Systematic Approach to Organizing Structural Alerts for Reactive

Perspective Cite This: Chem. Res. Toxicol. 2018, 31, 389−411

pubs.acs.org/crt

Systematic Approach to Organizing Structural Alerts for Reactive Metabolite Formation from Potential Drugs Alf Claesson* and Alexander Minidis Awametox AB, Lilldalsvägen 17 A, SE-14461 Rönninge, Sweden

Chem. Res. Toxicol. 2018.31:389-411. Downloaded from pubs.acs.org by MCMASTER UNIV on 06/20/18. For personal use only.

S Supporting Information *

ABSTRACT: Reactive metabolites are widely accepted as playing a pivotal role in causing idiosyncratic adverse drug reactions (IDR). However, much is unknown about the biological mechanisms of IDR, although the initiating event in most cases is an attachment of a reactive intermediate to macromolecules leading to immune-mediated responses. Reactive metabolites are also involved in many mutagenesis/carcinogenesis events by reacting with DNA. Drug designers thus have reasons to make large efforts to avoid making test compounds having a liability to generate reactive metabolites. In this Perspective we argue for using structural alerts (SA) as the most straightforward way to link forecasting about chemical hazards of planned test compounds to the accumulated knowledge base. Although many SAs have been widely recognized and reviewed previously, there are also a lot of observations that have no readily discernible chemical interpretation. For drug designers to benefit from all published data, the knowledge has to be organized in a way that is readily searchable starting with a query structure. We propose that an increased number of structural alerts with more details should be applied to obtain improved decision support. The association of selected SAs with reference drugs, whose proposed or hypothesized activation mechanisms build the knowledge base, should be readily available in a format comprising of small summaries with included hyperlinks for quick access to the original literature, as outlined in the TOC illustration. Since some structural alerts are present in drugs that do not cause idiosyncratic adverse reactions or drug−drug interactions, it is important to elaborate on the reasons for this discrepancy as much as possible.

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CONTENTS

1. Introduction 2. Factors Influencing the Awareness/Avoidance Process 3. Principles for Selecting SAs 4. Recognized SAs as the Basis for an Expert System 5. Principles for Setting up a Searchable SA System 6. Selecting and Describing the Most Important SAs 6.1. Benzene and Naphthalene 6.2. Halobenzenes 6.3. Nitrogen-Substituted Benzenes 6.4. Oxygen-Substituted Benzenes/Aromatics: Precursors of Quinones 6.5. Benzenes/Aromatics Concurrently Substituted with Oxygen and Nitrogen 6.6. Precursors of Quinone Methides (Quinomethanes) and Imines 6.7. Heteroaromatics © 2018 American Chemical Society

6.7.1. Six-Membered Rings 6.7.2. Five-Membered Heterocycles (FMHs) 6.7.3. Fused Heterocyclic Ring Systems 6.8. Alkyl on Aromatics 6.9. Other Types of Unsaturation 6.10. c-Propylamines 6.11. Other Elimination Reactions 6.12. Geminal Dihaloalkyl 6.13. Carboxylic Acids 6.14. Hydrazine and Derivatives 6.15. Thioamides, Thioureas, and Other Sulfur Compounds 6.16. Other Structural Alerts 7. Protein Kinase Inhibitors Highlighted 8. Knowledge Creates New Hypotheses

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Received: February 21, 2018 Published: May 10, 2018 389

DOI: 10.1021/acs.chemrestox.8b00046 Chem. Res. Toxicol. 2018, 31, 389−411

Chemical Research in Toxicology

Perspective

Chart 1. Structures Mentioned in the Text

9. Discussion 9.1. Dominance of Aromatics, but They Are Not Alone 9.2. Keep It Simple: Embracing the SA 10. Concluding Remarks Associated Content Supporting Information Author Information Corresponding Author ORCID Notes Biographies Abbreviations References

individuals affected. These are often referred to as Type B toxicities;2,3 Type A is largely predictable from dose level. Within the fields of drug metabolism and toxicity there is agreement that these events are frequently initiated by covalent attachment of a RM to proteins which may then be recognized as foreign. In turn, these are hence able to initiate an immune response.4 In addition to these and general tissue necrotic effects, RMs can also cause mutations by reacting with DNA as well as inhibit metabolizing enzymes, usually cytochrome P450. The latter causes time-dependent inhibition, which is behind many drug−drug interactions.5 They can also exert noncovalent oxidative toxicities, especially when they contain a quinoid type of structure, such as quinones, quinoneimines, quinone methides, and similar species, a very common type of RM.6 A number of reports and discussion papers published during the past decade have focused on the understanding and insight into what might be achieved when aiming to minimize risks and hazards of drugs entering clinical development, see, for example, ref 7−10. Risk assessment, which takes the dose and degree of bioactivation into account, is a key concept of this discourse. Many discussions and results therefore aim at learning from the effects of drugs that have been withdrawn or have restricted use, attempting to shed new light on these effects from a different perspective. In several cases this has involved new investigations comprising of trapping experiments and covalent binding of radioactively labeled material, see references in Thompson et al.8 A common theme of these reports might be understood as attempts to create a decision tree for devising relevant in vitro assays so that a clinical

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1. INTRODUCTION A crucial aspect of drug safety involves drug metabolism related problems that give rise to reactive metabolites (RM) causing considerable harm to patients, in particular drug-induced liver injuries (DILI) and a range of hypersensitivity reactions. Concerns regarding compounds in development that exhibit such safety issues are still leading to costly withdrawals from clinical trials. The latest case that we are aware of is Lilly’s termination of the development of the mPGES inhibitor LY3031207 (Chart 1) in Phase 1 when two subjects experienced drug-induced liver injury (DILI).1 Especially problematic are idiosyncratic adverse drug reactions (IDR) that are rare but can be devastating for the 390



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Figure 1. Felbamate is metabolized in three steps to a reactive 2-phenyl-acrylic aldehyde.

When selecting any kind of chemical leads and eventually a CD, a multitude of other factors play significant roles in the decision process: most significantly the therapeutic area and length of dosing have proven to be exceedingly influential in the decision making about safety versus benefit. For example, it would be highly improbable to let a clearly RM-generating antidiabetic compound progress into the clinic, whereas quite a few anticancer compounds with hepatotoxic issues have recently been launched (see Section 7).

candidate drug (CD) with a tolerable RM profile can be safely selected based on levels of estimated RM exposure.11,12 The activities regarding best practice seem to have resulted in converging reasoning within several pharmaceutical companies, further stimulating development of new test systems for hazard identification and risk assessment.13−15 Progress in understanding of interindividual differences has increased by better understanding the roles of the innate and adaptive immune systems in IDR, which has also helped to explain differences in organ toxicities such as skin and hepatotoxicity.4 In contrast to these reports on safety methodology and studies of activities/effects of test compounds, the current perspective will focus on the structural features that are central to the awareness/avoidance process of filtering chemical compounds for preclinical pharmacological evaluation. The key concept is data-driven hazard awareness which then is followed up by an analytical process of risk assessment where pharmacokinetics, toxicology, and safety pharmacology play the major roles.

3. PRINCIPLES FOR SELECTING SAs In the following we will focus on what to base the selection of the most important SAs for formation of RMs, which will influence decisions in the drug design process. R&D pharmaceutical companies maintain their own watch lists of selected toxicophores, which most often also include intrinsically reactive compounds not requiring metabolic activation (see Section 4). In the process of selecting a lead compound as a starting point, many other types of unfit compounds have to be filtered away for a number of reasons that may not relate to toxicity.16,17 In this Perspective only alerts that give rise to reactive metabolites will be considered, aiming at a new expanded watch list of SAs for RMs and bringing forward some new refinements to the field. Use of known SAs as selected by experts represents the currently most successful route for raising awareness of potential problems regarding RM formation, resulting in proper actions and precautions before entering development. Other types of (current) approaches to identify structural alerts for toxicity based on computational analysis of structures for hazard identification (see review18) will not be a topic here since they largely have been applied to a wide variety of compound types including intrinsically reactive ones. Also, many attempts at using computational methods rely on the simpler concept of mutagenicity19 which is close to the current discussion but yet does not embrace the many complexities of the RM field. That said, more sophisticated rule-based approaches involving machine learning for hazard identification in structures should have a noticeable future impact. A caveat for exaggerated optimism in this case though is a similar scarcity of solid data. The common possible existence in one structure of more than one structural liability plus the many unknowns regarding current structure-toxicity relationships, especially IDRs, will inevitably slow the development of rules. Furthermore, it is highly desirable that future systems incorporate a tentative risk assessment involving a quantitative measure of RMs formed as well as what type of effect the intermediates could exert, a quite challenging problem.20,21

