. Indeed, 2– 4% of a dose of 4.161 were accounted for by N-acetyl-N-desisopropylpropranolol (4.292) in the urine of humans administered thedrug [193]. There were indications that the total formation of the conjugate may havebeen even higher given its further oxidative metabolism. Another interesting finding isthe evidence that NAT1 was the enzyme involved in the reaction. The N-acetylation ofacidic NH2 groups is also documented in a few cases. This is the case of theanticonvulsant agent zonisamide (4.293), whose N-Ac conjugate, 4.294, accounted forca. 8% of a dose in rats, and was a minor metabolite in humans [194]. It is interesting tonote that when acetylation targets slightly acidic NH2 groups (pKa of zonisamide is ca.10), it increases acidity and hence ionization and hydrosolubility (pKa of N-acetylzonisamide is ~ 5). This contrasts with the acetylation of basic groups, wherebasicity is canceled. A different example is provided by cyanamide (4.295), a potentinhibitor of aldehyde dehydrogenase and an alcohol deterrent used as the citratedcalcium salt. Cyanamide (4.295) is isoelectronic with the sulfonamido group, and itsacidic amino group is a good target of N-acetyltransferases [195]. N-Acetylcyanamide(4.296) is the major metabolite of cyanamide in humans, rabbits, rats, and dogs. This last

finding was largely unexpected, given that dogs as a rule are poor acetylators.


Fig. 4.78. A number of industrial arylamines are known for their carcinogenicpotential, following metabolic toxification in which reactions of N- and O-acetylationhave a role to play [183b] [196 –198]. As summarized here using fluoren-2-amine(4.297) as a prototypal example, such xenobiotic arylamines can be N-oxidized to an N-arylhydroxylamine (e.g., 4.299) by cytochromes P450 (mainly CYP1A2) or theperoxidase function of prostaglandin G/H synthase (see Part 2). O-Sulfonation to 4.301(Chapt. 4.3) or NAT-catalyzed O-acetylation to 4.302 (R¼H) forms a labile ester ableto undergo heterolytic cleavage to form a highly reactive, adduct-forming nitreniumcation 4.303 (R¼H). Interestingly, quantum-mechanical calculations have shown thatthe heterolytic cleavage of an N,O-sulfate ester is markedly easier when the hydrogensulfate anion (HOSO�3 ) rather than the sulfate anion (SO2�

4 ) is the leaving group [198].This appears linked to the bladder carcinogenicity of several such arylamines. Thereactions so far discussed are marked with red arrows to signify that they are thepreferred pathways of toxification of carcinogenic arylamines. Indeed, direct N-acetylation of xenobiotic arylamines to form, e.g., 4.298, is considered in a number ofcases to be a reaction of detoxification. Nevertheless, the possibility remains in othercases of N-oxidation of an arylacetamide to an N-aryl-N-hydroxyacetamide such as4.300. The latter can also be activated to a reactive O-sulfate or O-acetate, andultimately to a nitrenium cation (e.g., 4.301, 4.302, and 4.303, resp.; R¼Ac). Oneimportant reaction is not explicit here, namely N,O-acetylation whose mechanism is

presented in more details in the next Figure.


Fig. 4.79. Like the O-acetylation of xenobiotic N-arylhydroxylamines (�NHOH) andN-arylhydroxylamides (arylhydroxamic acids; �N(OH)acyl), the reactions of N,O-acetylation (in fact, N,O-transacetylations) are catalyzed by arylamine acetyltransfer-ases. However, the nature of the Ac donor in reactions of N,O-acetylation differs fromthat in N- and O-acetylations [199]. To help the reader, we begin here by summarizingin Box A the reactants and products of O-acetylation, where the Ac moiety istransferred from acetyl-CoA to a generic N-arylhydroxylamine 4.304, the productsbeing the generic O-acetyl-N-arylhydroxylamine 4.305 and coenzyme A. In contrast,the Ac donor in reactions of N,O-acetylation (Boxes B – D) is an N-aryl-N-hydroxyacetamide, 4.306, which transfers its Ac group to a cysteinyl residue in theenzyme protein, thus forming an acetyl thioester (NAT-S-acetyl) and liberating the N-arylhydroxylamine 4.307 (Box B). Two options exist following this first step. In theintramolecular reaction of N,O-acetylation (Box C), the N-arylhydroxylamine 4.307just formed remains bound to the enzyme and captures the acetyl group to yield the O-acetyl-N-arylhydroxylamine 4.308. In the intermolecular reaction of N,O-acetylation(Box D), another N-arylhydroxylamine, 4.309, binds to the enzyme and is acetylated to

the O-acetyl-N-arylhydroxylamine 4.310.


Fig. 4.80. In Part 3, we illustrated the fatty acyl esterification of low-molecular-weightalcohols such as ethanol. These reactions are independent of coenzyme A andcatalyzed by hydrolases (EC 3) such as fatty-acyl-ethyl ester synthase (FAEES).Mention was made of the different esterification pathways open to large lipophilicalcohols, mainly endogenous and exogenous steroids, which we briefly present here[8] [200]. The major enzyme involved in the fatty acyl esterification of such steroids issterol O-acyltransferase (EC 2.3.1.26; cholesterol acyltransferase (ACAT)), a micro-somal enzyme of which two genes (SOAT1 and SOAT2) exist in humans. Anotherenzyme catalyzing the same reaction is lecithin:cholesterol acyltransferase (EC 2.3.1.43;human gene LCAT) found in blood serum, and whose acyl donor is not acyl-CoA but aphospholipid [201]. Our first illustration is 17b-estradiol (4.18) which we encounteredearlier in connection with catechol O-methylation (Sect. 4.2.1) and glucuronidation(Sect. 4.4.1). This endogenous steroid is also frequently used as a drug; its conjugationto fatty acids such as palmitic, stearic, oleic, palmitoleic, linoleic, and arachidonic acidshas been demonstrated in rat liver microsomes supplemented with the correspondingacyl-CoA [202]. Such highly lipophilic metabolites (e.g., the oleyl conjugate 4.311) arestored in tissues and act as natural slow delivery devices. The same is true of theantiasthmatic glucocorticoid budesonide (4.312) [203]. Its incubation in ATP- andCoA-fortified human lung and liver microsomes yielded a variety of fatty acylconjugates such as the palmitic ester 4.313. Following its inhalation, budesonide isextensively conjugated and retained as fatty acyl esters in airway tissues from which it is

slowly released by hydrolysis, explaining its long duration of action.


Fig. 4.81. The coupling of xenobiotic acids to coenzyme A, and the numerousmetabolic pathways open to such acyl-CoA cofactors, form a complex and insufficientlyunderstood field of research at the interface of lipid biochemistry and xenobioticmetabolism [204]. The key compound here is coenzyme A (4.273 in Fig. 4.71), on whichboth the previous Chapt. 4.5 and the present one are centered [205]. However, there isa major difference between the two Chapters, as schematized in this introductoryFigure. Whereas the previous Chapter dealt with endogenous acids (acetic acid andfatty acids) bound to coenzyme A and transferred to a xenobiotic acceptor, the presentChapter has xenobiotic acids as substrates. Their conjugation to coenzyme A iscatalyzed by ligases, yielding xenobiotic acyl-CoA cofactors which are a crossroads toan unusual variety of further metabolic pathways, most or all of which are catalyzed byenzymes whose physiological function is in lipid biochemistry. These pathways will bepresented, in turn; many of them are synthetic (anabolic) ones, an obvious exceptionbeing b-oxidation. Before discussing these pathways according to the classificationshown, we examine the ligases involved in xenobiotic acyl-CoA formation and some

properties of the resulting cofactors.


Fig. 4.82. The coupling of carboxylic acids to coenzyme A is catalyzed by enzymesknown as acid-thiol ligases (EC 6.2.1), three of which play a leading role in formingxenobiotic acyl-CoA cofactors [204 –207]. As shown, these are the short-chain acyl-CoA ligase, the medium-chain acyl-CoA ligase, and the long-chain acyl-CoA ligase,which includes one or more very long-chain acyl-CoA ligases. There is an overlap in thesubstrate specificity of these enzymes, all the more so, since they are in fact familieswith various isozymes as documented by the human genes reported in the Figure. Thesubcellular and tissular localizations differ among enzymes. The enzymatic reactionstake their energy from the splitting of ATP into AMP and pyrophosphate, with themagnesium cation acting as a cofactor in the reaction. As we shall see, a large variety ofxenobiotic carboxylic acids are substrates of these ligases, but each of the resulting acyl-CoA conjugates is then processed by only a few among the pathways depicted in the

previous Figure.


Fig. 4.83. The main focus of this Chapter is on the metabolic reactions typical ofxenobiotic acyl-CoA cofactors. In addition and as a result of their intrinsic reactivity[208], acyl-CoA conjugates also undergo a variety of reactions summarized in thisFigure. Thus, acyl-CoA thioesters react as electrophiles with H2O (chemical hydrolysis)and with glutathione (see Chapt. 4.7) to form glutathione conjugates as shown [209].Interestingly, a series of xenobiotic acyl-CoA conjugates revealed a correlationbetween their rates of hydrolysis and their rates of transacylation with glutathione[209a]. Acyl-CoA conjugates are also specific substrates of acyl-CoA thioesterases (EC3.1.2.1 and mainly 3.1.2.2) [210]. A worrying property is the structure-dependentcapacity of some xenobiotic acyl-CoA conjugates to react as electrophiles withproteins, thereby forming acylated proteins with immunogenic potential [211] [ 212].On a more positive side, some xenobiotic acyl-CoA conjugates are pharmacologicallyactive. Thus, the (S)-enantiomers of the well-known non-steroidal anti-inflammatorydrugs (NSAIDs) ibuprofen and ketoprofen (see Sect. 4.6.4) are the active compoundsthat inhibit cyclooxygenase (COX), but there is proof that the acyl-CoA conjugate ofthe �inactive� (R)-enantiomers also contributes to the therapeutic effects by inhibitingCOX-2 [213]. There is also evidence that clofibric acid and other fibrate drugs used ashypolipidemic peroxisome proliferators act as their acyl-CoA conjugates to inhibitfatty-acid synthesis [214]. Finally, the inhibition of fatty-acid b-oxidation is documentedfor some medicinal arylalkanoic acids, particularly profens; the mechanism of thisinhibition does not involve their acyl-CoA conjugates but appears to be due to acombination of coenzyme A sequestration and direct inhibition of acyl-CoA ligases

[205] [215].


