Metabolismo de fármacos- Burger

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432 Principles of Drug Metabolism

3.3.2Sulfation Reactions,4523.4Glucuronidation and Glucosidation, 453

3.4.1Introduction, 4533.4.2Glucuronidation Reactions, 454

3.4.3Glucosidation Reactions, 4563.5Acetylation and Acylation, 456

3.5.1Acetylation Reactions, 457

3.5.2Other Acylation Reactions, 458

3.6Conjugation with Coenzyme A and

Subsequent Reactions, 459

3.6.1Conjugation with Coenzyme A, 4593.6.2Formation of Amino Acid Conjugates,

459

3.6.3Formation of Hybrid Lipids and Sterol

Esters, 459

3.6.4Chiral Inversion of ArylpropionicAcids, 460

3.6.5 P-Oxidation and 2-Carbon ChainElongation, 460

3.7 Conjugation andRedox

Reactionsof Glutathione, 461

3.7.1Introduction, 461

3.7.2Reactions of Glutathione, 462

3.8Other Conjugation Reactions,464

4 Biological Factors Influencing Drug Metabolism,

466

4.1 Interindividual Factors, 4664.1.1Animal Species, 466

4.1.2Genetic Factors-Polymorphism inMetabolism,467

4.1.3Genetic Factors-Polymorphism inAbsorption, Distribution, and Excre-

tion, 472

1 INTRODUCTION

Xenobiotic metabolism, which includes drug

metabolism, has become a major pharmaco-

logical science with particular relevance to bi-ology, therapeutics, and toxicology. Drug me-tabolism is also of great importance in

medicinal chemistry because it influences (inqualitative, quantitative, and kinetic terms)the deactivation, activation, detoxification,

and toxification of the vast majority of drugs.As a result, medicinal chemists engaged indrug discovery (lead finding and lead optimi-zation) should be able to integrate metabolicconsiderations into drug design. To do so,however, requires a good knowledge of xeno-biotic metabolism.

This chapter, which is written by medicinalchemists for medicinal chemists, offers knowl-

4.1.4Ethnic Differences,472

4.1.5Gender Differences, 473

4.2 Intraindividual Factors, 473

4.2.1Age, 473

4.2.2Biological Rhythms, 474

4.2.3Disease, 474

4.2.4Enzyme Inhibition, 475

4.2.5Enzyme Induction, 476

5 Drug Metabolism and the Medicinal Chemist,

477

5.1SMRs,478

5.1.1Introduction, 478

5.1.2Chirality and Drug Metabolism, 478

5.1.3Qualitative Relations Between

Metabolism and Lipophilicity, 4795.1.4Quantitative Relations Between

Metabolism and Lipophilicity, 480

5.1.5The Influence of Electronic Factors,

480

5.1.63D-QSMRs and Molecular Modeling,481

5.1.7Global Expert Systems to Predict

Biotransformation, 483

5.2Modulation of Drug Metabolism by

Structural Variations, 483

5.2.1Overview, 483

5.2.2Principles of Prodrug Design, 484

5.2.3Chemical Aspects of Prodrug Design,

486

5.2.4Multistep Prodrugs, 487

5.3The Concept of Toxophoric Groups, 490

6 Concluding Remarks, 492

edge and understanding rather than encyclope-dic information. Readers wanting to go furtherin the study of xenobiotic metabolism may con-sult various classic or recent books 1-10).

1.1 Definitions and Concepts

Drugs are but one category among the manyxenobiotics (Table 13.1) that enter the bodybut have no nutritional or physiological value(11). The study of the disposition-or fate--of

xenobiotics in living systems includes the con-

sideration of their absorption into the organ-ism, how and where they are distributed andstored, the chemical and biochemical transfor-mations they may undergo, and how and bywhat

route s)

they are finally excreted and re-turned to the environment. As for "metabo-lism," this word has acquired two meanings,being synonymous with disposition i.e., the


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introduction

Table 13.1 Major Categories of Xenobiotics(modified from Ref. 11)

rws

Food constituents devoid of physiological rolesFood additives (preservatives, coloring and

flavoring agents, antioxidants, etc.)

Chemicals of leisure, pleasure, and abuse (ethanol,coffee and tobacco constituents, hallucinogens,

doping agents, etc.)

Agrochemicals (fertilizers, insecticides, herbicides,

etc.)

Industrial and technical chemicals (solvents, dyes,

monomers, polymers, etc.)Pollutants of natural origin (radon,

sulfur

dioxide,

hydrocarbons, etc.)Pollutants produced by microbial contamination

e.g.,

aflatoxins)

Pollutants produced by physical or chemicaltransformation of natural compounds polycyclic

aromatic hydrocarbons by burning, Maillardreaction products by heating,

etc.)

sum of the processes affecting the fate ofachem-icalsubstance in the body) and withbiotransfor-mation

as understood in this chapter 12).In pharmacology, one speaks of pharmaco-

dynamic effects to indicate what a drug does tothe body and pharmacokinetic effects to indi-

cate what the body does to a drug; two aspectsof the behavior of xenobiotics that are stronglyinterdependent. Pharmacokinetic effects willobviously have a decisive influence on the in-tensity and duration of pharmacodynamic ef -

fects, whereas metabolism will generate newchemical entities (metabolites) that may havedistinct pharmacodynamic properties of their

own. Conversely, by its own pharmacody-namiceffects,acompound may affect the stateof the organism e.g., hemodynamic changes,enzyme activities) and therefore the organ-ism's capacity to handle xenobiotics. Only a

systemic approach can help one appreciate theglobal nature of this interdependence 13).

1.2 Types of Metabolic Reactions Affecting

Xenobiotics

A f is t discrimination that can be made amongmetabolic readions is based on the nature of the

catalyst. Reactions of xenobiotic metabolism,like other biochemical reactions, are catalyzedby enzymes. However, while the vast majority ofreactions of xenobiotic metabolism are indeed

enzymatic ones, some nonenzymatic reactionsare also well documented. This is because a va-riety of xenobiotics have been found to be labile

enough to read nonenzymatically under biolog-

ical conditions of pH and temperature 14).Butthere is more. In a normal enzymatic reaction,metabolic intermediates exist en route to theproduct s) and do not leave the catalytic site.However, many exceptions to this rule areknown, with the metabolic intermediate leavingthe active site and reacting with water, an en-

dogenous molecule or macromolecule, or a xeno-biotic. Such reactions are also ofanonenzymatic

nature but are better designated as postenzy-matic reactions (14).

The metabolism of drugs and other xenobi-otics is often a biphasic process in which thecompound may first undergo a functionaliza-tion reaction (phase I reaction) of oxidation,reduction, or hydrolysis. This introduces orunveils a functional group such as a hydroxyor amino group suitable for coupling with anendogenous molecule or moiety in a second

metabolic step known as a conjugation reac-tion (phase I1reaction). In a number of cases,phase I metabolites may be excreted beforeconjugation, whereas many xenobiotics can bedirectly conjugated. Furthermore, reactions offunctionalization may follow some reactions ofconjugation, e.g., some conjugates are hydro-lyzed and/or oxidized before their excretion.

Xenobiotic biotransformation thus pro-duces two types of metabolites, namely func-

tionalization products and conjugates. Butwith the growth of knowledge, biochemistsand pharmacologists have progressively cometo recognize the existence of a third class ofmetabolites, namely xenobiotic-macromole-cule adducts, also called macromolecular con-

jugates (15). Such peculiar metabolites areformed when axenobiotic binds covalently to abiological macromolecule, usually followingmetabolic activation i.e., postenzymatically).

Both functionalization products and conju-gates have been found to bind covalently tobiological macromolecules; the reaction is of -ten toxicologically relevant.

