Evidence for the in Vitro Bioactivation of Aminopyrazole Derivatives

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Evidence for the in vitro bioactivation of aminopyrazole derivatives: trapping of reactive aminopyrazole intermediates using glutathione ethyl ester in human liver microsomes. Eileen Ryan, Benjamin J. Morrow, Catherine F. Hemley, Jo-Anne Pinson, Susan A. Charman, Francis C. K. Chiu, and Richard Charles Foitzik Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.5b00202 • Publication Date (Web): 28 Aug 2015 Downloaded from http://pubs.acs.org on September 2, 2015

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Chemical Research in Toxicology

Evidence for the in vitro bioactivation of aminopyrazole derivatives: trapping of reactive aminopyrazole intermediates using glutathione ethyl ester in human liver microsomes.

Eileen Ryan,† Benjamin J. Morrow,‡,§ Catherine F. Hemley,‡,§ Jo-Anne Pinson,‡,§ Susan A. Charman,† Francis C.K. Chiu† and Richard C. Foitzik‡,§*

†

Centre for Drug Candidate Optimisation and ‡Medicinal Chemistry Theme, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia

§

Cancer Therapeutics CRC, 343 Royal Parade, Parkville, Victoria 3052 Australia

*

To whom correspondence should be addressed: Richard Foitzik, 381 Royal Parade,

Parkville, Victoria 3052, Australia. Tel: +613 99039142; Fax: +613 99039143; email:[email protected].

1 Environment ACS Paragon Plus


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Table of contents graphic


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Abstract Drug-induced toxicity is a leading cause of drug withdrawal from clinical development and clinical use and represents a major impediment to the development of new drugs. The mechanisms underlying drug-induced toxicities are varied, however metabolic bioactivation to form reactive metabolites has been identified as a major contributor.1,

2

These electrophilic species can covalently modify important

biological macromolecules and thereby increase the risk of adverse drug reactions or idiosyncratic toxicity. Consequently, screening compounds for their propensity to form reactive metabolites has become an integral part of drug discovery programs. This screening process typically involves identification of “structural alerts” as well as the generation of reactive metabolites in vitro in subcellular hepatic fractions, followed by trapping the reactive species with nucleophiles, and characterisation via LC/MS. This paper presents evidence for the bioactivation of a series of aminopyrazole derivatives via LC/MS detection of glutathione ethyl ester trapped reactive intermediates formed in human liver microsomal incubations. These results indicate that the aminopyrazole motif, within specific contexts, may be considered a new structural alert for the potential formation of reactive metabolites. Keywords:

aminopyrazole,

reactive

metabolites,

glutathione,

iminopyrazole, thiol trapping


bioactivation,


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Introduction Drug-induced toxicity has been estimated to account for the attrition of approximately one-third of drug candidates and is a major contributor to the high cost of drug development, particularly when not recognised until late in clinical development or post-marketing.3 The application of early high-throughput assays for identifying the potential for drug toxicity is therefore of great interest to the pharmaceutical industry. One concept that has being linked to drug toxicity is the metabolic “bioactivation” of drugs to form reactive metabolites (RMs), which are invariably electrophilic in nature and may covalently modify important biological macromolecules.4-7 Although the exact relationship between RM formation and druginduced toxicity is complex and involves a cascade of biological responses that are not well understood,8, 9 screening for RMs has become an integral part of the drug discovery process to reduce toxicological risk.10 The tendency of a drug candidate to undergo bioactivation to a RM will depend on several factors including: (1) whether the molecule possesses a functionality and/or architecture that is susceptible to bioactivation, (2) the presence of alternate metabolic “soft” spots within the molecule that compete with bioactivation, and (3) the efficiency of detoxification of a RM and/or its precursor by metabolizing enzymes.11, 12 The earliest indication for known RM formation is often provided by software packages such as DEREK (deductive estimation of risk from existing knowledge),13, 14 or by visual inspection of the molecule for “structural alerts” which are known to generate RMs in certain configurations. Of these structural alerts, those frequently associated with severe toxicities include anilines and anilides; arylacetic and arylpropionic acids; hydrazines and hydrazides; thiophenes; nitroaromatics; and structures that either contain or form α,β-unsaturated carbonyl-