Fig. 4.84. In contrast to the acyl-CoA cofactors whose lability and metabolicintermediacy prevents their excretion from the body, most of the subsequentmetabolites are sufficiently stable and hydrophilic to be excreted. Exceptions are thelipid and sterol conjugates, which are too lipophilic to be excreted. Here, we begin withamides formed from an endogenous amino acid and a xenobiotic acyl-CoA cofactor[206] [216]. The most common amino acid in such reactions is glycine, and itsprototypal substrate is benzoic acid (4.314), more precisely its benzoyl-CoA cofactor[217]. The enzymes catalyzing the formation of N-benzoylglycine (4.315) are mainlyglycine N-benzoyltransferase (EC 2.3.1.71) and glycine N-acyltransferase (EC 2.3.1.13;human genes GLYAT, GLYATL1, and GLYATL2). Note that N-benzoylglycine isbetter known under its trivial name �hippuric acid�, since it was first characterized in1829 by Liebig in the urine of horses [218]. Hippuric acid (4.315) is thus considered bymany to be the first xenobiotic metabolite ever characterized. Numerous ring-substituted derivatives and analogues of benzoic acid form a glycine conjugate [219],giving insight into informative structure – metabolism relationships [132] [220]. Furtherexamples of medicinal interest include the hypolipidemic drug nicotinic acid (4.316)whose conjugation to N-nicotinoyl glycine (4.317; nicotinuric acid) is a major metabolicpathway [221]. The same is true for acetylsalicylic acid (4.318, Aspirin); in humans,over 90% of a dose is hydrolyzed to salicylic acid (4.192), most of which (depending on

the dose) is eliminated as salicyluric acid (4.319) [222].


Fig. 4.85. The previous Figure suggests that only benzoic acids and small analoguesyield glycine conjugates in humans. This appears indeed as a rule, allowing for possibleexceptions (see later). In contrast, the formation of glutamine conjugates presentedhere favors larger substrates. Two enzymes are the main catalysts of the reaction,namely glutamine N-phenylacetyltransferase (EC 2.3.1.14) which shows a broadsubstrate specificity, and glutamine N-acyltransferase (EC 2.3.1.68) which does not acton benzoic acids. Our first example is 4-phenylbutanoic acid (4.320), an effective drugused in patients with hyperammonemia due to inborn errors in urea synthesis. Indeed,the metabolism of 4-phenylbutanoic acid (4.320) helps nitrogen excretion in the form ofits glutamine conjugate 4.321, a major metabolite in humans (20 – 25% of a dose) [223].There is more to the metabolism of this drug, since it undergoes C2 chain shortening byb-oxidation (see Sect. 4.6.4) to form 2-phenylacetic acid also excreted as the glutamineconjugate 4.322 (30– 35%). These metabolites are not formed in the rat, whichproduces high levels of acyl glucuronides, plus N-(2-phenylacetyl)glycine (4.323) notfound in humans. The Figure also shows one of the many metabolic pathways of theantiepileptic drug valproic acid (4.324). This compound formed only traces of itsglycine conjugate in epileptic patients, while its glutamine conjugate 4.325 and glutamicacid conjugate 4.326 were produced in somewhat higher amounts [224]. The lattermetabolite is a unique observation in humans, being formed either by conjugation with

glutamic acid or by deamidation of the glutamine conjugate.


Fig. 4.86. Taurine (H2N�CH2CH2�SO3H) is another amino acid which, together withglycine, shares an important physiological function in the conjugation of biliary acids.In humans, the conjugation of bile acid-CoA with taurine or glycine is catalyzed by thecytosolic enzyme bile acid-CoA:amino acid N-acyltransferase (EC 2.3.1.65; humangene BAAT) [8] [225]. There have been a number of recent reports of xenobioticcarboxylic acids yielding taurine conjugates. Thus, 2-(4-chloro-2-methylphenoxy)aceticacid (4.327), a plant growth regulator used as herbicide, formed both the taurineconjugate 4.328 and the glycine conjugate 4.329 [226]. Both metabolites were of markedimportance in the dog. In contrast, no taurine conjugate and only traces of the glycineconjugate were formed and excreted in rats, which excreted most of a dose unchanged.Another interesting example is that of MRL-II (4.330), an agonist of the a-peroxisomeproliferator-activated receptor (aPPAR) of interest as a cholesterol- and triglyceride-lowering agent [227a]. When administered to dogs, dose-dependent amounts of theacylglucuronide and taurine conjugates were excreted in the bile. Low doses favoredthe taurine conjugate, whose proportion decreased at higher doses. In contrast to 2-(4-chloro-2-methylphenoxy)acetic acid (4.327), the retinoid X-receptor ligand andantitumor agent targretin (4.331) did form taurine conjugates in rats from theunchanged drug and from two oxidized metabolites [227b]. There are few reports ofxenobiotic taurine conjugates being formed in humans. One nice example is that of theNSAID ibuprofen (4.332). Its taurine conjugate 4.333 was a minor urinary metabolite(1 – 2% of a dose) excreted almost exclusively as the (S)-enantiomer [228], in contrastto the withdrawn NSAID benoxaprofen whose taurine conjugate was mainly the (R)-

enantiomer in rats (see also Sect. 4.6.4).


Fig. 4.87. The esters formed by the acylation of endogenous hydroxy compounds withxenobiotic acyl-CoA cofactors are summarized in Fig. 4.81. Based on their lipophilicityand resulting behavior in the body, the resulting conjugates are classified as morelipophilic than the parent xenobiotic carboxylic acids (hybrid lipid, sterol esters, andethyl esters), or more hydrophilic (carnitine esters). Here, we summarize the majortypes of mixed (i.e., hybrid) glycerides and sterol esters formed from xenobiotic acids[204] [229] [230]. The formation of hybrid triglycerides and sterol esters has beenreported for a number of xenobiotic carboxylic acids using, e.g., human or animalhepatocytes or adipocytes [230]. In vivo evidence in animals is also available, mostly inrats [231]. These conjugates include hybrid triglycerides such as the genericdipalmitoylglyceride conjugate 4.334, hybrid phospholipids such as the generic 1-acyl-2-palmitoyl-3-phosphocholine 4.335, and cholesteryl esters 4.336. There are alsoexamples of xenobiotic carboxylic acids being coupled to the 3-OH group of bileacids [232]. A number of enzymes are known or believed to catalyze the formation oflipid conjugates, most notably diacylglycerol O-acyltransferase (EC 2.3.1.20; humangenes DGAT1 and DGAT2), 2-acylglycerol O-acyltransferase (EC2.3.1.22; humangenes MOGAT1, MOGAT2, and MOGAT3), 1-acylglycerophosphocholine O-acyl-transferase 1 (EC 2.3.1.23; human gene PLCAT1), sterol O-acyltransferase (seeFig. 4.80), and acylglycerol-3-phosphate O-acyltransferases (EC 2.3.1.51 and -52;

human gene root AGPAT) [8].


Fig. 4.88. A marked number of xenobiotic carboxylic acids have been reported to formhybrid lipid conjugates [229– 231], as illustrated here. Benzoic acid (4.314) is unusual,since the vast majority of known substrates are larger molecules. Several drugs aresubstrates, for example, the antiepileptic valproic acid (4.324), which was incorporatedinto phospholipids in cultured neurons [233]. Other drugs include NSAIDs such asibuprofen (4.332) and ketoprofen [231], and some hypolipidemic drugs such as lifibrol(4.337) [234]. Some agrochemicals are also substrates, witness the herbicide 4-(2,4-dichlorophenoxy)butanoic acid (4.338 ; 2,4-DB) [230]. In this case, triacylglycerolanalogues were the major products formed in cultured adipocytes, while some mixeddiacylglycerols and phosphatidylcholines (i.e., containing one fatty acid and onexenobiotic acid) were also observed. An example of cholesteryl ester formation isprovided by the hypolipidemic agent 4-[10-(4-chlorophenoxy)decyloxy]benzoic acid(4.339) [235]. Following oral administration to rats, the cholesteryl ester was formedand accumulated in the liver, being neither metabolized further nor transported bylipoproteins. Accumulation in the liver and other tissues is certainly a factor of concern,as illustrated by the fate of fenvalerate (4.340) in rats and mice [236]. This pyrethroidinsecticide exists as four stereoisomers. Following administration of the isomericmixture, a lipophilic residue was identified in all analyzed tissues and found to be thecholesteryl ester of (2R)-2-(4-chlorophenyl)isovaleric acid (4.341). Remarkably, thisconjugate was shown to be the causative agent of microgranulomatous changes in theliver of mice chronically fed fenvalerate (4.340). A few studies have investigated therate of elimination of hybrid lipid conjugates from tissues and found it to be markedly

slower than that of their natural counterparts [231b] [237].


Fig. 4.89. There are only few examples of the coenzyme A-dependent esterification ofxenobiotic carboxylic acids with short-chain alkanols. In fact and to the best of ourknowledge, the only example of medicinal interest is that of etretinate (4.342), a highlyteratogenic retinoid used cautiously for the treatment of severe psoriasis resistant toother forms of therapy. Etretinate is an ethyl ester prodrug whose hydrolase-catalyzedhydrolysis yields the active carboxylic acid known as acitretin (4.343). In patients underlong-term therapy, plasma etretinate levels remain detectable for over two yearsfollowing discontinuation of treatment, a pharmacokinetic result incompatible witheffective hydrolysis and further degradation and elimination of acitretin (4.343) [238].The explanation was found with the discovery that part of the metabolically formed4.343 forms the acitretinoyl-CoA thioester conjugate 4.344, a reaction catalyzed byhuman liver microsomal long-chain fatty acid-CoA ligase (see Fig. 4.82). This thioesteris then transesterified to etretinate by an insufficiently characterized �ethanolacyltransferase� which can also use other short-chain alkanols [239]. The resulting�futile� metabolic cycle is in fact far from pharmacologically futile, the highly lipophilicetretinate being stored in adipose tissues and serving as a reservoir for the activeacitretin (4.343). Interestingly, the CoA-thioester 4.344 is also the intermediate in theformation of side-chain-shortened metabolites such as 4.345 (resulting from an initial C1

shortening in a 3-methyl-branched carboxylic acid¼a-oxidation) and 4.346 (resultingfrom subsequent C2 shortening¼b-oxidation ; discussed below). As an aside, we notethat another interesting metabolite of acitretin is (Z)-acitretin (4.347) whose reversibleformation is presumably catalyzed by the retinoid cis-trans isomerases EC 5.2.1.3 and

5.2.1.7.


Fig. 4.90. l-Carnitine ((R)-(3-carboxy-2-hydroxypropyl)(trimethyl)ammonium) is ad-amino acid whose essential role is to allow membrane translocation of fatty acidsfollowing their conjugation to acyl-carnitines. The formation of the latter is catalyzedby carnitine O-acyltransferases, in particular carnitine O-acetyltransferase (EC 2.3.1.7;human gene CRAT) which acts with C2 – C10 alkanoic acids, carnitine O-octanoyl-transferase (EC 2.3.1.137; human gene CROT) which acts with C4 – C16 alkanoic acids,and carnitine O-palmitoyltransferase (EC 2.3.1.21; human genes CPT1A, CPT1B,CPT1C, and CPT2) having a broad substrate specificity in the C6 – C20 range [8] [240].While Gly, Glu, and taurine form amide conjugates with xenobiotic acids, carnitineforms esters via its OH group, since its trimethylammonium group is not available forconjugation. Another physicochemical characteristic of carnitine conjugates is the factthat the negative charge in the parent carboxylate anion is replaced by a zwitterionicfunction capable of forming an internal ionic bond.

Two medicinal examples of carnitine conjugates are shown here. Valproic acid(4.324), whose capacity to form hybrid glycerides was presented above, also formsvalproylcarnitine (4.348) as a minor metabolite in humans, as seen in the urine ofepileptic patients treated with the drug [241]. The anti-inflammatory agent tolmetin(4.195), whose acylglucuronide was discussed in Chapt. 4.4, also forms amino acidconjugates. In rats, for example, the relative quantitative importance of conjugates wasacyl glucuronide> taurine conjugate 4.349>carnitine conjugate 4.350>glycine con-

jugate [211a].


