Liabilities Associated with the Formation of âHardâ Electrophiles in

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Perspective

Liabilities Associated with the Formation of “Hard” Electrophiles in Reactive Metabolite Trapping Screens Amit S. Kalgutkar Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.6b00332 • Publication Date (Web): 02 Nov 2016 Downloaded from http://pubs.acs.org on November 6, 2016

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Chemical Research in Toxicology

Liabilities Associated with the Formation of “Hard” Electrophiles in Reactive Metabolite Trapping Screens Amit S. Kalgutkar* Pharmacokinetics, Dynamics, and Metabolism – New Chemical Entities, Pfizer Worldwide Research and Development, 610 Main St, Cambridge, Massachusetts 02139, United States

ACS Paragon Plus Environment

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ABSTRACT: Soft electrophiles (e.g., epoxides, quinones, quinone-imines, quinone-methides, etc.) generated via the oxidative bioactivation of phenyl, phenolic, amino- and alkylphenolic substituents can be trapped with nucleophiles of comparable softness (e.g., glutathione or cysteine) in reactive metabolite screens. In contrast, hard nucleophiles such as cyanide and amines are frequently utilized to trap hard electrophiles (e.g., iminiums and aldehydes) that result from the oxidative bioactivation of cyclic (or acylic) amines and primary alcohols. In some instances, soft sulfydryl nucleophiles have also been utilized to trap aldehydes to yield cyclized thiazolidine adducts. Case studies where hard electrophiles are thought to be responsible for cytochrome P450 inactivation, genotoxicity and/or target organ toxicity in animals have been presented. The association of hard electrophiles with immune-mediated idiosyncratic adverse drug reactions is less clear given the paucity of available examples, and the fact that several marketed drugs containing cyclic amine motifs can generate hard electrophiles via α-carbon ring oxidation. The perspective examines available data associating toxicity with the formation of hard electrophilic intermediates from small molecule drugs/drug candidates. Pragmatic risk mitigation strategies around unwarranted idiosyncratic toxicity risks with drug candidates that generate hard electrophiles are also discussed against the backdrop of marketed agents that possess analogous cyclic amine framework.


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Table of Contents Graphic


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CONTENTS 1. Introduction

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2. Reaction of Aldehyde with Cysteine or GSH

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3. Literature Examples Pertaining to Cyclized Cysteinyl Adducts

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4. Case Studies on the Formation of Electrophilic Iminium/Aldehyde Metabolites and Toxicological Consequences

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5. Adverse Drug Reactions Linked to Iminium/Aldehyde Formation

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5.1 Mianserin (97)

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5.2 Abacavir (32)

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6. Perspective

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Author Information

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Corresponding Author

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Biography

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Notes

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Acknowledgments

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Dedication

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Abbreviations

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References

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1. INTRODUCTION Lenz et al.1 recently published a manuscript describing the characterization of an unusual CysGly adduct 4 derived from the oxidative bioactivation of a homomorpholine derivative AZX (1) in glutathione (GSH)– and NADPH–supplemented human liver microsomes (HLM). Mass spectrometry analysis of 4 using accurate mass measurement was consistent with the thiazolidine structure shown in Scheme 1. A proposed mechanism for the formation of 4 involves α-carbon oxidation of the homomorpholine ring in 1 by cytochrome P450 (P450) to a carbinolamine intermediate 2 followed by ring-opening to the aminoaldehyde 3. Reaction of 3 with the cysteinyl portion of GSH is expected to yield the cyclized Cys-Gly thiazolidine metabolite 4 with the net loss of a water molecule and the γ-glutamic acid residue. Replacement of GSH with methoxylamine as an exogenous trapping agent in NADPH-supplemented HLM incubations of 1 led to the formation of the aldoxime adduct 5 (Scheme 1), which provides further evidence for the formation of aldehyde 3 as an electrophile. Failure to detect an intact AZX-GSH conjugate in the microsomal incubations suggested that hydrolytic cleavage of GSH to Cys-Gly occurs prior to cyclization. Scale-up and purification attempts on 4 for 1H NMR characterization proved unsuccessful due to rapid degradation of 4 to the aminoethanol derivative 6, via an unknown mechanism. According to the hard and soft acids and bases theory2,3, aminoaldehyde 3 is categorized as a hard electrophile and should preferentially react with nucleophiles of comparable hardness (e.g., reaction with methoxylamine to afford 5). Therefore, the formation of the Cys-Gly adduct 4 is somewhat unexpected considering that it is derived from the reaction of the hard electrophile 3


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with GSH, which is considered to be a soft nucleophile.3 This finding raises several questions such as, how common is the reaction of GSH with aldehydes? What is the mechanism leading to the cyclized thiazolidine product formation? Should all cyclic tertiary amine motifs be categorized as structural alerts given their propensity to react with GSH? What are the general toxicological implications of hard electrophiles arising from the process of metabolism? With these objectives in mind, a retrospective evaluation of the “reactive metabolite” literature was conducted on the generation of hard electrophiles in a preclinical discovery setting, the trapping techniques utilized, and the toxicological implications associated with this phenomenon with a particular emphasis on immune-mediated idiosyncratic adverse drug reactions (IADRs). The analysis was restricted to compounds that exclusively afford hard electrophilic intermediates. Thus, five-membered heterocyclic rings (e.g., furan and thiophene) were excluded from this analysis since their bioactivation by P450 yields reactive species containing both hard (aldehyde) and soft (olefin) electrophilic centers (i.e., α,β-unsaturated aldehydes), which preferentially react with soft nucleophiles such as GSH in a 1,4-Michael fashion.4,5 Likewise, functional groups such as ortho- or para-aminophenols and bis-anilines that are bioactivated by P450 to GSHreactive ortho- or para-quinone-imine or bis-imine intermediates, respectively, were also omitted from this examination.5 The results of the analysis are presented herein, and discussed against the backdrop of several marketed therapeutic agents that can (or are known to) yield hard electrophiles in the course of drug metabolism but are devoid of toxicity.

2. REACTION OF ALEDHYDES WITH CYSTEINE OR GSH The chemical reaction of simple alkyl aldehydes (e.g., formaldehyde and acetaldehyde) with the amino acid cysteine to form thiazolidine-4-carboxylic acid derivatives is a well-established


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process,6–10 which is in addition to the known susceptibility of aldehydes to form Schiff bases upon condensation with amine nucleophiles.3 The mechanism of cyclization is thought to occur via two plausible pathways; one (pathway A, Scheme 2) involving the nucleophilic addition of the cysteinyl-NH2 group to the aldehyde carbonyl and the other (pathway B, Scheme 2) involving nucleophilic attack on the carbonyl by the cysteinyl thiol. The first pathway generates a carbinolamine intermediate 7, which is converted to the corresponding Schiff base (8, iminium species) upon elimination of a water molecule. Nucleophilic attack of the cysteinyl-SH moiety on the electrophilic iminium species furnishes thiazolidine-4-carboxylic acid (11) as a product. The second pathway yields a thiohemiacetal 9, which loses a water molecule to generate the sulfonium ion intermediate 10 followed by nucleophilic addition of the cysteinyl-NH2 group on the electrophilic sulfonium ion center to yield the cyclized product 11.

