designing drugs to avoid toxicity

1 Designing Drugs to Avoid Toxicity

GRAHAM F. SMITH

Central Chemistry Team Lead, Merck Research Laboratories Boston,33 Avenue Louis Pasteur, Boston, MA 02115, USA

INTRODUCTION 1

THE SAFETY WINDOW 2

COMMON SAFETY RISKS AND THEIR SAR 2Toxicity associated with the liver 2Cardiovascular toxicity ( h ERG, ETC.) 13Genotoxicity/mutagenicity 16Phospholipidosis 39Phototoxicity 40Idiosyncratic toxicity 42

CONCLUSIONS 42

REFERENCES 43

INTRODUCTION

Two thousand four data from the Centre for Medicines Research show that toxicity is nowthe leading cause of failure of compounds in clinical development. With the improvedsystemic exposure, which came with better understanding of drug metabolism and phar-macokinetics (DMPK), came increased observations of dose-limiting toxicity [1]. Theleading causes of drug failure are now tied at 30%, with toxicity as likely to be the demiseof a drug’s development as lack of efficacy, (PK-related attrition now stands at 10%).Nevertheless, most safety-related attrition (70%) occurs pre-clinically following candidateselection, suggesting that we are still in need of better predictive models of in vivo toxicity.Where in vitro assays, or simple in vivo experiments, are predictive of adverse events inhumans, then these are increasingly carried out earlier in the drug discovery cycle.

The structure–toxicity relationships for mutagenicity and hepatotoxicity are already wellestablished owing to robust in vitro assays which translate well to clinical outcomes. Theseassays have frequently been used to implicate common alerting structures or so-called‘structure alerts’. Identifying structural alerts for toxicity, and high-throughput assays forearly indicators of toxicity issues in vivo, have become a normal part of early drug discovery.Regulatory authorities require that these robust assays be run on all new chemical entitiesbefore entering first-in-human trials.

Progress in Medicinal Chemistry – Vol. 50Edited by G. Lawton and D.R. WittyDOI: 10.1016/B978-0-12-381290-2.00001-X

1 � 2011, Elsevier B.V.All rights reserved.

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http://dx.doi.org/10.1016/B978-0-12-381290-2.00001-X

Sometimes, inadvertently, medicinal chemists do introduce toxicophores into drug mole-cules. Most often their reactive nature is produced or enhanced in vivo during normal meta-bolic processes. Wherever possible this review elaborates the biochemical mechanism attrib-uted to this type of toxicity. This allowsmedicinal chemists to validate the mechanism in theirown case and also to contextualize their ownmolecules in terms of their likelihood to undergosimilar biotransformation. Some successfully marketed drugs are positive in glutathionebinding assays [2], however, it is well established that the toxicities of known compoundswith chemically reactive metabolites can be correlated with the generation of hepatic proteinadducts and/or the detection of stable phase II metabolites such as glutathione conjugates.

The genotoxic carcinogens have the unifying feature that they are either electrophiles per seor can be activated to form electrophilic reactive intermediates. Hard electrophiles generallyreact with hard nucleophiles such as functional groups in DNA and lysine residues in proteins.Soft electrophiles react with soft nucleophiles, which include cysteine residues in proteins andin glutathione. Glutathione has a concentration of approximately 10 mM in the liver. Freeradicals can also react with lipids and initiate lipid peroxidative chain reactions [3]. Thepresence of a toxicity risk, or even the confirmation of a metabolic pathway to known toxicity,does not preclude a molecule from entering development. The risks are evaluated in thecontext of the body’s highly developed ability to clear toxic molecules from circulation and torecover from damage.

THE SAFETY WINDOW

All drugs are toxic at some level and so a major challenge in drug discovery is to find amargin of efficacy, over adverse events or toxicities, sufficient to provide clinical benefit topatients whilst avoiding putting them at unnecessary risk. The therapeutic index (TI) iscommonly used in the pharmaceutical industry and is the ratio of the no observable adverseevent level (NOAEL) divided by the human efficacious exposure level (Ceff) or exposure atthe maximum anticipated human dose (Cmax).

To determine margin, it is recommended to compare plasma Cmax from animal pharma-cology and toxicity studies with (predicted) human pharmacokinetic Cmax data usingunbound free fraction [4]. Depending on the disease target and nature of toxicity Ctrough

or area under the curve (AUC) can also be used to determine margins. Ideally these marginswould be around 10-fold or more over a reversible toxicity outcome which is observed inanimal testing, but which can also be clinically monitored easily in humans.

COMMON SAFETY RISKS AND THEIR SAR

TOXICITYASSOCIATED WITH THE LIVER

CYP inhibition

One of the liver’s main physiological roles is the clearance and metabolism of xenobioticsinto hydrophilic metabolites in order to facilitate their excretion. The liver has an abundanceof xenobiotic metabolizing enzymes and a high capacity for both phase I and phase II

2 DESIGNING DRUGS TO AVOID TOXICITY

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biotransformation. It receives more than 80% of its blood flow from the portal vein intowhich drugs are absorbed from the gastrointestinal tract and therefore liver is often a primarytarget for chemical-induced toxicity. There is the possibility that reactions catalysed bycytochrome p450 (CYP) enzymes may generate metabolites that are not only more toxic butalso more reactive than the original xenobiotic. Drug-induced liver injury is the mostfrequent reason for the withdrawal of an approved drug from the market. Drug-inducedliver injury has now become the leading cause of liver failure in the Unites States and resultsin at least 2700 deaths per annum [5].

Time-dependent inhibition (TDI) of CYPs refers to a change in potency during an in vitroincubation or dosing period in vivo, as opposed to a normal reversible inhibitor doseresponse. Inhibition of specific CYP enzymes by a drug can lead to pharmacokineticchanges in another drug, or so-called drug–drug interactions [6]. When inhibition affectsthe major metabolic route of another enzyme, and therefore alters (usually increases)exposure, this leads to unpredictable exposure levels and often to unacceptable risks topatients. In common with other proteins, CYPs are eventually metabolized and replaced ifthey are irreversibly inhibited. CYP enzymes have a turnover of the order of 1–2 days.However, TDI is often associated with bioactivation to electrophilic species which have thepotential for a number of toxic pathways beyond the simple inhibition of CYPs.

There are several known mechanisms of CYP inhibition:

* Competing enzyme substrates affecting the turnover of other drugs.* Competitive inhibitors such as quinidine which are not substrates.* Haem ligands: non-selective metal chelators such as the imidazole antifungals.* Metal inhibitor complex forming drugs such as erythromycin.* Inactivation or suicide inhibitors such as tienilic acid.

There are good methods of in vitro assessment of CYP inhibition and induction. Theoutcome of this is that common motifs and SAR for these toxic mechanisms exist.The following structure classes have well-established mechanisms for CYP inhibition.

AlkynesMechanism-based CYP inhibition (MBI) can arise from the covalent attachment of alkynemetabolites to the CYP protein. The formation of these adducts is described in Scheme 1.1.[(Scheme_1)TD$FIG]

Scheme 1.1 Oxidation of alkynes to electrophilic species

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Reactive metabolites can also be generated which may form covalent adducts with CYPproteins or other proteins leading to toxicity [7–10]. Generation of alkyne-CYP intermediate(A) can lead directly to the haem-bound product (B). For example, oxirene (C) (derivedfrom ring closure of A) can react with a CYP haem nitrogen generating B, and can also reactwith other nucleophilic sites in the CYP protein. Ketene D (formed by the migration of theR2 group in intermediate A) can also react with CYP and other proteins to form potentiallytoxic conjugates.

Gestodene (1) is one of many synthetic steroid drugs, including oral contraceptives,which contain an acetylene moiety. This drug was shown to be a mechanism-based inhibitorof CYP3A4 and 3A5. Avariety of other alkyne-containing steroids have also been evaluatedand show differing degrees of activity [11]. 17a-Ethynylestradiol (2) is a common compo-nent of oral contraceptives and taken by millions of women worldwide. This steroid hasbeen shown to be a mechanism-based inhibitor of CYP3A4 in vitro[12]. However, admin-istration of 17a-ethynylestradiol to women has been shown to have no impact on eitherintestinal or hepatic CYP3A4 activity [13]. This is most likely to be due to the very lowdoses required to achieve effective contraception, thereby mitigating the potential drug–drug interaction risk. [(Fig._1)TD$FIG]

CYP inhibition is the most common toxicity associated with alkyne-containingdrugs. Therefore, early investigation of metabolic routes (in vitro and in vivo), coupledwith reactive metabolite screening, is warranted for medicinal chemists studyingalkynes. Compounds should be evaluated across a range of CYP enzymes/species(with and without pre-incubation) to ensure that the potential for inhibition is fullyevaluated.

ThiophenesThe thiophene ring is susceptible to hepatic oxidation by CYP and undergoes epoxida-tion, followed by epoxide ring opening with nucleophilic biomolecules, to give adducts[14–22] (Scheme 1.2). Alternatively the epoxide can open to give a (-thionoenal whichcan also undergo adduct formation. The thiophene sulphur can also undergo oxidation,thus activating the ring towards nucleophilic addition of biomolecules. Peroxidase addi-tion of chlorine to the thiophene sulphur can also activate the ring towards nucleophilicaddition. Both the epoxide and the S-oxides have been postulated as reactive intermedi-ates. Identification of any of these metabolites therefore implies formation of reactiveintermediates.

Tienilic acid (3), a diuretic, is a mechanism-based inhibitor of CYP2C9 and seemsto inactivate it stoichiometrically. The molecule was launched onto the market and


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then subsequently withdrawn in 1982 due to a link with hepatitis [23–25]. The non-steroidal anti-inflammatory suprofen (4) showed nephrotoxicity in the clinic and is amechanism-based inhibitor of CYP2C9. It was marketed and subsequently withdrawndue to cases of acute renal failure [26, 27]. The antiplatelet drug panaldine(Ticlopidine) (5) shows TDI of CYP2B6; because it is linked with increased risk ofagranulocytosis its use has been replaced by clopidogrel (Plavix) (6) [28]. OSI-930 (7)was being developed for oncology when it was discovered that the molecule reactedvia the sulphoxide to form adducts with CYPs 3A4 and 2D6 [29]. In all of these casessulphoxide and glutathione adducts of the thiophene moiety have been detected andare postulated to cause the time-dependent inhibition of CYP enzymes and furtherrelated toxicities.

One way to reduce or inactivate this pathway is to introduce 2,5-substitution on thethiophene ring. Alternatively, the ring can be deactivated towards nucleophilic attackthrough introduction of adjacent functionality. Introduction of an alternative metabolicweak point elsewhere in the molecule may also reduce toxic exposure overall.Examples of these strategies can be seen in Zyprexa (8) and Plavix (6) [31–35],two commercially successful, widely marketed drugs. It appears that a small structuralchange between panaldine (5) and Plavix, which introduces an additional metabolicroute, reduces thiophene-related hepatotoxicity. Panaldine generates about 20 metabo-lites, some of which covalently bind to proteins, while the primary metabolic fate ofPlavix is hydrolysis of the methyl ester and some glucuronidation of the resultingacid. Plavix is dosed at 75 mg QD, while panaldine is dosed at 250 mg BID, so thedose difference between panaldine and Plavix may also be a potential mitigatingfactor. [( F i g. _ 1 )T D $ FI G ]

[(Scheme_2)TD$FIG]

Scheme 1.2 Metabolism of thiophenes

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smgraham

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[30-35]

[ ( Fi g . _1 ) T D$ F I G]

FuransIn a similar manner to that for thiophenes, furan toxicity occurs via furan epoxidationfollowed by epoxide ring opening to a g-keto aldehyde which in turn forms adducts withbiomolecules and induces toxicity [36, 37] (Scheme 1.3). Alternatively, the epoxide canultimately give rise to a lactone which can also form adducts. The epoxide has beenpostulated as the reactive intermediate common to all observed metabolites. Identificationof any of these metabolites therefore implies formation of the epoxide.

The clinical development of the 5-lipoxygenase inhibitor L-739,010 (9) was discontinuedby Merck due to hepatotoxicity; the compound is a mechanism-based inhibitor of CYP3A4


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[38–40]. Upon incubation with recombinant CYP3A4, a covalently bound adduct of thecompound was formed, which was identified using mass spectrometry.

The HIV protease inhibitor L-754,394 (10) showed hepatotoxicity via potent mechanism-based inhibition of CYP3A4 and its clinical development was discontinued. It also has beenshown subsequently for L-756,423 (11) that attachment of benzofuran through the 2-position, potentially blocking epoxidation, results in the removal of the furan-associatedtoxicity [41–43]. The fungal pneumotoxin Ipomeanol (12) was also developed for oncologyand then halted due to hepatotoxicity. Upon activation of Ipomeanol with rabbit CYP4B1 inthe presence of N-acetyl cysteine and N-acetyl leucine a major product (13) consistent withfuran epoxide formation was observed and characterized [44–46].

It is interesting to note that there are examples of 2,5-disubstituted benzofurans such asranitidine (14) [47] which do not undergo typical furan metabolism. This is probably due totheir low lipophilicity, low dose and additional substitution. Substituted benzofurans havebeen observed to undergo metabolism. Benzofuran itself undergoes the typical furanhydroxylation at the 2-position, possibly through direct hydroxylation and also potentiallythrough epoxidation, followed by ring opening to generate 2-hydroxyphenylacetic acid [48].[(Fig._1)TD$FIG]

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BenzodioxolanesThe benzodioxolane moiety is associated with mechanism-based irreversible inhibitionand/or induction of CYPs. In addition some compounds that contain the benzodioxolanemoiety are associated with hepatotoxicity. CYP-dependent oxidation of the methyleneleads to both a reactive carbene intermediate (A) which can form irreversible adducts withthe haem of CYPs (metal inhibitor complex) or the catechol (B) which is an ortho-quinone precursor and known toxin through redox chemistry. The mechanism of toxicityand CYP inhibition of benzodioxolane compounds has been discussed in detail [49–51](Scheme 1.4).