Fig. 4.91. This Figure exemplifies cases where the substrate of carnitine conjugation isnot the xenobiotic per se but a metabolite thereof. A medicinally relevant situation isthat of pivampicillin (4.351), the (pivaloyloxy)methyl ester of the b-lactam antibioticampicillin (4.352). Bio-hydrolysis of pivampicillin liberates the promoiety pivalic acid(4.353), a known substrate of the carnitine conjugation pathway. However, theformation and excretion of pivaloylcarnitine (4.354) in humans and rats dosed withpivalic acid or pivampicillin is limited by the body reserves of carnitine. Humanssupplemented with carnitine excreted up to 90% of the administered amount of pivalicacid as pivaloylcarnitine [242]. The second example is that of cycloprate (4.355), amiticide agent whose hydrolysis liberates cyclopropanecarboxylic acid (4.356). Thelatter is a good substrate of carnitine O-acetyltransferase, as shown in rats receivinghigh doses of cycloprate [243]. In rat hepatocytes, the substrate specificity of carnitineester formation was cyclopropanecarboxylic acid (4.356)>cyclobutanecarboxylic

acid>cyclohexanecarboxylic acid¼benzoic acid>pivalic acid.


Fig. 4.92. In this last Section of Chapt. 4.6, we survey reactions of xenobiotic acyl-CoAconjugates that modify the acyl moiety by oxidizing it, lengthening its chain, orracemizing it. Most available examples concern b-oxidations, which in eukaryotesoccur in peroxisomes (for very-long-chain fatty acids) and mitochondria, theirphysiological products being acetyl-CoA and ATP. Extensive investigations with w-phenylalkanoic acids 4.358 have revealed that such xenobiotic carboxylic acids are alsogood substrates of the b-oxidation enzymatic machinery [8] [244] [245]. Thus, 12-phenyllauric acid forms its CoA-conjugate (C12-4.358-CoA), a reaction catalyzed bylong-chain acyl-CoA ligase (EC 6.2.1.3; see Fig. 4.82). The sequential b-oxidation stepsinvolve 2,3-dehydrogenation catalyzed by acyl-CoA oxidase (EC 1.3.3.6), hydration tothe (S)-3-hydroxyacyl intermediate catalyzed by enoyl-CoA hydratase (EC 4.2.1.17),dehydrogenation to the 3-oxoacyl intermediate catalyzed by 3-hydroxyacyl-CoAdehydrogenase (EC 1.1.1.35), and oxidative cleavage with loss of acetyl-CoA catalyzedby 3-oxoacyl-CoA thiolase (EC 2.3.1.16). The resulting 10-phenyldecanoyl-CoA (C10-4.358-CoA) can be hydrolyzed by fatty acyl-CoA hydrolase (EC 3.1.2.2; see Fig. 4.83)to 10-phenyldecanoic acid (C10-4.358). In addition, it is substrate of a subsequent cycleof b-oxidation to 8-phenyloctanoic acid, and further on all the way to 2-phenylaceticacid. See also Fig. 4.85 in which the b-oxidation to 4-phenylbutanoic acid (4.320) was

alluded to.


Fig. 4.93. A more complex case of b-oxidation is presented by the thromboxane A2

receptor antagonist known as (þ)-S-145 (4.359) [246]. When incubated in rathepatocytes and rat liver peroxisomes, its fatty-acyl side chain indeed underwent b-oxidation. Interestingly, two independent pathways of b-oxidation were characterizeddue to the presence of the (Z)-configured C¼C bond at the 5-position of the side chain.In the first pathway, this C¼C bond was reduced by an NADPH-dependent reductaseto yield 4.360 before b-oxidation produced the C2-shortened metabolite 4.361. In thesecond pathway, the parent compound was substrate of a first cycle of b-oxidation toyield the C2-shortened metabolite 4.362. Further b-oxidation of the latter to produce theC4-shortened metabolite 4.363 first necessitated reduction (or perhaps 3!2 displace-ment) of the C¼C bond. A recent example of b-oxidation is provided by[99mTc]tricarbonyl(15-cyclopentadienyl pentadecanoic acid)technetium (4.364 ; abbre-viated as [99mTc]CpTT-PA) [247]. This compound has been developed as a potentialdiagnostic tool in heart diseases. And, indeed, it was incorporated in rat myocardium,recognized as a fatty acid, and metabolized as such by b-oxidation. The C12-shortenedanalogue (produced by six cycles of b-oxidation) was the major metabolite found in

heart lipids and heart perfusate.


Fig. 4.94. The two previous Figures illustrate the b-oxidation of xenobiotic carboxylicacids. However, these are not the only cases of interest here, since w-oxidation of analkyl side chain in a xenobiotic compound may also yield a metabolite able to undergob-oxidation. This situation is exemplified here with 2,2-dimethyl-N-(2,4,6-trimethoxy-phenyl)dodecanamide (4.365 ; CI-976), a highly effective inhibitor of cholesterol O-acyltransferase (EC 2.3.1.26; ACAT) with potential as an inhibitor of cholesterolabsorption and deposition [248]. When incubated with liver microsomes from humansor animals, the compound was oxidized at various positions in its alkyl side chain,yielding ketones and the carboxylic acid metabolite 4.366. In rats in vivo and in vitro,the latter metabolite underwent three cycles of b-oxidation to produce the C6-shortened carboxylic acid 4.367. In addition, a C5-shortened homologue was produced asa minor metabolite, but it was not seen upon incubations of 4.366 or its w-OHprecursor. This intriguing metabolite further illustrates the complexity of theendogenous metabolic pathways which xenobiotic carboxylic acids may enter. Asomewhat different picture emerges with the statins, two of which are used in theirlactone, prodrug form, namely lovastatin and simvastatin (4.181; see Fig. 4.53), whilemost others are used as the active hydroxy acids. Several statins including simvastatinand lovastatin have been shown to undergo one or two cycles of b-oxidation [249], andindeed their hydroxy-acid form is known to yield an acyl-CoA thioester. However, the(R)-configured 3-OH group in statins prevents b-oxidation (see Fig. 4.92); an inversionof configuration is necessary and occurs via dehydration at C(2) and C(3), as shown

here.


Fig. 4.95. The chain elongation by C2 units is known for a small number of substrates[204]. An example is that of benzoic acid (4.314) which in the horse yielded smallamounts of 3-hydroxyphenylpropionic acid (4.370) and possibly also 3-oxo-3-phenyl-propanoic acid (4.369) [250]. These metabolites were also produced from exogenous(radiolabeled) benzoic acid, and they represented stable intermediates in a cycle of C2

addition. Even more informative is the case of 5-(3-carboxypropyl)picolinic acid(4.372). This metabolite was formed in rats by the oxidative dechlorination of thedopamine b-hydroxylase inhibitor 5-(4-chlorobutyl)picolinic acid (4.371), and it wasexcreted mainly as the C2-elongated metabolites 4.373 to 4.376 [204] [251]. The fourmetabolites are arranged here in a biochemically logical sequence allowing comparisonwith b-oxidation (Fig. 4.92). This reveals the remarkable fact that several enzymes inb-oxidation also catalyze the reverse reaction. Indeed, acetyl-CoA transfer to the CoA-conjugate of 4.372 to produce 4.373 was most likely catalyzed by EC 2.3.1.16 (hereknown as acetyl-CoA C-acyltransferase). Reduction of 4.373 to 4.374 is catalyzed by EC1.1.1.35 acting as a hydrogenase, while dehydration of 4.374 to 4.375 is expected to becatalyzed by EC 4.2.1.17 acting as a dehydrase. The last step is a hydrogenationcatalyzed by the NADPH-dependent trans-2-enoyl-CoA reductase (EC 1.3.1.38), anenzyme distinct from the flavoprotein acyl-CoA oxidase (EC 1.3.3.6) which initiates b-oxidation. Note that the elongation of 4.372 was also seen in other animal speciesincluding humans. Also worthy of note is the fact that all known reactions of chainelongation involve a single C2 unit, except for cyclopropanecarboxylic acid (4.356)

which was elongated up to C16 depending on animal species [204].


Fig. 4.96. An intriguing metabolic reaction has attracted much interest since the 1970s,namely the inversion of sense of chirality of non-steroidal anti-inflammatory 2-arylpropanoic acids (i.e., profens), the most investigated of which is ibuprofen (4.332),an extensively used drug [252] [253]. It took many years and a number of researchteams to characterize the enzymology and mechanism of the reaction, which are nowfairly well understood. As shown here, the first step is the formation of an acyl-CoAintermediate, a reaction catalyzed by long-chain acyl-CoA ligase (EC 6.2.1.3; Fig. 4.82)[206] [254]. This reaction is enantioselective in that it shows a marked or practicallyexclusive preference (depending on animal species) for the inactive (�)-(R)-profens[255]. In other words and in the case of ibuprofen, the ligase forms only or mainly the(R)-ibuprofenoyl-CoA. Once formed, this intermediate is the substrate of a reactionunder inversion of configuration catalyzed by 2-methylacyl-CoA 2-epimerase (EC5.1.99.4; a-methylacyl-CoA racemase; human gene AMACT), a peroxisomal andmitochondrial enzyme catalyzing an essential step in the oxidation of cholesterol tocholic acid [256] [257]. This reaction is one of racemization when considering only thesubstrate moiety, but, in strictly correct terms, it is one of epimerization, since the acyl-CoA conjugate contains several stereogenic centers (see Fig. 4.71). As a result ofepimerization, the ibuprofenoyl moiety now exists in the (R)- and (S)-forms, and acyl-CoA thioesterases act on both (R)-ibuprofenoyl-CoA and (S)-ibuprofenoyl-CoA toliberate the corresponding ibuprofen enantiomer. In the metabolic scheme shown here,(S)-ibuprofen is thus an end-point only, not an entry point; in contrast, (R)-ibuprofen

is both.


Fig. 4.97. A few profens beside ibuprofen have been shown to undergo inversion ofconfiguration in humans, namely ketoprofen (4.377), fenoprofen, and benoxaprofen[258] [259]. In vivo and in vitro results in a variety of animal species indicate that theextent of inversion of profen depends strongly on substrate as well as on species[252] [260]. Furthermore, inversion of configuration is not restricted to 2-methyl-substituted arylacetic acids, witness KE-748 (4.378), the active metabolite of theantirheumatic agent KE-298 [261]. Like ibuprofen, KE-748 underwent extensive (R)-to-(S) inversion when administered to rats or incubated with rat hepatocytes. Anapparently unconnected example is that of phytanic acid (4.379), namely(3RS,7R,11R)-3,7,11,15-tetramethylhexadecanoic acid. This branched fatty acid ispresent in various dietary sources, in particular in the fat of ruminant animals where itaccumulates as a metabolite of phytol, itself a decomposition product of chlorophyll. Inhumans and animals, phytanic acid is conjugated to coenzyme A, but its 3-methylsubstitution forbids subsequent b-oxidation [244]. Instead, the phytanoyl-CoAintermediate is substrate of a-oxidation to form the coenzyme A conjugate of pristanicacid, more accurately the two epimers (2S)- and (2R)-pristanoyl-CoA (4.380). b-Oxidation to form propanoyl-CoA is now possible, but only for (2S)-pristanoyl-CoA;2-methylacyl-CoA 2-epimerase is necessary to catalyze the inversion of configuration of(2R)-pristanoyl-CoA and avoid accumulation of (2R)-pristanic acid [256] [257] [262].To us, phytanic acid (4.379) thus appears as an example at the interface of xenobiotics

and alimentary compounds.