1.3 Specificities and Selectivities in

Xenobiotic Metabolism

The words "selectivity"and "specificity" maynot have identical meanings in chemistry and


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Principles of DrugMetabolism

biochemistry. In this chapter, the specificity ofan enzyme is taken to mean an ensemble ofproperties, the description of which makes it

possible to specify the enzyme's behavior. Incontrast, the term selectivity is applied to met-abolic processes, indicating that a given meta-bolic reaction or pathway is able to select somesubstrates or products from a larger set. Inother words, the selectivity of a metabolic re-action is the detectable expression of the spec-ificity of an enzyme. Such definitions may not

be universally accepted, but they have themerit of clarity.

What, then, are the various types of selec-

tivities (or specificities) encountered in xeno-

biotic metabolism? What characterizes an en-zyme from a catalytic viewpoint is first itschemospecificity, i.e., its specificity in terms ofthe type s) of reaction it catalyzes. When twoor more substrates are metabolized at differ-ent rates by a single enzyme under identicalconditions, substrate selectivity is observed.In such a definition, the nature of the prod-

uct (~ )and their isomeric relationship are notconsidered. Substrate selectivity is distinctfrom product selectivity, which is observedwhen two or more metabolites are formed atdifferent rates by a single enzyme from a sin-gle substrate. Thus, substrate-selective reac-tions discriminate between different com-pounds, whereas product-selective reactionsdiscriminate between different groups or posi-

tions in a given compound.

The substrates being metabolized at differ-ent rates may share various types of relation-ships. They may be chemically dissimilar orsimilar e.g., analogs), in which case the termof substrate selectivity is used in a narrowsense. Alternatively, the substrates may beisomers such as positional isomers regioiso-mers) or stereoisomers, resulting in substrateregioselectivity or substrate stereoselectivity.Substrate enantioselectivity is a particular

case of the latter (see Section 5.1.2 .Products formed at different rates in prod-uct-selective reactions may also share varioustypes of relationships. Thus, they may be an-alogs, regioisomers, or stereoisomers, result-ing in product selectivity (narrow sense),product regioselectivity or product stereose-

lectivity e.g., product enantioselectivity).Note that the product selectivity displayed by

two distinct substrates in a given metabolicreaction may be different; in other words, theproduct selectivity may be substrate-selective.

The term substrate-

product selectivity can beused to describe such complex cases, which areconceivable for any type of selectivity but havebeen reported maihly for stereoselectivity.

1.4 Pharmacodynamic Consequences of

Xenobiotic Metabolism

The major function of xenobiotic metabolismcan be seen as the elimination of physiologi-cally useless compounds, some of which may

be harmful as witnessed by the tens of thou-sands of toxins produced by plants. The func-

tion of toxin inactivation justifies the designa-tion of detoxification originally given toreactions of xenobiotic metabolism. However,the possible pharmacological consequences ofbiotransformation are not restricted to detox-ification. In the simple case of a xenobiotichaving a single metabolite, four possibilitiesexist:

1. Both the xenobiotic and its metabolite aredevoid of biological effects (at least in theconcentration or dose range investigated);such a situation has no place in medicinalchemistry.

2. Only the xenobiotic elicits biological ef:fects; a situation which in medicinal chem-istry is typical of, but not unique to, soft

drugs.

3. Both the xenobiotic and its metabolite arebiologically active; the two activities being

comparable or different either qualita-tively or quantitatively.

4. The observed biological activity is causedexclusively by the metabolite; a situationwhich in medicinal chemistry is typical ofprodrugs.

Whenadrug or another xenobiotic is trans-

formed into a toxic metabolite, the reaction isone of toxification (16).Such a metabolite mayact or react in a number of ways to elicit avariety of toxic responses a t different biologi-cal levels (17, 18).However, it is essential tostress that the occurrence of a reaction of toxi-fication i.e., toxicity at the molecular level)doesnot necessarily imply toxicityat the levels


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Introduction

Table 13.2 Metabolism-Related Questions

Answered in lead discovery and optimizationSusceptibility to metabolism?

Expected rate of metabolism?Nature of major metabolites?Enzymeslisozymes involved?Potential for enzyme inhibition?

Answered in thepreclinical an d clinical phases

Nature and relative formation of major and

minor metabolites?Enzymes/isozymesand tissues involved?

Influence of genetic factors? Influence of otherfactors?

Distribution and elimination of metabolites?

Activities and toxicities of metabolites?Activity of drug and metabolites as inducer,

autoinducer, andlor inhibitor?

Potential for and occurrence of drug-drug

interactions?

of organs and organisms. This will be dis-cussed later in this text.

1.5 Setting the Scene

In drug research and development, metabo-lism is of pivotal importance because of theinterconnectedness between pharmacokineticand pharmacodynamic processes (Table 13.2).In vitro metabolic studies are now initiatedvery early during lead optimization to assessthe overall rate of oxidative metabolism, toidentify the metabolites, to obtain primary in-formation on the enzymes involved, and to

postulate metabolic intermediates. Based onthese findings, the metabolites must be syn-thetized and tested for their own pharmaco-logical and toxicological effects. In preclinicaland early clinical studies, many pharmacoki-netic data must be obtained and relevant cri-teria must be satisfied before a drug candidatecan enter large-scaleclinical trials 19,201.Asaresult of these demands, the interest of medici-nal chemists for drug metabolism has grown re-

markably in recent years (Table 13.3) (21).As will become apparent, the approach fol-

lowed in this chapter is an analytical one,meaning that the focus is on metabolic reac-tions, the target groups they affect, and theenzymes by which they are catalyzed. This in-formation provides the foundations of drugmetabolism, but i t must be complemented by asynthetic view to allow a broader understand-

Table 13.3 Aspects of DrugMetabolism ofMajor Interest to Medicinal Chemists (21)

The chemistry and biochemistry of metabolic

reactionsPredictions of drug metabolism based on

quantitative structure-metabolism relationshipsQSMRs),expert systems, and molecular

modeling of enzymatic sitesThe consequences of such reactions on activation

and inactivation, toxification, and detoxification

Prodrug and soft drug design

Changes in physicochemical properties pKa,lipophilicity, etc.) resulting from

biotransformation

The potential for drug-

drug interactions(inhibition andlor induction)

The potential for genetic polymorphism

ing and meaningful predictions. Two steps arerequired to approach these objectives, namely(1) the elaboration of metabolic schemeswhere the competitive and sequential reac-tions (Sections 2 and 3) undergone by a given

drug are ordered, and (2)an assessment of thevarious biological factors (Section 4) that in-fluence such schemes both quantitatively andqualitatively. As an example of a metabolicscheme, Fig. 13.1 presents the biotransforma-tion of propranolol (1) in humans (22). Thereare relatively few studies as comprehensiveand clinically relevant as this one, which re-mains as current today as it was when pub-lished in 1985. Indeed, over 90% of a dose was

accounted for and consisted mainly of prod-

ucts of oxidation and conjugation. The missing10% may represent other minor and presum-ably quite numerous metabolites, e.g., thoseresulting from ring hydroxylation at other po-sitions or from the progressive breakdown ofglutathione conjugates.

A large variety of enzymes and metabolicreactions are presented in Sections 2 and 3.Aswill become clear, some enzymes catalyze only

a single type of reaction e.g., N-acetylation),whereas others use a basic catalytic mecha-nism to attack a variety of moieties and pro-duce different types of metabolites e.g., cyto-chromes P450). As an introduction to theseenzymes and reactions, we present an esti-mate of their relative importance in drug me-tabolism (Table 13.4). In this table, the corre-spondence between the number of substrates


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GLUC GLUC

HOSULF

GLUC SULF

GLUC

GLUC

SULF

Figure 13.1. The metabolism ofpropranolol 1)

in humans, accounting for more than 90% of the

dose.GLUC, glucuronide s);SULF, sulfate s)(22).


