Metabolism by Aldehyde Oxidase - American Chemical Society

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Metabolism by aldehyde oxidase: drug design and complementary approaches to challenges in drug discovery Nenad Manevski, Lloyd King, William Ross Pitt, Fabien Lecomte, and Francesca Toselli J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00875 • Publication Date (Web): 06 Aug 2019 Downloaded from pubs.acs.org on August 6, 2019

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Journal of Medicinal Chemistry

Title page and Abstract Metabolism by aldehyde oxidase: drug design and complementary approaches to challenges in drug discovery Nenad Manevski,*1 Lloyd King,1 William R Pitt,1 Fabien Lecomte,1 and Francesca Toselli2 1UCB

Celltech, 208 Bath Road, Slough, SL13WE, United Kingdom. 2UCB BioPharma, Chemin du

Foriest 1, 1420 Braine-l'Alleud, Belgium *corresponding author

Abstract Aldehyde oxidase (AO) catalyzes oxidations of azaheterocycles and aldehydes, amide hydrolysis, and diverse reductions. AO substrates are rare among marketed drugs and many candidates failed due to poor pharmacokinetics, interspecies differences, and adverse effects. As most issues arise from complex and poorly understood AO biology, an effective solution is to stop or decrease AO metabolism. This perspective focuses on rational drug design approaches to modulate AO-mediated metabolism in drug discovery. AO biological aspects are also covered, as they are complementary to chemical design and important when selecting the experimental system for risk assessment. The authors’ recommendation is an early consideration of AO-mediated metabolism supported by computational and in vitro experimental methods, but not an automatic avoidance of AO structural flags, many of which are versatile and valuable building blocks. Preferably, consideration of AO-mediated metabolism should be part of the multiparametric drug-optimization process, with the goal to improve overall drug-like properties.

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Graphical abstract Aldehyde oxidase Biology of AO (right species) Experimental assessment (right assay)

N



N

Substrate

Replace



HO

Computational assessment (right model)

S

S Mo

N

R

N H

N H ↓ Ring size Block Saturate R = -Me, -F, -CN... EDG or EWG Bulky group

+H2O

S

N

O Molybdenum cofactor (enzyme active site)

Complementary approaches (decision support)

N

O N H Metabolite

D

Kinetic deuterium effect

Remove nitrogen

N

N

Modulate electronics

Steric hindrance

Drug design approaches (avoidance and decrease)

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1


Introduction

Aldehyde oxidase (AO, EC 1.2.3.1) is a cytosolic molybdenum-containing enzyme involved in the biotransformation of numerous drugs and xenobiotics.1-5 Although structurally similar to xanthine oxidoreductase (XO),6 AO has a significantly broader substrate selectivity and can catalyze four distinct reactions types: oxidations of azaheterocycles,7 oxidations of aldehydes,8 hydrolysis of amides,9 and various reductions.10-11 Each reaction type presents a unique set of challenges and opportunities in drug discovery and development (Table 1). Oxidations of azaheterocycles have been reported to contribute to high metabolic clearance and interspecies differences, resulting in the termination of several drug discovery programs in the past, including carbazeran,12 BIBX1382,13 and Lu AF09535.14 AO-mediated oxidations of aldehydes in vitro have been described previously, both for endogenous and xenobiotic substrates, but their impact on drug disposition was recognized only recently (e.g. imrecoxib).8, 15 Hydrolysis of amides by AO, first described in 2015,9 has also been found to contribute to high metabolic clearance and, depending on the drug of interest, a release of aryl-amine fragments that may be prone to bioactivation. Finally, AO-catalyzed reductions are diverse, affecting mainly nitro, N-oxide and isoxazole moieties, and are mainly of interest from the perspective of bioactivation.16 Table 1. Selected examples of AO-related challenges experienced in drug discovery and development. Compound Carbazeran BIBX1382 FK3453 Lu AF09535 RO-1

Reaction type

References 12

Oxidation of azaheterocycle

High metabolic clearance in humans due to oxidations by AO.

13 17 14 18

JNJ-38877605 Oxidation of azaheterocycle SGX523

Observed challenges

Renal toxicity in humans due to accumulation of insoluble AO metabolite. Renal toxicity had not been observed in non-clinical studies in rats and dogs. High clearance in humans due to AO metabolism. Renal toxicity in humans due to accumulation of insoluble AO metabolite.