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like structures, including quinones and quinone-methides.15-19 In addition, it is essential to determine experimentally whether these functional groups, if present in a candidate of interest, are susceptible to metabolic activation. For example, thiophene, a well-known RM forming motif, may not be accessible to drug metabolising enzymes when incorporated in certain configurations and therefore be silent with respect to bioactivation.20 Since most RMs are short-lived and not detectable in the circulation, in vitro studies in subcellular hepatic fractions are generally used to trap RMs with a suitable nucleophile thereby forming stable adducts that can be characterised by LC/MS.11 The current list of structural alerts, albeit extensive, is knowledge-based and thus will continue to evolve as new functionalities are found to undergo bioactivation.2 This paper presents evidence that the aminopyrazole motif, at least in specific structural contexts, might need to be considered a novel structural alert for the formation of RMs. Aminopyrazoles are versatile heterocyclic building blocks often employed in medicinal chemistry programs due to their favourable physical and chemical properties as compared to chemically similar moieties such as anilines21 It is generally thought that reducing lipophilicity of molecules potentially decreases the risk of off target activity, also decreasing the formation of RMs potentially reduces the risk of idiosyncratic toxicological effects such as those associated with anilines.10, 22-25

There is an increasing number of drug discovery programs that are using

aminopyrazoles as a versatile functional group;21, 26-35 to the best of our knowledge there are no approved drugs that contain this chemotype however they are present in compounds that are currently in clinical trials.26-28 Herein we disclose the ability of a series of aminopyrazole derivatives to generate reactive intermediates that form conjugates with glutathione ethyl ester (GSH-EE) in vitro following incubation with



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human liver microsomes and outline a suggested substitution pattern that may impede the formation of these conjugates and in turn, improve the stability of the aminopyrazole group. Experimental Procedures Materials. Reduced GSH-EE and 1-aminobenzotriazole (ABT) were obtained from Sigma Aldrich (St Louis, MO, USA). Human liver microsomes (Lot #1210057, pool of 200 (100 male and 100 female)) were obtained from Xenotech (Lenexa, Kansas, USA). Stock solutions of compounds were prepared in DMSO (10 mg/mL) with subsequent dilution with 50% acetonitrile/water. Stock solutions of GSH-EE and ABT (50 mM) were prepared on the day of the experiment in 10% acetonitrile/water. Microsomal incubations were conducted in 0.1 M phosphate buffer (pH 7.4) containing an NADPH-regenerating system (1 mg/mL NADP, 1 mg/mL glucose-6phosphate, 1 U/mL glucose-6-phosphate dehydrogenase) and MgCl2.6H2O (0.67 mg/mL). Synthesis of aminopyrazole derivatives. Synthesis and characterisation of the aminopyrazole compounds are described in the supporting information. LC/MS conditions. Sample analyses were conducted on a Waters Xevo G2 QTOF time-of-flight mass spectrometer coupled to a Waters Acquity UPLC (Waters Corporation, Milford, MA, USA) operating in positive mode electrospray ionisation at 100 °C source temperature, 650 °C desolvation temperature, 3.5 kV capillary voltage, 30 V cone voltage, desolvation gas flow (nitrogen) 1000 L/h and cone gas flow (nitrogen) 100 L/h. Data acquisition was performed employing MassLynx software in MSE mode over a mass range of 60 to 1400 Daltons. Mass calibration was