Paroxetine (15) is a marketed selective seratonin reuptake inhibitor (SSRI) with aknown CYP2D6 inhibition profile; it is both a reversible and a time-dependentCYP2D6 inhibitor. This results in significantly increased exposure to co-medicationsthat are metabolized by CYP2D6. Metabolism of the benzodioxolane group has beenstrongly implicated in the CYP2D6 inhibition shown by paroxetine [52–56] and recentstudies have shown that the potency of paroxetine as a CYP2D6 inhibitor in vitroincreases eightfold following pre-incubation [57]. The increase in potency was asso-ciated with the formation of a CYP mechanism-based inhibitor complex.Administration of paroxetine has been shown to convert some volunteers who areextensive CYP2D6 metabolisers to a poor metaboliser phenotype [58]. In additionparoxetine inhibits its own metabolism leading to non-linear time-dependent pharma-cokinetics. The half-life of paroxetine after single doses of 20 mg/day is 10 h but after

[(Scheme_3)TD$FIG]

Scheme 1.3 Furan oxidative metabolism

[(Scheme_4)TD$FIG]

Scheme 1.4 Metabolism of benzodioxolane


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multiple doses of 20 mg/day this increases to 24 h [59]. While, the fate of the benzo-dioxolane is well established in vitro, it is of note that most SSRIs are relatively potent,reversible inhibitors of CYP2D6.

Niperotidine (16) is an H2 antagonist structurally related to ranitidine. Twenty-five casesof acute hepatitis (including one death from fulminant hepatitis) associated with niperotidineuse were reported in Italy between March and August 1995 and the drug was withdrawnfrom the market. The methylenedioxy group of niperotidine (absent in ranitidine) is knownto undergo metabolism to catechol and quinone metabolites [60, 61].

Methylenedioxymethamphetamine (MDMA) (17) has been shown to inhibit CYP2D in atime-dependent manor through a mechanism producing a UVabsorption spectrum consis-tent with a carbene formation [6]. MDMA causes liver damage in humans.[(Fig._1)TD$FIG]

Fortunately, several viable isosteric replacements are available for the benzodioxo-lane structure. Replacement of one of the oxygen atoms with a methylene results in adihydrobenzofuran moiety, which may often show similar pharmacology to a benzo-dioxolane. The dihydrobenzofuran system can be rather susceptible to oxidative metab-olism, and this should be checked promptly when this group is employed (Scheme 1.5).The difluorobenzodioxolane group is a metabolically blocked at the ‘methylene’ car-bon, and this does not undergo the same metabolic reactions as the methylenedioxygroup. The difluorobenzodioxolane group is rather unusual as it is considerably morelipophilic than the methylenedioxy group. There are no drugs in the MDDR (moleculardetection of drug resistance) drug database containing this moiety. The methylenecarbon may also be blocked with other groups, for example as a dimethylketal, althoughthe stability of such groups towards acid-catalysed hydrolysis needs to be carefullyassessed.

There are many additional groups that have the potential to mimic a benzodioxolane.Owing to their instability towards hydrolysis in dilute aqueous acid, benzoxazoles shouldalso be employed with caution, if at all. The benzodioxane ring-expanded system appearsnot to be implicated in the same kinds of toxicity/mechanism-based CYP inhibition as thebenzodioxolane group.

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Haem LigandsThe previous examples of potential liver toxins all form covalent inhibitor complexes withCYPs and other proteins. Another commonly encountered class of inhibitors is the haemligands which offer lone pair donation, usually from nitrogen, to stabilize the iron in thehaem complex. These molecules have an affinity for the active site of CYPs in both theoxidized and reduced forms but are reversible inhibitors (Scheme 1.6). Many heterocycles

[(Scheme_5)TD$FIG]

Scheme 1.5 Some potential benzodioxolane isosteres

[(Scheme_6)TD$FIG]

Scheme 1.6 Haem ligands


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frequently used in drug-like molecules are capable of performing this role, for examplepyridines, azines and azoles [62].

The 11-b-hydroxylase inhibitor metyrapone (18) is an inhibitor of cortisol synthesis andof CYP3A4 [63, 64]. Metyrapone also causes induction of CYP3A4 synthesis in hepato-cytes. The HIV protease inhibitor ritonavir (19) [65, 66] contains two 5-substituted thia-zoles. Ritonavir is a potent inhibitor of CYP3A-mediated biotransformations (e.g. nifedi-pine oxidation and terfenadine hydroxylation). Ketoconazole (20) is a member of theantifungal imidazole drugs. Ketoconazole strongly inhibits CYP3A4 selectively [67].Sulconazole (21), another member of the antifungal imidazole derivatives, strongly inhibitsmost CYPs (1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4). [(Fig._1)TD$FIG]

CYP induction

CYP induction occurs when a drug or chemical causes an increase in enzyme activity,usually via increased gene transcription [68–70]. In many cases, inducers are also hepato-toxic. CYP induction can lead to a reduction in efficacy of co-medications and also to anincrease in reactive metabolite-induced toxicity. CYP induction is therefore a metabolicliability in drug therapy and it is highly desirable to develop new drug candidates that are notpotent CYP inducers.

Most commonly, ligand activation of key receptor transcription factors leads toincreased transcription. In the human liver, some of these enzymes, but not all, areinducible. Human CYP1A, CYP2A, CYP2B, CYP2C, CYP2E and CYP3A enzymesare currently known to be inducible. CYP gene families 2 and 3 have a similarmechanism of gene activation through a ligand-activated nuclear receptor constitutiveandrostane receptor or constitutively active receptor CAR and/or pregnane X receptor(PXR). CYP3A4 is the most highly expressed CYP enzyme representing up to 28% ofall CYPs and is highly inducible by a wide variety of xenobiotics. CYP3A4 has beenimplicated in the metabolism of more than 50% of prescribed pharmaceuticals [71].

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CYP1A genes belong to the Per-Arnt-Sim (PAS) family of transcription factors andrequire the aliphatic hydrocarbon receptor (AhR). CYP1A2 is also one of the majorCYPs in human liver, accounting for approximately 10% of total amount of hepaticCYPs.

There are four main mechanisms of CYP induction [72]:

1. PXR upregulates the important CYP3A and 2C enzymes. PXR is referred to as themaster regulator of CYP enzymes. The classic substrate for PXR is the antibioticrifampicin (22). Similarly, the glucocorticoid anti-inflammatory and immunosuppres-sant dexamethasone (23) has been reported to be a substrate [73]. It has been hypoth-esized that unwanted activation of the PXR is responsible for approximately 60% of allobserved drug–drug interactions [74]. Today, many drug companies routinely includethe PXR reporter gene assay at the drug discovery stage as part of the selection processesof drug candidates for clinical development.

2. Aliphatic hydrocarbon (Ah) or aryl hydrocarbon receptor (AhR) induces CYP1Aenzymes 1 and 2; certain polycyclic aromatic hydrocarbons in the diet and environ-ment induce their own metabolism, for example hydrocarbons in cigarette smoke,charbroiled meats and cruciferous vegetables. 2,3,7,8-Tetrachlorodibenzo-p-dioxin(TCDD) (24) and the related TCDF (25) are the prototypical CYP1A inducers.Tryptophan derivatives, caffeine, eicosanoids and some prostaglandins are alsoAhR substrates.

3. CAR induces CYP2B and CYP3A enzymes. Typical substrates are barbiturates such asphenobarbital (26).

4. Peroxisome proliferator-activated receptors (PPARs) upregulate CYP4A. Typical exam-ples include the fibrates, PPAR alpha receptor agonists such as clofibrate (27). Thethiazolidinedione antidiabetic agents such as rosiglitazone (28) act as PPAR gammaagonists.

Transcription factors such as HNF4a are also involved and there is also significant post-translational regulation of protein half-life, especially of CYP2E1. The glucocorticoidreceptor (GR) and estrogen receptor (ER) may also be involved. Two other nuclearreceptors, designated LXR and FXR, which are respectively activated by oxysterolsand bile acids, also play a role in liver CYP7A1 induction [75]. Together all of thesereceptors are able to sense a great variety of xenobiotics and consequently regulatenumerous phase I and phase II drug-metabolizing enzymes and drug transporters. In thisway they attempt to adjust the body’s metabolic response to the challenges of thechemical environment.

To avoid toxicity associated with potential CYP induction, it is important to divertthe structure–activity relationship of interest from that of the nuclear receptor which isalso being activated. The screening approaches to avoiding CYP induction arereviewed by Pelkonen et al.[75]. It is possible to establish in vitro assays for AhR,CAR, PPAR gamma and PXR, and SAR from these assays may be used to refine aQSAR model. In this way in silico models have been developed for all of thesereceptors using QSAR and docking approaches, some of which reach up to 80%successful prediction.[ ( Fi g . _1 ) T D$ F I G]


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CARDIOVASCULAR TOXICITY (hERG, ETC.)

Virtually all cases of extended QT interval are traced to the inward rectifying potassiumion channel (IKr) related gene known as hERG (human ether-a-go-go-related gene),which encodes the protein Kv 11.1. Inhibition of the cardiac IKr current leads to pro-longation of the QT interval and to a risk of lethal ventricular arrhythmia (torsade depointes (TdP)) [76–78]. The electrocardiogram (ECG) traces in Figure 1.1 show theprolongation of QT leading to TdP. Once hERG involvement in inherited long QT wasestablished, QT-prolonging TdP-prone drugs began to be tested on hERG. This showedhERG to be a major contributor to drug-acquired QT prolongation. This phenomenonwas once considered a trivial finding, in fact IKr was a valid drug target for the class IIIarrhythmic drugs, but more recently QT prolongation has become a major regulatory

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issue. Since 2005, the FDA has required that all new drug candidates are evaluated todetermine the drug’s effect on the QT interval. Other channels which may play a moreminor role include Nav1.5 and Ca2+.

QT prolongation can routinely lead to a drug being withdrawn from the market or fromdevelopment as happened in the cases of the antihistamine terfenadine (29) and the gastricprokinetic cisapride (30). Astemizole (31) a long duration antihistamine drug, the anti-psychotic sertindole (32) and the quinolone antibacterial grepafloxacin (33) were also allwithdrawn post-launch over concerns about life threatening TdP.

Today nearly all drug discovery programmes include an early assessment of hERGliabilities, including an in vitro primary radioligand binding assay in the IKr ion channel[79]. In addition to IKr, early assessment of Nav1.5 and Cav1.2 channels is also beingconducted earlier. Functional alternatives to these binding assays are patch clamp and patchexpress. Apart from an earlier and cheaper alert to hERG toxicity these high-throughputassay data provide excellent data for validating structure–activity relationships and buildingcomputational models.

Cavalli et al.[80] was able to build a 3D QSAR model (Figure 1.2) based onknown drugs. This model is often used as a first pass design tool to avoid hERGactivity. The empirically based model has been validated and enhanced by homologymodels related to known crystal structures of four other bacterial potassium channels[81–83]. These models have been used successfully in the development of drugs suchas maraviroc (34) to overcome hERG binding issues encountered in the discoveryphase [84].

[(Fig._1)TD$FIG]

Fig. 1.1 A: Normal ECG. B: Long QT syndrome. C: Ventricular arrhythmia (torsade de pointes).


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Workers fromMerck showed that bio-isosteres which might improve IKr profile based onprevious pairwise analysis of molecules assayed can be used to computationally point theway towards reduced hERG affinity [85]. Bell and Bilodeau [86] recently gave a goodoverview of medicinal chemistry tricks to avoid hERG SAR. Techniques usually involvereducing basicity and lipophilicity (Scheme 1.7). The IKr channel seems to have high affinityfor many types of lipophilic bases, therefore adding polar groups, for example alcohols orethers, removing hydrophobic groups, reducing Pi-stacking interactions and removing ormodifying aryl rings are all good approaches chemically. [(Fig._1)TD$FIG]

[(Fig._2)TD$FIG]

Fig. 1.2 The Cavalli hERG pharmacophore model.

[(Scheme_7)TD$FIG]

Scheme 1.7 Simple modifications which often reduce the risk of hERG activity

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GENOTOXICITY/MUTAGENICITY

Genotoxicity describes a deleterious action on a cell’s genetic material affecting itsintegrity. The term genotoxicity includes DNA reactivity, resulting in mutation, and alsointeraction with various protein targets, for example spindle microtubules, leading tonumerical chromosome changes or aneuploidy. It is regulatory practice to view DNA-reactive effects as having no acceptable threshold (or no-effect level), whereas reactionwith protein targets might have an acceptable threshold and potential to establish a safetymargin as is the case for other toxicities. Genotoxic substances are potentially mutagenicor carcinogenic. This definition includes both some classes of chemical compounds andcertain types of radiation.

Typical genotoxins such as aromatic amines are believed to cause mutations because theyare nucleophilic and form strong covalent bonds with DNA, resulting in the formation ofaromatic amine-DNA adducts and preventing accurate replication. Genotoxins affectingsperm and eggs can pass genetic changes to descendants who have never been exposed tothe genotoxin. As many mutations can contribute to the development of cancer, manymutagens are carcinogens. So-called spontaneous mutations are also known to occur dueto errors in DNA replication, repair and recombination, and the many endogenous productsof cellular metabolism such as oxygen radicals.