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2 FunctionalizationReactions 437

Table 13.4 Estimate of the Relative Contributions of Major Drug-Metabolizing Enzymesa

Overall

Contribution

Number of to DrugEnzymes Substratesb Metabolism

Cytochromes P450 (Section 2.2.1)Dehydrogenases and reductases (Section 2.2.2)

Flavin-containing monooxygenases (Section 2.2.2)

Hydrolases (Section 2.2.3)

Methyltransferases (Section 3.2)

Sulfotransferases (Section 3.3)

Glucuronyltransferases (Section 3.4)

N-Acetyltransferases (Section 3.5)

Acyl-coenzyme A synthetases (Section 3.6)

Glutathione S-transferases (Section 3.7)

Phosphotransferases (Section 3.8)

a

low, ** intermediate, *** high, very high.

bIncludingdrug metabolites.

and the overall contribution to drug metabo-lism does not need to be perfect, because someenzymes show a limited capacity e.g., sulfo-transferases), whereas others make a signifi-

cant contribution to the biotransformation oftheir substrates e.g., hydrolases).

2 FUNCTIONALIZATION REACTIONS

2.1 Introduction

Reactions of functionalization are comprisedof oxidations (electron removal, dehydrogena-tion, and oxygenation), reductions (electron

addition, hydrogenation, and removal of oxy-gen), and hydrationsldehydrations (hydrolysisand addition or removal of water). The reac-tions of oxidation and reduction are catalyzedby a very large variety of oxidoreductases,whereas various hydrolases catalyze hydra-tions.A large majority of enzymes involved inxenobiotic functionalization are briefly re-viewed in Section 2.2 23).Metabolic reactionsand pathways of functionalization constitute

the main body of Section 2.

2.2 Enzymes Catalyzing Functionalization

Reactions

2.2.1 Cytochromes P450 Monooxygenationreactions are of major significance in drug me-tabolism and are mediated by various enzymesthat differ markedly in their structure and

properties. Among these, the most important

as far as xenobiotic metabolism is concernedare the cytochromes P450 EC 1.14.14.1,

1.14.15.1, and 1.14.15.3-1.14.15.6). a very

large group of enzymes belonging to heme-coupled monooxygenases 7,24-28). The cyto-

chrome P450 enzymes CYPs)are encoded by

the CYPgene superfamily and are classified in

families and subfamiliesassummarized in Ta-

ble 13.5. Cytochrome P450 is the major drug-

metabolizing enzyme system, playing a keyrole in detoxification and toxification, and is of

additional significance in medicinal chemistry

because several CYP enzymes are drug tar-gets, e.g., thromboxane synthase CYP5) and

aromatase CYP19). The three CYP familiesmostly involved in xenobiotic metabolism are

CYP1-CYP3, whose relative importance is

shown in Table 13.6.Examples of the many drugs interacting

with cytochromes P450 as substrates, inhibi-tors, or inducers will be considered later (seeTable 13.9 in Section 4), whereas this section

focuses on the metabolic reactions. An under-

standing of the regiospecificity and broad re-activity of cytochrome P450 requiresa presen-tation of its catalytic cycle (Fig. 13.2). Thiscycle involves a number of steps that can besimplified as follows:

1. The enzyme in its ferric (oxidized) form ex-ists in equilibrium between two spin states,


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Table 13.5 The Human CYPGene Superfamily:ATable of the Families and Subfamilies ofGene Products 7,24-28)

Families Subfamilies Representative Gene Products--

P4501Family Aryl hydrocarbonhydroxylases; xenobiotic metabolism;

inducible bypolycyclic aromatichydrocarbons)P450 2 Family (Xenobiotic metabolism;

constitutive and xenobiotic-

inducible)

P450 3 Family (Xenobiotic and steroidmetabolism; steroid -inducible)

P450 4 FamilyPeroxisome

proliferator -inducible)

P450 5 FamilyP450 7 Family (Steroid

7 -hydroxylases)P450 8 Family

P45011Family (Mitochondria1 steroidhydroxylases)

P450 17 Family (Steroid17a-hydroxylase)

P450 19 Family (Steroid aromatase)P450 21 Family (Steroid

21-hydroxylases)P450 24 Family 25-HydroxyvitaminD

24-hydroxylase)P450 26 Family

P450 27 Family (Mitochondria1 steroidhydroxylases)

P450 39 FamilyP450 46 FamilyP450 51 Family

-

P4501A

SubfamilyP450

1B

Subfamily

P450 2A SubfamilyP450 2B Subfamily(Includes

phenobarbital-

inducible forms)P450 2C Subfamily

(Constitutive forms; includessex-specific

forms)P450 2D SubfamilyP450 2E Subfamily

Ethanol-

inducible)P450 2F SubfamilyP450 25 SubfamilyP450 3A Subfamily

P450 A Subfamily

P450 4B SubfamilyP450 4F SubfamilyP450 5A SubfamilyP450 7A SubfamilyP450 7B SubfamilyP450 8A SubfamilyP450 8B SubfamilyP450

11A

SubfamilyP450

11B

Subfamily

(Steroidhydroxylases)

P450 26ASubfamily

P450 27ASubfamilyP450 27B

Subfamily

CYP2F1

cYP2J2

CYP3A4 CYP3A5 CYP3A7 fital CYP

enzyme), CYP3A43CYP4A11 (Fatty acid and ~ 1 ) -

hydroxylases)

CYP4B1

CYP4F2 CYP4F3 CYP4F8 CYP4F12

CYP5A1 TXAsynthase)CYP7A1

CYP7B1

CYP8Al Prostacyclin synthase)CYP8B1

C Y P l l A l (Cholesterol side-chaincleavage)

CYPllBl CYP11B2

CYP39

CYP46

CYP51 Lanosterol 14a-demethylase)

This list reports all human YPs with known substra te s) andlor inhibitor s1. At the time of writing, human CYPs ofunknown function were 2Al,2R1,2 51,2Ul, 2W1,4A20, 4A22 ,4Fl l, 4F22,4V2,4Xl, 20,26B1,26Cl, and 27C1 28).


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Principles of Drug Metabolism

XOH

Hz0

Figure13.2. Catalytic cycle of cytochrome P450 associated with monooxygenase reactions.Fe3+,

ferricytochrome P450; hs, high spin; Is, low spin; Fez+, ferrocytochrome P450; F,,, flavoprotein

1-NADPH-cytochrome P450 reductase; F,,, NADH-cytochrome b, reductase; cyt b,, cytochrome

b,;XH,

substrate (modified from Ref. 6).

of lesser relevance (31). Aldehyde reduc-

tases are widely distributed in nature and oc-

cur in a considerable number of mammalian

tissues. Their subcellular location is primarily

cytosolic, and in some instances is also mito-chondrial. The so-called ketone reductases in-

clude a- and P-hydroxysteroid dehydroge-

nasese.g.,

EC 1.1.1.50 and EC1.1.1.51),

various prostaglandin ketoreductases e.g.,

prostaglandin-F synthase, EC 1.1.1.188; pros-

taglandin-E, 9-reductase, EC 1.1.1.189), and

many others that are comparable with alde-

hyde reductases. One group of particular

importance are the carbonyl reductases

(NADPH; EC 1.1.1.184). Furthermore, themany similarities (including some marked

overlap in substrate specificity) between mo-nomeric, NADPH-dependent aldehyde reduc-

tase AKRl),aldose reductase AKR2),and car-

bony1

reductase A m ) have led to theirdesignation as

aldo-ketoredudases

AKRs)(32).

