BURGER'S MEDICINAL
`CHEMISTRY
`AND DRUG DISCOVERY
`Fifth Edition
`Volume I: Principles and Practice
`
`'
`
`Edited by
`
`Manfred E. Wolff
`
`lmmunoPharmaceutics , Inc.
`San Diego, California
`
`A WILEY-INTERSCIENCE PUBLICATION
`
`JOHN WILEY & SONS, Inc., New York · Chichester · Brisbane · Toronto · Singapore
`
`Apotex Ex. 1021
`
`Apotex v. Auspex
`IPR2021-01507
`
`

`

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`Notice Concerning Trademark or Patent Rights.
`The listing or discussion in this book of any drug in
`respect to which patent or trademark rights may exist
`shall not be deemed, and is not intended as
`a grant of, or authority to exercise, or an
`infringement of, any right or privilege protected by
`such patent or trademark.
`
`This text is printed on acid-free paper.
`Copyright © 1995 by John Wiley & Sons, Inc.
`
`All rights reserved . Published simultaneously in Canada.
`
`Reproduction or translation of any part of this work beyond
`that permitted by Section 107 or 108 of the 1976 United
`States Copyright Act without the permission of the copyright
`owner is unlawful. Requests for permission or further
`information should be addressed to the Permissions Department,
`John Wiley & Sons, 605 Third Avenue , New York, NY
`10158-0012.
`
`Library of Congress Cataloging in Publication Data:
`Burger, Alfred, 1905-
`[Medicinal chemistry]
`Burger's medicinal chemistry and drug discovery. •· 5th ed .
`edited by Manfred E. Wolff.
`p.
`cm.
`" A Wiley-Interscience publication. "
`Contents: v. 1. Principles and practice
`Includes bibliographical references and index .
`ISBN 0-471-57556-9
`1. Pharmaceutical chemistry.
`I. Wolff, Manfred E .
`III. Title: Medicinal chemistry and drug discovery.
`RS403.B8 1994
`615 '. 19--dc20
`
`II . Title.
`
`94-12687
`
`Printed in the United States of America
`
`10 9 8 7 6 5 4 3 2 1
`
`Apotex Ex. 1021
`
`

`

`CHAPTER SIX
`
`Drug Metabolism
`
`BERNARD TESTA
`
`lnstitut de Chimie Therapeutique, Ecole de
`Pharmacie, Universite de Lausanne
`Lausanne, Switzerland
`
`CONTENTS
`
`1 Introduction , 130
`1.1 Definitions and concepts, 131
`1.2 Types of metabolic reactions affecting
`xenobiotics, 131
`1.3 Specificities and selectivities in xenobiotic
`metabolism , 132
`1.4 Pharmacodynamic consequences of xenobiotic
`metabolism, 133
`1.5 Biological factors affecting drug
`metabolism, 133
`2 Functionalization Reactions , 134
`2.1 Introduction , 134
`2.2 Enzymes catalyzing functionalization
`reactions, 134
`2.2.1 Oxidoreductases, 134
`2.2.2 Hydrolases , 137
`2.3 Oxidation and reduction of carbon atoms, 137
`2.3.1 sp 3-Carbon atoms, 137
`2.3.2 s/- and sp-Carbon atoms, 140
`2.4 Oxidation and reduction of nitrogen
`atoms, 141
`2.5 Oxidation and reduction of sulfur and other
`atoms, 143
`2.6 Oxidative cleavage reactions , 146
`2.7 Hydration and hydrolysis, 147
`3 Conjugation Reactions, 147
`3.1 Introduction , 147
`3.2 Methylation , 148
`3.2.1 Introduction , 148
`3.2.2 Methylation reactions , 148
`3.3 Sulfation, 150
`3.3.1 Introduction , 150
`3.3.2 Sulfation reactions, 150
`3.4 Glucuronidation and glucosidation, 152
`
`129
`
`Burger's Medicinal Chemistry and Drug Discovery,
`Fifth Edition , Volume 1: Principles and Practice,
`Edited by Manfred E. Wolff.
`ISBN 0-471-57556-9 © 1995 John Wiley & Sons, Inc.