19

20-21



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Momelotinib


BILR 355


Disproportionate human metabolite due to oxidations by AO [metabolites in safety testing (MIST) issue] Disproportionate human metabolite due to oxidations by AO (MIST issue)


Rapid metabolism by AO in rats prevents pharmacological studies.

24


Rapid metabolism by AO in rats prevents toxicological studies.

7

Imidazoquinolinecontaining Cancer Osaka thyroid kinase inhibitors Pyridine-containing toll-like receptor subtype 7 agonist VX-509 (decernotinib)


Dantrolene

Reduction of nitro group

Imrecoxib

Aldehyde oxidation

GDC-0834

Hydrolysis of amide

AO metabolite is a time-dependent inhibitor of CYP3A4, leading to clinically relevant drug-drug interactions. Bioactivation via AO-mediated nitro reduction and subsequent liver injury. In vitro–in vivo disconnect in metabolite abundance due to AO-mediated aldehyde oxidation. High metabolic clearance in humans due to AOmediated amide hydrolysis.

22

23

25

26

8

9

The challenge represented by AO-mediated metabolism is driven by several overlapping factors, including the complex biology of the enzyme (section 7), the widespread use of structural motifs that are AO substrates (e.g. azaheterocycle and amides), and common approaches to reduce cytochrome P450 (CYP)-mediated metabolism [e.g. reduction of logD and introduction of electron-withdrawing groups (EWG)]. The limited understanding of AO biology compared to other drug-metabolizing enzymes, such as CYPs or UDP-glucuronosyltransferases (UGTs), makes it a challenge to accurately predict human pharmacokinetics (PK) of drug candidates primarily metabolized by AO, which is reflected by the small number of marketed drugs that are AO substrates. Until a better understanding of AO activity in various tissues, and of how this is affected by genetic polymorphisms and extrinsic factors, is established for humans and non-clinical species, compounds with lower AO-mediated clearance and diversified biotransformation pathways are more likely predicted to have an acceptable human PK and safety. Therefore, it is essential to identify, assess and modify structural features of


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drug candidates that lead to AO-mediated metabolism, so that elimination pathways can be diversified to include other xenobiotic-metabolizing enzymes. This perspective focuses on rational drug design approaches to control AO metabolism, either by mitigation (e.g. stop, decrease, diversify) or utilization (e.g. design of a prodrug or a modified scaffold) of the enzymatic reaction. AO-mediated xenobiotic metabolism has been extensively reported previously by various groups, but only a few reviews and perspectives focused on drug design have been published,7, 27-28 often discussing exclusively AO-mediated oxidations. The authors’ motivation arose from our experience at UCB and published examples showing that AO-mediated metabolism is best approached at the drug design stage,7, 24, 29 as an integral part of the multi-parametric optimization process to increase the overall drug-like properties of the compound. Later, during the drug development stage, approaches to assess and predict AO-mediated metabolism in humans are also possible and have been applied, but success has been limited due to an insufficient understanding of AO biology, which has often resulted in poor predictions of human PK by in vitro–in vivo extrapolation (IVIVE) or allometric scaling. The rational drug design aspect of this review focuses on arguably the most common reaction catalyzed by AO in the drug-like chemical space,28 namely the AO-mediated oxidations of electrondeficient azaheterocycles via a nucleophilic mechanism (see graphical abstract). Oxidations of azaheterocycles were historically perceived mainly as a risk for high metabolic clearance in humans. Recent examples have significantly broadened this view, to include evidence for renal toxicity due to the formation of insoluble crystalline metabolites,19-20 metabolites in safety testing (MIST)-related issues,22, 30 interspecies differences,7, 24 as well as AO-derived metabolites leading to time-dependent inactivation of CYP3A4 (vide infra).25 Despite these discouraging examples, AO should not be perceived as an enemy of drug design teams. Oxidative metabolites formed by AO are often 5 ACS Paragon Plus Environment