In chemical reactivity studies conducted under physiological conditions (pH 7.4 phosphate buffer), aldehyde derivatives tend to display significantly greater reactivity with cysteine relative to GSH, a phenomenon possibly related to: (a) pKa differences (GSH = 9.3 versus cysteine = 8.33),11,12 which leads to a greater fraction of the cysteine thiolate anion being present at pH 7.4 and/or (b) the ready availability of the free NH2 group in cysteine for cyclization to the stable thiazolidine adduct.13,14 In the case of GSH, the proximal NH2 residue is not readily available for cyclization due its participation in the γ-glutamyl-cysteine peptide bond, and this characteristic possibly leads to a weak and reversible thiohemiacetal bond upon reaction of the available free thiol moiety with the aldehyde functionality.14,15 Inclusion of γglutamyltranspeptidase (GGT) in incubations of acetaldehyde and GSH results in a facile cleavage of the γ-glutamate residue in GSH to yield dipeptide Cys-Gly, which displays equimolar reactivity with acetaldehyde at rates comparable to those noted with cysteine.15 In the


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absence of GGT, GSH appears to be relatively inert in forming thiazolidine conjugates with acetaldehyde.15 Consistent with this hypothesis, Anni et al.16 demonstrated rapid conjugation of synthetic Cys-Gly with acetaldehyde in phosphate buffer to yield 2-methylthiazolidine-4carbonylglycine (see Scheme 2) in near quantitative fashion. A rapid reactivity of acetaldehyde has also been noted with cysteine and the structurally related chelating agent D-penicillamine under stoichiometric conditions to yield the corresponding cyclized thiazolidine adducts.15 Low nonenzymatic degradation of GSH to CysGly has also been noted in the literature,17,18 which suggests that low levels of aldehyde consumption upon treatment with GSH in buffer can be the result of reaction with the liberated Cys-Gly residue to afford the cyclized thiazolidine conjugate. Some noteworthy differences between these observations and the work of Lenz et al.1 also become apparent from this analysis. In contrast with the facile reaction of simple alkyl aldehydes with cysteine, no thiazolidine adducts of AZX were detected in liver microsomal incubations containing cysteine (or γGlu-Cys); thiazolidine adduct formation appeared to rely on the presence of a terminal glycine or a lysine group. Furthermore, GSH analogs with γ-glutamate replacements (e.g., α-glutamate or β-asparate) also formed identical Cys-Gly adducts suggesting that hydrolysis of α-glutamate or β-asparate to Cys-Gly occurred readily. The reason(s) for these discrepancies remains unclear and warrant further investigation including studies examining the effect of GGT inhibition on AZX-Cys-Gly formation in HLM with the nucleophiles used in the study. Recently, Inoue et al.19 exploited the dual reactivity of cysteine (and the related derivative homocysteine) by examining the formation of both soft (e.g., quinones, epoxides, quinoneimines, etc.) and hard electrophiles (e.g., aldehyde) generated in HLM incubations. Chemical


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reactivity studies with model aldehydes, however, revealed lower reactivity with cysteine and homocysteine as compared with aldoxime formation in the presence of traditional aldehyde trapping agents such as methoxylamine or semicarbazide.20–22 Nonetheless the ability of cysteine and homocysteine to form stable 5- or 6-membering ring adducts upon reaction with aldehydes confirmed their potential utility as bifunctional trapping agents in reactive metabolite assays used in drug discovery to examine bioactivation potential of new chemical entities.

In hindsight, the bifunctional reactivity of cysteine (and related derivatives) is not altogether surprising. For instance, the catechol metabolite 13, derived from the P450 2D6 mediated methylenedioxyphenyl ring opening in the antidepressant drug paroxetine (12), undergoes further two-electron oxidation to an electrophilic ortho-quinone 14, which reacts with GSH in HLM incubations to yield three distinct GSH conjugates (15, 17, and 20) (Scheme 3).23 The formation of GSH conjugate 15 is consistent with Michael addition of GSH to the ortho-quinone species 14, whereas 17 is formed by the addition of a second molecule of GSH to the orthoquinone species 16 derived from a two-electron oxidation of GSH conjugate 15. GSH conjugate 20 was shown to be a Cys-Gly conjugate whose formation can be rationalized via the generation of an ortho-quinone intermediate 18 from GSH adduct 17, loss of the γ-glutamic acid residue from one of the adducted GSH molecules to yield 19, and intramolecular condensation of the cysteinyl primary amine group with an adjacent carbonyl in 19 to form the orthobenzoquinoneimine/GSH conjugate 20. Structurally similar Cys-Gly adducts have also been observed with para-aminophenol and catechol derivatives.24–28

Similarities can be drawn between the mechanism of thiol adduction to aldehydes and the Pinner-type reaction involving reaction of nitriles (compound 21) with cysteine to yield stable


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4,5-dihydrothiazole-5-carboxylic acid cyclic adducts (compound 23) via the initial thioimidate intermediate 22 (Scheme 4).29–31 Analogous to the observations with aldehyde derivatives, chemical reactivity studies in pH 7.4 phosphate buffer indicate that structurally diverse nitriles display ~ 15-fold lower reactivity towards GSH in comparison to cysteine.32 Furthermore, the degree to which 4,5-dihydrothiazole adducts are generated from the reaction of several nitrilecontaining compounds with GSH is far greater in HLM than in pH 7.4 phosphate buffer, and inclusion of the GGT inhibitor acivicin in the microsomal incubations results in significant inhibition of adduct formation.33 These observations provide compelling evidence for the involvement of GGT in the cleavage of the γ-glutamate residue from GSH during the cyclization process to the 4,5-dihydrothiazole adducts. The Pinner reaction of nitriles is also known to occur with catalytically active cysteine and serine residues in proteins to yield reversible thioimidate or imidate adducts (Scheme 4),34 a feature that has been exploited in the design of targeted reversible covalent inhibitors. For example, cyanopyrrolidine analogs saxagliptin and vildaglitpin (Scheme 4) are marketed anti-diabetic drugs, which inhibit dipeptidyl peptidase IV by covalent adduction with an active site serine residue to generate slowly reversible imidate adducts.35,36 Likewise, odanacatib, currently in phase III clinical trials for the treatment of osteoporosis, is an α-amidoacetonitrile derivative that forms a thioamidate adduct with an active site cysteine in cathepsin K.37,38

3. LITERATURE EXAMPLES PERTAINING CYCLIZED CYSTEINYL ADDUCTS

TO

Apart from the publication of Lenz et al., a survey of the literature revealed few additional cases involving the formation of thiazolidine conjugates from aldehyde metabolites. The first example pertains to the indole neurotransmitter 5-hydroxytryptamine (24, serotonin (5-HT),


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Scheme 5). The major metabolic pathway of 24 in the central nervous system involves oxidative deamination by monoamine oxidase (MAO) to afford 5-hydroxyindole-3-acetaldehyde (25), which is converted to 5-hydroxyindole-3-acetic acid (26) by aldehyde dehydrogenase (ALDH). A minor pathway involves reduction of 25 to the primary alcohol 5-hydroxytryptophol (27) by aldehyde reductase. Singh and Dryhurst39 demonstrated that incubation of 24 with NADPH- and L-cysteine (or

GSH)-supplemented pig or bovine brain microsomes or with synaptosomes

resulted in the formation of the (2R,4R)- and (2S,4R)-epimers of 2-[(5-hydroxy-1H-indol-3yl)methyl]thiazolidine (compounds 28 and 29, respectively) or α-amino-4[[(carboxymethyl)amino]carbonyl]-2-[(5-hydroxy-1H-indol-3-yl)methyl-δ-oxo-3thiazolidinepentanoic acid (compounds 30 and 31, respectively) from a reaction of cysteine or GSH with the aldehyde 25. While the rates of formation of thiazolidine adducts in brain microsomes or synaptosomes were relatively faster with L-cysteine than with GSH, it is interesting to note that the γ-glutamate residue was retained in the GSH portion of the adducts 30 and 31.

