Is there enough evidence to classify cycloalkyl amine substituents as structural alerts?

Journal Pre-proofs Commentary Is There Enough Evidence to Classify Cycloalkyl Amine Substituents as Structural Alerts? Amit S. Kalgutkar, James P Driscoll PII: DOI: Reference:

S0006-2952(20)30006-X https://doi.org/10.1016/j.bcp.2020.113796 BCP 113796

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Biochemical Pharmacology

Received Date: Accepted Date:

19 November 2019 7 January 2020

Please cite this article as: A.S. Kalgutkar, J.P. Driscoll, Is There Enough Evidence to Classify Cycloalkyl Amine Substituents as Structural Alerts?, Biochemical Pharmacology (2020), doi: https://doi.org/10.1016/j.bcp. 2020.113796

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Is There Enough Evidence to Classify Cycloalkyl Amine Substituents as Structural Alerts? Amit S. Kalgutkar*1 and James P Driscoll2 1Medicine

Design, Pfizer Worldwide Research, Development and Medical, 1 Portland Street,

Cambridge, MA 02139 2Drug

Metabolism and Pharmacokinetics, Myokardia, Inc., South San Francisco CA 94080

Corresponding Author: Amit S. Kalgutkar, PhD, Medicine Design, Pfizer Worldwide Research, Development and Medical, 1 Portland Street, Cambridge, MA 02139, USA; Telephone: (617)551-3336; Email: [email protected] ABSTRACT Basic amine substituents provide several pharmacokinetic benefits relative to acidic and neutral functional groups, and have been extensively utilized as substituents of choice in drug design. On occasions, basic amines have been associated with off-target pharmacology via interactions with aminergic G-protein coupled receptors, ion-channels, kinases, etc. Structural features associated with the promiscuous nature of basic amines have been well-studied, and can be mitigated in a preclinical drug discovery environment. In addition to the undesirable secondary pharmacology, -carbon oxidation of certain secondary or tertiary cycloalkyl amines can generate electrophilic iminium and aldehyde metabolites, potentially capable of covalent adduction to proteins or DNA. Consequently, cycloalkyl amines have been viewed as structural alerts (SAs), analogous to functional groups such as anilines, furans, thiophenes, etc., which are oxidized to reactive metabolites that generate immunogenic haptens by covalently binding to host proteins. Detailed survey of the literature, however, suggests that cases where preclinical or 1

clinical toxicity has been explicitly linked to the metabolic activation of a cycloalkyl amine group are extremely rare. Moreover, there is a distinct possibility for the formation of electrophilic iminium/amino-aldehyde metabolites with numerous cycloalkyl amine-containing marketed drugs, since stable ring cleavage products have been characterized as metabolites in human mass balance studies. In the present work, a critical analysis of the evidence for and against the role of iminium ions/aldehydes as mediators of toxicity is discussed with a special emphasis on often time overlooked detoxication pathways of these reactive species to innocuous metabolites. Keywords: Cyclic amine, structural alert, iminium, aldehyde, reactive metabolite, cyanide, amine, IADRs, cytochrome P450, aldehyde oxidase. Abbreviations: ACSLI, acyl-CoA synthetase-1; ALT, alanine aminotransferase; AO, aldehyde oxidase; AST, aspartate aminotransferase; BSA, bovine serum albumin; BBW, black box warning; CYP, cytochrome P450; DILI, drug-induced liver injury; GSH, glutathione; IADRs, idiosyncratic adverse drug reactions; MAO, monoamine oxidase; MPTP, N-methyl-4-phenyl1,2,3,6-tetrahydropyridine; MPDP+, N-methyl-4-phenyl-2,3-dihydropyridinium; MPP+, Nmethyl-4-phenylpyridinium; SA, structural alert; SAR, structure-activity relationship; ULN, upper limit of normal.

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1.

Introduction

The process of metabolic activation (also referred to as bioactivation) of small molecule drugs into electrophilic reactive metabolites has been widely accepted as the rate-limiting step for the occurrence of idiosyncratic adverse drug reactions (IADRs), especially the ones that possess an immune component [1]. Covalent adduction of reactive species with endogenous host proteins in target tissue (e.g., liver in the case of drug-induced liver injury (DILI)) is believed to cause direct toxicity by inhibiting critical protein function or an immunogenic reaction through the presentation of hapten-carrier conjugates as noted with several drugs that have withdrawn and/or are associated with a black box warning (BBW) label for IADRs [2]. Since the downstream mechanisms by which reactive metabolites cause IADRs remain unclear, efforts to avoid, or at least reduce exposure to, such species embody a practicable approach for resolving questions around the toxicity potential of investigational drug candidates [2]. A general abstinence from using functional groups or substructures (referred to as structural alerts (SAs)) frequently found in drugs associated with toxicity is thought to be a pragmatic starting point for mitigating IADR risks in drug discovery [2]. 2.

Why are Cycloalkyl Amines Considered as Structural Alerts?

The cataloging of functional groups (e.g., anilines/anilides, hydrazines, benzenoid, nitroaromatics, 5-membered heteroaromatics, carboxylic acids, etc.) as SAs has been summarized in several accounts. Secondary and tertiary cycloalkyl amines have also been considered in this list, since these ring systems can be oxidatively metabolized to electrophilic species [3,4]. Bioactivation of cycloalkyl amines (e.g., compound 1) occurs through a cytochrome P450 (CYP) or monoamine oxidase (MAO) mediated ring -carbon oxidation to an iminium metabolite (2), which is in equilibrium with the corresponding enamine (3), 3

carbinolamine (4) and the amino-aldehyde (5) species (Fig. 1). The iminium species 2 is a hard electrophile, which can be trapped with a hard nucleophile such as potassium cyanide to yield cyanide conjugate 6 [3, 5–7]. The amino-aldehyde intermediate 5 also possesses electrophilic character and can react with both hard (e.g., amines such as methoxylamine) and soft (e.g. glutathione (GSH)) nucleophiles to generate aldoxime (7) and cyclized cysteine-glycinethiazolidine (8) conjugates, respectively [8]. In some instances, intramolecular trapping of 2 has been demonstrated, which further confirms their electrophilic nature. For example, -carbon oxidation on the serotonin 4 receptor partial agonist 9 generates iminium 10, which undergoes an intramolecular reaction with a neighboring alcohol group to form the cyclized oxazolidine metabolite 11 [9]. In principle, alkyl (and cycloalkyl) amines containing electron-deficient nitrogen atoms (e.g., amides, aminopyrimidines, etc.) such as 12 can also be oxidized to afford carbinolamides derivatives (e.g., compound 13) as stable metabolites [10]. In some cases, carbinolamides be hydrolyzed to electrophilic aldehyde species (e.g., compound 14) (see Fig. 1). CYP- or MAO-catalyzed -carbon oxidation is also observed with numerous primary, secondary and/or tertiary acyclic amine-containing xenobiotics and drugs (e.g., selective serotonin reuptake inhibitors such as fluoxetine and the triptan class of migraine drugs which includes sumatriptan and zolmitriptan). In the case of acyclic amines such as sumatriptan (15), the stable end-products of -carbon oxidation are the amine (18), carboxylic acid (19) and alcohol (20) derivatives (Fig. 1) [11]. There are no published accounts on the trapping of the intermediate iminium (16) and aldehyde (17) intermediates with nucleophiles (e.g., cyanide, amines, and/or GSH). Although in the case of the anesthetic lidocaine (21), an acyclic tertiary amine, the putative iminium intermediate 22, which is obtained from the -carbon oxidation on

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the N-ethyl moiety, is trapped in an intramolecular fashion by a adjacent amide nitrogen to afford the corresponding imidazolidinone derivative 23 as a metabolite of 21 in humans [12]. Instances of protein covalent modification by electrophilic species derived from the metabolism of cycloalkyl amines have also been described. For example, a CYP catalyzed sixelectron oxidation of the 1,3-disubstituted piperazine ring in the melanocortin agonist MB243 (24) (Fig. 2) to an electrophilic imine-methide 25 resulted in extensive covalent binding (> 2000 pmol equivalent/mg of microsomal protein) to liver microsomes from rat and human [13]. Inclusion of GSH in the microsomal incubations results in the formation of the sulfydryl conjugates 27 and 28, which are derived from the chemical rearrangement of the initial GSH adduct 26. Introduction of alkyl groups on the 1,3-piperazine ring in 24 proved to be an effective strategy in reducing protein covalent binding as evident with the a bridged isoquinuclidine derivative 29, which exhibited considerably less covalent binding (~ 26 pmol equivalent/mg of microsomal protein) to microsomal protein in comparison to 24, and was also orally active as a melanocortin receptor agonist. A second example relates to the CYP mediated piperidine ring bioactivation of the cannabinoid type 1 receptor antagonist rimonabant (30) (Fig. 2) in human liver microsomes, which leads to high levels of protein covalent binding and time-dependent irreversible inactivation of human CYP3A4 [14]. An electrophilic iminium 31 is the principal metabolite of 30 in human liver microsomes and can be trapped with cyanide to yield adduct 32 [14]. Consistent with this observation, microsomal covalent binding by 30 is reduced by ~ 40% upon coincubation with cyanide. Inclusion of methoxylamine in the human liver microsomal incubations with 30 also led to ~ 30% reduction in protein covalent binding but no corresponding aldoxime adduct(s) expected from reaction of the amine nucleophile with the putative amino5