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The international test guidelines require a bacterial mutagenicity test (the Ames test)and an in vitro test for chromosome aberrations or for mutation in a mouse lymphoma cellline, before the first human clinical trials. An in vivo test for chromosome damage(typically a micronucleus test) must be done before phase II clinical trials [87]. Manycompanies also use early versions of these regulatory assays for screening, or some of thewide range of relatively high-throughput screening assays available for early detection ofgenotoxicity. The Ames test is a bacterial assay that allows the detection of strong earlysignals of mutagenicity [88–90]. Ames tests use a histidine-free medium with a genet-ically engineered strain of bacteria that can only proliferate into colonies after certainmutations restore their ability to synthesize histidine. It has been established that thepredictive power of positive Ames test results for rodent carcinogenicity is high, rangingfrom 60 to 90% depending on the compound set examined. An assay is also used thatidentifies chromosomal damage, either visible as chromosome breaks at metaphase, or asmicronuclei (chromatin that is left outside the main nucleus and comprises either frag-ments of broken chromosomes (clastogenicity), or whole chromosomes, indicatingpotential for aneuploidy). An in vitro and in vivo chromosomal aberration assay isrequired before first-in-human studies; these studies are often conducted in the presenceof metabolic activation in order to assess the toxicity of any metabolites which may beformed.

Following extensive testing, the validation of structural types leading to mutagenicity iswell established. The development of the so-called ‘structure alerts’ related to mutage-nicity from the 1950s to the current day is well reviewed by Benigni and Bossa [91].In general the alkylation of DNA by electrophilic chemicals leads to mutagenicity. Theother mechanism is via molecules which intercalate with DNA, changing its tertiarystructure, and interfering with normal DNA function and replication. In this sectionbiochemical pathways which explain the reactivity of these groups are elaborated, sothat they might be more appropriately used and modified by medicinal chemists to reducethe risk of mutagenicity.

Electrophiles not requiring metabolic activation

During the course of in vitro testing in research programmes, certain chemical inter-mediates and mild electrophiles find their way into the screening cascade by design orby accident. Despite some of these being perfectly stable chemicals in bufferedsolution, it must be noted that the body is perfectly able to find nucleophiles withsufficient potency such as amines and thiols which will unselectively react withthese electrophiles. The toxicity of these functional groups will in general be relatedto their chemical reactivity. Figure 1.3 shows a set of common electrophiles whichshould be avoided unless targeting a specific drug–protein covalent interaction is thedesired goal.

Alkyl halides and sulphonates

Alkyl halides and sulphonates are susceptible to nucleophilic attack by a cysteine-SH orother bio-molecule nucleophiles to form adducts [92]. Their toxicity is directly related to

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their chemical reactivity. Leaving groups beta to an electron-withdrawing group (EWGsuch as carbonyls, aryl groups, nitriles, etc.) are also of concern due to possible elimi-nation to form a Michael acceptor molecule. Mammalian response to such agents in-volves elevation of activity of phase II detoxifying enzymes [93, 94]. An SN1 mechanismfor the substitution is also possible. For example mono alkyl fluorides are less susceptibleto nucleophilic attack, but are likely to be converted via cationic (SN1-like) mechanismswhere possible.

Toremifene (Fareston) (35) is an oral anti-estrogen drug for the treatment of metastaticbreast cancer. There are numerous adverse events and toxicities reported with the use oftoremifene as described in the pharmaceutical documentation ring (PDR) entry. However,many of these may be due to its estrogenic activity rather than the presence of an alkylhalide. Many alkyl halides and sulphates have been reported as anticancer agents. Theirdesigned mode of action is alkylation of DNA, and hence they are cytotoxic with many sideeffects. In these cases, the genetic toxicity is incorporated by design and part of the riskanalysis for development and usage.[(Fig._1)TD$FIG]

In addition to mechanisms seen for the other halides, organic iodides can cause hypo-thyroidism, hyperthyroidism, phototoxicity, photosensitivity and skin sensitization.

[(Fig._3)TD$FIG]

Fig. 1.3 Some common electrophiles encountered in medicinal chemistry.


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Exposure to iodides can cause iodism, which is poisoning by iodine. Hepatic andgenetic toxicity has been observed for some aryl iodides, but a specific toxic structuralmoiety has not yet been established. Aromatic iodo compounds inhibit 5’-monodeio-dinase, the enzyme which catalyses the peripheral conversion of T4 (thyroxine) toT3 (triiodothyronine) (Scheme 1.8). The resulting decrease in circulating T3 levelsstimulates thyroid-stimulating hormone (TSH) production by the pituitary gland [95–98]. Chronic TSH stimulation of the thyroid gland in this way may lead to follicularcell hypertrophy, hyperplasia and ultimately neoplasia. Pituitary hyperplasia and neo-plasia have also been associated with the chronic secretion of TSH. Humans are muchless susceptible to the effects of chronic thyroid stimulation than are rodents.Additionally, if compounds containing an iodide travel to the skin, and the patientis exposed to sunlight, there is the potential for a radical-mediated generation ofmolecular iodine, which can then react with biomolecules, such as proteins and nucleicacids.

Cordarone (amiodarone) (36) inhibits peripheral conversion of T4 to T3 and maycause increased thyroxine levels or decreased T3 levels [99, 100]. It is also a potentialsource of large amounts of inorganic iodine. Cordarone can cause either hypothyroid-ism or hyperthyroidism. Because of the slow elimination of cordarone and its meta-bolites, high plasma iodide levels, altered thyroid function and abnormal thyroid-function tests may persist for several weeks or even months following cordaronewithdrawal. Photosensitization, which results in a blue-grey discolouration of theexposed skin, occurs in 10% of patients. The radiographic contrast agent sodiumiopanoate (37) results in perturbation of serum thyroid hormone levels in humans[101]. Synthroid (levothyroxine (T4)) (38) is a relatively safe (produced endogenously)thyroid hormone developed and marketed by Abbott (PDR). Synthetic levothyroxine isidentical to that produced in the human thyroid gland. Overdoses of synthroid lead topredictable thyroid side-effects [98]. The antiviral idoxuridine (Apridin) (39), used fortreatment of herpes simplex virus, is an effective radiosensitizer but its clinical devel-opment for this use has been limited by toxicity. Prolonged intravenous infusions ofidoxuridine (39) are necessary for optimal tumour uptake but cause dose-limitingmyelosuppression [101].

[(Scheme_8)TD$FIG]

Scheme 1.8 Metabolism of thyroxine

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Organic iodine can be replaced with bromine as the functional group with a highestsimilarity to it in terms of lipophilicity and polarizability. Branched alkyl substituents arealso alternatives although with a different electrostatic potential.[(Fig._1)TD$FIG]

Epoxides and aziridines

Nucleophilic attack by the cysteine-SH or other bio-molecule nucleophiles at epoxides andaziridines can form protein adducts [102] (Scheme 1.9). Reactivity considerations aretherefore similar to the alkyl halides and sulphate esters. Non-sterically hindered epoxidesappear to be of special concern. An SN1 mechanism is also possible.

Taisho has discontinued development of aloxistatin (40), a thiol protease inhibitor, forthe potential treatment of muscular dystrophy. In clinical trials it did not have a positiveeffect on Duchenne muscular dystrophy and chronic administration produced necrosis ofthe liver [103]. Both fumagillin (41) and TNP-470 (42) are irreversible inhibitors of type

[(Scheme_9)TD$FIG]

Scheme 1.9 Mechanism of epoxide toxicity


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2 methionine aminopeptidase (MetAP2) and were being developed to treat musculardystrophy [104, 105]. The less hindered epoxide ring in both molecules covalentlymodifies His-231 of MetAP2. In this specific case, the covalent modification of anenzyme is part of the mechanism of action. In most other cases, the covalent modificationby an epoxide of a protein would be reason for toxicological concern. Therefore, unlessthe epoxide is incorporated by design to provide irreversible inhibitors, this type ofreactivity is undesirable.[(Fig._1)TD$FIG]

Michael acceptors

All non-aromatic double bonds connected to an EWG can potentially undergo Michaeladdition in vivo with nucleophiles such as glutathione. EWGs typically include -CO2R,-CONR2, -CN, -SO2C, -SO2NR2, epoxides and ketones. Ortho- and para-quinones arealso susceptible [108, 109]. Toxicity occurs by non-specific nucleophilic attack by acysteine-SH to form a covalent DNA adduct thus causing mutagenicity or loss ofprotein function or immunogenic response. Michael acceptors with a b-nitrogen tiedinto a ring system containing the alkene moiety are less capable of glutathione orcysteine addition into the double bond. These vinylogous amides (or ureas) are exem-plified by Norvasc (43).

The Pfizer compound CI-1033 (Canertinib) (44) [110] is a pan-erbB tyrosine kinaseinhibitor that is presently in phase II clinical development [111]. The investigators notedreversible dose-limiting hypersensitivity at high doses. The compound was designedto bind to Cysteine-773 of the erbB1 kinase. The corresponding erbB1-CI-1033

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[108]

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adduct is presently being used as a marker in the clinical development of this compound[112].[ ( Fi g . _1 ) T D$ F I G]

Quinones, 1,2- and 1,4-diphenols

1,2- and 1,4-diphenols can be readily oxidized to quinones. Quinones are Michael acceptorsand cellular damage can occur through alkylation of crucial cellular proteins or DNA(Scheme 1.10). In addition, they are highly redox active molecules which can redox cyclewith their hydroquinone (HQ) and semiquinone radical sisters, leading to the formation ofreactive oxygen species including superoxide, hydrogen peroxide and the hydroxyl radical.These species in turn lead to oxidative stress and the formation of oxidized cellular macro-molecules [111, 112].

The parent p-benzoquinone (p-BQ) itself has been proven to form DNA adducts. DNAadduct formation and cytotoxicity in HL-60 cells treated with either HQ or p-BQ has beenexamined. Treatment of HL-60 cells with either HQ or p-BQ produced the same DNA

[(Scheme_0)TD$FIG]

Scheme 1.10 Mechanism of diphenol toxicity


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[110]

adduct. The DNA adduct level varied from 0.05 to 10 adducts per 107 nucleotides as afunction of treatment time and concentration for both compounds. To achieve the sameDNA adduct level required higher concentrations and longer treatment times with HQcompared to p-BQ. The p-BQ was also more cytotoxic to HL-60 cells than HQ [113].

The issue of hepatotoxicity with the use of the catechol-O-methyl transferase(COMT) inhibitors tolcapone (45) and entacapone (46) has also been examined.Neither drug caused hepatotoxicity in pre-clinical toxicity testing. However, in clinicaltrials of tolcapone, liver chemical test results were elevated to more than three timesabove the upper limit of normal [114, 115]. Post-marketing surveillance studies notedthree instances of acute liver failure with death after 60,000 patients had receivedtolcapone for a total of 40,000 patient-years. For this reason, the drug was withdrawnfrom the market in Europe and Canada, and a black box warning issued in the UnitedStates. In contrast, clinical trials with entacapone demonstrated no increase in liverenzymes above those observed with placebo. It has been shown that tolcapone ismetabolized to amine and acetylamine metabolites in humans, but the analogous meta-bolites were not detected in a limited human study of entacapone metabolism. Thus, ithas been hypothesized that one or both of these metabolites could be oxidized toreactive species and that these reactive metabolites might play a role in tolcapone-induced hepatocellular injury [116].

When a-methyldopa (47) is incubated with rat liver microsomes in the presence of anNADPH-generating system a quinone is formed in the presence of NADPH and O2. Thebinding was inhibited by a carbon monoxide atmosphere indicating the involvement of CYPsbut the mechanism involves CYP superoxide generation [117]. a-Methyldopa was primarilymetabolized to a GSH adduct. It was also metabolized to a product which was identified asthe cysteinyl adduct [118]. Troglitazone (Rezulin) (48), the treatment for type II diabetes, actsas a masked quinone that is revealed upon in vivometabolism [119, 120]. After FDA approvalin 1997, it was withdrawn after severe hepatotoxicity was seen in a number of patients.

[(Fig._1)TD$FIG]

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Polycyclic aromatic compounds

It has been shown that polycyclic aromatic compounds are able to intercalate with DNA andcause frame shift errors in replication [121, 122]. This structure type does not need bioacti-vation to show its toxicity and binds reversibly and not covalently unlike many of theelectrophiles. This class of compound is least clearly defined structurally, but seems to showthe strongest correlation to in vitromutagenicity. Many members of this structural class areDNA clastogens, which are mutagens causing chromosome effects including breaks, rear-rangements and changes in number. Kazius et al.[123] showed that the consensus structurecould be defined best by 11 planar atoms connected as shown in Figure 1.4. This substruc-ture describes a polycyclic planar system consisting of at least three rings whichmay containheteroatoms.

Aflatoxin B1 (49), thienoquinolone (50), the flavanoid claidzein (51), 9-aminoacridine(52) and 2-aminofluorine (53) are all clastogenic and mutagenic to mammals. Thistoxicity may be avoided by reducing the planarity of the system or reducing the numberof rings.

