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1 Review Article 2 Linking Aromatic Hydroxy Metabolic Functionalization of Drug Molecules to 3 Structure and Pharmacologic Activity

4 Babiker M. El-Haj 1,*, Samrein B.M. Ahmed 2, Mousa A. Qarawi 2,, Heyam S.Ali4

5 1 Department of Pharmaceutical Sciences, College of Pharmacy, Ajman University, UAE; b.elhag.ajman.ac.ae 6 2 College of Medicine, Sharjah Institute for Medical Research, University of Sharjah, UAE; 7 samreinahmed@sharjah. ac.ae 8 3 Department of Pharmaceutical Sciences, College of Pharmacy, Ajman University, UAE; 9 [email protected] 10 4 Department of Pharmaceutics, Dubai Pharmacy College, UAE; [email protected] 11 12 * Correspondence: e-mail: b.elhag.ajman.ac.ae; Tel.: 0071567204338 13

14 Abstract: Drug functionalization through formation of hydrophilic groups is the norm in phase I 15 of drugs for modification of drug action. The reactions involved are mainly oxidative 16 catalyzed mostly by CYP isoenzymes. The ring, as phenyl or fused with other rings, is the 17 most common hydrophobic pharmacophoric moiety in drug molecules. On the other hand the 18 (mainly methoxy) bonded to the benzene ring assumes an important and sometimes 19 essential pharmacophoric status in some drug classes. Upon metabolic oxidation, both moieties, i.e. 20 the benzene ring and the alkoxy group, produce hydroxy groups; the products are arenolic in 21 nature. Through a pharmacokinetic effect, the hydroxy group enhances the water and 22 elimination of the metabolite with the consequent termination of drug action. However, through 23 bonding, the hydroxy group may modify the pharmacodynamics of the interaction of the 24 metabolite with the site of parent drug action (i.e. the receptor). Accordingly, the expected 25 pharmacologic outcome will be enhancement, retaining, attenuation, or loss of activity of the 26 metabolite relative to the parent drug. All the above issues have been presented and discussed in 27 this review using selected members of different classes of drugs with inferences regarding 28 mechanisms, drug design and drug development.

29 Keywords: Aromatic hydroxy metabolites; arenolic drug metabolites; metabolic O-dealkylation; 30 metabolic aromatic ring ; primary and auxiliary pharmacophores; auxophores; 31 metabolic modification of drug activity.

32 Introduction 33 Phase I metabolism of drugs (also known as the non-synthetic or functionalization phase) is 34 mainly brought about by microsomal by adding hydrophilicity to hydrophobic moieties in 35 drug molecules. In most cases, this is attained metabolically by revealing or introducing the 36 hydrophilic hydrogen bonding hydroxyl group. Generally, metabolic interference may take place at 37 pharmacophoric and/or auxophoric groups. Accordingly, the location in a drug molecule at which 38 the metabolic change takes place may determine the pharmacologic outcome of the metabolite. 39 In any case, the resulting drug metabolite may be pharmacologically inactive, less active, 40 equiactive, or even more active with respect to the parent drug. In other cases, an inactive parent 41 drug is metabolically converted to the active form, in which case the inactive parent drug is known 42 as ‘prodrug.’ Cases where the metabolic changes are intermediary in drug activation are also known. 43 For some drug metabolites, the pharmacologic outcomes - pharmacodynamic, 44 pharmacokinetic, and toxicologic - have proven to be substantially more favorable compared to their 45 parent drugs. In such cases, the metabolites have been developed into drugs of their own rights. In 46 this review, such drug metabolites will be referred to as “metabolite drugs”. 47 Generally, in drug development, the pharmacophoric or auxophoric roles of functional groups 48 in drug compounds or drug prototypes are modified in the synthetic laboratory in some ways.

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49 Introduction of a group in the framework, reduction of a group to framework status, or replacement 50 of a group by another are the main strategies followed. Through biotransformation, the body may 51 play an analogous in vivo role by changing pharmacophoric and/or auxophoric groups into others 52 or by introducing groups at certain positions of the drug molecule with concomitant changes in 53 pharmacodynamic and/or pharmacokinetic properties of the metabolite relative to the parent drug. 54 Such metabolic changes of a drug molecule may lead to a variety of pharmacologic outcomes. The 55 dependence of the metabolic change on the structure of the drug molecule and the pharmacologic 56 outcomes of the resulting metabolites are the subjects of this review. 57 58 Objective of the Review 59 The objective of this review is to investigate the relationship between the metabolic change and 60 modification of pharmacodynamic and/or pharmacokinetic properties of the drug in an endeavor to 61 explain when the pharmacologic activity of the metabolite is retained, decreased, lost, or even 62 enhanced with respect to the parent drug. The link of the metabolic functionalization and 63 pharmacologic activity to the structure of the drug is also a prime interest. Such information, as 64 described above, will be useful in drug designing, when contemplating new drug entities. As well, it 65 should be useful in drug development when considering what chemical changes need to be made in 66 a lead or prototype for better drug efficacy and metabolic stability 67 68 Methodology 69 The inclusion criteria of the selection of drugs to be reviewed include 70 (a) drugs that are metabolized by O-dealkylation or aromatic ring hydroxylation; and 71 (b) availability of data regarding the pharmacologic activity of the major 72 metabolite(s). 73 The sources of information include: 74  the published literature; 75  drug manufacturers’ data sheets; and 76  reference books on and activity of metabolites 77 The selection of the case study drugs has been based on varying the pharmacologic and 78 chemical classes, as will be described in each section. 79 80 Review Strategy 81 Although the selected drugs are mostly metabolized by multi routes, only metabolic oxidative 82 reactions that result in one chemical functionality, the aromatic hydroxy group, will be considered. 83 The representative drugs are selected based on both chemical structure and pharmacologic action. 84 Each study/metabolite(s) case will be presented in a figure including the following information 85 subject to availability in the literature: 86  the chemical structure of the drug and metabolite(s), and the enzymes or isoenzymes 87 involved; 88  the status of the pharmacologic activity of the metabolite; 89  the percentage concentration of the metabolite(s) with respect to the parent drug. 90 Each parent drug will be briefly reviewed. The arguments for the links of metabolic changes to 91 pharmacologic activity and structure will be discussed for some drug/metabolite cases as they are 92 presented with an overall discussion given for all the cases at the end of the section. 93 94 Aromatic-Hydroxy (Arenolic) Metabolites 95 Arenolic metabolites can result from one of two metabolic reactions: O-dealkylation of aralkoxy 96 groups or hydroxylation of aromatic rings (mainly benzene); in the latter case, the positions of the 97 hydroxy groups are determined by the prevailing electronic and on the aromatic ring. 98 The benzene ring may be present in the drug molecule as a separate entity (i.e. as a ), or 99 it may be fused to another benzene ring, or to an alicyclic or a heterocyclic ring. When the benzene

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100 ring is fused to either an alicyclic or heterocyclic ring the resulting ring system may be described as 101 benzenoid. Arenolic metabolites will be presented and discussed in two sections, 1 and 2. 102 103 Section 1. Arenolic Metabolites Resulting from O-Dealkylation of Aralkoxy Groups 104 Metabolic CYP-catalyzed O-dealkylation of aralkoxy groups involves, as a first step, the 105 hydroxylation of the carbon atom of the group that is linked to the atom. This 106 hydroxylated metabolite is unstable. It breaks spontaneously into two molecules: the dealkylated 107 metabolite (e.g., an or a ), and an (e.g., formaldehyde after demethylation, 108 or acetaldehyde after deethylation, etc.). The reaction is shown for a general case in Fig. 1.1., where 109 the group R can be aliphatic or aromatic [1]. 110 O-dealkylation of methyl R OH An alcohol OH or a phenol CYP 450 R O CH3 R O CH2 O Methyl ether H C 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 111 H 112 Fig. 1.1. CYP-catalyzed O-dealkylation of alkyl or aralkyl 113 114 O-dealkylation of aralkyl ethers results in the production of arenolic metabolites of varying 115 pharmacologic activities, as will be reviewed for the following selected cases: 116 (1) Enhancement of activity occurring in the narcotic/ drug and its 117 semisynthetic and synthetic congeners, all containing an aryl-, and the 118 analgesic/ drug , which contains an aryl-ethoxy group 119 (2) Retaining of activity in the SSRI antidepressant venlafaxine, containing an aryl-methoxy 120 group 121 (3) Loss of activity in the COX1/COX2-inhibiting NSAIDS , indomethacin, and 122 , each containing an aryl-methoxy group 123 124 1.1. Metabolic Aralkoxy Cleavage Resulting in Enhancement of Pharmacologic Activity: 125 and Phenacetin 126 1.1.1. Opioids. Opioid drugs can be defined as those acting on the different types of opioid 127 receptors (mu, kappa, and delta) to produce a variety of biologic effects. Mu-receptor stimulation is 128 associated with analgesic (antinociceptive) effects, respiratory depression, reduced GI motility 129 (constipation), and euphoric and dependence effects, while kappa-receptor stimulation is associated 130 with analgesia, psychomimetic effects, dysphoria, and diuresis [2, 3]. 131 For the sake of discussion, methoxy-group-containing opioid drugs can be classified with 132 regard to source and chemical build-up. Source includes natural origin (e.g. codeine), semisynthetic 133 (e.g. codeine congeners), and synthetic (e.g. ). Tramadol is considered to be a 134 synthetic-codeine congener since it was developed using codeine as template. Chemically, codeine 135 and its semisynthetic congeners are classified as pentacyclic or tetracyclic morphinans (Fig. 1.2.). On 136 the other hand, tramadol is a non-morphinan bicyclic opioid devoid of rings B and C, as shown in 137 Fig. 1.2.