aldehyde intermediate 34 (in equilibrium with carbinolamine 33) were detected. Subsequent incubation of electrochemically oxidized [14C]-30 with tryptic bovine serum albumin (BSA) digest and model peptides such as leucine-enkephalinamide and angiotensin II resulted in the characterization of peptide adducts (e.g., compound 40) derived from reaction of the peptide Nterminal amino group with an electrophilic metabolite of 30 [15]. The proposed bioactivation pathway involved oxidation of 31 to a dihydropyridinium species 36 (via the enamine 35) followed by a sequential -carbon oxidation and hydrolytic step to yield the electrophilic dialdehyde metabolite 39. Independent incubations of 39 with model peptides or BSA, which yielded 40, confirmed the proposed bioactivation mechanism [15]. A third example involves a sphingosine-1-phosphate receptor agonist MRL-A (41) (Fig. 2), which covalently modified hepatic and renal proteins in rats after in vivo administration of radiolabeled 41 [16]. Proteolytic digestion of proteins from target tissue generated a single major amino acid adduct 45. The proposed bioactivation pathway involves initial oxidative cleavage of the azetidine ring to the amino-aldehyde intermediate 43 (in equilibrium with the carbinolamine 42), followed by oxidation to an α,β-unsaturated aldehyde 44, which reacts with an ɛ-amino group within a lysine residue. Further digestion of rat liver homogenates led to the identification of the adducted peptide 45, which originated from covalent labeling of acyl-CoA synthetase-1 (ACSL1). These examples, while limited in nature, provide sufficient evidence in support of the hypothesis that bioactivation (-carbon oxidation) of cycloalkyl amines can yield reactive metabolites capable of adducting to proteins in vitro (and in vivo). 3.

Toxicological Consequence of Cycloalkyl Amine Bioactivation

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There are four published accounts on the covalent modification of proteins/DNA by cycloalkyl amine-derived reactive species as a cause for toxicity. The first two examples involving a selective dual A2A/A1 adenosine receptor antagonist 46 (pyrrolidine ring-based) and a 5-HT2C agonist 55 (piperazine ring-based) invoke the formation of reactive iminium and aminoaldehyde metabolites as an explanation for the genotoxicity observed in the bacterial Salmonella Ames reverse mutation and mouse Lymphoma L5178Y assays, since the mutagenic signals only surfaced in incubations containing a source of CYP enzymes (rat liver S9/NADPH) [17,18]. Both compounds covalently adducted to calf-thymus DNA in CYP dependent fashion, implying that they were oxidized to a reactive metabolite(s) that covalently adducted to DNA. Incubations of 46 or 55 with rat liver S9 fraction/NADPH in the presence of potassium cyanide or methoxylamine resulted in the formation of cyanide (51-53 and 61) or aldoxime (50 and 58) adducts, derived from addition of the nucleophiles to the electrophilic iminium (47 and 52), nitrone (60) and amino-aldehyde (49 and 57) intermediates. Moreover, in both instances, a reduction in the mutagenic response in the Ames assay and DNA covalent binding was observed upon co-incubations with cyanide and/or methoxylamine, which suggested that electrophilic species were responsible for the genotoxic effects of 46 and 55. From a structure-activity relationship (SAR) perspective, compound 54, which does not contain the pyrrolidine ring present in 46, was not genotoxic. Likewise, the lack of mutagenicity with 62 (an alkyl piperazine variant of 55) appeared to be consistent with a reduction in piperazine ring opening, presumably due to the presence of the metabolically labile methyl group [17]. The third example pertains to a zwitterionic piperidine derivative 63 (Fig. 3), which demonstrated elevations in liver enzymes and liver necrosis in single dose exploratory toxicology studies in monkeys but not in rats and dogs [19]. The excessive CYP-dependent protein covalent binding in liver microsomes

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from monkey (but not rat and human) was significantly reduced upon co-treatment with cyanide but not with GSH or methoxylamine, which was also consistent with the detection of a cyanide conjugate 68, derived from addition of the hard nucleophile to a mono-hydroxylated metabolite of 63. The bioactivation pathway (Fig. 3) involves an -carbon oxidation on the piperidine ring in 63 to the iminium 64 as a rate-limiting step. Because the monkey was not representative of the metabolism of 63 in humans, it was speculated that non-human primates would have been a poor choice as a non-rodent species for preclinical toxicity studies. To date, there is only one example which discusses bioactivation of a cycloalkyl amine motif as a contributing factor in IADRs. Immune-mediated IADRs such as blood dyscrasias and DILI, have been reported during the clinical use of the tetracyclic antidepressant mianserin (69) [20], have been linked to a CYP catalyzed bioactivation of 69 and its human metabolites 8hydroxymianserin (70) and N-desmethylmianserin (71) to electrophilic iminium ions 72-74 in human liver microsomes (Fig. 4) [21–23]. The characterization of cyanide adducts in incubations of 69 with human liver microsomes in the presence of cyanide is consistent with the formation of iminium species. Studies with radiolabeled 69 and its metabolites also resulted in covalent binding to microsomal protein and cytotoxicity towards human mononuclear leukocytes, which were included in the microsomal incubation. An electrophilic iminium species 75 and its corresponding cyanide adduct 76 were also characterized in incubations of 69 in human neutrophils and in incubations with horseradish peroxidase/H2O2 [24]. Bioactivation of 69 in human neutrophils was also associated with apoptosis in a peroxide-dependent fashion [24]. SAR studies indicate that replacement of the nitrogen atom from the N5 position in 69 with a methine group (compound 77) reduces cytotoxicity as does substituting a methyl group for a

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hydrogen atom at position C14b (compound 78) (Fig. 4), which lends additional support to the potential role of the iminium in the IADRs associated with 69 [23]. It is premature to qualify the cycloalkyl amine group as a SA given the paucity of available examples, particularly ones related to idiosyncratic toxicity. The case studies discussed above with marketed drugs also raise several questions. For example, rimonabant (30) was withdrawn after two years in the European Union market due to serious psychiatric side effects (e.g., depressive disorder, mood alterations) and not due to prototypic IADRs (e.g., immune-mediated DILI) that can be attributed to protein-conjugates derived from piperidine ring oxidation products such as 39. As such, there is no known DILI risk associated with 30 based on clinical experience to-date. A low daily dose (≤ 50 mg) is thought to be a crucial mitigating factor for IADR risks, especially for drugs that form reactive metabolites [2]. Rimonabant (30) fulfils this requirement, since its daily dose for the treatment of obesity was 20 mg. The circumstantial evidence supporting the contribution of electrophilic iminium metabolites in the agranulocytosis encountered with 69 can also be challenged based on the good safety record of the corresponding 6-aza analog mirtazapine (79) (Fig. 4), which has been marketed as an antidepressant in the United States since 1996. Clinical use of 79 is rarely associated (1 in 1000) with the agranulocytosis noted with 69, despite similarities in metabolic profile of the two drugs in humans and comparable daily doses (45 mg and 60 mg for 69 and 79, respectively) [25]. Whether oxidation of 79 and its human metabolites 8-hydroxymirtazapine (80) and Ndesmethylmirtazapine (81) results in the formation of iminium species analogous to 69 is currently not known. In addition to reactive species arising from -carbon oxidation, a new bioactivation pathway was described for 69, which involves CYP mediated oxidation of 8hydroxymianserin 70 and a para-hydroxyphenyl metabolite (82) of N-desmethylmianserin (71), 9

respectively, to quinone-imine intermediates 83 and 84, which are trapped as the corresponding GSH-ethyl ester conjugates (compounds 85 and 86) in human liver microsomes (see Fig. 4) [26]. Whether the pyridine nitrogen in 79 prevents the formation of electrophilic iminium ion and/or quinone-imine species that are detected with 69 will need to be studied, which may qualify (or disqualify) the present weight of evidence supporting bioactivation as a causative factor in the agranulocytosis with 69. Finally, it is worth commenting on the unique metabolism-dependent mutagenicity with 46 and 55 in the Salmonella Ames assay. There are no other published accounts around DNA reactivity of electrophilic iminium and/or aldehyde intermediates arising from the metabolism of cyclic (or acyclic) amine-containing investigational (or marketed) drugs. The DNA affinity of the electrophilic metabolites of 46 and 55 may be due to the flatness of the chemical structures of the parent compounds (and reactive metabolites), since such a trend is also noted with procarcinogens (e.g., aflatoxin B1 and polycyclic aromatic hydrocarbons) whose carcinogenic properties are dependent on CYP catalyzed oxidations to reactive metabolites that covalently adduct to DNA. 4.