2. FACTORS INFLUENCING THE AWARENESS/AVOIDANCE PROCESS The actual selection process of compounds to test in clinical trials usually starts with a large number of diverse hit/lead compounds that are scrutinized and tweaked in a process that normally takes several years. In the end of this process a limited number of structures optimized for potency, ADME properties, and a lack of acute toxicity should have emerged. Of particular relevance to the current topic is that at the point of CD selection, it should be reasonable to expect that structural features of the CD that have precedence with regards to activation to reactive species have been identified. The underlying knowledge may have originated from a xenobiotic or any other chemical that is mentioned to have undergone an enzymatic conversion to verified or postulated reactive intermediates. This recognition of structural alerts (SA) might seem a tough requirement on any project team, but should nevertheless be seriously considered. The majority of reactive species formations can, at least in principle, be predicted based on the accumulated knowledge of reported cases as well as theoretical reasoning regarding bond-making/ -breaking processes, be it via enzymes or in other ways, such as direct electron-transfer processes. An entirely different notion is assessing the risk versus benefit since that has to do with considerations of probability for an activation actually taking place to any significant extent in humans. This aspect combined with the dose/exposure will determine the body burden, at this stage largely guessed, and all taken together will determine how serious a presence of a SA may be weighed. 391



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Figure 2. Illustration of useful schematics for matching planned test compounds against current knowledge. The schematics has been implemented in the application SpotRM+.35

The chemical classes of compounds that have been associated with RM formation and especially with IDR are still relatively few, and the outcomes vary regarding clinical symptoms, making correlation between structural features and toxic symptoms hard to establish, especially when the number of investigated examples is low. The difficulties of extrapolations are also based on the often poorly elucidated routes by which RMs arise and what biological processes they affect. This belongs in the large, extremely difficult area of structure toxicity relationships in general, which involves the many human offtargets, frustratingly unknown and creating an enormous black box for drug designers. Toxicity originating in metabolic conversions can be very hard to investigate, and it is not unusual to have many uncertainties remaining even after large efforts. Certain routes to RMs can be represented by very few drug examples, creating problems of weighing structurally distinctive features versus generalizations. Such cases require investigations of several analogs for proper elucidation of structure reactivity relationships. One example is felbamate, which requires a threestep conversion to the reactive species (Figure 1).22 Another often cited example is the antiepileptic drug valproic acid (Chart 1) which undergoes complicated transformations that involve paths of lipid biochemistry.23 Prediction of this type of activation is, however, within reach of rule-based systems, although the small number of verified examples complicates the creation of accurate rules. Of utmost importance is the rationale as why to add a certain SA to the watch list. The first, most compelling evidence, which can be associated with a SA, is having observed clinical manifestation(s) of side effects of the types: • Hepatotoxicity

As mentioned, the major difficulty is yet to convincingly identify the offending substructure(s), a task that can present considerable problems due to insufficient knowledge of all metabolic transformations. In addition, the difficulties often involve uncertainties regarding the types and extent of clinically observed adverse reactions, which usually include mixed types and tend to be generally underreported. Furthermore, it is not unusual to have a drug with more than one suspect substructure, complicating the analysis and necessitating more experimental evidence. Just to mention one example: In benzbromarone (Chart 1) at least three substructures that could form RMs can be identified based on solid precedence.25 In many cases laboratory experiments can underscore a link to a reasonably well-defined structural feature for which a mechanistic rationale can be conceived. Although desirable in principle, clinical evidence for SA selection can be bypassed when the propensity of a certain test compound for forming RMs has been duly affirmed in vitro, for example, in the widely used glutathione (GSH) trapping test in (human) liver microsomes.14 Because addition products as mechanismindicating markers often can be identified in these cases, the mechanism of activation also becomes more clear-cut and easier to assess. The important aspect of how to define a SA by delimiting a proper substructure will be discussed later on.

4. RECOGNIZED SAS AS THE BASIS FOR AN EXPERT SYSTEM Judging from the recent literature on RM issues, one might be misled to think that a consensus list of SAs already exists that can be easily applied to new test compounds. True, one can readily find in the many reviews on this topic the structural alerts mostly paid attention to during the last two to three decades, which are not overwhelmingly many. They cannot, however, be said to be well described and properly delimited for immediate use in medicinal chemistry. In 2005 Kalgutkar et al. published “A comprehensive listing of bioactivation pathways of organic functional groups”26 and a related large review.27 This has helped to improve the general awareness situation regarding the most common SAs which have then spread into many review articles,28 of which a few are cited here9,29,30 and thereby formed the basis for creating in-house systems in the industry. In addition, major pharmaceutical companies have internal lists of SAs, additionally often curated by their own in vitro experiments, which are incorporated into in-house cheminformatics systems for structural evaluation

• Blood dyscrasias, for example, agranulocytosis • Anaphylaxis and hypersensitivity reactions • Cutaneous reactions, for example, Stevens−Johnson syndrome • Carcinogenicity • Drug−drug interactions caused by time-dependent inhibition of cytochrome P450 enzymes When attempting to learn from the drugs causing DILI, the FDA drug labeling system is useful. Based on this, specialists have extracted information and created DILIrank, a list of drugs ranked by the risk for developing drug-induced liver injury in humans.24 392



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Table 1. Color Classification of Example Drugs Largely Based on Clinical/Safety Data classification

based on

drug examples

red

The drug has demonstrated clinical adverse effects that have a proven or likely association with bioactivation to RMs or a preclinical compound has shown extensive RM formation.

yellow

The drug contains an alert (or related) substructure, and there have been some reports of adverse effects that have been discussed in terms of RM formation or a preclinical compound has displayed RM formation to some degree

green

Despite having an alert, the drug has been used clinically without reported findings of adverse effects that can be associated with RM formation. The explanations for vindication typically include low dosage or a very low degree of metabolism that involves the relevant substructure. The last-mentioned case often depends on metabolism in other parts of the molecule (“soft spots”). In spite of having an alert in the structure, the clinical information is insufficient to classify the drug into any of the other categories. This might be because the drug has only recently been introduced or is not widely used, and therefore sufficient safety data have not been accumulated.

sudoxicam, felbamate, amodiaquine, lamotrigine, and ritonavir. mirtazapine, zolpidem, atorvastatin, tolmetin and phenazone rivaroxaban, cefuroxime, aripiprazole, and tolterodine formoterol, delamanid, and ranolazine

neutral

search results. This might be a preferred approach when, in rare cases, the example drug has a unique route for bioactivation that is difficult to generalize. For illustrating the problem at hand, a known culprit in the context of RMs is thiophene.36 Having as the only depiction of thiophenes a naked corestructure would result in loss of fine-tuning since it would be the only entry into all of the more than 15 thiophenecontaining drugs mentioned, or not mentioned, in the RM literature (if these were included as the drug examples in the database), even including benzothiophenes as well. The solution should rather be to have a more fine-tuned selection via a set of slightly varying and specific thiophene substructures, coded as separate SMARTS strings. Given the prerequisite in our approach that a certain SMARTS is hardwired to the examples best illustrating the case, the user hence would not be overburdened by having too many widely different examples to consider. A SMARTS string can be quite restrictive and match solely what is very close to the input structure pattern (the query), thereby limiting the number of examples to precisely the few most relevant ones. In parallel one might devise a SMARTS string to result in broad matches but then just have a few cherry-picked examples linked to that (though many more might be relevant). Attention to other relevant examples can be achieved by having a cross-reference system linking to other monographs and microreviews as depicted in Figure 2. A delimited description of an alert might also be preferred when, in rare cases, the example drug/compound has a unique route of bioactivation that is difficult to generalize. The example of a 6-aminopyrazolo[2,3-a]pyrimidine substructure of the candidate drug OT-7100 (Chart 1) can illustrate this. It represents an alert that will be an extremely rare hit by probed compounds, though on the other hand there is very little collateral cost of having it included, linked to only one example drug (which is hydroxylated in the 3-position forming a quinoid RM).37 As pointed out above, evidence regarding the substructures involved in RM formation might not always be possible to establish, and if one is not able to pick confidently a SA in a certain molecule such a drug (with its nonidentified RM mechanism) might have to be excluded from the example collection. On the other hand, it is usually possible to suggest a hypothesis about a certain mechanism, and depending on the level of expertise of the person(s) drafting summaries, it might be deemed acceptable to include an interesting idea. The lack of distinct SAs in a drug that gives rise to DILI or other ADRs might also reflect a general “ugliness” of the drug, for example, having several suspect subgroups as occurring for example in many antibiotics where alkenes, phenols, and intrinsically reactive groups such as quinones can be found in the same

during the design and synthesis process. In 2011 Stepan et al., at Pfizer, published an analysis of the performance of the SA concept applied to the top-selling 200 drugs.31 There, the authors recognized 26 drugs with SAs among the 31 drugs recalled due to IDRs, whereas of the 37 drugs associated with black box warnings (BBW), the Pfizer authors listed 29 with identifiable SAs. However, some of the SAs listed actually were intrinsically reactive groups, such as Michael acceptors or nitrogen mustards. All drug designers should benefit from easy access to the most common SAs for checking planned compounds against a range of alerts, that is, found in a searchable database. However, the only open access resource providing RM alerts with searching possibilities based on input structures seems to be the online service ToxAlerts. The mentioned review by Kalgutkar et al. served as inspiration for their implemented SA set, which lists 35 alerts32 in the SMARTS format33,34 (all of them referring back to the review, not to the original literature). This short list, however, also includes a few non-RM-forming structures that are intrinsically reactive such as, for example, 2and 4-fluoropyridines. Related commercial applications are listed in the review by Kirchmair et al.20