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NCH3 NCH3 N(CH3)2

B C B C A D A D A E O HO H3CO H CO 3 H3CO

Pentacyclic morphinan Tetracyclic morphinan Tramadol 138 (Codeine and its congeners) () 139 Fig. 1.2. of methoxy-group-containing morphinan and non-morphinan 140 opioids 141 142 1.1.1.1. Codeine. Codeine (Fig. 1.3) is an opium alkaloid; ii is the methyl ether of and is 143 present in opium in 1-3% concentration [4]. It is obtained semisynthetically on a large scale by the 144 methylation of morphine. Codeine is a weak mu-receptor agonist, being only about 0.1% as active as 145 morphine [5]. The analgesic activity of codeine has been attributed mainly to its metabolites. 146 Codeine is metabolized per the pathways shown in Fig. 1.3 to codeine-6-glucuronide, morphine, and 147 norcodeine. The resulting morphine is further metabolized to the 3- and 6-glucuronide conjugates 148 [6, 7]. As indicated in Fig. 1.3, reports in the literature on the concentrations of codeine metabolites 149 are variable. Further, reports on the mu-receptor affinity and analgesic activity of codeine 150 metabolites are even contradictory. It is a longstanding belief that the mu-receptor affinity and 151 analgesic activity of codeine are mediated through its metabolite morphine, which has an affinity for 152 the mu-opioid receptor 200-fold greater than that of codeine [8]. The analgesic activity of codeine has 153 been suggested to be largely due its metabolite morphine [8-11]. However, of late, Vree et al. (2000) 154 have developed a different view of codeine’s affinity to the mu-opioid receptor and its analgesic 155 activity. Here, we quote the abstract of their paper: 156 Eighty per cent of codeine is conjugated with glucuronic to codeine-6-glucuronide. Only 157 5% of the dose is O-demethylated to morphine, which in turn is immediately glucuronidated at the 158 3- and 6-positions and excreted renally. Based on the structural requirement of the molecule 159 for interaction with the mu-receptor to result in analgesia, codeine-6-glucuronide, in analogy to 160 morphine-6-glucuronide, must be the active constituent of codeine. Poor metabolizers of codeine, 161 those who lack the CYP450 2D6 isoenzyme for the O-demethylation to morphine, experience 162 analgesia from codeine-6-glucuronide. Analgesia of codeine does not depend on the formation of 163 morphine and the metabolizer phenotype. [12] 164 The literature’s contradictory evidence on the mu-receptor affinity and the analgesic activity of 165 codeine and its metabolites will be weighed in the discussion section. 166 Morphie-3- NCH3 NCH 3 glucuronide %) (60 (Inactive) 1 8 CYP2D6 2 7 UGT2B7 6 0-15% 3 4 5 H CO O O 3 OH HO OH ( 5-1 Codeine 0% Morphine (active) ) Morphie-6- glucuronide 50-70% UGT2B7 (active)

Codeine-6-glucuronide 167 (active) 168 169 Fig. 1.3: Metabolic pathways of codeine 170 171 It may be of interest to note that codeine’s metabolic pathways cover almost the entire spectrum 172 of biological activity by including reactions that are augmenting, activating, inactivating, and 173 attenuating of the analgesic activity of the metabolite with respect to the parent drug. 174 1.1.1.2. Codeine congeners. Codeine synthetic pentacyclic-morphinan congeners include 175 , , and the tetracyclic morphinan congener levomethorphan, which all have

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176 3-arylmethoxy groups. On the other hand, tramadol may be considered to be a non-morphinan 177 codeine analogue since its development has been based on codeine as template. Tramadol contains 178 an arylmethoxy group relative in position to that of codeine. While hydrocodone, oxycodone, and 179 tramadol are currently clinically used in pain management [13, 14], levomethorphan has never been 180 used. The four drugs are metabolized by the isozyme CYP2D6 to their corresponding O-desmethyl 181 phenolic counterparts as shown in the corresponding figures: 1.4, 1.5, 1.6, and 1.7 [15-17]. Due to 182 their substantially higher mu-receptor affinities and, accordingly, their higher analgesic activities 183 compared to their parent drugs [15, 16], the four phenolic metabolites have been developed into 184 drugs of their own rights. Presumably, the increase in mu-receptor affinity and analgesic activity is a 185 result of the phenolic hydroxy groups in the four-metabolite drugs. We will present possible 186 explanation of why this is the case in the discussion of this section.

NCH3 NCH3

CYP2D6 UGT7B Hydrmorphone glucuronide

CH3O O O HO O O 187 Hydrocodone

188 Fig. 1.4.: Metabolic pathways of hydrocodone 189

NCH3 NCH3 NCH3 OH OH OH CYP2D6 UGT2B7

O O H3CO O HO O Glu-O O O Oxycodone Oxymorphone 190 glcuronide 191 192 Fig. 1.5. Metabolic pathways of oxycodone 193 CH3 CH 3 CH3 N N N H CO CH3 CH3 3 HO glu-O CH3 CYP2D6 UGT2B7

HO HO HO Tramadol O-Desmethyltramadol O-Desmethyltramadol 194 g lucuronide 195 Fig. 1.6. Metabolic pathways of tramadol 196 NCH3 NCH3

CYP2D6 UGT2B7 O-glucuronidation

H3CO HO 197 Levomethorphan 198 199 Fig. 1.7. Metabolic pathways of levomethorphan 200 201 Discussion of Section 1.1.1 on Opioids 202 Section 1.1.1 on the metabolic O-demethylation of the methoxy-group-containing opioids is 203 discussed separately because of the availability of substantial supportive evidence. 204 A number of theories have been proposed to account for the role of the phenolic-hydroxy group 205 in mu-receptor interaction and the consequent production of analgesia. 206 Foye (2013) has classified opioid drugs into two categories according to the pharmacophore 207 responsible for mu-receptor interaction: the rigid multicyclic (morphinan) opioids (exemplified by 208 morphine, codeine, and codeine congeners), and the flexible opioids (exemplified by

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209 4-arylpyridinepethidine) [18]. To the latter category, we may add arylcyclohexylmethylamine, 210 which is found in tramadol. The structures of the three categories are depicted in Fig. 1.8. 211 N H N D B A C A 4 1 N R A E O 4-A rylpiperidine M orphinan Arylcyclohexyl d im ethyl - R = M ethyl or ethy l m ethylam ine

212 Flexible non-m orphinan opioids 213 214 Fig. 1.8. Structures of the opioid drugs pharmacophores 215 216 Foye (2013) has argued the importance of a phenolic-hydroxy group on ring A (Fig. 1.1.8) to the 217 mu-receptor interaction and analgesic activity of the rigid multicyclic morphinan opioids such as 218 morphine and the O-desmethyl metabolites of codeine congeners [18]. The author also maintained 219 that a phenolic-hydroxy group is not a requirement for the mu-receptor interaction and analgesic 220 activity of the flexible non-morphinan opioids. It should be noted that none of the latter groups of 221 drugs contain a phenolic hydroxy group and that, to our knowledge, aromatic-ring hydroxylation 222 has not been reported as a metabolic route of any of them. 223 Foye’s theory of “phenolic OH binding liability” for the flexible opioids (18) may be contested 224 based on observations from the metabolic activity of O-desmethyltramadol (a metabolite of 225 tramadol) (Fig. 1.6) and ketomebidone (an analogue of ) (Fig. 1.9). Both drugs can be 226 categorized as non-rigid flexible non-morphinan opioids. O-Desmethyltramadol exhibits a 200-fold 227 increase in mu-receptor affinity and analgesic activity relevant to tramadol [19]. Ketomebidone, on 228 the other hand, has four-fold mu-receptor affinity and analgesic activity compared to pethidine 229 [20].The enhanced mu-receptor affinity and analgesic activity of both O-desmethyltramadol and 230 ketomebidone can be attributed to the phenolic hydroxy group, which is reminiscent in position to 231 that of morphine. CH3 CH3 N N (CH ) N 3 2 (CH3)2N