`
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`130
`
`Drug Metabolism
`
`3.4.1 Introduction , 152
`3.4.2 Glucuronidation reactions , 152
`3.4.3 Glucosidation reactions, 156
`3.5 Acetylation and acylation, 156
`3.5.1 Acetylation reactions , 156
`3.5.2 Other acylation reactions, 157
`3.6 Conjugation with coenzyme A and subsequent
`reactions , 158
`3.6.1 Conjugation with coenzyme A , 158
`3.6.2 Formation of amino acid
`conjugates, 159
`3.6.3 Formation of hybrid lipids and sterol
`esters , 160
`3.6.4 Chiral inversion of arylpropionic
`acids, 161
`3.6.5 {:l-Oxidation and 2-carbon chain
`elongation, 161
`3.7 Conjugation and redox reactions of
`glutathione, 163
`3.7.1 Introduction , 163
`3. 7 .2 Glutathione reactions, 164
`3.8 Other conjugation reactions, 167
`4 The Significance of Drug Metabolism in Medicinal
`Chemistry, 168
`4.1 Structure-metabolism relationships, 168
`4.1.1 Metabolic schemes, 168
`4.1.2 The influence of configurational
`factors, 170
`4.1.3 Quantitative structure-metabolism
`relationships : the influence of electronic
`factors and lipophilicity , 170
`4.2 Metabolism and drug design, 171
`4.2.1 Modulation of drug metabolism by
`structural variations, 171
`4.2.2 Principles of prodrug design , 172
`4.2.3 Examples of prodrugs and chemical
`delivery systems, 174
`4.3 The concept of toxophoric groups, 177
`5 Concluding Remarks , 178
`
`1 INTRODUCTION
`
`includes
`Xenobiotic metabolism , which
`drug metabolism , has become an important
`pharmacological science with particular re(cid:173)
`levance to biology , therapeutics , and tox(cid:173)
`icology. Drug metabolism also is of great
`importance in medicinal chemistry, because
`it influences in qualitative, quantitative ,
`and kinetic terms the deactivation, activa(cid:173)
`tion, detoxication , and toxication of the
`vast majority of drugs. As a result, medici-
`
`nal chemists engaged in drug discovery
`(lead finding and optimization) must be
`able to integrate metabolic considerations
`into drug design. To do so, however, re(cid:173)
`quires a fair or even good knowledge of
`xenobiotic metabolism.
`This chapter presents knowledge and
`understanding
`rather
`than encyclopedic
`information. Readers wanting to go fur(cid:173)
`ther
`in
`the study of xenobiotic meta(cid:173)
`bolism should consult available references
`(1-3).
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`1 Introduction
`
`1. 1 Definitions and Concepts
`
`Drugs are but one category of the many
`xenobiotics (Table 6.1) that enter the body
`but have no nutritional or physiological
`value. The study of the disposition (or fate)
`of xenobiotics in living systems includes the
`consideration of their absorption into the
`organism , how and where they are distrib(cid:173)
`uted and stored , the chemical and bio(cid:173)
`chemical transformations they may under(cid:173)
`go , and how and by what route(s) they are
`finally excreted and returned to the en(cid:173)
`vironment. The word metabolism has ac(cid:173)
`quired two meanings: it is synonymous with
`(1) disposition (i.e. , the sum of the pro(cid:173)
`cesses affecting the fate of a chemical
`substance in the body) and (2) biotrans(cid:173)
`formation as understood in this chapter (5).
`In pharmacology , one speaks of phar(cid:173)
`macodynamic effects to indicate what a
`drug does to the body and pharmacokinetic
`effects to indicate what the body does to a
`drug ; these two aspects of the behavior of
`xenobiotics are strongly
`interdependent.
`Pharmacokinetic effects will obviously have
`a decisive influence on the intensity and
`duration of pharmacodynamic effects,
`while metabolism will generate new chemi(cid:173)
`cal entities (metabolites) that may have
`distinct pharmacodynamic properties of
`their own . Conversely , by its own phar-
`
`Table 6.1 Major Categories of Xenobiotics"
`
`131
`
`macodynamic effects , a compound may
`the state of the organism (e .g.,
`affect
`hemodynamic changes and enzyme ac(cid:173)
`tivities) and hence its capacity to handle
`xenobiotics. Only a systemic approach can
`help one appreciate the global nature of
`this interdependence (6) .
`
`1.2 Types of Metabolic Reactions Affecting
`Xenobiotics
`
`A first discrimination that can be made
`among metabolic reactions is based on the
`nature of their catalysts. Reactions of xeno(cid:173)
`biotic metabolism , like other biochemical
`reactions , are catalyzed by enzymes. How(cid:173)
`ever, while the vast majority of reactions of
`xenobiotic metabolism are
`indeed en(cid:173)
`zymatic ones, some nonenzymatic reactions
`are also well documented. This is due to
`the fact that a variety of xenobiotics have
`been found to be labile enough to react
`nonenzymatically under biological condi(cid:173)
`tions of pH and temperature (7). But there
`is more. In a normal enzymatic reaction,
`metabolic intermediates exist en route to
`the product(s) and do not leave the cata(cid:173)
`lytic site . However, many exceptions to this
`rule are known: the metabolic intermediate
`leaves the active site and reacts with water,
`an endogenous molecule or macromole-
`
`Drugs
`Food constituents devoid of physiological roles
`Food additives (preservatives, coloring and flavoring agents, antioxidants, etc.)