A second example is evident with the nucleoside analog and reverse transcriptase inhibitor abacavir (32, Scheme 6), which is used to treat human immunodeficiency virus/acquired immune deficiency syndrome. Abacavir is primarily metabolized in humans through modifications on the primary β,γ-alcohol moiety to a glucuronide conjugate and a carboxylic acid derivative 34 that is formed via the action of cytosolic alcohol dehydrogenase (ADH) (Scheme 6).40,41 In vitro incubations of [14C]-abacavir in β-NAD-supplemented human liver cytosol results in protein covalent binding that is thought to be mediated by an electrophilic β,γ-unsaturated aldehyde species 33, formed in the course of the oxidative process. The characterization of aldoxime (35) and hydrazide (36) derivatives in cytosolic incubations of abacavir and trapping agents


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methoxylamine and semicarbazide, respectively (Scheme 6) provided support for the formation of the aldehyde intermediate 33.19,41 Upon incubation of abacavir in human liver cytosol/β-NAD in the presence of cysteine or homocysteine, corresponding five (thiazolidine-4-carboxylic acid, compound 37) and six (1,3-thiazine-4-carboxylic acid, compound 38) membered adducts of 33 (see Scheme 6) were also detected.19 While the thiazolidine adduct 37 was not detected when cysteine was replaced with S-methylcysteine, it is interesting to note that aldoxime conjugates expected from the reaction of 33 with the primary amine group in S-methylcysteine were also not observed.

Two additional illustrations describing thiazolidine ring metabolites derived from the oxidative bioactivation of piperazine derivatives were also evident in the literature analysis.42,43 MB243 (39), a 1,3-disubstituted piperazine derivative and selective melanocortin receptor subtype-4 agonist, demonstrated extensive covalent binding to liver microsomal proteins from rats and humans in a NADPH-dependent fashion.42 Inclusion of GSH to the microsomal incubations resulted in a reduction of protein covalent binding and the formation of novel Cys-Gly (44) and Cys (45) adducts of 39. The mechanism for their formation (Scheme 7) is thought to involve piperazine ring oxidation to a reactive imine-amide intermediate 40, which is trapped by GSH. Hydrolysis of the glutamic acid residue (presumably by GGT) in the initial GSH adduct 41 furnishes the Cys-Gly adduct 42 that undergoes internal aminolysis through the cysteinyl amino group resulting in opening of the piperazine ring to afford 43, which is followed by ring closure to the imidazoline-Cys-Gly adduct 44. Hydrolytic cleavage of the glycine motif in 44 generates the imidazoline-Cys conjugate 45. Administration of [3H]-39 to bile-duct cannulated rats also led to the detection of 44 and 45 in isolated rat bile. An analogous bioactivation pathway was also deciphered with an N-substitutedpiperazine derivative 46 (Scheme 7),43 involving piperazine


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ring oxidation to the imine-amide 47, reaction with GSH followed by loss of the glutamate residue and aminolysis by the cysteinyl amine to 48, and aromatization of the imidazoline to the imidazole-Cys-Gly and imidazole-Cys adducts 49 and 50, respectively.

4. CASE STUDIES ON THE FORMATION OF ELECTROPHILIC IMINIUM/ALDEHYDE METABOLITES AND ASSOCIATED LIABILITIES Based on the analysis thus far, it is clear that the electrophilic species in the metabolism studies is in fact an aldehyde derivative, irrespective of the techniques used to trap this species (GSH, cysteine, amines and/or peptide-based nucleophiles44) and the subtle variations noted in the types of conjugates formed (GSH, Cys-Gly, and/or Cys conjugates) with soft sulfydryl amino acid trapping agents. In the case of cyclic amines, the proposed cleavage of the α-carbon-nitrogen bond in the homomorpholine ring in 1 (Scheme 1) is generally consistent with the known oxidative (P450- or MAO-mediated) biotransformation pathways of cyclic tertiary amines, which involve an initial α-carbon oxidation on the cycloalkylamine framework to afford a cyclic iminium species followed by hydrolysis to the corresponding aminoaldehyde derivative, which is in equilibrium with the carbinolamine form (see illustration in Scheme 8).45–49

The electrophilic nature of the aldehyde functionality in aminoaldehyde intermediates is evident with the 4-benzyloxy-1-methyl-1,2,3,6-tetrahydropyridine analog 51 (Scheme 8), which is metabolized by MAO-B to the corresponding 2,3-dihydropyridinium (iminium) metabolite 52.50 Hydrolysis of the iminium bond in 52 results in the aminoaldehyde 54 (via the intermediate carbinolamine 53), followed by spontaneous rearrangement to the β-keto aldehyde 55 through intramolecular nucleophilic attack of the terminal secondary amine group on the aldehyde functionality. In MAO incubations of 51 fortified with hydrazine, the β-keto aldehyde


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55 was characterized as the pyrazole derivative 57, which is obtained from cyclization of the hydrazide intermediate 56. Likewise, the cyclized metabolite 62 observed in human plasma during radiolabeled human mass balance studies with the anti-diabetic drug [14C]-sitagliptin (58)51 can be generated via an initial ring hydroxylation to carbinolamide 59, followed by spontaneous intramolecular condensation of the corresponding aminoaldehyde tautomer 60 with the pendant primary amine functionality.

The iminium species generated during α-carbon oxidation of cyclic amines is also considered as a hard electrophile, which can be trapped with a hard nucleophile such as potassium cyanide.49,52,53 Intramolecular trapping of iminium ions generated in the course of metabolism has also been documented with a 5HT4 partial agonist 6354; two-electron oxidation of the piperidine ring in 63 (Scheme 9) yields the putative iminium species 64, which reacts with a pendant tertiary alcohol group to yield a cyclized oxazolidine metabolite 65, which incidentally also retains pharmacological activity. Analysis of circulating metabolites from first-in-human pharmacokinetic studies revealed that 65 was the major metabolite in systemic circulation.54

Cases linking the generation of iminium ion metabolites with protein covalent binding have also been reported in the literature with xenobiotics such as phencyclidine and nicotine.55–57 In addition, there are examples where oxidative bioactivation of cyclic secondary and tertiary amine functionality has resulted in toxicity arising via covalent adduction of electrophilic iminium and/or aminoaldehyde intermediates to DNA and/or protein targets. Examples include the 5HT2C agonist 66 and the selective dual A2A/A1 adenosine receptor antagonist 73, which demonstrated genotoxicity in the bacterial Salmonella Ames reverse mutation and/or mouse Lymphoma L5178Y assays in an Aroclor 1254–induced rat liver S9/NADPH–dependent


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fashion.58–60 In both cases, irreversible and dose–dependent incorporation of radioactivity in calf thymus DNA was observed in incubations with [14C]-66 or [3H]-73 in an Aroclor 1254-induced rat liver S9/NADPH-dependent manner, implying that the two compounds were bioactivated by P450 enzyme(s) into DNA–reactive metabolites. The Salmonella assay is an integral part of drug safety evaluation, and is required for regulatory filing of new drug applications. Because positive findings in the Salmonella reverse mutation assay have demonstrated a good correlation with the outcome of rodent carcinogenicity testing,61,62 a positive result often leads to the discontinuation of development, particularly for drugs intended to treat non-life-threatening diseases as was the case with 66 and 73.