CYP2D6 O OH OH O O

H3CO HO HO Tramadol O-Desmethyltramadol 232 Pethidine Ketomebidone 233 234 Fig.1.9. Tramadol/O-desmethyltramadol and pethidine/ketomebidone 235 236 Another opioid drug-receptor interaction theory has been suggested by Beckett and Casy(1959) 237 [21] who stated that the macromolecule with which the analgesic interacts has, or attains, a certain 238 conformation into which the phenolic group must fit before the biological effect (of analgesia) can 239 occur. A “three-point” attachment of the macromolecule to a substrate’s flat aromatic moiety, its 240 basic center, and its area was postulated. 241 Glucuronide and acetyl derivatives of morphine provide substantiating evidence of the 242 phenolic hydroxy group’s involvement in strengthening the mu-receptor affinity and, accordingly, 243 the analgesic activity of the morphinan-opioid drugs that contain it, either intrinsically or 244 metabolically. Further discussion of this point will follow. 245 By virtue of its phenolic-hydroxy group at position 3 and alcoholic hydroxy group at position 6 246 (Fig. 1.10), morphine forms two glucuronide conjugates: morphine-3-glucuronide and 247 morphine-6-glucuronide (Fig.1.3). While morphine-6-glucuromide is a far more potent mu-receptor 248 agonist and analgesic than morphine [22–24], morphine-3-glucuronide is devoid of both effects [25]. 249 Furthermore, the O-glucuronide conjugates of both O-desmethyltramadol (Fig. 1.6) and levorphanol

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250 (Fig. 1.7) are devoid of mu-receptor agonistic effects and, accordingly, analgesic activity [26-27]. The 251 above data may be explained based on size and hydrogen-bonding ability differences between the 252 hydroxy and glucuronide groups. In morphine, the hydrogen bonding provided by the 6-OH group 253 is important for mu-receptor fitting and thus analgesic activity. With three hydroxy moieties, the 254 glucuronide group at position 6 of morphine is capable of establishing more hydrogen bonds than 255 the 6-OH. Further stronger interactions of the glucuronide group with the mu receptor involve 256 -ion and ion-dipole binding provided by the carboxylate (COO-) moiety. Such state of affairs 257 will most probably lead to stronger mu-receptor fit and hence higher analgesic activity of 258 morphine-6-glucuronide than morphine. On the other hand, the size factor favors the hydroxy 259 group at position 3 of morphine to anchor the aromatic ring in its hydrophobic pocket in the mu 260 receptor rather than the considerably bigger glucuronide group. With reference to the tug-of-war 261 hypothesis (to be discussed in section 1.1.3), the glucuronide group may even cause the dislodging 262 of the aromatic ring from its hydrophobic pocket. 263 Upon comparing the mu-receptor affinity and analgesic activity of morphine, 264 6-acetylmorphine, and diamorphine () (Fig. 1.10), 6-acetylmorphine has been found to be the 265 most active of the three . It is four times as active as morphine. Heroin is also more active than 266 morphine by a factor of two but less active than 6-acetylmorphine [28-31]. 267

NCH3 NCH3 NCH3

1 esterase 1 esterase

6 3 O 6 O CH3CO 3 O OCCH 3 HO OCCH3 HO OH

O O O Morphine 6-Acetylmorphine Diamorphine 268 (Heroin) 269 270 Fig. 1.10. Diamorphine, 6-acetylmorphine, and morphine 271 272 A plausible explanation of the above data is as follows: being more lipophilic than morphine, 273 both 6-acetylmorphine and heroin (Fig. 1.1.10) will cross the blood-brain barrier faster and in higher 274 concentrations than morphine. On the other hand, having a free phenolic hydroxy group, 275 6-acetylmorphine will interact with the opioid mu receptor more efficiently than diamorphine.In 276 diamorphine, the free hydroxy group is generated metabolically in the brain by esterase hydrolysis, 277 which will lead to a delayed effect and reduced efficacy. The effects of morphine and the two 278 acetylated opiates (diamorphine and 6-acetylmorphine) can therefore be explained by a combination 279 of pharmacodynamic and pharmacokinetic influences. 280 Extra substantiating evidence for the role of the free phenolic hydroxy group may be obtained 281 from levomethorphan and its O-desmethyl metabolite levorphanol (Fig. 1.7). These two tetracyclic 282 opiate drugs lack the 6-hydroxy group and hence the ability to form glucuronide conjugates at that 283 position; yet, levorphanol has a stronger affinity for the mu receptor and analgesic activity than its 284 parent drug levomethorphan [32]. 285 In conclusion, we may summarize the role of the hydroxy group in arenolic opioidsinthe 286 following statement: ‘As shown in the opioid pharmacophores in Fig. 1.8, the aromatic rings labeled 287 A, present in both morphinan and non-morphinan opioids, are essential components of the 288 pharmacophore of the opioid-mu-receptor interaction. The high affinity of the phenolic opioids for 289 the mu receptor is an indication of a logistic role played by the hydroxy group. Through hydrogen 290 bonding with an adjacent amino-acid residue in the mu receptor, the hydroxy group plays the 291 logistic role of anchoring the aromatic ring to the assigned hydrophobic pocket in the receptor, thus 292 enhancing both affinity and efficacy. Substantiating evidence to the above statement is provided by 293 the work of Sahu et al (2008) on the HIV-1-NNRT inhibitors, tetrahydroimidazobenzodiazepinones 294 [33]. In this class of compounds, the predominant hydrophobic proper orientation for maximum 295 effect has been found to be enhanced by hydrogen bonding and polar interactions.

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296 Vree’s et al assertion that the analgesic activity of codeine being entirely due to its glucuronide 297 conjugate [12] may now be reconsidered in view of the evidence so far presented about the role of 298 phenolic hydroxy group in the opioid drugs. It is important to emphasize that Vree’s et al. 299 conclusion was abstract rather than experimental and, as such, may be subject to difference of 300 opinion. 301 Because both codeine and tramadol have intrinsic analgesic activities, they can be viewed as 302 prodrugs of morphine and O-desmethyltramadol respectively, both having sustained-release effects. 303 Both morphine and O-desmethyltramadol are strong mu-receptor agonists used in the management 304 of severe pain but having the disadvantage of causing dependence. Therefore, for the management 305 of mild to moderate pain it would be advisable to use the corresponding prodrugs, codeine and 306 tramadol, which have the advantage of sustained release. However, some people who are poor 307 CYP2D6 metabolizers do not make use of the beneficial prodrug-sustained-release effect. For such 308 people, it is advisable to adjust the dose of the parent drug, give the O-desmethyl metabolites, or 309 seek alternative therapies. The frequency of the phenotype of poor metabolizers differs among 310 ethnic groups. Less than 1% of Asians, 2-5% of African Americans, and 6-10% of Caucasians are poor 311 metabolizers of CYP2D6 [34-36]. 312 In addition to the pharmacodynamic receptor interactions of the methoxy opioids (occurring 313 mainly through their polar hydroxy metabolites), the analgesic effects of hydrophobic opioid drugs, 314 such as , , , and pethidine (Fig. 1.11), have mainly been 315 explained by pharmacokinetic effects. These drugs cross the blood-brain barrier more efficiently 316 and, accordingly, they reach the mu-receptors in the brain in higher concentrations than the 317 methoxy-group-containing members [18, 37]. 318 O O N N N O O O O Fentanyl N

N Dextropropoxyphene 319 Methadone Pethidine 320 Fig. 1.11. Highly hydrophobic opioids 321 322 1.1.2. Acetanilide/Phenacetin/. The story of the development of the most 323 commonly used analgesic antipyretic drug, paracetamol, as a metabolite drug of acetanilide and 324 phenacetin, is depicted in Fig 1.12. Both the latter two drugs were once used as and 325 . Paracetamol results from phenacetin by metabolic O-deethylation [38] and from 326 acetanilide by metabolic para-hydroxylation of the aromatic ring [39]. Compared to its two 327 precursors, paracetamol has been found to have superior pharmacologic profiles, including toxicity 328 and therapeutic index (38, 39). The free hydroxy group seems to give paracetamol the edge in 329 inhibiting, through hydrogen-bonding interaction, two H2 synthases, now believed to 330 be involved in the mechanism of action of paracetamol [40]. Besides, the hydroxy group may play 331 the logistic role of anchoring the aromatic ring in the proper geometric orientation in the 332 active cavity. O H O H o rg a n ic sy n th e sis