3-Alkyl indoles and azaindoles

3-Methylindole is a known pneumotoxin and shown to be potentially mutagenic by theformation of DNA adducts in vitro[124]. CYP-mediated oxidation of the 3-alkyl groupeither directly or via epoxidation of 2,3-double bond leads to reactive metabolites such asepoxides, Michael acceptors and vinylogous imines [125] which cause toxicity (Scheme1.11). The existence of the epoxide has been indirectly shown by labelling studies [126].Incorporation of leaving groups at the C-3 methyl (or secondary alkyl) position (e.g. OH,OR, NR1R2) allows for potential gramine-like cleavage, resulting in electrophilicintermediates.

[(Fig._4)TD$FIG]

Fig. 1.4 Polycyclic aromatic compounds.


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Indole-3-carbinol (54) is a natural component of brassica vegetables and is beingconsidered as an anticancer agent. The primary metabolic fate of this compound is viaformation and subsequent nucleophilic attack of the 3-methyleneindolenine and so thesespecies are now thought to provide a clue to the compound’s mode of action [127, 128].The dopamine D4-selective antagonist L-745,970 (55) was being studied by Merck as apotential treatment for schizophrenia. The N-acetylcysteine-S-yl adduct on the 3-alkylsubstituent was detected in vivo in rat, monkey and humans [129]. The leukotrieneantagonist asthma drug zafirlukast (Accolate) (56) was shown to be oxidized in vitroand in vivo by CYP3A4 to give GST adducts on the 3-alkyl substituent. Zafirlukast is amechanism-based inhibitor of CYP3A4 and shows idiosyncratic hepatotoxicity inpatients [130].

There is less evidence that N-alkyl indoles and azaindoles can form the sameelectrophilic intermediates. Nonetheless, it may be prudent to screen such compoundsfor TDI and reactive metabolite formation. Gramine-like cleavages are possible in otheraminomethyl heterocycles, for example imidazoles (Scheme 1.12). Hence consideration

[(Scheme_1)TD$FIG]

Scheme 1.11 Mechanism of 3-methylindole toxicity

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should be given to any electron-rich heteroaromatic systems toward gramine-likefragmentation. [ ( F ig . _ 1) T D $F I G ]

Aromatic and secondary aliphatic nitro compounds

Toxicological properties of nitro groups have been the subject of study for the past 50years. Most nitro-containing compounds can cause methaemoglobinemia and arepotentially mutagenic [131]. Metabolites of nitro-aromatic compounds have beenshown to bind covalently to DNA. The nitro group can be reduced to the same reactivenitroso toxicophore as would be formed by the oxidation of the corresponding aromaticamine.

The pathway to aniline transformation would be reductive [132]. Three sequentialreduction products observed are aromatic nitroso (ArN=O), aromatic hydroxylamines(ArNHOH) and anilines (ArNH2) (Scheme 1.13), all of which are toxicophores in theirown right (vide infra). The nitroso aromatics are carcinogens and covalently labelled

[(Scheme_2)TD$FIG]

Scheme 1.12 Other potential gramine-like cleavage reactions


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proteins (e.g. aromatic nitroso labelling of C-93 in haemoglobin). Hydroxylaminesare known to covalently modify DNA via electrophilic nitrenium formation from O-sulphated or acetylated metabolites. In humans, xanthine oxidase and microsomalNADPH-cytochrome c have been identified as enzymes involved in nitro reduction.Most of the nitro reduction occurs in the gut by anaerobic bacteria [133]. Oxidativepathways also contribute to the metabolism of many of these compounds [134]. Thecytochrome CYP family of enzymes is primarily responsible for the oxidative metab-olism of these compounds.

Simple aliphatic secondary nitroalkanes are known carcinogens (Scheme 1.14).2-Nitropropane has been shown to be hepatotoxic in rats and rabbits and carcinogenicin rats [135]. In male Sprague-Dawley rats, it induces characteristic base modifications inrat liver DNA and RNA [136, 137]; primary nitroalkanes by contrast are apparentlyneither mutagenic [138–140] nor carcinogenic [141]. Primary nitroalkanes do not pro-duce base modifications in rat liver DNA and RNA. It is not clear at the moment whyprimary nitroalkanes are not subject to the same metabolic activation as secondarynitroalkanes.

It has been suggested that in the antibacterial agent chloramphenicol (57), which containsan aromatic nitro group and causes aplastic anaemia, a nitrosochloramphenicol may beinvolved as a toxic intermediate [142, 143]. In this case, chloramphenicol is reduced toaminochloramphenicol by intestinal bacteria, which in turn is N-oxygenated by liver micro-somes to the nitroso group. The Km and Vmax values are similar to those reported for anilineN-oxygenation.

Several nitrofuran derivatives (nitrofurantoin (58), furazolidone (59) and nitrofurazone(60)) have been used clinically as antibiotics for the treatment of urinary tract infections[144] and also as topical agents. Although these compounds inhibit a wide variety ofenzymes, their ability to cause DNA damage appears to be the primary event that leadsto cell death. Nitrofurantoin has a high renal clearance and most of the compound is deli-vered into the urinary tract, that is the site of infection, and systemic exposure is thereforelimited. Nitrofurantoin has rarely been associated with either acute or chronic types of liverinjury [145]. Nitrofurantoin also causes pulmonary reactions. However, when nitrofuran-toin is used for continuous long-term therapy, chronic pulmonary injury (which is rare)occurs via lipid peroxidation which could be consistent with generation of superoxide fromnitro reduction [146]. [(Fig._1)TD$FIG]

[(Scheme_3)TD$FIG]

Scheme 1.13 Reduction pathways involved in vivo for the activation of nitroaromatics

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Metronidazole (61), tinidazole (62), nimorazole (63) and ornidazole (64) have been usedfor the treatment of anaerobic and protozoal infections (e.g. amoebiasis). As metroindazolehas been shown to be carcinogenic in mice and rats, and is mutagenic in bacteria, there has

[(Scheme_4)TD$FIG]

Scheme 1.14 Bioactivation of secondary nitroalkanes


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been concern regarding possible long-term effects in humans. It also produces tumours inmice and rats [147, 148]. These agents are most often limited to urinary infections since theyhave poor systemic exposure. The limited exposure and rapid urinary clearance may wellserve to limit the risk of the nitro group. [(Fig._1)TD$FIG]

The electron-withdrawing properties of the nitro group on the aromatic nucleus can bemimicked with various other EWGs such as SO2, COR, CN and nitrogen within the ringsuch as pyridines. However, it may be difficult to replace the electronic properties of thenitro group itself. It is worth noting that the nitro group is a very poor hydrogen bondacceptor in contrast to the excellent H-bonding capacity of the deceptively similar carbox-ylic acid group.

Anilines

Aromatic amines can cause methaemoglobinemia, agranulocytosis, aplastic anaemia,hepatotoxicity, skin hypersensitivity and increased risk of mutagenicity.

There are two principal mechanisms of aniline toxicity. The first is oxidation of thearomatic ring ortho or para to the aniline nitrogen as in Scheme 1.15. This leads to ortho-and para-hydroxy anilines, respectively. These species are themselves precursors to highlyelectrophilic ortho- and para-iminoquinones.

The second pathway is oxidation of the aniline nitrogen to hydroxylamine, nitroso,nitro and related species as in Scheme 1.16. The nitroso species is a reactive metab-olite in its own right; the hydroxylamine species undergo acetylation or sulphation todeliver a good leaving group which leads to reactive metabolites. Redox cyclingbetween species (e.g. nitroso and nitro) leads to reactive oxygen species. For the firststep of this oxidation to the hydroxylamine to occur there must be at least one protonon the anilinic nitrogen. This oxidation has been observed for N-acetyl aniline species[149–152].

For the full oxidation to the nitroso to occur there must be two protons on the anilinicnitrogen. The greater the number of metabolic steps required to cleave the aniline nitrogen

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substituents yielding the NH and NH2 aniline, or the difficulty of these metabolic steps, thelower is the likelihood of N-oxidation.

Sabbioni et al. studied the haemoglobin binding, mutagenicity and carcinogenicity of 36substituted anilines with differing electronic properties [150]. This paper concludes that theamount of haemoglobin binding decreases with the oxidizability of the aniline amino groupwhereas the mutagenicity and carcinogenicity increases with oxidizability of the anilineamino group.

Formation of N-hydroxy species from diphenyl anilines has also been observed in humanhepatocytes [151, 152]. Indeed one hypothesis for the drug-induced toxicity of diclofenac(65) (see examples below) is via formation of the N-hydroxy diphenylaniline species(Scheme 1.17). AU1

[(Scheme_5)TD$FIG]

Scheme 1.15 Aryl oxidation of anilines

[(Scheme_6)TD$FIG]

Scheme 1.16 N-oxidation of anilines


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[154, 155]

Carbutamide (66) caused bone marrow toxicity in humans whereas tolbutamide (67),which is the direct analogue lacking the aniline functionality, is devoid of such toxicity[153]. Practolol (68) caused severe skin and eye lesions in some patients which led to itswithdrawal from the market. Evidence points to hydrolysis of the amide and oxidation of theaniline [154–156]. Atenolol (69), the direct analogue which lacks the anilinic nitrogen, has acleaner profile.[(Fig._1)TD$FIG]

There are many examples of successful drugs developed which contain the anilinefragment, so for this fragment particularly, a case by case assessment is required.Successful drugs often use aminoheterocycles and groups which block the phenyl ring toortho or para oxidation. When in doubt, it may be possible to try removing the anilinicnitrogen altogether as in Scheme 1.18.

[(Scheme_7)TD$FIG]

Scheme 1.17 Oxidation of diphenylamines

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Additionally there are forms of the aniline substructure which are not capable ofbeing metabolized to the reactive intermediate. These blocked anilines are shown inScheme 1.19.

Thioureas

Hepatotoxicity, pulmonary toxicity and cytotoxicity are common toxicities associatedwith metabolic activation of thioureas. Cytotoxicity has been observed with a series ofmono- and disubstituted thiourea-containing compounds in freshly isolated rat hepa-tocytes [160, 161]. Thioureas inhibit thyroid peroxidase, the enzyme that catalyses thesynthesis of T4 and T3 in the thyroid gland [162]. Tumours of the thyroid and liver

[(Scheme_8)TD$FIG]

Scheme 1.18 Aniline alternatives

[(Scheme_9)TD$FIG]

Scheme 1.19 Anilines blocked to oxidative metabolism


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have been reported in rodents for a number of thioureas. Desulphurization of thethioureas in vivo occurs by oxidation followed by nucleophilic substitution or elimi-nation reactions [165–167] (Scheme 1.20). Reaction of the sulphonic acids with wateror hydroxide via nucleophilic substitution at carbon leads to the corresponding ureavia loss of sulphate (Pathway A). Alternatively elimination pathways B and C lead tothe cyanamide or carbodiimide products in compounds with any free NH groups onthe thiourea nitrogens. Observation of the urea, nitrile or carbodiimides as metabolitesis indicative of formation of the reactive intermediates. A study of the metabolism ofthioureas led to the conclusion that the more readily desulphurized compounds are theones showing more toxicity. The oxidation of the sulphur atom may be catalysed byflavin-containing monooxygenases (FMO) and/or CYP isoenzymes to lead to sulphinicand sulphonic acids [168].

Thioperamide (70) is a histamine H3 antagonist for the treatment of psychiatric disorderand cognitive disorder. It was discontinued due to liver toxicity [160]. Afeletecan (BAY-38-3441) (71) is a topoisomerase I inhibitor, for the treatment of cancer [169]. Toxicity resultsof several phase I trials showed that at doses ranging between 295 and 470 mg/m2, adverseevents included skin, gastrointestinal and haematological toxicity. Development of thiscompound was discontinued. [(Fig._1)TD$FIG]

[(Scheme_0)TD$FIG]

Scheme 1.20 Metabolism of thioureas

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[163, 164]

Bioisosteric replacement of thioureas has been studied with ureas, sulphamides, cyano-guanidines and guanidines with varied success [170] (i.e. biological activity is often lost).One report by Petersen et al. [171] shows the replacement of thioureas with cyanoguani-dines to give a dramatic increase in biological activity. Although 2-aminothiazoles areconsidered thiourea isosteres they are not suggested as a safe alternative to thioureas sincethey display their own inherent toxicity.

Thiazoles and aminothiazoles

The predominant fate of the thiazole ring is its oxidative ring scission catalysed by P450enzyme formation of the corresponding a-dicarbonyl metabolites and thioamide derivatives[172, 173] (Scheme 1.21). The well-established toxicity associated with thioamidesand thioureas has led to the speculation that thiazole toxicity is attributed to ring

[(Scheme_1)TD$FIG]

Scheme 1.21 Metabolism of thiazoles


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scission yielding the corresponding thioamide metabolite [174]. Ring opening hasalso been observed in benzothiazoles. For instance, benzothiazole itself is convertedto S-methylmercaptoaniline.

Sudoxicam (72) is the first NSAID reported to have anti-inflammatory activity in animals[175]. Its development was stopped because of adverse effects reported in clinical trials.SM-8849 (73) (Sumitomo Pharmaceuticals Co. Ltd.) is a bone resorption inhibitor andimmunosuppressant that was discontinued after phase II clinical trials for the treatment ofrheumatoid arthritis [176]. [(Fig._1)TD$FIG]

To avoid this metabolism, medicinal chemists could try replacing the ring structure withan isoxazole or isothiazole ring which are more likely to undergo reductive metabolism thanoxidation. It has been noted that imidazole, oxazole and thiazole rings are substrates forCYP oxidation. In contrast, the pyrazole ring seems to be the most metabolically stable ofthe five-membered heterocycles.