`Chemicals of leisure, pleasure, and abuse (ethanol , coffee and tobacco constituents, hallucinogens,
`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 chemical transformation of natural compounds (polycyclic
`aromatic hydrocarbons from burning, Maillard reaction products from heating, etc.)
`
`0 Modified from Ref. 4.
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`132
`
`Drug Metabolism
`
`cule, or a xenobiotic. Such reactions are
`also nonenzymatic but are better desig(cid:173)
`nated as postenzymatic reactions (7).
`The metabolism of drugs and other
`xenobiotics is typically a biphasic process in
`which
`the compound first undergoes a
`functionalization
`reaction
`(phase
`I
`re(cid:173)
`action) of oxidation, reduction, or hydrol(cid:173)
`ysis. This introduces or unveils a functional
`group such as a hydroxyl or amino suitable
`for linkage with an endogenous molecule or
`moiety in the second metabolic step known
`as a conjugation reaction (phase II re(cid:173)
`action). In a number of cases, phase I
`metabolites may be excreted before conju(cid:173)
`gation, while many xenobiotics can be
`directly conjugated. And what is more ,
`functionalization
`reactions may
`follow
`some conjugation reactions, e.g., some
`conjugates are hydrolyzed and/or oxidized
`before their excretion.
`Xenobiotic biotransformation thus pro(cid:173)
`duces two types of metabolites: function(cid:173)
`alization products and conjugates. How(cid:173)
`ever, with the growth of knowledge, bio(cid:173)
`chemists and pharmacologists have pro(cid:173)
`gressively come to recognize the existence
`of a third class of metabolites: xenobiotic(cid:173)
`macromolecule adducts, also called macro(cid:173)
`molecular conjugates (8). Such peculiar
`metabolites are formed when a xenobiotic
`binds covalently to a biological macromole(cid:173)
`cule , usually following metabolic activation
`(i.e. , postenzymatically). Both function(cid:173)
`alization products and conjugates have
`been found to bind covalently to biological
`macromolecules, the reaction often being
`toxicologically relevant.
`
`1.3 Specificities and Selectivities in
`Xenobiotic Metabolism
`
`The words selectivity and specificity may
`not have identical meanings in chemistry
`and biochemistry.
`In
`this chapter,
`the
`specificity of an enzyme will be taken to
`mean an ensemble of properties , the de-
`
`scnption of which makes it possible to
`specify the enzyme's behavior. In contrast,
`the term selectivity will be applied to meta(cid:173)
`bolic processes , indicating that a given
`metabolic reaction or pathway is able to
`select some substrates or products from a
`larger set. In other words, the selectivity of
`a metabolic reaction is the detectable ex(cid:173)
`pression of the specificity of an enzyme.
`Such definitions may not be universally
`accepted, but they have the merit of clarity.
`What , then , are the various types of
`selectivities (or specificities) encountered in
`xenobiotic metabolism? What characterizes
`an enzyme from a catalytic viewpoint is first
`its chemospecificity, i.e., its specificity in
`terms of the type(s) of reaction it catalyzes.
`When two or more substrates are metabo(cid:173)
`lized at different rates by a single enzyme
`under identical conditions, substrate selec(cid:173)
`tivity is observed. In such a definition , the
`nature of the product(s) and their isomeric
`relationship are not considered. Substrate
`selectivity is distinct from product selectivi(cid:173)
`ty , which is observed when two or more
`metabolites are formed at different rates by
`a single enzyme from a single substrate.
`Thus substrate-selective reactions discrimi(cid:173)
`nate between different compounds, where(cid:173)
`as product-selective reactions discriminate
`between different groups or positions in a
`given compound.
`The substrates being metabolized at dif(cid:173)
`ferent rates may share various types of
`relationships . Chemically, they may be very
`or slightly different (e.g., analogs),
`in
`which case the term substrate selectivity is
`used in a narrow sense. Alternatively , the
`substrates may be isomers such as position(cid:173)
`al isomers (regioisomers) or stereoisomers,
`resulting in substrate regioselectivity or
`substrate stereoselectivity. Substrate enan(cid:173)
`tioselectivity is a particular case of the
`latter.
`Products formed at different rates in
`product-selective reactions may also share
`various types of relationships. Thus they
`may be analogs, regioisomers , or stereo-
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`I Introduction
`
`133
`
`in product selectivity
`resulting
`isomers,
`(narrow sense), product regioselectivity, or
`product stereoselectivity (e.g., product en(cid:173)
`antioselectivity). Note
`that the product
`selectivity displayed by two distinct sub(cid:173)
`strates in a given metabolic reaction may be
`different ; in other words , the product selec(cid:173)
`tivity may be substrate selective. The term
`substrate-product selectivity can be used to
`describe such complex cases, which are
`conceivable for any type of selectivity but
`have been reported mainly for stereoselec(cid:173)
`tivity.