Other reductases that have a role to playin drug metabolism include glutathione reduc-

tase NADPH:oxidized-glutathioneoxidore-

ductase; EC 1.6.4.2) and quinone reductase

[NAD P)H: quinone acceptor) oxidoreduc-tase; DT-diaphorase; EC 1.6.99.21.

Aldehyde dehydrogenases [ALDHs; alde-hyde:NAD PIf

oxidoreductases; EC 1.2.1.3and EC 1.2.1.51 exist in multiple forms in the

cytosol, mitochondria, and microsomes ofvarious mammalian tissues. It has been pro-

posed that ALDHs form a superfamily of re-

lated enzymes consisting of class 1ALDHs

(cytosolic), class 2 ALDHs (mitochondrial),and class 3 ALDHs (tumor-associated and

other isozymes). In all three major classes,

constitutive and inducible isozymes exist.In a proposed nomenclature system, the hu-

man ALDHs are designated as 1A1, 1A6,1B1,

2,3A1, 3A2, 3B1, 3B2, 4A1, 5A1, 6A1,

7A1,8A1,

and9A1 33-35).

Dihydrodiol dehydrogenases trans-1,2-

dihydrobenzene-1,2-diol:NADP

oxidoreduc-

tase; EC 1.3.1.20) are cytosolic enzymes; sev-

eral of which have been characterized.Al-


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drogenation (not shown). In the latter cases,however, the hydroxylated metabolite is usu-ally unstable and undergoes a rapid, postenzy-

matic elimination (reaction 4-B). Dependingon the substrate, this pathway produces a sec-ondary or primary amine, an alcohol or phe-nol, or a thiol, while the alkyl group is cleavedas an aldehyde or a ketone. Reaction 4 consti-tutes a very common and frequent pathway asfar as drug metabolism is concerned, becauseit underlies some well-known metabolic reac-tions of N-C cleavage discussed later. Notethat the actual mechanism of such reactions is

usually more complex than shown here andmay involve intermediate oxidation of theheteroatom.

Aliphatic carbon atoms bearing one ormore halogen atoms (mainly chlorine or bro-mine) can be similarly metabolized by hy-droxylation and loss of HX to dehalogenatedproducts (reactions 5-A and 5-B; see below).Dehalogenation reactions can also proceed re-ductively or without change in the state of ox-

idation. The latter reactions are dehydrohalo-genations (usually dehydrochlorination ordehydrobromination) occurring nonenzymat-i d l y (reaction6).Reductive dehalogenationsinvolve replacement of a halogen by a hydro-gen (reaction 7) or vie-bisdehalogenation (re-action 8 . Some radical species formed as inter-mediates may have toxicological significance.

Reactions 1-A, 1-B, 3,4-A, and 5-A are cat-alyzed by cytochromes P450. Here, the iron-

bound oxene (Section 2.2.1) acts by a mecha-nism known as "oxygen rebound, whereby aH atom is exchanged for a OH group. In sim-plified terms, the oxene atom attacks the sub-strate by cleavinga C-H bond and removingthe hydrogen atom (hydrogen radical). Thisforms an iron-bound HO. species and leavesthe substrate as a C-centered radical. In thelast step, the iron-bound HO. species is trans-ferred to the substrate.

Halothane (2)offers a telling example of themetabolic fate of halogenated compounds of me-dicinal interest. Indeed, this agent undergoestwo major pathways, oxidative dehalogenationleading to trifluoroacetic acid (3)and reductionproducing a reactive radical (4)(Fig. 13.4).

2.3.2 sp2- and sp arbon Atoms. Reactionsat sp2-carbons are characterized by their own

Figure 13.4. Halothane (2) and two of its metabo-lites, namely trifluoroacetic acid (3)produced by ox-idation and a reactive radical (4)produced by reduc-

tion.

pathways, catalytic mechanisms, and prod-ucts (Fig. 13.5). Thus, the oxidation of aro-matic rings generates a variety of (usually sta-ble) metabolites. Their common precursor isoften a reactive epoxide (reaction 1-A), whichcan either be hydrolyzed by epoxide hydrolase(reaction 1-B) to a dihydrodiol or rearrangedunder proton catalysis to a phenol (reaction1-C).The production ofa phenol is a very com-mon metabolic reaction for drugs containing

one or more aromatic rings. Thepara-positionis the preferred position of hydroxylation forunsubstituted phenyl rings, but the regios-electivity of the reaction becomes more com-plex with substituted phenyl or with other ar-omatic rings.

Dihydrodiols are seldom observed, as arecatechol metabolites produced by their dehy-drogenation catalyzed by dihydrodiol dehy-drogenase (reaction 1-D). It is interesting to

note that this reaction restores the aromatic-ity that had been lost on epoxide formation.The further oxidation of phenols and phenolicmetabolites is also possible, the rate of reac-tion and the nature of products depending onthe ring and on the nature and position of itssubstituents. Catechols are thus formed by re-action 1-E,whereas hydroquinones are some-times also produced (reaction 1-F).

In a few cases, catechols and hydroqui-

nones have been found to undergo further ox-idation to quinones (reactions 1-G and 1-11.Such reactions occur by two single-electronsteps and can be either enzymatic or nonenzy-matic i.e., resulting from autoxidation andyielding as by-product the superoxide anion-radical 0,'-). The intermediate in this reac-tion is a semiquinone. Both quinones andsemiquinones are reactive, particularly to-


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R'

\R

/OH R

c a(2) R'-CH-CH-R

\=

.-IR

IR R

R '

R R

R Pf

R OH

Figure13.5. Major functionalization reactions involving an sp2- or spcarbon in substrate mole-cules. These reactions are oxidations (oxygenationsand dehydrogenations), reductions (hydrogena-

tions), and hydrations, plus some postenzymatic rearrangements.

ward biomolecules, and have been implicatedin many toxification reactions. For example,

the high toxicity of benzene for bone marrowis believed to be a result of the oxidationof catechol and hydroquinone catalyzed by

myeloperoxidase.

The oxidation of diphenols to quinones is

reversible (reactions1 H

and1-J);a variety ofcellular reductants are able to mediate the re-duction of quinones either by a two-electron

mechanism or by two single-electron steps.

The two-electron reduction can be catalyzed

by carbonyl reductase and quinone reductase,

whereas cytochrome P450 and some flavoproteins act by single-electron transfers. The

nonenzymatic reduction of quinones can oc-cur, for example, in the presence of 0, - or

some thiols such as glutathione.Olefinic bonds in xenobiotic molecules can

also be targets of cytochrome P45O catalyzed

epoxidation (reaction 2-A). In contrast to

arene oxides, the resulting epoxides are fairly


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2 unctionalization Reactions

Figure 13.6. Carbamazepine (5)and its 10,ll-ep -oxide metabolite (6).

stable and can be isolated and characterized.But like arene oxides, they are substrates ofepoxide hydrolase yielding dihydrodiols (reac-tion 2-B). This is exemplified by carbamaz-epine 5), whose l0,ll-epoxide (6) is a majorand pharmacologically active metabolite inhumans and is further metabolized to the in-active dihydrodiol 44) (Fig. 13.6).

The reduction of olefinic groups (reaction2-C)isdocumented for a few drugs bearing ana,P-ketoalkene function. The reaction isthought to be catalyzed by various NAD P)Hoxidoreductases.