Hydrazines

Hydrazines and hydrazides are known human carcinogens [177]. Hydrazines also causehepatotoxicity, neurotoxicity, lupus-like syndrome and non-SLE hypersensitivity.Monosubstituted alkyl hydrazine drugs such as the antidepressant phenelzine (74) appearto be oxidized to reactive intermediates, such as diazonium ions, yielding radicals that cancause haemolysis [178]. Reactions of alkyl disubstituted hydrazines such as the antineo-plastic agent procarbazine (Mutulane) (75) can generate azoxy compounds from diazines,which then undergo oxidative dealkylation and elimination resulting in diazonium alkylat-ing agents. Hydrazides can be hydrolysed to hydrazines (Scheme 1.22) or they may befurther oxidized to acylonium ions as shown in Scheme 1.23.

WhenR1 is an alkyl or aromatic group andR2 is hydrogen, the reaction appears to proceedthrough radical intermediates. When both R1 and R2 are alkyl or aralkyl groups, the reactionappears to proceed through the formation of the azoxy compounds. When R1 is an acylgroup and R2 is a hydrogen or an alkyl group, hydrolysis of the acyl group may occur eitherbefore or after oxidation to a diazine, which may then undergo direct nucleophilic substi-tution or formation of radicals.

The association of example drugs in this class such as hydralazine (Apresoline) (76) andisoniazid (Nydrazid) (77) with lupus-like syndrome may be related to oxidation by macro-phages and the myeloperoxidase system of neutrophils. A combined genomics, proteomicand metabonomic study of hydrazine-treated rats identified changes in glucose metabolism,lipid metabolism and oxidative stress, providing potential biomarkers of hydrazine-induced

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toxicity [179–181]. Procarbazine was found to covalently bind to proteins when fortifiedwith microsomes and NADPH, quite possibly via free radical intermediates [182].Procarbazine has been found to be mutagenic, carcinogenic and teratogenic in several assaysystems in vitro and in vivo. Procarbazine is oxidatively metabolized both in vitro and in vivoto its azo and azoxy derivatives, as is the potent model carcinogen 1,2-dimethylhydrazine.The azometabolite is capable of generating radical intermediates, and the azoxy isomers canpotentially be further oxidized to diazonium species [159]. AU2The hepatic necrosis elicitedduring therapeutic administration of isoniazid and iproniazid (78) has been attributed tocovalent binding to proteins of acetyl and isopropyl radicals formed from hydrazines thatwere liberated metabolically [183]. Isoniazid has been found to carry out a specific proteindegradation in vitro via generation of oxygen radicals [184]. [(Fig._1)TD$FIG]

[(Fig._1)TD$FIG]

[(Scheme_2)TD$FIG]

Scheme 1.22

[(Scheme_3)TD$FIG]

Scheme 1.23 Oxidation of hydrazines


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Some compounds may contain an embedded hydrazine that does not formally fit thedefinition of the alert, however, metabolic processing in vivo may release a hydrazinecovered in the alert. Due diligence should be taken to understand the metabolic processingof the embedded hydrazine-like functionality and data should be provided to demonstratethat the risk due to metabolic processing is minimal [185].

Hydroxamic acids

Hydroxamic acids have been shown to be mutagenic via acylation or sulphation to theO-esters by endogenous enzymes, followed by Lossen rearrangement giving isocyanateswhich could serve as electrophiles for carbamoylation of DNA and other nucleophilicproteins. Several studies support the suggestion that activation processes of this class ofmutagens involve the Lossen rearrangement of O-acylated hydroxamic acids (Scheme 1.24)[186, 187].

Studies show that the relative mutagenicity of such a compound closely follows theability of the R group on the hydroxamic acid to migrate [188]. N-methylation of thehydroxamoyl group decreases mutagenicity, probably because in this case the Lossenrearrangement is not possible. Electron-withdrawing substituents on the aromatic groupmake the Lossen rearrangement of hydroxamic acids slower.

The metabolite analysis of Pfizer MMP-13 inhibitor CP-544439 (79) shows that alkylhydroxamic acids can also be converted into the reactive toxic isocyanate intermediatethrough a Lossen rearrangement [189]. SomeMMP inhibitors containing alkyl hydroxamic

[(Scheme_4)TD$FIG]

Scheme 1.24 Metabolism of hydroxamic acids to isocyanates

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acids have shown clastogenicity. Workers from Pfizer also showed that HIV integraseinhibitors containing hydroxamic acids such as compound 2c (80) could be mutagenic inthe AMES assay [190].[(Fig._1)TD$FIG]

Most of the substrates found in the literature are aryl hydroxamic acids [191]although the Lossen rearrangement from benzylic hydroxamic acids has also beendemonstrated [192]. Bioisosteres which are sometimes used to replace the hydroxamicacid group are carboxylic acids, acylsulphonamides, phosphinic acids and phosphonicacids.

Aminotriazoles

The parent structure 3-amino-1,2,4-triazole, widely used as a herbicide, is teratogenic,goitrogenic and carcinogenic requiring no metabolic activation. The parent is excretedlargely intact. 3,5-Diamino-1,2,4-triazole and 3-amino-5-mercapto-1,2,4-triazole alsoshow the same toxicity. Aminotriazole induces thyroid tumours in mice and rats by anon-genotoxic mechanism, which involves inhibition of thyroid peroxidase via a suicidemechanism [193], resulting in a reduction of circulating thyroid hormone and increasedsecretion of TSH. There is inadequate evidence in humans for the carcinogenicity ofaminotriazole. Any compound which may be metabolized to produce 3-amino-1,2,4-triazole, 3,5-diamino-1,2,4-triazole or 3-amino-5-mercapto-1,2,4-triazole may havepotential teratogenic, goitrogenic and carcinogenic effects. No aminotriazoles are cur-rently on the market as pharmaceuticals.

Sufotidine (AH25352) (81) is a histamine H2 receptor antagonist and has been discon-tinued by GSK from phase III clinical trials as an antiulcerant [194] based on the appearanceof carcinoid tumours in long-term toxicity testing in rodents. GSK also discontinueddevelopment of loxtidine (82), a histamine H2 receptor antagonist with antiulcer activity[195–197], because of treatment-related differentiated adenocarcinomas in animal studies.In rats, oral administration of loxtidine for 2 years and 3 months produced diffuse, differ-entiated adenocarcinomas probably caused by achlorhydria. This effect was treatmentrelated, not dose related.

Any modification that results in compounds that cannot be metabolized to generate3-amino-1,2,4-triazole, 3,5-diamino-1,2,4-triazole or 3-amino-5-mercapto-1,2,4-triazole asbreakdown products can be envisaged as potential isosteres; examples include C-linkedtriazoles or the use of other five-membered heterocycles [198].[(Fig._1)TD$FIG]


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PHOSPHOLIPIDOSIS

Phospholipidosis (PL) is a phospholipid storage disorder, resulting in excessive accumula-tion of phospholipids in lysosomes of various tissue types [199, 200]. There is a generalagreement that inhibition of lysosomal phospholipase A1, A2 and/or C contributes to theaccumulation of cationic amphiphilic drug (CADs)–phospholipid complexes. The prevail-ing scientific opinion is that PL by itself is not adverse; however, some regulatory authoritiesconsider PL to be adverse because a small number of chemicals are able to cause PL andconcurrent organ toxicity. Drugs that cause PL make up over 5% of currently approveddrugs. However, of all drugs reported to cause PL, 70% also cause QT prolongation [201,202]. Thus drugs which induce PL often are scrutinized more closely by the regulatorybodies. Until a greater understanding of PL emerges, a well-thought-out risk managementstrategy for PL will increase confidence in safety and improve selection and development ofnew drugs.

The SSRI antidepressant fluoxetine (83) and the antimalarial chloroquine (84) are asso-ciated with PL and also QT prolongation. The antiestrogen tamoxifen (85) is also associatedwith the induction of PL. All of these drugs contain the typical cationic amphiphilicpharmacophore typically associated with PL.[(Fig._1)TD$FIG]

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Most often, PL is detected histologically via electron micrography during the postmortem pathology analysis of pre-clinical exploratory in vivo toxicology studies. Thesestudies are expensive and are not suitable as a first line assay for PL SAR. Once PL isdetected as a programme toxicity issue, it is possible to run in vitro assays for PL [203].Recently workers at Nextcea [204] have proposed a simpler biomarker 22:6-BMP which isassociated with phospholipid metabolism which could be used as a more convenientprimary assay in vitro. Ploemen et al.[205] have created an in silico model which predictsPL-inducing potential using two simple physicochemical properties, pKa and ClogP(Figure 1.5).

Pelletier et al.[206] have refined these calculations to increase their concordance from 75to 80%with a 201 compound data set (Figure 1.6). Once an issue of PL has been highlightedin a drug discovery programme these rules can be used to rapidly assess and prioritizesimilar molecules for exploratory in vivo toxicology studies.

PHOTOTOXICITY

Phototoxicity covers several toxic conditions mediated by drugs and UV visible light. Thisarea has been well reviewed byMoore and Quintaro [207, 208]. Phototoxicity testing is nowa requirement of all regulatory guidance [209, 210]. Further photosafety evaluation isrecommended for molecules which absorb light energy in the UV visible region (290–700 nm) and may be unstable. Workers from Pfizer [211] showed that many phototoxiccompounds were also photo-unstable but established no direct link. They also noted thatmolecules with a molar extinction coefficient (MEC) of more than 1000 l/mol�cm in the UVvisible range were more likely to be toxic. Phototesting in vitro is now commonplace usingthe 3T3 neutral red phototoxicity test [212]. The 3T3 neutral red phototoxicity test is arelatively new assay that was recently adopted by regulatory agencies such as OECD andFDA as an available method for the assessment of phototoxic potential of developmentcompounds.

The HOMO–LUMO gap in molecules is correlated with phenoxy radical toxicity towardL1210 leukemia cells [213, 214] and DNA single-strand photocleavage by methylbenz[a]anthracenes [215]. It is sometimes difficult to apply this parameter, as the differences inHOMO–LUMO gap values between chemical congeners can be subtle.

Amiodarone (36), a class III antiarrhythmic agent, has been linked to several cases ofbasal cell carcinoma in patients receiving long-term treatment [216, 217]. Norfloxacin (86),a member of the new class of fluoroquinolone antibiotics [218], displays toxicity which isthought to arise from radical formation by photodefluorination. Demeclocycline [219] (87)and other members of the tetracycline antibiotics were cited in the 1950s as causing rapid

[(Fig._5)TD$FIG]

Fig. 1.5 Ploemen’s in silico model for predicting phospholipidosis.


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onset localized burning and itching, like exaggerated sunburn. Furosemide [220] (88) is acommonly used diuretic in the treatment of hypertension. A high dosage of furosemidecauses phototoxic blisters. This may be due to the covalent binding of furosemide and itsglucuronide to HSA and/or other endogenous substances. Again a photodechlorination isthought to occur producing an intermediate alkyl radical which goes on to alkylate otherbiomolecules. 8-Methoxypsoralen [221] (89) is a natural furanocoumarin present in manyfoodstuffs such as parsnips and parsley and is used in combination with long wavelengthultraviolet light to treat psoriasis, vitiligo and Tcell lymphoma [222]. The usefulness of (89)in treating these diseases resides in its ability to be photoactivated to a species capable ofbinding covalently to nucleic acids and lymphocytes by which DNA synthesis and cellularproliferation are inhibited [223, 224]. [(Fig._1)TD$FIG]

In general, phototoxicity in molecules seems to be linked to aryl-halogen bonds whichcleave to form radicals or to unusual conjugated systems where the excited triplet state cantake part in degradation or alkylation reactions.

[(Fig._6)TD$FIG]

Fig. 1.6 Pelletier’s modified in silico model for predicting phospholipidosis.

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IDIOSYNCRATIC TOXICITY

Idiosyncratic toxicity is also referred to as idiosyncratic adverse drug reaction (IADR), type-B reactions, hypersensitivity reactions and allergic reactions [225, 226]. They are oftenserious or fatal immune-mediated responses. Idiosyncratic toxicity occurs unpredictably in asmall percentage of the treated population (<1/5000 patients) who probably display poor orunusual metabolizing genotypes and/or have rare/incompatible immune system genotypes.Either of these mechanisms probably gives rise to an immune response to a drug metaboliteprotein adduct which can either generate antibodies to drug or self (autoimmunity). Inseveral cases they involve antibodies being generated to the CYPwhich produces the reactivemetabolite [227]. They are not yet well predicted by animal toxicology studies [228]. Thetoxic event usually occurs after more than one week and can often take several months tomanifest. Because of the infrequent nature of these reactions they are often undetected untilphase III clinical trials and beyond, by which time there are sufficient patients to see an effectstatistically. At this time the patient population includes diverse phenotypes (e.g. 2D6�/�)and patients on co-medications which can exacerbate the effect of the drug [229]. Around10% of new drugs post-1975 had either to be withdrawn or given a black box warning due toidiosyncratic toxicity. Most drugs that cause idiosyncratic toxicity form electrophilic reactivemetabolites which are themselves responsible for the observed toxicity [230].

It is important to note that there is a significantly greater risk of idiosyncratic drug-inducedliver injury with oral medications whose dose is greater than 50 mg/day [231]. This impliesthat the body’s natural defence and clearance mechanisms are able to deal with many of thecauses of idiosyncratic toxicity so long as they are not overwhelmed by high drug exposure.There are many cases of very similar structures where the lower dose compound avoids,while the higher dose compound triggers, idiosyncratic toxicity. The analysis of idiosyncratictoxicity by Li [232] shows the common properties of causative agents to be:

* Formation of reactive metabolites;* Metabolism by high risk CYP isoforms (2D6, 2C19);* Induction of CYP enzymes;* Significant drug–drug interactions with co-administered drugs.