m e ta b o lic-rin g (C H 3 C O ) 2 O

h y d ro x y la tio n N HC O CH 3 N H 2 P a ra cetam o l p -A m in o p h e n o l

m e ta bo lic N HC O CH 3 O -d e e th y latio n A ce tan ilid e

O C 2 H 5

P h en a cetin m e d ic in a l c he m istry 333 N HC O CH 3

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334 335 Fig. 1.12: From acetanilide to phenacetin to paracetamol 336 337 1.2. Metabolic O-Dealkylation of Aralkoxy Groups Resulting in Retaining of Pharmacologic 338 Activity: Venlafaxine/Desvenlafaxine 339 Venlafaxine is an antidepressant in the SSRI category. It is mainly metabolized by 340 O-demethylation to equiactive desvenlafaxine (O-desmethylvenlafaxine) (Fig. 1.13). Venlafaxine is 341 further metabolized through N-demethylation and glucuronide conjugation to inactive products 342 (Fig.1.1.13) [41-44]. Desvenlafaxine has been developed into a drug of its own right and approved by 343 the FDA in the US for the treatment of major depressive disorder (MDD), similarly to its parent 344 drug, venlafaxine, which is used for major depressive and anxiety disorders. However, the 345 European Medicines Agency (EMA) had second thoughts and did not approve desvenlafaxine as a 346 drug. They argue that venlafaxine is almost all metabolized to desvenlafaxine and that both 347 compounds have essentially the same pharmacologic and pharmacokinetic profiles; hence, there is 348 no strong reason to use desvenlafaxine as a separate drug [45]. 349 Venlafaxine carries structural similarity to the analgesic drug tramadol. The latter drug exerts 350 its analgesic effect mainly through -norepinephrine reuptake inhibition (SNRI) [46] and to 351 a minor extent through blocking oh the mu-receptor. O-Demethylation of both venlafaxine and 352 tramadol has resulted in active metabolites that have been developed into drugs of their own rights. 353 The fact that neither venlafaxine nor desvenlafaxine has analgesic effects, and that neither tramadol 354 nor O-desmethyltramadol has antidepressant effects, emphasizes the close link between drug action 355 and drug structure. 356 CH3 CH3 N N CH3 OH CH3 OH CYP2D6 UGT2B7 Desvenlafaxine glucuronide H3CO HO Venlafaxine Desvenlafaxine 357 (O-Desmethylvenlafaxine) 358 359 Fig.1.13: Metabolic pathway of venlafaxine 360 361 1.3. Metabolic O-Dealkylation of Aralkoxy Groups Resulting in Loss of Pharmacologic 362 Activity: NSAIDS (Naproxen, Indomethacin and Nabumetone) 363 NSAIDS fall into five chemical classes: arylalkanoic , salicylates, fenamates, 364 (cyclic ), and diarylheteroaromatics. The benzene ring, either as a separate entity or 365 fused with other rings, constitutes an integral part of the pharmacophore in all the NSAIDS chemical 366 classes. The carboxyl group (in the form of carboxylate ion COO-) forms the other part of the 367 pharmacophore in the arylalkanoic acid NSAIDS. With the exception of , the mechanism of 368 action of the NSAIDS involves the competitive inhibition of , the precursor of 369 , from accessing the COX active cavity. The binding of the arylalkanoic acid NSAID to 370 the amino-acid moieties in COX active cavity involves ion-ion and ion-dipole through the 371 carboxylate group (COO-) and hydrophobic through alkyl and aryl groups [47-50] as depicted in 372 Fig. 1.14. O O - O O-

N H 3CO O

Arachidonic acid Cl NSAID (Indomethacin) COX 373 (Allosteric pocket)

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374 375 Fig. 1.14: An NSAID (Indomethacin) inside the COX receptor (adapted from [50]) 376 377 Naproxen, indomethacin, and nabumetone (Figs. 1.15, 1.16, and 1.17 respectively) are NSAIDS 378 that act through COX1/COX2 inhibition. To our knowledge, they are about the only three 379 arylalkanoic acid NSAIDS that contain aromatic methoxy groups. Further, nabumetone is a prodrug 380 NSAID, which must first be activated by metabolic oxidation to the carboxy derivative, 381 6-methoxynaphthylacetic acid, as shown in Fig. 1.17. Naproxen, indomethacin, and the active form 382 of nabumetone are mainly metabolized through O-demethylation to give O-desmethylnaproxen, 383 O-desmethylindomethacin, and 6-hydroxynaphthylacetic acid, respectively (Figs. 1.15, 1.16, and 384 1.17). The first two O-desmethyl metabolites are devoid of COX inhibitory effect and, accordingly, of 385 NSAID activity [51-53]. By analogy, 6-hydroxynaphthylacetic acid, the O-desmethyl metabolite of 386 nabumetone, is expected to be devoid of NSAID activity. According to Duggan et al. (1972), the 387 p-hydroxy groups in the O-desmethyl metabolites place polar, -donating properties 388 within otherwise entirely aromatic-hydrophobic-pharmacophoric groups in the parent drugs [54]. 389 Presumably, a poor metabolite-COX fit would result with a subsequent loss of pharmacologic 390 activity. The loss of pharmacologic activity in the O-desmethyl arylalkanoic acid NSAIDS is further 391 reflected on in the discussion section 392 CH3 CH3 OH CYP3A4 OH UGT2B7 O O O6-Desmethylnaproxen CYP1A2 CH3O HO diglucuronide Naproxen 393 O6-Desmethylnaproxen 394 395 Fig. 1.15: Metabolic pathway of naproxen (51) 396 H 3CO C OOH H O COOH N C H3 Indom ethacin d CY P2C 9 N C H3 U G T2B 7 diglucuronide O O conjugate

C l C l

In dom ethacin O -D esm ethy lindo m ethacin 397 (50 % , m ajor m etabolite, inactive) 398 399 Fig. 1.16: Metabolic pathway of indomethacin (53, 54, 55) 400 O

OH OH CH3 CYP2D6 O O H3CO HO H3CO Nabumetone 6-Methoxynphthylacetic acid 6-Hydroxynaphthylacetic acid 401 (Active metabolite) 402 403 Fig. 1.17: Metabolic pathways of nabumetone (56, 57) 404 405 Overall Discussion of Section 1 406 Phase I metabolic O-dealkylation of aralkoxy groups almost invariably occurs in all the drugs 407 containing these moieties. 408 From the cited drug examples in this section, metabolic O-dealkylation of aralkoxy groups has 409 resulted in three situations regarding pharmacologic activity: 410 (1) Enhancement of activity is exhibited by the O-desmethyl morphinan opioids, 411 O-desmethyltramadol and O-desethyl phenacetin, 412 (2) Retaining of activity is exhibited by O-desmethyl venlafaxine, and

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413 (3) Loss of activity is exhibited by O-desmethyl naproxen and O-desmethyl indomethacin. 414 Some inferences can be made from the effect on pharmacologic activity in the three NSAID 415 cases upon considering the new state of structural affairs created by the loss of the hydrophobic 416 and generation of the hydrophilic hydroxy group. The effect mostly depends on the 417 site of drug action involved. The cases where the activity is enhanced involve the opioid drugs 418 acting on the mu-receptor. In these drugs, the aromatic ring A, an integral part of the 419 pharmacophore (Fig. 1.14), binds to the receptor through hydrophobic forces of attraction. The 420 hydroxy group on the aromatic ring plays the logistic role of optimally anchoring the hydrophobic 421 ring in its hydrophobic pocket in the mu-receptor [53, 58]. The same theory can be extended to the 422 acetanilide/phenacetin/paracetamol case, where the sites of action are the H2 synthase enzymes used 423 in the formation of prostaglandin, as has been recently proposed [39]. 424 The loss of pharmacologic activity upon O-demethylation of the aryl-methoxy group is mainly 425 observed in the NSAIDS indomethacin and naproxen and inferred for nabumetone. In this class of 426 arylalkanoic acid NSAIDS, a hydrophobic moiety that fits into a hydrophobic pocket in COX is an 427 essential requirement for activity as depicted in Fig. 1.14 (44, 50). O-demethylation of the 428 aryl-methoxy group in these drugs results in two situations that may be explained in 429 pharmacodynamic terms. Firstly, loss of the methoxy methyl group, which represents a small but 430 essential hydrophobic interacting moiety with COX. Secondly, formation of a hydrophilic hydroxy 431 group, which through hydrogen bonding with an adjacent amino-acid residue in COX can dislodge 432 the hydrophobic moiety in the NSAID from its hydrophobic pocket in the enzyme. An analogy can 433 be made to the tug-of-war hypothesis, as depicted in Fig. 1.18. The hydrogen-bonding and the 434 hydrophobic binding site in COX respectively pull on the hydroxy group and the aromatic ring in 435 the metabolite. Hence, both the hydroxy group and the aromatic ring in the metabolite represent the 436 rope, while the two binding sites represent the players. The hydrogen-bonding interaction, being the 437 stronger, wins the game and dislodges the aromatic ring out of the plane of interaction with the 438 hydrophobic site in the enzyme. The situations described above will act synergistically with the loss 439 of the methoxy methyl group to cause significant weakening of the drug-COX hydrophobic 440 interaction, thus leading to loss of activity. However, a pharmacokinetic effect may not be excluded. 441 Aromatic hydroxy groups are almost invariably metabolized in phase II to the highly water-soluble 442 and rapidly eliminated glucuronide conjugates. Such effect will lead to reduction of the effective 443 concentration of the metabolite at the receptor or enzyme active cavity thus resulting in curtailing or 444 loss of activity. According to Fura (2006), attenuation or loss of pharmacologic activity is associated 445 with biotransformation of pharmacophoric groups and the possible accompanying changes in 446 physicochemical properties [59]. 447

