Linking Aromatic Hydroxy Metabolic Functionalization of Drug Molecules to Structure and Pharmacologic Activity

molecules

Review Linking Aromatic Hydroxy Metabolic Functionalization of Drug Molecules to Structure and Pharmacologic Activity

Babiker M. El-Haj 1,* ID , Samrein B. M. Ahmed 2, Mousa A. Garawi 1 and Heyam S. Ali 3

1 Department of Pharmaceutical Sciences, College of Pharmacy, Ajman University, Ajman, UAE; [email protected] 2 College of Medicine, Sharjah Institute for Medical Research, University of Sharjah, Sharjah, UAE; [email protected] 3 Department of Pharmaceutics, Dubai Pharmacy College, Dubai, UAE; [email protected] * Correspondence: [email protected]; Tel.: +9715-6-720-4338

Received: 7 July 2018; Accepted: 13 August 2018; Published: 23 August 2018

Abstract: Drug functionalization through the formation of hydrophilic groups is the norm in the phase I metabolism of drugs for the modiﬁcation of drug action. The reactions involved are mainly oxidative, catalyzed mostly by cytochrome P450 (CYP) isoenzymes. The benzene ring, whether phenyl or fused with other rings, is the most common hydrophobic pharmacophoric moiety in drug molecules. On the other hand, the alkoxy group (mainly methoxy) bonded to the benzene ring assumes an important and sometimes essential pharmacophoric status in some drug classes. Upon metabolic oxidation, both moieties, i.e., the benzene ring and the alkoxy group, produce hydroxy groups; the products are arenolic in nature. Through a pharmacokinetic effect, the hydroxy group enhances the water solubility and elimination of the metabolite with the consequent termination of drug action. However, through hydrogen bonding, the hydroxy group may modify the pharmacodynamics of the interaction of the metabolite with the site of parent drug action (i.e., the receptor). Accordingly, the expected pharmacologic outcome will be enhancement, retention, attenuation, or loss of activity of the metabolite relative to the parent drug. All the above issues are presented and discussed in this review using selected members of different classes of drugs with inferences regarding mechanisms, drug design, and drug development.

Keywords: aromatic hydroxy metabolites; arenolic drug metabolites; metabolic O-dealkylation; metabolic aromatic-ring hydroxylation; primary and auxiliary pharmacophores; auxophores; metabolic modiﬁcation of drug activity

1. Introduction Phase I metabolism of drugs (also known as the non-synthetic or functionalization phase) is mainly brought about by microsomal enzymes adding hydrophilicity to hydrophobic moieties in drug molecules. In most cases, this is attained metabolically by revealing or introducing the hydrophilic hydrogen-bonding hydroxyl group. Generally, metabolic interference may take place at pharmacophoric and/or auxophoric groups. Accordingly, the location in a drug molecule at which the metabolic change takes place may determine the pharmacologic outcome of the metabolite. In any case, the resulting drug metabolite may be pharmacologically inactive, less active, equiactive, or even more active with respect to the parent drug. In other cases, an inactive parent drug is metabolically converted to the active form, in which case the inactive parent drug is known as a “prodrug”. Cases where the metabolic changes are intermediary in drug activation are also known.

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For some drug metabolites, the pharmacologic outcomes—pharmacodynamic, pharmacokinetic, and toxicologic—prove to be substantially more favorable compared to their parent drugs. In such cases, the metabolites were developed into drugs of their own rights. In this review, such drug metabolites are referred to as “metabolite drugs”. Generally, in drug development, the pharmacophoric or auxophoric roles of functional groups in lead drug compounds or drug prototypes are modiﬁed in the synthetic laboratory in some way. The introduction of a group in the framework, the reduction of a group to framework status, or the replacement of a group by another are the main strategies followed. Through biotransformation, the body may play an analogous in vivo role by changing pharmacophoric and/or auxophoric groups into others, or by introducing groups at certain positions of the drug molecule with concomitant changes in pharmacodynamic and/or pharmacokinetic properties of the metabolite relative to the parent drug. Such metabolic changes of a drug molecule may lead to a variety of pharmacologic outcomes. The dependence of the metabolic change on the structure of the drug molecule and the pharmacologic outcomes of the resulting metabolites are the subjects of this review. In its interactions with drugs, the body discriminates between different chemical structures, as well as between stereoisomers of the same drug. In stereoisomers of the same drug, the atoms and groups have the same connectivity but differ in spatial arrangement, i.e., three-dimensional (3D) structures. Drug molecules that contain chiral centers exist as stereoisomers known as enantiomers. Enantiomers are designated in two ways: (a) as R or S, depending on the atomic numbers of the atoms bonded to the chiral center, or (b) as dextro (+) or levo (−) depending on the direction in which they rotate the plane of polarized light. Enantiomers of a chiral drug have identical chemical and physical properties; however, they differ in their pharmacodynamic and/or pharmacokinetic properties when they interact with the chiral environment of the body [1]. Where applicable, the pharmacodynamic differences of enantiomers are highlighted for the chiral drugs and their metabolites, which are presented as examples in this review as classes of drugs or as individual drugs.

2. Objective of the Review The objective of this review is to investigate the relationship between the metabolic change and the modiﬁcation of pharmacodynamic and/or pharmacokinetic properties of drugs in an endeavor to explain when the pharmacologic activity of the metabolite is retained, decreased, lost, or even enhanced with respect to the parent drug. The link between the metabolic functionalization and pharmacologic activity to the structure of the drug is also of prime interest. Such information, as described above, will be useful in drug design when contemplating new drug entities. Additionally, it should be useful in drug development when considering what chemical changes are required in a lead drug compound or prototype for better drug efﬁcacy and metabolic stability.

3. Methodology The inclusion criteria of the selection of drugs to be reviewed include the following:

(a) Drugs that are metabolized by O-dealkylation or aromatic ring hydroxylation; (b) Availability of data regarding the pharmacologic activity of the major metabolite(s).

The sources of information include the following:

• The published literature; • Drug manufacturers’ data sheets; • Reference books on drug metabolism and activity of metabolites.

The selection of the case-study drugs was based on varying the pharmacologic and chemical classes, as described in each section. Molecules 2018, 23, 2119 3 of 30 Molecules 2018, 23, x FOR PEER REVIEW 3 of 29

4. Review Strategy Although the selected drugs are mostlymostly metabolizedmetabolized by multimulti routes,routes, onlyonly metabolicmetabolic oxidativeoxidative reactions that that result result in in one one chemical chemical functionality, functionality, the the aromatic aromatic hydroxy hydroxy group, group, were were considered. considered. The Therepresentative representative drugs drugs were were selected selected based based on both on bothchemical chemical structure structure and pharmacologic and pharmacologic action. action. Each Eachstudy/metabolite(s) study/metabolite(s) case is case presented is presented in a in figure a ﬁgure including including the the following following information information subject subject to availability inin thethe literature:literature: • • TheThe chemicalchemical structurestructure ofof thethe drugdrug andand metabolite(s),metabolite(s), andand thethe enzymesenzymes oror isoenzymesisoenzymes involved;involved; • • TheThe statusstatus ofof thethe pharmacologicpharmacologic activityactivity ofof thethe metabolite;metabolite; • • TheThe percentagepercentage concentrationconcentration ofof thethe metabolite(s)metabolite(s) withwith respectrespect toto thethe parentparent drug.drug. Each parent drugdrug isis brieﬂybriefly reviewed.reviewed. The argumentsarguments forfor thethe linkslinks betweenbetween metabolicmetabolic changeschanges and pharmacologicpharmacologic activity and structurestructure areare discusseddiscussed forfor somesome drug/metabolitedrug/metabolite cases, as well as being presentedpresented inin anan overalloverall discussiondiscussion givengiven forfor allall thethe casescases atat thethe endend ofof thethe section.section.

5. Aromatic Hydroxy (Arenolic) Metabolites Arenolic metabolites can result from oneone of two metabolic reactions: O-dealkylation of aralkoxy groups or hydroxylationhydroxylation of aromaticaromatic rings (mainly benzene); in the latter case, the positions of thethe hydroxy groups are determined by the prevailing electronic and steric effects on the aromatic ring. TheThe benzenebenzene ringring maymay bebe present present in in the the drug drug molecule molecule as as a separatea separate entity entity (i.e., (i.e., as as a phenyla phenyl group), group), or itor may it may be be fused fused to to another another benzene benzene ring, ring, or or to to an an alicyclic alicyclic or or a a heterocyclic heterocyclic ring. ring. WhenWhen thethe benzenebenzene ringring isis fusedfused toto eithereither anan alicyclicalicyclic oror heterocyclicheterocyclic ring,ring, thethe resultingresulting ringring systemsystem maymay bebe describeddescribed asas benzenoid. Arenolic metabolites are presentedpresented andand discusseddiscussed inin SectionsSections 5.15.1 andand 5.25.2..

5.1. Arenolic Metabolites Resulting from thethe O-DealkylationO-Dealkylation ofof AralkoxyAralkoxy GroupsGroups The metabolicmetabolic cytochromecytochrome P450P450 (CYP)-catalyzed (CYP)-catalyzedO O-dealkylation-dealkylation of of aralkoxy aralkoxy groups groups involves, involves, as as a ﬁrsta first step, step, the the hydroxylation hydroxylation of of the the carbon carbon atom atom of of the thealkyl alkyl groupgroup thatthat isis linkedlinked to the oxygen atom. ThisThis hydroxylatedhydroxylated metabolite metabolite is unstable;is unstable; it breaks it breaks spontaneously spontaneously into two molecules:into two themolecules: dealkylated the metabolitedealkylated (e.g., metabolite an alcohol (e.g., or an a phenol)alcohol andor a an phenol) aldehyde and (e.g., an aldehyde formaldehyde (e.g., afterformaldehyde demethylation, after acetaldehydedemethylation, after acetaldehyde deethylation, after etc.) deethylation, [2]. The reaction etc.) is [2]. shown The forreaction a general is shown case in for Figure a general1, where case the in RFigure group 1, can where be aliphaticthe R group or aromatic. can be aliphatic or aromatic.

O-dealkylation of methyl ether R OH An alcohol OH or a phenol CYP 450 R O CH3 R O CH2 O Methyl ether HC Formaldehyde Intermediate H

O-dealkylation of ethyl ether R OH OH CYP 450 R O CH2CH3 R O CHCH3 O Ethyl ether CH3 C Acetaldehyde Intermediate H Figure 1. Cytochrome P450 (CYP)-catalyzed O-dealkylation-dealkylation ofof alkylalkyl oror aralkylaralkyl ethers.ethers.

The O-dealkylation ofof aralkoxyaralkoxy groupsgroups resultsresults inin thethe formation of arenolic metabolites ofof varying pharmacologicpharmacologic activities,activities, asas isis reviewedreviewed forfor thethe followingfollowing selectedselected cases:cases: (1)(1) EnhancementEnhancement of of activity occurring in thethe opioidopioid narcotic/analgesicnarcotic/analgesic drug, codeine, and its semisyntheticsemisynthetic and and synthetic co congeners,ngeners, all containing an aryl-methoxy group, and the analgesic/antipyreticanalgesic/antipyretic drug, drug, phenacetin, phenacetin, which which contains contains an an aryl-ethoxy aryl-ethoxy group; group; (2) Retention of activity by venlafaxine, (a selective-serotonin-reuptake-inhibitor (SSRI) antidepressant, containing an aryl-methoxy group);

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Molecules 2018, 23, x FOR PEER REVIEW 4 of 29 (2) Retention of activity by venlafaxine, (a selective-serotonin-reuptake-inhibitor (SSRI)

(3) Attenuationantidepressant, or containingloss of activity an aryl-methoxy by COX1/C group);OX2-inhibitors, -naproxen, indomethacin, and nabumetone-each containing an aryl-methoxy group. (3) Attenuation or loss of activity by COX1/COX2-inhibitors, -naproxen, indomethacin, and nabumetone-each containing an aryl-methoxy group. 5.1.1. Metabolic Aralkoxy Group Cleavage Resulting in the Enhancement of Pharmacologic 5.1.1.Activity: Metabolic Opioids Aralkoxy and Phenacetin Group Cleavage Resulting in the Enhancement of Pharmacologic Activity: Opioids and Phenacetin Opioids Opioids Opioid drugs can be defined as those acting on the various types of opioid receptors (mu, kappa, and delta)Opioid to drugsproduce can a range be defined of biologic as those effects. acting Mu-receptor on the various stimulation types is ofassociated opioid receptorswith analgesic (mu, kappa,(antinociceptive) and delta) effects, to produce respiratory a range depression, of biologic reduced effects. gastrointestinal Mu-receptor motility stimulation (constipation), is associated and witheuphoric analgesic and dependence (antinociceptive) effects, effects, while respiratorykappa-recep depression,tor stimulation reduced is associated gastrointestinal with analgesia, motility (constipation),psychomimetic and effects, euphoric dysphoria, and dependence and diuresis effects, [3,4]. while kappa-receptor stimulation is associated withFor analgesia, the sake psychomimetic of discussion, effects, methoxy-group-cont dysphoria, and diuresisaining opioid [3,4]. drugs can be classified with regardsFor theto their sake ofsource discussion, and chemical methoxy-group-containing build-up. Source opioid includes drugs natural can be origin classified (e.g., with codeine), regards tosemisynthetic their source (e.g., and chemicalcodeine congener build-up.s), Source and synthetic includes (e.g., natural tramadol). origin (e.g.,Tramadol codeine), is considered semisynthetic to be (e.g.,a synthetic codeine codeine congeners), congener and since synthetic it was (e.g.,developed tramadol). using Tramadolcodeine as is template. considered Chemically, to be a synthetic codeine codeineand its semisynthetic congener since congeners it was developed are classified using as pe codeinentacyclic as or template. tetracyclic Chemically, morphinans codeine (Figure and 2). On its semisyntheticthe other hand, congeners tramadol are is classifieda non-morphinan as pentacyclic bicyclic or tetracyclic opioid devoid morphinans of rings (Figure B and2 C,). Onas shown the other in hand,Figure tramadol 2. is a non-morphinan bicyclic opioid devoid of rings B and C, as shown in Figure2.

NCH3 NCH3 N(CH3)2

B C B C A D A D A E O HO H3CO Pentacyclic morphinan Tetracyclic morphinan Tramadol (Codeine and its congeners) (Levomethorphan)

FigureFigure 2. ChemicalChemical classification classification of methoxy-group-containingof methoxy-group-containing morphinan morphinan and non-morphinan and non-morphinan opioids. opioids. (1) Codeine (1) Codeine Codeine (Figure 33)) isis anan opiumopium alkaloid;alkaloid; itit isis thethe methylmethyl etherether ofof morphinemorphine andand isis presentpresent inin opium in a 1–3% concentration [[5].5]. It is obtained semisynthetically on a large scale by the methylation of morphine.morphine. CodeineCodeine isis aa weak weak mu-receptor mu-receptor agonist, agonist, being being only only about about 0.1% 0.1% as activeas active as morphineas morphine [6]. The[6]. The analgesic analgesic activity activity of codeine of codeine is mainly is mainly attributed attributed to its metabolites.to its metabolites. Codeine Codeine is metabolized is metabolized as per theas per pathways the pathways shown inshown Figure in3 Figureto codeine-6-glucuronide, 3 to codeine-6-glucuronide, morphine, morphine, and norcodeine. and norcodeine. The resulting The morphineresulting morphine is further metabolizedis further metabolized to the 3- and to 6-glucuronidethe 3- and 6-glucuronide conjugates [conjugates7,8]. As indicated [7,8]. As in indicated Figure3, reportsin Figure in 3, the reports literature in the on literature the concentrations on the concentrations of codeine of metabolites codeine metabolites are variable. are variable. Further, Further, reports onreports the mu-receptor on the mu-receptor affinity and affinity analgesic and activity analge ofsic codeine activity metabolites of codeine are evenmetabolites contradictory. are even It is acontradictory. longstanding It beliefis a longstanding that the mu-receptor belief that the affinity mu-receptor and analgesic affinity activity and analgesic of codeine activity are of mediated codeine throughare mediated its metabolite, through its morphine, metabolite, which morphine, has an whic affinityh has for an the affinity mu-opioid for the receptor mu-opioid 200-fold receptor greater 200- thanfold greater that of codeinethan that [9 of]. Thecodeine analgesic [9]. The activity analgesic of codeine activity was of suggestedcodeine was to besuggested largely due to be its largely metabolite due morphineits metabolite [9–12 morphine]. However, [9–12]. of late, However, Vree et al.of (2000)late, Vree developed et al. (2000) a different developed view ofa codeine’sdifferent view affinity of tocodeine’s the mu-opioid affinity receptorto the mu-opioid and its analgesic receptor activity. and its The analgesic authors activity. argued The that authors the analgesic argued activity that the of codeineanalgesic was activity due to of its codeine glucuronide was conjugatedue to its ratherglucuronide than to conjugate its O-desmethyl rather metabolite, than to its morphine O-desmethyl [13]. metabolite,The literature’s morphine contradictory [13]. evidence on the mu-receptor affinity and the analgesic activity of codeineThe and literature’s its metabolites contradictory are weighed evidence in the on discussionthe mu-receptor section. affinity and the analgesic activity of codeineIt may and be its of metabolites interest to noteare weighed that codeine’s in the metabolicdiscussion pathways section. cover almost the entire spectrum of biologicalIt may be activity, of interest including to note reactions that codeine’s that are me augmenting,tabolic pathways activating, cover inactivating, almost the entire and attenuating spectrum of thebiological analgesic activity, activity ofincluding the metabolite reactions with that respect are to augmenting, the parent drug. activating, inactivating, and attenuating of the analgesic activity of the metabolite with respect to the parent drug.