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metabolically stable (authors’ unpublished observations) and, if pharmacologically active, may provide valuable inspiration for novel scaffolds and improved drug design.31 On the other hand, high turnover of AO substrates enables the potential for a prodrug approach, as exemplified by 5-fluoro-2pyrimidinone,32 famciclovir,33 and fasudil.34 In addition, the current perspective will also cover reactions of aldehyde oxidation, amide hydrolysis and reductions, albeit in less detail, proportional to the number of examples available in the literature. For these reactions, focus will often be placed on safety and drug developability (e.g. interspecies difference and MIST), not high metabolic clearance per se. Although designing away from, or at least reducing, the propensity of drug candidates to be metabolized by AO is desirable in drug discovery, the task faced by design teams is difficult. Firstly, AO structural flags are very common (e.g. azaheterocycles and amides), usually covering a large proportion of the chemical space (see Section 3.1). Secondly, due to opposing catalytic mechanisms (e.g. electrophilic vs. nucleophilic oxidation), common strategies to reduce oxidative metabolism (especially if CYP-mediated) will usually favor AO-mediated metabolism.7 Thirdly, directly blocking sites susceptible to attack by AO with bulky groups may alter key pharmacophores and erode ligand efficiency by increasing the molecular weight and logD, ultimately degrading the overall drug-like properties.35 Therefore, designing away from AO-mediated metabolism requires a thoughtful approach based on understanding and application of several overlapping factors (Figure 1), including the catalytic mechanism, substrate specificity, structure-activity relationship (SAR) with the therapeutic target, AO biology, and various supporting tools (e.g. computational and experimental methods to assess metabolism by AO). Although the focus is on human AO, examples of AOmediated metabolism in non-clinical species are also provided, especially when available human data are limited and interspecies differences played a major role in the decision-making. Emphasis is also 6 ACS Paragon Plus Environment

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placed on preservation of the key drug-like properties, including lipophilicity, solubility, permeability, ligand efficiency, and recognition and (if possible) removal of known structural alerts. N Abundance (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


S

H

N

100

O



O

N

75 N

50

N N

0 0

5

10

15

20

25

30

Time (min)

Experimental assessment (section 8)

Biology of AO (section 7)



S S

H

OH

25

Mo

N

Enzyme structure and catalytic mechanism (section 2)

N Substrate specificity and reactions catalyzed (section 3)

Drug design approaches to AO-mediated metabolism (section 5)

Computational assessment of AO metabolism (section 4)

Examples of discovery programs (section 6)

1 enzyme 4 enzymes

N

N

N

N

X

N N

OH

Figure 1. Factors influencing AO-mediated metabolism and drug design. Representation of AOcatalyzed sites of oxidation on quinazoline is prepared based on transition state energies calculated in Montefiori et al. 2017.36 Drawings of human body and mouse are authored by Mikael Häggström and David Liao, respectively, and are freely provided for reuse under the Creative Commons license. Drug disappearance and metabolite appearance curves are simulated with GraphPad Prism 8.1 (GraphPad Software). Representation of AO active site is prepared based on published co-crystal structure of human AO (PDB ID: 4UHX)37 using the PyMOL 2.3 software (Schrödinger, Inc.). A summary of factors influencing AO drug design is presented in Figure 1. Section 2 covers the structure and catalytic mechanisms of AO, providing a rational basis for further discussion. Reactions 7 ACS Paragon Plus Environment


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catalyzed by AO and their substrate specificity are covered in section 3, including examples of oxidation, hydrolysis and reduction. Section 4 explores computational approaches to evaluate metabolism by AO, even before the compound is synthetized. Section 5 summarizes key chemical design approaches to control AO-mediated metabolism, including methods to stop, decrease, or utilize AO-catalyzed reactions. Section 6 is dedicated to examples of drug discovery programs that tackled AO metabolism, providing a valuable insight into challenges, pitfalls and opportunities encountered by design teams. Finally, sections 7 and 8 provide an in-depth information about AO biology, including reported suggestions of AO as a therapeutic target, and experimental assessment, complementing the drug design approaches.


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2


Enzyme structure and catalytic mechanism of AO

Understanding of AO structure and catalytic mechanism is important to support rational drug design. Additionally, it is beneficial to appreciate differences and similarities between AO and other key drugmetabolizing enzymes (e.g. CYPs and various hydrolases) so that medicinal chemistry design can be driven on a balance of overall biotransformation liabilities. 2.1

Structure of human AO

Human AO is a butterfly-shaped homodimer with approximate dimensions of 150 Å x 90 Å x 65 Å (Figure 2 A).2-3,

37

Each monomer can be further divided into three distinct domains: (I) a small

N-terminal domain (20 kDa), which harbors the two [2Fe-2S] clusters, (II) the central domain with flavin adenine dinucleotide (FAD) cofactor stacked between two leucine residues (40 kDa), and (III) the large C-terminal domain (90 kDa) containing the molybdenum pyranopterin cofactor (MoCo) and the substrate binding site (for reactions of oxidation and hydrolysis; reductions likely occur at the FAD-binding site3, 10). The three domains are connected by two linker regions bridging domains I / II and domains II / III.2