The avoidance of these features in new drug candidates by careful consideration of thestructure–toxicity relationships associated with these properties should reduce the risk ofidiosyncratic toxicity.

CONCLUSIONS

A quality development candidate compound after a positive proof of concept clinical trialhas a good chance of making it to be a marketed drug. It is our role as medicinal chemists tochoose the right compounds to design and synthesize. Although toxicity is now the jointprimary cause of failure of clinical candidates, there is a growing body of information aboutstructure–toxicity relationships even as those relate to complex bioactivation pathways[233–235]. With this ever growing knowledge, it should be possible for talented medicinalchemists to design inherently safer molecules. Furthermore, the introduction of a broader


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array of in vitro toxicology studies earlier in the drug discovery process should result in highquality development candidates entering the clinic and a richer database from which futurestructure–toxicity relationships may be discovered.

As part of medicinal chemistry design, we predict the properties of the molecules wemake and take decisions based on those predictions. From measured physical and in vitroproperties we can also predict pharmacokinetic and pharmacodynamic data to estimate thedose of development compounds.Medicinal chemistry design therefore determines the dosesize and influences the non-mechanism based TI. Good medicinal chemistry design, takinginto account both potency at the target and also the predictive toxicology discussed in thisreview, increases the probability of a project’s success and heavily influences speed ofreaching a clear proof of concept outcome.

UNCITED REFERENCES

[30, 106, 107, 157, 158, 163, 164].

REFERENCES

[1] Kola, I. and Landis, J. (2004) Nat. Rev. Drug Discov. 3, 711–715.[2] Kramer, J.A., Sagartz, J.E. and Morris, D.L. (2007) Nature (London) 6, 636–649.[3] Park, B.K., Kitteringham, N.R., Maggs, J.L., Pirmohamed, M. and Williams, D.P. (2005) Annu. Rev.

Pharmacol. Toxicol. 45, 177–202.[4] Napier, C. and Wallis, R. (2009) Safety Pharmacology Society, 9th Annual Meeting, Poster 35.[5] Bell, L.N. and Chalasani, N. (2009) Semin. Liver Dis. 29(4); 337–347.[6] Riley, R.J., Grime, K. and Richard Weaver, R. (2007) Exp. Opin. Drug Metab. Toxicol. 3(1); 51–66.[7] Ortiz de Montellano, P.R. and Kunze, K.L. (1980) J. Biol. Chem. 255, 5578–5585.[8] Ortiz de Montellano, P.R. and Kunze, K.L. (1981) Arch. Biochem. Biophys. 209, 710–712.[9] Komives, E.R. and Ortiz de Montellano, P.R. (1987) J. Biol. Chem. 262, 9793–9802.

[10] Roberts, E.S., Pernecky, S.J., Alworth, W.L. and Hollenberg, P.F. (1996) Arch. Biochem. Biophys. 331,170–176.

[11] Guengerich, P.F. (1990) Chem. Res. Toxicol. 3, 363–371.[12] Lin, H.L., Kent, U.M. and Hollenberg, P.F. (2002) J. Pharmacol. Exp. Ther. 301, 160–167.[13] Belle, D.J., Callaghan, J.T., Gorski, J.C., Maya, J.F., Mousa, O., Wrighton, S.A. and Hall, S.D. (2002) J.

Clin. Pharmacol. 53, 67–74.[14] Bray, H.G., Carpanini, M.B. and Waters, B.D. (1971) Xenobiotica 1, 157–168.[15] Dansette, P.M., Thang, D.C., El Amri, H. and Mansuy, D. (1992) Biochem. Biophys. Res. Commun. 186,

1624–1630.[16] Munsuy, D., Valadon, P., Erdelmeier, I., Lopez-Garcia, P., Amar, C., Girault, J.-P. andDansette, P.M. (1991)

J. Am. Chem. Soc. 113, 7825–7826.[17] Liu, Z.C. and Eutrecht, J.P. (2000) Drug Metab. Dispos. 28, 726–730.[18] Mosier, P.D., Jurs, P.C., Custer, L.I., Durham, S.K. and Pearl, G. (2003) Chem. Res. Toxicol. 16, 721–732.[19] Valadon, P., Dansette, P.M., Girault, J.-P., Amar, C. and Mansuy, D. (1996) Chem. Res. Toxicol. 9,

1403–1413.[20] Dreiem, A. and Fonnum, F. (2004) Neurotoxicology 25, 959–966.[21] Fontana, E., Dansette, P.M. and Poli, S.M. (2005) Curr. Drug Metab. 6(5); 413–454.[22] Treiber, A., Dansette, P.M., El Amri, H., Girault, J.-P., Ginderow, D., Mornon, J.-P. and Mansuy, D. (1997)

J. Am. Chem. Soc. 119, 1565–1571.

GRAHAM F SMITH 43

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12345678910111213141516171819202122232425262728293031323334353637383940414243444546

[23] Mansuy, D. (1997) J. Hepatol. 26(S2); 22.[24] Dansette, P.M., Bonierbale, E.,Minoletti, C., Beaune, P.H., Pessayre, D. andMansut, D. (1998)Eur. J. Drug

Metab. Pharmacokinet. 23, 443–451.[25] Oker-Blom, C., Makinen, J. and Gothoni, G. (1980) Toxicol. Lett. 6, 93–99.[26] Luke, D.R. and Berens, K.L. (1992) Renal Failure 14, 187–192.[27] Abraham, P.A., Halstenson, C.E., Opsahl, J.A., Matzke, G.R. and Keane, W.F. (1988) Am. J. Nephrol. 8,

90–95.[28] Dalvie, K.D. and O’Connell, T.N. (2004) Drug Metab. Dispos. 32(1); 49–57.[29] Medower, C., Wen, L. and Johnson, W.W. (2008) Chem. Res. Toxicol. 21, 1570–1577.[30] Beasley, C.M., Tollefson, G.D. and Tran, P.V. (1997) J. Clin. Psychiatry AU358(S10); 13–17.[31] Gardner, I. (1998) Mol. Pharmacol. 53, 991.[32] Gardner, I. (1998) Mol. Pharmacol. 53, 999.[33] Herbert, J.M. (1994) Exp. Opin. Investig. Drugs 3, 449.[34] Kaufman, J.S., Foire, L., Hasbargen, J.A., O’Connor, T.Z. and Perdriset, G. (2000) J. Thromb.

Thrombolysis 10, 127–131.[35] Richter, T.,Murdter, T.E., Heinkele, G., Pleiss, J., Tatzel, S., Schwab,M., Eichelbaum,M. and Zanger, U.M.

(2004) J. Pharm. Exp. Ther. 308, 189–197.[36] Byrns, M.C., Predecki, D.P. and Peterson, L.A. (2002) Chem. Res. Toxicol. 15, 373–379.[37] Ravindranath, V., Burka, L.T. and Boyd, M.R. (1984) Science (Washington DC) 224, 884–886.[38] Molon-Noblot, S., Gillet, J.-P., Durand-Cavagna, G., Huber, A.C., Patrick, D.H. and Duprat, P. (1996)

Toxicol. Pathol. 24, 231–237.[39] Chauret, N., Nicoll-Griffith, D., Friesen, R., Li, C., Trimble, L., Dube, D., Fortin, R., Girard, Y. and Yergey,

J. (1995) Drug Metab. Dispos. 23, 1325–1334.[40] Zhang, K.E., Naue, J.A., Arison, B. and Byas, K.P. (1996) Chem. Res. Toxicol. 9, 547–554.[41] Dorsey, B.D., McDonough, C., McDaniel, S.L., Levin, R.B., Newton, C.L., Hoffman, J.M., Darke, P.L.,

Zugay-Murphy, J.A., Emini, E.A., Schleif,W.A., Olsen, D.B., Stahlhut,M.W., Rutkowski, C.A., Kuo, L.C.,Lin, J.H., Chen, I.-W., Michelson, S.R., Holloway, M.K., Huff, J.R. and Vacca, J.P. (2000) J. Med. Chem.43, 3386–3399.

[42] Sahali-Sahly, Y., Balani, S.K., Lin, J.H. and Baille, T.A. (1996) Chem. Res. Toxicol. 9, 1007–1012.[43] Bateman, K.P., Baker, J., Wilke, M., Lee, J., LeRiche, T., Seto, C., Day, S., Chauret, N., Ouellet, M. and

Nicoll-Griffith, D.A. (2004) Chem. Res. Toxicol.1356–1361[44] Alvarez-Diez, T.M. and Zheng, J. (2004) J. Chem. Res. Toxicol. 17, 150–157.[45] Wolf, C.R., Statham, C.N., McMenamin, M.G., Bend, J.R., Boyd, M.R. and Philpot, R.M. (1982) Mol.

Pharmacol. 22, 738–744.[46] Baer, B.R., Rettie, A.E. and Henne, K.R. (2005) Chem. Res. Toxicol.855–864[47] Garcıa Rodrıguez, L.A., Wallander, M.-A. and Stricker, B.H.C. (1997) Br. J. Clin. Pharmacol. 43,

183–188.[48] Connelly, J.C., Connor, S.C., Monte, S., Bailey, N.J.C., Borgeaud, N., Holmes, E., Troke, J., Nicholson, J.

K. and Gavaghan, C.L. (2002) Drug Metab. Dispos. 30(12); 1357–1363.[49] Kumagai, Y., Fukuto, J.M. and Cho, A.K. (1994) Curr. Med. Chem. 3(1); 254–261.[50] Murray, M. (2000) Curr. Drug Metab. 1, 67–84.[51] Anders, M.W., Sunram, J.M. and Wilkinson, C.F. (1984) Biochem. Pharmacol. 33(4); 577–580.[52] Kaye, C.M., Haddock, R.E., Langley, P.F., Mellows, G., Tasker, T.C.G., Zussman, B.D. and Greb, W.H.

(1989) Acta Phsychiatr. Scand. 80(S350); 60–75.[53] Begre, S., Von Bardeleben, U., Ladewig, D., Jaquet-Rochat, S., Cosendai-Savary, L., Golay, K.P., Kosel,

M., Baumann, P. and Eap, C.B. (2002) J. Clin. Psychopharmacol. 22(2); 211–215.[54] Ruwe, F.J.L., Smulders, R.A., Kleijn, H.J., Hartmans, H.L.A. and Sitsen, J.M.A. (2001) Hum.

Psychopharmacol. 16(6); 449–459.[55] Ozdemir, V., Naranjo, C.A., Herrmann, N., Reed, K., Sellers, E.M. and Kalow,W. (1997)Clin. Pharmacol.

Ther. 62(3); 334–347.[56] Bloomer, J.C.,Woods, F.R., Haddock, R.E., Lennard,M.S. and Tucker, G.T. (1992)Br. J. Clin. Pharmacol.

33(5); 521–523.


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[57] Bertelsen, K.M., Venkatakrishnan, K., von Moltke, L.L., Obach, R.S. and Greenblatt, D.J. (2003) DrugMetab. Dispos. 31, 289.

[58] Alfaro, C.L., Lam, Y.W.F., Simpson, J. and Ereshefsky, L. (1999) J. Clin. Psychopharmacol. 19, 155.[59] Preskorn, S.H. (1997) Clin. Pharmacokinet. 32(1); 1–21.[60] Walgren, J.L., Mitchell, M.D. and Thompson, D.C. (2005) Crit. Rev. Toxicol. 35, 325–361.[61] Gasbarrini, G., Gentiloni, N., Febbraro, S., Gasbarrini, A., Di Campli, C., Cesana, M., Miglio, F., Miglioli,

M., Ghinelli, F., D’Ambrosi, A., Amoroso, P., Pacini, F. and Salvadori, G. (1997) J. Hepatol. 27(3);583–586.

[62] Testa, B. and Jenner, P. (1981) Drug Metab. Rev. 12(1); 1–117.[63] Murray, M., Sefton, R.M., Martini, R. and Butler, A.M. (1998) Chem.-Biol. Interact. 113, 161–173.[64] Harvey, J., Paine, A., Maurel, P. and Wright, M. (2000) Drug Metab. Dispos. 28, 96–101.[65] Kumar, G.N., Rodrigues, D., Buko, A.M. and Denissen, J.F. (1996) J. Pharmacol. Exp. Ther. 277, 423.[66] Eagling, V.A., Back, D.J. and Barry, M.G. (1997) Br. J. Clin. Pharmacol. 44, 190.[67] Zhang, W., Ramamoorthy, Y., Kilicarslan, T., Nolte, H., Tyndale, R.F. and Sellers, E.M. (2002) Drug

Metab. Dispos. 30(3); 314–318.[68] Tompkins, L.M. and Wallace, A.D. (2007) J. Biochem. Mol. Toxicol. 21(4); 176–181.[69] Urquhart, B.L., Tirona, R.G. and Kim, R.B. (2007) J. Clin. Pharmacol. 47, 566–657.[70] Wrighton, S.A., Van den Branden, M. and Ring, B.J. (1996) J Pharmacokinet. Biopharm. 199(24);

461–473.[71] Lin, J.H. (2006) Pharm. Res. 23(6); 1089–1116.[72] Drocourt, L., Pascussi, J.M., Assenat, E., Fabre, J.M., Maurel, P. and Vilarem, M.J. (2001) Drug Metab.