Fig. 4.98. This Figure opens an important Chapter dedicated to glutathione (4.385 ;GSH), a tripeptide playing an essential role in the protection of organisms against avariety of environmental insults. We initiate this Chapter with the biosynthesis ofglutathione (4.385) as schematized in the left part of the Figure [263] [264]. Thereaction begins with the formation of g-glutamyl-cysteine (4.383) from l-cysteine(4.381) and l-glutamic acid (4.382), as catalyzed by g-glutamylcysteine synthetase (EC6.3.2.2). Glycine (4.384) is introduced in the second step catalyzed by glutathionesynthetase (EC 6.3.2.3); both reactions are ATP-dependent. The all-important andlimiting component of GSH is cysteine, whose SH group is responsible for itsconjugating and antioxidant capacities.

Indeed, glutathione is not only an important conjugating compound, it is also anessential antioxidant agent, so essential that its physiological concentrations in cells andplasma are in the 0.5 – 10 mm and micromolar range, respectively [265]. In the body,GSH exists in a redox equilibrium with an oxidized form known as glutathione disulfide(GSSG) [266]. Glutathione acts mainly as a radical scavenger as summarized in theright side of the Figure [267 – 269]. Like other endogenous thiols including serumalbumin, glutathione scavenges free radicals, in particular C-centered radicals(Reaction 1) and reactive oxygen species (ROSs) such as the HO. radical, superoxide,and peroxide radicals (Reactions 2 –5). These reactions transform GSH into theglutathionyl radical (GS .) which is detoxified by reacting with a second GS . radical toyield GSSG (Reaction 6). The latter is recycled to GSH by glutathione disulfidereductase (EC 1.8.1.7; Reaction 7) [263] [264]. As such, GSH plays a critical role in

cellular protection against oxidative stress and radiations [270].


Figs. 4.99 and 4.100. Whatever the significance of radical scavenging reactions in thecellular protection, our scope remains on xenobiotic metabolism, and specifically hereon GSH-dependent conjugations. These are catalyzed by glutathione S-transferases(EC 2.5.1.18; GSTs), a large and diverse group of enzymes present in the cytoplasm, inthe endoplasmic reticulum, in mitochondria, and in peroxisomes [10] [271– 274]. Notone but two superfamilies of genes exist in animals, namely those coding for thecytoplasmic superfamily of enzymes (GSTs in the narrow sense), and those coding forthe microsomal superfamily of enzymes (designated as MGSTs when distinguishedfrom the cytoplasmic GSTs). In humans, the microsomal enzymes are the products ofthree MGST genes, and they function as homotrimers. The cytoplasmic (i.e., soluble)enzymes are products of the GST genes, a large number of which are known. Theseenzymes function as homodimers or heterodimers as shown. The main functions ofthese enzymes are the formation of conjugates of endogenous and exogenouscompounds, and these are the relevant functions we will present here. In addition,these enzymes perform other physiological functions such as isomerization, reduction,

thiolysis, and transport which fall outside our scope.


Fig. 4.100.


Figs. 4.101 – 4.103. Fig. 4.101 focuses on some common features in the catalyticmechanism of glutathione S-transferases [275] [276]. A main element in thismechanism is the acidity of the SH group in glutathione, which in aqueous solutionhas a pKa of 9.0 and is thus poorly ionized at physiological pH. Significantly, SH acidityis markedly increased (pKa in the range 6 – 7) when GSH is bound to glutathione S-transferases [277]. This 100- to 1000-fold increase in acidity results from a considerablestabilization of the ionized thiol, a stabilization achieved in particular by an H-bonddonation from a neighboring tyrosine residue. This increased acidity is translated intoan increased nucleophilicity toward reactive electrophiles, and the thiolate anion in GS�

is thus the catalytically active group in GST-bound glutathione. In turn, the reactivity ofthe substrate is increased by polarization of its electrophilic center, as shown inFig. 4.101 for 4-phenylbut-3-en-2-one (Panel a). This Panel shows the initial catalyticstep in reactions of glutathione addition, whose main types of substrates and productsare reviewed in Fig. 4.102. Panel b in Fig. 4.101 schematizes the transition state in areaction of glutathione addition– elimination whose substrate here is 1-chloro-2,4-dinitrobenzene. The main types of substrates and products of these reactions arereviewed in Fig. 4.103. Both Panels a and b are highly simplified representations(modified from [275a]) which do not incorporate further residues involved in catalysisand/or substrate binding by hydrophobic and electrostatic interactions [278].Furthermore, the high structural flexibility of glutathione S-transferases (GSTs) is

not taken into account here [279].


Fig. 4.102.

Fig. 4.103.


Fig. 4.104. An intriguing aspect of glutathione conjugation is the potential occurrenceof nonenzymatic reactions [280]. In an enlightening and still current review, Kettererhas highlighted the role of nonenzymatic glutathione conjugation reactions inxenobiotic metabolism, offering a cogent classification into four possible scenarios[280a]. Thus, a given chemical containing a potential target group may be a good or apoor substrate of glutathione transferases; it may also be able or unable to reactspontaneously (i.e., nonenzymatically) with glutathione in solution. The four resultingcombinations are shown here; the first two cases involve reactions which, in vitro and invivo, will be mediated mainly (Case 1) or solely (Case 2) by GSTs. Case 3 involvesmainly or exclusively nonenzymatic reactions, whereas Case 4 comprises chemicalsresistant to GSH conjugation. The remainder of the Figure compares the nonenzymaticvs. enzymatic GSH conjugation of five xenobiotics; the reactions were monitoredspectrophotometrically and involved a 1-min incubation of the xenobiotic with GSH inthe absence of enzymes, followed by the addition of an aliquot of rat liver cytosol and afurther 1-min incubation [280a]. 4-Nitrobenzyl chloride (4.386) and 1-chloro-2,4-dinitrobenzene (4.387) are seen to be good GST substrates and to react spontaneouslywith GSH. 1,2-Dichloro-4-nitrobenzene (4.388) was a fair GST substrate and reactedspontaneously with GSH. 1-(4-Nitrophenoxy)propane 2,3-oxide (4.389) was a poorGST substrate but reacted spontaneously with GSH, whereas (E)-4-phenylbut-3-en-2-

one (4.390) was almost inert.


Fig. 4.105. A convenient mechanism accounting for the nonenzymatic reactions ofglutathione is based on electrophilicity – nucleophilicity considerations [281]. Indeed,electrophiles and nucleophiles are ranked according to their softness or hardness. Thus,hard electrophilic sites have a highly localized positive charge (i.e., a high chargedensity) which is poorly polarizable (i.e., remains highly localized during the approachof the reaction partner). In contrast, soft electrophilic sites have a delocalized positivecharge (i.e., a low charge density) which is readily polarizable by the approachingreaction partner. For the definition of hard and soft nucleophilic sites, the reader justneeds to replace �electrophilic� with �nucleophilic� and �positive� with �negative� in thetwo previous sentences. Glutathione (in protonated and ionized form) is a softnucleophile, and as such will react relatively easily with soft electrophiles; thisspontaneous reactivity will allow a small (Case 1 in Fig. 4.104) or large (Case 3 inFig. 4.104) percent of the conjugate to be formed nonenzymatically, depending onenzymatic efficiency. In the presence of hard electrophiles, spontaneous reaction is

precluded (Cases 2 and 4 in Fig. 4.104).


Fig. 4.106. Glutathione and glutathione transferases have evolved as a major (in fact,THE major) chemical protection against reactive xenobiotics and reactive compoundsproduced during the metabolism of endogenous and exogenous compounds. Examplesinclude products of lipid peroxidation and radiation damage (see also Fig. 4.98)[282] [283]. The Table shown in this Figure (taken from [281a]) points to the fact thatthiols are among the softest endogenous nucleophiles, implying that GSH will reactpreferentially with the soft electrophiles on the right of the Table. Were it not forcatalytic facilitation by GSTs, GSH would not be able to detoxify the harderelectrophiles listed here. Significantly, these harder electrophiles are precisely the onesthat best react spontaneously with various nucleophilic sites in nucleic acids, being thusable to induce DNA damage.

Because things are never simple in nature, the neat detoxification mechanisms ofGSH-GSTs may sometimes backfire and yield reactive metabolites [284]. This will be

illustrated in some of the following Figures.


Fig. 4.107. Once formed, glutathione conjugates 4.391 are extensively processed in thebody through transport and degradation. Thus, they are substrates of a number oftransporters (carriers and export pumps) which mediate their transport across cellmembranes in liver, intestine, and kidney, among others organs [285] [286]. Moresignificant in our context is their stepwise enzymatic breakdown to their majorexcretion products. Their degradation begins with the cleavage of the glutamyl residueby g-glutamyltranspeptidase (EC 2.3.2.2; human genes GGT1, GGT3, GGT5, andGGT6). These are membranal enzymes present in the liver, kidneys, and other organs.The resulting cysteinylglycine conjugate 4.392 loses its glycyl moiety by the action ofvarious dipeptidases (EC 3.4.13). The cysteine conjugate 4.393 so produced undergoes areaction of N-acetylation catalyzed by cysteine S-conjugate N-acetyltransferase (EC2.3.1.80), a microsomal enzyme found mainly in the kidney. The product is an N-acetylcysteine conjugate known as a mercapturic acid (4.394), a major urinaryexcretion product of glutathione conjugates. The N-acetylation of cysteine conjugatesis reversible due to the involvement of various amidases (EC 3.5.1). More importantly,both the cysteine and the N-acetylcysteine conjugates are substrates of cysteine S-conjugate b-lyase (EC 4.4.1.13; human genes CCBL1 and CCBL2), a mainly renal andhepatic enzyme which cleaves the S�C bond in the cysteinyl moiety, thus liberating athiolated metabolite, 4.395, of the parent xenobiotic [8] [287– 290]. The latter can befurther S-methylated by thiol S-methyltransferase (EC 2.1.1.9; TMT; see Chapt. 4.2) toform 4.396. Both 4.395 and 4.396 are substrates of cytochromes P450 and flavin-containing monooxygenases which catalyze their S-oxygenation (see Part 2

[2] [4] [291]).