A) Overview of human AO structure in surface and ribbon representations. FAD binding site (reduction site)

MoCo active site (oxidation and hydrolysis site)

Domain I (20 kDa)

Linker 2

Allosteric inhibition site (thioridazine binding) Linker 1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Domain II (40 kDa) FAD (flavin adenine dinucleotide)

two [2Fe-2S] clusters

H N

O HO N H2N N

N N

O

OH

HO

N

O O O P P O HO OH

MoCo (molybdopterin cofactor) O

H2N

N

N

H H N O

O

N H H

HN

N

HO O

Domain III (90 kDa)

CH3

OH CH3

HO OH P O O

S S

Mo O

S OH

B) Active site of human AO showing substrate phthalazine, MoCo, and key amino acid residues thought to be important in substrate binding and catalytic mechanism.

Figure 2. Structure of human AO (A) and its MoCo active site (B). Representations are prepared based on published co-crystal structure of human AO (PDB ID: 4UHX)37 using the PyMOL 2.3 software (Schrödinger, Inc.). 10 ACS Paragon Plus Environment

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Substrate access to and product release from the MoCo active site are governed by a funnel that leads into the active site cavity.37 Features of this funnel are defined by two flexible loops on the surface of the protein, gates 1 and 2, and the amino acid composition of these loops may explain unique substrate specifies of various mammalian AO enzymes.3, 37 Gate 1 is predominantly composed of hydrophobic residues whereas gate 2 is a short, dynamic loop characterized by the presence of residue Phe885, the second residue after the gate, which interacts with the substrate molecule, orientating it towards the catalytic site.3, 37 At the MoCo active site (reactions of oxidations and hydrolysis), important and conserved residues are Glu1270, Phe923, Lys893 and Gln776.37 Glu1270 and Lys893 are believed to be directly involved in the catalytic mechanism by providing hydrogen bonding to stabilize the transition state (see section 2.2). On the other hand, two conserved aromatic residues, Phe885 and Phe923, are likely important for the substrate orientation towards the MoCo.37 In addition to the MoCo active site, human AO is thought to have an allosteric inhibitor binding site (occupied by thioridazine in the available crystal structure)37 and FAD active site responsible for reduction reactions.3, 10 The structure, sequence conservation, single nucleotide polymorphisms, and evolution of mammalian AO and related proteins have been extensively reviewed elsewhere.3-4, 38 2.2

Catalytic mechanism of AO

Although catalyzed by the same enzyme, reactions of oxidation, hydrolysis, and reduction by AO have a few differences that deserve to be clarified. Moreover, it is worth highlighting differences between AO and other common drug-metabolizing enzymes, especially CYPs, flavin-containing monooxygenases (FMOs), and hydrolytic enzymes. Reactions of oxidation. In the first step, the site of oxidation, i.e. the electrophilic carbon of the azaheterocycle or aldehyde, is attacked by the nucleophilic -OH ligand of the MoCo ligand (MoVI), likely further activated by Glu1270 (Figure 3 A; step i). According to the concerted mechanism 11 ACS Paragon Plus Environment


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hypothesis, hydride transfer to MoCo’s sulfido ligand occurs simultaneously (via a tetrahedral intermediate) to form a transition state stabilized by hydrogen bonding from Glu1270 and Lys893.3940

The concerted mechanism is supported by the enzyme kinetic studies (N-substituted heterocycles

and deuterium isotope effects) and density functional theory calculations36, 40-41 (see section 4 for more details). Hydride abstraction and transfer to S of MoCo is likely the rate-limiting step in the formation of the transition stage,42-43 opening a possibility for the kinetic deuterium isotope effects (KDIE) to decrease the rate of AO-catalyzed oxidations (section 5.3). In the second step (Figure 3 B; step ii), a molecule of water hydrolyzes the transition state intermediate, releasing the oxidized product and the reduced MoCo (MoIV). Within each catalytic cycle, oxygen from MoCo is incorporated into the substrate and additional oxygen from H2O replenishes the vacant coordination position on the MoCo. As illustrated in Figure 3, the oxygen that is incorporated into the product is derived from one of two sources: the initial -OH ligand of the MoCo from AO in the first-turnover reaction, and from H2O via MoCo in the subsequent reactions. The origin of oxygen from H2O enables experimental approaches that use 18O from H218O (see sections 8.1 and 8.3). The catalytic cycle is closed by the re-oxidation of the MoCo (MoIV→MoVI) (Figure 3 A; step iii). Two reducing equivalents (2e−) are transferred via two [2Fe-2S] clusters and the FAD cofactor to molecular oxygen as their final acceptor, to generate superoxide and/or hydrogen peroxide.44 In addition to molecular oxygen, the electron acceptor can be another molecule, including a xenobiotic, leading to AO-catalyzed reductions (see section 3.4 below).