Detoxication of Reactive Metabolites Derived from the Bioactivation of Cycloalkyl

Amines Trapping of iminium species with cyanide was first described in studies aimed at understanding the toxicities of xenobiotics such as the Parkinsonian-inducing neurotoxin N-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP, 87) and the tobacco alkaloid nicotine (92) (Fig. 5) at the molecular level. Both 87 and 92 are metabolized by CYP and/or MAO to stable iminium metabolites N-methyl-4-phenyl-2,3-dihydropyridinium (MPDP+, 88) and nicotine-Δ1′ (5′)-iminium

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(93), which can also be derivatized with cyanide to the respective conjugates 89 and 94 in human liver microsomes, liver mitochondria and/or recombinant MAO incubations [27,28]. However, a frequently overlooked detoxication pathway of iminium metabolites is their facile oxidation by aldehyde oxidase (AO) to non-reactive lactam metabolites [29]. Since AO resides in the cytosol, the detoxication pathway can only be studied in human liver cytosol, S-9 fractions, and/or hepatocytes. Consistent with this behavior, the iminium metabolites 88 and 93 are efficiently detoxicated by AO in liver cytosol and/or human hepatocytes to lactams 90 and 95, respectively [28,30]. In fact, the metabolic conversion of 92 to the lactam cotinine (95) represents its principal clearance mechanism in humans [28]. The iminium metabolite 88 generated by brain MAO-B undergoes further oxidation to yield the ultimate neurotoxin N-methyl-4phenylpyridinium (MPP+, 91), which inhibits complex I of the mitochondrial electron transport chain leading to cessation of oxidative phosphorylation and depletion of ATP [3]. Because of the lack of AO activity in the brain, the detoxication pathway involving the conversion of 88 to 90 only occurs in the liver [30]. Detoxication of electrophilic iminium metabolites has also been demonstrated in two recent examples involving momelotinib (96), a small molecule inhibitor of Janus kinase 1/2 and clinical candidate for the treatment of myeloproliferative neoplasms and a multikinase inhibitor KW2449 (100) (Fig. 5) [31,32]. In human liver microsomes, CYP enzymes oxidize the morpholine ring in 96 to the iminium 97, which is trapped with cyanide to afford 98. However, in human hepatocytes, 97 is also oxidized by AO to the lactam 99, which retains biological activity as a Janus kinase 1/2 inhibitor and is observed as a major circulating metabolite of 96 in humans. Likewise, the iminium metabolite 101, derived from an MAO-B catalyzed oxidation of the piperazine ring in 100, is trapped with cyanide to furnish adduct 102 in human liver 11

microsomes/mitochondria. However, in human liver S-9 or human hepatocytes, 100 is almost exclusively converted to the lactam 103 in a AO-dependent fashion. The lactam 103 is the principal circulating metabolite of 100 in humans, with circulating metabolite concentrations ~ 10 times higher than those of the parent compound. Moreover, in the case of rimonabant (30), circulating metabolites derived from multistep oxidations on the piperidine moiety have been characterized in humans, which include a lactam metabolite (SR90161), derived from the detoxication of the electrophilic iminium metabolite 31 by AO (https://www.ema.europa.eu/en/documents/scientific-discussion/acomplia-epar-scientificdiscussion_en.pdf). The detoxication pathways of amino-aldehydes (and all aldehydes in general) are primarily mediated by oxidative and reductive phase I enzyme catalyzed reactions to form non-reactive carboxylic acid and/or alcohol derivatives as metabolites. Enzymes capable of oxidizing aldehydes include CYP, AO (and xanthine oxidase), and aldehyde dehydrogenase (ALDH) [33– 35]. The ALDH superfamily play a particularly important role in the cellular protection against potentially toxic aldehydes as evidenced by the fact that mutations and polymorphisms in ALDH genes which leads to impaired aldehyde metabolism are the molecular basis of several disease states [35]. Like AO, ALDH enzymes are expressed in the cytosol as well as in the mitochondria, and therefore, assessment of aldehyde detoxication requires metabolism studies in human liver S-9 fraction or in human hepatocytes. Reduction of aldehydes (including aminoaldehyde intermediates) to the corresponding alcohols is mediated via the non-CYP reductive enzyme systems alcohol dehydrogenase, aldo-keto reductase, and short-chain dehydrogenase/reductase, which are localized in microsomes, cytosol, and blood [34].

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5.

Analysis of Idiosyncratic Toxicity Trends for Marketed Drugs, Which Contain a

Cycloalkyl Amine Motif Basic amine centers (both acyclic and cyclic) are widely exploited in drug design to improve aqueous solubility and/or tissue penetration (e.g., brain penetration) in addition to general improvements in pharmacologic potency [36]. The metabolic process of ring -carbon oxidation to stable non-reactive metabolites has been demonstrated with several marketed drugs that contain a cycloalkyl amine substituent. As per the catalytic mechanism of -carbon oxidation, the biotransformation pathway is bound to generate the expected iminium/amino-aldehyde derivatives as reactive species. For example, sildenafil (104) (Fig. 6), which is used to treat erectile dysfunction, is cleared via oxidative metabolism by CYP3A4 on several sites in the molecule including the piperazine ring [37]. In human mass balance studies, piperazine ring hydroxylation and cleavage products constitute ~ 50% of the administered radioactivity. The formation of the major fecal metabolite 105 can be rationalized in terms of -carbon oxidation on both piperazine ring nitrogen atoms to yield iminium/amino-aldehyde intermediates followed by liberation of an electrophilic glyoxal derivative to afford 105. To further examine how common this phenomenon is, we decided to gather additional evidence with regards to the ring -carbon oxidation potential of cycloalkyl amine-containing marketed drugs (2009–present) (https://www.fda.gov/). Visual examination of the structural features of the marketed drugs revealed 50 examples where a cycloalkyl amine/amide substituent was present as part of the chemical architecture. The drugs are segregated on the basis of their IADR risks, and in addition, information pertaining to the indication, daily dose, cycloalkyl amine/amide ring type, and presence of alternate SAs is also provided (Tables 1 and 2). Out of

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the 50 structurally diverse cycloalkyl amine/amide-containing drugs, 28 were devoid of idiosyncratic toxicities (e.g., DILI, agranulocytosis, and cutaneous reactions) based on safety information gathered from their respective package inserts (Table 1). The daily doses of the drugs ranged from 1–560 mg, which is consistent with the assortment of their primary pharmacologic targets. Moreover, 12 out of 28 compounds were also found to possess alternate SAs. Overall, these observations re-emphasize the notion that not all SA-containing drugs are toxic. A crucial requirement for metabolism-driven toxicity is the bioactivation of the SA into a reactive metabolite. Eight out of the 28 drugs were also found to undergo cycloalkyl ring carbon oxidations as evident by the formation of stable downstream oxidation products (lactams, amino-carboxylic acids or -alcohols) as metabolites (Table 1). For example, avanafil, a phosphodiesterase 5 inhibitor used to treat erectile dysfunction, is cleared predominantly in humans via metabolism by CYP3A4 and to a lesser extent by CYP2C9 [39]. Amino-carboxylic acid M16 (Fig. 6) is the major metabolite of avanafil with circulating concentrations ~ 29% that of the parent compound [39]. M16 is obtained from the pyrrolidine ring -carbon oxidation to carbinolamine 106, followed by ALDH mediated oxidation of its tautomeric aldehyde (ringopened) form 107. There are no reports on the trapping of iminiums and/or amino-aldehyde metabolites, which are expected to be formed during the metabolism of avanafil and for that matter sildenadil (104). However, it is more than likely that cyanide, amine or GSH conjugates will be detected if these two compounds are incubated in human liver microsomes and/or recombinant CYP3A4 in the presence of exogenous nucleophiles, especially when considering that ring -carbon oxidation constitutes their major route of metabolism. Both 104 and avanafil possess excellent safety records with respect to IADRs at their recommended daily doses of 100 mg and 200 mg, respectively.

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Based on the safety information presented in the package inserts, 22 out of the 50 cycloalkyl amine/amide-containing drugs were associated with IADRs, primarily involving liver and skin as target tissue (Table 2). The daily doses of these drugs ranged from 3–1200 mg. Anticancer agents, which represented the vast majority (18 out of the 22 drugs), were the ones in the high daily dose category (45–1200 mg). Despite IADR risks, all of the 22 drugs remain on the market, which speaks to their favorable benefit-risk ratio in the treatment of cancer. Moreover, 16 out of the 22 drugs were also found to possess alternate SAs. The aniline/anilide motif was a prominent fixture in most of the anticancer drugs, and 3 out of the 18 anticancer drugs (acalabrutinib, carfilzomib, dacomitinib) were targeted covalent inhibitors of tyrosine kinases. Out of 22 drugs, 13 were metabolized on their cycloalkyl rings as exemplified with alectinib, cobimetinib, and fedratinib, where metabolites arising from CYP3A4 catalyzed -carbon oxidation also constituted the major clearance route in humans (Fig. 6). The second generation anaplastic lymphoma kinase inhibitor alectinib is oxidized on its morpholine ring to form multiple hydroxylated-morpholine isomers (M5), which undergo ring scission to form M1a, M4, and M6 as cleavage products [67,68]. Metabolite M4 is the major circulating metabolite of alectinib in humans [67]. Cobimetinib is a mitogen-activated protein kinase inhibitor, which contains a piperidine and a azetidinone substituent. While the azetidinone functionality is resistant to oxidative metabolism, several oxidative metabolites have been observed, which include piperazine ring cleavage products (M32 and M40) and the nitrone (M19) metabolite. In addition, a lactam metabolite (M12) potentially obtained by an AO mediated oxidation of an iminium intermediate has also been observed in circulation and excreta. In the case of the Janus kinase 2 inhibitor fedratinib, the pyrrolidinone derivative (SAR317981), which can be obtained via oxidation of the initial iminium intermediate by AO, is the major 15

circulating metabolite (accounting for ~ 9% of the area under the plasma concentration versus time curve). SAR317981 also retains Janus kinase 2 inhibitory activity of the parent, albeit with some loss of potency. A second circulating metabolite is the N-butyric acid derivative SAR318031, which can be generated via hydrolysis of the putative iminium intermediate, followed by subsequent oxidation [77]. Because the majority of the cycloalkyl amine drugs associated with IADRs contained additional SAs (16 out of 22), it is difficult to formally (or even circumstantially) associate toxicity with the formation of reactive species via ring -carbon oxidation. For example, in the case of the anaplastic lymphoma kinase inhibitor crizotinib (Table 2), which is associated with rare cases of reversible DILI (grade 3 or 4 elevations in alanine aminotransferase (ALT) in 4–7 % of the patients), human mass balance/metabolite identification studies indicate that the lactam derivative (M10), derived from piperazine ring oxidation, is its major circulatory metabolite (Fig. 7) [75]. Although crizotinib does not contain a prototypic SA, CYP3A4 catalyzed O-dealkylation on crizotinib and M10 yields the hydroxyaminopyridine metabolites M4 and M2, respectively, which can be considered as SAs because of their propensity to form reactive quinone-imine metabolites. Consistent with this hypothesis, a cysteine conjugate (M6) of M2, presumably derived from GSH trapping of the putative quinoneimine metabolite, was detected as a minor urinary metabolite in humans [75]. A second example deals with the tyrosine kinase inhibitor imatinib (Fig. 7), which is a potent mechanism-based inactivator of CYP3A4 activity in human liver microsomes [88], and is also associated with DILI [89]. Reactive metabolite trapping studies with stable labeled potassium cyanide and methoxylamine led to the detection of seven cyanide conjugates and one aldoxime adduct [6], derived from piperazine ring -carbon oxidation. However, the speculation [7] that piperazine ring bioactivation in imatinib, and for that matter, in crizotinib, could potentially account for