Incubations of 66 or 73 with rat liver S9/NADPH in the presence of methoxylamine and potassium cyanide led to the detection of aldoxime and cyano adducts derived from oxidative bioactivation of the piperazine and pyrrolidine rings in 66 and 73, respectively. Mass spectral data for the aldoxime conjugates 69 (for compound 66) and 77 (for compound 73) was consistent with the condensation of methoxylamine with the aminoaldehyde metabolites 68 and 76, which are derived from hydrolytic cleavage of the nitrogen-α-carbon bond in 66 and 73 via the intermediate carbinolamines 67 and 75, respectively (Scheme 10). In the case of 66, the cyano conjugate 72 was obtained from a bioactivation pathway involving N-hydroxylation of the secondary piperazine nitrogen (compound 70) in 66 followed by two-electron oxidation to yield an electrophilic nitrone 71, which reacted with cyanide. In contrast, cyanide trapping studies with 73 revealed the presence of a bis-cyano adduct 80, which can be generated via the addition of cyanide to the two-electron oxidation product 79 of the initial mono-cyano adduct 78. Consistent with the output from the reactive metabolite trapping efforts, significant reduction in mutagenic response in the Ames test and/or covalent binding to DNA was noted with 66 and 73


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upon co-incubation with methoxylamine and/or cyanide, suggesting that the aminoaldehyde and iminium intermediates are likely candidates responsible for genotoxicity. Finally, in the case of 73, analog 81 (Scheme 10), which does not contain the pyrrolidine ring, was not genotoxic. In the case of 66, P450 phenotyping studies in HLM established that the piperidine ring in 66 is mainly metabolized by P450 3A and 1A isoforms;60 observations that are partially consistent with metabolism-dependent genotoxic response of 66, since P450 1A1/1A2 expression is significantly induced in rats upon exposure to Aroclor 1254.63

Evidence for piperidine ring bioactivation has also been demonstrated with the cannabinoid type 1 receptor antagonist rimonabant (82, Scheme 11), which was approved in 2006 for the treatment of obesity but was withdrawn in 2008 due to serious drug-related psychiatric disorders, including anxiety and depression. In vitro metabolism studies in HLM with 8264 resulted in high level of NADPH-dependent covalent binding to microsomal protein and time-dependent irreversible inactivation of human P4503 A4 isoform, which is also responsible for the metabolism of rimonabant in humans. Excessive protein covalent binding and cytotoxicity were also noted in incubations of 82 with human hepatocytes and SV40-T-antigen-immortalized human liver epithelial derived cells selectively expressing P4503 A4.65 An electrophilic aminoaldehyde (84) (in equilibrium with the corresponding carbinolamine 83) and/or iminium (85) ion formed via P4503 A4-mediated piperidine ring α-carbon oxidation in 82 were proposed as likely contributors to the observed covalent binding in human hepatic tissue.64,65 Moreover, liver microsomal covalent binding of 82 was reduced by ~ 40% upon addition of cyanide, with little to no effect of GSH. This observation was consistent with the finding that the electrophilic iminium ion 85 is a major metabolite of 82 in NADPH-supplemented HLM, and is amenable to trapping with cyanide to yield adduct 86.64 Interestingly, liver microsomal covalent binding was


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also reduced by approximately 30% upon co-incubation with methoxylamine but no corresponding aldoxime adducts expected from reaction with the putative aminoaldehyde intermediate 84 were detected in HLM.64 Subsequent incubation of electrochemically oxidized [14C]-82 with tryptic BSA digest and model peptides such as leucine-enkephalinamide or angiotensin II resulted in the characterization of a peptide adduct (compound 90) derived from reaction of the peptide N-terminal amino group with a electrophilic metabolite of 82.66 The peptide adduct, which contained a C5H4 fragment originating from the aminopiperidine motif in 82, was also generated in rimonabant and leucine-enkephalinamide co-incubations in HLM suggesting that reactive metabolite(s) of 82 identical to electrochemically generated species are also present in HLM incubations.66 Based on the insights gained from the mass spectral characterization of electrochemical oxidation products including the peptide adducts, a pathway leading to the formation of the peptide adducts was postulated and involved further oxidation of the iminium ion 85 to short-lived intermediates (e.g., enamine 87 and dihydropyridinium species 88), which ultimately degraded to the protein reactive pentanedial metabolite 89 (Scheme 11). Independent incubations of 89 with model peptides or BSA also led to the formation of 90,66 which supported the proposed bioactivation pathway.

A final example is evident with the zwitterionic piperidine derivative 91 (Scheme 12), which demonstrated elevations in liver enzymes and liver necrosis in single dose exploratory toxicology studies in non-human primates.67 In contrast, corresponding in vivo toxicity studies in rats and dogs showed no evidence of liver injury. A role for metabolism-dependent toxicity in primates could be inferred for 91 based on the finding that rapid metabolism and high levels of protein covalent binding were noted in NADPH-supplemented liver microsomes from monkeys than in microsomal preparations from rat, dog, or human. Monkey liver microsomal covalent binding of


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[3H]-91 was significantly attenuated upon co-treatment with cyanide ion but not with GSH or methoxylamine. Consistent with this finding, a cyanide adduct 96 of a mono-hydroxylated metabolite of 91 was also observed in monkey liver microsomes in a NADPH-dependent fashion, which allowed an insight into the non-human primate-specific bioactivation pathway of 91 (Scheme 12). Compound 91 was thought to undergo P450 mediated two-electron oxidation on the piperidine ring to yield iminium ion 92, which is in equilibrium with the corresponding enamine form 93. P450 catalyzed epoxidation of 93 would generate the unstable epoxide 94, which could undergo spontaneous ring opening to the dipolar oxy-anion 95 that ultimately is trapped by cyanide to yield adduct 96. In accordance with this proposed scheme, one or more electrophilic species arising from piperidine ring oxidation could be responsible for the observed protein covalent binding of 91. Because the monkey was not representative of the metabolism of 91 in humans, this animal species would have been a poor choice as the second non-rodent species for preclinical toxicological assessment.