5. PRINCIPLES FOR SETTING UP A SEARCHABLE SA SYSTEM Any meaningful evaluation of planned test compounds based on SAs should be supported by a validation against a reasonable interpretation of real examples from the clinic additionally supported with in vitro test results. As a start, we will outline the following suggestion for analysis or diagnosis of potential test compounds. The same concept has also been implemented in the application SpotRM+.35 As schematically described in Figure 2, assessment of hazard is based on (1) literature reports on selected examples of drugs or other xenobiotics with reported or likely (as judged by us) RM problems that (2) are linked to one or several SAs (which in rare cases may represent only a new hypothesis). Refinement and expansion are possible by adding example drugs without having a link to a specific alert just to have it included and searchable by structure and keywords. Relevant information can be as simple as a link to an internet reference. We propose (Table 1) to convey an indication of the hazard associated with a given drug, and indirectly to its structural alert(s), by classifying the drugs as red, yellow, green, or neutral.35 The chemical description of an alert should be as detailed (less generalized) as possible to result in fewer, but more specific examples, in other words, to reduce the noise level of 393



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Naphthalene itself, like benzene, is a toxic and carcinogenic hydrocarbon which is more easily oxidized by CYPs than benzene. Unsubstituted naphthyl is found only in a low number of drugs. All of these are linked to RM issues, for example, duloxetine39 and pronethalol; the latter was withdrawn from clinical trials in 1965 (Chart 1). Agomelatine (Chart 1) is an example of a methoxy-substituted naphthalene; it is under review by EMA for adverse events of hepatotoxicity. Currently, medicinal chemists wisely seem to avoid incorporation of naphthalene into test substances, and this substructure is well deserved to get a warning flag in a SA system. A list of SAs could simply start with the benzene and naphthalene substructures, although most real drugs with RM problems have substituents that promote the problems. 6.2. Halobenzenes. The toxicity of halobenzene compounds that originates from formation of RMs is directly related to oxidations of the aromatic ring, not the halogen (with iodine posing a possible exception). This in contrast to most other benzene derivatives where the attached group is metabolized. A reason to examine halobenzenes separately in the current RM awareness/avoidance setting is their wide use by medicinal chemists, which reflects more likely accessibility and need of compound property variations than safety considerations.40 We found 303 fluorobenzene, 385 chlorobenzene, and 37 bromobenzene drugs (groups of approved and investigational) in the database DrugBank (www.drugbank. ca) as of July 2017. Toxicity by bioactivation of simple halogenated benzene compounds is not unknown, as revealed by many studies published a few decades ago,41 and it would not be surprising to see the same biotransformations occurring in drugs. Since introduction of fluorine on benzene is shown to slow oxidation on the ring by the CYP enzymes, this has become a frequently used attempt to try to increase bioavailability of new test compounds.42 However, this design trick has less convincing basis in reality. An excerpt from a review: “The incorporation of F may indeed block metabolic sites, as there are numerous anecdotal examples; however, in the overall matched pair analysis, there is no general trend that supports this notion (43% Ph → p-F-Ph improve metabolic clearance, 39% of matched pairs have worse metabolic clearance)”.40 This approach becomes more or less futile when the aromatic ring is activated by suitably placed amino groups (and possibly oxy).43,44 There are several cases, however, when introduction of fluorine has been shown to improve metabolic stability. One recent example is fluorinated benzbromarone (Chart 1), which has considerably lowered metabolic epoxidation and adduction with N-acetylcysteine than the parent.25 Introduction of the other types of halogens, which has for a long time been commonplace in medicinal chemistry,40 has very mixed influences on metabolic stability and, of most importance, can rarely suppress oxidative bioactivation if the benzene ring already is in an exposed position. The higher lipophilicity caused by introduction of chlorine or bromine also supports increased metabolic clearance as has been well established.45 The halobenzenes, like their nonhalogenated analogs, give rise to epoxides, phenols, and, eventually, benzoquinones. There is a strong influence on product composition when epoxidation occurs at the halogen-bearing carbon (always leading to a phenol)46 or more subtle influences when that is not the case. Different reactivity of the formed epoxides should include rates of rearrangement to phenols, of enzymatic

molecule. Also for many types of compounds having alkyls and mixed aromatic−aliphatic heterocycles with several types of heteroatoms, it might be hard to spot the problem. These types of structures are less suited as drug examples, as they often lack well described case histories.

6. SELECTING AND DESCRIBING THE MOST IMPORTANT SAs Another important aspect of the procedure to select a certain SA is the possibility to define it with a proper SMARTS string so that it will match the liabilities in queried compounds in a way that the drug designer finds helpful. This is an extremely difficult part that will determine much of the success of setting up an expert system comprising SAs validated by relevant examples. It is futile to aim at achieving a perfect system. Mismatches with the drug designer’s expectations are inevitable since too many unknowns prevail and robust knowledge is less abundant than the opposite. In the following, the unavoidable subjectivity of linking specific SAs with drug examples will also be evidenced since alerts will often overlap each other on the drug structure, especially on substituted benzenes. 6.1. Benzene and Naphthalene. The vast majority of substructures giving rise to RMs belong in the aromatics class, both carboaromatic (benzene and naphthalene), heteroaromatic, and mixed. With benzene being such a widely used and easily incorporated building block, it would be rather difficult to convince medicinal chemists to refrain from using it, despite its long history of known potential issues, not only regarding safety aspects.38 Therefore, structures containing benzene will form a large part of a SA system, and it will be important to attempt to classify in some detail the large number of benzene-based substructures that have been used in investigational and approved drugs. The involvement of the simple unsubstituted phenyl group, connected via a carbon to the rest of the molecule, is welldocumented in several cases of drugs showing hepatotoxicity and/or hypersensitivity reactions. For example, phenytoin (Figure 3) contains two such phenyl groups which give rise

Figure 3. Established route to an o-benzoquinone from phenytoin.

to arene oxides/o-benzoquinones and can be applied as a good example of a drug classified as red. Many more examples of the unsubstituted phenyl class can be hardwired to the SMARTS description [cH]1[cH][cH][cH][cH][c]1-[#6] but, as discussed above, care should be exercised to limit the number of examples to proven oxidations resulting in ortho-benzoquinones (via initial hydroxylation/epoxidation) or a proven capture of an arene oxide with a nucleophile. 394



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Table 2. A Few Structural Alerts That Can Be Used in SMARTS Format with Examples of Drugs Containing the Alert (Full Table of Proposed Alerts Is Found in Table S1)

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Table 2. continued

a Entry number corresponds to entry number in the complete table as found in the Supporting Information. bStructures shown are depicted in a more “human” readable fashion. Structural annotations loosely follow SMARTS nomenclature, that is, n = aromatic nitrogen N = aliphatic nitrogen; bond with a second dashed bond = aromatic bond.

hydrolysis by epoxide hydrolases, and of ring-opening reactions with nucleophiles like glutathione. In addition, the quinones that retain halogens at a double bond are more reactive toward nucleophiles and will give faster and different adductions with proteins and DNA.41,47 To summarize, halogenated benzenes/aromatics from a safety perspective raise complex questions that for now are ill suited for quick decisions in the awareness/avoidance situation. As is valid for other benzene-containing compounds, the medicinal chemist should as much as possible steer away from exposing these parts of the structure to (extensive) biotransformation. The substructure Ph-[Cl,Br,I] (Supporting Information, Table S1, entries 23−24, 56) should be listed as a

structural alert for RM formation with a limited number of available examples. In the application SpotRM+,35 which forms the basis of this Perspective, we list two iodobenzenes, seven bromobenzenes, and five 4-halophenyl-C drugs as examples. The literature does not provide clear evidence of the importance of the halo substituent as root to the problem for any of the drugs. In contrast, 4-F-benzene-N specifically justifies listing as an alert since there are several relevant drug examples (Table 2, entry 33). 6.3. Nitrogen-Substituted Benzenes. Additional examples of benzene (and other aromatic) derivatives as structural alerts have to take on the nitrogen-substituted benzenes, which comprise the anilines, the anilides, and the nitro aromatics since 396



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Figure 4. Generation of quinone methides by benzylic oxidation of alkylphenols.