Fig. 4.108. With this Figure, we begin our survey of the various types of substrates andreactions involved in glutathione conjugation. The Sect. 4.7.2 is dedicated to reactionsof additions as summarized in Fig. 4.102, whereas reactions of addition –elimination(summarized in Fig. 4.103) will be presented in Sect. 4.7.3. A first class of reactions ofaddition are on saturated C-atoms, the substrates being the so-called �nitrogenmustards� (this Figure) and epoxides (Figs. 4.109 – 4.111). Nitrogen mustards areantitumor alkylating agents containing a (2-chloroethyl)amino function, as exemplifiedhere with mechlorethamine (4.397) [292] [293]. Other drugs include chlorambucil,melphalan, cyclophosphamide, and ifosfamide. The global reaction of conjugation mayappear as the substitution of a Cl-atom with a glutathionyl moiety, but its mechanism isnot a genuine substitution, since spontaneous dechlorination first forms an aziridiniumion, 4.398, which is the actual alkylating species. This reactive intermediate alsoundergoes enzymatic and nonenzymatic conjugation with GSH, yielding first theconjugate 4.399, then the diconjugate 4.403 from aziridinium 4.401. Some spontaneoushydrolytic dechlorination also occurs to produce the (2-hydroxyethyl)amino deriva-

tives 4.400, 4.404, and 4.405.


Fig. 4.109. Epoxides are conveniently classified into the reactive arene oxides and themore stable alkene oxides (see Parts 2 and 3 [2 – 4]). Numerous drugs and otherxenobiotics contain a phenyl moiety whose oxidation yields an arene oxide of genericstructure 4.406. Nucleophilic attack by the glutathionyl anion at either of the twooxirane C-atoms occurs with inversion of configuration (i.e., an SN2 mechanism) toyield the glutathionyl conjugate 4.407. In vivo, the latter is processed as presented inFig. 4.107 to yield the N-acetylcysteinyl conjugate, 4.408, known as a premercapturicacid. Compared to the general case in Fig. 4.107, an additional step is required toproduce a mercapturic acid, 4.409, namely aromatization by dehydration [283] [294]. Amore detailed presentation of the stereochemistry of the reaction is exemplified withphenanthrene 9,10-epoxide (4.410) [275a] [295]. This substrate is a symmetric meso-(R,S)-compound, but GSH attack on either oxirane C-atom cancels the plane ofsymmetry and renders the products chiral. Due to inversion of configuration at the C-atom undergoing substitution, the two metabolites are the (9R,10S)- and (9S,10R)-conjugates, 4.411. The product stereoselectivity of the reaction depends on theglutathione S-transferase involved; thus, rat M1-1 gave an approximately equalmixture of the two products, whereas rat M2-2 was essentially stereospecific for the

(9R,10S)-conjugate [275a].


Fig. 4.110. The glutathione conjugation of alkene oxides is illustrated here with twoindustrial examples, namely butadiene and styrene. Both xenobiotics are oxygenatedby CYP2E1 (see Part 2 [2] [4]) to their respective epoxides, namely butadienemonoepoxide and styrene oxide. Buta-1,3-diene monoepoxide (4.412 ; see also Part 3[3] [4]) is substrate of a number of metabolic pathways including GSH conjugation[296]. When incubated with human placental GST, the compound underwent GSHattack at both C-atoms of the oxirane ring, namely at C(1) (Reaction a) and at C(2)(Reaction b) [297]. The conjugate 4.414 resulting from the attack at C(2) provedchemically stable. In contrast, a fast equilibrium was found between conjugate 4.413(resulting from the attack at C(1)) and two thiirane products resulting from anintramolecular rearrangement, namely the thiirane 4.415 and the episulfonium ion4.416. Buta-1,3-diene monoepoxide is a chiral compound, yet styrene oxide (4.417)appears better suited to discuss product regio- and stereoselective glutathioneconjugation [298]. This compound is a metabolite of styrene, a potential carcinogen.When incubated with liver cytosol from Wistar rats, pure (R)-4.417 gave (S)-4.418 and(R)-4.419 in a 6 :1 ratio, whereas pure (S)-4.417 gave (R)-4.418 and (S)-4.419 in a 1 : 32ratio. In other words, the (R)-enantiomer was conjugated preferentially at C(1),whereas the (S)-enantiomer was conjugated almost exclusively at C(2). Results withSprague – Dawley rat liver cytosol were different, since both styrene oxide enantiomerswere conjugated predominantly at C(1). Substrate enantioselectivity was also observedin that (R)-styrene oxide was conjugated ca. twice as fast as (S)-styrene oxide. Thisobservation was confirmed with rat purified glutathione S-transferases M, especially

M3-3.


Fig. 4.111. Two medicinally relevant examples are shown here, one dealing with theGSH conjugation of an arene oxide, the other with that of an alkene oxide. The in vivometabolism of the COX-2 inhibitor valdecoxib (4.420) was examined in great details inmice, revealing a total of 16 metabolites which accounted for practically the totality ofits in vivo metabolism [299]. Of relevance here was the methyl sulfone 4.422 whoseformation was quite reasonably postulated to have involved phenyl epoxidation,glutathione conjugation to 4.421, and finally the various metabolic steps summarized inFig. 4.107. Another example is simvastatin (4.181) whose glutathione conjugate 4.424was characterized as a major biliary metabolite in rats administered the drug [300].This conjugate was most probably formed from the 4a’,5’-epoxy-6’-hydroxy inter-

mediate 4.423.


Fig. 4.112. Polarized alkenyl moieties make good targets for enzymatic and non-enzymatic GSH conjugation by a Michael reaction. The initial catalytic step in reactionhas been summarized in Fig. 4.101 using 4-phenylbut-3-en-2-one (4.425), an a,b-unsaturated ketone, as an example. Returning to the same substrate, we note that thereaction is regioselective for the C(b)-atom. Furthermore, the substrate has twoenantiotopic faces such that the saturation of and addition to the C(b)-atom creates astereogenic center. Class mu enzymes appear particularly effective in catalyzing theGSH conjugation of phenylbutenone, with the preferential stereoisomer formed being(R)-4.426 [301]. An important feature of the GSH conjugation of polarized alkenes isits reversible nature, the reverse reaction (elimination) being also catalyzed by mu classGSTs. However, and as a rule, this reversibility is modest and its in vivo relevance smallcompared with that of the glutathione conjugates of isocyanates and isothiocyanates, aswe shall see later [302]. The lower part of the Figure presents a selection of polarizedalkenyl moieties known to undergo glutathione conjugation [303]. Shown here arethree types of a,b-unsaturated aldehydes, two types of a,b-unsaturated ketones, a,b-unsaturated esters, a,b-unsaturated amides, a,b-unsaturated nitriles, and alkenylgroups polarized by two different aryl moieties. Some of these moieties are exemplified

in the next two Figures.


Fig. 4.113. Many different classes of chemicals are detoxified by GSH conjugation; forexample, toxic endogenous products of radical reactions and lipid peroxidation such asN-propenal purines and pyrimidines, and 4-hydroxyalkenals [303a]. Here, we focus onxenobiotic toxins, namely the two important industrial chemicals acrylamide (4.427)and acrylonitrile (4.431) [304]. Rats administered the two xenobiotics togetherexcreted three mercapturic acids as major urinary metabolites of each chemical. Bothacrylamide and acrylonitrile underwent direct glutathione conjugation which resulted inthe excretion of the mercapturates 4.428 and 4.432, respectively. Furthermore, theyboth formed the corresponding epoxide. In the case of acrylamide (4.427), its epoxideformed the two mercapturic acids 4.429 and 4.430 which resulted from GSH addition tothe C(a)- and C(b)-atom, respectively. In the case of the epoxide of acrylonitrile, thesame two positions were targets of GSH conjugation, with the attack at C(a) producingthe mercapturate 4.433. As for the conjugate produced by the attack at C(b), itunderwent spontaneous decyanylation before being transformed into the mercapturate

4.434. For both epoxides, C(b)-conjugation predominated over C(a)-conjugation.


Fig. 4.114. A few examples of medicinal relevance are presented here, the brokenarrows pointing to the C-atom attacked by GSH. We begin with morphinone (4.435), arather unstable metabolite of morphine produced by dehydrogenation of its secondaryalcohol group [305]. Morphinone was suspected of being responsible for the rapid,dose-dependent decrease in hepatic glutathione content caused by morphine. Thehypothesis gained likelihood when the glutathione conjugate of morphinone wasunambiguously identified as a biliary metabolite of morphine in guinea pigs,accounting for almost 10% of a dose. The glutathione conjugate of the diuretic drugethacrynic acid (4.436), a well-known metabolite, is formed both enzymatically andnonenzymatically by attack at the electron-deficient exo-methylidene C-atom [306]. Amore intriguing example is that of verlukast (4.437), a leukotriene D4 antagonist thatwas under development for the treatment of bronchial asthma [307]. The compound isof interest, because it lacks a polarizing carbonyl group, this role being played here bythe quinolin-2-yl moiety. Although there was spontaneous GSH addition to verlukast(4.437), the reaction was clearly enzyme-catalyzed in rat liver and renal cytosol. In ratsin vivo, the biliary excretion of the glutathione conjugate and its breakdown productsaccounted for ca. 25% of a dose. Our last example is quite unusual, namely theglutathione-dependent activation of potential prodrugs of cytotoxic thiopurines. Thus,trans-6-[2-acetylethenyl)sulfanyl]guanine (4.438) features a butenone promoiety astarget for GSH conjugation [308]. The transient conjugate 4.439 spontaneouslyeliminated 6-thioguanine (4.440). GST M1-1 and A4-4 were the most active enzymes

among the 13 human GSTs examined.


Fig. 4.115. The formation and reactivity of quinones has been discussed repeatedly inPart 2, where their glutathione conjugation was alluded to. We now take a closer look atthis reaction, which also proceeds by a mechanism of Michael addition. In biology,quinones play important roles in electron transfer and oxygen activation [309]. Theyare also significant in a toxicological perspective, as the oxidation of some drugsproduces quinones or quinone-imines which may form adducts with biomacromole-cules (toxification). This capacity for adduct formation is correlated with theirreactivity toward glutathione (detoxification) [283a]. We begin here with naturalcatechols such as dihydrocaffeic acid (4.441). Oxidation by cytochrome P450 and/orperoxidases yielded the highly reactive ortho-quinone 4.442 which readily formed thethree GSH conjugates 4.443, 4.444, and 4.445, the former seemingly being thepredominant one [310]. A comparable activation to an ortho-quinone appears to beinvolved in the neurotoxicity of (methylenedioxy)amphetamine and the infamous�ecstasy� [311]. An example of a para-quinone is provided by the antioxidant 2-(tert-butyl)hydroquinone (4.446 ; TBHQ) [312]. At high doses, this compound has beenshown to promote kidney and bladder carcinogenicity in the rat. When administered torats, the compound was metabolized to three biliary glutathione conjugates. These are,in increasing quantitative importance, the two monoconjugates formed from thequinone 4.447, namely 6-glutathionyl-TBHQ (4.448) and 5-glutathionyl-TBHQ, andthe diconjugate 3,6-diglutathionyl-TBHQ (4.450). The latter diconjugate proves thatthe monoconjugate 4.448 is itself a substrate for oxidation – conjugation involving theintermediacy of the glutathionyl-quinone 4.449. This further sequence of oxidation isparticularly significant in a toxicological perspective, since it often follows transport of

the monoconjugate to target tissues [313].