Despite the commonality in bioactivation mechanisms of these cyclic amine ring systems, noteworthy differences are also apparent for which there is no clear explanation. First is the failure to detect the expected cyanide adduct of the putative iminium ion intermediate of AZX in HLM/NADPH incubations supplemented with potassium cyanide, which has led the authors to speculate that the equilibrium favored carbinolamine/aminoaldehyde forms rather than the iminium. Second, aldoxime conjugates were detected with AZX, 66 and 73, but not with 82 or 91. Third, there is no description of the corresponding thiazolidine adducts (derived from adduction of GSH to aminoaldehydes 68, 76, and 84 analogous to the study by Lenz et al.1), despite the fact that GSH was included as a trapping agent in the in vitro liver microsomal incubations and all incubations were subjected to full scan nominal mass analysis for


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characterization of adducts (as opposed to the neutral loss of pyroglutamate residue (129 Da) technique used in high-throughput reactive metabolite screens.68,69

5. ADVERSE DRUG REACTIONS IMINIUM/ALDEHYDE FORMATION

LINKED

TO

5.1 Mianserin (97). Clinical use of the tetracyclic antidepressant 97 is associated with adverse reactions including hepatotoxicity and blood dyscrasias.70–72 Detection of specific mianserin antibodies against platelets in the serum of a patient with thrombocytopenia is suggestive of an immune-mediated component in the development of mianserin-induced agranulocytosis.73 Circumstantial evidence has been presented, which links toxicity to the P450-mediated bioactivation of the parent compound 97 and its primary urinary metabolites74,75 8hydroxymianserin (98) and N-desmethyl-mianserin (99) to the corresponding reactive iminium ions 100-102 in HLM (Scheme 13).76–80 The detection of cyanide adduct(s)49,52 in incubations of 97 in HLM containing NADPH and cyanide is consistent with these observations. Studies with radiolabeled mianserin and its metabolites also resulted in the NADPH-dependent, irreversible incorporation of radiolabel in microsomal protein and cytotoxicity towards human mononuclear leukocytes included in the microsomal incubation.76,80 A reactive iminium species 103 and its corresponding cyanide adduct 104 have also been detected in incubations of mianserin human neutrophils and in incubations with horseradish peroxidase/H2O2.81 Significant apoptosis was also observed upon incubation of 97 with human neutrophils in a peroxide-dependent fashion.81 Wen and Fitch82 have also characterized GSH-ethyl ester conjugates (compounds 107 and 108) derived from addition of the thiol nucleophile to quinone-imine intermediates 105 and 106, which in turn are obtained from the two-electron oxidation of 8-hydroxymianserin 98 and a para-hydroxyphenyl metabolite of 99, respectively, in HLM/NADPH incubations. Structure-


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metabolism studies have also been conducted in an attempt to understand the structural requirements for mianserin bioactivation.83 Replacement of the nitrogen atom from the N5 position with a methine group (compound 109) in mianserin significantly reduces cytotoxicity as does substituting a methyl group for a hydrogen atom at position C14b (compound 110) in 97 (see Scheme 13), which reaffirms iminium ion formation as the putative pathway leading to toxicity.

5.2 Abacavir (32). While generally well tolerated, the clinical use of 32 has been associated with significant cases of hypersensitivity (e.g., fever, skin rash etc.) in approximately 4% of the patient population, which can be severe and, in rare cases, fatal. Hypersensitivity with abacavir has been linked to mutations in the HLA gene, particularly HLA-B*5701, for which genetic testing is now available in most western countries.84–86 Prior screening for the HLA-B*5701 mutation has been shown to reduce the incidence of abacavir hypersensitivity reactions.86 Evidence for bioactivation of the pendant primary alcohol group in 32 to the electrophilic aldehyde 33 was presented in Scheme 6, which provides a plausible explanation for the immunemediated IADRs observed with the clinical use of abacavir. An additional mechanistic possibility for the in vitro covalent binding of 32 to cytosolic protein involves the rapid isomerization of the initial β,γ-unsaturated aldehyde species 33 to a more thermodynamically stable conjugated α,β-unsaturated aldehyde species 111, which is a Michael acceptor.41,87–89 To investigate this possibility, Charneira et al.87 synthesized both aldehyde isomers (33 and 111) of 32 and investigated their chemical reactivity with the α-amino group of ethyl valinate (Scheme 14). The resulting adducts (e.g., compound 112 in Scheme 14) arising from the chemical reactions were stabilized by reduction with sodium cyanoborohydride (e.g., compound 113) and derivatized with phenylisothiocyanate, leading in both instances to the formation of a single


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phenylthiohydantoin derivative 114. In vitro incubations of synthetic 111 with human hemoglobin followed by postmodifications (reduction and Edman degradation) also generated the phenylthiohydantoin 114, which is the expected product of reaction between 111 and the Nterminal valine residue of hemoglobin. Likewise, incubations of 32 in human liver cytosol/βNAD/ethyl valinate, which were subjected to reduction with sodium cyanoborohydride and derivatization with phenylisothiocyanate yielded high levels of the thiohydantoin derivative 114, which further confirmed the isomerization of 33 to 111 in human hepatic tissue. Recently, abacavir-derived Edman adducts with the N-terminal valine of hemoglobin were also detected in HIV patients on abacavir regimen, which provides circumstantial evidence for the potential involvement of a reactive aldehyde metabolite in the clinical hypersensitivity associated with abacavir.89 Interestingly, reaction of synthetic 111 with cysteine exclusively afforded the Michael-type 1,4-addition product 115 (Scheme 14),87 which is in contrast to the detection of the thiazolidine adduct 37 derived from reaction of cysteine with β,γ-unsaturated aldehyde species 33 (see Scheme 6).

5.3 Ethanol. Ethanol is a dose-dependent hepatotoxin that causes significant reduction in hepatic GSH levels in alcoholics with liver disease,90,91 and supplementation with precursors of cysteine (e.g., S-adenosylmethionine) reduces early stage alcohol-induced hepatotoxicity in humans and experimental animals.92,93 Exposure of primary rat hepatocytes to ethanol also leads to reduction in GSH levels in a dose-dependent fashion with maximal depletion noted at ethanol concentrations approximating 20 mM.94 Pyrazole, a specific inhibitor of ADH, prevents ethanolinduced GSH depletion, whereas disulfiram, an inhibitor of ALDH, further potentiates the decrease in GSH concentration induced by ethanol.94 These observations suggest that the mechanism of hepatic GSH depletion involves the reaction of the ethanol metabolite


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acetaldehyde with the GSH breakdown product Cys-Gly to yield 2-(methylthiazolidine-4carbonyl)glycine derivative (Scheme 2).15,16,18 The thiazolidine conjugate has been detected in rat bile after chronic ethanol treatment, which incidentally lead to high systemic acetaldehyde concentrations (~ 50 µM),16 comparable to the values (~ 100 µM) observed in some Japanese alcoholic patients.95,96 Administration of large doses of L-cysteine or the thiol-based chelator Dpenicillamine effectively protects ethanol-treated rats against acetaldehyde toxicity with the concomitant formation of the anticipated cyclized condensation product between D-penicillamine and acetaldehyde.97–99 Non-enzymatic conjugation of acetaldehyde with free α-amino terminus, ε-amine side groups of lysine residues and sulfhydryl groups in proteins (e.g., hemoglobin and serum albumin) have been reported in vitro, and in vivo in alcoholics.100-108 Conjugates of acetaldehyde with proteins trigger the generation of antibodies against de novo acetaldehyde epitopes.109,110 Quantification of acetaldehyde-protein conjugates, directly or via their antibodies, has been used to evaluate chronic alcohol intake.107,111,112

Chronic alcohol consumption is a strong risk for the development of certain cancers including those of the upper gastrointestinal tract, the liver, and the female breast.113 The carcinogenicity of acetaldehyde has been demonstrated in animals,114 and stable DNA adducts of acetaldehyde have been detected in different organs of alcohol-fed rats and in leukocytes of alcoholics.115–118 The concentration of acetaldehyde in target tissues depends on the formation of this metabolite by ADH and subsequent oxidation (detoxication) of this metabolite by ALDH. Polymorphisms in genes encoding ADH and ALDH that cause elevated acetaldehyde concentrations in vivo are known to be associated with increased cancer risk.119 For example, ~ 40% of Japanese, Chinese, or Korean population carry heterozygous ALDH2*2 allele, which codes for an ALDH enzyme with significantly impaired catalytic activity leading to high acetaldehyde concentrations after


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the consumption of even small amounts of ethanol. Chronic ethanol consumption by individuals with this allele results in a heightened risk for upper alimentary tract and colorectal cancer. In Caucasians, ADH1C*1 allele produces ~ 2.5-fold more acetaldehyde than the corresponding allele ADH1C*2. In studies with moderate to high alcohol intake, ADH1C*1 allele frequency and rate of homozygosity was found to be significantly associated with an increased risk of several types of cancers. These observations in humans underline the important role of the hard electrophile acetaldehyde in ethanol-mediated carcinogenicity.