Figure 5. Phencyclidine and flindokalner both generate quinone methides, but the intermediate phenols are formed in different ways.

they are behind the majority of the first identified adverse drug reactions that were associated with RMs, as mentioned in the Introduction. The mechanisms of bioactivation of simple anilines and nitroarenes are well established and do not require repetition here.48 The SAs that we find relevant to list in this context are (Table S1, entries 1−5): True anilines; 4-H-benzene-N-C (not acyl); 1,4-diaminobenzene (not depicted); 4-OH-benzene-N; 4-alkoxybenzene-N; 4-H-anilide (4-H-benzene-N-acyl). Other benzene-N compounds have additional substituents that influence RM formation: p-alkylbenzene-N and o-alkylarylN can form methides and are related to the corresponding oxygen compounds (Section 6.6). The alerts 4-OH-benzene-N and 4-alkoxybenzene-N are also mentioned in Section 6.5 dealing with these mixed drug examples. 6.4. Oxygen-Substituted Benzenes/Aromatics: Precursors of Quinones. Certain benzenes substituted with oxygen (and no nitrogen) should also be included since quite a few examples of drug structures validate these as SAs. Unlike nitrogen, the principle route for an oxygen to activate a benzene is to initiate the introduction of another ortho or para oxygen which then leads to generation of quinones. Therefore, an isolated phenol substructure or its immediate precursor, an alkoxybenzene, can be considered as SAs, although clear-cut drug examples are not obvious. A special case of phenyl ethers has been described on a few occasions where an alkyl phenyl ether, having free para position

and with the carbon alpha to the ether oxygen blocked to oxidation (tert-alkyl or aryl), acts as a softspot for metabolic oxidation to reactive species. A noteworthy example is a taranabant analog (Chart 1) that gives very high levels of covalent binding in liver microsomes that has been firmly linked to the phenoxy group.49 Relevant SA listings: 4-H-Phenyl ether, w q-C (Table S1, entry 36); phenyl-OH; alkyl phenyl ether. 6.5. Benzenes/Aromatics Concurrently Substituted with Oxygen and Nitrogen. The existence of both oxygen and nitrogen on a benzene core has often been found to be a toxic combination, for example, in phenacetin. Since the SMARTS that describe benzenes singly substituted with oxygen or nitrogen (Sections 6.3 and 6.4) in some cases exclude the hits having both these substituents (because of a forced Hsubstituent in the para position), it is recommended to add SMARTS containing both oxygen and nitrogen, for example, like in Table S1, entries 5 and 12. The corresponding SAs of ortho-[N,O]-benzenes are not listed because published drug examples with good evidence of ortho-quinoneimine formation are scarce. However, several anticancer protein kinase inhibitors (PKIs) (at http://www. brimr.org/PKI/PKIs.htm, accessed on February 12, 2018), often having adverse effects from likely RM interference, have this configuration of elements, and there is little reason to give it a clean bill from an RM perspective solely due to the absence of documentation. 397



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Figure 6. Most common FMHs with two heteroatoms in drugs.

6.6. Precursors of Quinone Methides (Quinomethanes) and Imines. The most common precursor of a quinone methide is a 4- or 2-alkylphenol, such as in the simple parent cresols (Figure 4A), while the most cited example of a real drug having this kind of liability is troglitazone (Figure 4C).50 The initiating chemical event is a CYP-mediated hydroxylation on the benzylic carbon giving rise to a labile benzylic alcohol. Both the ortho and para phenolic SMARTS should be included in the database (Table S1, entries 8−9). Many natural products can form quinone methides, for example, the toxic alkaloid dauricine (Figure 4B).51 In this context, it is obvious that any alkyl (excluding tertiary ones) phenyl ether having an alkyl substituent (with a benzylic H) in an ortho or para position can be an immediate precursor of a 2- or 4-alkylphenol leading further to the reactions described; this substructure should be included in the watch list as well (Table S1, entry 49 for the ortho example). The reverse order of events to the one just described is the generation of a phenol, which already has a leaving group in place on the benzylic carbon. There are two ways of generating this state (Figure 5): (1) hydroxylation of the benzene ring, as in phencyclidine,52 and (2) release of the phenol by dealkylation of an aryl alkyl ether (an ester can serve as an excellent precursor as well) as in the case of flindokalner53 cf. Table S1, entries 20−22. These two situations will be discussed later in terms of retrospectively looking for older drugs that might undergo these types of bioactivation but which have not yet been recognized as such. 6.7. Heteroaromatics. Drug design aspects of mitigating metabolism of saturated as well as aromatic heterocycles have been reviewed elsewhere.54 6.7.1. Six-Membered Rings. The most abundant sixmembered aromatic ring in drug compounds after benzene is pyridine.55 In itself this heterocycle, unlike benzene, does not raise any concerns regarding RMs (only CYP inhibition is a recurrent issue). Oxidation on nitrogen instead of the ring carbons is highly preferred creating an N-oxide which is more polar (and thus hydrophilic) than the parent compound and is only slightly reactive as an electrophile. Therefore, there is no reason to list the pyridine core as a SA. The other most common nitrogen-containing six-membered rings, pyrimidine, pyrazine, and pyridazine (additionally, a few s-triazine drugs are anticancer agents), have not in themselves been implicated in causing RM problems, but, as expected, the substituents on these heterocycles will greatly influence the metabolic processes. The same patterns of substituent reactions outlined for benzene can be seen with these aromatics. However, an amino group on pyridine and pyrimidine behaves differently from benzeneamines by being less sensitive to oxidations on the amine.56 In contrast, diaminopyridines with an ortho relationship of the amino groups have been reported to give rise to quinonediimines (Table 2, entry 50).57 One can, in principle, construct SMARTS that in one go include certain unfavorable substitution patterns on benzene as well as six-membered nitrogen heterocycles. However, they become harder to write for 1,4-disubstituted six-membered heterocycles, and we therefore recommend to include only

various 1,2-disubstituted substructures in more generally encompassing SMARTS. Each of these can easily be written to encompass any type of aromatic compound. An example describing such a situation is nevirapine, where a quinoneimine methide is enzymatically generated as readily as in any benzenebased analog (Chart 1).58 The ortho substructure listed in Table 2, entry 12, matches this compound as well. 6.7.2. Five-Membered Heterocycles (FMHs). There are many more types of FMHs used in drugs than there are sixmembered ones. The rings (hetero atoms ≤2) listed in Figure 6 represent the vast majority of the ones used (including presence in fused rings). To these come the abundant tetraand triazoles, which are rarely oxidized on the ring (the metabolism of FMHs was reviewed by Dalvie et al.);59 oxadiazoles with increasing use can also be mentioned. As is the case with pyridine, imidazole is not easily oxidized by CYP enzymes on the ring but likewise binds to the iron in the CYP heme and inhibits the enzyme via its electron pair on nitrogen. Although there are reports of ring oxidations, the products from imidazoles in most cases appear to be relatively innocuous ureas and hydantoins.59 However, results that were reported in 2018 have to modify this picture. The mPGES inhibitor LY3031207 (Chart 1) in Phase 1 clinical trials was found to cause DILI.1 Preclinical models indicated formation of an epoxide on the 3,4 double bond that gave rise to adducts with GSH.60 For these reasons, a 2-aminoimidazole becomes a new and relevant SA. As we discuss alkyl-substituted FMHs, other imidazoles will recur as RM precursors. In some contrast, oxidations of other rings, in particular thiophene and furan, give rise to less innocuous products, which have been repeatedly shown to cause health problems, regarding furan not least originating from natural products. The dialdehyde (or other 1,4-diketo-2-alkenes) from furan(s) reacts with primary amino groups in proteins to generate a pyrrole product that can transform the protein into an antigen or make it nonfunctional.61 From a thiophene, reactive S-oxides and/or epoxides may be formed in addition to other less well characterized products, for example, sulfenic acids.36 These two FMHs therefore deserve to be stand-alone SMARTS; their presence always raising warning flags requiring action. In rare cases, reactivity of metabolites from thiophene-containing drugs forms the basis of the drugs’ therapeutic activity, for example, the antiplatelet agent ticlopidine (Chart 1) and analogs. Some of the RMs are believed to form disulfide bonds with the P2Y12 receptor as the drugs’ mechanism of action. Pyrroles behave much like furans as RM precursors and are not considered attractive as building blocks, with only three drugs (nonindoles) on the market in the FDA Orange Book in 2013. In the context of drugs and RMs, pyrrole seems to be completely dominated by its conspicuous role as the heterocyclic part of indole (next section). Substituents that together with the FMH ring may generate RMs are much the same as those seen on benzene. Amino groups on thiophene and furan are rare due to their destabilizing influence toward oxidation in general. Some of the other FMHs can sustain amino substituents, and there are several drugs within this class, mostly within the aminothiazoles, some of which, for example, Ro-0281675 (Chart 1), 398