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DILI is questionable, considering that several tyrosine kinase inhibitors that do not contain a cycloalkyl amine functionality (e.g., lapatinib, pazopanib, sunitinib, regorafenib, etc.) are also associated with hepatotoxicity [90]. Moreover, human mass balance studies with [14C]-imatinib indicate that the major metabolite is derived from piperidine ring N-demethylation with minimal contribution (< 2% of the total circulating radioactivity in plasma or excreta) from piperidine ring oxidation products [91]. Another interesting structure-metabolism relationship also evident with the quinolone antibiotics and piperazine-containing drugs ciprofloxacin and levofloxacin (Fig. 7), both of which associated with hypersensitivity and severe DILI incidences. One could attribute ciprofloxacin DILI to reactive metabolites obtained from the metabolism of its piperazine ring, since piperazine ring scission products, including the lactam, have been observed as biliary metabolites in humans [92]. However, such a hypothesis could be easily challenged when considering that the major clearance mechanism of levofloxacin in humans involves renal excretion in unchanged form (87% of the administered dose) as depicted in its package insert. 6.

Critical Examination of Standalone Iminium/Aminoaldehyde Trapping Assays

Collectively, if all 50 drugs (especially the 28 drugs that are not associated with prototypic IADRs) included in this analysis were to be subjected to reactive metabolite trapping assays utilizing GSH, cyanide, and amine as trapping agents, it is very likely that most of these marketed agents will demonstrate a positive signal for conjugate formation with one or more nucleophiles, which we suspect will be independent of their IADR risk profile. This is because the assays, which are limited to incubations in human liver microsomes, are bound to generate false positive signals, since they cannot inspect aforementioned detoxication pathways involving contributions from cytosolic and/or mitochondrial enzymes such as glutathione transferases, AO, 17

ALDH, and keto reductases, etc. There is an additional complication around interpretation of data around extremely low levels of reactive metabolite conjugates observed in liver microsomes. For instance, low levels of several cyanide adducts have been detected in human liver microsomal incubations with the hepatotoxic antidepressant nefazodone (Fig. 7), which are derived from the -carbon oxidation on its piperazine ring and its downstream metabolites [93]. However, the extensive CYP-dependent protein covalent binding that is observed with 14Cnefazodone in human liver microsomes is not impacted by the inclusion of cyanide or semicarbazide in the incubation mixtures [94]. A dramatic decrease (69–85%) in protein covalent binding to human liver microsomes or liver S-9 fractions is observed only in the presence of GSH, which is consistent with a previously detailed bioactivation sequence involving CYP mediated hydroxylation of the 3-chlorophenylpiperazine motif in nefazodone to p-hydroxynefazodone, a circulating metabolite of nefazodone in humans, followed by its oxidation to quinone-type reactive metabolites amenable to trapping with GSH [93]. Moreover, failure to detect metabolites derived from the -carbon oxidation on the piperazine ring in nefazodone in human mass balance studies [95] further discounts the low levels of cyanide conjugates detected in the in vitro microsomal incubations with nefazodone. Finally, some discussion is warranted on the nature of the reactive species that is capable of irreversible protein modification. Reaction of protein amino acid residues such as cysteine and lysine with reactive iminiums will yield the corresponding hemithioaminal or hemiaminal intermediates, which are likely to be unstable, and revert back to the iminium species (and free protein). If so, then the electrophilic amino-aldehyde metabolite, derived from the hydrolysis of the iminium intermediate, is a likely candidate for reaction with nucleophiles such as GSH, DNA bases and/or lysine residues on proteins as noted in the examples discussed in this perspective. 18

Since the formation of an iminium species precedes that of the amino-aldehyde intermediate, it is obvious that cyanide anion will prevent the toxicological outcome (e.g., DNA or protein covalent modification by the amino-aldehyde) associated with a cycloalkyl amine derivative. Ultimately, this begs the question whether a reactive metabolite trapping assay with cyanide is even required in drug discovery, especially when the amino-aldehyde metabolite can be trapped with an appropriate amine nucleophile. In fact, we propose a shift in paradigm from high-throughput screening for electrophilic iminium/amino-aldehyde intermediates in human liver microsomes to a more rounded characterization of the major metabolites in hepatocytes for representative members of a new chemical series, which will offer a more holistic comparison of the overall metabolic disposition pathways, involving detoxication of reactive species (iminium → lactams by AO and amino-aldehyde → amino-carboxylic acids or -alcohols by aldehyde dehydrogenases/reductases) derived from -carbon oxidation of cycloalkyl amines and possibly other SAs (e.g., anilines), which are bioactivated to soft electrophiles (e.g., quinone-imines), capable of forming conjugates with endogenous GSH in hepatocytes. Mianserin (69) remains the only example where idiosyncratic toxicity in humans has been circumstantially attributed to the metabolism of its cycloalkyl amine substituent into an electrophilic iminium, and more recently, due to the generation of an electrophilic quinoneimine. As noted earlier, mirtazapine (79) is structurally similar to 69, but is not associated with blood dyscrasias and/or DILI despite administration at comparable daily doses. Hence, a detailed comparison of the in vitro metabolic and bioactivation fate of these two antidepressants in NADPH-supplemented human liver microsomes (± CN, ± GSH) and in human hepatocytes (± the AO inhibitor hydralazine to block the iminium detoxication pathway leading to the lactam derivative [96]) is warranted as a pragmatic starting point towards validating the use of 19

hepatocytes to study cycloalkyl amine ring bioactivation/detoxication. If the corresponding radiolabeled versions of 69 and 79 are readily available (or can be synthesized), then comparative protein covalent binding studies in NADPH-supplemented human liver microsomes (± CN, ± GSH) and in human hepatocytes (± hydralazine) should also prove fruitful in estimating the metabolic intrinsic clearance that arises from protein covalent modification by reactive metabolites, and eventually lead to estimates of the body burden to reactive metabolite exposure expressed as a function of the total daily dose [94, 97–99]. In addition, a side-by-side comparison of the principal metabolic pathways and propensity to form both hard (cycloalkyl amine/amide bioactivation) and soft (bioactivation on other SAs such as anilines) reactive metabolites is needed for the 50 drugs (examined in this paper) in liver microsomes and hepatocytes. Corresponding studies with radiolabeled versions (if and when available) of these drugs should facilitate the estimation of the overall metabolic intrinsic clearance as well as the intrinsic clearance arising from reactive metabolite formation (characterized through protein covalent binding studies). Such a dataset will also enable the determination of the total body burden due to electrophilic metabolite exposure in relation to the daily doses of the respective drugs. The combined mechanistic and quantitative covalent binding dataset may shed additional light regarding the propensity of the 28 drugs (Table 2) to cause DILI relative to the other compounds examined, as was demonstrated in previous studies to discriminate between hepatotoxins and non-hepatotoxins [94, 97–99]. Moreover, the data can also be utilized for a simple correlation of the IADR potential (e.g., DILI) with the degree of CYP catalyzed metabolic intrinsic clearance in relation to the lipophilicity and the daily doses of the drugs. Several studies have demonstrated that the risk of DILI with withdrawn and/or marketed drugs

20

increases significantly for drugs with substantial CYP mediated clearance in the liver, daily doses ≥ 100 mg, and octanol-water partition coefficient of ≥ 3 [100–102]. With respect to studies aimed at examining iminium detoxication by AO, it is also important to note that AO expression in animals and human is the highest in the liver, and oxidative metabolism by AO is largely dominated by hepatic AO activity [35]. Immunohistochemical analysis of several human tissues has demonstrated that excretory organs such as the gastrointestinal tract, lung and kidney also express catalytically active AO [103], which can further serve to detoxify iminium species that are generated extrahepatically via the action of CYP enzymes. Marked species differences in the expression and catalytic activity of AO have also been noted across mammals [104]. AO activity generally appears to be high in monkeys and humans and low in rats. In contrast, dogs are largely deficient in AO. As a consequence, iminium detoxication is likely to be compromised in animal species other than non-human primates. Thus, it is tempting to speculate that if toxicity (e.g., DILI) was to occur via protein covalent modification by iminium intermediates, dogs would be the most susceptible species since they do not express AO. As such, there are no published accounts to substantiate or refute this hypothesis. Cycloalkyl amines such as the N-substituted-4-aryl-1,2,3,6-tetrahydropyridine and 4-aryl-Nsubstitutedpiperidin-4-ol will require additional scrutiny with respect to their CYP and/or MAO mediated -carbon oxidation to cyclic dihydropyridinium and stable pyridinium metabolites, which have been linked to neurodegenerative effects in animals and humans as illustrated with the nigrostriatal neurotoxin 87. It is therefore necessary that the N-substituted-4-aryl-1,2,3,6tetrahydropyridine and 4-aryl-N-substitutedpiperidin-4-ol motifs remain in the list of SAs. For compounds that contain such sub-structures, demonstration of their metabolic conversion to 21

stable dihydropyridinium and pyridinium metabolites in liver, and most importantly in brain tissue, should be considered as evidence for replacing these substituents during the pharmacology SAR campaign, which will avoid unnecessary cumbersome in vivo toxicity studies. As such, the Parkinsonian-like effects of 87, observed in humans, can be replicated in chronic (and acute) toxicology studies in mice and monkeys, should drug discovery teams chose to further interrogate the toxicological consequences of pyridinium metabolite formation in a preclinical setting, especially when the precursor motifs cannot be easily replaced with alternate substituents [105,106]. Conflicts of Interest: The authors declare no conflict of interest in relation to the contents of this work. References [1] [2]