`
`1.4 Pharmacodynamic Consequences of
`Xenobiotic Metabolism
`
`The major function of xenobiotic metabo(cid:173)
`lism can be seen as the elimination of
`physiologically useless compounds, some of
`which may be harmful; witness the tens of
`thousands of toxins produced by plants.
`The function of toxin inactivation justifies
`the designation of detoxication originally
`given to reactions of xenobiotic metabo(cid:173)
`lism. However , the possible pharmacologi(cid:173)
`cal consequences of biotransformation are
`not restricted to detoxication. In the simple
`case of a xenobiotic having a single metab(cid:173)
`olite, four possibilities exist:
`
`1. Both the xenobiotic and its metabolite
`are devoid of biological effects (at least
`in the concentration or dose range in(cid:173)
`vestigated); such a situation has no
`place in medicinal chemistry.
`2. Only the xenobiotic elicits biological
`effects, a situation that in medicinal
`chemistry is typical of but not unique to
`soft drugs.
`3. Both the xenobiotic and its metabolite
`are biologically active, the two activities
`being comparable or different either
`qualitatively or quantitatively.
`is
`4. The observed biological activity
`caused exclusively by the metabolite, a
`
`situation that in medicinal chemistry is
`typical of prodrugs.
`
`When a drug or another xenobiotic is
`transformed into a toxic metabolite, the
`reaction is one of toxication (9). Such a
`metabolite may act or react in a number of
`ways to elicit a variety of toxic responses at
`different biological levels (10, 11). How(cid:173)
`ever, it is essential to stress that the occur(cid:173)
`rence of a reaction of toxication (i.e .,
`toxicity at the molecular level) does not
`necessarily imply toxicity at the levels of
`organs and organisms, as discussed in sec(cid:173)
`tion 4.3.
`
`1.5 Biological Factors Affecting Drug
`Metabolism
`
`A variety of physiological and pathological
`factors
`influence xenobiotic metabolism
`and may thus affect the wanted and un(cid:173)
`wanted activities of drugs. It is customary
`to distinguish between interindividual and
`intraindividual
`factors,
`depending
`on
`whether they vary between or within given
`organisms or
`individuals,
`respectively
`(Table 6.2).
`The interindividual factors by definition
`remain constant during the life span of an
`organism or individual. Species differences
`in xenobiotic metabolism are important in
`
`Table 6.2 Biological Factors Affecting Xeno(cid:173)
`biotic Metabolism
`
`Interindividual
`Factors
`
`Animal species
`Genetic factors
`Sex
`
`Intraindividual
`Factors
`
`Age
`Biological rhythms
`Pregnancy
`Stress
`Nutritional factors
`Enzyme induction
`Enzyme inhibition
`Diseases
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`134
`
`Drug Metabolism
`
`the extrapolation of animal data to humans
`(12-14). Genetic differences result from
`the fact that some xenobiotic-metabolizing
`enzymes are defective
`in a number of
`individuals (15). The consequences of these
`genetic polymorphisms include an impaired
`metabolism of the drugs that are substrates
`of such enzymes, with a marked risk of
`overdose or therapeutic failure in affected
`individuals. This is
`the realm of phar(cid:173)
`macogenetics. Sex is also a factor of signifi(cid:173)
`cance in a number of cases, being related to
`hormonal influences on enzyme activities.
`Intraindividual factors express physio(cid:173)
`logical changes or pathological states affect(cid:173)
`ing, e.g., the hormonal balance and immu(cid:173)
`nological reactions of individuals. The age
`of a person may markedly influence his or
`her ability to metabolize drugs, especially
`at the extremes of life. The influence of
`biological
`rhythms on pharmacokinetics
`and pharmacodynamics is
`the object of
`chromopharmacology (16). Pregnancy af(cid:173)
`fects drug metabolism due to the profound
`hormonal and physiological changes in the
`woman, but the intrinsic activity of the
`placenta must not be forgotten ( 17). Much
`information is available on the influence of
`disease, but relatively few rationalizations
`and explanatory mechanisms have been
`proposed (18).
`Nutritional factors such as diet, nutri(cid:173)
`ents, and starvation influence xenobiotic
`metabolism. As far as drug therapy and
`toxicology are concerned, factors of even
`greater significance are enzyme induction
`and enzyme inhibition (19-21). Enzyme
`inducers act by increasing the concentration
`and hence activity of some enzymes or
`isozymes, while
`inhibitors decrease
`the
`activity of some enzymes or isozymes by
`reversible inhibition or irreversible inactiv(cid:173)
`ation. Enzyme induction and enzyme inhi(cid:173)
`bition by coadministered drugs are two of
`the major causes of drug-drug interactions.