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Molecules 2018, 23, x FOR PEER REVIEW 5 of 29 Morphine-3- Molecules 2018, 23, x FOR PEER REVIEW 5 of 29 NCH ) glucuronide Molecules 2018, 23, x FOR PEER REVIEW 3 NCH3 % 5 of 29 (60 Morphine-3-(inactive) 9 H NCH 9 H ) glucuronideMorphine-3- 3 NCH3 % 1 8 60 14NCH CYP2D6 14 ( CYP2B7) Morphine-3-glucuronide(inactive) 9 3 NCH3 % 13H 139 H 60 NCH6 ( ) glucuronideMorphine-6-(inactive) 1 9 3 8 NCH63 % 14 H CYP2D60-15% 14 9 H (56CYP2B7-0 3 O 5 O 5 ( 10 glucuronide(inactive) H3CO1 139 OH8 HO13 OH %) 14 HH6 CYP2D6 149HH CYP2B7 Morphine-6- 1 13 8 6 (active) 145 0-15% 14 13 (5CYP2B7-1 H CO3 O 6 OH CYP2D6 O 5 6 0% glucuronideMorphine-6- 3 Codeine13H HOMorphineH OH ) 5 0-15% 13 (5-1 H CO3 O 6 OH O 5 6 0% Morphine-6-glucuronide(active) 3 H HOH OH ) 5 0-15% (5-1 H CO3 CodeineO OH MorphineO 5 0% glucuronide(active) (50-70%)3 CYP2B7H HOH OH ) Codeine Morphine (active) (50-70%)Codeine-6-glucuronideCodeineCYP2B7 Morphine (50-70%) CYP2B7 (active) (50-70%)Codeine-6-glucuronideCYP2B7 Codeine-6-glucuronide(active) FigureFigure 3. 3. Metabolic pathways of codeine. Codeine-6-glucuronide(active) Metabolic pathways of codeine. (active)Figure 3. Metabolic pathways of codeine. (2) Codeine Congeners Figure 3. Metabolic pathways of codeine. (2) Codeine Congeners Figure 3. Metabolic pathways of codeine. (2) Codeine syntheticCongeners pentacyclic-morphinan congeners include hydrocodone, oxycodone, and the (2) Codeine Congeners tetracyclicCodeine(2) Codeinesynthetic morphinan Congenerssynthetic pentacyclic-morphinan congener, pentacyclic-morphinan levomethorphan, congenerscongen whichers all include include have 3-arylmethoxy hydrocodone, hydrocodone, groupsoxycodone, oxycodone, Figures and 4–6. the and the tetracyclicOntetracyclic theCodeine morphinanother morphinan hand, synthetic tramad congener, congener, pentacyclic-morphinanol (Figure levomethorphan, levomethorphan, 6) may be consideredcongen which ers all include alla nohave haven-morphinan 3-arylmethoxy hydrocodone, 3-arylmethoxy codeine groupsoxycodone, groupsanalog Figures since and Figures 4–6. theits 4–6. Codeine synthetic pentacyclic-morphinan congeners include hydrocodone, oxycodone, and the On thedevelopmentOntetracyclic otherthe other hand, morphinan hand,was tramadol basedtramad congener, onol (Figure (Figurecodeine levomethorphan,6 6) )as maymay template. be be considered considered whichTramadol all a haveno a containsn-morphinan non-morphinan 3-arylmethoxy an arylmethoxy codeine groups codeine analog Figuresgroup analog since in4–6. its a since tetracyclic morphinan congener, levomethorphan, which all have 3-arylmethoxy groups Figures 4–6. its developmentpositiondevelopmentOn the other reminiscent hand, waswas based basedtramad to that onol of (Figurecodeine codeine. 6) as asmayWhile template. template. be hy considereddrocodone, Tramadol Tramadol a nooxycodone, containsn-morphinan contains an and arylmethoxy an codeinetramadol arylmethoxy analog are group currently since group in its a in a On the other hand, tramadol (Figure 6) may be considered a non-morphinan codeine analog since its clinicallypositiondevelopment reminiscent used wasin pain based to management that on of codeine codeine. [14,15], as While template. levomethorphan hydrocodone, Tramadol hasoxycodone, contains never been an and arylmethoxyused. tramadol The four are group drugscurrently in are a positiondevelopment reminiscent was to based that ofon codeine.codeine as While template. hydrocodone, Tramadol oxycodone,contains an arylmethoxy and tramadol group are currentlyin a metabolizedclinicallyposition reminiscent used by in the pain isozyme to management that ofCYP2D6 codeine. [14,15], to Whiletheir levomethorphan corresponding hydrocodone, hasOoxycodone,-desmethyl never been and phenolic used. tramadol The counterparts, four are drugscurrently are as clinicallyposition used reminiscent in pain managementto that of codeine. [14, 15While], levomethorphan hydrocodone, oxycodone, has never and been tramadol used. are The currently four drugs shownmetabolizedclinically in usedthe by correspondingin the pain isozyme management CYP2D6 Figures [14,15], to4–7 their [16–18].levomethorphan corresponding Due to their hasO-desmethyl never substantially been phenolic used. higher The counterparts, four mu-receptor drugs are as are metabolizedclinically used by in the pain isozyme management CYP2D6 [14,15], to theirlevomethorphan corresponding has neverO-desmethyl been used. phenolicThe four drugs counterparts, are affinities,shownmetabolized in andthe by accordingly,corresponding the isozyme their CYP2D6 Figures higher to4–7 analgesictheir [16–18]. corresponding ac Duetivities to comparedtheir O-desmethyl substantially to their phenolic parent higher counterparts, drugs mu-receptor [15,16], as as shownmetabolized in the by corresponding the isozyme CYP2D6 Figures to4 –their7[ 16 corresponding–18]. Due to O their-desmethyl substantially phenolic highercounterparts, mu-receptor as theaffinities,shown four in phenolic andthe accordingly,corresponding metabolites their Figureswere higher developed 4–7 analgesic [16–18]. into ac Duetivities drugs to compared theirof their substantially ownto their rights. parent higher Presumably, drugs mu-receptor [15,16], the affinities,shown and in the accordingly, corresponding their Figures higher 4–7 analgesic [16–18]. activities Due to their compared substantially to their higher parent mu-receptor drugs [15 ,16], increasetheaffinities, four in phenolicand mu-receptor accordingly, metabolites affinity their wereand higher analgesic developed analgesic activi into actytivities drugsis a result compared of theirof the own tophenolic their rights. parent hydroxy Presumably, drugs groups [15,16], the in affinities, and accordingly, their higher analgesic activities compared to their parent drugs [15,16], the fourtheincreasethe four-metabolite phenolicfour inphenolic mu-receptor metabolites metabolitesdrugs. affinity We were present wereand developed analgesic developeda possible into activi explaninto drugsty drugsisation ofa result their ofof why theirof own thethis own rights.phenolic is the rights. case Presumably, hydroxy inPresumably, the discussion groups the increasethe in the four phenolic metabolites were developed into drugs of their own rights. Presumably, the in mu-receptorsection.theincrease four-metabolite in mu-receptor affinity drugs. and affinity We analgesic present and analgesic a activity possible activi is explan aty result isation a result ofof why the of the phenolicthis phenolic is the case hydroxy hydroxy in the discussion groupsgroups in in the increase in mu-receptor affinity and analgesic activity is a result of the phenolic hydroxy groups in four-metabolitesection.the four-metabolite drugs. Wedrugs. present We present a possible a possible explanation explanation of why of why this isthis the is casethe case in the in the discussion discussion section. the four-metabolite drugs. WeNCH present a possible explanation of why this is theNCH3 case in the discussion section. 3 NCH3 9 section. 9 9 NCH3 NCH3 NCH3 1 1 14 1 14 CYP2D6 14 UGT27B NCH3 NCH3 NCH3 139 139 139 1 NCH3 6 1 14NCH3 66NCH3 149 9 CYP2D6 1 149 UGT27B 3 O 3 O 3 O Gluc-O 513 O H3CO 1 14513 O HO 513 O 1 149 9 CYP2D6 1 149 UGT27B 6 661 13 1 1413 1 13 3 O14 3 O CYP2D6 3 O14 UGT27B Gluc-O 5 6O H3CO 5 66O HOHydromorphone5 O Hydromorphone13 Hydrocodone13 13 3 O 3 O 3 O Gluc-O glucuronide5 6O H3CO 5 66O HO 5 O Gluc-OHydromorphone3 O O H CO Hydrocodone3 O 5 O Hydromorphone3 O 5 3 HO 5 O glucuronide HydrocodoneFigure 4. MetabolicHydromorphone pathways of hydrocodone.Hydromorphone glucuronide Hydrocodone Hydromorphone Hydromorphone Figure 4. Metabolic pathways of hydrocodone. glucuronide NCH NCH FigureFigure3 4. 4.Metabolic Metabolic pathwayspathways3 of of hydrocodone. hydrocodone. NCH3 9FigureOH 4. Metabolic pathways9 OH of hydrocodone. 9 OH NCH3 NCH3 NCH3 1 14 CYP2D6 1 14 UGT27B 14 NCH3 NCH3 1 NCH3 139 OH 139 OH 9 OH NCH NCH NCH13 1 14 3 1 14 3 6 3 9 OH6 CYP2D6 9 OH UGT27B 1 149 OH6 35O 35O H3CO 1 1413 O HO 1 1413 O Gluc-O 35O O 9 OH CYP2D6 9 OH UGT27B 1 14139 OH 1 14 13 6 1 1413 6 13 6 35O CYP2D6 35O UGT27B 1 14 H3CO O HO O Gluc-OOxymorphone35O O Oxycodone13 6 Oxymorphone13 6 13 6 35O 35O O H3CO 6O HO 6O Gluc-O glucuronide356O 35O 35O Oxymorphone H3CO Oxycodone O HO Oxymorphone O Gluc-O 35O O glucuronide OxycodoneFigure 5. MetabolicOxymorphone pathways of oxycodone.Oxymorphone glucuronide Oxycodone Oxymorphone Oxymorphone Figure 5. Metabolic pathways of oxycodone. glucuronide NCH3 NCH3 Figure 5. MetabolicB pathways of oxycodone. Figure 5. Metabolic pathways* 9 of oxycodone. FigureNCH3 5. Metabolic1 pathwaysNCH83 of oxycodone. D * UGT2B7 CYP2D6 B NCH14 NCH3 A * 9 3 O-glucuronidation C NCH 1 B D NCH* 8 3 CYP2D6 * 9 3 UGT2B7 31 5 146 H3CO HO AB D * 8 O-glucuronidation CYP2D6 * 9 C UGT2B7 1 14 8 3A D 5* 6 UGT2B7 O-glucuronidation HLevomethorphanCO CYP2D6 HOLevorphanol14C 3 A O-glucuronidation 3 5 C 6 H3CO HO TheLevomethorphan ring-letter designation, the carbon-atomLevorphanol3 5 numbering 6 and the chiral-center H CO HO asterisk3Levomethorphan designation in levomethorphanLevorphanol is the same as for levorphanol. TheLevomethorphan ring-letter designation, the carbon-atomLevorphanol numbering and the chiral-center asteriskThe ring-letter Figuredesignation designation, 6. Metabolicin levomet the horphancarbon-atom pathways is the numbering sameof levomethorphan. as for and levor thephanol. chiral-center asterisk designation in levomethorphan is the same as for levorphanol. The ring-letter designation, the carbon-atom numbering and the chiral-center asterisk designationFigure 6. inMetabolic levomethorphan pathways is the same of levomethorphan. as for levorphanol. Figure 6. Metabolic pathways of levomethorphan. Figure 6. Metabolic pathways of levomethorphan. Figure 6. Metabolic pathways of levomethorphan.

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CH3 CH3 CH3 N N N H CO CH3 HO CH3 glu-O CH3 3 2 CYP2D6 1 * UGT2B7 *

HO HO HO Tramadol O-Desmethyltramadol O-Desmethyltramadol (active) glucuronide (inactive)

Figure 7. Metabolic pathways of tramadol.

The subjectsubject of of stereochemistry stereochemistry is ofis paramountof paramount importance importance when when considering considering drug structure–activity drug structure– relationshipactivity relationship of chiral drugs.of chiral Of thedrugs. four codeineOf the four congeners, codeine tramadol congeners, and levorphanol tramadol and present levorphanol the most interestingpresent the features. most interesting features.

Stereochemistry of thethe MorphinanMorphinan OpioidsOpioids The stereochemistrystereochemistry of of the the pentacyclic pentacyclic morphinan morphinan opiates, opiates, to which to which belong belong morphine morphine and codeine, and iscodeine, complicated is complicated by their rigid by their ring systemrigid ring and system the presence and the of presence multi-chiral of multi-chiral centers. Morphine centers. and Morphine codeine bothand codeine contain both five contain chiral centers, five chiral as indicatedcenters, as by in thedicated wedged by the bonds wedged in Figure bonds3 in. Assigning Figure 3. Assigning absolute configurationabsolute configuration to each chiral to each center chiral and center then an and overall then absolutean overall configuration absolute configuration to either a codeine to either or a morphinecodeine or molecule a morphine is a molecule challenge. is With a challenge. a total number With a oftotal 32 enantiomers,number of 32 the enantiomers, task is overwhelming. the task is However,overwhelming. for the However, purpose of for designation the purpose of of chirality, designatio opticaln of activity chirality, may optical be used. activity In multi-chiral may be used. center In molecules,multi-chiral optical center activity molecules, is an optical additive activity function is an of additive the rotations function at the ofcomponent the rotations asymmetric at the component centers. Inasymmetric morphine, centers. the most In important morphine, member the most of theimpo pentacyclicrtant member morphinans, of the pent theacyclic chiral centersmorphinans, at C5, the C6, andchiral C9 centers (Figure at3 )C5, rotate C6, theand plane C9 (Figure of polarized 3) rotate light the to plane the left of (polarizedlevo (−)) whilelight to the the remaining left (levo ( centers−)) while at C13the remaining and C14 do centers so to the at rightC13 and (dextro C14(+)) do [ 19so]. to Depending the right on(dextro the overall(+)) [19]. optical Depending rotation, on the the analgesic overall activityoptical rotation, of natural the morphine analgesic is activity attributed of natural to levomorphine morphine [ 20is ].attributed to levomorphine [20]. As shown in Figure6 6,, thethe tetracyclictetracyclic morphinan,morphinan, levorphanol,levorphanol, hashas twotwo chiralchiral centerscenters atat C9C9 andand C14, in addition toto exhibitingexhibiting ciscis//trans isomerism. Despite thethe factfact thatthat thethe B/CB/C ciscis (14(14RR)) configuration configuration is two-foldtwo-fold lessless activeactive thanthan thethe B/CB/C transtrans isomer,isomer, it it is is the one that is clinically used as a narcoticnarcotic analgesic [21]. [21]. The The additive additive optical optical rotation rotation of of the the pl planeane of of polarized polarized light light of ofthe the two two chiral chiral centers centers in inlevorphanol levorphanol must must be beto tothe the left, left, thus thus accounting accounting for for the the origin origin of of the the prefix prefix levo. The The fact thatthat enantiomers of chiral drugs may have different actions is demonstrated by the dextro counterpart of levorphanol, i.e., dextrophan, whichwhich isis used as an antitussive, and is devoid of the narcotic analgesic activity characteristic of the levolevo enantiomerenantiomer [[22].22]. The non-morphinan opioid drug, tramadol,tramadol, contains two chiral centers, as indicated by asterisks in FigureFigure7 7,, and and accordingly, accordingly, it it exists exists in in four four enantiomeric enantiomeric forms. forms. InIn addition,addition, becausebecause itit containscontains aa cyclohexane ring, tramadoltramadol existsexists asas ciscis//transtrans isomers.isomers. Despite Despite the the fact fact that tramadol is marketed as the racemate of thethe ciscis isomer,isomer, itsits enantiomersenantiomers andand activeactive OO-desmethyl-desmethyl metabolitemetabolite havehave significantsignificant selectivity in theirtheir analgesicanalgesic mechanismmechanism ofof action.action. Specifically,Specifically, thethe 11SS,2,2SS-(-(−−) enantiomerenantiomer inhibitsinhibits norepinephrine reuptake, while thethe 11RR,2,2RR-(-(−−)) enantiomer enantiomer inhibits inhibits 5-HT 5-HT (serotonin) reuptake to produce analgesic activity. On thethe otherother hand,hand, thethe dextrorotatorydextrorotatory OO-desmethyltramadol-desmethyltramadol produces a six-fold greater analgesicanalgesic activityactivity thanthan tramadoltramadol viavia stimulatingstimulating thethe mumu receptorsreceptors [[23].23].

Discussion of Opioids The metabolicmetabolicO -demethylationO-demethylation of theof methoxy-group-containingthe methoxy-group-containing opioids isopioids discussed is separatelydiscussed becauseseparately of thebecause availability of the availability of substantial of substantial supportive supportive evidence. evidence. A number of theories were proposed to account for the role of thethe phenolic–hydroxyphenolic–hydroxy group in mu-receptor interaction and thethe consequentconsequent productionproduction ofof analgesia.analgesia. When discussing the structure–activity relationship of opioids, Foye (2013) [[24]24] classiﬁedclassified opioid drugs into two categories according to the pharmacophorepharmacophore responsible for mu-receptor interaction: the rigid multicyclic (morphinan) opioids (exemplified by morphine, codeine, and codeine congeners), and the flexible opioids (exemplified by 4-arylpyridinepethidine). To the latter category,

Molecules 2018, 23, 2119 7 of 30

Moleculesthe rigid 2018 multicyclic, 23, x FOR (morphinan)PEER REVIEW opioids (exemplified by morphine, codeine, and codeine congeners),7 of 29 andMolecules the 2018 flexible, 23, x opioids FOR PEER (exemplified REVIEW by 4-arylpyridinepethidine). To the latter category, we may7 addof 29 wearylcyclohexylmethylamine, may add arylcyclohexylmethylamine, which is found which in tramadol. is found Thein tramadol. structures The of thestructures three categories of the three are categoriesdepictedwe may add in are Figure arylcyclohexylmethylamine,depicted8. in Figure 8. which is found in tramadol. The structures of the three categories are depicted in Figure 8.