[3]

[4] [5]

[6]

T. Cho, J. Uetrecht, How reactive metabolites induce an immune response that sometimes leads to an idiosyncratic drug reaction, Chem. Res. Toxicol. 30 (2017) 295–314. A. F. Stepan, D. P. Walker, J. Bauman, D. A. Price, T. A. Baillie, A. S. Kalgutkar, M. D. Aleo, Structural alert/reactive metabolite concept as applied in medicinal chemistry to mitigate the risk of idiosyncratic drug toxicity: a perspective based on the critical examination of trends in the top 200 drugs marketed in the United States, Chem. Res. Toxicol. 24 (2011) 1345–1410. A. S. Kalgutkar, I. Gardner, R. S. Obach, C. L. Shaffer, E. Callegari, K. R. Henne, A. E. Mutlib, D. K. Dalvie, J. S. Lee, Y. Nakai, J. P. O’Donnell, J. Boer, S. P. Harriman, A comprehensive listing of bioactivation pathways of organic functional groups, Curr. Drug Metab. 6 (2005) 161–225. J. Bolleddula, K. DeMent, J. P. Driscoll, P. Worboys, P. J. Brassil, D. L. Bourdet, Biotransformation and bioactivation reactions of alicyclic amines in drug molecules, Drug Metab. Rev. 46 (2014) 379–419. D. Argoti, L. Liang, A. Conteh, L. Chen, D. Bershas, C. P. Yu, P. Vouros, E. Yang, Cyanide trapping of iminium ion reactive intermediates followed by detection and structure identification using liquid chromatography-tandem mass spectrometry (LCMS/MS) Chem. Res. Toxicol. 18 (2005) 1537–1544. J. R. Kenny, S. Mukadam, C. Zhang, S. Tay, C. Collins, A. Galetin, S. C. Khojasteh, Drug-drug interaction potential of marketed oncology drugs: in vitro assessment of timedependent cytochrome P450 inhibition, reactive metabolite formation and drug-drug interaction prediction, Pharm. Res. 29 (2012) 1960–1976. 22

[7] [8] [9]

[10] [11] [12] [13]

[14]

[15] [16] [17]

[18]

[19]

A. C. Li, E. Yu, S. C. Ring, J. P. Chovan, Structural identification of imatinib cyanide adducts by mass spectrometry and elucidation of bioactivation pathway, Rapid Commun. Mass Spectrom. 28 (2014) 123–134. K. Inoue, K. Fukuda, T. Yoshimura, K. Kusano, Comparison of the reactivity of trapping reagents toward electrophiles: Cysteine derivatives can be bifunctional trapping reagents, Chem. Res. Toxicol. 28 (2015) 1546–1555. A. Sawant-Basak, K. J. Coffman, G. S. Walker, T. F. Ryder, E. Tseng, E. Miller, C. Lee, M. A. Vanase-Frawley, J. W. Wong, M. A. Brodney, T. Rapp, R. S. Obach, Metabolism of a serotonin-4 receptor partial agonist 4-{-40[4-tetrahydrofuran-3-yloxy)benzo[d]isoxazol-3-yloxymethyl]-piperidin-1-ylmethyl}-tetrahydropyran-4-ol (TBPT): Identification of an unusual pharmacologically active cyclized oxazolidine metabolite in human, J. Pharm. Sci. 102 (2013) 3277–3293. L. Perrin, N. Loiseau, F. André, M. Delaforge, Metabolism of N-methyl-amide by cytochrome P450s: formation and characterization of highly stable carbinol-amide intermediate, FEBS J. 278 (2011) 2167–2178. A. S. Kalgutkar, D. K. Dalvie, N. Castagnoli Jr, T. J. Taylor, Interactions of nitrogencontaining xenobiotics with monoamine oxidase (MAO) isozymes A and B: SAR studies on MAO substrates and inhibitors, Chem. Res. Toxicol. 14 (2001) 1139–1162. G. D. Breck, W. F. Trager, Oxidative N-dealkylation: a mannich intermediate in the formation of a new metabolite of lidocaine in man, Science 173 (1971) 544–546. G. A. Doss, R. R. Miller, Z. Zhang, Y. Teffera, R. P. Nargund, B. Palucki, M. K. Park, Y. S. Tang, D. C. Evans, T. A. Baillie, R. A. Stearns, Metabolic activation of a 1,3disubstituted piperazine derivative: evidence for a novel ring contraction to an imidazoline, Chem. Res. Toxicol. 18 (2005) 271–276. A. J. Foster, L. H. Prime, F. Gustafsson, D. G. Temesi, E. M. Isin, J. Midlöv, N. Castagnoli Jr, J. G. Kenna, Bioactivation of the cannabinoid receptor antagonist rimonabant to a cytotoxic iminium ion metabolite, Chem. Res. Toxicol. 26 (2013) 124– 135. A. Thorsell, E. M. Isin, U. Jurva, Use of electrochemical oxidation and model peptides to study nucleophilic biological targets of reactive metabolites: the case of rimonabant, Chem. Res. Toxicol. 27 (2014) 1801–1820. H. Aloysius, V. W. Tong, J. Yabut, S. A. Bradley, J. Shang, Y. Zou, R. A. TschirretGuth, Metabolic activation and major protein target of a 1-benzyl-3-carboxyazetidine sphingosine-1-phosphate-1 receptor agonist, Chem. Res. Toxicol. 25 (2012) 1412–1422. A. S. Kalgutkar, J. N. Bauman, K. F. McClure, J. Aubrecht, S. R. Cortina, J. Paralkar, Biochemical basis for differences in metabolism-dependent genotoxicity by two diazinylpiperazine-based 5-HT2C receptor agonists, Bioorg. Med. Chem. Lett. 19 (2009) 1559–1563. H. K. Lim, J. Chen, C. Sensenhauser, K. Cook, R. Preston, T. Thomas, B. Shook, P. F. Jackson, S. Rassnick, K. Rhodes, V. Gopaul, R. Salter, J. Silva, D. C. Evans, Overcoming the genotoxicity of a pyrrolidine substituted arylindenopyrimidine as a potent dual adenosine A(2A)/A(1) antagonist by minimizing bioactivation to an iminium ion reactive intermediate, Chem. Res. Toxicol. 24 (2011) 1012–1030. T. A. Baillie, Metabolism and toxicity of drugs. Two decades of progress in industrial drug metabolism, Chem. Res. Toxicol. 21 (2008) 129–137.

23

[20] [21] [22] [23] [24]

[25]

[26] [27] [28] [29] [30] [31]

[32]

[33] [34] [35]

K. Otani, S. Kaneko, H. Tasaki, Y. Fukushima, Hepatic injury caused by mianserin, BMJ 299 (1989) 519. R. J. Riley, J. L. Maggs, C. Lambert, N. R. Kitteringham, B. K. Park, An in vitro study of the microsomal metabolism and cellular toxicity of phenytoin, sorbinil and mianserin, Br. J. Clin. Pharmacol. 26 (1988) 577–588. R. J. Riley, P. Roberts, N. R. Kitteringham, B. K. Park, B. K. Formation of cytotoxic metabolites from phenytoin, imipramine, desipramine, amitriptyline and mianserin by mouse and human hepatic microsomes, Biochem. Pharmacol. 39 (1990) 1951–1958. P. Roberts, N. R. Kitteringham, B. K. Park, Elucidation of the structural requirements for the bioactivation of mianserin in-vitro, J. Pharm. Pharmacol. 45 (1993) 663–665. S. Iverson, N. Zahid, J. P. Uetrecht, Predicting drug-induced agranulocytosis: characterizing neutrophil-generated metabolites of a model compound, DMP 406, and assessing the relevance of an in vitro apoptosis assay for identifying drugs that may cause agranulocytosis, Chem. Biol. Interact. 142 (2002) 175–199. L. P. C. Delbressine, M. E. G. Moonen, F. M. Kaspersen, G. N. Wagenaars, P. L. Jacobs, C. J. Timmer, J. E. Paanakker, H. J. M. van Hal, G. Voortman, Pharmacokinetics and biotransformation of mirtazapine in human volunteers, Clin. Drug Invest. 15 (1998) 45– 55. B. Wen, W. L. Fitch, Screening and characterization of reactive metabolites using glutathione ethyl ester in combination with Q-trap mass spectrometry, J. Mass Spectrom. 44 (2009) 90–100. E. Wu, T. Shinka, P. Caldera-Munoz, H. Yoshizumi, A. Trevor, N. Castagnoli Jr, Metabolic studies on the nigrostriatal toxin MPTP and its MAO B generated dihydropyridinium metabolite MPDP+, Chem. Res. Toxicol. 1 (1988) 186–194. J. Hukkanen, P. Jacob 3rd, N. L. Benowitz, Metabolism and disposition kinetics of nicotine, Pharmacol. Rev. 57 (2005) 79–115. S. Brandange, L. Lindblom, The enzyme “aldehyde oxidase” is an iminium oxidase. Reaction with nicotine delta 1’(5’) iminium ion, Biochem. Biophys. Res. Commun. 91 (1979) 991–996. S. Yoshihara, S. Ohta, Involvement of hepatic aldehyde oxidase in conversion of 1methyl-4-phenyl-2,3-dihydropyridinium (MPDP+) to 1-methyl-4-phenyl-5,6-dihydro-2pyridone, Arch. Biochem. Biophys. 360 (1998) 93–98. J. Hosogi, R. Ohashi, H. Maeda, S. Tashiro, E. Fuse, Y. Yamamoto, T. Kuwabara, Monoamine oxidase B oxidizes a novel multikinase inhibitor KW-2449 to its iminium ion and aldehyde oxidase further converts it to the oxo-piperazine form in human, Drug Metab. Pharmacokinet. 32 (2017) 255–264. J. Zheng, Y. Xin, J. Zhang, R. Subramanian, B. P. Murray, J. A. Whitney, M. R. Warr, J. Ling, L. Moorehead, E. Kwan, J. Hemenway, B. J. Smith, J. A. Silverman, Pharmacokinetics and disposition of momelotinib revealed a disproportionate human metabolite-resolution for clinical development, Drug Metab. Dispos. 46 (2018) 237–247. F. P. Guengerich, Common and uncommon cytochrome P450 reactions related to metabolism and chemical toxicity, Chem. Res. Toxicol. 14 (2001) 611–650. V. Vasiliou, A. Pappa, T. Estey, Role of human aldehyde dehydrogenases in endobiotic and xenobiotic metabolism, Drug Metab. Rev. 36 (2004) 279–299. D. Dalvie, L. Di, Aldehyde oxidase and its role as a drug metabolizing enzyme, Pharmacol. Ther. 201 (2019) 137–180. 24