`Many examples are known of drugs inhib(cid:173)
`iting the metabolism of other drugs and
`thus intensifying and prolonging their ef-
`
`is
`induction
`In contrast, enzyme
`fects.
`frequently accompanied by a decrease in
`efficacy.
`
`2 FUNCTIONALIZATION REACTIONS
`
`2.1 Introduction
`
`Functionalization reactions comprise oxida(cid:173)
`tions (electron removal, dehydrogenation,
`and oxygenation), reductions (addition of
`electrons, hydrogenation, and removal of
`oxygen), and hydrations-dehydrations (hy(cid:173)
`drolysis and addition or removal of water).
`The reactions of oxidations and reductions
`are catalyzed by a large variety of oxido(cid:173)
`reductases, while various hydrolases cata(cid:173)
`lyze hydrations. A large majority of en(cid:173)
`zymes recognized to be involved in xeno(cid:173)
`biotic functionalization are briefly reviewed
`in section 2.2 (22), and metabolic reactions
`and pathways are also addressed in section
`2. Catalytic mechanisms, however , fall out(cid:173)
`side the scope of this chapter and will not
`be discussed.
`
`2.2 Enzymes Catalyzing Functionalization
`Reactions
`
`2.2.1 oxmoREDUCTASES. Alcohol dehy(cid:173)
`drogenases (ADH; alcohol:NAD + oxido(cid:173)
`reductase; EC 1.1.1.1) are zinc enzymes
`found in the cytosol of the mammalian liver
`and in various extrahepatic tissues. Mam(cid:173)
`malian
`liver
`alcohol
`dehydrogenases
`(LADHs) are dimeric enzymes. The human
`enzymes belong to three different classes:
`class I, comprising the various isozymes
`that are homodimers or heterodimers of
`the a, {3, and -y subunits (e.g., the aa,
`/3 1/3 1 , a{32 and {3 1-y
`isozymes); class II,
`comprising the 1T1T enzyme; and class III,
`comprising the xx enzyme (23).
`Enzymes categorized as aldehyde reduc(cid:173)
`tases
`include
`alcohol
`dehydrogenase
`(NADP+) [aldehyde reductase (NADH);
`
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`2 Functionalization Reactions
`
`135
`
`EC
`oxidoreductase;
`alcohol:NADP +
`1.1.1.2], aldehyde reductase [alditol:NAD(cid:173)
`(P +) 1-oxidoreductase ; aldose reductase;
`EC 1.1.1.21], and many others of lesser
`relevance (24). Aldehyde reductases are
`widely distributed in nature and occur in a
`considerable number of mammalian tissues.
`Their subcellular location is primarily cyto(cid:173)
`solic and in some instances also mitochon(cid:173)
`drial. The so-called ketone reductases in(cid:173)
`clude a- and J3-hydroxysteroid dehydrogen(cid:173)
`ases (e.g., EC 1.1.1.50 and EC 1.1.1.51) ,
`various prostaglandin ketoreductases (e.g. ,
`prostaglandin F synthase, EC 1.1.1.188;
`prostaglandin
`E2
`9-reductase ,
`EC
`1.1.1.189), and many others that are com(cid:173)
`parable to aldehyde reductases. One group
`of particular importance is the carbonyl
`(NADPH)
`(EC 1.1.1.184).
`reductases
`Furthermore , the many similarities (includ(cid:173)
`ing some marked overlap
`in substrate
`specificity) between monomeric, NADPH(cid:173)
`dependent aldehyde reductase (AKRl),
`aldose reductase (AKR2) , and carbonyl
`reductase (AKR3) have led to their de(cid:173)
`signation as aldoketo reductases (AKRs)
`(25) .
`Other reductases that play a role in drug
`metabolism include glutathione reductase
`(NADPH :oxidized-glutathione oxidoreduc(cid:173)
`tase; EC 1.6.4.2) and quinone reductase
`(NAD(P)H:(quinone acceptor) oxidore(cid:173)
`ductase; DT-diaphorase; EC 1.6.99.2).
`Aldehyde
`dehydrogenases
`(ALDHs;
`aldehyde:NAD(Pt oxidoreductases ; EC
`1.2.1.3 and EC 1.2.1.5) exist in multiple
`forms in the cytosol , mitochondria , and
`microsomes of various mammalian tissues.
`It has been proposed that aldehyde dehy(cid:173)
`drogenases form a superfamily of related
`enzymes consisting of class 1 ALDHs (cyto(cid:173)
`solic), class 2 ALDHs (mitochondrial), and
`class 3 ALDHs
`(tumor-associated and
`other isozymes). In all three major classes ,
`constitutive and inducible isozymes exist
`(26).