NCH3 N NCH3 H N B D H A B D C 4 1 N R E H A C 4 1 N R O H E H O H 4-Arylpiperidine Arylcyclohexyl dimethyl- Morphinan 4-Arylpiperidine Arylcyclohexyl methylamine dimethyl- Morphinan methylamine FigureFigure 8. StructuresStructures of of the the opioid opioid drug pharmacophores. Figure 8. Structures of the opioid drug pharmacophores. Foye (2013) [24] argued the importance of a phenolic–hydroxy group on ring A (Figure 8) to the Foye (2013) [24] argued the importance of a phenolic–hydroxy group on ring A (Figure8) to the mu-receptorFoye (2013) interaction [24] argued and theanalgesic importance activity of aof phenolic–hydroxy the rigid multicyclic group morphinan on ring A opioids(Figure 8)such to theas mu-receptor interaction and analgesic activity of the rigid multicyclic morphinan opioids such as morphinemu-receptor and interaction the O-desmethyl and analgesic metabolites activity of codeine of the rigidcongener multicyclics. The author morphinan also maintained opioids such that as a morphine and the O-desmethyl metabolites of codeine congeners. The author also maintained that a phenolic–hydroxymorphine and the Ogroup-desmethyl is not ametabolites requirement of forcodeine the mu-receptor congeners. Theinteraction author alsoand maintainedanalgesic activity that a phenolic–hydroxy group is not a requirement for the mu-receptor interaction and analgesic activity of ofphenolic–hydroxy the flexible non-morphinan group is not opioids.a requirement It should for thbee notedmu-receptor that none interaction of the latter and analgesicgroups of activity drugs the flexible non-morphinan opioids. It should be noted that none of the latter groups of drugs contains containsof the flexible a phenolic non-morphinan hydroxy groupopioids. and It shouldthat arom be atic-ringnoted that hydroxylation none of the latteris not groups reported of drugs as a a phenolic hydroxy group and that aromatic-ring hydroxylation is not reported as a metabolic route metaboliccontains a route phenolic for any hydroxy of them. group and that aromatic-ring hydroxylation is not reported as a for any of them. metabolicFoye’s route theory for of any “phenolic of them. OH binding liability” for the flexible opioids [24] may be contested Foye’s theory of “phenolic OH binding liability” for the flexible opioids [24] may be contested basedFoye’s on observations theory of “phenolicfrom the metabolic OH binding activity liability” of O-desmethyltramadol for the flexible opioids (a metabolite [24] may beof tramadol;contested based on observations from the metabolic activity of O-desmethyltramadol (a metabolite of tramadol; Figurebased on 7) andobservations ketobemidone from the (an metabolic analog of activitypethidine; of OFigure-desmethyltramadol 9). Both drugs can (a metabolite be categorized of tramadol; as non- Figure7) and ketobemidone (an analog of pethidine; Figure9). Both drugs can be categorized as rigidFigure flexible 7) and ketobemidonenon-morphinan (an opioids. analog ofO -Desmethyltramadolpethidine; Figure 9). Bothexhibits drugs a can200-fold be categorized increase inas non-mu- non-rigid flexible non-morphinan opioids. O-Desmethyltramadol exhibits a 200-fold increase in receptorrigid flexible affinity non- andmorphinan analgesic activityopioids. relevant O-Desmethyltramadol to tramadol [25]. exhibits Ketobemidone, a 200-fold on increasethe other in hand, mumu-receptor affinity and analgesic activity relevant to tramadol [25]. Ketobemidone, on the other hand, hasreceptor a four-fold affinity mu-receptor and analgesic affinity activity and relevant analgesic to activity tramadol compared [25]. Ketobemidone, to pethidine [26].on the The other enhanced hand, has a four-fold mu-receptor affinity and analgesic activity compared to pethidine [26]. The enhanced mu-receptorhas a four-fold affinity mu-receptor and analgesic affinity activity and analgesic of both activity O-desmethyltramadol compared to pethidine and ketobemidone [26]. The enhanced can be mu-receptor affinity and analgesic activity of both O-desmethyltramadol and ketobemidone can be attributedmu-receptor to theaffinity phenolic and analgesichydroxy group,activity which of both is inO -desmethyltramadola position reminiscent and to ketobemidonethat of morphine. can be attributed to the phenolic hydroxy group,group, whichwhich isis inin aa positionposition reminiscentreminiscent toto thatthat ofof morphine.morphine. CH 3 CH3 CH N 3 CH3 4 N (CH ) N 3 3 2 4 5 (CH3)2N N 2 2 N (CH ) N 3 3 2 1 (CH3)2N H CO 2 6 5 1 HO 3 HO 2 O 1 O H CO OH 6 1 HO 3 HO OH O O O OH OH O (1R,2R)-Tramadol (1R,2R)-O-Desmethyl- Pethidine Ketobemidone tramadol (1R,2R)-Tramadol (1R,2R)-O-Desmethyl- Pethidine Ketobemidone tramadol Figure 9. Tramadol/O-desmethyltramadol and pethidine/ketobemidone. FigureFigure 9.9. Tramadol/Tramadol/O-desmethyltramadol andand pethidine/ketobemidone.pethidine/ketobemidone. Another opioid drug–receptor interaction theory was suggested by Beckett and Casy (1959) [27] Another opioid drug–receptor interaction theory was suggested by Beckett and Casy (1959) [27] who Anotherstated that opioid the drug–receptormacromolecule interaction with which theory the wasanalgesic suggested interacts by Beckett has, or and attains, Casy (1959)a certain [27] who stated that the macromolecule with which the analgesic interacts has, or attains, a certain conformationwho stated that into the which macromolecule the phenolic with group which must the fit before analgesic the interactsbiological has, effect or (of attains, analgesia) a certain can conformation into which the phenolic group must fit before the biological effect (of analgesia) can occur.conformation A “three-point” into which attachment the phenolic of the group macromolecul must fite before to a substrate’s the biological flat aromatic effect (of moiety, analgesia) its basic can occur. A “three-point” attachment of the macromolecule to a substrate’s flat aromatic moiety, its basic center,occur. Aand “three-point” its hydrocarbon attachment area was of thepostulated. macromolecule to a substrate’s flat aromatic moiety, its basic center, and its hydrocarbon area was postulated. center,Glucuronide and its hydrocarbon and acetyl area derivatives was postulated. of morphine provide substantiating evidence of the phenolic Glucuronide and acetyl derivatives of morphine provide substantiating evidence of the phenolic hydroxyGlucuronide group’s andinvolvement acetyl derivatives in strengthening of morphine the provide mu-receptor substantiating affinity, evidence and accordingly, of the phenolic the hydroxy group’s involvement in strengthening the mu-receptor affinity, and accordingly, the analgesichydroxy group’sactivity involvementof the morphinan-opioid in strengthening drugs the that mu-receptor contain it, affinity, either andintrinsically accordingly, or metabolically the analgesic analgesic activity of the morphinan-opioid drugs that contain it, either intrinsically or metabolically produced.activity of theFurther morphinan-opioid discussion of this drugs point that follows. contain it, either intrinsically or metabolically produced. produced. Further discussion of this point follows. FurtherBy virtue discussion of its of phenolic–hydroxy this point follows. group at position 3 and alcoholic hydroxy group at position By virtue of its phenolic–hydroxy group at position 3 and alcoholic hydroxy group at position 6, morphineBy virtue (Figure of its 3) forms phenolic–hydroxy two glucuronide group conjugates: at position morphine-3-glucu 3 and alcoholicronide hydroxy and morphine-6- group at 6, morphine (Figure 3) forms two glucuronide conjugates: morphine-3-glucuronide and morphine-6- glucuronideposition 6, morphine (Figure 3). (Figure While 3morphine-6-glucuromide) forms two glucuronide is conjugates: a far more potent morphine-3-glucuronide mu-receptor agonist andand analgesicglucuronide than (Figure morphine 3). While [28–30], morphine-6-glucuromide morphine-3-glucuronide is ais far devoid more of potent both effects mu-receptor [31]. Furthermore, agonist and theanalgesic O-glucuronide than morphine conjugates [28–30], of mobothrphine-3-glucuronide O-desmethyltramadol is devoid (Figure of 6) both and effects levorphanol [31]. Furthermore, (Figure 7) arethe devoidO-glucuronide of mu-receptor conjugates agonistic of both effects, O-desmethyltramadol and accordingly, of(Figure analgesic 6) and activity levorphanol [32,33]. The(Figure above 7) dataare devoid may be of explained mu-receptor based agonistic on size effects,and hydrogen-bonding and accordingly, ability of analgesic differences activity between [32,33]. the The hydroxy above anddata glucuronidemay be explained groups. based In onmorphine, size and hydrogen-bondingthe hydrogen bonding ability provided differences by between the 6-OH the group hydroxy is and glucuronide groups. In morphine, the hydrogen bonding provided by the 6-OH group is

Molecules 2018, 23, 2119 8 of 30 morphine-6-glucuronide (Figure3). While morphine-6-glucuromide is a far more potent mu-receptor agonist and analgesic than morphine [28–30], morphine-3-glucuronide is devoid of both effects [31]. Furthermore, the O-glucuronide conjugates of both O-desmethyltramadol (Figure6) and levorphanol (Figure7) are devoid of mu-receptor agonistic effects, and accordingly, of analgesic activity [ 32,33]. MoleculesThe above 2018 data, 23, x mayFOR PEER be explained REVIEW based on size and hydrogen-bonding ability differences between8 of 29 the hydroxy and glucuronide groups. In morphine, the hydrogen bonding provided by the 6-OH importantgroup is important for mu-receptor for mu-receptor fitting, and fitting, thus, and analge thus,sic analgesic activity. activity. With Withthree threehydroxy hydroxy moieties, moieties, the glucuronidethe glucuronide group group at position at position 6 of 6 ofmorphine morphine is isca capablepable of of establishing establishing more more hydrogen hydrogen bonds bonds than the 6-OH. 6-OH. Further Further stronger stronger interactions interactions of ofthe the glucuronide glucuronide group group with with the mu the receptor mu receptor involve involve ion– ionion–ion and ion–dipole and ion–dipole binding binding provided provided by the bycarboxylate the carboxylate (COO−) (COOmoiety.−) Such moiety. a state Such of affairs a state will of mostaffairs probably will most lead probably to stronger lead mu-receptor to stronger mu-receptorfit, and hence, fit, higher and hence, analgesic higher activity analgesic of morphine-6- activity of glucuronidemorphine-6-glucuronide than morphine. than On morphine. the other On hand, the other the si hand,ze (steric) the size factor (steric) favors factor the favorshydroxy the group hydroxy at positiongroup at 3 position of morphine 3 of morphine to anchor tothe anchor aromatic the ri aromaticng in its ring hydrophobic in its hydrophobic pocket in pocketthe mu in receptor, the mu ratherreceptor, than rather the considerably than the considerably bigger glucuronide bigger glucuronide group. group. Upon comparing the mu-receptor affinity affinity and analgesic analgesic activity activity of of morphine, morphine, 6-acetylmorphine, 6-acetylmorphine, and diamorphine (heroin) (Figure 10),10), 6-acetylmorphi 6-acetylmorphinene was found to be the most active of the three opiates; it is fourfour timestimes asas activeactive asas morphine. morphine. Heroin Heroin is is also also more more active active than than morphine morphine by by a factora factor of oftwo, two, but but less less active active than than 6-acetylmorphine 6-acetylmorphine [34 [34,35].,35].

NCH NCH3 3 NCH3

9 H 9 H 9 H 1 1 14 8 esterase14 8 esterase 1 14 8 13 13 13 6 6 6 3 O H3CCO 5 OCCH3 3 O 5 HO 3 O 5 OH H HO H OCCH3 H O O O Morphine Diamorphine 6-Acetylmorphine (Heroin)

Figure 10. Diamorphine, 6-acetylmorphine, and morphine.

A plausible explanation of the above data is as follows: being more lipophilic than morphine, A plausible explanation of the above data is as follows: being more lipophilic than morphine, both 6-acetylmorphine and heroin (Figure 10) will cross the blood–brain barrier faster and in higher both 6-acetylmorphine and heroin (Figure 10) will cross the blood–brain barrier faster and in concentrations than morphine. On the other hand, having a free phenolic hydroxy group, 6- higher concentrations than morphine. On the other hand, having a free phenolic hydroxy group, acetylmorphine will interact with the opioid mu receptor more efficiently than diamorphine. In 6-acetylmorphine will interact with the opioid mu receptor more efficiently than diamorphine. diamorphine, the free hydroxy group is generated metabolically in the brain by esterase hydrolysis, In diamorphine, the free hydroxy group is generated metabolically in the brain by esterase hydrolysis, which will lead to a delayed effect and reduced efficacy. The effects of morphine and the two which will lead to a delayed effect and reduced efficacy. The effects of morphine and the two acetylated opiates (diamorphine and 6-acetylmorphine) can, therefore, be explained by a acetylated opiates (diamorphine and 6-acetylmorphine) can, therefore, be explained by a combination combination of pharmacodynamic and pharmacokinetic influences. of pharmacodynamic and pharmacokinetic influences. Additional substantiating evidence for the role of the free phenolic hydroxy group may be Additional substantiating evidence for the role of the free phenolic hydroxy group may be obtained from levomethorphan and its O-desmethyl metabolite, levorphanol (Figure 7). These two obtained from levomethorphan and its O-desmethyl metabolite, levorphanol (Figure7). These two tetracyclic-morphinan opiate drugs lack the 6-hydroxy group, and hence, the ability to form tetracyclic-morphinan opiate drugs lack the 6-hydroxy group, and hence, the ability to form glucuronide glucuronide conjugates at that position; yet, levorphanol has a stronger affinity for the mu receptor conjugates at that position; yet, levorphanol has a stronger affinity for the mu receptor and analgesic and analgesic activity than its parent drug, levomethorphan [36]. activity than its parent drug, levomethorphan [36]. In conclusion, we may summarize the role of the hydroxy group in arenolic opioids in the In conclusion, we may summarize the role of the hydroxy group in arenolic opioids in the following statement: as shown in the opioid pharmacophores in Figure 8, the aromatic rings labeled following statement: as shown in the opioid pharmacophores in Figure8, the aromatic rings A, present in both morphinan and non-morphinan opioids, are essential components of the labeled A, present in both morphinan and non-morphinan opioids, are essential components of pharmacophore of the opioid–mu-receptor interaction. The high affinity of the arenolic opioids for the pharmacophore of the opioid–mu-receptor interaction. The high affinity of the arenolic opioids for the mu receptor is an indication of a logistic role played by the hydroxy group. Through hydrogen the mu receptor is an indication of a logistic role played by the hydroxy group. Through hydrogen bonding with an adjacent amino-acid residue in the mu receptor, the hydroxy group plays the logistic bonding with an adjacent amino-acid residue in the mu receptor, the hydroxy group plays the logistic role of anchoring the aromatic ring to the assigned hydrophobic pocket in the receptor, thus role of anchoring the aromatic ring to the assigned hydrophobic pocket in the receptor, thus enhancing enhancing both affinity and efficacy. Substantiating evidence to the above statement is provided by both affinity and efficacy. Substantiating evidence to the above statement is provided by the work the work of Sahu et al. (2008) [37] on tetrahydroimidazobenzodiazepinones (the human of Sahu et al. (2008) [37] on tetrahydroimidazobenzodiazepinones (the human immunodeficiency immunodeficiency virus 1 (HIV-1) non-nucleoside reverse transcriptase (NNRT) inhibitors). In this class of compounds, the predominant hydrophobic proper orientation for maximum effect was found to be enhanced by hydrogen bonding and polar interactions. The assertion by Vree et al. of the analgesic activity of codeine being entirely due to its glucuronide conjugate [13] may now be reconsidered in view of the evidence so far presented on the role of the arenolic hydroxy group in opioid drugs. It is important to emphasize that the conclusion by Vree et al. was abstract rather than experimental, and as such, may be subject to a difference of opinion.

Molecules 2018, 23, 2119 9 of 30 virus 1 (HIV-1) non-nucleoside reverse transcriptase (NNRT) inhibitors). In this class of compounds, the predominant hydrophobic proper orientation for maximum effect was found to be enhanced by hydrogen bonding and polar interactions. The assertion by Vree et al. of the analgesic activity of codeine being entirely due to its glucuronide conjugate [13] may now be reconsidered in view of the evidence so far presented on the role of the arenolic hydroxy group in opioid drugs. It is important to emphasize that the conclusion by Vree et al. Molecules 2018, 23, x FOR PEER REVIEW 9 of 29 was abstract rather than experimental, and as such, may be subject to a difference of opinion. BecauseBecause bothboth codeinecodeine andand tramadoltramadol havehave intrinsicintrinsic analgesicanalgesic activities,activities, they can bebe viewedviewed asas prodrugsprodrugs ofof morphine and O-desmethyltramadol, respectively, both having sustained- sustained-releaserelease effects. effects. BothBoth morphinemorphine andand OO-desmethyltramadol-desmethyltramadol areare strongstrong mu-receptormu-receptor agonistsagonists usedused inin thethe managementmanagement ofof severesevere pain,pain, butbut havehave thethe disadvantagesdisadvantages ofof causingcausing dependencedependence andand tolerance.tolerance. Therefore,Therefore, forfor thethe managementmanagement of mild-to-moderate pain, it would be advisable to use the corresponding prodrugs, codeinecodeine andand tramadol,tramadol, which which have have the the advantage advantage of of sustained sustained release. release. However, However, some some people people who who are poorare poor CYP2D6 CYP2D6 metabolizers metabolizers do not do make not usemake of theuse beneﬁcialof the beneficial prodrug prodrug sustained-release sustained-release effect. For effect. such people,For such it ispeople, advisable it is toadvisable adjust the to doseadjust of the the dose parent of drug, the parent to administer drug, toO administer-desmethyl O metabolites,-desmethyl ormetabolites, to seek alternative or to seek therapies. alternative The therapies. frequency Th ofe thefrequency phenotype of the of poorphenotype metabolizers of poor differs metabolizers among ethnicdiffers groups.among Lessethnic than groups. 1% of Asians,Less than 2–5% 1% of of African Asians, Americans, 2–5% of African and 6–10% Americans, of Caucasians and 6–10% are poor of metabolizersCaucasians are of poor CYP2D6 metabolizers [38–40]. of CYP2D6 [38–40]. InIn additionaddition toto thethe pharmacodynamicpharmacodynamic receptorreceptor interactionsinteractions ofof thethe methoxymethoxy opioidsopioids (occurring(occurring mainlymainly through through their their polar polar hydroxy hydroxy metabolites), metabolites), the theanalgesic analgesic effects effects of hydrophobic of hydrophobic opioid opioiddrugs, drugs,such as such fentanyl, as fentanyl, dextropropoxyphene, dextropropoxyphene, methadone, methadone, and pethidine and pethidine (Figure 11), (Figure were mainly11), were explained mainly explainedby pharmacokinetic by pharmacokinetic effects. Th effects.ese drugs These cross drugs the cross blood–brain the blood–brain barrier barrier more moreefficiently, efﬁciently, and andaccordingly, accordingly, they theyreach reachthe mu-receptors the mu-receptors in the brain in the in brain higher in concentrations higher concentrations than the methoxy- than the methoxy-group-containinggroup-containing members members[41]. [41].