Dispos. 29, 1325–1331.[73] Evans, R.M. (2005) Mol. Endocrinol. 19, 1429–1438.[74] Waxman, D.J. (1999) Arch. Biochem. Biophys. 369(1); 11–23.[75] Pelkonen, O., Turpeinen, M., Hakkola, J., Honkakoski, P., Hukkanen, J. and Raunio, H. (2008) Arch.

Toxicol. 82, 667–715.[76] Sanguinetti, M.C. and Tristani-Firouzi, M. (2006) Nature (London) 44(23); 463–469.[77] Jamieson, C., Moir, E.M., Rankovic, Z. and Wishart, G. (2006) J. Med. Chem. 49(17); 5029–5046.[78] Pearlstein, R., Vaz, R. and Rampe, D. (2003) J. Med. Chem. 6(11); 2017–2022.[79] Stansfeld, P.J., Sutcliffe, M.J. and Mitcheson, J.S. (2006) Exp. Opin. Drug Metab. Toxicol. 2(1); 81–94.[80] Cavalli, A., Poluzzi, E., De Ponti, F. and Recanatini, M. (2002) J. Med. Chem. 45, 3844–3853.[81] Mitcheson, J.S. (2008) Chem. Res. Toxicol. 21(5); 1005–1010.[82] Perry, M., de Groot, M.J., Helliwell, R., Leishman, D., Tristani- Firouzi, M., Sanguinetti, M.C. and

Mitcheson, J. (2004) Mol. Pharmacol. 66, 240–249.[83] Doyle, D.A.,Morais, C.J., Pfuetzner, R.A., Kuo, A., Gulbis, J.M., Cohen, S.L., Chait, B.T. andMacKinnon,

R. (1998) Science (Washington DC) 280, 69.[84] Price, D.A., Armour, D., de Groot, M., Leishman, D., Napier, C., Perros, M., Stammen, B.L. andWood, A.

(2006) Bioorg. Med. Chem. Lett. 16(17); 4633–4637.[85] Sheridan, R.P., Hunt, P. and Culberson, J.C. (2006) J. Chem. Inf. Model. 46, 180–192.[86] Bell, I.M. and Bilodeau, M.T. (2008) Curr. Top. Med. Chem. 8, 1128–1139.[87] Kirkland, D., Aardema, M., Henderson, L. and Muller, L. (2005) Mutat. Res. 584, 1–256.[88] Ames, B.N. (1984) Cancer 53, 2030–2040.[89] Maron, D.M. and Ames, B.N. (1983) Mutat. Res. 113, 173–215.[90] Zeiger, E. (1987) Cancer Res. 47, 1287–1296.[91] Benigni, R. and Bossa, C. (2008) Mutat. Res. 659, 248–261.[92] DePierre, J.W. (2003) Handbook of Environmental Chemistry AU4Vol. 3(Pt. R); pp. 205–251.[93] Prestera, T., Holtzclaw, W.D., Zhang, Y. and Talalay, P. (1993) Proc. Natl. Acad. Sci. U.S.A. 90(7);

2965–2969.[94] Talalay, P., Fahey, J.W., Holtzclaw, W.D., Prestera, T. and Zhang, Y. (1995) Toxicol. Lett. 82–83, 173–179.[95] Hill, R.N., Erdreich, L.S., Paynter, O.E., Roberts, P.A., Rosenthal, S.L. and Wilkinson, C.F. (1989) Fund.

Appl. Toxicol. 12, 629–697.[96] Capen, C.C. (1997) Toxicol. Pathol. 25, 39–48.

GRAHAM F SMITH 45

12345678910111213141516171819202122232425262728293031323334353637383940414243444546

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Replacement Text

DePierre, J.W. (2003) Mamalian Toxicity of Organic Compounds of Bromine and Iodine. Ian T. Cousins and Anna Palm (Eds.), 'Handbook of Environmental Chemistry'. Vol. 3 (Pt. R); . Springer-Verlag Berlin / Heidelberg, pp. 205 -251. DOI: 10.1007/b11992

[97] McClain, R.M. (1995) Mutat. Res. 333, 131–142.[98] Burgi, H., Wimpfheimer, C., Burger, A., Zaunbauer, W., Rosler, H. and Lemarchand-Beraud, T. (1976) J.

Clin. Endocrinol. Metab. 43, 1203–1210.[99] Spaniol, M., Bracher, R., Ha, H.R., Follath, F. and Kr€ahenb€uhl, S. (2001) J. Hepatol. 35(5); 628–636.[100] Wiersinga, W.M. (1997) Amiodarone and the thyroid; pharmacotherapeutics of the thyroid gland.

Weetman, A.P., Grossman, A. (Eds.), ‘Handbook of Experimental Pharmacology’. Vol. 128, Springer-Verlag, Berlin, pp. 225–287.

[101] Harrington, K.J., Syrigos, K.N., Uster, P.S., Zetter, A., Lewanski, C.R., Gullick, W.J., Vile, R.G. andStewart, J.S. (2004) Br. J. Cancer 91(2); 366–373.

[102] Melnick, R.L. (2002) Ann. N.Y. Acad. Sci. 982, 177–189.[103] Fukushima, K., Arai,M., Kohno, Y., Suma, T. and Sotah, T. (1990) Toxicol. Appl. Pharmacol. 105(1); 1–12.[104] Griffith, E.C., Su, Z., Satomi Niwayama, S., Ramsay, C.A., Chang, Y.-H. and Liu, J.O. (1998) Proc. Natl.

Acad. Sci. U.S.A. 95(26); 15183–15188.[105] Sin, N., Meng, L., Wang, M.Q.W., Wen, J.J., Bornmann, W.G. and Crews, C.M. (1997) Proc. Natl. Acad.

Sci. U.S.A. 94(12); 6099–6103.[106] P�erez-Garrido, A., Helguerac, A.M., López, G.C., Nat�alia, M., Cordeiroe, D.S. and Escudero, A.G. (2010)

Toxicology 268, 64–77.[107] Schultz, T.W., Yarbrough, J.W., Hunter, R.S. and Aptula, A.O. (2007) Chem. Res. Toxicol. 20, 1359–1363.[108] Mcintyre, J.A., Castaner, J. and Leeson, P.A. (2005) Drug Future AU530(8); 771–779.[109] Janne, P.A., von Pawel, J., Cohen, R.B., Crino, L., Butts, C.A., Olson, S.S., Eiseman, I.A., Chiappori, A.A.,

Yeap, B.Y., Lenehan, P.F., Dasse, K., Sheeran, M. and Bonomi, P.D. (2007) J. Clin. Oncol. 25, 3936–3944.[110] Campos, S., Hamid, O., Seiden, M.V., Oza, A., Plante, M., Potkul, R.K., Lenehan, P.F., Kaldjian, E.P.,

Varterasian, M.L., Jordan, C., Charbonneau, C. and Hirte, H. (2005) J. Clin. Oncol. 23(24); 5597–5604.[111] Bolton, J.L., Trush, M.A., Penning, T.M., Dryhurst, G. and Monks, T.J. (2000) Chem. Res. Toxicol. 13,

135–160.[112] Monks, T.J. and Jones, D.C. (2002) Curr. Drug Metab. 3, 425–438.[113] Levay, G., Pongracz, K. and Bodell, W.J. (1991) Carcinogenesis 12, 1181–1186.[114] Watkins, P. (2000) Neurology 55(4); S51–S52.[115] Nutt, J.G. (2000) Neurology 55(4); S33–S37.[116] Smith, K.S., Smith, P.L., Heady, T.N., Trugman, J.M., Harman, W.D. and Macdonald, T.L. (2003) Chem.

Res. Toxicol. 16, 123–128.[117] Dybing, E., Nelson, S.D., Mitchell, J.R., Sasame, H.A. and Gillette, J.R. (1976) Mol. Pharmacol. 12,

911–920.[118] Patel, N., Kumagai, Y., Unger, S.E., Fukuto, J.M. and Cho, A.K. (1991) Chem. Res. Toxicol. 4, 421–426.[119] Kassahun, K., Pearson, P.G., Tang, W., McIntosh, I., Leung, K., Elmore, C., Dean, D., Wang, R., Doss, G.

and Baillie, T.A. (2001) Chem. Res. Toxicol. 14, 62–70.[120] Smith, M.T. (2003) Chem. Res. Toxicol. 16, 679–687.[121] Wolfe, A., Shimer, G.H. and Meehan, T. (1987) Biochemistry 26, 6392–6396.[122] Ferguson, L.R. and Denny, W.A. (2007) Mutat. Res. 623, 14–23.[123] Kazius, J., Nijssen, S., Kok, J., Back, T. and Ijzerman, A.P. (2006) J. Chem. Inf. Model. 46(2); 597–605.[124] Regal, K.A., Laws, G.M., Yuan, C., Yost, G.S. and Skiles, G.L. (2001) Chem. Res. Toxicol. 14(8);

1014–1024.[125] Bray, T.M. and Emmerson, K.S. (1994) Annu. Rev. Pharmacol. Toxicol. 34, 91–115.[126] Skordos, K.W., Skiles, G.L., Laycock, J.D., Lanza, D.L. and Yost, G.S. (1998) Chem. Res. Toxicol. 11(7);

741–749.[127] Staub, R.E., Feng, C., Onisko, B., Bailey, G.S., Firestone, G.L. and Bjeldanes, L.F. (2002) Chem. Res.

Toxicol. 15(2); 101–109.[128] Stresser, D.M.,Williams, D.E., Griffin, D.A. and Bailey, G.S. (1995)DrugMetab. Dispos. 23(9); 965–975.[129] Zhang, K.E., Kari, P.H., Davis, M.R., Doss, G., Baillie, T.A. and Vyas, K.P. (2000)DrugMetab. Dispos. 28

(6); 633–642.[130] Kassahun, K., Skordos, K., McIntosh, I., Slaughter, D., Doss, G.A., Baillie, T.A. and Yost, G.S. (2005)

Chem. Res. Toxicol. 18(9); 1427–1437.


12345678910111213141516171819202122232425262728293031323334353637383940414243444546

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I

[131] Porohit, V. and Basu, A. (2000) Chem. Res. Toxicol. 13(8); 673–692.[132] Uetrecht, J. (2002) Drug Metab. Rev. 34(3); 651–665.[133] Scheline, R.R. (1973) Pharmacol. Rev. 25, 451–523.[134] Zbaida, S. (2002) Nitroreductases and azidoreductases AU6. In: Ioannides, C. (Ed.), ‘Enzyme Systems that

Metabolise Drugs and Other Xenobiotics’ Chapter 16. Wiley.[135] IARC International Agency for Research on Cancer. (1982)Monographs on the Evaluation of Carcinogenic

Risk of Chemicals to Man, Some Industrial Chemicals and Dyestuffs, Vol. 29, pp. 331–343. WHO, Lyon.[136] Fiala, E.S., Conway, C.C. and Mathis, J.E. (1989) Cancer Res. 49, 5518–5522.[137] Conway, C.C., Nie, G., Hussain, N.S. and Fiala, E.S. (1991) Cancer Res. 51, 3143–3147.[138] Conway, C.C., Hussain, N.S., Way, B.M. and Fiala, E.S. (1991) Mutat. Res. 261, 197–207.[139] Andrae, U., Homfeldt, H., Vogl, L., Lichtmannegger, J. and Summer, K.H. (1988) Carcinogenesis 9,

811–815.[140] Fiala, E.S., Sodum, R.S., Hussain, N.S., Rivenson, A. and Dolan, L. (1995) Toxicology 99, 89–97.[141] Fiala, E.S., Czerniak, R., Castonguay, A., Conaway, C.C. and Rivenson, A. (1987) Carcinogenesis 8,

1947–1949.[142] Ascheri, M., Eyer, P. and Kampffmeyer, H. (1985) Biochem. Pharmacol. 34(20); 3755–3763.[143] Yunis, A.A. (1988) Ann. Rev. Pharmacol. Toxicol. 28, 83–100.[144] Scholar, E.M. and Pratt, W.B. (2000) The Antimicrobial Drugs, 2nd edn. Oxford University Press, Oxford,

p. 245.[145] Goldstein, L.I., Ishak, K.G. and Burns, W. (1974) J. Digest. Dis. 19, 987–998.[146] Holmes, C. and Thurlbeck, W.M. (1979) Am. Rev. Respir. Dis. 120, 1125–1136.[147] Rustia, M. and Shubik, P. (1972) J. Natl. Cancer Inst. 48, 721–729.[148] Rustia, M. and Shubik, P. (1979) J. Natl. Cancer Inst. 63, 863–868.[149] Veronese, M.E. and McLean, S. (1991) Eur. J. Clin. Pharmacol. 40, 547–552.[150] Veronese, M.E., McLean, S., D’souza, C.A. and Davies, N.W. (1985) Xenobiotica 15, 929–940.[151] Lynn, R.K., Garvie-Gould, C., Milam, D.F., Scott, K.F., Eastman, C.L. and Rodgers, R.M. (1983) Drug

Metab. Dispos. 11, 109–114.[152] Hart, S.J., Tontodonati, R. and Calder, I.-C. (1981) J. Chromatogr. 225, 387–405.[153] Sabbioni, G. and Sepai, O. (1995) Chimia 49, 374–380.[154] Bort, R., Mace, K., Boobis, A., Gomez-Lechon, M.-J., Pfeifer, A. and Castell, J. (1999) Biochem.

Pharmacol. 58, 787–796.[155] Bort, R., Pondosa, X., Jover, M., Gomez-Lechon, M.-J. and Castell, J. (1999) J. Pharmacol. Exp.