`Dihydrodiol dehydrogenases (trans-1 ,2-
`dihydrobenzene-1,2-diol :N ADP + oxidore-
`
`ductase; EC 1.3.1.20) are cytosolic en(cid:173)
`zymes, several of which have been char(cid:173)
`acterized. Although the isozymes are able
`to use NAD +, the preferred cofactor is
`NADP +. Other oxidoreductases that play a
`major or less important role in drug metab(cid:173)
`olism are hemoglobin; monoamine oxidases
`(EC 1.4.3.4; MAO-A and MAO-B), which
`are essentially mitochondrial enzymes; the
`cytosolic molybdenum hydroxylases (xanth(cid:173)
`ine oxidase, EC 1.1.3.22; xanthine dehy(cid:173)
`drogenase , EC 1.1.1.204; and aldehyde
`oxidase , EC 1.2.3.1); and the broad group
`of copper-containing amine oxidases (EC
`1.4.3.6) (27-30).
`Monooxygenation reactions are of major
`significance in drug metabolism and are
`mediated by various enzymes that differ
`markedly in their structure and properties.
`Among these , the most important as far as
`xenobiotic metabolism is concerned are the
`cytochromes P450 (EC 1.14.14.1, also EC
`1.14.15.1, 1.14.15.3 , 1.14.15.4, 1.14.15.5 ,
`1.14.15 .6) , a
`large group of enzymes
`belonging to heme-coupled monooxygen(cid:173)
`ases (31, 32) . The cytochrome P450 (CYP)
`enzymes are encoded by the CYP gene
`superfamily and are classified in families
`and subfamilies (Table 6.3). The cyto(cid:173)
`chrome P450 is perhaps the major drug(cid:173)
`metabolizing enzyme system, playing a key
`role in detoxication and toxication , and is
`of additional significance
`in medicinal
`chemistry because several CYP enzymes
`are drug targets, e.g. , TX synthase (CYP5)
`and aromatase (CYP19). Other monox(cid:173)
`ygenases of importance are the flavin-con(cid:173)
`taining monooxygenases ( dimethylaniline
`monooxygenase
`(N-oxide-forming); EC
`1.14.13.8) and dopamine J3-hydroxylase
`(dopamine
`J3-monooxygenase;
`EC
`1.14.17.1).
`Various peroxidases are progressively
`being recognized as important enzymes in
`drug metabolism. Several cytochrome P450
`enzymes have been shown to have per(cid:173)
`oxidase activity (33) . Prostaglandin-endo(cid:173)
`peroxide synthase (EC 1.14.99.1) is able to
`
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`

`

`136
`
`Drug Metabolism
`
`Table 6.3 The Human CYP Gene Superfamilya
`
`Families (P450)
`
`Subfamilies (P450)
`
`Representative Gene Products
`(CYP)
`
`1 (mammalian aryl
`hydrocarbon hydroxylases;
`xenobiotic metabolism
`inducible by polycyclic
`aromatic hydrocarbons)
`2 (mammalian ; xenobiotic and
`steroid metabolism;
`constitutive and xenobiotic(cid:173)
`inducible)
`
`lA
`
`2A
`
`2B (includes phenobarbital(cid:173)
`inducible forms)
`2C (constitutive forms ;
`includes sex-specific forms)
`2D
`2E (ethanol inducible)
`2F
`3A
`
`4A
`
`4B
`4F
`
`llA (cholesterol side-chain
`cleavage)
`llB (steroid 11/3-
`hydroxylases)
`
`21A
`
`3 (mammalian; xenobiotic and
`steroid metabolism; steroid(cid:173)
`inducible)
`4 (mammalian fatty acid w(cid:173)
`and (w-1)-hydroxylases;
`peroxisome proliferator
`inducible)
`
`5 (TXA synthase)
`7 (mammalian cholesterol 7a(cid:173)
`hydroxylase
`11 (mammalian mitochondrial
`steroid hydroxylases)
`
`17 (mammalian steroid 17a(cid:173)
`hydroxylase)
`19 (mammalian steroid
`aromatase)
`21 (mammalian steroid 21-
`hydroxylases)
`27 (mammalian steroid
`hydroxylase; mitochondrial)
`
`"From Ref. 32.