The few drugs that contain an acetylenicmoiety are also targets for cytochrome P450-catalyzed oxidation. Oxygenation of the triplebond (reaction 3-A) yields an intermediatethat, depending on the substrate, can react ina number of ways, for example, binding co-valently to the enzyme or forming a highlyreactive ketene whose hydration produces asubstituted acetic acid (reactions 3-B and 3-C).

2.4 Reactions of Nitrogen Oxidation

and Reduction

The main metabolic reactions of oxidation andreduction of nitrogen atoms in organic mole-cules are summarized in Fig. 13.7. The func-tional groups involved are amines and amides

and their oxygenated metabolites, as well as1,4-dihydropyridines, hydrazines, and azo

compounds. In many cases, the reactions can

be catalyzed by cytochrome P450 andlor fla-vin-containing monooxygenases. The first ox-

ygenation step in reactions 1-4 and 6 have

frequently been observed.

Nitrogen oxygenation is a (apparently)

straightforward metabolic reaction of tertiary

amines (reaction 1-A), whether they are ali-

phatic or aromatic. Numerous drugs undergo

this reaction and the resulting N-oxide metab-

olite is more polar and hydrophilic than the

parent compound. Identical considerations

apply to pyridines and analogous aromaticazaheterocycles (reaction 2-A). Note that

these reactions are reversible; a number of re-ductases are able to deoxygenate N-oxides

back to the amine i.e., reactions 1-Band 2-B).

Secondary and primary amines also un-

dergo N-oxygenation and the first isolable me-tabolites are hydroxylamines (reactions 3-A

and 4-A, respectively). Again, reversibility isdocumented (reactions 3-B and 4-B). These

compounds can be aliphatic or aromatic

amines, and the same metabolic pathway oc-

curs in secondary and primary amides i.e., R

= acyl), whereas tertiary amides seem to be

resistant to N-oxygenation. The oxidation ofsecondary amines and amides usually stops at

the hydroxylamine hydroxylamide level, but

formation of short-lived nitroxides (not shown)

has been reported.As opposed to secondary amines and

amides, their primary analogs can be oxidized

to nitroso metabolites (reaction 4-C), but fur-

ther oxidation of the latter compounds to nitro

compounds does not seem to occur in vivo In

contrast, aromatic nitro compounds can be re-

duced to primary amines through reactions

4-E, 4-D, and finally 4-B. This is the case for

numerous chemotherapeutic drugs suchas

metronidazole.

Note that primary aliphatic amines having

a hydrogen on the alpha-carbon can displayadditional metabolic reactions, shown as reac-tion 5 in Fig. 13.5. Indeed, N-oxidation mayalso yield imines (reaction 5-A), whose degreeof oxidation is equivalent to that of hydrox-ylamines (45). Imines can be further oxidized


15/25


a c eR-NH2 R-NHOH R-N=O -NO2

b d

a d

R

-

NH

-

NH

-

R'R-N=N-R R-N=N-R

b e

Figure 13.7. Major functionalization reactions involving nitrogen atoms in substrate molecules.The reactions shown here are mainly oxidations (oxygenations and dehydrogenations) and reduc-

tions (deoxygenations and hydrogenations).

to oximes (reaction 5-C), which are in equilib

-

rium with their nitroso tautomer (reactions

5-F and 5-G).1,4-Dihydropyridines,and particularly cal-

cium channel blockers such as nivaldipide (7)(Fig. 13.8 , are effectively oxidized by cyto-

chrome P450. The reaction is one of aromati-

zation (reaction 6 in Fig. 13.7 , yielding thecorresponding pyridine.

Dinitrogen moieties are also targets of oxi-doreductases. Depending on their substitu-

ents, hydrazines are oxidized to azo com-

pounds (reaction 7-A), some of which can be

oxygenated to azoxy compounds (reaction

1-D . Another important pathway of hy-drazines is their reductive cleavage to primaryamines (reaction 7-C). Reactions 7-A and 7-D

are reversible and the corresponding reduc-


16/25

2 unctionalizationReactions

7)

Figure 13.8. Nivaldipine 7).

tions (reactions 7-B and 7

-E) are mediated by

cytochrome P450 and other reductases. A tox-icologically significant pathway thus exists forthe reduction of some aromatic azo com-

pounds to potentially toxic primary aromaticmines (reactions 7-B and 7-C).

2.5 Reactions of Oxidation and Reduction

of Sulfur and Other Atoms

A limited number of drugs contain a sulfur

atom, usually as a thioether. The major redoxreactions occurring at sulfur atoms in organiccompounds are summarized in Fig. 13.9.

Thiol compoundscan be oxidized to sulfenicacids (reaction 1-A), then to sulfinic acids (reac-tion 1-E),and finally to sulfonic acids (reaction13 . Depending on the substrate, the pathwayis mediated by cytochrome P450 andlor flavin-containing monooxygenases. Another route ofoxidation of thiols is todisulfideseither directly

(reaction 1-Cthrough thiyl radicals) or by dehy-

dration between a thiol and a sulfenic acid (re-

action 1-B .However, our understanding of sul-

fur biochemistry is largely incomplete, and

much remains to be learned. This is particularlytrue for reductive reactions. Whereas it is well

known that reaction 1-Cis reversible i.e.,reac-

tion 1-D),the reversibility of reaction 1-A is un-

clear and reduction of sulfinicand sulfonic acids

seems unlikely.

The metabolism of sulfides (thioethers) is

rather straightforward. Besides the S-deal-kylation reactions discussed earlier, thesecompounds can also be oxygenated by mono-

oxygenases to sulfoxides (reaction 2-A) and

then to sulfones (reaction 2-C). Here, it isknown with confidence that reaction 2-A is in-deed reversible, as documented by many ex-amples of reduction of sulfoxides (reaction2-B), whereas the reduction of sulfones hasnever been found to occur.

Thiocarbonyl compounds are also substratesof monooxygenases, forming S-monoxides(sulfines, reaction 3-A) and then S-dioxides

(sulfenes, reaction 3-C).As a rule, these metab-olites cannot be identified as such because oftheir reactivity. Thus, S-monoxides rearrangeto the correspondingcarbonyl by expelling a sul-fur atom (reaction3-D).This reaction is knownasoxidative desulfuration and occurs in thioarn-ides and thioureas e.g., thiopental). As for theS-dioxides, they react very rapidly withnucleo-philes and particularly withnucleophilicsites inbiological macromolecules. This covalent bind-

ing results in the formation of adductsof toxico-

a e

(1)R SH

R-SOH R S02H S03H

\-

R-S-S-R'

S

I I a(3) R-C-R'

R-CO-R'

Figure13.9. Major reactions

of oxidation and reduction in-

volving sulfur atoms in or-ganic compounds.


17/25

Principlesof DrugMetabolism

NHCOCH3 NCOCH3 redox reactions include silicon, phosphorus,arsenic, and selenium (Fig. 13.11). Note how-

Q

-

l $

ever that the enzymology and mechanisms of

these reactions are insufficiently understood.For example,a few silanes have been shown toyield silanols in v vo (reaction 1).The same

OH 0 applies to some phosphines, which can be ox-8)

9) ygenated to phosphine oxides by monooxygen-

ases (reaction 2).Figure 13.10. Paracetamol 8) and its toxic qui-noneimine metabolite 9).