Therapeut. 288, 65–72.[156] Renner, H.W. and M€unzner, R. (1980) Mutat. Res.349–355[157] Orton, T.C., Lewerey, C. and Lindup, W.E. (1977) Br. J. Pharmacol. 60(2); 319P.[158] Amos, H.E., Lake, B.G. and Atkinson, A.C. (1977) Clin. Exp. Allergy 7(5); 423.[159] Nelson, S.D. (1982) J. Med. Chem. 25, 753–761.[160] Onderwater, R.C.A., Commandeur, J.N.M., Groot, E.J., Sitters, A., Menge, W.M.P.B. and Vermeulen, N.P.

E. (1998) Toxicology 125(2,3); 117–129.[161] Onderwater, R.C.A., Commandeur, J.N.M., Menge, W.M.P.B. and Vermeulen, N.P.E. (1999) Chem. Res.

Toxicol. 12(5); 396–402.[162] Thomas, G.A. and Williams, E.D. (1991) Mutat. Res. 248, 357–370.[163] Capen, C.C. (1997) Toxicol. Pathol. 25, 39–48.[164] World Health Organization. (1974) IARC Monographs on the Evaluation of Carcinogenic Risk of

Chemicals to Man, ‘Some Anti-Thyroid and Related Substances, Nitrofurans and Industrial Chemicals,’Vol. 7. International Agency for Research on Cancer, Lyon.

[165] Miller, A.E., Bischoff, J.J. and Pae, K. (1988) Chem. Res. Toxicol. 1(3); 169–174.[166] Svarovsky, S.A., Simoyi, R.H. and Makarov, S.V. (2001) J. Phys. Chem. 105, 12634–12643.[167] Hillhouse, J.H., Blair, I.A. and Field, L. (1986) Phosphorus Sulfur Relat. Elem. 26, 169–184.[168] Henderson,M.C., Kreuger, S.K., Stevens, J.F. andWilliams, D.E. (2004)Chem. Res. Toxicol. 17, 633–640.[169] Pommier, Y.G., Barceló, J., Furuta, T., Takemura, H., Sordet, O. and Liao, Z.-Y. (2005) Cancer Drug

Discov. Dev. 1, 61–107.

GRAHAM F SMITH 47

12345678910111213141516171819202122232425262728293031323334353637383940414243444546

12345678910111213141516171819202122232425262728293031323334353637383940414243444546

[170] Hogberg, M., Engelhardt, P., Vrang, L. and Zhang, H. (2000) Biorg. Med. Chem. Lett. 10, 265–268.[171] Petersen, H.J., Nielsen, C.K. and Arrigoni-Martelli, E. (1978) J. Med. Chem. 21, 773–781.[172] Mitzutani, T., Yoshida, K. and Kawazoe, S. (1994) Drug Metab. Dispos. 22, 750–755.[173] Mitzutani, T. and Suzuki, K. (1996) Toxicol. Lett. 85, 101–105.[174] Dalvie, D.K., Kalgutkar, A.S., Khojasteh-Bakht, S.C., Obach, R.S. and O’Donnell, J.P. (2002) Chem. Res.

Toxicol. 15, 269–299.[175] Hobbs, D. and Twomey, T. (1977) Drug Metab. Dispos. 5, 75–81.[176] Yabuki, M., Shimakura, J., Ito, M., Kanamura, H., Iba, K. and Nakatsuka, I. (1997) Eur. J. Drug Metab.

Pharmacokinet. 22, 25–33.[177] Gannett, P.M. and Powell, J.H. (2002) J. Environ. Pathol. Toxicol. Oncol. 21(1); 1–31.[178] Gamberini, M., Cidade, M.R., Valotta, L.A., Armelin, M.C.S. and Leite, L.C.C. (1998) Carcinogenesis 19

(1); 147–155.[179] Uetrecht, J. (2006) Drug Metab. Rev. 38, 745–753.[180] Kleno, T.G., Leonardsen, L.R., Kjeldal, H.O., Laursen, S.M., Jensen, O.N. and Baunsgaard, D. (2004)

Proteomics 4, 868–880.[181] Kleno, T.G., Kiehr, D., Baunsgaard, D. and Sidelmann, U.G. (2004) Biomarkers 9(2); 116–138.[182] Maloney, S.J., Wiebkin, P., Cummings, S.W. and Prough, R.A. (1985) Carcinogenesis 6(3); 397–401.[183] Timbrell, J.A. (1979) Drug Metab. Rev. 10, 125–147.[184] Zolla, L. and Timperio, A.M. (2005) Biochem. Cell Biol. 83, 166–175.[185] Lindon, J.C., Nicholson, J.K., Holmes, E., Antti, H., Bollard, M.E., Keun, H., Beckonert, O., Ebbels, T.,

Reily, M.D., Robertson, D., Stevens, G.J., Luke, P., Breau, A.P., Cantor, G.H., Bible, R.H., Niederhauser,U., Senn, H., Schlotterbeck, G., Sidelmann, U.G., Laursen, S.M., Tymiak, A., Car, B.D., Lehman-McKeeman, L., Colet, J.-M., Loukaci, A. and Thomas, C. (2003) Toxicol. Appl. Pharmacol. 187,137–146.

[186] Skipper, P., Tannenbaum, S.R., Thilly, W.G., Furth, E.E. and Bishop, W.W. (1980) Cancer Res. 40,4704–4708.

[187] Lee, M.S. and Isobe, M. (1990) Cancer Res. 50, 4300–4307.[188] Lipczynska-Kochany, E., Iwumura, H., Takahashi, K., Hakura, A. andKawazoe, Y. (1984)Mutat. Res. 135,

139–148.[189] Dalvie, D., Cosker, T., Boyden, T., Zhou, S., Schroeder, C. and Potchoiba,M.J. (2008)DrugMetab. Dispos.

36(9); AU71869–1883.[190] Plewe, M.B., Butler, S.L., Dress, K.R., Hu, Q., Johnson, T.W., Kuehler, J.E., Kuki, A., Lam, H., Liu, W.,

Nowlin, D., Peng, Q., Rahavendran, S.V., Tanis, S.P., Tran, K.T.,Wang, H., Yang, A. and Zhang, J. (2009) J.Med. Chem. 52, 7211–7219.

[191] Bauer, L. and Exner, O. (1974) Angew. Chem. Int. 13, 376–384.[192] Hirrlinger, B. and Stolz, A. (1997) Appl. Environ. Microbiol. 63(9); 3390–3393.[193] Doerge, D.R. and Niemczura, W.P. (1989) Chem. Res. Toxicol. 2(2); 100–103.[194] Smith, J.T.L. and Pounder, R.E. (1988) Gastroenterology 94[195] Bayliss, M.K., Bell, J.A., Jenner, W.N., Park, G.R. and Wilson, K. (1999) Xenobiotica 29, 253–268.[196] Brittain, R.T., Jack, D., Reeves, J.J. and Stables, R. (1985) Br. J. Pharmacol. 85, 843–847.[197] Gatehouse, D., Wedd, D.J., Paes, D., Delow, G., Burlinson, B., Pascoe, S., Brice, A., Stemp, G. and Tweats,

D.J. (1988) Mutagenesis 3, 57–68.[198] Lipinski, C.A., LaMattina, J.L. and Oates, P.J. (1986) J. Med Chem. 29, 2154–2163.[199] Chatman, L.A., Morton, D., Johnson, T.O. and Anyway, S.D. (2009) Toxicol. Pathol. 37, 997–1005.[200] Ratcliffe, A.J. (2009) Curr. Med. Chem. 16, 2816–2823.[201] Reasor, M.J., Hastings, K.L. and Ulrich, R.G. (2006) Exp. Opin. Drug Saf. 5(4); 567–583.[202] Willard, J. (2008) FDA phospholipidosis working group preliminary results and developing opinions.

Presented at the Society of Toxicology Pathology (STP) Continuing Education Course on Drug-InducedPhospholipidosis, June 2008.

[203] Fujimura, H., Dekura, E., Kurabe, M., Shimazu, N., Koitabashi, M. and Toriumi, W. (2007) Exp. Toxicol.Pathol. 58(6); 375–386.

[204] Tengstrand, E.A., Miwa, G.T. and Hsieh, F.Y. (2010) Exp. Opin. Drug Metab. Toxicol. 6(5); 555–570.


12345678910111213141516171819202122232425262728293031323334353637383940414243444546

12345678910111213141516171819202122232425262728293031323334353637383940414243444546

[205] Ploemen, J.-P.H.T.M., Kelder, J., Hafmans, T., Van de Sandt, H., vanBurgsteden, J.A., Salemink, P.J.M. andvan Esch, E. (2004) Exp. Toxicol. Pathol. 55, 347–355.

[206] Pelletier, D.J., Gelhaar, D.J., Tilloy-Ellul, A., Johnson, T.O. and Greene, N.J. (2007) J. Chem. Inf. Model.47, 1197–1205.

[207] Moore, D.E. (2002) Drug Safety 25, 345–372.[208] Quintaro, B. and Miranda, M.A. (2000) Ars Pharmaceut. 41(1); 27–46.[209] FDA, CDER. (2003) Guidance for Industry: Photosafety Testing. May 2003.[210] OECD Guideline for Testing Chemicals. In Vitro 3T3 NRU Phototoxicity Test. Draft Document, February

2002.[211] Henry, B., Foti, C. and Alsante, K. (2009) J. Photochem. Photobiol. 96, 57–62.[212] Jones, P.A. and King, A.V. (2003) Toxicol. In Vitro 17, 703–708.[213] Siraki, A.G., Chevaldina, T., Moridani, M.Y. and O’Brien, P.J. (2004) Curr. Opin. Drug Discov. Dev. 7(1);

118–125.[214] Moridani, M.Y., Siraki, A. and O’Brien, P.J. (2003) Chem.-Biol.. Interact. 145, 213–223.[215] Dong, S., Fu, P.P., Shirsat, R.N., Hwang, H.-M., Leszczynski, J. and Yu, H. (2002) Chem. Res. Toxicol. 15,

400–407.[216] Zachary, C.B., Slater, D.N., Holt, D.W., Storey, G.C.A. andMacDonald, D.M. (1984) Br. J. Dermatol. 110,

451–456.[217] Dootson, G. and Byatt, C. (1994) Clin. Exp. Dermatol. 19, 422–424.[218] Sortino, S. (2006) Photochem. Photobiol. 82, 64–70.[219] Wainwright, N.J., Collins, P. and Ferguson, J. (1993) Drug Safety 9, 437–440.[220] Moore, D.E. and Sithipitaks, V. (1983) J. Pharm. Pharmacol. 35, 489–493.[221] Anderson, T.F. and Voorhees, J.J. (1980) Annu. Rev. Pharmacol. Toxicol. 20, 235–257.[222] Edelson, R., Berger, C., Gasparro, F., Jegasothy, B., Heald, P., Wintroub, B., Vonderheid, E., Knobler, R.,

Wolff, K., Plewig, G.,McKiernan, G., Christiansen, L., Oster, M., Honigsman, H.,Wilford, H., Kokoschka,E., Rehle, T., Perez, M.D., Stingl, G. and Laroche, L. (1987) N. Engl. J. Med. 316, 297–303.

[223] Dalla Via, L., Gia, O., Magno, S.M., Santana, L., Teijeira, M. and Uriarte, E. (1999) J. Med. Chem. 42(21);4405–4413.

[224] Gunther, E.J., Yeasky, T.M., Gasparro, F.P. and Glazer, P.M. (1995) Cancer Res. 55(6); 1283.[225] Uetrecht, J. (2001) Curr. Opin. Drug Discov. Dev. 4, 55–59.[226] Uetrecht, J. (2007) Annu. Rev. Pharmacol. Toxicol. 47, 513–539.[227] Uetrecht, J. (2009) Semin. Liver Dis. 29(4); 383–392.[228] Shenton, J.M., Chen, J. and Uetrecht, J.P. (2004) Chem.-Biol. Interact. 150, 53–70.[229] Ulrich, R.G. (2007) Annu. Rev. Med. 58, 17–34.[230] Walgren, J.L., Mitchel, M.D. and Thompson, D.C. (2005) Crit. Rev. Toxicol. 35, 325–361.[231] Lammert, C., Einarsson, S., Saha, C., Niklasson, A., Bjornsson, E. and Chalasani, N. (2009) Hepatology

47, 2003–2009.[232] Li, A.P. (2002) Chem.-Biol. Interact. 142, 7–23.[233] Kalgutkar, A.S., Gardner, I., Obach, S.R., Shaffer, C.L., Callegari, E., Henne, K.R.,Mutlib, A.E., Dalvie, D.

K., Lee, J.S., Nakai, Y., O’Donnell, J.P., Boer, J. and Harriman, S.P. (2005) Curr. Drug Metab. 6, 161–225.[234] Kalgutkar, A.S. and Soglia, J.R. (2005) Exp. Opin. Drug Metab. Toxicol. 1(1); 91–142.[235] Kalgutkar, A.S. and Didiuk, M.T. (2009) Chem. Biodiv. 6, 2115–2137.

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Journal: Progress in Medicinal Chemistry, 50Article No.: Chapter 1

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This is fine - GFS

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Springer-Verlag Berlin / Heidelberg, pp. 205 -251. DOI: 10.1007/b11992

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See renumbering p30-34

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this is 2nd citation of 159 no change needed

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OK but Corrected McIntyre

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John Wiley & Sons, Ltd, Chichester, UK. doi: 10.1002/9780470846308

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yes 2008 is correct

designing drugs to avoid toxicity

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