`
`lAl , 1A2
`
`2A6, 2A7
`
`2B6
`
`2C8, 2C9, 2C10, 2C18 , 2C19
`
`2D6
`2El
`2Fl
`3A3 , 3A4, 3A5
`
`4A9, 4All
`
`4B1
`4F2
`5
`7
`
`llAl
`
`llBl , 11B2
`
`17
`
`19
`
`21A2
`
`27
`
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`

`2 Functionalization Reactions
`
`137
`
`use a number of xenobiotics as cofactors in
`a reaction of cooxidation (34) . And finally,
`a variety of other peroxidases may oxidize
`drugs, e.g. , catalase (EC 1.11.1.6) and
`myeloperoxidase
`( donor: hydrogen-perox(cid:173)
`ide oxidoreductase; EC 1.11.1.7) (35).
`
`2.2 .2 HYDROLASES. Hydrolases constitute
`a complex ensemble of enzymes, many of
`which are known or suspected to be in(cid:173)
`volved in xenobiotic metabolism. Relevant
`enzymes among the serine hydrolases in(cid:173)
`clude carboxylesterases
`(carboxylic-ester
`hydrolase; EC 3.1.1.1), arylesterases (aryl(cid:173)
`ester hydrolase; EC 3.1.1.2) , cholinesterase
`(acylcholine acylhydrolase; EC 3.1.1.8) ,
`and a number of serine endopeptidases
`(EC 3.4.21). The roles of arylsulfatases
`(EC 3.1.6.1) , aryldialkylphosphatases (EC
`3.1.8.1) , ,B-glucuronidases (EC 3.2.1.31) ,
`and epoxide hydrolases (EC 3.3.2.3) are
`worth noting. Some cysteine endopeptid(cid:173)
`ases (EC 3.4.22) , aspartic endopeptidases
`(EC 3.4.23) , and metalloendopeptidases
`(EC 3.4.24) are also of potential interest.
`
`2.3 Oxidation and Reduction of Carbon
`Atoms
`
`When examining reactions of carbon oxida(cid:173)
`tion (oxygenations and dehydrogenations)
`and carbon reduction (hydrogenations) , it
`is convenient from a mechanistic viewpoint
`- , sp 2
`to distinguish between sp 3
`- and sp(cid:173)
`carbon atoms.
`
`2.3.1 sp 3
`- CARBON ATOMS. Reactions of
`reduction of sp 3 -carbon
`oxidation and
`atoms are schematized in Figure 6.1 and
`will be discussed sequentially below. In the
`simplest cases, a nonactivated carbon atom
`in an alkyl group undergoes cytochrome
`P450-catalyzed hydroxylation , the terminal
`and penultimate positions being the pre(cid:173)
`ferred but not exclusive sites of attack
`(reactions 1-A and 1-B , respectively) . De(cid:173)
`hydrogenation by dehydrogenases can then
`
`yield a carbonyl derivative (reactions 1-C
`and 1-E) , which is either an aldehyde or a
`ketone . Note that reactions 1-C and 1-E act
`not only on metabolites but also on xeno(cid:173)
`biotic alcohols and are reversible (i .e. ,
`reactions 1-D and 1-F), because dehydro(cid:173)
`genases catalyze the reactions in both direc(cid:173)
`tions. And while a ketone is seldom oxid(cid:173)
`ized further , aldehydes are good substrates
`of aldehyde dehydrogenases or other en(cid:173)
`zymes and lead irreversibly to carboxylic
`acid metabolites (reaction 1-G). A classical
`example is that of ethanol, which in the
`in
`redox equilibrium with
`body exists
`acetaldehyde; this metabolite is rapidly and
`irreversibly oxidized to acetic acid.
`Recent evidence indicates that for a
`number of substrates, the oxidation of
`primary and secondary alcohols and of
`aldehydes can also be catalyzed by cyto(cid:173)
`chrome P450. A typical example is the
`C(lO)-demethylation of androgens and ana(cid:173)
`logues catalyzed by aromatase (CYP19). A
`special case of carbon oxidation, recog(cid:173)
`nized only recently and of underestimated
`significance ,
`is desaturation of a di(cid:173)
`methylene unit by cytochrome P450
`to
`produce an olefinic group (reaction 2). An
`interesting example is provided by testos(cid:173)
`terone , which among many cytochrome
`P450-catalyzed reactions undergoes allylic
`hydroxylation
`to 6,B-hydroxytestosterone
`and desaturation
`to 6,7-dehydrotestos(cid:173)
`terone (36) (Fig. 6.2).
`is a known regioselectivity in
`There
`cytochrome P450-catalyzed hydroxylations
`for carbon atoms adjacent (a) to an unsatu(cid:173)
`rated system (reaction 3) or an heteroatom
`such as N, 0, or S (reaction 4-A) (see Fig.