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A)

O

O S

S

Mo

S

VI

H

O

S

N

S i

Glu 1270

S

–

N C N

H

O S

H

O

N

H

O

S

Mo

Tetrahedral intermediate

SH

Mo

N

O

IV

N

H

O

H

O

+

HN

Glu 1270

O

H

Lys893 Transition state

iii FAD

[2Fe-2S]

+H2O

-2e

O O2

ii

-

S

H2O2

HO SH

Mo

S

IV

OH

Cycle 1:

N

O

N

H N N

MoCo-†OH + S + H2‡O ↔ MoCo-‡OH + S-†OH

Cycles 2-n: MoCo-‡OH + S + H2‡O ↔ MoCo-‡OH + S-‡OH B)

R

R

CH3

CH3

NH

NH

O GDC-0834

H+

O S

–

O

S SH S Mo S IV

HO

HO SH IV Mo OH S S

R= O H3C

NH

N

O

N R CH3 H2O

N H3C

CH3

N

NH2

O

M1

O

O HO S

HO IV S Mo SH S

S

M2

Figure 3. Proposed concerted catalytic mechanisms of AO-catalyzed oxidations40-41, 45 (A) and amide hydrolysis9, 28 (B). Amino acid residues are numbered according to the human AO sequence.37



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Although both AO and CYPs catalyze C-atom hydroxylations, it is important to emphasize that catalytic mechanisms are different (or even contrasting), leading to unique substrate specificity and site of metabolism (SOM) on the drug-like compounds. Whereas AO-catalyzed oxidations occur by nucleophilic attack of the -OH group of the MoCo ligand (Figure 3 A; step i), CYP-catalyzed oxidations are driven by the electrophilic attack of the activated oxygen species (perferryl oxygen complex FeO3+ or oxene, an electrophilic atom with only six electrons in the outer layer).46-47 Similarly, FMOs catalyze oxidations of soft nucleophiles (e.g. basic amines and sulfides) by electrophilic attack of the 4a-hydroperoxyflavin moiety (FADH−OOH complex).48 These differences play a profound role in drug design: whereas electron-deficient moieties will be largely protected from CYP- and FMOcatalyzed oxidations, this may leave them vulnerable to AO-catalyzed oxidations (see section 5). Examples of both CYPs and AO catalyzing the same reaction do exist, however: O6-benzylguanine is oxidized at the position 8 of the guanine by both CYP1A2 and AO.49 Another interesting and useful difference between CYP- and AO-catalyzed oxidations is the origin of oxygen atom: whereas CYPs utilize oxygen from atmospheric O2,46 AO uses oxygen from H2O.50 This unique feature of the AO catalytic mechanisms can be used for analytic purposes, for example in experiments with H218O to identify metabolites formed by AO.51 Reactions of hydrolysis. Based on the example of the amide hydrolysis of GDC-0834,9,

28

the

following catalytic mechanism was proposed (Figure 3 B). The carbonyl group of the amide is attacked by the nucleophilic hydroxymolybdenum moiety to form a tetrahedral intermediate.9 As this step requires a relatively strong nucleophile, authors proposed the MoIV oxidative state,9 instead of the MoVI involved in the AO-catalyzed oxidations (see above). This proposed mechanism, like AOcatalyzed reductions, would require accumulation of the enzyme in the MoIV oxidative state, probably by the previous oxidation of another compound, either endogenous or exogenous.9 The formed 14 ACS Paragon Plus Environment

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tetrahedral intermediate yields the free amine (M1) and possibly ester bound to molybdenum, which is further hydrolyzed to release the free acid metabolite (M2) and the free MoIV hydroxymolybdenum (Figure 3 B).9,