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has been spotted as being associated with RM problems in clinical trials.62 Alkyl-substituted FMHs are treated in Section 6.8, among them certain notorious 1-alkyl-2-hydroxyalkylpyrroles. 6.7.3. Fused Heterocyclic Ring Systems. Although the number of licensed drugs having fused heterocyclic ring systems is large, the number of represented ring systems is considerably lower. Many of these systems, to be sure, are not truly aromatic since they are rather unsaturated cyclic amides via tautomerism, as in many purine derivatives. Relatively well recognized patterns of bioactivation are associated with the traditional systems of benzene fused with electron-rich FMHs such as pyrrole (in indole), thiophene, imidazole, thiazoles, and oxazoles that activate the benzene ring toward oxidation. As a prudent measure, the indole, benzofuran, benzimidazole, benzothiophene, and benzothiazole ring systems (that one may call scaffolds) should be put on an alert list while knowing that their substituents will be highly decisive regarding extent and type of bioactivation.54 However, if the chosen substituent patterns of the already SA-listed pyrrole, furan, and thiophene (as SMARTS strings) leave open the possibility of ring fusions, it might be unnecessary to list the fused ring systems. Benzimidazole, on the other hand, is here selected to be on our alert list (Table 2, entry 35) and is not represented only by its imidazole substructure since this FMH by itself is not a SA. The scarcity of reports of other FMHs, such as isoxazole and pyrazole (in indazole) fused with benzene, as generating RMs currently does not seem to warrant these fused rings in an alert list. However, one might note that the hydrazine-like character of indazole sometimes can come into the open when it is oxidized in the 3-position.63 Also, it can be noted that work on 2-amino-1,3,4-oxadiazoles was diverted because of fear of hydrazide formation by ringopening oxidations.64 Heteroaromatics fused with other heteroaromatics instead of benzene are plentiful in medicinal chemistry even when they are not very common in licensed drugs.55 Most often a particular ring system, for example, pteridine, is unique to one class of similarly substituted drugs that would be liable to the same bioactivation steps. Information of this type of liability of common fused heterocycles is scarce. Instead, what seems to characterize such systems is sensitivity to oxidation by the molybdoenzyme aldehyde oxidase,65,66 which typically produces a tautomeric heterocyclic arenone/hydroxy-arene structure. This oxidation may well lead further to quinoid RMs, but examples are not readily found. The fused nitrogen heterocycles that share a nitrogen between two rings are few when it comes to number of drugs containing them. Before 2014 only one case each for imidazo[1,2-a]pyridine, pyrazolo[2,3-a]pyrimidine, and imidazo[1,2-b]pyridazine (Chart 2) was found in a marketed drug, while none of the many isomeric systems was represented. RM problems were found early on in the first mentioned ring system when alpidem (Chart 2), for treatment of insomnia, was associated with several cases of acute hepatitis (daily dose 50− 150 mg) and was withdrawn in 1995. It gave GSH adducts at the 7-position of its chlorinated imidazo[1,2-a]pyridine ring, and formation of an epoxide was postulated.67 The analogous low-dose (5−10 mg/day) zolpidem (Chart 2) was not similarly afflicted. Hence the ring imidazo[1,2-a]pyridine is a clear alert to be included as a SMARTS string (Table S1, entry 32).

Chart 2. More Compounds Mentioned in the Text

The propensity for ring oxidation (epoxidation) of other similar systems is an interesting question that, so far, has not been addressed. Not considering the cost, it should be possible to use quantum mechanical calculations to identify sensitive sites of these other systems. 6.8. Alkyl on Aromatics. Alkyl groups on benzene (including polycyclics) and heteroaromatics that have a benzylic hydrogen are precursors of benzylic alcohol metabolites via enzymatic oxidation, mostly CYP-mediated. As is wellknown from organic chemistry, benzylic carbenium ions are stabilized by suitably placed electron-donating groups. Such “long-lived” ions, which cannot expel a proton to generate a methide, may play a role as RMs from certain drugs, particularly when the precursor alcohol is converted to a sulfate68 via sulfotransferases as indicated in Figure 7, which depicts a palkoxy-stabilized carbenium ion as an example. An equally reactive radical can also be formed by direct reaction on the benzylic carbon by certain CYP enzymes, a reaction that can 399



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Figure 7. Benzylic oxidation generating a stabilized carbenium ion (depicted) or a radical.

noted that a 3-hydroxymethylfuran would be equally exposed to generate a labile sulfate, so a specific SMARTS for a precursor structure of that should be designed as well. Yet another FMH that can strongly support a benzylic positive charge is pyrrole. Certain 1-alkyl-2-hydroxyalkylpyrroles are very reactive toward nucleophiles as evidenced by the carcinogenic pyrrolizidine alkaloids’ reactions with DNA.73 It should be noted that even the seemingly detoxified pyrrolizidine structure GS-DHP (Chart 2) is reactive toward DNA. GS-DHP is generated from a dehydrogenated alkaloid (dehydromonocrotaline) by simply incubating this alcohol with GSH in a buffered water solution. To summarize, there are three principle in vivo scenarios for a benzylic type alcohol that can lead to a reactive intermediate. (1) In extreme cases, formation of a carbenium ion (much more likely after sulfate formation), as mentioned for the alcohol dehydromonocrotaline.73 Formation is strongly influenced by the electron-donating ability of the substituents on the benzylic carbon. (2) Formation of a sulfate, which might be able to generate a carbenium ion within an appropriate time frame; much depending on the substitution pattern on the aromatic. Benzyl sulfate itself is probably not sufficiently reactive. (3) When having the prerequisites (Section 6.6), formation of a quinone methide, an imine methide (or equivalent 3methylene-species in a pyrrole or similar ring) by elimination of water. There are at least a couple of divertive routes into detoxification: form a glucuronide (and be excreted as such) and be oxidized to an aldehyde (and carboxylic acid) or ketone. Regarding “alkyls on aromatics”, the appropriate SAs that are most urgent to include as warning flags of potential RM problems should be the alkyls ortho or para to activating nitrogen and oxygen substituents on benzene and sixmembered heterocycles (for example, Table 2, entries 10 and 12). Regarding FMHs, the SAs that highlight liabilities of this type should include an alkyl positioned to give the most resonance stabilized carbenium ions. Ritonavir is an example (Table 2, entry 14) supporting these criteria. 6.9. Other Types of Unsaturation. Olefins are often involved in the generation of RMs via either epoxidation of the double-bond or allylic oxidation, leading to an allylic alcohol that can be further oxidized to a ketone or aldehyde acting as a Michael acceptor (Figure 9C), or it can be sulfated. An existing allylic alcohol can of course undergo these conversions directly. We have chosen the allyl-H subgroup as a SMARTS (Table S1, entry 39) since it includes both possibilities as warning flags. Homoallylic alcohols will also match this SMARTS when they have an allylic hydrogen; abacavir is the example often referred to in this regard (Chart 2). When it is oxidized to an aldehyde, the double-bond will shift to a conjugated position. Olefins are ubiquitous in natural products, and many of these compounds are associated with hypersensitivity reactions, for example, eugenol, α-terpinene, safrole, and limonene. Some

easily lead to inactivation of the processing enzyme. Gemfibrozil (Chart 2) was suggested to inactivate CYP2C8 in such a process.69 As detailed in Section 6.5, when the benzylic type alcohol or sulfate is suitably disposed, it can also form a quinone methide or a quinone imine methide by elimination of water or hydrogen sulfate. Most of the published examples from the literature of true benzylic carbenium ions causing toxicity are from the field of polycyclic aromatic hydrocarbons (see review),70 as indicated in Figure 8.

Figure 8. Sulfate ester of 7-hydroxymethylbenz[a]anthracene is strongly mutagenic, whereas the sulfate of 7-(2-hydroxyethyl)benz[a]anthracene is not.

Many FMHs show great ability to support a carbenium ion on a carbon (benzylic type) attached to the ring. This may lead to labile alcohols requiring low activation energy to form a carbenium ion (or, as mentioned, eliminate water directly when that possibility exists). The classical example is the very labile alcohol formed from 3-methylindole (skatole), which eliminates water to form a methide (Figure 9A). In fact, all the

Figure 9. 3-Alkylindoles are notorious for forming reactive methides (A); 5-hydroxymethylfurfural forms a reactive sulfate (B); the allyl-H group can be oxidized all the way to a reactive Michael acceptor via an allylic alcohol, which concurrently can form a labile sulfate ester (C).

possible six hydroxymethylindoles are to some extent somewhat labile since the precursor methylindoles all lead to adducts with GSH when incubated with CYP enzymes.71Another reported example is the food ingredient 5-hydroxymethylfurfural (Figure 9B), formed from reducing sugars by acidcatalyzed dehydration or in the Maillard reaction, which is mutagenic and carcinogenic via sulfate formation.72 It may be 400



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olefins, such as citronellol, are allylic alcohols and are only one step from the actual unsaturated sensitizing ketone, aldehyde, or carbenium ion (via a sulfate ester). Benzylic (Section 6.8) and allylic alcohols as drugs or metabolites share the same type of weakness by readily forming carbenium ions (after sulfation). Alkynes are not readily epoxidized when the triple bond is disubstituted. In contrast, an alkyne that is terminal and does not have propargylic hydrogens is often attacked by the powerful CYP enzymes concurrently generating reactive intermediates; the classical example involving the CYP inhibition by a RM of ethinylestradiol, revealed in 197974 (among the first examples of suicide inhibition). A terminal alkyne is therefore an obvious SA to list (Table S1, entry 40) So far, we have not included propargyl-H (analogously to allyl-H) as an alert in SpotRM+ since drug examples are lacking. But since this substructure should behave like an allylH (Figure 9C), it could very well defend a position in a SA list. 6.10. c-Propylamines. The cyclopropyl group per se is not particularly sensitive toward oxidation by CYP enzymes and therefore does not have to be included in a watch list. The situation is very different when it has a nitrogen directly bound and is an amine or amide overall. There are numerous drug examples of cyclopropyl amines that give rise to reactive species via oxidations by CYP or, in particular, MAO. The list of examples that are bioactivated includes tranylcypromine (Table S1, entry 18) and trovafloxacin, whereas certain drugs, like nevirapine (Chart 1) and abacavir (Chart 2), which have the cPr bound to an “aniline type” nitrogen have no reported indications of RMs originating from that group. 6.11. Other Elimination Reactions. Certain drugs have a unique mechanism of forming RMs. Felbamate is an antiepileptic drug, still in use although restricted, which causes hepatotoxicity and blood dyscrasias by a proven mechanism (Figure 1). A hydrolysis initiates the activation which proceeds via oxidation to an aldehyde, leading to the final elimination reaction that generates a reactive acrylic aldehyde. A relevant description of a SA SMARTS derived from this observation should clearly capture the possibility of a ketone instead of aldehyde formation, though what to include in addition, especially regarding the leaving group, is less obvious. A simple 3-hydroxyaldehyde might be sufficiently stable (cf. properties of endogenous glyceraldehyde) not to automatically warrant an alert listing. Also an absence of a 2-aryl, a group that certainly acts to lower the activation energy for elimination, should be taken into account. We suggest a structural description (Chart 2), where X, Ar, and R have the following characteristics for translation into a SMARTS string: X = halogen, acyloxy, aryloxy; Ar = any aryl; R = any group linked via O, N, or C (see also Table S1, entry 37). The above mechanism of felbamate activation is not as unique as suggested above since certain fluorinated compounds have been shown to be activated in an analogous way.75−77 The general reaction to give a Michael acceptor can be written as shown in Figure 10A, and a drug candidate, a dipeptidyl peptidase-IV inhibitor, is shown in Figure 10B to follow this route generating an unsaturated aldehyde as a RM.76 A SMARTS can be written to also include cases like these fluoro compounds, in addition to the “felbamate alert”, but there is no real disadvantage of having a separate encoding for these fluoro-substituted systems (Table S1, entry 57 with gosogliptin as a neutral example, on the RYGN scale). 6.12. Geminal Dihaloalkyl. Alkyl halides other than fluoroalkyls are unusual as drugs but yet exist. One class of