`6.1). In the former cases , hydroxylation
`can easily be followed by dehydrogenation
`(not shown). In the latter cases , however,
`the hydroxylated metabolite is usually un(cid:173)
`stable and undergoes a
`rapid , post(cid:173)
`enzymatic cleavage of a hydrolytic nature
`(reaction 4-B) . Depending on the sub(cid:173)
`strate, this pathway produces a secondary
`or primary amine, an alcohol or phenol, or
`
`Apotex Ex. 1021
`
`

`

`138
`
`R"
`I
`R'-?-CH2-CH3
`
`R
`
`R"
`I
`R' -C-CHr CH20H
`I
`R
`
`A
`
`B
`
`Drug Metabolism
`
`____Q___
`
`R"
`I
`R' -C-CH2-COOH
`I
`R
`
`(I)
`
`'
`
`H
`
`R' -t- CH2- C,,0
`
`___£___
`~
`
`R"
`
`I
`R
`
`R"
`I
`R' -C-CHOH-CH3
`I
`R
`
`____L__
`-i,-
`
`R" 0
`I
`II
`R' -?-C-CH3
`
`R
`
`R-CHi-CH2-R'
`
`R-Ol=CH-R'
`
`Y-CH2-CH3
`
`Y-CHOH-CH3
`
`y =aryl.
`
`'
`R"
`R'
`' .
`,
`c=c
`
`/
`R
`
`R-C:::C-
`
`R-X-CH2-R' ~ [R-X-CHOH-R'] _.!L._
`
`R-XH
`
`R'
`
`' Ol-X
`
`R/
`
`X = NR", 0 , S
`
`[ R~:<:H l
`
`X = halogen
`
`A
`
`B
`
`R'
`'c=o
`R/
`
`H X
`I
`I
`R'-C-C-R"
`I
`I
`R R"'
`
`R"
`R'
`'c=c'
`'
`/
`R
`R"'
`
`X = Cl , Br
`
`+ R·-cto
`H
`
`R"
`I
`R'-C-X
`I
`R
`
`X =Cl , Br
`
`R"
`I
`R' -C-H
`I
`R
`
`(7)
`
`X X
`I
`I
`R'-C-C-R"
`I
`I
`R R"'
`
`X = Cl , Br
`
`R"
`R'
`'c=c'
`'
`/
`R
`R"'
`
`(2)
`
`(3)
`
`(4)
`
`(5)
`
`(6)
`
`(8)
`
`Fig. 6.1 Major reactions of functionalization involving an sp 3-carbon in substrate molecules. The reactions shown
`here are mainly oxidations (oxygenations and dehydrogenations) and reductions (hydrogenations) plus some
`postenzymatic reactions of hydrolytic cleavage .
`
`Apotex Ex. 1021
`
`

`

`2 Functionalization Reactions
`
`139
`
`OH
`
`0
`
`(1)
`
`OH
`
`OH
`
`OH
`
`(2)
`
`(3)
`
`0
`
`0
`
`Fig. 6.2 Testosterone (1), 6,8-hydroxytestosterone (2) , and 6,7 dehydrotestosterone (3).
`
`a thiol, while the alkyl group is cleaved as
`an aldehyde or a ketone. Reactions 4
`constitute a frequent pathway as far as drug
`metabolism is concerned, because the path(cid:173)
`way underlies some well-known metabolic
`reactions of N-C cleavage (discussed in
`section 2.6). Note that the actual mecha(cid:173)
`nism of such reactions is usually more
`complex than shown here and may involve
`intermediate oxidation of the heteroatom.
`Aliphatic carbon atoms bearing one or
`more halogen atoms (mainly chlorine or
`bromine) can be similarly metabolized by
`hydroxylation and loss of HX to dehaloge(cid:173)
`nated products (reaction 5-A and 5-B) (see
`Fig. 6.1 and Section 2.6). Dehalogenation
`reactions can also proceed reductively or
`without change in the state of oxidation.
`The latter reactions are dehydrohalogena(cid:173)
`tions (usually dehydrochlorination or dehy(cid:173)
`drobromination) occurring nonenzymatical(cid:173)
`ly (reaction 6). Reductive dehalogenations
`involve replacement of a halogen by a
`hydrogen (reaction 7), or vic-bisdehaloge-
`
`nation (reaction 8). Some radical species
`formed as intermediates may have toxi(cid:173)
`cological significance.
`Halothan offers a telling example of the
`metabolic fate of halogenated compounds
`of medicinal interest. Indeed, this agent
`undergoes two major pathways: oxidative
`dehalogenation, leading to trifluoroacetic
`acid and reduction, producing a reactive
`radical (Fig. 6.3).
`
`CF3-CHCIBr
`(4)
`
`[ CF3-CHCI I
`
`(6)
`
`Fig. 6.3 Halo than ( 4) , trifluoroacetic acid ( 5) , and a
`reactive radical ( 6).
`
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`
`

`

`140
`
`Drug Metabolism
`
`quin