N O O O O O

N N N O N

Fentanyl Dextropropoxyphene Methadone Pethidine

FigureFigure 11.11. Highly hydrophobic opioids.opioids.

Acetanilide/Phenacetin/Paracetamol Acetanilide/Phenacetin/Paracetamol The story of the development of the most commonly used analgesic antipyretic drug, The story of the development of the most commonly used analgesic antipyretic drug, paracetamol, paracetamol, as a metabolite drug of acetanilide and phenacetin is depicted in Figure 12. Both the as a metabolite drug of acetanilide and phenacetin is depicted in Figure 12. Both the latter drugs latter drugs were once used as analgesics and antipyretics. Paracetamol results from phenacetin by were once used as analgesics and antipyretics. Paracetamol results from phenacetin by metabolic metabolic O-deethylation [42], and from acetanilide by metabolic para-hydroxylation of the aromatic O-deethylation [42], and from acetanilide by metabolic para-hydroxylation of the aromatic ring [43]. ring [43]. Compared to its two precursors, paracetamol was found to have superior pharmacologic Compared to its two precursors, paracetamol was found to have superior pharmacologic proﬁles, profiles, including those of toxicity and therapeutic index [42,43]. The free hydroxy group seems to including those of toxicity and therapeutic index [42,43]. The free hydroxy group seems to give give paracetamol the edge in inhibiting, through hydrogen-bonding interaction, two prostaglandin paracetamol the edge in inhibiting, through hydrogen-bonding interaction, two prostaglandin H2 H2 synthases, now believed to be involved in the mechanism of action of paracetamol [44]. Moreover, synthases, now believed to be involved in the mechanism of action of paracetamol [44]. Moreover, the hydroxy group may play the logistic role of anchoring the aromatic ring in the proper geometric the hydroxy group may play the logistic role of anchoring the aromatic ring in the proper geometric orientation in the enzyme active cavity. orientation in the enzyme active cavity.

OH OH organic synthesis

metabolic-ring (CH3CO)2O

hydroxylation NHCOCH3 NH2 Paracetamol p-Aminophenol

metabolic NHCOCH3 O-deethylation Acetanilide

OC2H5

Phenacetin medicinal chemistry NHCOCH 3 Figure 12. From acetanilide to phenacetin to paracetamol.

Molecules 2018, 23, x FOR PEER REVIEW 9 of 29

Because both codeine and tramadol have intrinsic analgesic activities, they can be viewed as prodrugs of morphine and O-desmethyltramadol, respectively, both having sustained-release effects. Both morphine and O-desmethyltramadol are strong mu-receptor agonists used in the management of severe pain, but have the disadvantages of causing dependence and tolerance. Therefore, for the management of mild-to-moderate pain, it would be advisable to use the corresponding prodrugs, codeine and tramadol, which have the advantage of sustained release. However, some people who are poor CYP2D6 metabolizers do not make use of the beneficial prodrug sustained-release effect. For such people, it is advisable to adjust the dose of the parent drug, to administer O-desmethyl metabolites, or to seek alternative therapies. The frequency of the phenotype of poor metabolizers differs among ethnic groups. Less than 1% of Asians, 2–5% of African Americans, and 6–10% of Caucasians are poor metabolizers of CYP2D6 [38–40]. In addition to the pharmacodynamic receptor interactions of the methoxy opioids (occurring mainly through their polar hydroxy metabolites), the analgesic effects of hydrophobic opioid drugs, such as fentanyl, dextropropoxyphene, methadone, and pethidine (Figure 11), were mainly explained by pharmacokinetic effects. These drugs cross the blood–brain barrier more efficiently, and accordingly, they reach the mu-receptors in the brain in higher concentrations than the methoxy- group-containing members [41].

N O O O O O

N N N O N

Fentanyl Dextropropoxyphene Methadone Pethidine Figure 11. Highly hydrophobic opioids.

Acetanilide/Phenacetin/Paracetamol The story of the development of the most commonly used analgesic antipyretic drug, paracetamol, as a metabolite drug of acetanilide and phenacetin is depicted in Figure 12. Both the latter drugs were once used as analgesics and antipyretics. Paracetamol results from phenacetin by metabolic O-deethylation [42], and from acetanilide by metabolic para-hydroxylation of the aromatic ring [43]. Compared to its two precursors, paracetamol was found to have superior pharmacologic profiles, including those of toxicity and therapeutic index [42,43]. The free hydroxy group seems to give paracetamol the edge in inhibiting, through hydrogen-bonding interaction, two prostaglandin H2 synthases, now believed to be involved in the mechanism of action of paracetamol [44]. Moreover, Moleculesthe hydroxy2018, 23group, 2119 may play the logistic role of anchoring the aromatic ring in the proper geometric10 of 30 orientation in the enzyme active cavity.

OH OH organic synthesis

metabolic-ring (CH3CO)2O

hydroxylation NHCOCH3 NH2 Paracetamol p-Aminophenol

metabolic NHCOCH3 O-deethylation Acetanilide

OC2H5

Phenacetin medicinal chemistry NHCOCH 3 Molecules 2018, 23, x FOR PEERFigure REVIEW 12. From acetanilide to phenacetin to paracetamol. 10 of 29

5.1.2. Metabolic O-Dealkylation ofof AralkoxyAralkoxy GroupsGroups ResultingResulting inin RetainingRetaining PharmacologicPharmacologic Activity:Activity: Venlafaxine/DesvenlafaxineVenlafaxine/Desvenlafaxine Venlafaxine is is an an antidepressant antidepressant in the in noradrenaline–serotoninthe noradrenaline–serotonin reuptake reuptake inhibitor inhibitor (NSRI) category. (NSRI) Beingcategory. chiral Being (Figure chiral 13), venlafaxine (Figure 13), exists venlafaxine as R and S enantiomers. exists as R Regarding and S neurotransmitter-reuptakeenantiomers. Regarding inhibition,neurotransmitter-reuptake some degree of selectivity inhibition, was some observed: degree the ofR- selectivityenantiomer was acted observed: as both a noradrenalinethe R-enantiomer and serotoninacted as both reuptake a noradrenaline inhibitor, while and sero the toninS-enantiomer reuptake acted inhibitor, only aswhile a serotonin the S-enantiomer reuptake inhibitoracted only [45 as]. However,a serotonin the reuptake drug is marketedinhibitor [45]. as the However, racemate. the drug is marketed as the racemate.

N N N OH OH OH CYP2D6 * UGT2B7 * * * * *

H3CO HO Gluc-O Desvenlafaxine Desvenlafaxine Venlafaxine glucuronide (O-Desmethylvenlafaxine)

Figure 13. Metabolic pathway of venlafaxine.

Venlafaxine is mainly metabolized by O-demethylation to equiactive desvenlafaxine (O- Venlafaxine is mainly metabolized by O-demethylation to equiactive desvenlafaxine desmethylvenlafaxine; Figure 13). Venlafaxine is further metabolized through N-demethylation and (O-desmethylvenlafaxine; Figure 13). Venlafaxine is further metabolized through N-demethylation glucuronide conjugation to inactive products (Figure 13) [46–48]. Desvenlafaxine was developed into and glucuronide conjugation to inactive products (Figure 13)[46–48]. Desvenlafaxine was developed a drug of its own right and approved by the Food and Drug Administration (FDA) in the United into a drug of its own right and approved by the Food and Drug Administration (FDA) in the States (US) for the treatment of major depressive disorder (MDD), similar to its parent drug, United States (US) for the treatment of major depressive disorder (MDD), similar to its parent drug, venlafaxine, which is used for major depressive and anxiety disorders. However, the European venlafaxine, which is used for major depressive and anxiety disorders. However, the European Medicines Agency (EMA) had second thoughts and did not approve desvenlafaxine as a drug. They Medicines Agency (EMA) had second thoughts and did not approve desvenlafaxine as a drug. They argued that venlafaxine is almost fully metabolized to desvenlafaxine, and that both compounds have argued that venlafaxine is almost fully metabolized to desvenlafaxine, and that both compounds essentially the same pharmacologic and pharmacokinetic profiles; hence, there is no strong reason to have essentially the same pharmacologic and pharmacokinetic proﬁles; hence, there is no strong use desvenlafaxine as a separate drug [49]. Whether the analogy of the selective activity of the R and reason to use desvenlafaxine as a separate drug [49]. Whether the analogy of the selective activity S enantiomers of venlafaxine may be extended to desvenlafaxine is a subject for experimental of the R and S enantiomers of venlafaxine may be extended to desvenlafaxine is a subject for investigation. experimental investigation. Venlafaxine carries structural similarity to the analgesic drug, tramadol. The latter drug exerts Venlafaxine carries structural similarity to the analgesic drug, tramadol. The latter drug exerts its analgesic effect mainly through serotonin–norepinephrine reuptake inhibition (SNRI) [50], and to its analgesic effect mainly through serotonin–norepinephrine reuptake inhibition (SNRI) [50], and to a minor extent, through blocking of the mu-receptor. The O-Demethylation of both venlafaxine and a minor extent, through blocking of the mu-receptor. The O-Demethylation of both venlafaxine tramadol resulted in active metabolites that were developed into drugs of their own rights. The fact and tramadol resulted in active metabolites that were developed into drugs of their own rights. that neither venlafaxine nor desvenlafaxine has analgesic effects, and that neither tramadol nor O- The fact that neither venlafaxine nor desvenlafaxine has analgesic effects, and that neither tramadol desmethyltramadol has antidepressant effects, emphasizes the close link between drug action and nor O-desmethyltramadol has antidepressant effects, emphasizes the close link between drug action drug structure. and drug structure. 5.1.3. Metabolic O-Dealkylation of Aralkoxy Groups Resulting in the Attenuation or Loss of Pharmacologic Activity: NSAIDs (Naproxen, Indomethacin, and Nabumetone) NSAIDs fall into five chemical classes: arylalkanoic acids, salicylates, fenamates, oxicams (cyclic sulfonamides), and diarylheteroaromatics. The benzene ring, either as a separate entity or fused with other rings, constitutes an integral part of the pharmacophore in all the NSAID chemical classes. The carboxyl group (in the form of a carboxylate ion, COO−) forms the other part of the pharmacophore in the arylalkanoic acid NSAIDs. With the exception of aspirin, the mechanism of action of the NSAIDs involves the competitive inhibition of arachidonic acid, the precursor of prostaglandins, from accessing the COX active cavity. The binding of the arylalkanoic acid NSAID to the amino-acid moieties in the COX active cavity involves ion–ion, ion–dipole, and hydrogen bonding through the carboxylate group (COO−) and hydrophobic interactions through alkyl and aryl groups [51–54]. The alkyl groups are those of the methoxy and propionic acid moieties. Naproxen, indomethacin, and nabumetone (Figures 14–16, respectively) are NSAIDs that act through COX1/COX2 inhibition. They are about the only three arylalkanoic acid NSAIDs that contain aromatic methoxy groups. Furthermore, nabumetone is a prodrug NSAID, which must first be

Molecules 2018, 23, 2119 11 of 30

5.1.3. Metabolic O-Dealkylation of Aralkoxy Groups Resulting in the Attenuation or Loss of Pharmacologic Activity: NSAIDs (Naproxen, Indomethacin, and Nabumetone) NSAIDs fall into ﬁve chemical classes: arylalkanoic acids, salicylates, fenamates, oxicams (cyclic sulfonamides), and diarylheteroaromatics. The benzene ring, either as a separate entity or fused with other rings, constitutes an integral part of the pharmacophore in all the NSAID chemical classes. The carboxyl group (in the form of a carboxylate ion, COO−) forms the other part of the pharmacophore in the arylalkanoic acid NSAIDs. With the exception of aspirin, the mechanism of action of the NSAIDs involves the competitive inhibition of arachidonic acid, the precursor of prostaglandins, from accessing the COX active cavity. The binding of the arylalkanoic acid NSAID to the amino-acid moieties in the COX active cavity involves ion–ion, ion–dipole, and hydrogen bonding through the carboxylate group (COO−) and hydrophobic interactions through alkyl and aryl groups [51–54]. The alkyl groups are those of the methoxy and propionic acid moieties. Naproxen, indomethacin, and nabumetone (Figures 14–16, respectively) are NSAIDs that act

Moleculesthrough 2018 COX1/COX2, 23, x FOR PEER inhibition. REVIEW They are about the only three arylalkanoic acid NSAIDs11 thatof 29 containMolecules 2018 aromatic, 23, x FOR methoxy PEER REVIEW groups. Furthermore, nabumetone is a prodrug NSAID, which11 must of 29 Molecules 2018, 23, x FOR PEER REVIEW 11 of 29 activatedﬁrst be activated by the bymetabolic the metabolic oxidation oxidation of the ofcarbonyl the carbonyl group group to the to thecarboxy carboxy derivative, derivative, 6- activated by the metabolic oxidation of the carbonyl group to the carboxy derivative, 6- methoxynaphthylaceticactivated6-methoxynaphthylacetic by the metabolic acid, acid, asoxidation as shown shown in inFigureof Figure the 16. carbonyl16 . group to the carboxy derivative, 6- methoxynaphthylacetic acid, as shown in Figure 16. methoxynaphthylacetic acid, as shown in Figure 16. CH3 CH3 CH3 OH CH3 OH CH3 CYP3A4 * OH CH3 UGT2B7 CYP3A4 * OH * O OH O UGT2B7 S-O6-Desmethylnaproxen CYP3A4CYP1A2 * OH CH3O * O HO O S-O -Desmethylnaproxen CYP1A2 * UGT2B7 diglucuronide6 CH O O HO O S-O -Desmethylnaproxen 3 S-(+)-Naproxen CYP1A2 6 diglucuronide(inactive) CH3O HOS-O6-Desmethylnaproxen S-(+)-Naproxen diglucuronide(inactive) S-O6-Desmethylnaproxen(inactive) S-(+)-Naproxen S-O6-Desmethylnaproxen (inactive) (inactive) Figure 14. Metabolic pathway(inactive) of naproxen [55] Figure 14. Metabolic pathway of naproxen [[55]55] Figure 14. Metabolic pathway of naproxen [55] H3CO COOH HO H3CO COOH COOH HO H3CO N CH3COOH HO COOH N CH3 UGT2B7 Indomethacin N CH3 CYP2C9 COOH N CH3 UGT2B7 diglucuronideIndomethacin O N CH3 CYP2C9 Indomethacin CYP2C9 O N CH3 UGT2B7 conjugatediglucuronide O diglucuronide O O conjugate(inactive) Cl O conjugate Cl (inactive) IndomethacinCl (inactive) Cl Cl Indomethacin O-DesmethylindomethacinCl Indomethacin O-Desmethylindomethacin (50O-Desmethylindomethacin %, major metabolite, inactive) (50 %, major metabolite, inactive) Figure 15. Metabolic(50 pathway %, major ofmetabolite, indomethacin inactive) [56]. Figure 15. Metabolic pathway of indomethacin [56]. Figure 15. Metabolic pathway of indomethacin [56]. [56]. O O O CH3 Nabumetone Nabumetone CH3 (prodrug) H3CO CH3 Nabumetone 2 (prodrug) H3COA 1 (prodrug) H COP 2 3 Y A 1 C P 2 A Y 1 C P Y C OH OH CYP2D6 OH OH O OH CYP2D6 O OH H CO HO 3 O CYP2D6 O H3CO O HO O 6-MethoxynaphthylaceticH3CO acid 6-HydroxynaphthylaceticHO acid 6-Methoxynaphthylacetic acid 6-Hydroxynaphthylacetic acid 6-Methoxynaphthylacetic(active metabolite) acid 6-Hydroxynaphthylacetic(inactive) acid (active metabolite) (inactive) Figure(active metabolite)16. Metabolic pathways of nabumetone(inactive) [57]. Figure 16. Metabolic pathways of nabumetone [57]. Figure 16. Metabolic pathways of nabumetone [57]. [57]. Stereochemistry of Arylalkanoic Acid NSAIDs Stereochemistry of Arylalkanoic Acid NSAIDs StereochemistryThe arylalkanoic of Arylalkanoic acid NSAIDs Acid presented NSAIDs in this section fall into two subclasses: arylpropionic The arylalkanoic acid NSAIDs presented in this section fall into two subclasses: arylpropionic acid NSAIDs,The arylalkanoic to which acid belongs NSAIDs naproxen presented (Figure in this 14), section and arylacetic fall into acid two NSAIDs,subclasses: to whicharylpropionic belong acid NSAIDs, to which belongs naproxen (Figure 14), and arylacetic acid NSAIDs, to which belong indomethacinacid NSAIDs, toand which 6-methoxynaphthylacetic belongs naproxen (Figure acid, 14the), activeand arylacetic metabolites acid of NSAIDs, nabumetone to which (Figures belong 15 indomethacin and 6-methoxynaphthylacetic acid, the active metabolites of nabumetone (Figures 15 andindomethacin 16, respectively). and 6-methoxynaphthylacetic Naproxen contains a chiral acid, center the active at the metabolites α-carbon of of the nabumetone propionic acid(Figures moiety 15 and 16, respectively). Naproxen contains a chiral center at the α-carbon of the propionic acid moiety asand indicated 16, respectively). by an asterisk Naproxen in Figure contains 14, and a chiral so do center other atmembers the α-carbon of the ofarylpropionic the propionic acid acid NSAIDs, moiety as indicated by an asterisk in Figure 14, and so do other members of the arylpropionic acid NSAIDs, suchas indicated as ibuprofen by an andasterisk ketoprofen. in Figure Accordingly, 14, and so do these other drugs members exist asof enantiomericthe arylpropionic pairs, acid R-( −NSAIDs,) and S- such as ibuprofen and ketoprofen. Accordingly, these drugs exist as enantiomeric pairs, R-(−) and S- (+).such The as ibuprofenCOX-inhibition and ketoprofen. activity of Accordingly,the arylpropionic these acid drugs NSAID exist assubclass enantiomeric was found pairs, to Rreside-(−) and in itsS- (+). The COX-inhibition activity of the arylpropionic acid NSAID subclass was found to reside in its S(+).-(+)-enantiomers The COX-inhibition while activity the R of-(− the)-enantiomers arylpropionic were acid inactiveNSAID subclass[58]. However, was found the to resideinactive in itsR S-(+)-enantiomers while the R-(−)-enantiomers were inactive [58]. However, the inactive R enantiomersS-(+)-enantiomers in some while members, the R-( −such)-enantiomers as naproxen were and inactive ibuprofen, [58]. areHowever, metabolically the inactive inverted R enantiomers in some members, such as naproxen and ibuprofen, are metabolically inverted unidirectionallyenantiomers in tosome the activemembers, S-enantiomers, such as naproxen and hence, and may ibuprofen, be considered are asmetabolically prodrugs. Generally, inverted unidirectionally to the active S-enantiomers, and hence, may be considered as prodrugs. Generally, whenunidirectionally the metabolic to the functionalization active S-enantiomers, of a chiral and drug hence, occurs may at be a distantconsidered group as from prodrugs. the chiral Generally, center, when the metabolic functionalization of a chiral drug occurs at a distant group from the chiral center, thewhen absolute the metabolic configuration functionalization of the enantiomers of a chiral of drug the parentoccurs drugat a distant may be group extended from tothe its chiral metabolite. center, the absolute configuration of the enantiomers of the parent drug may be extended to its metabolite. However,the absolute this configuration ought not to beof the caseenantiomers with optica of thl activity,e parent which drug ismay only be determined extended to experimentally. its metabolite. However, this ought not to be the case with optical activity, which is only determined experimentally. OfHowever, all the this arylalkanoic ought not toacid be theNSAIDs, case with only optica S-naproxenl activity, whichand its is onlysodium determined salt are experimentally.internationally Of all the arylalkanoic acid NSAIDs, only S-naproxen and its sodium salt are internationally marketedOf all the as arylalkanoic enantiopure aciddrugs. NSAIDs, In this context, only S S-naproxen-(+)-ibuprofen and andits Ssodium-(+)-ketoprofen salt are areinternationally marketed in marketed as enantiopure drugs. In this context, S-(+)-ibuprofen and S-(+)-ketoprofen are marketed in somemarketed countries. as enantiopure All the otherdrugs. arylpropionic In this context, acid S-(+)-ibuprofen NSAIDs are and marketed S-(+)-ketoprofen as racemates. are marketed When the in some countries. All the other arylpropionic acid NSAIDs are marketed as racemates. When the racematesome countries. of an enantiopureAll the other drug arylpropionic was either acid previously NSAIDs used are marketedor is currently as racemates. in circulation, When the racemate of an enantiopure drug was either previously used or is currently in circulation, the enantiopureracemate of drugan enantiopure is known as drug a chiral-switch was either drug. previously This definition used or appliesis currently to S-(+)-naproxen in circulation, whose the enantiopure drug is known as a chiral-switch drug. This definition applies to S-(+)-naproxen whose enantiopure drug is known as a chiral-switch drug. This definition applies to S-(+)-naproxen whose