Figure 10. Generalized reaction of 3-halo-amines/alcohols to give a reactive Michael acceptor (A) and a fluoropyrrolidine that follows this route (B).

drugs that has a multitude of combined fluorides/chlorides, and in one case a bromide, is the inhaled anesthetics. In this class, the bromide/chloride/fluoride compound halothane (Chart 2) is conspicuous by causing IADR manifested as serious hepatitis. The mechanism involves formation of a hydroxyl compound that forms an acid halide, which acylates proteins.78 The anesthetic enflurane, which is no longer in common use, has two oxidizable carbons both of which should give rise to acyl fluorides on oxidative metabolism (the more likely one is drawn in Figure 11). There is a reported 2% metabolic conversion of

Figure 11. Proposed CYP oxidation of enflurane.

enflurane in vivo, but adducts with proteins do not seem to have been characterized. This anesthetic carried warnings of liver injury on its insert label. Another drug giving rise to an acyl halide is the antibacterial chloramphenicol (Chart 2), which has an additional alert via its aromatic nitro group. The very reactive acyl chloride formed reacts with the oxidizing CYP protein and inhibits it.26 A SMARTS describing a 1,1-dihaloalkyl is clearly warranted as a warning flag (Table S1, entry 17). 6.13. Carboxylic Acids. Arguably, the functional group that during the last couple of decades probably has attracted the most attention as a precursor of reactive metabolites is the carboxylic acid (CA) group. This has caused some consternation in the medicinal chemistry community which has been accustomed to looking upon this group as an easily handled building block, a convenient attractor to positive charge in receptors, and also as a useful enhancer of solubility and hydrophilicity that entails different pharmacokinetic properties from a neutral compound. Formation of acyl glucuronides (AG) is the human body’s way of further solubilizing and enhancing excretion of CAs. The danger of having some AGs reacting as activated esters with proteins may be well understood as a basic chemical principle (Figure 12). However, our impression is that drug designers have found it difficult to obtain suitable guidance from the specialists in ADME and toxicology regarding the potential hazards of these in spite of numerous publications on the subject.79,80 Furthermore, it appears that there is considerably 401



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alkyl CAs89 and clinical warnings for formation of CoA derivatives seem to be unreported. 6.14. Hydrazine and Derivatives. Surprisingly many drugs can be classified into this group, ranging from the simple antidepressant phenelzine (a.k.a. phenethylhydrazine; still on the market in the US) to the antihypertensive hydralazine and the anti-TBC agent isoniazid. Very few drugs belonging here are without the types of side effects that can be suspected to be caused by RMs in the broad sense, involving also mechanismbased enzyme inhibition (not restricted to CYPs). Unsurprisingly, drug designers evade getting stuck in this murky area, with the exception of designing compounds for acute deadly diseases such as anticancer agents. However, surprisingly many academic researchers wanting to demonstrate interesting biological activity make use of these types of compounds from various sources. Many structures in the thiosemicarbazone and semicarbazone classes are each year published in medicinal chemistry journals, all with faint prospects of ever reaching the market. The thiosemicarbazone substructure, in addition to the hydrazine hazard, would seem to carry an increased risk because of the thiocarbonyl structure. However, exploration of mechanistic toxicological chemistry for thiosemicarbazones is minimal. Mechanisms of activation of hydrazines and derivatives usually seem to follow the mechanistic chemistry elucidated by organic chemists during the last century. Formation of carboncentered cations by expulsion of N2 seems to be commonplace, but there are also reports of reactions mediated more like what one sees in aryl diazonium salts, for example, when activated isoniazid acts as an acylating agent (Figure 13)

Figure 12. Acyl glucuronides and CoA derivatives can act as acylating agents (on amino groups).

more to CA activation than was discussed 10−20 years ago when the AGs as RMs were mainly in focus. Through studies by Weidolf’s research group81,82 and also by Grillo,83 plus reports by Skonberg and co-workers calling for heightened attention,84 it has become apparent that S-acyl-coenzyme A thioesters (CoA conjugates, Figure 12) play a role for hepatotoxicity that adds to and probably exceeds that of AGs. These CoA thioesters are more reactive than the corresponding AGs.81 They are also located differently from the AGs in the cell and therefore will encounter other reactants. Since CoA conjugates of CAs are important in fatty acid metabolism, it has been suggested that such drug metabolites can interfere with the normal biochemical processes.85,86 Studies published in 2015 reveal the causes of the large hepatotoxic difference between the withdrawn drug ibufenac and the commonly used ibuprofen (Chart 2) as likely based largely on the different extent of CoA conjugate formation.82 A further example of irreversible binding initiated by CoA formation of an intended drug molecule is provided by the azetidine-3-carboxylic acid MRL-A (Chart 2), which becomes covalently attached to the enzyme long chain acyl-CoA synthetase-1 when the enzyme attempts formation of a CoA derivative and β-oxidation of this derivative.87 The likely shortcomings of any general structural alert for CAs should warrant experimental assessment of acyl CoA formation of alkyl CAs.88,89 A summary of guidance gathered from the large number of reports published by specialists in the last 10 years may involve separate SA considerations for the following classes of CAs: • Arylacetic acids (i.e., α-unsubstituted), a definitive SA. • α-Alkyl arylacetic acids (also referred to as αarylpropionic acids when belonging to the NSAIDs) may not be completely harmless. General awareness and guiding by in vitro evaluations might be sufficient.89 • Aryloxyacetic acids, which are shown to give highly reactive AGs,90 have few relevant example drugs and largely represent unprecedented hazards. However, one may note that hepatotoxic tienilic acid (Table S1, entry 60) is an aryloxyacetic acid as well as a thiophene. • Alkyl CAs should be covered by a SA SMARTS (Table S1, entry 43) based on the valproic acid (Chart 1) precedence, that is, with an α-alkyl group. At present, not enough precedence among xenobiotics seems to be at hand for proper design of more general “fatty acid-like” SAs. However, it seems clear that many alkyl CA structures can be expected to interfere with endogenous lipid metabolism. • Aryl CAs should not have restrictions based on activation processes, that is, no SA required, since the AGs of aryl CAs are generally much more stable than those from

Figure 13. Isoniazid has been reported to acylate amino acids.

A SMARTS covering hydrazines and derivatives can be written very simply involving just a carbon linked to singly bound dinitrogen (C−N−N). The noncyclic diazo alert (C− NN) may be added as a separate alert (with links to anticancer agents such as dacarbazine). The FMH pyrazole might, arguably, be described as a “hidden hydrazine/hydrazone”, and it is interesting to note that outside of the anticancer drugs, this ring in its N-unsubstituted form is only found in allopurinol (Chart 2). This irreversible inhibitor of xanthine oxidase has a long track record of severe side effects, including hepatotoxicity, but no RM mechanism has been proposed to explain this. It might, however, be relevant to include the substructure shown in Chart 2 in a list of SAs just to call attention to the suspicions linked to the allopurinol structure. 6.15. Thioamides, Thioureas, and Other Sulfur Compounds. For many years, final compounds of these classes have been disregarded, and the thiocarbonyl subgroup has a natural place in a SMARTS list of alerts. An example of a failed thiourea drug is the histamine H2 antagonist metiamide (Table S1, entry 27) while tolrestat (Table S1, entry 28), an aldose reductase inhibitor, may represent the thioamides. Both drugs were stopped in clinical trials. The thiazolidine-2,4-dione ring has appeared in drugs of the past but is currently only found in the antidiabetic glitazones. 402



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Figure 14. Complicated rearrangement initiated by oxidation on the piperazine part of MB243.

Figure 15. 4-Substituted piperidines can be oxidized to reactive intermediates, probably dihydropyridine iminium ions eventually giving pyridinium ions or adducts with nucleophiles.