Fig. 4.116. The classical example of a quinoneimine undergoing glutathione conjuga-tion is that of N-acetyl-para-quinoneimine (4.451; see Part 2 [2] [4]), the toxicmetabolite of paracetamol (4.142 ; see Fig. 4.43). The GSH conjugation of this highlyreactive electrophile to form the conjugate 4.452 proceeds both enzymatically andnonenzymatically, and it is in direct competition with covalent binding to proteins[280a]. A more recent finding is that of the toxification of tolcapone (4.453), aninhibitor of catechol O-methyltransferase (COMT) whose therapeutic use is known tobe associated with a risk of liver disorders. A potential pathway of toxification hasemerged with the discovery of the primary amine 4.454 as a reduced metabolite oftolcapone [314]. Indeed, this aromatic amine was readily oxidized by peroxidases,human liver microsomes, or various CYPs to an intermediate that reacted withglutathione to yield the GSH conjugate 4.456. All evidence points to the ortho-quinoneimine 4.455 as the reactive species. A comparable story is seen with theantihelminthic drug thiabendazole, whose use is also associated with a risk of nephro-and hepatotoxicity. In fact, this risk appears related to the formation of the majormetabolite 5-hydroxythiabendazole (4.457) and to subsequent covalent binding tobiomacromolecules. Microsomal incubations of thiabendazole or its 5-OH metabolitein the presence of GSH afforded the glutathione conjugate 4.459 whose formation wasconsistent with the intermediate quinoneimine 4.458 [315]. The structure of thisintermediate is interesting in that the imino moiety is endocyclic, a feature shared by

other reactive intermediates [283a].


Fig. 4.117. Of further biological and toxicological interest is the oxidation of some 4-alkylphenols (or cyclic systems containing a 4-methylidenephenol moiety) to quinonemethides. These metabolites are strong alkylating agents which undergo Michaeladditions, thereby binding covalently to soluble and macromolecular nucleophiles[316]. Like other quinones, these quinonemethides are detoxified by glutathioneaddition, which occurs at the exocyclic methylidene C-atom in 4-alkylphenols, but mayoccur at other activated positions in more complex systems. Eugenol (4.460), a maincomponent of oil of clove and an antibacterial agent, was oxidized by rat liver and lungmicrosomes to the quinonemethide 4.461 which reacted nonenzymatically withglutathione [317]. The GSH conjugate 4.462 resulted from addition at the exocyclicCH C-atom, whereas the conjugate 4.463 had undergone addition at the terminal,conjugated CH2 C-atom. A more complex example is provided by the flavonoidquercetin (4.464), which in the presence of peroxidases is oxidized to a quinonemethideexisting as three resonance forms which all react with glutathione. The highlydelocalized resonance form 4.465 is the one predominating at neutral pH wherequercetin exists as an anion. This resonance form features two quinonemethide motifsas shown, but glutathione addition occurs only at ring A to yield the two conjugates

4.466 and 4.467 [318].


Fig. 4.118. In Fig. 4.33, we saw how the sulfate of N-(9H-fluoren-2-yl)-N-hydroxyacet-amide (4.110) can undergo heterolytic cleavage to generate a highly reactive, adduct-forming nitrenium ion. This story was left unfinished and continues here in the contextof detoxification by glutathione conjugation. As shown, the nitrenium ion (4.468) existsin a number of resonance forms, implying its stabilization by extended delocalization ofits positive charge. Interestingly, these resonance forms can help explain the non-enzymatic production of several glutathione conjugates observed when 4.468 wasreacted with glutathione [319]. The formation of four conjugates (4.469, 4.470, 4.471,and 4.472) can be understood as resulting from a direct nucleophilic attack on theelectron-poor sites N, C(1), C(3), and C(7). The case of the 4-glutathionyl conjugate4.474 is of particular interest, since its formation was shown to be an indirect one, thefirst step being a nucleophilic addition of the HO� anion to C(4a) to yield thequinolimide 4.473. The presence of the 4a-OH group enhances the electrophilicity ofC(4) and explains the regioselective addition of GSH; a final step of dehydration isneeded to restore aromaticity and yield 4.474. An important point to note is that the N-glutathionyl conjugate 4.469 was not isolated as such, but underwent GSH-dependent

reduction as discussed in Sect. 4.7.4.


Fig. 4.119. Haloalkenes are important substrates of glutathione transferases, theirconjugation with glutathione involving a mechanism known as nucleophilic vinylicsubstitution (the SNV reaction) [320– 322]. These are reactions of addition– eliminationwhich we will discuss in Sect. 4.7.3. However, a small sub-group of haloalkenes, i.e., 1,1-difluoroalkenes, tends to react by glutathione addition rather than by addition –elimination. This is exemplified here with tetrafluoroethene (4.475 ; tetrafluoroeth-ylene), 1-chloro-1,2,2-trifluoroethene (4.477), and 1-bromo-1-chloro-2,2-difluoroethene(4.479). Their glutathione conjugates (4.476, 4.478, and 4.480, resp.) result from areaction of addition which is not followed by elimination. An example of somemedicinal interest is provided by 1,1,3,3,3-pentafluoro-2-(fluoromethoxy)prop-1-ene(4.482), a potentially toxic breakdown product of the general anesthetic sevoflurane(4.481) generated by CO2 absorbents in anesthesia machines. This breakdown productis a substrate of GSH conjugation; as a 1,1-difluoroethene derivative, it does yield theaddition conjugate 4.483 [323]. And, in analogy with most haloalkenes, it also yieldsthe product of addition –elimination 4.484. The major toxicological interest of bothglutathione conjugates is their further biotransformation to reactive metabolitesidentified or inferred in patients and in rats. Indeed, the two glutathione conjugates aretransformed to cysteine conjugates and mercapturic acids which, in turn, undergo b-lyase-catalyzed cleavage to the thiols 4.485 and 4.486 (see Fig. 4.107). These thiolsspontaneously rearrange by loss of HF (in case of 4.485) or tautomerism (in case of4.486) to yield the highly reactive thioacyl fluoride 4.487. The latter can bind covalentlyto biomacromolecules, thus accounting for the nephrotoxicity of compound 4.482, or

react with H2O.


Fig. 4.120. A few alkynes (i.e., acetylenic derivatives) are known to undergoglutathione conjugation; these substrates have in common a C�C bond activated byelectron delocalization. A simple example is provided by dichloroethyne (4.487;dichloroacetylene), a product of the alkaline decomposition of trichloroethylene and apotent neurotoxin and nephrotoxin [324]. In rats exposed to low levels ofdichloroethyne vapors, the compound underwent glutathione conjugation as its majormetabolic pathway. The glutathione conjugate 4.488 and the mercapturic acid 4.489were major metabolites in bile and urine, respectively. Other metabolites werebreakdown products of the thiol 4.490. High covalent binding was seen to albumin,hemoglobin, renal DNA, and renal proteins, and was caused by reactive species such asthe thioacyl chloride 4.491. A medicinal example is that of the antifungal agentterbinafine (4.492). When incubated with human or rat liver microsomes, the drugunderwent CYP-catalyzed N-dealkylation to the secondary amine 4.493 and the highlyunsaturated aldehyde 4.494 [325]. The latter was trapped by GSH added to theincubates, yielding the glutathione conjugate 4.495 which retains a second reactiveelectrophilic site. This conjugate has thus been postulated to be excreted in the bile

where it could cause hepatobiliary dysfunction.


Fig. 4.121. Organic isocyanates (R�N¼C¼O) are used extensively to manufacturepaints, pesticides, and polyurethanes. Their reactivity can explain toxic reactions,namely a) massive damage to exposed areas (respiratory tract, eyes, etc.) in the case ofheavy accidental exposure, as exemplified by the effects of the infamous methylisocyanate [326]; and b) hypersensitivity of the respiratory tract, as seen in workersexposed to polluted atmospheres. At the molecular level, the toxicity of isocyanates is aconsequence of their reactivity as electrophiles toward biomacromolecules such asproteins (Reaction a). Their main pathway of detoxification is by hydrolysis (Reactionb), but the amine so liberated may produce hypersensitivity [107]. Isocyanates alsoreact enzymatically and spontaneously with glutathione, the reaction being reversible.As exemplified here with methyl isocyanate (4.496), its glutathione conjugate 4.497 canliberate the parent compound or be processed to cysteine conjugates (e.g., themercapturate 4.498) which also liberates 4.496 [327]. Because glutathione conjugatesare substrates of various transporters, they may reach deep compartments in the bodyand, if the conjugation is reversible, liberate therein a toxic isocyanate [328]. In otherwords, GSH conjugates of isocyanates are transport forms as much as products ofdetoxification. The lower part of the Figure presents other examples. Thus, 2,4-diisocyanatotoluene (4.499) is another chemical widely used in the paint and plasticindustries, and a cause of occupational asthma [329]. Isocyanates may also be formedas metabolites of xenobiotics, as exemplified with N-formylamphetamine (4.500), acontaminant in illicit preparations of amphetamine analogues, and the antitumor agentsulofenur (4.502). The former is oxidized by CYPs to the isocyanate 4.501 [330],whereas the latter undergoes general base-catalyzed cleavage to para-chlorophenyl

isocyanate (4.503) [331].


Fig. 4.122. A number of edible cruciferous vegetables such as broccoli, cabbage, andcauliflower contain organic isothiocyanates known to induce various glutathione S-transferases. The metabolism of these natural products is of interest due to theirchemoprotective properties. One such compound is sulforaphane (4.504), the metab-olism of which was carefully investigated in rats [332]. As shown here, sulforaphane(4.504) underwent both redox and conjugation reactions. A major route was sulfoxidereduction to erucin (4.505), whereas a minor pathway was dehydrogenation to 4.506.Three metabolites were identified in the bile, namely the glutathionyl conjugates oferucin and sulforaphane, i.e., 4.507 and 4.508, respectively, and the conjugate 4.509. Themercapturate metabolites of sulforaphane (4.504) and erucin (4.505) were the majorurinary metabolites. Other natural isothiocyanates known to undergo enzymatic andnonenzymatic glutathione conjugation are allyl isothiocyanate (4.510), benzyl isothio-cyanate (4.511), and phenethyl isothiocyanate (4.512) [333]. Like that of isocyanates,the glutathione conjugation of isothiocyanates is reversible, with the forward reactionbeing markedly faster than the reverse one. The GSTs A1-1, A2-2, M1a-1a, and P1-1

were shown to be catalytically competent.


Fig. 4.123. This Section presents the major reactions of glutathione conjugationinvolving nucleophilic substitution, better designated as nucleophilic addition–elimination. In a first part, we shall examine conjugation at sp3-C-atoms, and we beginwith simple haloalkanes, namely chloroalkanes and bromoalkanes [334]. Theirconjugation with glutathione occurs according to an SN2 mechanism, in other words,a nucleophilic substitution with inversion of configuration. This was nicely shown in anumber of studies, for example, with the model compound 2-bromo-3-phenylpropanoicacid (4.513) [335]. In the presence of rat liver cytosol, glutathione conjugation wassubstrate-enantioselective in that the (R)-enantiomer was a better substrate of GSTsthan the (S)-enantiomer. The reaction was also product-stereoselective in that the (R)-enantiomer was metabolized to the epimeric (S)-4.514 conjugate, whereas the (S)-enantiomer gave the (R)-4.514 conjugate. In a toxicological perspective, it isinformative to examine the conjugation of dichloromethane (4.515), a compoundrepresentative of toxic halomethanes [336] [337]. Nucleophilic substitution of the Cl-atom yields a conjugate, 4.516, reactive enough to form DNA adducts. This metabolitecan undergo nonenzymatic hydrolytic dechlorination to 4.517, itself an unstablemetabolite which loses GSH to yield the toxic formaldehyde (4.518). Another reactionworthy of mention is the GSH-dependent reduction of 4.517 to methanol (4.519), a

type of reaction to be discussed in Sect. 4.7.4 (see also Fig. 4.118).