Molecules 2018, 23, 2119 12 of 30

Stereochemistry of Arylalkanoic Acid NSAIDs The arylalkanoic acid NSAIDs presented in this section fall into two subclasses: arylpropionic acid NSAIDs, to which belongs naproxen (Figure 14), and arylacetic acid NSAIDs, to which belong indomethacin and 6-methoxynaphthylacetic acid, the active metabolites of nabumetone (Figures 15 and 16, respectively). Naproxen contains a chiral center at the α-carbon of the propionic acid moiety as indicated by an asterisk in Figure 14, and so do other members of the arylpropionic acid NSAIDs, such as ibuprofen and ketoprofen. Accordingly, these drugs exist as enantiomeric pairs, R-(−) and S-(+). The COX-inhibition activity of the arylpropionic acid NSAID subclass was found to reside in its S-(+)-enantiomers while the R-(−)-enantiomers were inactive [58]. However, the inactive R enantiomers in some members, such as naproxen and ibuprofen, are metabolically inverted unidirectionally to the active S-enantiomers, and hence, may be considered as prodrugs. Generally, when the metabolic functionalization of a chiral drug occurs at a distant group from the chiral center, the absolute conﬁguration of the enantiomers of the parent drug may be extended to its metabolite. However, this ought not to be the case with optical activity, which is only determined experimentally. Of all the arylalkanoic acid NSAIDs, only S-naproxen and its sodium salt are internationally marketed as enantiopure drugs. In this context, S-(+)-ibuprofen and S-(+)-ketoprofen are marketed in some countries. All the other arylpropionic acid NSAIDs are marketed as racemates. When the racemate of an enantiopure drug was either previously used or is currently in circulation, the enantiopure drug is known as a chiral-switch drug. This deﬁnition applies to S-(+)-naproxen whose racemate use was stopped, and to S-(+)-ibuprofen and S-(+)-ketoprofen whose racemates are currently in circulation. S-Naproxen, indomethacin, and the active form of nabumetone are mainly metabolized through O-demethylation to give 6-O-desmethylnaproxen, O-desmethylindomethacin, and 6-hydroxynaphthylacetic acid, respectively (Figures 14–16). The first twoO-desmethyl metabolites are devoid of COX inhibitory effects, and accordingly, of NSAID activity [59–61]. By analogy, 6-hydroxynaphthylacetic acid, the O-desmethyl metabolite of nabumetone [62], is expected to be devoid of NSAID activity. According to Duggan et al. (1972), the p-hydroxy groups in the O-desmethyl metabolites place polar, hydrogen-bond-donating properties within otherwise entirely aromatic, hydrophobic, pharmacophoric groups in the parent drugs [59]. Presumably, a poor metabolite–COX fit would result in subsequent loss of pharmacologic activity. The loss of pharmacologic activity in the O-desmethylarylalkanoic acid NSAIDs is further reflected upon in the discussion section.

5.2. Overall Discussion The phase I metabolic O-dealkylation of aralkoxy groups almost invariably occurs in all the drugs containing these moieties. From the cited drug examples in this section, the metabolic O-dealkylation of aralkoxy groups results in three situations regarding pharmacologic activity:

(1) Enhancement of activity is exhibited by the O-desmethyl morphinan opioids, O-desmethyltramadol and O-desethyl phenacetin; (2) Retention of activity is exhibited by O-desmethyl venlafaxine; (3) Signiﬁcant attenuation or loss of activity is exhibited by O-desmethyl naproxen and O-desmethyl indomethacin.

Some inferences can be made from the effect on pharmacologic activity in the three NSAID cases upon considering the new state of structural affairs created by the loss of the hydrophobic aralkoxy-alky group and the generation of the hydrophilic hydroxy group. The effect mostly depends on the site of drug action involved. The cases where the activity is enhanced involve the opioid drugs acting on the mu-receptor. In these drugs, the aromatic ring A, an integral part of the pharmacophore (Figure 14), binds to the receptor through hydrophobic forces of attraction. The hydroxy group on the aromatic ring plays the logistic role of optimally anchoring the hydrophobic aromatic ring in its hydrophobic pocket in the mu receptor [24]. The same theory may be extended to the acetanilide/phenacetin/paracetamol Molecules 2018, 23, 2119 13 of 30 case, where the sites of action are the H2 synthase enzymes used in the formation of prostaglandin, as was recently proposed [44]. The loss of pharmacologic activity upon O-demethylation of the aryl-methoxy group is mainly observed in the NSAIDs, naproxen and indomethacin, and is inferred for nabumetone. In this class of arylalkanoic acid NSAIDs, COX inhibition is due to hydrogen bonding and ion pairing due to the carboxyl group, and van der Waals contacts due to hydrophobic groups, mainly the aromatic rings. In addition, in the arylpropionic acid NSAIDs, the α-methyl group forms an essential hydrophobic interacting group. Furthermore, the methyl group in the methoxy moiety in naproxen, nabumetone, and indomethacin occupies a hydrophobic pocket in COX. Duggan et al. (2010) [59] reported that the methoxy group of naproxen is oriented toward the apex of the COX active site, and forms van der Waals interactions with Trp 387 and Tyr 385. The methoxy group in naproxen (as well as in nabumetone and indomethacin) is of special importance since its metabolic demethylation results in the polar, hydrogen-bonding hydroxyl group. According to Duggan et al. (2010) [59], the hydroxyl group in O-desmethylnaproxen places polar, hydrogen-bonding properties within an entirely hydrophobic pocket that was occupied by the methyl group of the methoxy moiety in naproxen, which is consistent with a reduction in COX inhibition. Furthermore, a pharmacokinetic effect is most probably involved in the attenuation of COX inhibition by the O-desmethyl metabolites of naproxen, 6-methoxynaphthylacetic acid, and indomethacin. In explanation, aromatic hydroxy groups are almost invariably metabolized in phaseMolecules II to 2018 the, 23 highly, x FOR PEER water-soluble REVIEW and rapidly eliminated glucuronide (and sometimes13 of sulfate) 29 conjugates. Such an effect will lead to a reduction in the effective concentration of the metabolite II to the highly water-soluble and rapidly eliminated glucuronide (and sometimes sulfate) conjugates. at theSuch receptor an effect or will enzyme lead to active a reduction cavity, in thus the effective resulting concentration in curtailing of orthe losingmetabolite activity. at the Accordingreceptor to Furaor (2006), enzyme attenuation active cavity, or loss thus of resu pharmacologiclting in curtailing activity or losing is associated activity. withAccording the biotransformation to Fura (2006), of pharmacophoricattenuation or groups loss andof pharmacologic the possible accompanying activity is associated changes inwith physicochemical the biotransformation properties of [61]. Thepharmacophoric retention of groups antidepressant and the possible activity accompanyi of desvenlafaxineng changes indicates in physicochemical a non-essential properties hydrophobic [61]. binding roleThe ofretention the methoxy of antidepressant methyl group activity in venlafaxine.of desvenlafaxine Nevertheless, indicates a ano hydrogen-bondingn-essential hydrophobic role may not bebinding excluded role sinceof the it methoxy is provided methyl by group the methoxy in venlafaxine. oxygen Nevertheless, in venlafaxine a hydrogen-bonding and by the hydroxy role group in desvenlafaxine;may not be excluded however, since this it is occursprovided more by the prominently methoxy oxygen in the in latter venlafaxine drug. and by the hydroxy group in desvenlafaxine; however, this occurs more prominently in the latter drug. 5.3. Arenolic Metabolites Resulting from Aromatic Ring Hydroxylation 5.3. Arenolic Metabolites Resulting from Aromatic Ring Hydroxylation The mechanism of metabolic aromatic-ring hydroxylation involves, as a ﬁrst step, the formation The mechanism of metabolic aromatic-ring hydroxylation involves, as a first step, the formation of anof epoxide an epoxide (arene (arene oxide) oxide) intermediate, intermediate, whichwhich rearrangesrearranges rapidly rapidly and and spon spontaneouslytaneously to the to arenol the arenol productproduct in most in most instances instances (Figure (Figure 17 17))[62 [62,63].,63].

R R R spontaneous [O] rearrangement

O OH

Arene Arene oxide Arenol (a phenol)

Aromatic-ring hydroxylation catalyzed by a variety of enzymes in different xenobiotic molecules Figure 17. Mechanism of aromatic-ring hydroxylation. Figure 17. Mechanism of aromatic-ring hydroxylation.

Metabolic aromatic-ring hydroxylation assumes its importance from the fact that a large number Metabolicof drug molecules aromatic-ring contain hydroxylationthe benzene ring assumes as a sepa itsrate importance entity (phenyl) from or the fused fact with that other a large rings, number of drugalicyclic molecules or heterocyclic. contain In the drug benzene molecules, ring aromatic as a separate rings serve entity the (phenyl) dual role orof providing fused with relatively other rings, alicycliclarge or hydrophobic heterocyclic. sites In for drug interaction molecules, with aromaticreceptors or rings acting serve as carriers the dual for roleother of functional providing groups. relatively largeHowever, hydrophobic not all sites of the for benzene interaction rings within drug receptors molecules or are acting subject as carriersto metabolic for other hydroxylation. functional This groups. However,is because notall certain of the electronic benzene and rings steric in drugeffects molecules dictate the are metabolic subject hydroxylation to metabolic hydroxylation.of aromatic rings. This is These effects include the following, with possible anomalous events [64,65]: because certain electronic and steric effects dictate the metabolic hydroxylation of aromatic rings. These(a) effects The includeleast substituted the following, aromatic with ring possiblewill be favo anomalousrably oxidized, events especially [64,65]: at the least hindered carbon atom; (b) The activated ring (i.e., the ring bearing an electron-donating group such as an alkyl) will be better oxidized; (c) Ring-deactivating groups (generally groups with negative inductive effects such as halo and nitro groups) discourage ring hydroxylation; (d) Being the farthest from steric effects in di-substituted benzene rings, the para position is the favored site of hydroxylation; (e) If two aromatic rings in a drug molecule have the same chemical environment, hydroxylation will occur in only one of them; (f) When the parent drug contains an aromatic hydroxy group, further metabolic hydroxylation is generally not favored even if there is more than one aromatic ring. Aromatic-ring hydroxylation of drugs leads to the formation of inactive metabolites, metabolites with attenuated activity, and metabolites that are equiactive with the parent drugs. These situations are reviewed using selected representative drugs. The basis for the choice of the candidate drugs is as follows: • Varying the chemical classes of the drugs; • Varying the pharmacologic class of the drugs, and accordingly, the type of drug-site-of-action interaction involved (i.e., the mechanism of action of the class of drugs);

Molecules 2018, 23, 2119 14 of 30

(a) The least substituted aromatic ring will be favorably oxidized, especially at the least hindered carbon atom; (b) The activated ring (i.e., the ring bearing an electron-donating group such as an alkyl) will be better oxidized; (c) Ring-deactivating groups (generally groups with negative inductive effects such as halo and nitro groups) discourage ring hydroxylation; (d) Being the farthest from steric effects in di-substituted benzene rings, the para position is the favored site of hydroxylation; (e) If two aromatic rings in a drug molecule have the same chemical environment, hydroxylation will occur in only one of them; (f) When the parent drug contains an aromatic hydroxy group, further metabolic hydroxylation is generally not favored even if there is more than one aromatic ring.

Aromatic-ring hydroxylation of drugs leads to the formation of inactive metabolites, metabolites with attenuated activity, and metabolites that are equiactive with the parent drugs. These situations are reviewed using selected representative drugs. The basis for the choice of the candidate drugs is as follows:

• Varying the chemical classes of the drugs; Molecules• Varying 2018, 23 the, x FOR pharmacologic PEER REVIEW class of the drugs, and accordingly, the type of drug-site-of-action14 of 29 interaction involved (i.e., the mechanism of action of the class of drugs); • • Varying the aromatic characteristics inin bothboth thethe numbernumber ofof ringsrings andand chemicalchemical environment.environment.

Metabolic aromatic-ringaromatic-ring hydroxylation hydroxylation leading leading to the to lossthe ofloss activity of activity is exemplified is exemplified by the following: by the following: (a) The central nervous system depressant anticonvulsant drugs, phenobarbital and phenytoin, of (a) The central nervous system depressant anticonvulsant drugs, phenobarbital and phenytoin, of the imide chemical class (Figure 18); the imide chemical class (Figure 18); (b) The CNS depressant tranquilizer benzodiazepines, diazepam (Figure 19) and estazolam (b) The CNS depressant tranquilizer benzodiazepines, diazepam (Figure 19) and estazolam (Figure (Figure 20); 20); (c) The arylalkanoic acid NSAIDs, diclofenac, ﬂurbiprofen, and ketorolac (Figures 21–23, (c) The arylalkanoic acid NSAIDs, diclofenac, flurbiprofen, and ketorolac (Figures 21–23, respectively), and the pyrazolone NSAID derivative, phenylbutazone (Figure 24); respectively), and the pyrazolone NSAID derivative, phenylbutazone (Figure 24); (d) TheThe anticoagulant anticoagulant warfarin warfarin (Figure (Figure 25).25). Metabolic aromatic-ring hydroxylation hydroxylation leading leading to to attenuation attenuation of of activity activity is is exemplified exempliﬁed by the CNS depressant and major tranquilizer, chlorpromazine (Figure(Figure 2626).).

NO O O NO 5 NH 5 NH 1 CYP2C9 1 glucuronide or sulfate O O conjugation at the pheolic OH group HO 4'-Hydroxy- Phenobarbital phenobarbital (inactive) H O H O N N O CYP2C9 O N N H H

Phenytoin OH 5-(4-Hydroxyphenyl)- 5-phenyl-hysantoin (inactive) Figure 18. Major metabolic pathways of phenobarbital and phenytoin. Figure 18. Major metabolic pathways of phenobarbital and phenytoin.