[36] [37] [38] [39] [40] [41] [42]

D. A. Smith, K. Beaumont, T. S. Maurer, L. Di, Volume of distribution in drug design, J. Med. Chem. 58 (2015) 5691–5698. D. K. Walker, M. J. Ackland, G. C. James, G. J. Muirhead, D. J. Rance, P. Wastall, P. A. Wright, Pharmacokinetics and metabolism of sildenafil in mouse, rat, rabbit, dog and man, Xenobiotica 29 (1999) 297–310. N. Raghavan, C. E. Frost, Z. Yu, K. He, H. Zhang, W. G. Humphreys, D. Pinto, S. Chen, S. Bonacorsi, P. C. Wong, D. Zhang, Apixaban metabolism and pharmacokinetics after oral administration to humans, Drug Metab. Dispos. 37 (2009) 74–81. https://www.vivus.com/docs/SpedraProductCharacteristics_SmPC.pdf S. F. van de Wetering-Krebbers, P. L. Jacobs, G. J. Kemperman, E. Spaans, P. A. Peeters, L. P. Delbressine, M. L. van lersel, Metabolism and excretion of asenapine in healthy male subjects, Drug Metab. Dispos. 39 (2011) 580–590. M. L. Sargentini-Maier, P. Espié, A. Coquette, A. Stockis, Pharmacokinetics and metabolism of 14C-brivaracetam, a novel SV2A ligand, in healthy subjects, Drug Metab. Dispos. 36 (2008) 36–45. L. Citrome, Cariprazine: chemistry, pharmacodynamics, pharmacokinetics, and metabolism, clinical efficacy, safety, and tolerability, Expert Opin. Drug Metab. Toxicol. 9 (2013) 193–206.

[43] https://www.accessdata.fda.gov/drugsatfda_docs/nda/2017/208082Orig1s000ClinPharm R.pdf [44] M. S. Bathala, H. Masumoto, T. Oguma, L. He, C. Lowrie, J. Mendell, Pharmacokinetics, biotransformation, and mass balance of edoxaban, a selective, direct factor Xa inhibitor, in humans, Drug Metab. Dispos. 40 (2012) 2250–2255. [45] J. L. Lam, A. Vaz, B. Hee, Y. Liang, X. Yang, M. N. Shaik, Metabolism, excretion and pharmacokinetics of [14C]glasdegib (PF-04449913) in healthy volunteers following oral administration, Xenobiotica 47 (2017) 1064–1076. [46] E. Scheers, L. Leclercq, J. de Jong, N. Bode, M. Bockx, A. Laenen, F. Cuyckens, D. Skee, J. Murphy, J. Sukbuntherng, G. Mannens, Absorption, metabolism, and excretion of oral 14C radiolabeled ibrutinib: an open-label, phase I, single-dose study in healthy men, Drug Metab. Dispos. 43 (2015) 289–297. [47] M. Francois-Bouchard, G. Simonin, M. J. Bossant, C. Boursier-Neyret, Simultaneous determination of ivabradine and its metabolites in human plasma by liquid chromatography-tandem mass spectrometry, J. Chromatogr. B Biomed. Sci. Appl. 745 (2000) 261–269. [48] K. A. Usmani, W. G. Chen, A. J. Sadeque, Identification of human cytochrome P450 and flavin-containing monooxygenase enzymes involved in the metabolism of lorcaserin, a novel selective human 5-hydroxytryptamine 2C agonist, Drug Metab. Dispos. 40 (2012) 761–771. [49] https://www.accessdata.fda.gov/drugsatfda_docs/nda/2010/200603Orig1s000PharmR.pdf [50] https://www.accessdata.fda.gov/drugsatfda_docs/nda/2014/206162Orig1s000PharmR.pdf [51] https://www.accessdata.fda.gov/drugsatfda_docs/nda/2016/207318Orig1s000ClinPharm R.pdf

25

[52] N. A. Farid, R. L. Smith, T. A. Gillespie, T. J. Rash, P. E. Blair, A. Kurihara, M. J. Goldberg, The disposition of prasugrel, a novel thienopyridine, in humans, Drug Metab. Dispos. 35 (2007) 1096-1104. [53] https://www.accessdata.fda.gov/drugsatfda_docs/nda/2018/210166Orig1s000Multidiscipl ineR.pdf [54] [55] [56] [57]

[58]

https://www.accessdata.fda.gov/drugsatfda_docs/nda/2018/210598Orig1s000Clin PharmR.pdf C. Weinz, T. Schwarz, D. Kubitza, W. Mueck, D. Lang, Metabolism and excretion of rivaroxaban, an oral, direct factor Xa inhibitor, in rats, dogs, and humans, Drug Metab. Dispos. 37 (2009) 1056–1064. Z. Y. Zhang, J. Wang, V. Kansra, X. Wang, Absorption, metabolism, and excretion of the antiemetic rolapitant, a selective neurokinin-1 receptor antagonist, in healthy male subjects, Invest. New Drugs 37 (2019) 139–146. S. H. Vincent, J. R. Reed, A. J. Bergman, C. S. Elmore, B. Zhu, S. Xu, D. Ebel, P. Larson, W. Zeng, L. Chen, S. Dilzer, K. Lasseter, K. Gottesdiener, J. A. Wagner, G. A. Herman, Metabolism and excretion of the dipeptidyl peptidase 4 inhibitor [14C]sitagliptin in humans, Drug Metab. Dispos. 35 (2007) 533–538. M. Zollinger, F. Lozach, E. Hurh, C. Emotte, H. Bauly, P. Swart, Absorption, distribution, metabolism, and excretion (ADME) of 14C-sonidegib (LDE225) in healthy volunteers, Cancer Chemother. Pharmacol. 74 (2014) 63–75.

[59] [60]

[61] [62]

[63] [64] [65] [66]

https://www.accessdata.fda.gov/drugsatfda_docs/nda/2018/211651Orig1s000Mult idisciplineR.pdf M. E. Dowty, J. Lin, T. F. Ryder, W. Wang, G. S. Walker, A. Vaz, G. L. Chan, S. Krishnaswami, C. Prakash, The pharmacokinetics, metabolism, and clearance mechanisms of tofacitinib, a janus kinase inhibitor, in humans, Drug Metab. Dispos. 42 (2014) 759–773. D. E. Grigoriadis, E. Smith, S. R. J. Hoare, A. Madan, H. Bozigian, Pharmacologic characterization of valbenazine (NBI-98854) and its metabolites, J. Pharmacol. Exp. Ther. 361 (2017) 454–461. N. J. Hewitt, K. U. Bühring, J. Dasenbrock, J. Haunschild, B. Ladstetter, D. Utesch, Studies comparing in vivo:in vitro metabolism of three pharmaceutical compounds in rat, dog, monkey, and human using cryopreserved hepatocytes, microsomes, and collagen gel immobilized hepatocyte cultures, Drug Metab. Dispos. 29 (2001) 1042–1050. G. Chen, A. M. Højer, J. Areberg, G. Nomikos, Vortioxetine: clinical pharmacokinetics and drug interactions, Clin. Pharmacokinet. 57 (2018) 673–686. A. A. Kadi, H. W. Darwish, H. A. Abuelizzi, T. A. Alsubi, M. W. Attwa, Identification of reactive intermediate formation and bioactivation pathways in abemaciclib metabolism by LC-MS/MS: in vitro metabolic investigation, R. Soc. Open Sci. 6 (2019) 181714. http://pi.lilly.com/ca/verzenio-ca-pm.pdf T. Podoll, P. G. Pearson, J. Evarts, T. Ingallinera, E. Bibikova, H. Sun, M. Gohdes, K. Cardinal, M. Sanghvi, J. G. Slatter, Bioavailability, biotransformation, and excretion of

26

[67]

[68]

the covalent bruton tyrosine kinase inhibitor acalabrutinib in rats, dogs, and humans, Drug Metab. Dispos. 47 (2019) 145–154. P. N. Morcos, L. Yu, K. Bogman, M. Sato, H. Katsuki, K. Kawashima, D. J. Moore, M. Whayman, K. Nieforth, K. Heinig, E. Guerini, D. Muri, M. Martin-Facklam, A. Phipps, Absorption, distribution, metabolism and excretion (ADME) of the ALK inhibitor alectinib: results from an absolute bioavailability and mass balance study in healthy subjects, Xenobiotica 47 (2017) 217–229. M. Sato-Nakai, K. Kawashima, T. Nakagawa, Y. Tachibana, M. Yoshida, K. Takanashi, P. N. Morcos, M. Binder, D. J. Moore, L. Yu, Metabolites of alectinib in human: their identification and pharmacological activity, Heliyon 3 (2017) e00354.