Fig. 4.124. Haloalkanes containing a 1,2-dihaloethyl motif are another class ofxenobiotics able to undergo GSH/GST-catalyzed toxification [338]. The nematocide1,2-dibromo-3-chloropropane (4.520) offers an eloquent example of alternating steps oftoxification and detoxification characteristic of the metabolism of numerous xeno-biotics. The compound is a persistent environmental pollutant that was used as a soilfumigant. Two major metabolic pathways lead to its toxification. The first pathway is aCYP-catalyzed oxidation to 2-bromoacrolein (4.521), followed by GSH conjugation to4.522, cyclization to the episulfonium species 4.523, hydrolytic ring opening of thethree-membered (thiirane) ring to 4.524, and reduction to the diol conjugate 4.525. Thesecond pathway is the direct, GST-catalyzed formation of the glutathione conjugate4.526 [339]. The latter is unstable by virtue of its vicinal halide-glutathione motif andundergoes spontaneous debromination to form an intermediate episulfonium species,4.527, able to bind covalently to DNA. This fact is indicated in the Figure by the redboxes in which the episulfonium formulae are embedded. This toxic species can also bedetoxified by hydrolytic ring opening to 4.528 and 4.529, or by formation of thediglutathionyl conjugate 4.530. However, two among the three resulting metabolites,i.e., 4.529 and 4.530, retain a vicinal halide-glutathione motif and can, in turn, form anepisulfonium ion, 4.531 and 4.532, respectively. These routes are followed by anotherround of detoxification by hydrolysis or GSH conjugation, yielding metabolites 4.525,

4.533, and 4.534, respectively.


Fig. 4.125. Halide atoms in haloalkanes are electron-withdrawing substituents whichactivate (render electrophilic) these compounds toward GSH conjugation. The samemechanism was presented in Figs. 4.30– 4.32 (Chapt. 4.3), where we saw how reactivearylmethanol sulfates are detoxified by glutathione conjugation. The benzylic C-atomin arylmethanol sulfates is strongly activated by the electron-withdrawing O-sulfatemoiety. Besides haloalkanes and arylmethanol sulfates, a few other types ofsubstituents facilitate GSH conjugation by addition – elimination at sp3-C-atoms,notably sulfoxide and sulfone groups as exemplified here. Diallyl sulfide (4.535), aflavor component of garlic, is considered a chemoprotective agent due to its inhibitionof CYP2E1-toxification of some carcinogenic chemicals. Rats dosed with 4.535excreted in their bile a variety of metabolites resulting from initial sulfoxidation andglutathione conjugation [340]. In vitro, diallyl sulfoxide (4.536) and diallyl sulfone(4.537) reacted spontaneously with glutathione to form the GSH conjugate 4.538. Themoieties eliminated, 4.539 and 4.540, respectively, were not characterized as such, but4.540 formed a glutathionyl disulfide, 4.541, according to a reaction discussed inFig. 4.130. Our next example is a medicinal one, namely pantoprazole (4.542), anirreversible proton pump inhibitor used to treat peptic ulcers and other related diseases[341]. As shown with Reaction a, the benzylic position is activated by the S¼O groupand is a good target for glutathione conjugation, as seen in rats dosed with the drug.Interestingly, the C(2)-atom of benzimidazole was also found to be a site of GSHaddition – elimination according to a mechanism discussed below (see Fig. 4.127). Notethat neither of the two GSH conjugates 4.543 and 4.544 was identified as such but as

further breakdown products.


Fig. 4.126. With the exception of 1,1-difluoroalkenes (see Fig. 4.119), haloalkenes tendto react with GSH/GSTs in a reaction of addition – elimination whose mechanism is anucleophilic vinyl substitution (SNV) [320] [321] [342]. This mechanism involves areaction intermediate where the target C-atom is in an sp3-configuration, in closeanalogy with the reaction of aromatic substitution to be presented in the next Figure.Haloalkenes have a number of industrial uses and are also environmental contami-nants. They undergo CYP-catalyzed oxidation and GST-catalyzed conjugation, theenzymes in the latter case being mainly microsomal glutathione S-transferases [319].Haloalkenes of relevance here include the much studied 1,1,2,2-tetrachloroethyleneand 1,1,2-trichloroethylene [343] [344]. However, we focus on 1,1,2,3,4,4-hexachloro-buta-1,3-diene (4.545), since it appears to be metabolized in vivo exclusively byglutathione conjugation [320]. As shown, the glutathione conjugate of hexachlorobu-tadiene, 4.546, is formed at C(1); it is then degraded to the cysteinyl and mercapturateconjugates, 4.547 and 4.548, respectively. The latter is preferentially excreted renally,while the former is a preferred substrate of b-lyase to yield the thiol metabolite 4.549.This thiol has an intrinsic reactivity which favors its isomerization to the thioacylchloride 4.550, and its loss of HCl to form the thioketene 4.551. Both are highly reactivemetabolites which react with nucleic acids and proteins, thereby accounting for thetoxicity and carcinogenicity of hexachlorobutadiene and other haloalkenes. However,detoxification of the thioacyl chloride 4.550 and the thioketene 4.551 is also well known,particularly by hydrolysis to the thio O-acid 4.552 (and to the corresponding carboxylic

acid).


Fig. 4.127. Addition– elimination reactions of glutathione to substituted aromatic ringsare comparable to those targeting haloalkenes, except among other things for a greaternumber of activating and displaceable groups. The mechanism of the reaction, asexemplified here with 1-chloro-2,4-dinitrobenzene (4.553 ; see also Fig. 4.101) beginswith the formation of a s-complex (also known as a Mesenheimer complex) [275a]. Thebetter the delocalization of the negative charge to electron-withdrawing ortho- andpara-substituents, the easier is the formation of the complex. The overall rate offormation of the GSH conjugate 4.554 will also depend on the relative ease ofelimination of the substituent, the formation of the s-complex being reversible. Anumber of chloro- and fluoroarenes are known to be substrates of the reaction, forexample 1,3,5-trifluoro-2-nitrobenzene (4.555) where any of the three F-atoms can bedisplaced [345]. A number of human GSTs catalyze the reaction, e.g., A1-1, A2-2,M1a-1a, and P1-1. Other substrates of the reaction include aryl sulfoxides (e.g., 4.556),aryl sulfones, and arylsulfonamides [346]. The GSH conjugation of the antihyperten-sive agent moxonidine (4.557) may appear unexpected until one realizes that a s-complex at C(4) has its negative charge delocalized to the three neighboring N-atoms[347]. A similar reasoning applies to chlorpyrifos (4.558), a widely used pesticide towhich many humans and animals are exposed [348]. This xenobiotic is conjugated byGSTs at C(6) (loss of the Cl� anion to yield conjugate 4.559) and C(2) (loss of thediethyl thiophosphate moiety to yield 4.560). In both cases, the geminal N-atom and an

ortho-Cl substituent delocalize the negative charge.


Fig. 4.128. A seemingly special case of glutathione conjugation has been reported forremoxipride (4.561) [349]. This atypical neuroleptic was withdrawn in 1993 due to a fewcases of aplastic anemia observed in patients receiving the drug. Based on in vitrostudies, a potential mechanism of toxification was deduced which implicated a knownhuman metabolite of the drug, namely its hydroquinone derivative 4.562. Whenincubated with stimulated human neutrophil granulocytes, this metabolite underwentperoxidase-catalyzed oxidation to the reactive quinone 4.563, which reacted withglutathione to form the glutathionyl conjugate 4.564. The formation of this metabolitewas demonstrated unambiguously; assuming its formation indeed involved thepostulated pathway, it would imply a reaction of addition – elimination resemblingthe GSH conjugation of both haloalkenes and haloarenes. The monoglutathionylconjugate 4.564 was substrate of a second round of oxidation to 4.565, followed by aGSH Michael addition to the diglutathionyl conjugate 4.566. The latter was oxidizedfurther to a quinone before undergoing cyclization (not shown). In summary, thereactive quinones detected in this study bring convincing evidence for multiple

peroxidase-catalyzed toxifications alternating with glutathione conjugations.


Fig. 4.129. Activated derivatives of carboxylic acids can behave as acylating agentstoward nucleophiles, and they tend to react enzymatically and/or spontaneously withglutathione. Acyl halides among the most reactive among such derivatives, andparticularly acyl chlorides used extensively in organic chemistry. An interestingexample of such a compound has been reported as a metabolite of 1,1-dichloroethene(4.567; 1,1-dichloroethylene) [350]. This chemical is extensively used in the manu-facture of plastics and is known to be a lung and liver toxicant. Its major route oftoxification in human lung and liver microsomes was found to involve CYP2E1oxidation to the epoxide 4.568, followed by GSH addition and HCl elimination togenerate the adduct-forming (S-glutathionyl)acetyl chloride (4.569). The latter reacted(presumably spontaneously) with GSH to form (S-glutathionyl)acetyl glutathione(4.570), and with water to form S-glutathionyl acetate (4.571). Other activated acylderivatives of distinct relevance in xenobiotic metabolism are acyl glucuronides (seeChapt. 4.4) and acyl-coenzyme A conjugates (see Chapt. 4.6), both being able to formglutathione conjugates in a reaction of transacylation (Figs. 4.50 and 4.83). Thus, thelipid-lowering agent clofibric acid (4.572) forms an acyl-CoA conjugate (4.573) and anacyl glucuronide 4.574, both of which are intermediates in the in vivo and in vitro

formation of clofibryl-S-acyl-glutathione 4.575 [351].


Fig. 4.130. Another reaction of GSH conjugation that can formally be considered as anaddition – elimination is the formation of mixed glutathionyl disulfides. One mechanismfor their formation is by reaction of the glutathionyl radical with another thiyl radical,in analogy with the formation of oxidized glutathione (GSSG; see Fig. 4.98). However,this mechanism does not appear to be effective compared to the xenobiotic thiol beingoxidized to a sulfenic acid (R�S�OH) prior to reacting with GSH. A classicalexample is that of the antimineral corticoid (diuretic) drug spironolactone (4.576). Thecompound has a complex metabolic fate, a major pathway of which is hydrolysis of thethioacetate ester group to the thiol metabolite 4.577, followed by S-methylation(Chapt. 4.2) to the thiomethyl derivative 4.578, a significant human metabolite. Inaddition, the thiol 4.577 is oxidized by CYP and/or flavin-containing monooxygenasesto the sulfenic acid 4.579 [352]. This reactive metabolite, when leaving the enzymecatalytic site, was trapped by GSH to form the glutathionyl-spironolactone disulfide

4.580 according to the mechanism shown.