CH3 CH3 CH3 O 1 O O N 9 N N CYP3A4 3 (major) 8 * OH A B 3 (minor) Cl N Cl 7 N Cl N 6 1' 1' 6' 2' C 5' 3' 4' 4' Temazepam Diazepam OH (active) 4'-Hydroxydiazepam (inactive) The two hydroxy metabolites are further metabolized by glucuronide conjugation in phase II. Figure 19. Metabolic pathways of diazepam.

N N N N N N N N N 4 CYP3A4 CYP3A4 * OH 4 Cl N (minor) Cl N (major) Cl N 1' 1" 2' 3' 4' 4-Hydroxyestazolam 4' OH (inactive) Estazolam 4'-Hydroxyestazolam (inactive)

The two hydroxy metabolites are further metabolized by glucuronide conjugation in phase II. Figure 20. Metabolic pathways of estazolam.

Molecules 2018, 23, x FOR PEER REVIEW 14 of 29 Molecules 2018, 23, x FOR PEER REVIEW 14 of 29 • Varying the aromatic characteristics in both the number of rings and chemical environment. • Varying the aromatic characteristics in both the number of rings and chemical environment. Metabolic aromatic-ring hydroxylation leading to the loss of activity is exemplified by the following:Metabolic aromatic-ring hydroxylation leading to the loss of activity is exemplified by the following: (a) The central nervous system depressant anticonvulsant drugs, phenobarbital and phenytoin, of (a) theThe imide central chemical nervous class system (Figure depressant 18); anticonvulsant drugs, phenobarbital and phenytoin, of (b) Thethe imide CNS depressantchemical class tranquilizer (Figure 18); benzodiazepines, diazepam (Figure 19) and estazolam (Figure (b) 20);The CNS depressant tranquilizer benzodiazepines, diazepam (Figure 19) and estazolam (Figure (c) The20); arylalkanoic acid NSAIDs, diclofenac, flurbiprofen, and ketorolac (Figures 21–23, (c) respectively),The arylalkanoic and theacid pyrazolone NSAIDs, NSAID diclofenac, derivative, flurbiprofen, phenylbutazone and ketorolac (Figure 24);(Figures 21–23, (d) Therespectively), anticoagulant and thewarfarin pyrazolone (Figure NSAID 25). derivative, phenylbutazone (Figure 24); (d) The anticoagulant warfarin (Figure 25). Metabolic aromatic-ring hydroxylation leading to attenuation of activity is exemplified by the CNS Metabolicdepressant aromatic-ring and major tranquilizer, hydroxylation chlorpromazine leading to attenuation (Figure 26). of activity is exemplified by the CNS depressant and major tranquilizer, chlorpromazine (Figure 26). NO O O NO NO O 5 NH O 5NO CYP2C9 NH 5 1 glucuronide or sulfate NH 5 NH1 O 1 CYP2C9 O 1 glucuronideconjugation orat thesulfate O O conjugationpheolic OH groupat the HO 4'-Hydroxy- pheolic OH group Phenobarbital HO 4'-Hydroxy-phenobarbital Phenobarbital phenobarbital(inactive) H O H O N (inactive) N H O H O N O CYP2C9 N O N O CYP2C9 N O H H N N H H

Phenytoin OH OH Phenytoin 5-(4-Hydroxyphenyl)- 5-(4-Hydroxyphenyl)-5-phenyl-hysantoin 5-phenyl-hysantoin(inactive) Molecules 23 2018, , 2119 (inactive) 15 of 30 Figure 18. Major metabolic pathways of phenobarbital and phenytoin. Figure 18. Major metabolic pathways of phenobarbital and phenytoin.

CH3 CH3 CH3 O CH O CH1 O CH3 N 3 9 N 3 N O O CYP3A4 O 3 (major) 8 9 1 N OH N N * A B 3 CYP3A4 3 (major) 8 (minor) Cl N* OH Cl 7 A B N 3 Cl N 6 (minor) 1' Cl N Cl 7 1' N Cl N 6 6' 2' 1'C 1' 6' 3'2' 5' C 4' 4' 5' 3' OH Temazepam Diazepam4' 4' Temazepam(active) Diazepam 4'-HydroxydiazepamOH (active) 4'-Hydroxydiazepam(inactive) (inactive) The two hydroxy metabolites are further metabolized by glucuronide conjugation in phase II. The two hydroxy metabolites are further metabolized by glucuronide conjugation in phase II. Figure 19. Metabolic pathways of diazepam. Figure 19. Metabolic pathways of diazepam.

N N N N N N N N N N N N N N N4 CYP3A4 CYP3A4 N * OH N 4 N 4 CYP3A4 CYP3A4 Cl *N OH (minor) Cl N 4 (major) Cl N Cl N (minor) Cl 1' N (major) Cl 1"N 1' 2' 1" 3'2' 4' 4-Hydroxyestazolam 4' 3' 4' OH 4-Hydroxyestazolam 4' (inactive) Estazolam OH 4'-Hydroxyestazolam (inactive) Estazolam 4'-Hydroxyestazolam(inactive) (inactive) The two hydroxy metabolites are further metabolized by glucuronide conjugation in phase II. The two hydroxy metabolites are further metabolized by glucuronide conjugation in phase II. Figure 20. Metabolic pathways of estazolam. Figure 20. Metabolic pathways of estazolam. Molecules 2018, 23, x FOR PEER REVIEWFigure 20. Metabolic pathways of estazolam. 15 of 29 Molecules 2018, 23, x FOR PEER REVIEW 15 of 29

Cl CH2COOH Cl H CH2COOH 1' HN 1 3' 1' N 1 3 3' 3 Diclofenac 4' Diclofenac 4' Cl 5 Cl C C 5 Y

4 Y C P

3A C Y 2C P P A4 Y P2 9

Y 3 2 C

C P 9 P C

Y 2

C 9 C 9

Cl CH2COOH Cl CH2COOH Cl CH2COOH Cl H CH2COOH Cl H CH2COOH Cl H CH2COOH HN NH HO 3' HN N N HO 3' N

HO Cl Cl Cl HO Cl Cl 5 Cl 5 4'-Hydroxydiclofenac OH 3'-Hydroxydiclofenac 4'-Hydroxydiclofenac OH 3'-Hydroxydiclofenac (20-30%) 5-Hydroxydiclofenac (20-30%) 5-Hydroxydiclofenac

Cl CH2COOH Cl H CH2COOH NH N 4',5-Dihydroxydiclofenac 4',5-Dihydroxydiclofenac HO Cl HO Cl OH OH Figure 21. Metabolic pathways of diclofenac. Figure 21. Metabolic pathways of diclofenac.

O O O O O N O N - OH N OH * O Gluc N OH CYP2C9 CYP2B7 N * O-Gluc CYP2C9 N OH CYP2B7 O O O O Gluc-O O HO O - Ketorolac HO Gluc O Ketorolac p-Hydroxyketorolac (10%) p-Hydroxyketorolac glucuronide p-Hydroxyketorolac (10%) p-Hydroxyketorolac glucuronide (inactive NSAID) (inactive) (inactive NSAID) (inactive) Figure 22. Metabolic pathways of ketorolac. Figure 22. MetabolicMetabolic pathways of ketorolac.

CH3 CH3 4;Hydroxy- HO CHCO2H 4;Hydroxy- HO *CHCO2H flurbiprofen * flurbiprofen

F (40-47%) i

F (40-47%) n

i a

P2C9 n c

Y a

P2C9 t c

C i

Y HO v 3' 2' CH3 CH t

C i 3 e

CH HO v

3' 2' 3 CH

m 3 e

3',4'-Dihydroxy- m CHCO2H HO CHCO2H e

3',4'-Dihydroxy- t e

1' CHCO2H HO CHCO2H flurbiprofen a t

* * b

1' a flurbiprofen o

* * b

F F l o

catechol (5%) i t

F l F e

catechol (5%) i

s e

Flurbiprofen s Flurbiprofen HO CH3 HO CH 3 3'-Hydroxy-4'- H3CO CHCO2H 3'-Hydroxy-4'- H3CO *CHCO2H methoxyflurbiprofen * methoxyflurbiprofen F (20-30%) F (20-30%)

Figure 23. Metabolic pathways of flurbiprofen. Figure 23. Metabolic pathways of flurbiprofen.

ng -ri g n ati in tio m ti-r la on NO aro a xy ati romdro l HO N NO a hy roxy HO N yd * h O * O

NO Oxyphenbutazone

N NO o Oxyphenbutazone N h me * ydom ga * hy ro eg -1 O dr xy a- O ox lat 1 yla ion Phenylbutazone tio Phenylbutazone n NO (developed into an anti-inflammatory N NO (developedmetabolite drug)into an anti-inflammatory N * metabolite drug) O * * O * OH OH gamma-Hydroxyphenylbutazone gamma-Hydroxyphenylbutazone (devoid of anti-inflammatory activity (devoid of anti-inflammatory activity but has urocosuric activity) but has urocosuric activity) Figure 24. Metabolism of phenylbutazone. Figure 24. Metabolism of phenylbutazone.

Molecules 2018, 23, x FOR PEER REVIEW 15 of 29 Molecules 2018, 23, x FOR PEER REVIEW 15 of 29

Cl CH2COOH Cl H CH COOH 1' N 1 2 H 3 3' 1' N 1 3' 3 Diclofenac 4' Cl Diclofenac 4' 5 Cl C

C Y Y 5 A4 C P2

3 C C

P P Y 9 4 Y P

CY A 2 2C 3 C

P P 9 2

CY 9 C 9

Cl CH2COOH Cl CH2COOH Cl CH2COOH H H Cl H CH COOH Cl CH2COOH Cl CH2COOH HO N 2 NH NH 3' H N N HO 3' N

HO Cl Cl Cl 5 HO Cl Cl Cl 4'-Hydroxydiclofenac OH5 3'-Hydroxydiclofenac 4'-Hydroxydiclofenac OH 3'-Hydroxydiclofenac (20-30%) 5-Hydroxydiclofenac (20-30%) 5-Hydroxydiclofenac

Cl CH2COOH H Cl CH2COOH NH N 4',5-Dihydroxydiclofenac 4',5-Dihydroxydiclofenac HO Cl HO Cl OH OH Figure 21. Metabolic pathways of diclofenac. Figure 21. Metabolic pathways of diclofenac.

O O O O N O N - OH O N OH * O Gluc N CYP2C9 CYP2B7 N - OH N OH * O Gluc O CYP2C9 CYP2B7 O O - O HO Gluc O O O - Ketorolac HO Gluc O Ketorolac p-Hydroxyketorolac (10%) p-Hydroxyketorolac glucuronide p-Hydroxyketorolac (10%) p-Hydroxyketorolac glucuronide (inactive NSAID) (inactive) Molecules 2018, 23, 2119 (inactive NSAID) (inactive) 16 of 30 Figure 22. Metabolic pathways of ketorolac. Figure 22. Metabolic pathways of ketorolac.

CH3 CH3 4;Hydroxy- HO CHCO2H 4;Hydroxy- HO *CHCO H flurbiprofen 2 flurbiprofen

F * (40-47%) i F n

(40-47%) a

P2C9 i c

Y n

t a

C P2C9 i

v HO c

3' 2' CH3 Y CH

t 3 e

C i

HO v

3' 2' CH3 CH m

3 3',4'-Dihydroxy- e e CHCO2H HO CHCO H

2 m t

1' flurbiprofen3',4'-Dihydroxy- a CHCO H e

* 2 HO *CHCO2H b

t o

1' flurbiprofen a l

F * F * b catechol i

(5%) o

t e

F F l s

catechol (5%) i t

Flurbiprofen e s Flurbiprofen HO CH3 HO CH3 3'-Hydroxy-4'- H3CO CHCO2H 3'-Hydroxy-4'- H CO *CHCO H methoxyflurbiprofen 3 2 methoxyflurbiprofen F * (20-30%) F (20-30%) Figure 23. Metabolic pathways of flurbiprofen. Figure 23. Metabolic pathways of flurbiprofen. ﬂurbiprofen.

g rin ti- gion ma rilnat NO aro tixy- ion mdaro lat HO N NO arhoy xy dro HO N * hy O * O NO Oxyphenbutazone N NO om Oxyphenbutazone N * hy eg d rom a- O hy oxe 1 * dr ylgaa O ox t-io1 yla n Phenylbutazone tio Phenylbutazone n NO (developed into an anti-inflammatory N NO metabolite(developed drug)into an anti-inflammatory N * metabolite drug) O * * * O OH OH gamma-Hydroxyphenylbutazone gamma-Hydroxyphenylbutazone (devoid of anti-inflammatory activity but(devoid has urocosuricof anti-inflammatory activity) activity but has urocosuric activity) Figure 24. Metabolism of phenylbutazone. Figure 24. Metabolism of phenylbutazone.

Molecules 2018, 23, x FOR PEER REVIEW 16 of 29

OH O 5 C9 CH P2 4 3 * 3 Y 7 phenyl C r ajo HO O O (m ) 1 ute OH O ro S-Warfarin 5 9 S-7-Hydroxywarfarin (inactive) 6 4 3 * 11 CH3 ca 10 12 rbo 1 red ny 7 O O uc l 2 tas OH 8 (m e OH in ro or 11 ute benzopyran ) * * CH3 (The S enantiomer is three- to five-fold O O as active as the R enantiomer.) * chiral center 11-Hydroxywarfarin (active)

Figure 25. Metabolic pathways of warfarin.

N N

N Cl N Cl 1 CYP2D6 1 UGT2B7 7-Hydroxychlorpromazine 7 3 3 S 5 HO 7 S 5 glucuronide Chlorpromazine 7-Hydroxychlorpromazine (major metabolite, moderately active) Figure 26. Metabolic pathways of chlorpromazine.

Metabolic aromatic-ring hydroxylation leading to the formation of equiactive products is exemplified by the following: (a) The beta-blocker, propranolol, of the chemical class, aryloxypropanolamine (Figure 27); (b) The β-hydroxy β-methylglutaryl coenzyme A reductase inhibitor, atorvastatin (used in lowering blood cholesterol level; Figure 28). Metabolic-ring hydroxylation leading to enhancement of activity is exemplified by acetanilide to paracetamol, which was already discussed in Section 5.1.1.

Figure 27. Metabolic pathways of propranolol.

Molecules 2018, 23, x FOR PEER REVIEW 16 of 29 Molecules 2018, 23, x FOR PEER REVIEW 16 of 29

OH O 5 OH O 9 CH 2C 5 4 3 * 3 YP 9 phenyl C C 7 4 CH3 P2 or 3O * O CY aj HO 7 1 phenyl (m r e) ajo ut HO O O OH O (m ro ) 1 S-Warfarin 5 ute S-7-Hydroxywarfarin (inactive) OH9 O ro 6 5 4 CH c S-7-Hydroxywarfarin (inactive) S-Warfarin 9 3 * 11 3 ar 10 12 r bo 6 4 1 3 * 11 CH3 caed ny 7 O O10 rbuc l 2 12 re ontas OH 8 1 (mdu yl e OH 7 O 2 O i ct r noas OH11 8 (mou r e OH inte) benzopyran ro or * 11 * CH3 ute benzopyran ) * * CH3 (The S enantiomer is three- to five-fold O O O O (Theas activeS enantiomer as the R is enantiomer.) three- to five-fold * chiral center 11-Hydroxywarfarin as active as the R enantiomer.) * chiral center 11-Hydroxywarfarin(active) Molecules 2018, 23, 2119 (active) 17 of 30 Figure 25. Metabolic pathways of warfarin. Figure 25. Metabolic pathways of warfarin.

N N N N N Cl N Cl 1 CYP2D6 1 N Cl N Cl UGT2B7 7-Hydroxychlorpromazine 7 1 3 CYP2D6 1 3 UGT2B7 S 5 HO 7 S 5 7-Hydroxychlorpromazine glucuronide 7 3 3 S 5 HO 7 S 5 glucuronide Chlorpromazine 7-Hydroxychlorpromazine Chlorpromazine 7-Hydroxychlorpromazine(major metabolite, (majormoderately metabolite, active) moderately active) Figure 26. Metabolic pathways of chlorpromazine. Figure 26. Metabolic pathways of chlorpromazine. Figure 26. Metabolic pathways of chlorpromazine. Metabolic aromatic-ring hydroxylation leading to the formation of equiactive products is Metabolic aromatic-ring hydroxylation leading to the formation of equiactive products is exemplifiedMetabolic by aromatic-ring the following: hydroxylation leading to the formation of equiactive products is exemplifiedexempliﬁed by by the the following: following: (a) The beta-blocker, propranolol, of the chemical class, aryloxypropanolamine (Figure 27); (a) The beta-blocker, propranolol, of the chemical class, aryloxypropanolamine (Figure 27); (a)(b) TheThe beta-blocker, β-hydroxy β propranolol,-methylglutaryl of the coenzyme chemical A class, reductase aryloxypropanolamine inhibitor, atorvastatin (Figure (used 27); in lowering β β (b)(b) ThebloodThe β-hydroxy -hydroxycholesterol β-methylglutaryl -methylglutaryllevel; Figure 28). coenzyme coenzyme A Areductase reductase inhibitor, inhibitor, atorvastatin atorvastatin (used (used in in lowering lowering bloodblood cholesterol cholesterol level; level; Figure Figure 28). 28 ). Metabolic-ring hydroxylation leading to enhancement of activity is exemplified by acetanilide to paracetamol,Metabolic-ringMetabolic-ring which hydroxylation hydroxylation was already lead leading discusseding to to enhancementenhancement in Section 5.1.1. of activity isis exempliﬁedexemplified byby acetanilide acetanilide to toparacetamol, paracetamol, which which was was already already discussed discussed in in Section Section 5.1.1 5.1.1..