6. PERSPECTIVE The examples discussed in this article suggest that formation of iminium ions and/or aldehyde intermediates from cyclic amine- and primary alcohol-containing compounds can be associated with liabilities such as mechanism-based inactivation of P450 and/or mutagenicity. In a preclinical drug discovery setting, information pertaining to irreversible inhibition of P450 or metabolism-dependent genotoxicity with new chemical entities will generally trigger mechanistic studies to investigate the role of reactive species as outlined with the case studies in Section 4. If reactive species are detected (inferred from reactive metabolite trapping studies), then bioactivation pathway(s) compatible with the structure of the reactive metabolite conjugate will be proposed, and appropriate medicinal chemistry strategies will be pursued to potentially eliminate the liability as outlined in previous publications on these topics.59,60,120

The potential role of hard electrophiles in the pathogenesis of IADRs, however, is less clear considering the limited number of available examples, which also raises numerous questions particularly from a structure-metabolism relationships perspective. Rimonabant (82) was never approved in the United States and was only marketed in the European Union as an anti-obesity


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drug for two years prior to its withdrawal. Whether immune-mediated IADRs could be triggered via protein-conjugates derived from piperidine ring oxidation products (e.g., 85 and/or 89) of 82 will remain unknown. Suffice to say, there is no known DILI risk for 82 based on the clinical experience to-date.121 The bioactivation sequence involving the oxidative deamination of serotonin (24) by MAO to the electrophilic aldehyde 25 can also occur with members of the “triptan” class of antimigraine drugs (Scheme 15) such as the acyclic tertiary amines sumatriptan (116) and rizatriptan (117). In humans, MAO-A is the principal enzyme responsible for the metabolism of 116 and 117 to the corresponding carboxylic acid metabolites 119 and 121 via the indole-3-aldehyde intermediates 118 and 120, respectively.122,123 Following intravenous or oral administration of [14C]-117, 121 is the major metabolite detected in human urine (35-51% of the dose).124 While the aldehyde intermediates 118 and 120 have not been detected, it is possible that inclusion of exogenous trapping agent (e.g., GSH and/or methoxylamine) in incubations of 116 or 117 in relevant matrices (e.g., liver mitochondria or hepatocytes) may lead to adducts (thiazolidine and/or aldoxime) derived from the reaction of the nucleophiles with the aldehyde intermediates. The characterization of the primary alcohol metabolite 122 in sumatriptan incubations in human liver preparation122 provides further support for the generation of the aldehyde metabolite 118. A similar possibility exists with structurally related zolmitriptan (123) where the P450 1A2-generated N-desmethyl metabolite 124 is oxidized by MAO-A to the corresponding carboxylic acid metabolite 126 via aldehyde intermediate 125.125,126 Cyclic tertiary amine eletriptan (127) is also extensively metabolized by P450 3A4 via biotransformation pathways involving N-demethylation, N-oxidation as well as the generation of a mixture of pyrrolidine ring hydroxylated metabolites.127 It is also noteworthy to point out that oxidative deamination of serotonin or triptans would involve α-carbon oxidation to the iminium


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ion, which is spontaneously hydrolyzed to the aldehyde species. However, there are no reports on cyanide trapping of iminium ions generated from the α-carbon oxidation of acyclic primary, secondary or tertiary amine-based drugs, presumably due to the instability of the iminium ion, which rapidly succumbs to hydrolysis. As such, triptans have been used for over a decade in the treatment of migraine headaches and are generally devoid of IADR liabilities.128 Low total daily dose has been proposed as a principal mitigation factor for lack of IADRs with numerous marketed drugs, particularly the ones that form reactive metabolites.129,130 In the case of triptans, efficacious total daily doses in human are fairly variable – zolmitriptan (5 mg), rizatriptan (30 mg), eletriptan (40–80 mg) and sumatriptan (200 mg).

Mirtazapine (128), the 6-aza analog of mianserin (97) (Chart 1), has been marketed as an antidepressant in the United States since 1996. Clinical use of 128 (maximum effective daily dose = 45 mg) is not associated with agranulocytosis noted with 97 (maximum effective daily dose = 60 mg), despite similarities in human metabolic profile of the two drugs.131 Whether twoelectron oxidation of 128 and its downstream human metabolites 8-hydroxymirtazapine and Ndesmethylmirtazapine by P450 or neutrophils can lead to reactive species analogous to 97 is currently not known. In vitro metabolism studies will be required to evaluate the influence of the pyridine nitrogen (in 128) on the formation of electrophilic iminium ion and/or quinone-imine species that were detected with 97.

Another example relates to the first rationally designed tyrosine kinase inhibitor imatinib (129, Chart 1) that is used in the treatment of chronic myeloid leukemia, advanced gastrointestinal stromal tumors, and other hematological disorders. Potent mechanism-based inactivation of human P450 3A4 by 129 has been determined in vitro studies in HLM, which explains the


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clinical drug-drug interactions observed with P450 3A4 substrates such as simvastatin.132,133 Reactive metabolite trapping studies with stable labeled potassium cyanide and methoxylamine in NADPH-supplemented HLM led to the detection of multiple cyanide conjugates and a aldoxime adduct,134 which are presumably derived from a P450 3A4-mediated piperazine ring α-carbon oxidation to electrophilic iminium and aminoaldehyde intermediates135 that inactivate the enzyme. The speculation135 that bioactivation of the piperidine ring in 129 to hard electrophiles could also potentially account for the hepatotoxicity of 129136 is questionable, considering that liver toxicity has also been noted with other marketed tyrosine kinase inhibitors that do not contain a cyclic tertiary amine functionality (e.g., lapatinib (130)).137 Furthermore, human mass balance studies with [14C]-129 indicate that the principal metabolite is derived from piperidine ring N-demethylation in the parent compound with little to no contribution from piperidine ring oxidation products.138

During studies evaluating protein covalent binding with hepatotoxic and non-hepatotoxic drugs, Usui et al.139 reported high levels of covalent binding of dexamethasone (131, Chart 1) to human hepatocytes and HLM supplemented with NADPH suggesting that the steroid is bioactivated to a protein-reactive species. Consistent with this hypothesis, Meneses-Lorente et al.140 demonstrated significant incorporation of [14C]-CN in HLM/NADPH incubations containing 131 and [14C]-potassium cyanide. The in vitro metabolic profile of dexamethasone in HLM141 does not provide any insights into the structural nature of the electrophilic species that reacts with cyanide and/or liver proteins. When normalized against its total daily dose (20 mg) and maximal plasma concentrations in humans, the risk of IADRs due to reactive metabolite formation with dexamethasone appears to be significantly lower than that associated with


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hepatotoxic drugs,139 which is consistent with the fact that dexamethasone is not associated with significant IADRs including drug-induced liver injury.