Arsenicals have received some attentionbecause of their therapeutic significance.Both

logical significance. Such a mechanism is be-lieved to account for the carcinogenicity of a

number of thioamides.Other elements besides carbon, nitrogen,

and sulfur can undergo metabolic redox reac-tions. The direct oxidation of oxygen atoms inphenols and alcohols is well documented forsome substrates. Thus, the oxidation of sec-ondary alcohols by some peroxidases can yielda hydroperoxyde and ultimately a ketone.Some phenols are known to be oxidized by cy-tochrome P450 to a semiquinone and ulti-

mately to a quinone. A classical example isthat of the anti-inflammatory drug paraceta-mol (8)(Fig. 13.10; acetaminophen), a minor

inorganic and organic arsenic compounds dis-play an As(II1)-As(V) edox equilibrium in the

body. This is shown with the arsine-arsine ox

-

ide and arsenoxide-arsonicacid equilibria (re-actions 3-A and 3-B and reactions 4-Band 44respectively). Another reaction of interest is

the oxidation of arseno compounds to arsenox-ides (reaction 4-A), a reaction of importance inthe bioactivation of a number of chemothera-peutic arsenicals.

The biochemistry of organoselenium com-pounds is of some interest. For example, a few

selenols have been seen to oxidize to selenenicacids (reaction 5-A) and then to seleninic acids(reaction 5-B).

fraction of which is oxidized b y h F 2 ~ 1to the2.6 Reactions of Oxidative Cleavage

highly reactive and toxic quinoneimine9.Additional elements of limited significance A number of oxidative reactions presented in

in medicinal chemistry that are able to enter the previous sections yield metabolic interme-

Figure 13.11. Some selected a b

reactions of oxidation and reduc- (4) R A s y A s R As=O c R As03H2

C

tion involving silicon, phospho-rus, arsenic, and selenium in xe-

5)

aR-SeH

bnobiotic compounds. SeOH --- R Se02H


18/25

2 unctionalizationReactions

Figure13.12. Fenfluramine lo),norfenfluraminel l) , m-trifluoromethy1)phenylacetone

12),

and

m-trifluoromethylbenzoic acid 13).

diates that readily undergo postenzymaticcleavage of a C-X bond X being an heteroa-tom).As briefly mentioned, reactions 4-A and

4-B in Fig. 13.3 represent important metabolic

pathways that affect many drugs. When X= N

(by far the most frequent case), the metabolicreactions are known as N-demethylations, N-dealkylations, or deaminations, depending on

the moiety being cleaved. Consider for exam-ple fenfluramine (10)(Fig. 13.12),which is N-deethylated to norfenfluramine l l ) ,an active

metabolite, and deaminated to (m-trifluoro-methy1)phenylacetone 121, an inactive me-

tabolite that is further oxidized to m-trifluoro-

methylbenzoic acid 13).When X = or S in reaction 4 (Fig. 13.31,

the metabolic reactions are known as O-deal-kylations or S-dealkylations, respectively. 0-

demethylation is a typical case of the formerreaction. And when X = halogen in reactions

5-A and 5-B (Fig. 13.3),loss of halogen can also

occur and is known as oxidative dehalogena-tion.

The reactions of oxidative C-X cleavage dis-

cussed above result from carbon hydroxyla-

tion and are catalyzed by cytochrome P450.

However, N-oxidation reactions followed by

hydrolytic C-N cleavage can also be catalyzed

by cytochrome P450 e.g., reactions 5-E and5-H in Fig. 13.7). The sequence of reactions

5-A and 5-E in Fig. 13.7 is of particular inter-

est because it is the mechanism by which

monoamine oxidase deaminates endogenous

and exogenous mines .

2.7 Reactions of Hydration and Hydrolysis

Hydrolases catalyze the addition of a molecule

of water to a variety of functional moieties.

Thus, epoxide hydrolase hydrates epoxides toyield trans-dihydrodiols (reaction 1-B in Fig.

13.5). This reaction is documented for many

arene oxides, particularly metabolites of aro-matic compounds, and epoxides of olefins.

Here, a molecule of water has been added tothe substrate without loss of a molecular frag-

ment, therefore the use of the term "hydra-tion" sometimes found in the literature.

Reactions of hydrolytic cleavage (hydroly-sis) are shown in Fig. 13.13. They are frequentfor organic esters (reaction I),inorganic esters

such as nitrates (reaction 2) and sulfates (re-action 3). and amides (reaction 4).These reac-

.,

tions are catalyzed by esterases, peptidases, orother enzymes, but nonenzymatic hydrolysis

is also known to occur for sufficiently labile

compounds under biological conditions of pH

and temperature. Acetylsalicylic acid, glyceroltrinitrate, and lidocaine are three representa-

tive examples of drugs undergoing extensive

cleavage of the organic ester, inorganic ester,or amide group, respectively. The reaction is of

particular significance in the activation of es-ter prodrugs (Section 5.2).

(1) R-COO-R'- R-COOH + R -OH

2) R-ON02 R-OH + HN03

3) R 0S03H

-OH H2S04 Figure 13.13. Major hydrolysis reactions

involving esters (organic and inorganic) and(4)

R-CONHR - R-COOH + R -NH2 -ides,


19/25

3 CONJUGATION REACTIONS

3.1 lntroduction

Principles of Drug Metabolis

As defined in the Introduction, conjugation re-actions (also infelicitously known as phase I1reactions) result in the covalent binding of anendogenous molecule or moiety to the sub-strate. Such reactions are of critical signifi-cance in the metabolism of endogenous com-pounds, as witnessed by the impressivebattery of enzymes that have evolved to cata-lyze them. Conjugation is also of great impor-tance in the biotransformation of xenobiotics,

involving parent compounds or metabolitesthereof (3).

Conjugation reactions are characterized bya number of criteria:

They are catalyzed by enzymes known astransferases.

They involve a cofactor that binds to theenzyme in close proximity to the substrateand carries the endogenous molecule ormoiety to be transferred.

The endogenous molecule or moiety ishighly polar (hydrophilic), and its size iscomparable with that of the substrate.

It is important from a biochemical andpractical viewpoint to note that these criteriaare neither sufficient nor necessary to defineconjugations reactions. They are not suffi-

cient, because in hydrogenation reactions i.e.,typical reactions of functionalization) the hy-dride is also transferred from a cofactorNADPH or NADH). And they are not neces-

sary, because all the above criteria suffer fromsome important exceptions mentioned below.

3.2 Methylation

3.2.1 lntroduction. Reactions of methyl-

ation imply the transfer of a methyl groupfrom the cofactor S-adenosyl-L-methionine(SAM) (14). As shown in Fig. 13.14, themethyl group in SAM is bound to a sulfoniumcenter, givingita marked electrophilic charac-

ter and explaining its reactivity. Furthermore,it becomes pharmacokinetically relevant todistinguish methylated metabolites in whichthe positive charge has been retained or lost.

Figure 13.14. S-adenosyl-L-methionine 14).

Anumber of methyltransferases are able tcmethylate small molecules (46, 47). Thus, re.actions of methylation fulfill only two of thethree criteria defined above, because themethyl group is small compared with the sub-strate. The main enzyme responsible for

0

methylation is catechol 0-methyltransferas(EC 2.1.1.6; COMT), which is mainly cytosolibut also exists in membrane-bound form. Sev-eral enzymes catalyze reactions of xenobioticN-methylation with different substrate speci-ficities, e.g., nicotinamide N-methyltransferase (EC 2.1.1.I), histamine methyltransferase EC 2.1.1.8), phenylethanolaminN-methyltransferase (noradrenalineN-methyltransferase; EC 2.1.1.28), and nonspecific

amine N-methyltransferase (arylamine Nmethyltransferase, tryptamine N-methyltransferase; EC 2.1.1.49) of which someisozymes have been characterized. Reactionsof xenobiotic S-methylation are mediated bythe membrane-bound thiol methyltransferas EC 2.1.1.9) and the cytosolic thiopurinemethyltransferase EC 2.1.1.67) (3).