28

Amide hydrolysis is also catalyzed by various hydrolytic enzymes, including

amidases, esterases and peptidases.52 Although hydrolases are a diverse group of enzymes, the general catalytic mechanism of hydrolysis is fairly preserved; a nucleophilic group of the enzyme (e.g. -OH of serine, -SH of cysteine, or activated H2O) attacks the carbonyl C-atom of the amide, while an electrophilic group at the active site polarizes the carbonyl C-atom and the proton donor (H−B) facilitates leaving of the cleaved amine.53 Reactions of reduction. In contrast to reactions of oxidation and hydrolysis, the catalytic mechanism of AO-catalyzed reductions is poorly understood. Historically, reactions of AO-mediated reduction were performed under specific incubation conditions: under anaerobic atmosphere and in the presence of electron donors such as 2-hydroxypyrimidine, N1-methylnicotine, benzaldehyde, or NADPH.54-55 More recent results show that reductions by AO can also occur under aerobic conditions.11 Regardless of the oxygen levels, AO-catalyzed reductions likely need to be coupled with an oxidation of another substrate (“electron donor”), either endogenous or exogenous.10, 26 Moreover, based on recent enzyme kinetic evidence, AO-mediated reductions likely occur at a site distinct from the oxidative MoCo site,10 either the FAD-binding site or near the [2Fe-2S] clusters.



3

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Substrate specificity and reactions catalyzed

As described above, AO catalyzes four known reaction types: oxidations of azaheterocycles, oxidations of aldehydes, hydrolysis of amides and various reductions. In this section we will review structural features leading to these reactions based on published reports. 3.1

Oxidation of azaheterocycles

Oxidations of azaheterocycle-containing drugs and drug candidates are the most common reactions catalyzed by AO (Table 1). Oxidations usually occur at the electron-deficient sp2 carbons adjacent to nitrogen, which are part of aromatic azaheterocycles (–CH=N– moiety) or non-aromatic charged azaheterocycles (–CH=N+< moiety; iminium ions). AO has a significantly broader substrate specificity than the closely related enzyme xanthine oxidoreductase (XO), which is mainly restricted to the metabolism of hypoxanthine and xanthine in catabolism of purines.56 Oxidations of aromatic azaheterocycles. A large variety of aromatic azaheterocycles and fused heteroaromatic systems were reported to be oxidized by AO, generally resulting in pyridone-like metabolites (Figure 4; Table 2). Although prevalence of AO structural alerts was not fully investigated, 56% of Pfizer’s kinase-targeted compounds57 and 36% of all ChEMBL-reported drugs (authors’ unpublished observations; https://www.ebi.ac.uk/chembl/g/#browse/drugs; last accessed in June 201858) were predicted to be potential substrates for AO. The sites of oxidations are predominantly carbons adjacent to nitrogen, although important exceptions are quinoline,30 pyridazine (authors’ observations), cinnoline,59 acridine,60 and cryptoleptine motifs,61 where oxidations are also observed at distant carbons (Figure 4). The susceptibility to oxidations by AO generally increases with nitrogen count, especially if the affected carbon is between two nitrogens; for instance, pyrimidine oxidations are more commonly reported than pyridine oxidations. Conversely, oxidations of phenyl analogues or heterocycles without nitrogen are not reported at all, even if electron-deficient carbons near electron16 ACS Paragon Plus Environment

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withdrawing group (EWG) are present. Based on literature reports, some common heterocycles like pyrimidine, quinoline and purine have a high likelihood of being AO substrates, indicating that such chemotypes should always be tested for AO activity (Table 2). The impact of overall molecular size and geometry is difficult to estimate without complementary docking approaches, but reported AO substrates have widely different sizes, ranging from 130 Da (quinoline) to 823 Da (quinolinecontaining azetidinyl ketolide).62 Analysis of published AO oxidative substrates also indicates that successful clinical progression is still possible (Table 2; marked in bold typeface), although comparatively rare (e.g. imatinib,63 AMG900,63 lenvantinib64 etc.).