[69] [70] [71] [72] [73] [74]

[75]

[76]

https://www.accessdata.fda.gov/drugsatfda_docs/nda/2013/022271Orig1s000Clin PharmR.pdf R. Abbas, P. H. Hsyu, Clinical pharmacokinetics and pharmacodynamics of bosutinib, Clin. Pharmacokinet. 55 (2016) 1191–1204. J. R. Enders, S. G. Reddy, E. C. Strickland, G. L. McIntire, Identification of metabolites of brexipiprazole in human urine for use in monitoring patient compliance, Clinical Mass Spectrometry 6 (2017) 21–24. Z. Wang, J. Yang, C. Kirk, Y. Fang, M. Alsina, A. Badros, K. Papadopoulos, A. Wong, T. Woo, D. Bomba, J. Li, J. R. Infante, Clinical pharmacokinetics, metabolism, and drugdrug interaction of carfilzomib, Drug Metab. Dispos. 41 (2013) 230–237. https://www.ema.europa.eu/en/documents/assessment-report/zykadia-epar-publicassessment-report_en.pdf R. H. Takahashi, E. F. Choo, S. Ma, S. Wong, J. Halladay, Y. Deng, I. Rooney, M. Gates, C. E. Hop, S. C. Khojasteh, M. J. Dresser, L. Musib, Absorption, metabolism, excretion, and the contribution of intestinal metabolism to the oral disposition of [14C]cobimetinib, a MEK inhibitor, in humans, Drug Metab. Dispos. 44 (2016) 28–39. T. R. Johnson, W. Tan, L. Goulet, E. B. Smith, S. Yamazaki, G. S. Walker, M. T. O’Gorman, G. Bedarida, H. Y. Zou, J. G. Christensen, L. N. Nguyen, Z. Shen, D. Dalvie, A. Bello, B. J. Smith, Metabolism, excretion and pharmacokinetics of [14C]crizotinib following oral administration to healthy subjects, Xenobiotica 45 (2015) 45–59. C. L. Bello, E. Smith, A. Ruiz-Garcia, G. Ni, C. Alvey, C. M. Loi, A phase I, open-label, mass balance study of [(14)C] dacomitinib (PF-00299804) in healthy male volunteers, Cancer Chemother. Pharmacol. 72 (2013) 379–385.

[77] [78] [79]

https://www.accessdata.fda.gov/drugsatfda_docs/nda/2019/212327Orig1s000Mult idisciplineR.pdf https://www.drugs.com/ppa/gilteritinib.html https://www.accessdata.fda.gov/drugsatfda_docs/nda/2018/210861Orig1s000_21 1710Orig1s000MultidisciplineR.pdf

[80] https://www.accessdata.fda.gov/drugsatfda_docs/nda/2018/210861Orig1s000_21 1710Orig1s000MultidisciplineR.pdf

27

[81] [82]

[83]

P. Stopfer, K. Rathgen, D. Bischoff, S. Lüdtke, K. Marzin, R. Kaiser, K. Wagner, T. Ebner, Pharmacokinetics and metabolism of BIBF 1120 after oral dosing to healthy male volunteers, Xenobiotica 41 (2011) 297–311. L. van Andel, Z. Zhang, S. Lu, V. Kansra, S. Agarwal, L. Hughes, M. M. Tibben, A. Gebretensae, L. Lucas, M. J. X. Hillebrand, H. Rosing, J. H. M. Schellens, J. H. Beijnen, Human mass balance study and metabolite profiling of 14C-niraparib, a novel poly(ADPRibose) polymerase (PARP)-1 and PARP-2 inhibitor, in patients with advanced cancer, Invest. New Drugs 35 (2017) 751–765. B. B. Chavan, S. Tiwari, G. Shankar, R. D. Nimbalkar, P. Garg, R. Srinivas, M. V. N. K. Talluri, In vitro and in vivo metabolic investigation of the palbociclib by UHPLC-QTOF/MS/MS and in silico toxicity studies of its metabolites, J. Pharm. Biomed. Anal. 157 (2018) 59–74.

[84] https://www.accessdata.fda.gov/drugsatfda_docs/nda/2012/203469Orig1s000Clin PharmR.pdf [85] [86] [87]

[88] [89] [90] [91] [92] [93]

[94]

https://www.accessdata.fda.gov/drugsatfda_docs/nda/2017/209092Orig1s000Mult idisciplineR.pdf P. Martin, S. Oliver, S. J. Kennedy, E. Partridge, M. Hutchinson, D. Clarke, P. Giles, Pharmacokinetics of vandetanib: three phase I studies in healthy subjects, Clin. Ther. 34 (2012) 221–237. H. Liu, M. J. Michmerhuizen, Y. Lao, K. Wan, A. H. Salem, J. Sawicki, M. Serby, S. Vaidyanathan, S. L. Wong, S. Agarwal, M. Dunbar, J. Sydor, S. M. de Morais, A. J. Lee, Metabolism and disposition of an novel B-cell lymphoma-2 inhibitor venetoclax in humans and characterization of its unusual metabolites, Drug Metab. Dispos. 45 (2017) 294–305. A. M. Filppula, J. Laitila, P. J. Neuvonen, J. T. Backman, Potent mechanism-based inhibition of CYP3A4 by imatinib explains its liability to interact with CYP3A4 substrates, Br. J. Pharmacol. 165 (2012) 2787–2798. T. J. Cross, C. Bagot, B. Portmann, J. Wendon, D. Gillett, Imatinib mesylate as a cause of acute liver failure, Am. J. Hematol. 81 (2006) 189–192. K. W. Lee, S. L Chan, hepatotoxicity of targeted therapy for cancer, Expert Opin. Drug Metab. Toxicol. 12 (2016) 789–802. H. P. Gschwind, U. Pfaar, F. Waldmeier, M. Zollinger, C. Sayer, P. Zbinden, M. Hayes, R. Pokorny, M. Seiberling, M. Ben-Am, B. Peng, G. Gross, Metabolism and disposition of imatinib mesylate in healthy volunteers, Drug Metab. Dispos. 33 (2005) 1503–1512. H. Tanimura, S. Tominaga, F. Rai, H. Matsumoto, Transfer of ciprofloxacin to bile and determination of biliary metabolites in humans, Arzneimittelforschung 36 (1986) 1417– 1420. J. N. Bauman, K. S. Frederick, A. Sawant, R. L. Walsky, L. M. Cox, R. S. Obach, A. S. Kalgutkar, Comparison of the bioactivation potential of the antidepressant and hepatotoxin nefazodone with aripiprazole, a structural analog and marketed drug, Drug Metab. Dispos. 36 (2008) 1016–1029. R. S. Obach, A. S. Kalgutkar, J. R. Soglia, S. X. Zhao, Can in vitro metabolismdependent covalent binding data in liver microsomes distinguish hepatotoxic from

28

[95] [96] [97]

[98]

[99]

[100] [101] [102] [103] [104] [105] [106]

nonhepatotoxic drugs? An analysis of 18 drugs with consideration of intrinsic clearance and daily dose, Chem. Res. Toxicol. 21 (2008) 1814–1822. R. F. Mayol, C. A. Cole, G. M. Luke, K. L. Colson, E. H. Kerns, Characterization of the metabolites of the antidepressant drug nefazodone in human urine and plasma, Drug Metab. Dispos. 22 (1984) 304–311. T. J. Strelevitz, C. C. Orozco, R. S. Obach, Hydralazine as a selective probe inactivator of aldehyde oxidase in human hepatocytes: estimation of the contribution of aldehyde oxidase to metabolic clearance. Drug Metab. Dispos. 40 (2012) 1441–1448. J. N. Bauman, J. M. Kelly, S. Tripathy, S. X. Zhao, W. W. Lam, A. S. Kalgutkar, R. S. Obach, Can in vitro metabolism-dependent covalent binding data distinguish hepatotoxic from nonhepatotoxic drugs? An analysis using human hepatocytes and liver S-9 fraction. Chem. Res. Toxicol. 22 (2009) 332–340. R. A. Thompson, E. M. Isin, Y. Li, L. Weidolf, K. Page, I. Wilson, S. Swallow, B. Middleton, S. Stahl, A. J. Foster, H. Dolgos, R. Weaver, J. G. Kenna, In vitro approach to assess the potential for risk of idiosyncratic adverse reactions caused by candidate drugs. Chem. Res. Toxicol. 25 (2012) 1616–1632. S. Nakayama, R. Atsumi, H. Takakusa, Y. Kobayashi, A. Kurihara, Y. Nagai, D. Nakai, O. Okazaki, A zone classification system for risk assessment of idiosyncratic drug toxicity using daily dose and covalent binding. Drug Metab. Dispos. 37 (2009) 1970– 1977. K. Yu, X. Geng, M. Chen, J. Zhang, B. Wang, K. Ilic, W. Tong, High daily dose and being a substrate of cytochrome P450 enzymes are two important predictors of druginduced liver injury. Drug Metab. Dispos. 42 (2014) 744–750. M. Chen, J. Borlak, W. Tong, High lipophilicity and high daily dose of oral medications are associated with significant risk for drug-induced liver injury. Hepatology 58 (2013) 388–396. K. McEuen, J. Borlak, W. Tong, M. Chen, Associations of drug lipophilicity and extent of metabolism with drug-induced liver injury. Int. J. Mol. Sci. 18 (2017) 1335. Y. Moriwaki, T. Yamamoto, S. Takahashi, Z. Tsutsumi, T. Hada, Widespread cellular distribution of aldehyde oxidase in human tissues found by immunohistochemistry staining. Histol. Histopathol. 16 (2001) 745–753. M. Strolin-Benedetti, R. Whomsley, E. Baltes, Involvement of enzymes other than CYPs in the oxidative metabolism of xenobiotics. Expert Opin. Drug Metab. Toxicol. 2 (2006) 895–921. G. J. Masilamoni, Y. Smith, Chronic MPTP administration regimen in monkeys: a model of dopaminergic and non-dopaminergic cell loss in Parkinson’s disease. J. Neural Transm. (Vienna) 125 (2018) 337–363. V. Jackson-Lewis, S. Przedborski, Protocol for the MPTP mouse model of Parkinson’s disease. Nat. Protoc. 2 (2007) 141–151.