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small hydrophobic H3C binding site large hydrophobic O binding site NSAID (naproxen, weak H-bond indomethacin or X ar nabumetone) f-w g-o Tu

vacant small hydrophobic H O binding site large hydrophobic binding site The strong H-bond pulls the H aromatic ring out of the plane (O-desmethyl metabolite of the hydrophobic site. strong H-bond of NSAID) X

The arrows indicate the direction of the pull on the hydroxyl group by the H-bonding sites and on the aromatic ring by the large hydrophobic site. The strong H-bond pull causes the dislodging of the aromatic ring out of the plane of the hydrophobic pocket. The combined effect of the dislodging of the aromatic ring from its binding site and the loss of the methyl-group hydrophobic binding lead to loss of affinity of the NSAID to COX and hence loss of efficacy. 448 449 450 Fig. 1.18: Tug-of-war game between COX-active sites and NSAID/O-desmethyl NSAID 451 452 The retaining of antidepressant activity of desvenlafaxine indicates a non-essential 453 hydrophobic binding role of the methoxy methyl group in venlafaxine. Nevertheless, a 454 hydrogen-bonding role may not be excluded since it is provided by the methoxy oxygen in 455 venlafaxine and by the hydroxy group in desvenlafaxine, however, with prominence in the latter 456 case. 457 458 Section 2. Arenolic Metabolites Resulting from Aromatic Ring Hydroxylation 459 The mechanism of metabolic aromatic-ring hydroxylation involves, as a first step, the formation 460 of an (arene ) intermediate, which rearranges rapidly and spontaneously to the arenol 461 product in most instances (Fig. 2.1) [60, 61]. 462 R R R spontaneous [O] rearrangement

O OH

Arene Arene oxide Arenol (a phenol)

Aromatic-ring hydroxylation catalyzed by a variety of enzymes 463 in different xenobiotic molecules 464 465 Fig. 2.1: Mechanism of aromatic-ring hydroxylation 466 467 Metabolic aromatic-ring hydroxylation assumes its importance from the fact that a large 468 number of drug molecules contain the benzene ring as a separate entity (phenyl) or fused with other 469 rings, alicyclic or heterocyclic. In drug molecules, aromatic rings serve the dual role of providing 470 relatively large hydrophobic sites for interaction with receptors or acting as carriers for other 471 functional groups. However, not all of the benzene rings in drug molecules are subject to metabolic 472 hydroxylation. This is because certain electronic and steric effects dictate metabolic hydroxylation of 473 aromatic rings. These effects include the following with possible anomalous events:

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474 (a) the least substituted aromatic ring will be favorably oxidized, especially at the least 475 hindered carbon atom; 476 (b) the activated ring (i.e., the ring bearing an electron-donating group such as alkyl) will be 477 better oxidized; 478 (c) ring-deactivating groups (generally groups with negative inductive effects such as halo and 479 nitro groups) discourage ring hydroxylation; 480 (d) being the farthest from steric effects in di-substituted benzene rings, the para position is the 481 favored site of hydroxylation; 482 (e) if two aromatic rings in a drug molecule have the same chemical environment, 483 hydroxylation will occur in only one of them; and 484 (f) when the parent drug contains an aromatic hydroxy group, further metabolic 485 hydroxylation is generally not favored even if there was more than one aromatic ring. 486 Aromatic-ring hydroxylation of drugs has led to the formation of inactive metabolites, 487 metabolites with attenuated activity, and metabolites that are equiactive with the parent drugs. 488 These situations have been reviewed using selected representative drugs. The basis for the choice of 489 the candidate drugs is as follows: 490  varying the chemical classes of the drugs; 491  varying the pharmacologic class of the drugs and, accordingly, the type of 492 drug-site-of-action interaction involved (i.e., the mechanism of action of the class of 493 drugs); and 494  varying the aromatic character in both number of rings and chemical environment. 495 Metabolic aromatic-ring hydroxylation leading to loss of activity is exemplified by: 496 (a) the CNS depressant anticonvulsant drugs, phenobarbital and phenytoin, of the 497 chemical class (Fig. 2.2); 498 (b) the CNS depressant tranquilizer benzodiazepines diazepam (Fig. 2.3) and estazolam (Fig. 499 2.4); 500 (c) the arylalkanoic acid NSAIDs , , and (Figs. 2.5-2.8) and the 501 pyrazolone derivative (Fig. 2.12); 502 (d) the anticoagulant warfarin (Fig. 2.9); and 503 (e) the proton-pump inhibitors (the ‘prazoles’). 504 Metabolic aromatic-ring hydroxylation leading to attenuation of activity is exemplified by the 505 CNS depressant and major tranquilizer chlorpromazine (Fig. 2.10). 506 Metabolic aromatic-ring hydroxylation leading to the formation of equiactive products is 507 exemplified by: 508 (a) the beta-blocker propranolol of the chemical class aryloxypropanolamine (Fig. 2.11); and 509 (b) the HMG-CoA reductase inhibitor atorvastatin (used in lowering blood level) 510 (Fig. 2.11). 511 Metabolic-ring hydroxylation leading to enhancement of activity is exemplified by acetanilide 512 to paracetamol, which has already been discussed in section 1.1. 513 514 2.1. Metabolic aromatic-ring hydroxylation leading to loss of activity 515 2.1.1. Phenobarbital/Phenytoin. The anticonvulsant drug phenobarbital (Fig. 2.2) has been 516 chosen as representative of the barbiturate group of drugs because it is the only member that 517 contains an aromatic benzene ring susceptible to metabolic hydroxylation. On the other hand, the 518 anticonvulsant drug phenytoin (Fig. 2.2) has been selected for its similarity to phenobarbital with 519 respect to chemical structure, pharmacologic activity, and even mechanism of action. Phenytoin 520 additionally contains two benzene rings with an identical chemical environment. Both phenobarbital 521 and phenytoin are metabolized by aromatic-ring hydroxylation (Fig. 2.2), a process that has led to 522 loss of pharmacologic activity [62-65]. 523

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H H 3 N O O O N 3 O 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 524 (inactive) 525 526 Fig. 2.2: Major metabolic pathways of phenobarbital and phenytoin 527 528 The barbiturates’ CNS depressant activity (sedative, hypnotic, and anticonvulsant) and its 529 termination are mainly dependent on the drugs’ lipophilicity, which is imparted by the aromatic 530 rings and the alkyl groups [66]. Lipophilicity helps the barbiturates to cross the blood-brain barrier, 531 exert their effects, and again facilitate their distribution to other tissues, thus reducing their effective 532 concentrations at the brain’s GABA receptors. This redistribution process of the barbiturates is 533 considered a deactivation process. In addition, metabolism plays a role in the deactivation of 534 barbiturates. All barbiturates contain two alkyl groups at carbon 5 of the barbituric acid ring (Fig.2.2) 535 with the exception of phenobarbital, which contains an alkyl group (ethyl) and a phenyl group. All 536 the alkyl groups in barbiturates are metabolized by oxidation in phase I at the ω or ω-1 carbons to 537 either primary or secondary , respectively. The primary alcoholic groups may further be 538 oxidized to carboxyl (COOH) groups. On the other hand, the phenyl group in phenobarbital is 539 metabolically oxidized to a phenolic group at the favored para position (Fig. 2.2). All such 540 hydrophilic functionalities are detrimental to the essential hydrophobicity of the alkyl and phenyl 541 groups and thus to the ability of the resulting metabolites to cross the blood-brain barrier. 542 Enhancement of the water solubility of the barbiturate metabolites and their subsequent, fast 543 elimination is a further cause of termination of pharmacologic activity resulting from the 544 introduction of hydrophilic functionalities. Even more, the phase I metabolites, either carboxylic or 545 phenolic, may further be conjugated in phase II to glucuronides and/or sulfates (Fig. 2.2) with a 546 consequent further reduction in solubility and inability to cross the blood-brain barrier. In 547 addition, enhancement of water solubility and rapid elimination through the kidneys is a further 548 deactivating process. 549 Despite the fact that the pharmacokinetics of the barbiturates play a major role in their 550 deactivation, a pharmacodynamic dimension cannot be excluded: the introduction of a 551 hydrogen-bonding functionality, such as the hydroxy (OH) group, on an essentially hydrophobic 552 site, will most probably negatively affect binding to the GABA receptor, resulting in loss of 553 pharmacologic activity. The tug-of-war analogy described for the NSAIDS in section 1.1.3 may be 554 extended to barbiturates and phenytoin. 555 The two aromatic rings in phenytoin have identical chemical environments, and only one of 556 them is hydroxylated, which is consistent with the rules of metabolic aromatic-ring hydroxylation. 557 The same reasoning that applies for the loss of activity of the phenolic metabolite of phenobarbital 558 discussed above should apply to the loss of activity of the phenolic metabolite of phenytoin. 559 560 2.1.2. Benzodiazepines: Diazepam and Estazolam. The benzodiazepines are CNS depressants 561 used as minor tranquilizers, sedatives, hypnotics, and anticonvulsants; in these respects, they have 562 largely superseded the barbiturates. Their mechanism of action involves binding to GABA receptors