*

*

*

N

* N * * N

*

Risk factors Increased nitrogen count 6-membered rings Electron-withdrawing groups

* * N *

*

N

N

*

N

pyridine pyridazine pyrimidine

pyrazine

* N

*

N quinoline

N

N

N

N

N N

N

N 1,5-naphthyridine

pyrido[3,4-d] pyrimidine

*

N N

CH3 N

N

pyrido[2,3-d]pyrimidine

N

N

pyrimido[5,4-d]pyrimidine

pteridine

*

N N

N

N

acridine

*

cryptoleptine N

N H

*

1H-indole

N

N H

N

1H-pyrrolo[2,3-b] pyridine (7-Azaindole)

N N N

N

N

pyrazolo[1,5-a] pyrimidine

* N N

[1,2,4]triazolo[4,3-b]pyridazine

*

N

N H

N

purine

adenine S N

*

1,3-benzothiazole

*

HN H2N

benzimidazole

* N N

N

imidazo[1,2-b] -[1,2,4]triazine X

N

N

O

*

X = O , guanine X = S, thioguanine

HN O

N H

H N N

xanthine

N

imidazo[1,2-a] pyrazine

N N

pyrazolo[1,5-d] -[1,2,4]triazine

H N N

N N

N H

N

N

NH2

N N H

N

N

N H

1H-pyrrolo[2,3-c] pyridine (6-Azaindole)

N

N N

phthalazine

quinoxaline

N

N

*

*

N *

*

N quinazoline

N

1,6-naphthyridine 1,8-naphthyridine N

N

cinnoline

N

N

N

*

N

isoquinoline

Beneficial factors Decreased nitrogen count 5-membered rings Electron-donating groups

*

*

*

Page 18 of 139

N

H N

N

N

N 1H-[1,2,3]triazolo [4,5-b]pyrazine O HN N

H N N

hypoxanthine

N O

1,3-oxazole

Figure 4. Examples of aromatic azaheterocycles oxidized by AO. Star denotes the site of oxidation by AO, if reported in the literature. See Table 2 for the list of published AO substrates.

Table 2. Selected examples of common azaheterocycles oxidized by AO. Exact sites of oxidation by AO, if specified in the literature, are marked with a star. Compounds that failed in human clinical trials due to poor PK or related issues are underlined. Compounds that have acceptable human PK despite being AO substrates are marked in bold. Azaheterocycle

Structure

Examples from the literature


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Pyridine

Imatinib,63 several pyridine analogues oxidized by rat AO.7, 65

* N * N

Pyrazine

Lu AF0953514 and LY260361866

N

*

PF-05190547,67 Lu AF09535,14 FK3453,17 AMG900,63 bafetinib,63 VU0409106,68 VU0424238 (auglurant),69 zebularine,70 RS-8359,71-72 5-fluoropyrimidin-2-one,32

N

Pyrimidine

*

N Pyridazine

Authors’ observation

N

N Quinoline

N * Isoquinoline

N N

Quinazoline

*

N

Zoniporide,73 A-77-1,63 lapatinib,a63 INCB28060,63 ML-347,63 LDN-193189,63, 74 A77-01,63 5-nitroquinoline,10 lenvatinib,a64 PF04217903,75 BILR 402,23 SB-277011,76 series of c-Met inhibitors,77 JNJ-38877605,78 capmatinib,79 cinchona alkaloids,72 SGX523.21 4,6-Disubstituted analogues,29 ripasudil,80 fasudil.a,b81

CL-387785,63 several analogues.82-83

* Cinnoline N

Stubley et al.59

N

N Quinoxaline

SB-525334,63 XK469,84 brimonidine.85

N * Phthalazine

N N

Carbazeran,12 several analogues.82-83

*

N 1,6-naphthyridine

2,8-Disubstituted analogues.29

N



Page 20 of 139

N 1,5-naphthyridine

PF-0497906486

N 1,8-naphthyridine

PF-945863,87 azetidinyl ketolides analogues.62

N Pyrido[3,4-d]pyrimidine

N

N

C8-substituted analogues88

N

N

* Pyrido[2,3-d]pyrimidine

N

RO-118

N Pyrimido[5,4-d]pyrimidine

N N

N

BIBX138213

N

N

N

N

Pteridine

Methotrexatea 89-90

N

N

* N-[(2dimethylamino)ethyl]acridine-4carboxamide (DACA)60

Acridine

N CH3 N

Cryptoleptine

Cryptoleptine and analogues61 N

Indole

Benzimidazole

*

*

N H N N H

3-methylindole91 (oxidation by pig AO)

*

Zoniporide analogue92


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H-pyrrolo[2,3-b]pyridine (7-Azaindole)

1H-pyrrolo[2,3-c]pyridine (6-Azaindole)