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performed using sodium formate adducts and leucine enkephalin as accurate mass reference (lockmass 556.2771). Chromatographic separation was achieved on a Supelco Ascentis Express RP Amide column (50 mm x 2.1 mm, 2.7 µm, Bellefonte, PA, USA) equipped with a Phenomenex Security Guard column with a Luna C8 cartridge (Torrance, CA, USA) and both were maintained at a temperature of 40 °C. Analysis was performed using an acetonitrile-water gradient containing 0.05% formic acid at a flow rate 0.4 mL/min and an injection volume of 5 µL. Mobile phase A contained 0.05% formic acid in MilliQ water and mobile phase B contained 100% acetonitrile. Chromatography was conducted using the following gradient with: 0% B from 0 to 0.2 min, 0% to 2% B from 0.2 to 0.8 min, 2% to 30% B from 0.8 to 3.0 min, 30% to 90% B from 3.0 to 4.7 min, 90% to 95% B from 4.7 to 4.8 min, at 95% B from 4.8 to 5.3 min and 95% to 0% B from 5.3 to 5.5 min and equilibrate at 0% B for 0.5 min prior to the next injection. Microsomal incubation conditions and reactive metabolite trapping. The reactivity of the aminopyrazole analogues with a thiol trapping reagent, GSH-EE, was examined in human liver microsomes at a substrate concentration of 10 µM, enzyme protein concentration of 1 mg/mL and GSH-EE concentration of 1 mM. To examine the role of cytochrome P450 (P450) enzymes in the bioactivation of these analogues, microsomes containing the NADPH-regenerating system were pre-incubated at 37 °C in a 96-well plate in the presence or absence of 1 mM ABT for 10 min. Thereafter, the metabolic reaction was initiated by the addition of the substrate with or without GSHEE and the samples were incubated for up to 120 min. Aliquots of the reaction mixture (100 µL) were quenched by the addition of ice-cold quench solution (150 µL acetonitrile containing 0.15 µg/mL diazepam as internal standard). The quenched samples were centrifuged at 4500 x g for 3 min at room temperature and the



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supernatant was transferred to a second 96-well plate for analysis. Control microsomal incubations containing no NADPH were included to monitor for potential non-NADPH dependent formation of GSH-EE conjugates whilst phosphate buffer incubations were included to monitor for potential non-enzymatic formation of GSHEE conjugates. To estimate the reactivity of the analogues, relative adduct formation was calculated using the peak area of the GSH-EE conjugate at the end of the two hour incubation with NADPH-supplemented human liver microsomes in the presence of GSH-EE, compared to the peak area of the parent at the start of the incubation. Results Detection of GSH-EE conjugates in human liver microsome incubations. In NADPH-supplemented human liver microsomes in the presence of GSH-EE, GSHEE conjugates were detected for six of the fifteen aminopyrazoles. One conjugate was detected for each of the 4-aminopyrazoles, 1, 3, 5, 7 and 11 whilst two adducts were detected for the 3-aminopyrazole, 13. No GSH-EE adducts were detected for the Nmethyl aminopyrazole analogues, 2, 4, 6, 8, 12, 14 and 15 or the 3,5-dimethyl analogue, 9, and its N-methyl derivative, 10 (Table 1). The formation of GSH-EE adducts was greatly inhibited following pre-incubation of NADPH-supplemented human liver microsomes with ABT (i.e. > 69% decrease in the peak area of the conjugate). In the absence of NADPH (control incubations) but in the presence of GSH-EE, the same GSH-EE conjugates were detected for 1, 3, 5 and 11, albeit to a much lesser extent (i.e. equivalent to < 15% of the conjugate peak area that was observed in NADPH-supplemented incubations). No GSH-EE conjugates were detected for 7 and 13 in control incubations. In the absence of human liver


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microsomes (i.e. in phosphate buffer), with and without cofactor, and in the presence of GSH-EE, no GSH-EE conjugates were detected for any of the aminopyrazole derivatives. In NADPH-supplemented human liver microsomes, all eight N-methyl aminopyrazoles, 2, 4, 6, 8, 10, 12, 14 and 15 were metabolised, to varying degrees, to their corresponding demethylated derivatives, 1, 3, 5, 7, 9, 11, 13 and 13, respectively and in the presence of GSH-EE, the same six conjugates of the corresponding demethylated analogues, 1, 3, 5, 7, 11 and 13 were observed as above. Pre-incubation of human liver microsomes with ABT inhibited the demethylation of the eight Nmethyl aminopyrazoles. Structural assignment of GSH-EE adducts. Formation of GSH-EE adducts was confirmed by accurate mass measurement and observation of the characteristic fragmentation of the glutamate side chain (loss of 129 Daltons) and GSH-EE (loss of 301 Daltons). LC/MS (Figure S1) and MS/MS data (Figure S2) are depicted in the supporting information. Estimation of the relative conjugate formation. Among the 4-aminopyrazoles, chlorination of the pyrimidine substituent in 3 appeared to increase reactivity when compared to the corresponding non-halogenated and trifluoromethyl analogues, 1 and 5 respectively (Table 1). Methyl substitution on the beta carbon or the presence of a heteroatom in the pyrazole beta to the amino group appeared to decrease the reactivity for the formation of GSH-EE adducts as seen for compounds 7 and 13.