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563 [67-69]. The benzodiazepines are metabolized through several phase I oxidative reactions with some 564 followed by phase II conjugative reactions. The metabolic pathways of the two benzodiazepine 565 representative members, diazepam [70, 71] and estazolam [72, 73], are shown Figs. 2.3 and 2.4, 566 respectively, with only aromatic-ring hydroxylation discussed in this section. The other metabolic 567 pathways of diazepam and estazolam will be discussed where relevant. 568 CH CH3 3 CH3 O O 9 1 O N N N 8 CYP3A4 3 OH A B 3 (minor) Cl N (major) Cl N N 7 6 5 Cl 1' 1' 6' 2' C 5' 3' 4' Temazepam 4' Diazaepam OH (active) 4'-Hydroxydiazepam (inactive) 569 The two hydroxy metabolites are further metabolized by glucuronide conjugation in phase II. 570 571 Fig. 2.3: Metabolic pathways of diazepam 572 2 N N 1 N N N 3 N N N N CYP3A4 CYP3A4 4 OH 4 Cl N (minor) Cl N (major) Cl N 1' 2' 3' 4' 4-Hydroxyestazolam 4' OH (inactive) Estazolam 4'-Hydroxyestazolam (inactive) 573 The two hydroxy metabolites are further metabolized by glucuronide conjugation in phase II. 574 575 Fig. 2.4: Metabolic pathways of estazolam 576 577 Due to favorable electronic and steric structural environments, diazepam and estazolam, to our 578 knowledge, are the only two members of the clinically used benzodiazepines to undergo metabolic 579 aromatic-ring hydroxylation at the 4’ position (Fig. 2.3 and Fig. 2.4). In most of the other members, 580 metabolic 4’-hydroxylationis is possibly disfavored by the presence of an electron-withdrawing halo 581 group at position 2’ (fluoro in flurazepam, flunitrazepam, and quazepam, and chloro in triazolam 582 and clonazepam). (For the numbering of the benzodiazepine ring system, refer to the structure of 583 diazepam in Fig. 2.3.) 584 4’-Hydroxylation of both diazepam [70, 71] and estazolam [72, 73] has resulted in inactive 585 metabolites. Foye (2013) has attributed the loss of sedative-hypnotic effects of 4’-hydroxyestazolam 586 to two factors [72]. The first factor is of pharmacodynamic nature. It is explained by the 4’-hydroxy 587 group weakening optimal binding of the aromatic ring to GABAA receptor by a steric effect. The 588 result is decreased receptor affinity and drug potency. Interestingly, in its essence, the steric 589 hindrance factor proposed by Foye (2013) [72] lends credence to our tug-of-war hypothesis discussed 590 in section 1.3. The second factor is of a pharmacokinetic disposition and results from decreased 591 hydrophobicity (i.e., increased hydrophilicity), which results in the decrease of the effective 592 concentrations of the circulating 4’-hydroxy metabolites due to enhanced polarity, water solubility, 593 and elimination, as per se and as glucuronide conjugates. In the absence of reports regarding the loss 594 of activity of 4’-hydroxydiazepam, an analogy may be extrapolated from that of 595 4’-hydroxyestazolam. 596 In contrast to 4’ hydroxylation, metabolic hydroxylation at position 3 of the diazepine ring in 597 diazepam (Fig. 2.3) has not affected activity but has rather introduced a pharmacokinetic dimension: 598 the 3-hydroxy metabolite is more hydrophilic and is subject to glucuronide conjugation with

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599 subsequent enhanced rate of elimination and hence shorter duration of action than diazepam. These 600 observations tend to consolidate a major pharmacodynamic role of 4’-aromatic-ring hydroxyl group 601 on causing loss of activity of diazepam as well as estazolam. The plausible explanation is that the 602 introduction of the hydrogen-bonding group (the hydroxy) into an essentially pharmacophoric 603 hydrophobic moiety (the benzene ring) is detrimental to the optimal GABAA receptor binding as can 604 be inferred from Foye’s (2013) explanation [72]. 605 A halo group at position 2’ of the benzodiazepine backbone (ring C, Fig. 2.3) serves three 606 purposes: it adds a welcomed hydrophobicity to the drug, disfavors metabolic ring hydroxylation, 607 and imparts a conformation-locking effect on the aromatic ring through a steric effect. The 608 consequence of these effects could be enhanced selectivity on drug-receptor interaction leading to 609 higher efficacy and possibly higher potency. Increased hydrophobicity would tend to enhance 610 blood-brain barrier penetration and therefore increased access to the GABAA receptor. 611 In the above context, it would be worthwhile to investigate the effect of another halo group at 612 position 6’ (Fig. 2.3) on the conformation locking of the aromatic rings in comparison to the NSAID 613 pair /diclofenac. In diclofenac (Fig. 2.5), the two ortho-positioned chloro groups have 614 resulted in a restricted conformation with consequent enhanced selectivity, efficacy, and potency 615 compared to fenoprofen, in which the two chloro groups are absent [74]. 616 617 2.1.3. NSAIDS. 618 The chemical classes of the NSAIDS have been discussed in section 1.3. 619 2.1.3.1. Diclofenac. Diclofenac is a phenylacetic acid NSAID. It was developed as a variant of 620 fenoprofen by introducing two ortho-positioned chloro groups in the anilino-aromatic ring to 621 restrict its free rotation (74). This restriction of rotation increases selectivity and hence potency with 622 respect to fenoprofen.The metabolism of diclofenac shown in Fig. 2.5 [75] represents one of the 623 anomalies of aromatic-ring hydroxylation in that hydroxylation occurs at the meta position of two 624 chloro groups. The hydroxy metabolites are pharmacologically inactive. 625 In accounting for the structure-activity relationship of diclofenac, Foye (2013) [75] has proposed 626 that the function of the two ortho chloro groups was to force the anilino-phenyl ring out of the plane 627 of the phenylacetic acid portion. Such twisting, as proposed by Foye (2013) [75], is important in the 628 binding of diclofenac to the of COX. The introduction of a hydroxy group in the 629 anilino-phenyl group would create a hydrogen-bonding character, which would weaken or hinder 630 the necessary twisting, thus resulting in attenuation or loss of pharmacologic activity. 631 Cl O H N OH Diclofenac Cl

C C 4 Y Y P A 2

3 P P C Y 2 9

C C 9

Cl O Cl O H H Cl O N N H OH OH HO N OH

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

Cl O H N 4',5-Dihydroxydiclofenac OH

HO Cl OH

632 (10-20%) 633 634 Fig. 2.5. Metabolic pathways of diclofenac 635 636 2.1.3.2. Ketorolac. Ketorolac (Fig. 2.6) is a pyrrole- derivative structurally related to 637 indomethacin and . It is metabolized to p-hydroxyketorolac (Fig. 2.6), which is inactive.

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638 Both the carboxy and phenolic hydroxy groups are further metabolized by glucuronide conjugation 639 to give pharmacologically inactive products [76]. In analogy to the previously discussed cases, the 640 loss of activity of 4-hydroxy ketorolac may be attributed to both pharmacodynamic and 641 pharmacokinetic effects. 642 O O O N OH N O-Gluc CYP2C9 N OH CYP2B7 O O O - HO Gluc O Ketorolac p-Hydroxyketorolac (10%) p-Hydroxyketorolac glucuronide 643 (inactive NSAID) (inactive) 644 645 Fig. 2.6. Metabolic pathways of ketorolac 646 647 2.1.3.3. Flurbiprofen. Flurbiprofen is an arylpropionic acid COX1/COX2-inhibitor NSAID. It is 648 mainly metabolized by aromatic-ring hydroxylation as shown in Fig.2.7with loss of activity [77]. The 649 metabolism of flurbiprofen shows a rather interesting pattern in that a catechol ring is formed in 650 which the 4’-position is anomalously methylated to yield a methoxy group with reduced polarity. 651 This metabolic route is reminiscent of that of adrenaline, which is metabolized by the methylation of 652 the para-hydroxy group by the enzyme catechol-O-methyl transferase (COMT). Furthermore, the 653 metabolic double hydroxylation of flurbiprofen to yield 3’,4’-dihydroxy flurbiprofen is also an 654 anomaly of metabolic-ring hydroxylation. We recall metabolic aromatic-ring dihydroxylation in the 655 same molecule is generally disfavored [60, 61]. 656 CH3 4;Hydroxy- HO CHCO2H flurbiprofen F (40-47% )