*

N H

N

N

JNJ-63623872 and analogues,93 VX-509 (decernotinib).25

6-Azaindole analogues51

N H * N

Thieno[2,3-c]pyridine

VU0467206 and analogues94

S

* N

Imidazo[1,2-a] pyrimidine

Several analogues95

N

N

N [1,2,4]triazolo[4,3b]pyridazine

N N

Imidazo[1,2-b][1,2,4]triazine

Pyrazolo[1,5-d][1,2,4]triazine

*

SGX52321

N

* N

N

N

N

INCB28060 (capmatinib)a 63 79

N

N

N GABAA inverse agonist.96-97

N [1,2,4]triazolo[4,3a]pyrimidine

N

*

N N

N

MET401 (c-Met inhibitor)98

N N

Pyrazolo[1,5-a]pyrimidine

Zaleplona 99-100

N 1H-[1,2,3]triazolo [4,5-b]pyrazine

1H-imidazo[4,5-b]pyrazine

* *

N

N

N

N H

N

N

N

N

N H

PF-4217903,75 various analogues.101

*

c-Met inhibitors102



H N

* Purine (7H and 9H)

N *

N X

Guanine (X = O) and thioguanine (X = S)

H N

HN H2N

2H-oxazole

Idelalisib1 (GS-1101),103-104 duvelisib,a 63 6-mercaptopurine,105 BIIB021,106 famciclovir,a,b 33, O -benzylguanine,107 caffeine.57 6

*

N

N

N

Page 22 of 139

*

O6-benzylguanine,49 thioguanine.108

N

*

Several 2H-oxazole analogues109 O

S Benzothiazole

N a

*

Benzothiazole itself studied in Guinea pig AO110

Drugs currently on the market

For fasudil34 and famciclovir34 (both prodrugs) high metabolic clearance by AO is not a liability as the AO-formed oxidative metabolite is active.

b

The size of an azaheterocyclic ring also plays an important role, especially in non-fused systems, with 6-membered rings being more prone to AO-mediated oxidations than 5-membered rings. Exceptions to this observation are the reported oxidations of oxazoles109 and benzothiazole.110 Various nitrogencontaining fused heteroaromatics, both in 6,6 and 6,5 arrangements, are also commonly reported as AO substrates (Figure 4; Table 2). The sites of oxidation by AO in such systems are less predictable and generally occur on carbons adjacent to nitrogens in both 6- and 5-membered rings. Oxidations of fused heteroaromatic 5,5 systems are not reported, however, indicating a possibility for AO-mitigating design of molecules. The susceptibility to oxidations by AO may be modulated by ring substitutions. Based on the nucleophilic catalytic mechanism and published SAR examples, consensus is that EWG and electrondonating (EDG) groups usually enhance and reduce oxidations by AO, respectively.28,

79, 82, 111

Examples of azaheterocycles where both EWG and EDG are present are more difficult to predict and 22 ACS Paragon Plus Environment

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complementary computational approaches are recommended (see Section 4), to help design and decision-making. However, some EDG ring substituents may enhance AO-mediated oxidations by facilitating binding to the enzyme, even if electronics of the affected carbon would suggest otherwise.77 Broad interspecies differences in azaheterocyclic substrate specificity between human and rodent AO have been reported, most likely due to differences in AO isoforms expressed, their tissue distribution and their binding site properties; this will be discussed in more detail in sections 7 and 8. Oxidation of non-aromatic azaheterocycles (iminium ions). In addition to the typical oxidations of aromatic heterocycles, several authors reported AO involvement in oxidations of non-aromatic heterocycles, often after initial oxidation by another drug-metabolizing enzyme like monoamine oxidase B (MAO-B) and CYPs. The piperazine ring of KW-2449, a multi-kinase inhibitor developed for treatment of leukemia, was oxidized to iminium ion by MAO-B and then by AO to the pharmacodynamically active piperazinone metabolite (Figure 5 A).112 The iminium ion intermediate of KW-2449 was also an irreversible inhibitor of AO, possibly through covalent binding to the enzyme.113 A similar metabolic pathway was observed for 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP), a dopaminergic pro-neurotoxin which induces parkinsonism in humans and other primates (Figure 5 B).114-115 The morpholine ring of momelotinib, an inhibitor of Janus kinase 1/2 and activin A receptor type 1, was first oxidized by CYPs to iminium ion and then by AO to the corresponding morpholin-3-one metabolite (Figure 5 C).22 In contrast to the low levels observed in rat and dog plasma (

Metabolism by Aldehyde Oxidase - American Chemical Society

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