Potential RM liabilities of piperidine and pyrrolidine in drugs, also by oxidation to carbinolamines/imines, have often been suggested,96 but considering the extensive current use of these amines in the pharma industry, it might be too early to raise a general warning flag for these as covalently binding agents. A case in point is phencyclidine (PCP; Figure 5), which was proposed97 to inactivate CYP2B enzymes via iminium intermediates. In spite of later extensive experimentation,98 it does not appear likely that this is a major mechanism (cf. discussion in Section 6.6).52 Only the substructure of an N-substituted 4-aryl-4-hydroxy(or F)-piperidine might provide cause for consideration because haloperidol, an analog of the neurotoxin MPTP (Figure 15), has been shown99 to form a pyridinium compound just like MPTP (cf. reviews on general bioactivation reactions26,27 and of alicyclic amine metabolism100). The pyridinium compounds are not in themselves reactive species, although the one from MPTP (Figure 15) has been shown to be the actual neurotoxin causing Parkinson’s. However, since MPTP can also be activated, suggested via MAO-B, to give covalent protein binding,123 this might also be the case with compounds in the 4-X-piperidine series. Reactive intermediates are probably the iminium ions (dihydropyridines) that should precede formation of the pyridinium compounds. It is unclear whether the aryl group plays a role in formation of a reactive intermediate since there are indications that certain 4-Fpiperidines lacking an aryl also give rise to RMs.124 The NK-2 antagonist UK-290795 (Chart 2) is an example of this type of structure. It was in development with Pfizer around year 2000 but was terminated very early for unknown reasons. (Parenthetically, no 4-F-piperidines were found among the 9935 structures in the DrugBank database, which lists many 4OH-piperidines.) It might be that any leaving group in the 4position of an N-substituted piperidine could form a similar intermediate (generalized alert in Chart 2 where the R-group, an implicit aryl, might be omitted). This would be in complete

Withdrawn troglitazone has been associated with formation of RMs at the thiazolidinedione ring (Figure 4C), and pioglitazone, which lacks the 2-methylphenol substructure of troglitazone, shows high covalent protein binding.91,92 Based on these results, this ring is included in the SA list. 6.16. Other Structural Alerts. It makes sense to presume that there are bioactivating reactions that for now are unknown to us. At the same time, some functional groups currently present uncertainties about their alleged role as RM precursors. For example, the potential role of certain aliphatic amines in RM-related side effects is problematic regarding its scope and potential mechanisms. Certain piperazines seem able to generate a distinct reactive intermediate that can bind to nucleophiles in proteins (more below). In addition, it has been suggested that other reactive intermediates, possibly aldehydes, might be formed from (Naryl)-piperazines such as ketoconazole93 and other drugs by CYP and FMO actions.94 Mechanistic details of this and extent of covalent binding to proteins do not seem to have been elucidated. Complicated rearrangements limited to particular substructures can contribute to the difficulties of RM/SA identification. One example is the final product formed from extensive oxidation of MB243, a piperazine (Figure 14), followed by internal rearrangements.95 Indirect identification of the RM had to rely on the structure determination of the final thiazolidinine-imidazoline product. The level of covalent binding to human liver microsomes of MB243 was reported to be very high, 2100 pmol/mg protein, making it justified to incorporate its unique piperazine substructure into a list of SAs (Table S1, entry 59). In view of the many indications of N-arylpiperazine bioactivation,94 it might be prudent to add the general substructure of N-arylpiperazine (Table S1, entry 63) to a list of SAs, in addition to that of MB243 (and in addition to the generic, already included benzeneamine/aniline structure). 403



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Figure 16. A number of PKIs have black box warnings, most of them originating from RM formation.

Part of the background to this unsatisfactory situation can probably be found in the restricted room for variations of inhibitor design inherent in the ATP-binding site. From an outside view, chemists seem to have been more or less forced to introduce structural elements that are far from optimal from a safety perspective. The following inhibitors with boxed warnings (Figure 16) may serve as examples of a few structural features that one should try to avoid as much as possible. Some of the structures also highlight the insufficient knowledge regarding assignments of SAs for RMs. Compounds mentioned (with daily dose) in the following are sunitinib (50 mg), pazopanib (800 mg), lapatinib (1250−1500 mg), ponatinib (max 45 mg/day), and regorafenib (160 mg). In addition to being associated with hepatotoxicity, most inhibitors show varying degrees of timedependent CYP inhibition.102 Sunitinib, which is used in a daily dose of 50 mg that in this context should be considered low, has clear warning signs when applying the SAs mentioned in this Perspective. Studies to pinpoint formation of RMs do not seem to have been performed, but the 5-fluorooxindole part should be prone to generate a quinoneimine (Figure 16). The dimethylpyrrole is another clear SA that can generate two isomeric methide type RMs by oxidation of the methyls as shown. The high-dose pazopanib, likewise, has a glaring liability in the 4-methylaniline, which generates a benzylic alcohol

agreement with the 3-F-pyrrolidines depicted in Figure 10, which can be described as 3-F-alkylamines as well. A few other functional groups not linked to the influence from aromaticity or olefins have also been suggested to be associated with RM formation.26 Among these are the sulfonylureas where a mechanism involving formation of isocyanates was proposed.101 In view of the uncertainties associated with this mechanism and adverse reactions of the sulfonylurea class, we do not suggest listing a SA for this.

7. PROTEIN KINASE INHIBITORS HIGHLIGHTED During the two decades preceding June 2017, more than 37 new drugs of the class PKIs targeted to the ATP-binding site have been launched, a large majority of these being tyrosine kinase inhibitors. While today many of the new drugs within this class that enter clinical trials largely illustrate basic RM awareness/avoidance principles, this has not been the case when looking back. More than half of the currently used PKIs cause hepatotoxicity and have a summary report in the NIH LiverTox database. An important caveat here is that the observed DILI is not necessarily caused by RMs since PKIs are polypharmacological drugs. Currently, at least six PKIs are given a black box warning. The site LiverTox has a special overview of hepatotoxicity of PKIs at https://livertox.nih.gov/ TyrosineKinaseReceptorInhibitors.htm 404



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Figure 17. Hypothesis of bioactivation of fluoxetine generating 4-trifluoromethylphenol.

8. KNOWLEDGE CREATES NEW HYPOTHESES Generalizations and categorization of reaction mechanisms for RM formation should provide an intellectual exercise and overview that might lead to new thoughts and hypotheses about suspected adverse drug metabolism. For example, formation of quinone methides from drugs and other xenobiotics is not reported or documented very often; see the handful of cases noted in Section 6.5. As mentioned there, the reaction of phencyclidine (PCP) to form a quinone methide (Figure 5) is initiated by a p-hydroxylation that leads to elimination of piperidine with a half-life of about 7 min at pH 7.4 and 37 °C.52 There are several other drugs, which have been associated with liver toxicity and where one could reason that they might undergo an analogous sequence of reactions, but have no such reaction reported in the literature. For example, ketamine (Chart 2) belongs to this class; there are reports of hepatotoxicity, but no metabolic mechanism has been suggested.111 One can note that the finding of dealkylation followed by elimination of hydrogen fluoride from flindokalner (Figure 5), which represents the initiating path b to quinone methide formation (Section 6.6), has not been followed-up by similar findings. There might, however, be reasons for paying more attention to this less noticed112 mechanism, which has mostly been associated with natural products (then having other leaving groups than fluoride). For example, 4-trifluoromethylphenol is released from the antidepressant fluoxetine (Figure 17) during its oxidative metabolism,113 initiated probably by benzylic oxidation, and is detectable in the urine of humans taking the drug. It was shown 70 years ago that this phenol and the ortho isomer are unstable since they can eliminate HF and generate the very reactive quinone methide shown, eventually giving rise to 2- or 4-hydroxybenzoic acids in water. The halflife of 2-trifluoromethylphenol was determined114 to be 6.9 h at 37 °C and pH 7.4, while Thompson et al.115 reported that 4trifluoromethylphenol has a three times shorter half-life, about 2.3 h. In the same paper they reported that the methide gave an adduct with thiol groups of proteins and glutathione and that the phenols are cytotoxic in liver slices. The relevance to side effects of fluoxetine caused by release of the phenolic quinone methide precursor 4-trifluoromethylphenol does not seem to have been addressed in the literature. However, one should note here that detoxification mechanisms of phenols by glucuronidation and sulfation are quite efficient.