HO 3' 2' CH3 CH3 3',4'-Dihydroxy- 4' CHCO 2H HO CHCO2H 1' flurbiprofen inactive F catechol F (5%) metabolites Flurbiprofen HO CH3 3'-Hydroxy-4'- H CO CHCO H 3 2 methoxyflurbiprofen F (20-30%) 657 658 Fig. 2.7. Metabolic pathways of flurbiprofen 659 660 2.1.4. Miscellaneous 661 662 2.1.4.1. Warfarin. Warfarin (Fig. 2.8) is an anticoagulant drug used as prophylactic in 663 preventing thrombus formation in susceptible patients. Warfarin is chiral with the S-enantiomer 664 having five-fold the activity of the R-enantiomer [78]. Warfarin contains two aromatic groups: a 665 benzopyran and a phenyl in addition to a 2-butanone chain (Fig. 2.8). The major metabolic pathway 666 of warfarin is through hydroxylation of the benzene ring of the benzopyran moiety, in addition to a 667 minor route through the reduction of the side-chain keto group to a secondary alcohol (Fig. 2.8) 668 [79-82]. The acidic hydroxy group at C4, being in conjugation with the benzene ring, possibly 669 explains the preference of metabolic hydroxylation of the benzopyran ring over the phenyl group 670 since it increases the electron density toward hydroxylation through a positive inductive effect. It 671 has been observed that the hydroxy group introduced metabolically on the aromatic ring has led to 672 loss of anticoagulant activity while the hydroxy group resulting from side-chain keto reduction has 673 resulted only in attenuation of activity [79]. Keto-group reduction and pharmacologic activity are 674 the subject of alcoholic metabolites to be discussed in part II of this review series. 675

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OH O 5 C9 CH P2 4 3 * 3 phenyl CY 7 jor H O O O ma 1 ( te) OH O rou S-W arfarin 5 9 7-H ydroxyw arfarin (inactive) 6 4 3 11 CH3 ca * 10 12 rbo 1 red ny 7 O O uc l 2 tas O H 8 (m e OH in ro or ute benzopyran ) * * CH3 11 O O (The S enantiomer is five-fold * chiral center 11-H ydroxyw arfarin as active as the R enantiom er.) 676 (active) 677 678 Fig. 2.8. Metabolic pathways of warfarin 679 680 2.2. Metabolic Aromatic-Ring Hydroxylation Resulting in Attenuation of Activity 681 682 2.2.1. Chlorpromazine. Chlorpromazine (Fig. 2.9) is a major tranquilizer used as an 683 antipsychotic. It is metabolized in humans by two major routes [83-87]: (a) aromatic-ring 684 hydroxylation at position 7 to a moderately active metabolite, and (b) sulfoxidation to an inactive 685 metabolite (Fig. 2.9). A minor deactivating route through N-demethylation also occurs. It should be 686 noted that the metabolic hydroxylation of chlorpromazine is in accordance with the rule that groups 687 with negative inductive effects, such as chloro, deactivate the ring to hydroxylation. 688

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, 689 moderately active) 690 691 Fig. 2.9: Metabolic pathways of chlorpromazine 692 693 2.3. Aromatic-ring hydroxylation resulting in parent-drug equiactive metabolites 694 2.3.1. Propranolol. Propranolol (Fig. 2.10) is an aryloxypropanolamine β–adrenoceptor blocker 695 used as an antihypertensive and antiangina agent. As shown in Fig. 2.10, it is metabolized in humans 696 to three major metabolites, two of which are inactive and one of which is as active as the parent drug. 697 In naphthoyloxyacetic acid (Fig. 2.10), the pharmacophore is ruptured, leading to loss of activity. In 698 metabolite (II), glucuronide conjugation of the side-chain hydroxy group has led to loss of activity 699 while in metabolite (III), activity is maintained upon introduction of a hydroxy group para to the 700 side chain. The glucuronide conjugate of the aromatic-ring hydroxy metabolite is inactive [88, 89]. 701 The metabolism of propranolol directs attention to two interesting points: (a) glucuronic-acid 702 conjugation of both alcoholic and phenolic hydroxy groups to loss of pharmacologic activity, a 703 phenomenon that is true for most cases, and (b) alkoxy groups are aromatic-ring activators and 704 para-directors in metabolic hydroxylation.

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OH

OCH2CHCH2NHCH(CH3)2 CYP1A2 CYP2D6

OH Naphthoyloxyacetic acid Propranolol OCH2CHCH2NHCH(CH3)2 (45-50%; inactive due to rupturing of pharmacophore) (III) (I)

OH Propranolol O-glucuronide CYP1A2 4-Hydroxypropranolol (40% , inactive) (17%; active and as potent (II) as propranolol) 705 706 707 Fig. 2.10: Metabolic pathways of propranolol 708 709 2.3.2. Atorvastatin. Atorvastatin (Fig.2.11) is a HMG-CoA (3-hydroxy-3-methyl-glutaryl-CoA) 710 reductase inhibitor used in lowering blood cholesterol and levels. Atorvastatin is 711 metabolized by CYP3A4 hydroxylation at the ortho- or para-position at ring D as shown in Fig. 2.11 712 [90, 91]. Being electron withdrawing, both the fluoro group and the pyrrole ring (C) disfavor 713 metabolic hydroxylation of rings A and B, respectively. In accordance with the rule, the 714 hydroxylation takes place at the least hindered benzene ring, i.e., ring D. The two metabolites are 715 equiactive with the parent drug and account for about 70% of its overall circulating activity [90, 91]. 716 The molecule of atorvastatin can be dissected into two parts: the dihydroxyheptanoic acid 717 moiety and the aromatic ring system with its . Dr. Philip Portoghese, a medicinal 718 chemist from the University of Minnesota, has developed a concept called “Message-Address,” 719 which conceptually breaks a drug molecule up into two components: one, which “finds” the active 720 site (the address), and the other, which actually delivers the drug’s chemical message (92). In 721 atorvastatin, the dihydroxyheptanoic acid moiety represents the message, while the aromatic-ring 722 system with substituents represents the address. When the address is substantially large, a 723 small-group metabolic change is not expected to result in a significant impact on its role.This is true 724 for atorvastatin, which contains a four-ring system that mainly interacts with the active site of the 725 enzyme via hydrophobic binding. 726 Since the term ‘pharmacophore’ is mostly used in the pharmacodynamics of drug action, we 727 propose, an adaptation of Dr. Portuguese’s concept by using the terms ‘primary pharmacophore’ 728 and ‘auxiliary (logistic) pharmacophore’ as equivalent terms to ‘message’ and ‘address’, 729 respectively. By binding to the receptor, the auxiliary (logistic) pharmacophore will facilitate the 730 anchoring of the primary pharmacophore prior to its binding and competitive inhibition of the 731 physiologic substrate. 732 HO Dihydroxyheptanoic acid moiety COOH OH (statin pharmacophore) F H

N HO COOH OH NH O F H HO A N CYP3A4 o-Hydroxyatorvastatin C HO NH COOH B D O OH

Atorvastatin F H N

NH OH O 733 p-Hydroxyatorvastatin 734

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735 Fig. 2.11: Metabolism of atorvastatin 736 737 2.3.3. Phenylbutazone. Two metabolites of Phenylbutazone (Fig. 2.12) which were isolated from 738 human urine possess some of the pharmacological activities of the parent drug. Metabolite I 739 (), formed by aromatic-ring hydroxylation, has the potent antirheumatic and 740 sodium-retaining effects of phenylbutazone; it has been developed into a drug of its own. On the 741 other hand, metabolite II, formed by the hydroxylation of the ω-1 carbon of the butyl side chain, also 742 possesses reduced sodium-retaining and antirheumatic properties, but it is a considerably more 743 potent uricosuric agent than phenylbutazone [93, 94]. 744

g rin ti- ion ma lat N O aro xy dro HO N hy O

N O Oxyphenbutazone N om hy e dr ga O ox -1 yl ati Phenylbutazone on N O (developed into an anti-inflammatory N metabolite drug) O OH gamma-Hydroxyphenylbutazone (devoid of anti-inflammatory activity 745 but has urocosuric activity) 746 747 Fig. 2.12: Metabolism of phenylbutazone 748 749 Phenylbutazone binds to and deactivates prostaglandin H synthase and synthase 750 through peroxide- (H2O2-) mediated deactivation. The reduced production of prostaglandin leads to 751 reduced inflammation in the surrounding tissues [94]. It is also pertinent to note that 752 γ-hydroxyphenylbutazone, which results from ω-1 hydroxylation of the butyl-side chain in 753 phenylbutazone, is devoid of anti-inflammatory activity [95]. Metabolic-aliphatic-hydroxylation and 754 pharmacologic activity of the resulting metabolites is the subject of Part II of this review series. 755 756 1.2.4. Discussion of Metabolic Aromatic-Ring Hydroxylation 757 For the drug-cases reviewed in this section, except for diclofenac, the structural features, either 758 electronic or steric, set above for the occurrence of aromatic-ring hydroxylation, conform well to the 759 rule of thumb. 760 Loss, decrease, or retention of pharmacologic activity upon aromatic-ring hydroxylation in the 761 cited cases may reflect the status of the ring in the parent drug regarding its mechanism of 762 interaction with the receptor. When hydrophobic binding of the aromatic ring with the receptor is 763 essential for activity, introduction of the hydrophilic-hydrogen-bonding hydroxy group will 764 compromise the fit and will not be tolerated. The established hydrogen bonding may force the ring 765 out of the plane of interaction with the receptor; the result will be loss of activity. This has been the 766 case with phenobarbital and phenytoin (Fig. 2.2), diazepam, (Fig. 2.3), estazolam (Fig. 2.4), NSAIDS 767 (Figs. 2.5, 2.6, and 2.7), and warfarin (Fig. 2.8). The tug-of-war hypothesis suggested for the 768 O-desmethyl-NSAIDS in section 1.1.3 may be extended to the above cases. Furthermore, the phase II 769 glucuronide conjugation of the aromatic-hydroxy group is an important factor in causing loss of 770 activity of the metabolite. It considerably enhances metabolite clearance and accordingly reduction 771 of its effective concentration at the receptor. 772 When aromatic-ring hydroxylation results in decreased activity, as in 773 7-hydroxychlorpromazine (Fig. 2.9), four inferences present themselves: (a) the hydroxy group has 774 resulted in increase of the optimum chlorpromazine molecular size; (b) the hydroxy group weakens