Fig. 4.131. A more unexpected example of a mixed glutathionyl disulfide is that of theantidiabetic troglitazone (4.581) whose use has been associated with cases ofhepatotoxicity. One pathway in its metabolic fate is scission of the thiazolidinedionering via an unstable S-oxide (4.582) to form the intermediate sulfenic acid 4.583 [353].The isocyanate group in the latter is broken down by hydration and loss of CO2,whereas the sulfenic group accounts for the formation of the glutathionyl disulfideconjugate 4.584. The formation of this metabolite was characterized unambiguously inhuman liver microsomes containing GSH. Another GSH conjugate (not shown) wasthe product of addition to the isocyanate group (see Fig. 4.121). Note that this reactionof glutathione with other thiols is not limited to xenobiotics, witness the process of S-glutathionylation [354]. This is a reversible post-translational modification of low-pKa

cysteinyl residues in proteins, with pi-class GST catalyzing the forward reaction. Theprocess plays a role in governing how cells respond to oxidative stress. To broaden theperspective further, we note that cysteinyl groups in accessible proteins may also bindsome xenobiotic thiols, as exemplified by the well-known angiotensin-convertingenzyme inhibitor captopril which, in vivo, forms a disulfide bond with human serum

albumin [355].


Fig. 4.132. Glutathione reacts with a number of metallic cations, forming thiolates(previously known as mercaptides) which are thermodynamically stable but kineticallylabile. In other words, the formation of thiolates is favored by their low energy, but theequilibrium constant is also markedly dependent on the relative concentrations of thecompeting thiol ligands. As such, thiolates can serve as transport forms of metals in theorganism [356]. Metallic cations forming thiolates include arsenic, cadmium, copper,gold, lead, mercury, platinum, and silver. Three such metals will be exemplified here,namely mercury, arsenic, and platinum. The bond between cationic Hg, As, or Pt andtheir counterions is more covalent than ionic (< 30%), explaining why theirconjugation with GSH is a reaction of substitution. Cationic Hg is well-known for itsstrong affinity for thiols, also known as mercaptans. Thus, mercury chloride (4.585)reacts spontaneously with glutathione to form mercurydiglutathione (4.586), whereasmethylmercury chloride (4.587) forms methylmercuryglutathione (4.588) [357]. Com-pound 4.587 also reacts rapidly with Cys-Gly, and the conjugate 4.589 was the mainbiliary metabolite in rats dosed with 4.587. As for arsenic, it is a worldwide naturalcontaminant and an occupational hazard (see Figs. 4.20 and 4.21). Like Hg, it has a highaffinity for endogenous thiol groups, and is for example highly bound to hemoglobinCys13a in rats [358]. Arsenic salts react spontaneously with glutathione in solution, asseen with methylarsonite (4.66) which formed the diglutathionyl complex 4.590[43] [47] [359]. Arsenical drugs used to treat some parasitic infections are alsoconjugated by GSH. Thus, rats dosed with melarsoprol (4.591) and its analog

trimelarsan (4.592) excreted the diglutathionyl conjugate 4.593 in their bile [360].


Fig. 4.133. Platinum complexes have considerable significance in cancer chemotherapy[361]. The kinetics of reaction of PtII complexes with glutathione [362], as well as theirhydration [3] [4] play a significant role in their efficacy and toxicity. While a globalpicture integrating adduct formation with DNA, biotransformation, and the role oftransporters is slow to emerge, it appears, for example, that GS –platinum complexesmay be excreted from cells by efflux pumps. Taking the archetypal drug cisplatin(4.594) as an example, we review here its major glutathione conjugates as formednonenzymatically in solution under physiological conditions of pH and temperature[363]. At GSH/cisplatin molar ratios equal or larger than 2 : 1, the 2 : 1 complex 4.595was formed. At a GSH/cisplatin molar ratio of 1 : 1, the 1 :1 complex 4.596 was formedfirst, to be progressively replaced by the 1 : 2 GS/Pt complex 4.597. These complexesindicate that the strongest Pt ligand in GSH is indeed its thiol group, followed by anamino and an amido group. The strength of the SH ligand is also obvious in complexesbetween cisplatin and N-acetylcysteine incubated in a 1 : 1 molar ratio. Here again, a 1 :1complex 4.598 was initially formed, to be progressively replaced by a 1 :2 complex 4.599having the S-atom bridging two Pt-atoms. The toxicity of the 2 : 1 GS/Pt complex wasdemonstrated in a cell-free system where it inhibited protein synthesis, and its transportacross tumor cell membranes was ATP-dependent. Solutions with a GSH/cisplatinmolar ratio of 1 :1 had a cytotoxicity that increased with increasing concentration of the1 : 1 complex 4.596, but decreased as the concentration of the 1 :2 complex 4.597

increased.


Fig. 4.134. In a number of cases, glutathione conjugates react with a molecule ofglutathione to yield glutathione disulfide (GSSG) and the reduced substrate. Thestructural and electronic conditions for such a reaction are schematized in the upperpart of the Figure using a generic R�Y�X substrate. First, the group X (often a leavinggroup) must be a good electroattractor able to activate the target atom Y toward GSHattack. Second, the target atom Y, once coupled to a glutathionyl moiety, must be ableto activate the S-atom toward attack by the second glutathione molecule. Heterolyticcleavage of the Y�S bond yields a molecule of GSSG and the R�Y� anion. Twoclasses of substrates are exemplified in this Figure, namely a-halo ketones andhydroperoxides. Several 2-chloroacetophenones such as 2,2’-dichloroacetophenone(4.600) were investigated as substrates for their GSH-dependent reduction [364].Glutathione addition and chloride elimination yielded a GS-conjugate (4.601) in whichelectronic delocalization activated the S-atom. The heterolytic cleavage of theCH2�SG bond allowed reduction of the substrate to 2’-chloroacetophenone (4.602).The initial conjugation step was catalyzed by GST O1-1. Our second example is of greatphysiopathological significance, since it implies the reduction of hydroperoxides 4.603.These compounds are reactive products of lipid peroxidation as well as intermediatesin the synthesis of some prostaglandins [152] [365]. Here, GSH attacks the proximal O-atom, but the resulting intermediate 4.604 reacts rapidly with a second glutathionethereby reducing the initial hydroperoxide to an alcohol, 4.605. The first conjugationstep was catalyzed by GSTs A1-1, A2-2, and P1-1, whose presence was confirmed in

human liver mitochondria [366].


Fig. 4.135. Nitrosoarenes, 4.608, occur as industrial chemicals or as metabolites formeda) by CYP-catalyzed oxidation of aromatic amines, 4.606, to aromatic hydroxylamines,4.607, followed by autoxidation or peroxidase-catalyzed oxidation, or b) by reductase-catalyzed reduction of nitroarenes [3] [4]. Nitrosoarenes react readily and nonenzy-matically with glutathione to form semimercaptals 4.609 [367] [368]. These aremetabolic crossroads to three distinct reduction pathways (a, b, and c) whose relativesignificance depends on experimental conditions (GSH concentration and pH) andsubstrate properties. Pathway a is a reductive thiolytic cleavage (Fig. 4.134); its productis an N-arylhydroxylamine 4.607. This pathway is favored at relatively high GSHconcentrations and for nitrosoarenes with electron-withdrawing substituents. Pathway bis favored by lower pH and relatively low GSH concentrations and for nitrosoareneswith electron-donating groups. It involves an intramolecular, proton-catalyzed rear-rangement of the semimercaptal 4.609 to the more stable sulfinanilide 4.610 whichtends to undergo proton-catalyzed hydrolysis to the aromatic amine 4.606 andglutathione sulfinic acid. In effect, Pathway b is a reduction of a nitrosoarene to anarylamine. The same is true for Pathway c, which may occur when excess GSH ispresent. The resulting mercaptal 4.611 then undergoes two GSH-reduction stepsproducing two GSSG molecules. Our last case is that of AsV salts (see also Asmethylation under Sect. 4.2.4). They react enzymatically (GST O1-1) and nonenzy-matically with glutathione as exemplified here by dimethylarsinic acid (4.67)[47] [48] [359]. The reaction, which is one of addition with elimination of a H2Omolecule, yields an intermediate GSH conjugate 4.612 whose reductive thiolytic

cleavage (Fig. 4.134) produces the AsIII species dimethylarsinous acid (4.68).


Fig. 4.136. In this last and short Chapter, we examine two unusual types of metabolicreactions, namely the nonenzymatic coupling of xenobiotic amines with endogenouscarbonyl compounds [369]. The first type of reaction is covered in the present Figureand involves the condensation of xenobiotic hydrazines and hydrazides with anendogenous ketone or aldehyde to form a Schiff base called a hydrazone. A well-knownexample is that of the antituberculosis drug isoniazid (4.284), which, besidesacetylation (see Chapt. 4.5), forms hydrazones with circulating pyruvic acid and 2-oxoglutaric acid to yield the conjugates 4.613 and 4.614, respectively. These areimportant in vivo metabolites of isoniazid, especially in slow acetylators [191]. Asecond relevant example is that of hydralazine (4.282), again a substrate of N-acetylation which forms hydrazones. Three such conjugates are formed in humans,namely the acetone hydrazone 4.615, the pyruvic acid hydrazone 4.616, and the 2-oxoglutaric acid hydrazone 4.617 [190]. Interestingly, the hydrolysis of the latter twohydrazones in biological media (buffer or plasma) was practically negligible, whereasthe former (4.615) did regenerate the parent drug [370]. Hydrolysis was even faster for

the acetaldehyde hydrazone, at best a very minor and occasional metabolite.


Fig. 4.137. Bicarbonate ions react with amines to form carbamic acids, a reversiblereaction whose rate constants and equilibrium constant depend largely on the reactivityof the amine, on the stability of the carbamic acid, and on external conditions such aspH and concentrations [371]. As shown for 2-haloethylamines such as 2-bromoethyl-amine (4.618), the reaction involves a nucleophilic attack by the unprotonated amine,meaning that the amine must be basic enough to have a well localized doublet ofelectrons, yet not too basic for the unprotonated species to be present in sufficientproportion. From the examples in the literature, it appears that a pKa value of ca. 8 isfavorable for a reaction under physiological conditions. The carbamic acid thus formed(4.619 in our example) was unstable and easily hydrolyzed back to the free amine. Theidentification of carbamic acids under physiological conditions is often prevented bythis lack of stability. However, the carbamic acid 4.619 reacted intramolecularly to formthe stable oxazolidin-2-one 4.620, which served as evidence of the intermediacy of thecarbamic acid [372].

A noteworthy number of medicinal amines are known to form carbamic acids invivo, a reaction greatly facilitated by the high level of endogenous bicarbonate inhuman blood (around 20 mm). Several of these carbamic acids would rapidly vanish byhydrolysis and remain unidentified, were it not for their capacity to serve as substratesof UDP-glucuronosyltransferases and yield carbonyloxy-b-d-glucuronides [373]. Weillustrate this pathway with mexiletine (4.621), an orally effective antiarrhythmic agent[374]. Its carbamic acid derivative 4.622 was not detected, in contrast to its carbamoylglucuronide 4.623 which was an important urinary metabolite in humans. Three other

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Received June 20, 2008


The Biochemistry of Drug Metabolism- an introduction

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