The above classification of enzymes makesexplicit the three types of functionalities un-

dergoing biomethylation, namely hydroxy(phenolic), amino, and thiol groups.

3.2.2 Methylation Reactions. Figure 13.15summarizes the main methylation reactionsseen in drug metabolism. 0-Methylation is acommon reaction of compounds containing acatechol moiety (reaction I), with a usual re-gioselectivity for the meta position. The sub


20/25

3 ConjugationReactions

,. ..

Figure 13.15. Major methylation reac-tions involving catechols, various amines,

5) R-SH R S CH3 and thiols.

strates can be xenobiotics, particularly drugs,with L-DOPA being a classic example. Morefrequently, however, 0-methylation occursasa late event in the metabolism of aryl groups,

after they have been oxidized to catechols (re-actions 1, Fig. 13.5). This sequence was seenfor example in the metabolism of the anti-in-flammatory drug diclofenac (15)(Fig. 13.161,

which in humans yielded 3'-hydroxy-4'-me-thoxy-diclofenac as a major metabolite with a

very long plasma half -life (48).Three basic types of N-methylation reac-tions have been recognized (reactions 2-4,Fig. 13.15).A number of primary amines e.g.,amphetamine) and secondary amines e.g.,tetrahydroisoquinolines) have been shown tobe in vitro substrates of amineN-methyltrans-ferase, whereas some phenylethanolaminesand analogs are methylated by phenyletha-nolamine N-methyltransferase (reaction 2).

However, such reactions are seldom of signif -icancein vivo presumably because of effective

oxidative N-demethylation. A comparable sit-

uation involves the N-H group in an imida-zole ring (reaction 3), exemplified by hista-

mine (49).A therapeutically relevant exampleis that of theophylline (16)whose N 9)-meth-ylation is masked by N-demethylation in adultbut not newborn humans.

N-

Methylation of pyridine-type nitrogenatoms (reaction 4, Fig. 13.15) seems to be of

greater i nvivo

pharmacological significancethan reactions 2 and 3 for two reasons. First,the resulting metabolites, being quaternaryamines, are more stable than tertiary or sec-ondary amines toward N-demethylation. Andsecond, these metabolites are also more polar

Figure 13.16. Diclofenac 15), theophylline 161,nicotinamide 17),and 6-mercaptopurine 18).


21/25


than the parent compounds, in contrast to theproducts of reactions 2 and 3. Good substratesare nicotinamide 17),pyridine, and a number

of monocyclic and bicyclic derivatives (49).S-Methylation of thiol groups (reaction 5)is documented for such drugs as 6-mercapto-purine 18) and captopril 50). Other sub-strates are metabolites (mainly thiophenols)resulting from the f3 C cleavage of (aromatic)glutathione and cysteine conjugates (see be-low). Once formed, such methylthio metabo-lites can be further processed to sulfoxides andsulfones before excretion i.e., reactions 2-A

and 2-C in Fig. 13.9).

From Fig. 13.15, it is apparent that meth-ylation reactions can be subdivided into twoclasses:

1. Those where the substrate and the producthave the same electrical state; a proton inthe substrate being exchanged for a posi-tively charged methyl group (reactions 1-3and 5).

2. Those where the product has acquired apositive charge, namely becomes a pyri-dine-type quaternary ammonium (reac-tion 4).

3.3 Sulfation

3.3.1 Introduction Sulfation reactions con-sist of a sulfate being transferred from the co-factor 3'-phosphoadenosine 5'-phosphosulfate

19) (PAPS; Fig. 13.17) to the substrate undercatalysis by a sulfotransferase. The three cri-teria of conjugation are met in these reactions.Sulfotransferases, which catalyze a variety ofphysiological reactions, are soluble enzymesthat include aryl sulfotransferase (phenol sul-fotransferase; EC 2.8.2.1), alcohol sulfotrans-ferase hydroxysteroid sulfotransferase; EC2.8.2.2), m i n e sulfotransferase arylaminesulfotransferase; EC 2.8.2.3), estrone sulfo-

transferase EC 2.8.2.4), tyrosine-ester

sulfotransferase (EC 2.8.2.9), steroid sulfo-transferase EC 2.8.2.151, and cortisol sulfo-transferase (glucocorticosteroid sulfotrans-ferase; EC 2.8.2.18). Among these enzymes,the former three are of particular significancein the sulfation of xenobiotics. Recent ad-vances in the molecular biology of theseenzymes has led to the recognition of three

Figure 13.17. 3'-Phosphoadenosine 5'- phospho-

sulfate(19)(PAPS).

human phenol sulfotransferases, the thermo-stable, phenol-preferring SULTlAl andSULTlA2 and the thermolabile, monoamine-preferring SULTlA3 (3, 51, 52).

The sulfate moiety in PAPS is linked to a

phosphate group by an anhydride bridgewhose cleavage is exothermic and supplies en-thalpy to the reaction. The electrophilic OHor NH site in the substrate will react with

the leaving SO,- moiety, forming an ester sul-fate or a sulfamate (Fig. 13.18). Some of these

conjugates are unstable under biological con-ditions and will form electrophilic intermedi-ates of considerable toxicological significance.

3.3.2 Sulfation Reactions. Sulfoconjugationof alcohols (reaction1 in Fig. 13.18) leads tometabolites of different stabilities. Endoge-nous hydroxysteroids i.e., cyclic secondary

alcohols) form relatively stable sulfates,whereas some secondary alcohol metabolitesof allylbenzenes e.g., safrole and estragole)form highly genotoxic carbocations (53). Pri-mary alcohols, e.g., methanol and ethanol, canalso form sulfates whose alkylating capacity iswell known (54). Similarly, polycyclic hy-droxymethylarenes yield reactive sulfates be-lieved to account for their carcinogenicity.

In contrast to alcohols, phenols form stable

sulfate esters (reaction 2). The reaction is usu-

ally of high affinity i.e., rapid), but the limitedavailability of PAPS restricts the amounts ofconjugate being produced. Typical drugs under-going limited sulfation include paracetamol (8)

(Fig. 13.10) and diflunisal 20)(Fig. 13.19).Aromatic hydroxylamines and hydroxyl-

amides are good substrates for some sulfo-transferases and yield unstable sulfate esters


22/25

3 Conjugation Reactions

1

R \

CH-OH\

CH-OS03 ---

R'/CH+

R

R / R

3)

R'\

N-OH\

N - O S 0 3 --

R'\

R R Figure 13.18. Major sulfation re-

(reaction 3 in Fig. 13.18). Indeed, heterolyticN - 0 cleavage produces a highly electrophilicnitrenium ion. This is a mechanism believedto account for part or all of the cytotoxicity ofarylamines and arylamides e.g., phenacetin).In contrast, significantly more stable productsare obtained during N-sulfoconjugation ofamines (reaction 4). Alicyclic amines, and pri-

actions involving primary and sec-ondary alcohols, phenols,

hydroxy-

lamines

and hydroxylamides, and

amines.

mary and secondary alkyl- and aryl-mines,

can all yield sulfamates (55). The significanceof these reactions in humans is still poorly

understood.

An intriguing and very rare reaction of con-

jugation occurs for minoxidil 21)(Fig. 13. g),

an hypotensive agent also producing hair

growth. This drug is an N-oxide, and the ac-

tual active form responsible for the different

therapeutic effects is the N,O-sulfate ester

3.4 Clucuronidation and Clucosidation

Figure 13.19. Diflunisal 201,minoxidil 21),and

itsN,O-sulfate

ester22).