Figure 27. Metabolic pathways of propranolol. Figure 27. Metabolic pathways of propranolol. Molecules 2018, 23, x FOR PEER REVIEWFigure 27. Metabolic pathways of propranolol. 17 of 29

HO COOH Dihydroxyheptanoic acid moiety * OH (statin pharmacophore) * F H

N HO 1 COOH 3 * * OH NH 5 O F H HO A N CYP3A4 3R,5S-o-Hydroxyatorvastatin C HO NH COOH B D * O * OH 3R,5S-A torvastatin F H (marketed enantiomer) N

NH OH O

3R,5S-p-Hydroxyatorvastatin Figure 28. Metabolism of atorvastatin. Figure 28. Metabolism of atorvastatin.

5.3.1. Metabolic Aromatic-Ring Hydroxylation Leading to Loss of Activity

Phenobarbital/Phenytoin The anticonvulsant drug, phenobarbital (Figure 18), was chosen as a representative of the barbiturate group of drugs because it is the only member that contains an aromatic benzene ring susceptible to metabolic hydroxylation. On the other hand, the anticonvulsant drug, phenytoin (Figure 18), was selected for its similarity to phenobarbital with respect to chemical structure, pharmacologic activity, and even mechanism of action. Phenytoin additionally contains two benzene rings with an identical chemical environment. Both phenobarbital and phenytoin are metabolized by aromatic-ring hydroxylation (Figure 18), a process that led to a loss of pharmacologic activity [64– 67]. The CNS depressant activity of barbiturates (sedative, hypnotic, and anticonvulsant) and its termination are mainly dependent on the drug lipophilicity, which is imparted by the aromatic rings and the alkyl groups [68]. Lipophilicity helps the barbiturates to cross the blood–brain barrier, exert their effects, and again facilitate their distribution to other tissues, thus reducing their effective concentrations at the brain’s gamma-aminobutyric acid receptors (GABA receptors). This redistribution process of the barbiturates is considered a deactivation process. In addition, metabolism plays a role in the deactivation of barbiturates. All barbiturates contain two alkyl groups at carbon 5 of the barbituric acid ring (Figure 18) with the exception of phenobarbital, which contains an alkyl group (ethyl) and a phenyl group. All the alkyl groups in barbiturates are metabolized by oxidation in phase I at the ω or ω-1 carbons to either primary or secondary alcohols, respectively. The primary alcoholic groups may further be oxidized to carboxyl (COOH) groups. On the other hand, the phenyl group in phenobarbital is metabolically oxidized to a phenolic group at the favored para position (Figure 18). All such hydrophilic functionalities are detrimental to the essential hydrophobicity of the alkyl and phenyl groups, and thus, to the ability of the resulting metabolites to cross the blood–brain barrier. Enhancement of the water solubility of the barbiturate metabolites and their subsequent, fast elimination is a further cause of termination of pharmacologic activity resulting from the introduction of hydrophilic functionalities. Even more, the phase I metabolites, either carboxylic or phenolic, may further be conjugated in phase II to glucuronides and/or sulfates (Figure 18) with a consequent further increase in water solubility, elimination, and termination of activity due to a reduction in the effective concentration at the receptor. Despite the fact that the pharmacokinetics of the barbiturates play a major role in their deactivation, a pharmacodynamic dimension cannot be excluded: the introduction of a hydrogen- bonding functionality, such as the hydroxy (OH) group, on an essentially hydrophobic site will most probably negatively affect binding to the GABAA receptor, resulting in a loss of pharmacologic

Molecules 2018, 23, 2119 18 of 30

5.3.1. Metabolic Aromatic-Ring Hydroxylation Leading to Loss of Activity

Phenobarbital/Phenytoin The anticonvulsant drug, phenobarbital (Figure 18), was chosen as a representative of the barbiturate group of drugs because it is the only member that contains an aromatic benzene ring susceptible to metabolic hydroxylation. On the other hand, the anticonvulsant drug, phenytoin (Figure 18), was selected for its similarity to phenobarbital with respect to chemical structure, pharmacologic activity, and even mechanism of action. Phenytoin additionally contains two benzene rings with an identical chemical environment. Both phenobarbital and phenytoin are metabolized by aromatic-ring hydroxylation (Figure 18), a process that led to a loss of pharmacologic activity [64–67]. The CNS depressant activity of barbiturates (sedative, hypnotic, and anticonvulsant) and its termination are mainly dependent on the drug lipophilicity, which is imparted by the aromatic rings and the alkyl groups [68]. Lipophilicity helps the barbiturates to cross the blood–brain barrier, exert their effects, and again facilitate their distribution to other tissues, thus reducing their effective concentrations at the brain’s gamma-aminobutyric acid receptors (GABA receptors). This redistribution process of the barbiturates is considered a deactivation process. In addition, metabolism plays a role in the deactivation of barbiturates. All barbiturates contain two alkyl groups at carbon 5 of the barbituric acid ring (Figure 18) with the exception of phenobarbital, which contains an alkyl group (ethyl) and a phenyl group. All the alkyl groups in barbiturates are metabolized by oxidation in phase I at the ω or ω-1 carbons to either primary or secondary alcohols, respectively. The primary alcoholic groups may further be oxidized to carboxyl (COOH) groups. On the other hand, the phenyl group in phenobarbital is metabolically oxidized to a phenolic group at the favored para position (Figure 18). All such hydrophilic functionalities are detrimental to the essential hydrophobicity of the alkyl and phenyl groups, and thus, to the ability of the resulting metabolites to cross the blood–brain barrier. Enhancement of the water solubility of the barbiturate metabolites and their subsequent, fast elimination is a further cause of termination of pharmacologic activity resulting from the introduction of hydrophilic functionalities. Even more, the phase I metabolites, either carboxylic or phenolic, may further be conjugated in phase II to glucuronides and/or sulfates (Figure 18) with a consequent further increase in water solubility, elimination, and termination of activity due to a reduction in the effective concentration at the receptor. Despite the fact that the pharmacokinetics of the barbiturates play a major role in their deactivation, a pharmacodynamic dimension cannot be excluded: the introduction of a hydrogen-bonding functionality, such as the hydroxy (OH) group, on an essentially hydrophobic site will most probably negatively affect binding to the GABAA receptor, resulting in a loss of pharmacologic activity. Furthermore, by replacing a hydrogen atom in the benzene ring, the hydroxyl group will create a steric effect and increase the metabolite molecule size, factors that are detrimental to the proper metabolite–receptor interactions, and thus, to a loss of pharmacologic activity. The two aromatic rings in phenytoin have identical chemical environments, and only one of them is hydroxylated, which is consistent with the rules of metabolic aromatic-ring hydroxylation. The same reasoning that applies to the loss of activity of the phenolic metabolite of phenobarbital discussed above should apply to the loss of activity of the phenolic metabolite of phenytoin. Further reasoning is considered in the discussion section.

Benzodiazepines: Diazepam and Estazolam The benzodiazepines are CNS depressants used as minor tranquilizers, sedatives, hypnotics, and anticonvulsants; in these respects, they largely supersede the barbiturates. Their mechanism of action involves binding to GABAA receptors [69–71]. The benzodiazepines are metabolized through several phase I oxidative reactions, with some followed by phase II conjugative reactions. The metabolic pathways of the two benzodiazepine representative members, diazepam [72,73] and estazolam [74,75], are shown Figures 19 and 20, respectively, with only aromatic-ring hydroxylation discussed in this section. The other metabolic pathways of diazepam and estazolam are discussed where relevant. Molecules 2018, 23, 2119 19 of 30

Due to favorable electronic and steric structural environments, diazepam and estazolam are about the only two members of the clinically used benzodiazepines to undergo metabolic aromatic-ring hydroxylation at the 40 position (Figures 19 and 20). In most of the other members, metabolic 40-hydroxylation is possibly disfavored by the presence of an electron-withdrawing halo group at position 20 (fluoro in flurazepam, flunitrazepam, and quazepam, and chloro in triazolam and clonazepam; for the numbering of the benzodiazepine ring system, reference should be made to the structure of diazepam in Figure 19). The 4’-hydroxylation of both diazepam [72,73] and estazolam [74,75] resulted in inactive metabolites. Foye (2013) attributed the loss of sedative, hypnotic effects of 4’-hydroxyestazolam to two factors [71]. The first factor is of a pharmacodynamic nature. It is explained by the 4’-hydroxy group weakening optimal binding of the aromatic ring to the GABAA receptor via a steric effect: the hydroxyl group is substantially bulkier than the hydrogen atom. The expected result is, therefore, decreased receptor affinity and drug potency. The second factor is of a pharmacokinetic disposition, and results from decreased hydrophobicity (i.e., increased hydrophilicity), which results in the decrease of the effective concentrations of the circulating 40-hydroxy metabolites due to enhanced polarity, water solubility, and elimination, as per se and as glucuronide conjugates. In the absence of reports regarding the loss of activity of 4’-hydroxydiazepam, an analogy may be extrapolated from that of 40-hydroxyestazolam. In contrast to 4’ hydroxylation, metabolic hydroxylation at position 3 of the diazepine ring in diazepam (Figure 19) does not affected activity; rather, it introduces a pharmacokinetic dimension: the 3-hydroxy metabolite is more hydrophilic and is subject to glucuronide conjugation with subsequent enhanced rate of elimination, and hence, a shorter duration of action than diazepam. These observations tend to consolidate a major pharmacodynamic role of the 4’-aromatic-ring hydroxyl group on causing loss of activity of diazepam, as well as of estazolam. The plausible explanation is that the introduction of the hydrogen-bonding group (the hydroxy) into an essentially pharmacophoric hydrophobic moiety (the benzene ring) is detrimental to the optimal GABAA receptor binding. A halo group at position 2’ of the benzodiazepine backbone (ring C, Figure 19) serves three purposes: it adds a welcomed hydrophobicity to the drug, disfavors metabolic ring hydroxylation, and imparts a conformation-locking effect on the aromatic ring through a steric effect. The consequence of these effects could be enhanced selectivity on drug–receptor interaction, leading to higher efficacy and possibly higher potency. Increased hydrophobicity would tend to enhance blood–brain barrier penetration, and therefore, increased access to the GABAA receptor. In the above context, it would be worthwhile to investigate the effect of another halo group at position 6’ (Figure 19) on the conformation locking of the aromatic rings in analogy with the NSAID pair, fenoprofen/diclofenac. In diclofenac (Figure 21), the two ortho-positioned chloro groups resulted in a restricted conformation with consequent enhanced selectivity, efficacy, and potency compared to fenoprofen, in which the two chloro groups are absent [76]. With respect to stereochemistry, both diazepam and estazolam are achiral. However, metabolic hydroxylation at C3 of diazepam and C4 of estazolam (Figures 19 and 20, respectively) results in chiral metabolites. The significance of such chirality as related to metabolite activity is discussed in Part 2 of this review series, which deals with metabolic aliphatic-ring hydroxylation.

NSAIDs The chemical classes of NSAIDs were discussed in Section 5.1.3.

(1) Diclofenac

Diclofenac is a phenylacetic acid NSAID. It was developed as a variant of fenoprofen by introducing two ortho-positioned chloro groups in the anilino-aromatic ring to restrict its free rotation [76]. This restriction of rotation increases selectivity, and hence, potency with respect to fenoprofen. The metabolism of diclofenac shown in Figure 21[ 77] represents one of the anomalies of Molecules 2018, 23, 2119 20 of 30 aromatic-ring hydroxylation in that hydroxylation occurs at the meta position of two chloro groups. The hydroxy metabolites are pharmacologically inactive. In accounting for the structure–activity relationship of diclofenac, Foye (2013) [78] proposed that the function of the two ortho chloro groups was to force the anilino-phenyl ring out of the plane of the phenylacetic acid portion. Such twisting, as proposed by Foye (2013) [78], is important in the binding of diclofenac to the active site of COX. The introduction of a hydroxy group in the anilino-phenyl group would create a hydrogen-bonding characteristic, which would weaken or hinder the necessary twisting, thus resulting in an attenuation or loss of pharmacologic activity.

(2) Ketorolac

Ketorolac (Figure 22) is a pyrrole-acetic acid derivative structurally related to indomethacin and tolmetin. It is metabolized to p-hydroxyketorolac (Figure 22), which is inactive. Both the carboxy and phenolic hydroxy groups are further metabolized via glucuronide conjugation to give pharmacologically inactive products [79,80]. In analogy to the previously discussed cases, the loss of activity of 4-hydroxy ketorolac may be attributed to both pharmacodynamic and pharmacokinetic effects. Pharmacodynamic effects are elaborated upon in the discussion section. Ketorolac contains a chiral center as designated by the asterisk in Figure 22. Its enantiomers differ in their pharmacodynamic effect: while the S-enantiomer acts as both a COX1/COX2 inhibitor and an analgesic, the R-enantiomer only retains the analgesic activity [80]. The stereochemical basis of the loss of activity caused by metabolic aromatic-ring hydroxylation of ketorolac is not known.

(3) Flurbiprofen

Flurbiprofen is an arylpropionic acid COX1/COX2-inhibitor NSAID. It is mainly metabolized by aromatic-ring hydroxylation, as shown in Figure 23, with loss of activity [81]. The metabolism of flurbiprofen shows a rather interesting pattern in that a catechol ring is formed in which the 40-position is anomalously methylated to yield a methoxy group with reduced polarity. This metabolic route is reminiscent of that of adrenaline, which is metabolized by the methylation of the para-hydroxy group by the enzyme catechol-O-methyl transferase (COMT) [82]. Furthermore, the metabolic double hydroxylation of flurbiprofen to yield 3’,4’-dihydroxy flurbiprofen is also an anomaly of metabolic-ring hydroxylation. We recall metabolic aromatic-ring dihydroxylation in the same molecule is generally disfavored [65,66]. Similar to naproxen, flurbiprofen interacts with the COX active site through ion pairing involving the carboxylate group and van der Waals contacts involving the α-methyl and phenyl groups. The metabolically introduced hydroxyl groups on the aromatic ring will be detrimental to the hydrophobic-pocket fit, thus leading to a loss of activity. Other factors are considered in the discussion section. Being an arylpropionic acid derivative, flurbiprofen is a chiral drug as indicated by the asterisk in Figure 23. As for all members of the arylpropionic acid NSAID subclass, the anti-inflammatory (COX-inhibiting) activity resides in S-(+)-flurbiprofen; R-(−)-flurbiprofen is expected to be the enantiomer with analgesic activity. Whether metabolic aromatic hydroxylation of flurbiprofen abolishes both anti-inflammatory and analgesic activities or exhibits selectivity is not known.

Miscellaneous: Warfarin Warfarin (Figure 25) is an anticoagulant drug used as a prophylactic in preventing thrombus formation in patients who are at high risk of developing thromboembolic disease. Warfarin is chiral with the S-enantiomer having three- to ﬁve-fold the activity of the R-enantiomer [83]. Warfarin contains two aromatic moieties, a benzopyran and a phenyl, in addition to a 2-butanone chain (Figure 25). The major metabolic pathway of warfarin is through the hydroxylation of the benzene ring of the benzopyran moiety, in addition to a minor route through the reduction of the side-chain keto group to a secondary alcohol (Figure 24)[84–87]. S-warfarin is metabolized by the CYP2C9 isoenzyme to S-7-hydroxywarfarin, while R-warfarin is metabolized by CYP1A2, CYP3A4, and CYP2C19 isoenzymes Molecules 2018, 23, 2119 21 of 30 to R-6, R-7, R-8, and R-10 hydroxy warfarins [88]. The acidic hydroxy group at C4, being in conjugation with the benzene ring, possibly explains the preference of metabolic hydroxylation of the benzopyran ring over the phenyl group since it increases the electron density toward hydroxylation through a positive inductive effect. Furthermore, both warfarin enantiomers are metabolized through reduction of the side-chain keto group by carbonyl reductase to a secondary alcohol (Figure 25)[85–88]. It was observed that the hydroxy group introduced metabolically on the aromatic ring led to a loss of anticoagulant activity, while the hydroxy group resulting from side-chain keto reduction resulted only in attenuation of activity [85]. In addition, the metabolic reduction of the side-chain keto group in warfarin created a second chiral center. Such a state of affairs may impact on the pharmacodynamic and pharmacokinetic effects of the 11-hydroxywarfarin metabolite (Figure 25). Keto-group reduction and pharmacologic activity are the subject of alcoholic metabolites to be discussed in Part 2 of this review series.

5.3.2. Metabolic Aromatic-Ring Hydroxylation Resulting in Attenuation of Pharmacologic Activity

Chlorpromazine Chlorpromazine (Figure 26) is a major tranquilizer used as an antipsychotic. It is metabolized in humans via two major routes [89–93]: (a) aromatic-ring hydroxylation at position 7 to a moderately active metabolite (Figure 26); and (b) sulfoxidation to an inactive metabolite. A minor deactivating route through N-demethylation also occurs. It should be noted that the metabolic hydroxylation of chlorpromazine is in accordance with the rule that groups with negative inductive effects, such as chloro, deactivating the ring to hydroxylation.

5.3.3. Aromatic-Ring Hydroxylation Resulting in Parent-Drug Equiactive Metabolites

Propranolol Propranolol (Figure 27) is an aryloxypropanolamine β-adrenoceptor blocker used as an antihypertensive and antiangina agent. As shown in Figure 26, it is metabolized in humans to three major metabolites, two of which are inactive and one of which is as active as the parent drug. In naphthoyloxyacetic acid (metabolite I, Figure 27), the pharmacophore is ruptured, leading to a loss of activity. In metabolite II, glucuronide conjugation of the side-chain hydroxy group led to a loss of activity, while in 4-hydroxypropranolol (metabolite III), activity was maintained. Furthermore, 4-hydroxypropranolol is metabolized in phase II to the inactive glucuronide conjugate [94,95]. The metabolism of propranolol directs attention to two interesting points: (a) glucuronic-acid conjugation of both alcoholic and phenolic hydroxy groups leads to a loss of pharmacologic activity, a phenomenon that is true for most cases; and (b) alkoxy groups are aromatic-ring activators and para-directors in metabolic aromatic-ring hydroxylation. Propranolol is a monochiral drug as indicated by the asterisk in Figure 27. For all the monochiral aryloxypropanolamines, the β-adrenoceptor-blocking activity resides mainly in the S-(−)-enantiomer, with the R-(+)-enantiomer exhibiting minimal activity [96]. Despite this fact, however, all the clinically used aryloxypropanolamine β-adrenoceptor-blockers, except S-(−)-timolol, are used as racemates. When the metabolic change occurs at a group distant from the chiral center, as in 4-hydroxypropranolol, extrapolation of the absolute enantiomeric designation from the parent drug to the metabolite is possible. Accordingly, 4-hydroxypropranolol may be assigned the S designation. However, optical activity designation and pharmacological activity may not be extrapolated from the parent drug and are only subjects of experimental veriﬁcation.