Two additional “false-positive” cases with respect to protein covalent binding mediated partially via hard electrophiles are evident with the anxiolytic drug buspirone (132) and the antihistamine diphenhydramine (133) (Chart 1) that are not associated with IADRs. Compound 132 demonstrated appreciable covalent binding to HLM in a NADPH-dependent fashion, which was attenuated by ~ 30% upon co-incubation with cyanide or GSH.142 In the case of 133, inclusion of GSH and semicarbazide also reduced covalent binding by approximately 80% and 50%, respectively.142 The findings suggest that 132 and 133 are bioactivated by P450 into protein-reactive soft and hard electrophiles that are trapped by GSH, cyanide, or semicarbazide. The major biotransformation pathways of 132 in humans do not involve oxidative metabolism on the piperazine ring,143 hence the origins of the reactive metabolite(s) that is trapped by cyanide is not known. Whether electrophilic intermediates are derived from the major diphenyhydramine biotransformation pathways involving N-demethylation and deamination reactions144 is not known. In both these examples, the level of microsomal covalent binding was comparable to that seen with prototypic hepatotoxic drugs such as acetaminophen.129 However, normalization of the protein covalent binding data with fraction of total metabolism that comprises covalent binding and total daily dose significantly improved the discrimination between the two compounds (132 and 133) and acetaminophen.142

Finally, several marketed drugs possess cyclic amine framework (piperidine, piperazine, pyrrolidine rings, etc.), and furthermore, some agents also undergo oxidative metabolism on these ring systems, yet reports of toxicity including IADRs are scarce. Noteworthy examples


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(see Chart 1) include the phosphodiesterase type 5 enzyme inhibitor sildenafil (134), which is used to treat erectile dysfunction and the oral direct factor Xa inhibitor rivaroxaban (135), which is used to treat thromboembolic disorders. In humans, the principal clearance mechanism of 134 involves oxidative metabolism by P450 3A4 on multiple regions in the molecule including the piperazine ring.145–147 Examination of fecal metabolites of [14C]-134 in humans reveal that piperazine ring hydroxylation and cleavage products constitute ~ 50% of the radioactivity. The formation of the major fecal metabolite 136 can be rationalized in terms of α-carbon oxidation on both piperazine ring nitrogen atoms to yield iminium/aminoaldehyde intermediates followed by liberation of an electrophilic glyoxal derivative to afford 136. Rivaroxaban (135) is cleared in humans principally through oxidative modifications on the morpholinone ring by P450 3A4.148,149 In the case of 135, α-carbon oxidation adjacent to the morpholine ring oxygen atom is expected to yield aldehyde intermediates en route to the final ring cleavage products of metabolism (compounds 137–139 in Chart 1). As such, there are no reports on trapping iminiums and/or aminoaldehyde metabolites with 134 or 135 in HLM incubations, nor is there any evidence of time-dependent inactivation of P4503A4 that is principally responsible for the metabolism of these drugs. However, it is possible that adducts with exogenously added nucleophiles (e.g., cyanide and/or methoxylamine) will be detected using modern state-of-the-art bioanalytical instrumentation that is currently employed in reactive metabolite trapping screens. As such, the maximum recommended daily dose of sildenafil and rivaroxaban is 100 mg and 20 mg, respectively.

The propensity of new chemical entities to generate hard (and soft) electrophiles is routinely examined in HLM supplemented with NADPH co-factor, which only assesses P450 mediated oxidative transformations. While cyclic amines can generate protein-reactive iminium and


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aminoaldehyde intermediates, it is important to note that iminium ions can also be oxidized by cytosolic aldehyde oxidase to yield non-reactive lactams as stable metabolites.150–152 Furthermore, electrophilic ring opened aminoaldehyde products can be oxidized to stable aminocarboxylicacid metabolites via cytosolic and/or mitochondrial ALDH enzymes as noted with the factor Xa inhibitor rivaroxaban (Chart 1). Similarly, the primary alcohol moiety in abacavir is bioactivated by liver cytosolic ADH to the electrophilic aldehyde metabolite with a competing metabolic pathway involving primary alcohol glucuronidation by microsomal uridine glucuronosyl transferase(s). The impact of the phase II glucuronidation metabolism on abacavir bioactivation to the protein-reactive aldehyde remains unclear. These observations suggest that additional metabolism studies should be conducted with compounds that form hard electrophiles in HLM (inferred from reactive metabolite trapping studies with cyanide, amines, or even GSH) in fully integrated systems such as human hepatocytes and human liver S-9 fractions that support phase I (P450- and non-P450 oxidations) and phase II (conjugation) pathways. This will allow an evaluation of competing detoxication pathways on the bioactivation potential of new chemical entities as measured through reactive metabolite trapping studies or protein covalent binding. For instance, the high protein covalent binding induced by P450 2D6 catalyzed bioactivation of paroxetine to quinone intermediates (Scheme 3) in human liver S-9 fractions can be dramatically reduced upon inclusion of S-adenosylmethionine (a co-factor for catechol-Omethyltransferase).23 This finding is consistent with O-methylation of paroxetine catechol by catechol-O-methyltransferase to the corresponding guaiacol regioisomers, which are the major metabolites of paroxetine in humans.153 Certain cyclic amine moieties such as the N-substituted4-aryl-1,2,3,6-tetrahydropyridine and 4-aryl-N-substitutedpiperidin-4-ol, however, will (and should) deserve special scrutiny with respect to their metabolism by MAO and P450 to cyclic


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dihydropyridinium and the stable pyridinium metabolites, which have been linked with neurodegenerative effects including parkinsonism and tardive dyskinesia in humans as illustrated with the nigrostriatal neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and the neuroleptic agent haloperidol and related analogs.47,48,154–160

From a standpoint of quantitative risk assessment of toxicity arising from aldehydes, the World Health Organization working group established a tolerable daily oral intake of the chemically reactive α,β-unsaturated aldehyde acrolein to be 7.5 µg/kg (~ 0.52 mg for a 70 kg human) body weight in humans.161 Acrolein can be viewed as an analog of the electrophilic α,β-unsaturated aldehyde metabolite 111 of abacavir that covalently reacts with the terminal valine residue in hemoglobin. In other studies, the maximal daily consumption of unsaturated aldehydes (e.g., acrolein) in humans was estimated to be as high as ~ 5 mg/kg, whereas that of saturated aldehydes (e.g., acetaldehyde) was ~ 2 mg/kg.162,163 If so, then extrapolating these observations to a drug discovery scenario implies that the total body burden to reactive aldehyde metabolites generated in the course of metabolism of primary alcohol or cyclic (and acyclic) amine motifs in most low daily dose drugs is unlikely to exceed a threshold needed for eliciting a toxicological response (e.g., hepatoxicity arising from GSH depletion and/or immune-mediated IADRs). However, further studies will be required to probe this speculation in the clinic with a candidate drug such as abacavir (daily dose = 600 mg) by monitoring systemic (and excreta) levels of the O-glucuronide and carboxylic acid metabolites relative to hemoglobin adducts (e.g., compound 114 in Scheme 14) of the α,β-unsaturated Michael acceptor 111 in patients sensitive to abacavir IADRs.