29

Figure Legends Figure 1. Reactive metabolites (iminium/amino-aldehyde) derived from the bioactivation of amines. Figure 2. Covalent modification of proteins by reactive species obtained from the bioactivation of cycloalkyl amines. Figure 3. Illustrative case studies of toxicity arising from the bioactivation of cycloalkyl amines. Figure 4. Bioactivation of the tetracyclic antidepressant mianserin (69) to reactive metabolites. Figure 5. Detoxication of electrophilic iminium metabolites by cytosolic aldehyde oxidase. Figure 6. -Carbon oxidation of the cycloalkyl amine motif in marketed drugs. Figure 7. Cycloalkyl amine ring scission metabolites of crizotinib and chemical structures of ciprofloxacin, levofloxacin, imatinib, and nefazodone, which are associated with DILI.

Table 1. Marketed drugs that contain cycloalkyl amine (or cycloalkyl amide) motifs, which are not associated with IADRs. Drug

Indication

Daily Dose (mg)a

SA Cyclic amine

Apixaban

Anticoagulant

10

Avanafil

Erectile dysfunction

200

Pyrrolidine

30

Cyclic amide Piperidinone

Metabolism on cyclic amine/amide Yes: Hydroxylation (M4/M7) – minor [38] Yes: Major circulating metabolites – M4 (pyrrolidine ring

D

N

N

Noc

A

No [41] No [42] No [43] No [44] Yes: Hydroxylation, enamine, lactam – all minor [45] Yes: Hydroxylation, ring opening – minor [46] No [47]

N A N N A

A N N N

N A

Asenapine

Antipsychotic

Brigatinib

Anticancer

20 (sublingual) 180

Brivaracetam Cariprazine Deutetrabenazine Edoxaban Glasdegib

Anticonvulsant Antipsychotic Chorea Anticoagulant Anticancer

100 6 48 125 100

Ibrutinib

Anticancer

560

Piperidinone

Ivabradine

Heart Failure

15

Benzazepin-2one

Letermovir Lorcaserin Lurasidone Olarapib

Antiviral Obesity Antipsychotic Anticancer

480 20 120-160 2

Piperazine Benzazepine Piperazine

Pimavanserin Prasugrel Prucalopride Revefenacin Rivaroxaban

Antipsychotic Anticoagulant Constipation COPD Anticoagulant

34 10 2 175 (g) 10-15

Piperidine Piperidine Piperidine Piperidine

Rolapitant

Chemotherapyinduced nausea

180

Piperidine

Sitagliptin

Antidiabetic

100

Sonidegib

Anticancer 200 Anticancer 1 Antiinflammatory 11

Morpholine

Tardive dyskinesia Antidepressant Antidepressant

80

Piperidine

Nod No [48] No [49] Yes: Dehydrogenation (enamine) [50] No [51] No [52] No [53] No [54] Yes: oxidative scission of morpholinone [55] Ambiguous; regiochemistry not established [56] Yes: Iminium species trapped intramolecularly [57] Yes: [58] No [59] Ambiguous; regiochemistry not established [60] No [61]

40 10

Piperazine Piperazine

No [62] No [63]

Talazoparib Tofacitinib Valbenazine Vilazodone Vortioxetine aDaily

Pyrrolidine

hydroxylation) and M16 (ring-opened) are approximately 23% and 29% that of the parent compound, respectively [39] No [40]

Piperazine & Piperidine Pyrrolidinone Piperazine Piperidine Piperidine Piperidine

Piperazinone

Morpholinone

Piperazinone

Piperidine Piperidinone

dose and IADR information extracted from package inserts. bSA presence deduced from

the chemical structure. The phenyl ring was not considered as a SA. cAccording to the package 31

A

N

N T A N T D N

N

A A N

N

insert, N-demethylation (AP26123) on the piperazine ring and cysteine conjugation are the primary metabolic pathways of brigatinib, and are mediated by CYP2C8 and CYP3A4. dAccording

to the package insert, letermovir is primarily metabolized via glucuronidation (on its

carboxylic acid moiety). Contribution of CYP enzymes towards metabolism is minimal.

Table 2. Marketed drugs that contain cycloalkyl amine (or cycloalkyl amide) motifs, which are associated with IADRs. Drug

Indication

Daily Dose (mg)a

SA Cyclic amine Piperazine

Metabolism on cyclic amine/amide

Ot

Yes: Hydroxylation, iminium, cyanide conjugation detected in rat liver microsomes [64]. Structural details on human metabolites is ambiguous [65].

No

Pyrrolidinone Yes: Multiple hydroxylations, dehydrogenation, ring scission [66]. Yes: Morpholine ring hydroxylation/ring scission [67,68].

Mi Ac

Cyclic amide

Abemaciclib

Anticancer

300

Acalabrutinib

Anticancer

200

Alectinib

Anticancer

1200

Piperidine, Morpholine

Alogliptin

Antidiabetic

25

Piperidine

No: Minimal metabolic elimination. Major route of clearance is renal elimination in unchanged form [69].

No

Bosutinib

Anticancer

500

Piperazine

No: [70]

An

32

An

Brexipiprazole

Antipsychotic

3–4

Piperazine

No: [71]

An Th

Carfilzomib

Anticancer

20-25 mg/m2 (IV)

Morpholine

No: [72]

Ep

Ceritinib

Anticancer

450

Piperidine

Yes: Minor route of metabolism involving hydroxylation/lactam formation [73].

An

Cobimetinib

Anticancer

60

Piperidine

Yes: Extensive oxidations on azetidine and piperidine rings [74].

An

Crizotinib

Anticancer

500

Piperidine

No

Dacomitinib

Anticancer

45

Piperidine

Yes: Piperidine ring oxidation to lactam is the major route of metabolism [75]. Yes: Piperidine ring oxidation (hydroxylation, ring scission and lactam formation) [76].

Fedratinib

Anticancer

400

Pyrrolidine

Yes: The predominant plasma metabolite (SAR317981, 9% circulating radioactivity) is the pyrrolidone (lactam)

An

33

Azetidinone

An Ac

Gilteritinib

Anticancer

120

Piperidine,

Piperazine 200

Pyrrolidine

derivative of unchanged drug. The second circulating metabolite (SAR318031)is a N‐butyric acid derivative of SAR317981, 6% of radioactivity) [77]. No detailed metabolism data available [78].

Larotrectinib

Anticancer

Lomitapide

Familial 60 hypercholesterolemia

Piperidine

Yes: Hydroxylation/lactam in parent and its Ndealkylated metabolite [80].

No

Nintedanib

Idiopathic pulmonary fibrosis

300

Piperazine

Yes: Piperazine ring hydroxylation – minor pathway [81]

An Ac

Niraparib

Anticancer

300

Piperidine

No: [82]

An

Palbociclib

Anticancer

125

Piperazine

No

Ponatinib

Anticancer

45

Piperazine

Yes: Multiple hydroxylations, ring scission, and lactam formation in human liver microsomes [83]. Yes: Hydroxylation, dehydrogenation, lactam formation [84].

Ribociclib

Anticancer

600

Piperazine

Unclear; N-demethylation is a metabolic pathway. In addition, reactive

Dia

34

Pyrrolidinone No detailed metabolism data available [79].

Dia

No

Alk An

Vandetanib

Anticancer

300

Piperidine

Venetoclax

Anticancer

400

Piperazine

aDaily

metabolites of ribociclib formed by CYP3A4, and to a lesser extent FMO-3, were shown to covalently bind to human liver microsomes and human hepatocytes [85]. No: [86]

Yes: Iminium formation and intramolecular cyclization, hydroxylation and lactam formation [87].

dose and IADR information extracted from package inserts. bSA presence deduced from

the chemical structure. The phenyl ring was not considered as a SA. ALT, alanine aminotransferase; AST, aspartate aminotransferase; ULN, upper limit of normal

CRediT Author Statement: Amit Kalgutkar: Conceptualization, Methodology, Data Curation and Investigation, Writing. James Driscoll: Data Curation and Investigation, Writing.

35

An

Nit

36