Atorvastatin Atorvastatin (Figure 28) is an HMG-CoA reductase inhibitor used in lowering blood cholesterol and triglyceride levels. Atorvastatin is metabolized by CYP3A4 hydroxylation at the ortho- or Molecules 2018, 23, 2119 22 of 30 para-position at ring D, as shown in Figure 28[ 97,98]. Being electron withdrawing, both the fluoro group and the pyrrole ring (C) disfavor metabolic hydroxylation of rings A and B, respectively. Accordingly, in accordance with the rule, hydroxylation takes place at the least hindered benzene ring, i.e., ring D. The two metabolites are equiactive with the parent drug and account for about 70% of its overall circulating activity [97,98]. As designated by the asterisks in Figure 28, atorvastatin is a bichiral drug, and hence, exists in four enantiomeric forms: 3R5R, 3R5S, 3S5R, and 3S5S [99]. The marketed enantiomer is 3R5S [100]. The hydroxy metabolites of atorvastatin exist in the same enantiomeric forms as the parent drug. In explaining the equiactivity of the atorvastatin hydroxy metabolites, the molecule of atorvastatin can be dissected into two parts: the dihydroxyheptanoic acid moiety and the aromatic ring system with its substituents. Dr. Philip Portoghese (1988) [101], a medicinal chemist from the University of Minnesota, developed a concept called “message address,” which conceptually breaks a drug molecule up into two components: one, which “finds” the active site (the address), and the other, which actually delivers the drug’s chemical message. In atorvastatin, the dihydroxyheptanoic-acid moiety represents the message, while the aromatic-ring system with substituents represents the address. When the address is substantially large, a small-group metabolic change is not expected to result in a significant impact on its role. This is true for atorvastatin, which contains a four-ring system that mainly interacts with the active site of the enzyme via hydrophobic binding. Since the term “pharmacophore” is mostly used in the pharmacodynamics of drug action, we propose an adaptation of Dr. Portoghese’s concept by using the terms “primary pharmacophore” and “auxiliary (logistic) pharmacophore” as equivalent terms to “message” and “address”, respectively. By binding to the receptor, the auxiliary (logistic) pharmacophore will facilitate the anchoring of the primary pharmacophore in the proper orientation in the receptor or enzyme active cavity. The primary pharmacophore will then compete with the physiologic substrate for the binding sites in the receptor or enzyme active site.

Phenylbutazone Two metabolites of phenylbutazone (Figure 24), which were isolated from human urine, possess some of the pharmacological activities of the parent drug. Metabolite I (oxyphenbutazone), formed by aromatic-ring hydroxylation, has the potent antirheumatic and sodium-retaining effects of phenylbutazone; it was developed into a drug of its own right. On the other hand, metabolite II, formed by the oxidation of the ω-1 carbon of the butyl side chain, also possesses reduced sodium-retaining properties, but it is a considerably more potent uricosuric agent than phenylbutazone [102,103]. Phenylbutazone binds to and deactivates prostaglandin H synthase and prostacyclin synthase through peroxide (H2O2)-mediated deactivation. The reduced production of prostaglandin leads to reduced inﬂammation in the surrounding tissues [103]. It is also pertinent to note that γ-hydroxyphenylbutazone, which results from ω-1 hydroxylation of the butyl side chain in phenylbutazone, is devoid of anti-inﬂammatory activity [104]. Metabolic, aliphatic hydroxylation and pharmacologic activity of the resulting metabolites is the subject of Part 2 of this review series. Despite phenylbutazone being a monochiral drug, the relationship between its stereochemistry and activity does not receive much interest, possibly because it was developed and used at a time when stereoselectivity of drug action was not a well-developed science.

5.4. Discussion of Metabolic Aromatic-Ring Hydroxylation For the drug cases reviewed in this section, except for diclofenac, the structural features, either electronic or steric, set above for the occurrence of aromatic-ring hydroxylation, conform well to the rule of thumb. Loss, decrease, or retention of pharmacologic activity upon aromatic-ring hydroxylation in the cited cases may reflect the status of the ring in the parent drug regarding its mechanism of interaction with the receptor. When hydrophobic binding of the aromatic ring with the receptor is essential for Molecules 2018, 23, 2119 23 of 30 activity, introduction of the hydrophilic hydrogen-bonding hydroxy group will compromise the fit and will not be tolerated. The established hydrogen bonding may force the ring out of the plane of interaction with the receptor; the result will be loss of activity. The aromatic-ring dislodging is also assisted by a steric effect caused by the bulkier hydroxyl group in the metabolite relevant to the hydrogen atom in the parent drug. Furthermore, an increase in size and surface area of the metabolite caused by the hydroxyl group relevant to the parent drug may be synergistic factors in the poor fit of the aromatic ring in its hydrophobic pocket in the receptor. This was the case with phenobarbital and phenytoin (Figure 18), diazepam, (Figure 19), estazolam (Figure 20), NSAIDs (Figures 21–23), and warfarin (Figure 25). Furthermore, the phase II glucuronide conjugation of the aromatic hydroxy group is an important factor in causing loss of activity of the metabolite. It considerably enhances metabolite clearance, and accordingly, reduces its effective concentration at the receptor or enzyme active cavity. When aromatic-ring hydroxylation results in decreased activity, as in 7-hydroxychlorpromazine (Figure 26), three inferences present themselves as possible explanations of the observation: (a) the hydroxylated ring is auxiliary pharmacophoric; (b) the hydroxy group results in an increase in the optimal molecular size in the metabolite relative to the parent drug; (c) the hydroxy group weakens optimal binding of the aromatic ring to the receptor via a steric effect relevant to the hydrogen atom. In addition, a reduction in the effective concentration of the hydroxy metabolite at the receptor through glucuronide conjugation may play an important role. When the hydroxy metabolite is equiactive with the parent drug, two inferences can be tentatively made. Firstly, the hydroxylated aromatic ring is auxiliary, i.e., it plays the role of the address. This is the case with the statin drug, atorvastatin (Figure 28), where the three-aromatic-ring system is auxiliary pharmacophoric, and therefore, is not involved in essential binding to the enzyme [105]. However, the role of the three-aromatic-ring system is logistic, that of proper anchoring of the drug in the enzyme active cavity for optimal interaction of the primary pharmacophoric groups with the enzyme to take place. The second inference is associated with 4-hydroypropanolol (Figure 27), the equiactive metabolite of propranolol. Propranolol is a nonselective β1/β2-adrenoceptor blocker; it belongs to the aryloxypropanolamine chemical class. In this class of compounds, a hydrophilic amide substitution at position 4 of the aromatic ring imparts β1 antagonistic selectivity [106], such as in atenolol and practolol (Figure 29). On the other hand, a hydrophilic hydroxy substitution at the same position reverses the activity altogether, i.e., from antagonistic to agonistic, such as in prenalterol (Figure 29)[107]. It can, hence, be concluded that the nature of the hydrophilic group substitution at position 4 of the aryloxypropanolamines significantly dictates the pharmacologic outcome of the β1-receptor interaction. Based on the above facts, it may be inferred that 4-hydroxypropranolol is a tentative β1-adrenoceptor agonistMolecules 2018 pending, 23, x FOR experimental PEER REVIEW verification. 23 of 29

OH OH 6 H 1 ON 6 H O 5 * 1 ON O 5 * 2 2 H2N 4 3 H3C N 4 Atenolol H 3 Hydrophilic Practolol Hydrophilic amide group (Selective beta-1- (Selective beta-1- amide group adrenoceptor blocker) adrenoceptor blocker)

OH 6 H 1 ON 5 * 2 HO 4 3 Prenalterol (Beta-1-adrenoceptor agonist)

Figure 29. Structures of atenolol and practolol.

The atorvastatin equiactive hydroxy metabolites furnish a useful inference: pharmacologic equiactivity of metabolites relative to the parent drug occurs when the metabolic functionalization takes place at the “address” or “auxiliary pharmacophore”. We will provide further examples of this phenomenon in Part 2 of this review series. Of the NSAIDs, phenylbutazone stands in a class of its own in that its mechanism of action does not involve the inhibition of COX, but rather the inhibition of prostaglandin H synthase. As shown in this section and in Section 5.1.4, a hydroxy group on the aromatic rings of arylalkanoic-acid COX1/COX2-inhibitor NSAIDs is detrimental to their pharmacologic activity. This is in contrast to phenylbutazone whose aromatic-hydroxy metabolite (oxyphenbutazone, Figure 24) is equiactive with the parent drug and was developed into an anti-inflammatory drug of its own right. The different mechanisms of action, and accordingly, the varying sites of drug action involved may explain the disparity between the activities of the hydroxyl metabolites of phenylbutazone and the arylalkanoic-acid NSAIDs. While pharmacodynamics may handsomely explain the pharmacological activity of drug hydroxy metabolites relative to the parent drugs, the role of the pharmacokinetics of these metabolites should not be excluded. In the cases where the pharmacologic activity of the hydroxy metabolite is either attenuated or lost relative to the parent drug, pharmacokinetic factors may come into perspective in two aspects. Firstly, the hydroxy metabolites are rapidly cleared by phase II conjugation, thus aiding in the termination of their action. Secondly, the hydroxy metabolites do not readily penetrate target tissues due to a reduction in membrane permeability caused by an increase in polar surface area, a limitation that affects their active concentrations at the target site [108].

6. Racemic Drugs versus Enantiopure Drugs Of all the chiral drugs presented as examples in this review, only levorphanol, S-(+)-naproxen, and 3R5S-atorvastatin are used clinically in enantiopure forms; the rest of the drugs are used as racemates. The use of a chiral drug as a racemate does not necessarily imply that the constituent enantiomers are identical in their pharmacodynamic, pharmacokinetic, or toxicologic properties. Difficulties in preparing the pure enantiomers via the separation from racemates or via enantioselective synthesis may be prohibitive factors due to a lack of technology and/or high cost. In some instances, however, despite the feasibility of separating or synthesizing the eutomer of a chiral drug, big international pharmaceutical companies seem not to be interested in the practice and they keep to the racemates. Certainly, they have their undeclared reasons for this. Nonetheless, the differences between the pharmacologic properties of the enantiomers of a chiral drug may seem insignificant to warrant their costly separation from the racemates or stereoselective synthesis. S-(+)- naproxen represents an example of chiral-switch drugs. A chiral-switch drug may be defined as “an enantiopure drug whose racemate was once used or is currently used clinically. On the other hand, levorphanol and 3R5S-atorvastatin represent examples of what may be described as “from-onset enantiopure drugs”. Some scientists [109], though acknowledging the importance of enantiopure drugs in advancing pharmacotherapy, expressed their reservations on the production and use of

Molecules 2018, 23, 2119 24 of 30

The atorvastatin equiactive hydroxy metabolites furnish a useful inference: pharmacologic equiactivity of metabolites relative to the parent drug occurs when the metabolic functionalization takes place at the “address” or “auxiliary pharmacophore”. We will provide further examples of this phenomenon in Part 2 of this review series. Of the NSAIDs, phenylbutazone stands in a class of its own in that its mechanism of action does not involve the inhibition of COX, but rather the inhibition of prostaglandin H synthase. As shown in this section and in Section 5.1.3, a hydroxy group on the aromatic rings of arylalkanoic-acid COX1/COX2-inhibitor NSAIDs is detrimental to their pharmacologic activity. This is in contrast to phenylbutazone whose aromatic-hydroxy metabolite (oxyphenbutazone, Figure 24) is equiactive with the parent drug and was developed into an anti-inﬂammatory drug of its own right. The different mechanisms of action, and accordingly, the varying sites of drug action involved may explain the disparity between the activities of the hydroxyl metabolites of phenylbutazone and the arylalkanoic-acid NSAIDs. While pharmacodynamics may handsomely explain the pharmacological activity of drug hydroxy metabolites relative to the parent drugs, the role of the pharmacokinetics of these metabolites should not be excluded. In the cases where the pharmacologic activity of the hydroxy metabolite is either attenuated or lost relative to the parent drug, pharmacokinetic factors may come into perspective in two aspects. Firstly, the hydroxy metabolites are rapidly cleared by phase II conjugation, thus aiding in the termination of their action. Secondly, the hydroxy metabolites do not readily penetrate target tissues due to a reduction in membrane permeability caused by an increase in polar surface area, a limitation that affects their active concentrations at the target site [108].

6. Racemic Drugs versus Enantiopure Drugs Of all the chiral drugs presented as examples in this review, only levorphanol, S-(+)-naproxen, and 3R5S-atorvastatin are used clinically in enantiopure forms; the rest of the drugs are used as racemates. The use of a chiral drug as a racemate does not necessarily imply that the constituent enantiomers are identical in their pharmacodynamic, pharmacokinetic, or toxicologic properties. Difficulties in preparing the pure enantiomers via the separation from racemates or via enantioselective synthesis may be prohibitive factors due to a lack of technology and/or high cost. In some instances, however, despite the feasibility of separating or synthesizing the eutomer of a chiral drug, big international pharmaceutical companies seem not to be interested in the practice and they keep to the racemates. Certainly, they have their undeclared reasons for this. Nonetheless, the differences between the pharmacologic properties of the enantiomers of a chiral drug may seem insignificant to warrant their costly separation from the racemates or stereoselective synthesis. S-(+)-naproxen represents an example of chiral-switch drugs. A chiral-switch drug may be defined as “an enantiopure drug whose racemate was once used or is currently used clinically. On the other hand, levorphanol and 3R5S-atorvastatin represent examples of what may be described as “from-onset enantiopure drugs”. Some scientists [109], though acknowledging the importance of enantiopure drugs in advancing pharmacotherapy, expressed their reservations on the production and use of chiral-switch drugs such as levocetirizine, esomeprazole, and escitalopram on the basis of the insignificant therapeutic advantages they present. To the list, we may add S-(+)-ibuprofen, (a chiral-switch drug marketed in some countries as dexibuprofen). The addition is made on the basis of the metabolic inversion of the inactive R-(−)-enantiomer to the active antipode, as well as the current marketing of the racemate by big international pharmaceutical companies.

Glucuronide Conjugation: Prevalence and Effect on Pharmacologic Activity Glucuronide conjugation is the most common phase II metabolic process [110], though certain structural features control its occurrence. It occurs with almost all aromatic hydroxy (arenolic) groups, most carboxyl groups, unhindered alcoholic hydroxy groups, and a few amino and sulfhydryl groups. Because of its relatively big size, glucuronic acid may not have easy access to active hydrogen-containing groups that are awkwardly situated in a molecule, i.e., sterically hindered groups. Molecules 2018, 23, 2119 25 of 30

For instance, tertiary alcoholic groups such as 14-hydroxy in oxycodone (Figure5) and 1 0-hydroxy in tramadol (Figure6) are sterically hindered, and hence, are not susceptible to glucuronide conjugation. With the exception of codeine glucuronide and morphine-6-glucuronide, glucuronide conjugation led to a loss of activity in all the cases presented in this review. With three hydroxy moieties and a completely ionized carboxyl moiety at physiologic pH, the glucuronide group considerably increases metabolite hydrophilicity, water solubility, elimination, and termination of action. In the end, the termination of activity is a consequence of the reduction in the effective concentration of the glucuronide conjugate at the receptor. With this pharmacokinetic concept of loss of activity for most drug glucuronide conjugates, the door is open only to pharmacodynamic speculation to explain the activity of codeine glucuronide and morphine-6-glucuronide.

7. Conclusions The hydroxy group is the most common hydrophilic group produced by metabolic functionalization in drug molecules. Aromatic hydroxy groups result in one of two ways: the O-dealkylation of aralkoxy groups or the hydroxylation of the aromatic ring. The O-dealkylation of aralkoxy groups in drug molecules is an invariably predictable metabolic route. On the other hand, metabolic aromatic-ring hydroxylation is governed by electronic and steric factors prevailing in the ring. The pharmacologic activity of the resulting arenolic metabolites resulting from both processes depends on the site of drug action (i.e., the receptor), as well as on the pharmacophoric or auxophoric status of the aromatic ring to which the alkoxy or hydroxy group is bonded. In general terms, phase I metabolic functionalization may reveal the status of a group in a drug molecule, whether primary or auxiliary pharmacophoric or auxophoric. Moreover, if there is previous knowledge of the pharmacophoric or auxophoric statuses of the rings, then the effect of the metabolically formed hydroxy group on the activity of the metabolite may be predicted. In addition to pharmacodynamic effects, the attenuation or loss of activity of polar metabolites may be explained by pharmacokinetic effects, which, by enhancing elimination, lead to a reduction in metabolite effective concentration at the receptor. When more active or equiactive metabolites showed favorable pharmacodynamic and pharmacokinetic properties, they were developed into drugs of their own rights. Nevertheless, not all equiactive metabolites were developed into drugs of their own rights, and hence, may be classified as drug-action-extension forms. Since, for the chiral drugs presented in this review, metabolic functionalization occurred at sites distant from the chiral center, absolute enantiomer configuration (as R or S) may be extrapolated from the parent drug to the hydroxy metabolite. However, optical activity designations (dextro or levo), as well as pharmacological activity, are the subject of experimental verification.

Funding: This research received no external funding. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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