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AUTHOR INFORMATION Corresponding Author

*610 Main St, Cambridge, MA 02139. Tel: 1-617-551-3336. E-mail: [email protected]

Notes The author is an employee and stockholder of Pfizer Inc.

DEDICATION This Perspective is dedicated to Professor Neal Castagnoli on his 80th birthday.

ABBREVIATIONS GSH, glutathione; HLM, human liver microsomes; P450, cytochrome P450; IADR, idiosyncratic adverse drug reactions; GGT, γ-glutamyltranspeptidase; 5-HT, 5-hydroxytryptamine (serotonin); MAO, monoamine oxidase; ALDH, aldehyde dehydrogenase; ADH, alcohol dehydrogenase.

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ischemic injury and blocks nitric oxide-induced PKCepsilon signaling and cardioprotection. J. Mol. Cell Cardiol. 44, 1016–1022.

Biography

Dr. Amit S. Kalgutkar is a research fellow in the Pharmacokinetics, Dynamics, and Metabolism Department at Pfizer, and an adjunct professor at the Department of Biomedical and Pharmaceutical Sciences, School of Pharmacy, University of Rhode Island. He received his Ph.D. in Chemistry from Virginia Tech in 1994, was a post-doctoral fellow at Vanderbilt University, and joined Pfizer in 1999. In his role as a drug metabolism scientist at Pfizer, he has been involved in the discovery and nomination of over a dozen clinical candidates spanning multiple therapeutic areas, and has authored more than 150 original papers and review articles, and is a co-inventor on several patents. He is currently on the editorial boards of Chemical Research in Toxicology, Drug Metabolism and Disposition, and Xenobiotica.


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Scheme 1. Proposed mechanism for the oxidative bioactivation of AZX (1) in HLM.

Scheme 2. Mechanisms for the formation of cyclized thiazolidine metabolites from the reaction of aldehydes with cysteine. R HN (A)

HN

OH

HO

- H2O O

R

R

H HO O

SH 7

O

H 8

S R HN

H

S

H NH

HO

HO (B)

O

R O

S

OH

HO

- H2O

HO O

9

COOH

H N O

NH2

11

S

NH2

HN

R

H

O

SH

H

NH

10

SH GGT

NH2

O

H

HN

O

COOH GSH

S

HN

S

HN

O

HOOC

O

COOH Cys-Gly

2-Methylthiazolidine-4carbonylglycine


58

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Scheme 3. Proposed mechanism for the generation of a cyclized ortho-benzoquinoneimine/GSH conjugate 20 in reactive metabolite trapping studies with the antidepressant paroxetine (12) with GSH.

Scheme 4. Pinner reaction of nitriles with cysteines.


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Scheme 5. Cyclized thiazolidine adducts derived from the oxidative deamination of serotonin (24) by MAO.


60

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Scheme 6. Oxidation of the nucleoside antiviral agent abacavir (32) to an aldehyde intermediate: Trapping studies with hard and soft nucleophiles.


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Page 62 of 68

Scheme 7. Generation of cyclized thiazolidine adducts during the oxidative bioactivation of piperazine rings. COOH H2N

H N O

[O]

HN R

COOH

H

H N

O

N H

HN

COOH

41

S

HN H HN

N

R

- Glu

COOH

O O

HN

GGT N

R 40

O

S

S HN

GSH

N

R

39

N H

O

N

N

O

N R

42

O - H2O

43

R1 O S

HN

O R=

N

HN NH

N

O

N O

R 44: R1 = NHCH2COOH 45: R1 = OH

F

N O N N

N [O]

N H

O

N N

46

HN

N O

Ar

47

Ar GSH

COOH

R

O

O

S

HN N

N

O

N N 48

S

HN

N

N Ar

O

N N

Ar 49: R = NHCH2COOH 50: R = OH


62

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Scheme 8. Intramolecular trapping of iminium ions obtained from the α-carbon oxidation of cyclic amines.

Scheme 9. Intramolecular trapping of an iminium species derived from the α-carbon oxidation of 5HT4 partial agonist 63.


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Page 64 of 68

Scheme 10. Proposed mechanisms of oxidative bioactivation with piperazine (66) and pyrrolidine (73) analogs leading to metabolism-dependent mutagenic response in Salmonella Ames test.

NH2

NH R

N

R

OH

N

R

R

O

N

N O

Cl

69

68

67 NH

NH2

CH3ONH2

R=

S9/NADPH

N

O 66

N R

OH

N

N

R 70

O

N

CN

N

R

N

N

71

N

OH CN

72 N

CN

R N 74

S9/NADPH

R N 73

80

79

78 75

HO

Ames Positive

CH3ONH2

76

O

O

N R=

R R NH

R N

N

N

N R N

CN

R N

R N

N

R NH N O 77

81

H2N

N

Ames Negative


64

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Scheme 11. Oxidative metabolism of the piperidine ring in the cannabinoid type 1 receptor antagonist rimonabant (82) to cyanide- and protein-reactive hard electrophiles.

Scheme 12. Proposed bioactivation pathway for the piperidine derivative 91 to protein-reactive metabolites.


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Page 66 of 68

Scheme 13. Oxidative bioactivation of the tetracyclic antidepressant mianserin (97) to proteinreactive metabolites. R N O

N

N CN

O NC

N

N N

N

SG-EE OH

104

103 107: R2 = CH3 108: R2 = H

109 Neutrophils or H2O2/HRP

GSH-EE

N N

R2 N

R N

R2 N 110

N

P450

O 105: R = CH3 106: R = H

N

P450

N

R1 Mianserin (97): R1 = H; R2 = CH3 8-Hydroxymianserin (98): R1 = OH; R2 = CH3 N-Desmethylmianserin (99): R1 = H; R2 = H

Iminium ions

R1

Mianserin (100): R1 = H; R2 = CH3 8-Hydroxymianserin (101): R1 = OH; R2 = CH3 N-Desmethylmianserin (102): R1 = H; R2 = H

Scheme 14. Rearrangement of the β,γ-unsaturated aldehyde metabolite 33 of abacavir to the protein-reactive 1,4-Michael acceptor 111.


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Scheme 15. Oxidative deamination of the triptan class of antimigrane drugs by MAO.


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Page 68 of 68

Chart 1. N

N

N

N

H N

N

N

H N

O

O

S O O

N N

N

H N

N N

N HN Cl

Mianserin (97)

Mirtazapine (128)

Imatinib (129)

OH

O

N

H

O N N

F

O

F N

OH

HO

Lapatinib (130)

N N

N

H

O

O

O

Diphenhydramine (133)

Buspirone (132)

Dexamethasone (131)

N

O

N N

Cl

O S

N

O

HN HN

N

O

R O

R

O Rivaroxaban (135): R =

S

O

O N

Sildenafil (134): R = 136: R =

N O

O

N H

N

137: R =

O

N

138: R =

139: R =

OH OH

O

HN

OH

HN


N OH OH

68

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