3.4.1 Introduction. Glucuronidation is a

major and very frequent reaction of conjuga-

tion. It involves the transfer to the substrate

of a molecule of glucuronic acid from thecofactor uridine-5'-diphospho-a-D-glucuronic

acid (23) (UDPGA; Fig. 13.20). The enzyme

catalyzing this reaction is known as UDP-glu-

curonyltransferase UDP-glucuronosyltrans-

ferase; EC 2.4.1.17, UDPGT) and consists of a

number of proteins coded by genes of theUGT

superfamily. The human UDPGT known to

metabolize xenobiotics is the product of two

gene families,UGTl

andUGT2

These en-zymes include UGTlAl (bilirubin UDPGTs)

and several UGTlA, aswell as numerous phe-

nobarbital-inducible or constitutively ex-pressed UGT2B 57-61).

In addition to glucuronidation, this sectionbriefly mentions glucosidation, a minor meta-bolic reaction seen for a few drugs. Candidateenzymes catalyzing this reaction could be


23/25


COOH

Figure 13.20. Uridine-5'-diphospho-a-E-glucuronicacid(23)(UDPGA).

phenol pgluoosyltransferase EC 2.4.1.351,

arylarnine glucosyltransferase EC 2.4.1.71), andnicotinate glucosyltransferase EC 2.4.1.196).

3.4.2 GlucuronidationReactions. Glumnicacid exists in UDPGA in the la-configuration,but the products of conjugation are p-gluc-uronides. This is because the mechanism ofthe reaction is a nucleophilic substitution withinversion of configuration. Indeed, and asshown in Fig. 13.21, all functional groups able

to undergo glucuronidation are nucleophiles,a common characteristic they share despitetheir great chemical variety.Asa consequenceof this diversity, the products of gluc-uronidation are classified as 0- N-. 5 -. andC-glucuronides.

0-Glucuronidation is shown in reactions1-5 (Figure 13.21).A frequent metabolic reac-tion of phenolic xenobiotics or metabolites istheir glucuronidation to yield polar metabo-

lites excreted in urine and/or bile. O-Gluc-uronidation is often in competition with O-sulfation (see above), with the latter reactionpredominating at low doses and the former athigh doses. In biochemical terms, glucu-ronidation is a reaction of low affinity and highcapacity, wheras sulfation displays high affin-ity and low capacity. A typical drug undergo-ing extensive glucuronidation is paracetamol(8) (Fig. 13.10). Another major group of sub-

strates are alcohols: primary, secondary, ortertiary (reaction 2, Fig. 13.21). Medicinal ex-amples include chloramphenicol and oxaze-Pam. Another important example is that ofmorphine, which is conjugated on its phenolicand secondary alcohol groups to form the 3-0-glucuronide (a weak opiate antagonist) andthe 6-0-glucuronide (a strong opiate agonist),respectively (62).

An important pathway of O-glucuronida-

tion is the formation of acyl-glucuronides (re-

action 3). Substrates are arylacetic acids e.g.,

diclofenac) (15)(Fig. 13.16) and aliphatic ac-ids e.g., valproic acid). Aromatic acids are sel-

dom substrates; a noteworthy exception is di-

flunisal 20)(Fig. 13.191, which yields both the

acyl and phenolic glucuronides. The signifi-

cance of acyl glucuronides has long been un-

derestimated, perhaps because of analytical

difficulties. Indeed, these metabolites are quite

reactive, rearranging to positional isomers andbinding covalently to plasma and seemingly also

tissue proteins 63).Thus, acyl glucuronide for-

mation cannot be viewed solely as a reaction of

inactivation and detoxification.

A special class of acyl glucuronides are the

carbamoyl glucuronides (reaction 4 in Fig.13.21). A number of primary and secondary

amines have been found to yield this type of

conjugate, whereas, as expected, the interme-diate carbamic acids are not stable enough to

be characterized. Carvedilol (24) (Fig. 13.22)is one drug exemplifying the reaction, in addi-tion to formingan 0-glucuronideon its alcoholgroup anda carbazole-N-linkedglucuronide (seebelow) (64). Much remains to be understoodconcerning the chemical and biochemical reac-tivity of carbamoyl glucuronides.

Hydroxylamines and hydroxylamides may

also form 0-glucuronides (reaction 5, Fig.13.21). Thus, a few drugs and a number ofaromatic amines are known to be N-hydroxyl-ated and then 0-glucuronidated. The gluc-uronidation of N OH groups competes with0-sulfation, but the reactivity ofN-0-glucuro-nides to undergo heterolytic cleavage andform nitrenium ions does not seem to be wellcharacterized.


24/25

R-COOH

R'\N-OH

/

R

H

IR-CO-N-R'

R-SH

R-CSSH

R \ R \

N-COOH N-CO-0-GLU

R

R

GLU

IR-CO-N-R'

GLU

IR-SO2-N-R

R-S-GLU

R-CS-S-GLU

GLUI

(14) R-CO-CH2-CO-R - R-CO-CH-CO-R

COOH

GLU=

Figure 13.21. Major glucuronidation reactions involving phenols, alcohols, carboxylic acids, car-bamic acids, hydroxylamines and hydroxylamides, carboxamides, sulfonamides, various amines, thi-ols, dithiocarboxylic acids, and 1 3-dicarbonyl compounds.


25/25


Figure 13.22.Carvedilol

241,

phenytoin

25),

and sulfadimethoxine 26).

Second in importance to 0-glucuronides

are the N-glucuronides formed by reactions6-11 in Fig.13.21: amides (reactions 6 and 7),

amines of medium basicity (reactions8and 9),

and basic amines (reactions 10 and 11). The

N-glucuronidation of carboxamides (reaction6) is exemplified by carbamazepine (5) (Fig.13.6) and phenytoin (25) (Fig. 13.22). In the

latter case, N-glucuronidation was found to

occur atN

(3). The reaction has special signif -

icance for sulfonamides (reaction 7) and par-ticularly antibacterial sulfanilamides such as

sulfadimethoxine (26) (Fig. 13.22),because it

produces highly water-soluble metabolitesthat show no risk of crystallizing in the

kidneys.

N-Glucuronidation of aromatic amines (re-action8,Fig. 13.21) has been observed in onlya few cases e.g., conjugation of the carbazole

nitrogen in carvedilol) (24). Similarly, thereare a number of observations thatpyridine-

type nitrogens and primary and secondary ba-sic amines can be N-glucuronidated (reactions

9 and 10, respectively). As far as human drugmetabolism is concerned, another reaction of

significance is the N-glucuronidation of li-

pophilic, basic tertiary amines containing one

or two methyl groups (reaction 11 (65, 66).

More and more drugs of this type e.g., anti-

histamines and neuroleptics), are found to un-dergo this reaction to a marked extent in hu-

mans,e.g.,

cyproheptadine27)

in Fig. 13.23).Third in importance are the S-glucuro-

nides formed from aliphatic and aromaticthiols (reaction 12 in Fig. 13.21) and from di-

thiocarboxylic acids (reaction 13) such as di-

ethyldithiocarbamic acid, a metabolite of dis-

ulfiram. As for C-

glucuronidation (reaction14),this reaction has been seen in humans for

l,3-dicarbonyl drugs such as phenylbutazone

and sulfinpyrazone (28) (Fig. 13.23).

3.4.3 Clucosidation Reactions. A few drugshave been observed to be conjugated to glucose

in mammals (67). This is usually a minor path-way in the cases where glucuronidation is pos-sible.An interesting medicinal example is that

of some barbiturates such as phenobarbital,which yield the N-glucoside.

3.5 Acetylation and Acylation

All reactions discussed in this section involvethe transfer of an acyl moiety to an acceptorgroup. In most cases, an acetyl is the acyl moi-ety being transferred, while the acceptor

group may be an amino or hydroxy function.

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