(mentioned in the submission folder to FDA) that can readily eliminate water to form a quinoneimine (which can probably also be formed by direct oxidation). It can be noted that dasatinib (Figure 16) is another PKI that has been shown to form a benzylic alcohol as well as a phenol, which both can give rise to RMs.103 Masitinib, which is used in dogs only, has the same liability.104 Lapatinib, which is given in a high daily dose of up to 1500 mg, has been reported to form a quinoneimine, as one might have predicted.105 The potential role of the furan part in formation of RMs has not been elucidated. The SAs of ponatinib, except the anilide (acylaniline), are not obvious when matching with the most common alerts mentioned above. Ponatinib, according to the NDA files from the applicant,106 is metabolized by CYPs (largely CYP3A4) and amide-cleaving enzymes, while other enzymes, like aldehyde oxidase, which might be expected to attack the heterocyclic ring system,107,108 are not mentioned. Since ponatinib, in spite of its low to moderate dose, displays a considerable number of serious side effects including DILI, it should be an attractive target for detailed structure-toxicity effects. This actually seems to have been initiated. It was recently reported that CYP1A1 oxidizes ponatinib on the 4methylbenzamide and the imidazo-pyridazine parts to reactive metabolites leading to non-identified GSH adducts.109 The metabolism of the multikinase inhibitor regorafenib (Figure 16), which has been associated with severe DILI, has not been reported on the detailed level of RM formation. It is mentioned here because of its large number of negative structural features such as two aniline fragments that can give rise to various quinoneimine structures while still being part of the whole molecule. Aniline structures with well-known RM liabilities will additionally be formed if enzymatic hydrolysis of the urea occurs. In addition to the structures discussed, it should be noted that most other PKIs have raised concerns from an IDR/DILI perspective.110 All the FDA-approved PKIs with links to labeling are found at http://www.brimr.org/PKI/PKIs.htm (latest update in December 2017). Again, it has to be emphasized that other off-target pharmacological actions of the PKIs might lead to the observed DILI. Strict selectivity for the targeted kinase is a rarity. The antibiotics are another class of drugs for life-threatening disease that likewise holds many drugs with hepatotoxicity. In December 2016 the FDA rejected the new semisynthetic ketolide solithromycin on the ground of its unacceptable hepatotoxicity. This compound, from a safety perspective, surprisingly displays a “true aniline”, which might be suspected to be involved in the hepatotoxicity.

9. DISCUSSION 9.1. Dominance of Aromatics, but They Are Not Alone. As is clear from the above listing of SAs, aromatics unsurprisingly dominate the picture of RM formation, a fact that has been pointed out previously.116 This is both by 405



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SMARTS derived from “singletons” will not entail noise since they will rarely find matches in the probed structures. Relevant to discussions of learning processes is also the training of new and existing medicinal chemists. In the context of new (academic) medicinal chemists, Rafferty has proposed that programs at (US) universities do not sufficiently stress understanding of physicochemical profiles of compounds while paying too much attention to maximizing target potency.122 Also, too much emphasis on sophisticated and new organic synthesis (of natural products) might divert attention from what characterizes new and safe drugs. To this we wish to add that attention to both safety and off-target toxicity of drugs often seems to be in much neglect in a number of academic programs. One could envision that the departments of organic chemistry should be able to contribute to a large extent to elucidating novel mechanisms of RM formation, which are perfectly suited to the expertise of mechanistic and physical organic chemistry. The strong linkage to biochemistry and life sciences should further increase the attractiveness, but this has so far not really caught on. There are many older drugs that deserve a closer look at how and by which enzymes they are activated. Here, theoretical mechanistic thinking can anticipate identification of potential routes to RMs by knowledge and by calculations of potential reaction paths. Additionally one might attempt to assess and rank the empirical capability of substructures (or SAs), via their RMs, to cause harm/toxicity. This could be in resemblance to the RYGN color scheme mentioned above for classifying drug examples. The question is whether it is useful and has a basis in clinical observations. A rationale for starting an attempt as a thought process could be that a certain path to a known RM proceeds in several steps and therefore has less probability to occur. However, a rule-based system containing such elements is far from straightforward to implement in a rigorous way, and it has to be preceded by a thorough analysis of existing data.

influence from substituents that steer oxidations on the rings and by the aromatic’s influence on the substituent. Furthermore, substituents may be given the possibility to interact with each other, for example, to take part in quinone methide formation or by stabilizing a benzylic carbenium ion. As noted by others, aromatics largely present an impediment to the ease of developing drugs,38,117 and there is a higher attrition rate for “flat compounds”.118 Safety plays a large role in this as indicated by investigations of the causes for drug attrition in four large pharmaceutical companies.119 Among the other most negative properties entailed by aromaticity, one can note reduced overall solubility. It has previously been suggested that more “natural productlike” test compounds made by chemists would increase the chances of greater success in development.118,120 The proposers mostly had in mind improvements in pharmaceutical (mainly solubility) and ADME properties, but it appears likely that safety aspects would also get a boost by following the advice of avoiding aromaticity and unsaturation as much as possible. Efforts in this direction are underway by trying to develop sp3 isosteres of the phenyl group (cf. review by Meanwell).38 A range of other functional groups not linked to the influence of aromaticity are also associated with RM formation. Amines and carboxylic acids are very common functionalities in drugs and can also be metabolites themselves; therefore, they have to be scrutinized in their roles as RM precursors. From a RM perspective, the abundant amines do not seem to present a general problem, although there have been reports of mutagenicity caused by the pyrrolidine part of a potential CD.121 Evidence was presented that mutagenic effects were abolished when reactive imines or aldehydes were trapped with cyanide or methoxylamine. However, more evidence would probably be needed for drug designers to pay attention. Regarding CAs, it has become apparent during the past few years that acyl-CoA formation likely plays a larger role for observed side effects than previously assumed (Section 6.13). As discussed there, the past few years’ of increased insight should lead to a better balanced view regarding assessment of hazards posed by CAs where potential interference with the endogenous lipid metabolism would weigh in regarding risk assessment. Selected SAs present on a list will have to reflect this. 9.2. Keep It Simple: Embracing the SA. By listing the most common SAs in a more detailed way than what we have seen attempted previously, we hope to increase the understanding of where to find the most obvious hazards.35 One can assume that with a good training process, designers should be able to easily spot these hazards. In addition, having a system where researchers can probe their thinking against the literature should dramatically increase the speed of learning the basics about RMs. New findings of compounds that give rise to GSH adducts are commonplace within the research departments of large pharmaceutical companies. When GSH trapping is run as a quick filtering process, there is a risk that not much work is spent on researching deeper into the mechanisms of bioactivation. This is clearly an impediment for the learning process of all drug designers who would benefit from fast access to new important findings where new examples can be added to an in-house database. The simple use of SAs as described here can greatly facilitate this process by adding SMARTS for the new alerts. Introduction into the database of more detailed

10. CONCLUDING REMARKS Based on knowledge of currently recognized SAs, which we have tried to organize in a structured way here, and of the current possibilities to predict metabolic and other conversions of drugs, it might be relatively straightforward to spot the major known types of hazards in existing and new drug structures. As a base of learning, the listed SAs with their relevant drug examples should also help to spot liabilities of combinations of substructures, which by themselves appear innocuous. Our list as presented here, although more detailed and longer than what we have seen elsewhere, is only a small start of a more comprehensive listing (and hopefully of building expert programs applying machine learning). Also, it only provides the drug designer with a point of departure since there are many considerations left regarding selection of the way forward when having identified a hazard: stay with the scaffold and modify substituents?; tweak the scaffold?; introduce metabolic soft spots?; and much more. It is obvious that we have an incomplete assessment of how many of the current drugs that give rise to reactive species, which in turn might be the cause of more or less noticed adverse events and drug−drug interactions. Considering the incredibly large number of novel chemical structures that potentially can become drugs, new not readily predicted conversions of novel compounds will likely come as unpleasant surprises, as seen recently.60 Nonetheless, metabolic pathways and relevant chemistry are accessible for theoretical and 406



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experimental studies and much more can and should be done to increase our knowledge in chemical toxicology. We believe that the attention on RM issues might have had a positive impact in the pharmaceutical industry regarding attrition rate. This might not be immediately apparent based on the drugs, especially PKIs that have entered the market during the recent few years but hopefully will be on the drugs to come. The risk analysis of a potential hazard follows its identification but is outside the scope of the current Perspective. Suffice it to say that chemistry always will have to strive to lower the exposure of the human body to xenobiotics, which also includes making more potent drugs that automatically carry a lower risk of generating harmful amounts of RMs and other off-target toxicities.

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Dr. Alexander Minidis received a Ph.D. in organic chemistry from the University of Basel, Switzerland in 1997 in conjunction with studies at the Max-Planck-Institute for Coal Research, Germany. After two postdocs, he joined AstraZeneca R&D Södertälje, Sweden. He was promoted several times during his tenure of more than a decade. He developed and honed skills in medicinal and computational chemistry with focus on cross-functional aspects foremost within CNS and pain following suite within other areas such as cancer and infectious diseases. After several short term endeavors, he now works as an independent consultant with his own company PharmaKarmaConsulting and with Awametox AB.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.8b00046. Table S1 provides the full range of proposed SAs (PDF)

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AUTHOR INFORMATION

ABBREVIATIONS ADR, adverse drug reaction; AE, adverse event; AG, acyl glucuronide; BBW, black box warning from FDA; CA, carboxylic acid; CD, candidate drug; DDI, drug−drug interaction; DILI, drug-induced liver injury; EMA, European Medicines Agency; FDA, Food and Drug Administration (USA); FMH, five-membered heterocycle; FMO, flavincontaining monooxygenase; IDR, idiosyncratic drug reaction; MAO, monoamine oxidase; MBI, mechanism-based inhibition; PKI, protein kinase inhibitor; RM, reactive metabolite; SULT, sulfonyl transferase; TDI, time-dependent inhibition

Corresponding Author

*Tel: +46-(0)70-553-7131. E-mail: alf.claesson@awametox. com. ORCID

Alf Claesson: 0000-0002-4571-5662 Notes

The authors declare no competing financial interest. Biographies

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REFERENCES

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