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775 optimal binding of the aromatic ring to the receptor by a steric effect (c) the tug-of-war effect is only 776 partially operative; and (d) the hydroxylated ring is playing an auxiliary role. In addition, reduction 777 of effective concentration of the hydroxy metabolite at the receptor through glucuronide conjugation 778 may be playing an important role. 779 When the hydroxy metabolite is equiactive with the parent drug, two inferences can be 780 tentatively made. Firstly, the hydroxylated aromatic ring is auxiliary; i.e., it plays the role of the 781 address. This is the case with the statin drug atorvastatin (Fig. 2.11), where the three aromatic rings 782 are not part of the primary pharmacophore and are therefore not involved in essential binding to the 783 enzyme [96]. Their role, however, is logistic, that of proper anchoring of the drug in the enzyme 784 active cavity for optimum interaction of the primary pharmacophoric groups with the enzyme to 785 take place. The second inference is associated with 4-hydroypropanolol (Fig. 2.10), the equiactive 786 metabolite of propranolol. Propranolol is a nonselective β1/β2-adrenoceptor blocker; it belongs to 787 the aryloxypropanolamine chemical class. In this class of compounds, hydrophilic 788 substitution at position 4 of the aromatic ring imparts β1 antagonistic selectivity [97], such as in 789 atenolol and practolol (Fig. 2.13). On the other hand, hydrophilic hydroxy substitution at the same 790 position reverses the activity altogether, i.e., from antagonistic to agonistic, such as in prenalterol 791 (Fig. 2. 13) [98]. It can hence be concluded that the nature of the hydrophilic group substitution at 792 position 4 of the aryloxypropanolamines significantly dictates the pharmacologic outcome of the 793 β1-receptor interaction. Based on the above findings, it may be inferred that 4-hydroxypropranolol is 794 a tentative β1-adrenoceptor agonist pending experimental testing. 795 OH 2 H O N CH3 OH O 3 H 1 2 O N CH3 O 6 CH3 3 1 H2N 4 5 6 CH3 Atenolol H3C N 4 (Hydrphilic H 5 amide group) Selective bera-1- Practolol adrenoceptor blocker (Hydrphilic Selective bera-1- amide group) adrenoceptor blocker OH 2 H O N CH 3 3 1 6 CH3 HO 4 5 Prenalterol 796 Beta-1-adrenoceptor agonist 797 798 Fig. 2.13: Structures of atenolol and practolol 799 800 The atorvastatin equiactive hydroxy metabolites furnish a useful inference: pharmacologic 801 equiactivity of metabolites relative to the parent drug occurs when the metabolic change takes place 802 at the “address” or “auxiliary pharmacophore”. We will provide further examples in review 2 of this 803 series. 804 Of the NSAIDS, phenylbutazone stands in a class of its own in that its mechanism of action does 805 not involve inhibition of COX but rather the inhibition of prostaglandin H synthase. As has been 806 shown in this section and in section 1.3, a hydroxy group on the aromatic rings of arylalkanoic-acid 807 COX1/COX2-inhibitor NSAIDS is detrimental to their pharmacologic activity. This is in contrast to 808 phenylbutazone whose aromatic-hydroxy metabolite (oxyphenbutazone (Fig. 2.12)) is equiactive 809 with the parent drug and has been developed into an anti-inflammatory drug of its own right. The 810 different mechanisms of action and, accordingly, the varying sites of drug action involved may 811 explain the disparity between the activities of the hydroxy-metabolites of phenylbutazone and the 812 arylalkanoic-acid NSAIDS. 813 As pharmacodynamics may handsomely explain the pharmacological activity of drug hydroxy 814 metabolites relative to the parent drugs, the role of the pharmacokinetics of these metabolites should 815 not be excluded. In the cases where the pharmacologic activity of the hydroxy metabolite is either 816 attenuated or lost relative to the parent drug, pharmacokinetic factors may come into perspective in 817 two aspects. Firstly, the hydroxy metabolites are rapidly cleared by phase II conjugation, thus aiding 818 in the termination of their action. Secondly, the hydroxy metabolites do not readily penetrate target

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819 tissues due to a decrease in membrane permeability caused by an increase in polar surface area, a 820 limitation that affects their active concentrations at the target site [99]. 821 822 Glucuronide conjugation: prevalence and effect on pharmacologic activity 823 Glucuronide conjugation is the most common phase II metabolic process [100] though certain 824 structural features control its occurrence. It occurs with almost all aromatic hydroxy (arenolic) 825 groups, most carboxyl groups, unhindered alcoholic hydroxy groups and a few amino and 826 sulfhydryl groups. Because of its relatively big size, glucuronic acid may not have easy access to 827 active-hydrogen containing groups that are awkwardly situated in a molecule, i.e. sterically 828 hindered groups. For instance, tertiary alcoholic groups such as 14-hydroxy in oxycodone (Fig.1.5) 829 and 1’-hydroxy in tramadol (Fig. 1.6) are sterically hindered and are hence not susceptible to 830 glucuronide conjugation. 831 With the exception of codeine glucuronide and morphine-6-glucuronide, glucuronide 832 conjugate has led to loss of activity of all the cases presented in this review. With three hydroxy 833 moieties and a completely ionized carboxyl moiety at physiologic pH, the glucuronide group 834 considerably increases metabolite hydrophilicity, water solubility, elimination and termination of 835 action. With this pharmacokinetic concept of loss of activity of most drug glucuronide conjugates, 836 the door is open only to pharmacodynamic speculation to explain the activity of codeine 837 glucuronide and morphine-6-glucuronide. 838 839 Conclusion 840 841 The hydroxy group is the most common hydrophilic group produced by metabolic 842 functionalization in drug molecules. Aromatic hydroxy groups result in one of two ways: 843 O-dealkylation of aralkoxy groups or hydroxylation of the aromatic ring. O-dealkylation of aralkoxy 844 groups in drug molecules is an invariably predictable metabolic route. On the other hand, metabolic 845 aromatic-ring hydroxylation is governed by electronic and steric factors prevailing in the ring. The 846 pharmacologic activity of the resulting arenolic metabolites resulting from both processes depends 847 on the site of drug action (i.e. the receptor) as well as on the pharmacophoric or auxophoric status of 848 the aromatic ring to which the alkoxy or hydroxy group is bonded. In general terms, phase I 849 metabolic functionalization may reveal the status of a group in a drug molecule, whether primary or 850 auxiliary pharmacophoric or auxophoric. As well, if there is pre-knowledge of the pharmacophoric 851 or auxophoric statuses of the rings, then the effect of the metabolically formed hydroxy group on 852 activity of the metabolite may be predicted. In addition to pharmacodynamic effects, the 853 attenuation or loss of activity of polar metabolites may be explained by pharmacokinetic effects, 854 which by enhancing elimination lead to decreased metabolite effective concentration at the receptor. 855 When more active or equiactive metabolites have shown favorable pharmacodynamic and 856 pharmacokinetic properties, they have been developed into drugs of their own rights. Nevertheless, 857 not all equiactive metabolites have been developed into drugs of their own rights and may hence be 858 classified as drug-action-extension forms. 859 860 Acknowledgement. We would like to thank Dr. J. DeRuiter for the use of the diagram in Fig. 50. We 861 also would like to apologize to Dr. DeRuiter for using the diagram without securing his/her consent 862 since we were not able to track his/her address. 863 864 References 865 1. Williams D.A. Drug Metabolism.In: Foye’s Principles of Medicinal Chemistry.7th ed.; Lemke T.L., 866 Williams D.A., Roche V.F., Zito S. W., Eds.; Wolters Kluwer and Lippincott Williams and Wilkins, 867 London, UK, 2013; p. 135; ISBN-13: 978-1-60913-345-0 868 2. Williams D.A., Roche V.E., Roche E.B. Central Analgesics, Opioid receptors. In: Foye’s Principles 869 of Medicinal Chemistry. 7th ed.; Lemke T.L., Williams D.A., Roche V.F., Zito S.W., Eds.; Wolters

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