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Discussion Reactive metabolite formation is considered a significant contributor to druginduced toxicities necessitating the early detection of risks associated with reactive functional groups.13, 36 One of the most common screening methods utilised is the in vitro thiol trapping of RMs using glutathione or its ethyl ester derivative, GSH-EE. Thiols are classified as ‘soft’ nucleophiles37 and will therefore react with ‘soft’ electrophiles such as quinones, quinone imines, iminoquinone methides, epoxides, arene oxides and nitrenium ions.38 Reaction of ‘hard’ electrophiles with GSH-EE is an inefficient process and therefore these types of RMs will not be reliably identified in such a screen.38 The bioactivation of five membered aromatic heterocycles that contain one heteroatom such as furan, thiophene and pyrrole, has been well documented39, 40 and primarily proceeds via epoxidation of the electron rich 2,3-double bond followed by either epoxide ring opening to yield a reactive aldehyde intermediate or further oxidation to a lactone, both of which form adducts.15, 39, 41, 42 Pyrazole, on the other hand, is relatively stable with respect to oxidative metabolism owing to the additional heteroatom in its five membered ring. The extra nitrogen atom results in a less πelectron rich system while also providing a site for protonation that can alter the available metabolic bioactivation pathways.39 Analysis of the aminopyrazoles in this study showed that both the 3- or 4aminopyrazole can result in the formation of GSH-EE conjugates. One conjugate was detected for each of the 4-aminopyrazoles, 1, 3, 5, 7 and 11 whilst, two isomeric adducts were detected for the 3-aminopyrazole, 13.

Relative adduct formation,

whereby the LC/MS signal of each adduct is normalised to its parent, is reported as an attempt to broadly estimate the susceptibility of each of the analogues to GSH-EE


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conjugation. Based on this data, it appears that formation of the aminopyrazole conjugates are largely independent of substitution (with H, Me, Cl or CF3 groups) on the adjacent pyrimidine core and that 4-aminopyrazoles are likely to be more reactive than 3-aminoprrazoles. The risk of oxidative metabolism of electron rich ring systems can often be reduced by substitution with electron withdrawing or bulky substituents. For example, substitution at all four positions of the pyrrole ring in the lipid lowering agent Atorvastatin protects the drug from oxidative metabolism of the pyrrole ring.43 In the current study, the presence of pyrazole N-methylation in the analogues, 2, 4, 6, 8, 12, 14 and 15 mitigates the formation of GSH-EE adducts. The N-methylation is possibly blocking the iminopyrazole formation by removing the pyrazole NH, thus stopping the elimination and hence the nucleophilic attack from GSH-EE. Alternatively, the pyrazole NH could directly be the point of oxidation, however due to the decreased πelectron rich pyrazole system it is likely that the N-OH formation occurs on the amine. The 3,5-dimethyl analogue, compound 9, demonstrates that substituting both pyrazole C-H carbons with a methyl group also hinders GSH-EE conjugation. In contrast, adduct formation is observed for analogue 7, indicating that methylation of only one of the pyrazole 3 and 5 carbons possibly leads to a decrease in steric interaction when compared to compound 9, and a nucleophilic attack from the GSHEE to the reactive imine. Taken together, we propose that the mechanism of aminopyrazole bioactivation involves oxidation of the aminopyrazole, resulting in the formation of a reactive iminopyrazole intermediate which undergoes nucleophilic attack from GSH-EE (Figure 1). Since putative epoxide conjugates were not observed for the current aminopyrazole series, the proposed mechanism of aminopyrazole bioactivation more closely resembles that of p-hydroxylated anilines such as



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acetaminophen (APAP), which undergoes oxidation to the chemically reactive quinone-imine species, N-acetyl-p-benzoquinone-imine (NAPQI),15 rather than via the epoxide intermediate as reported for pyrrole, furan and thiophene.15,

39, 41, 42

Recently, Srivastava et al. reported aminoimidazoles as potential toxicophores and in addition to identifying reactive epoxide intermediates, the authors described the formation of imine-methide and quinone-imine species.44 For the most part, it is believed that RMs are generated by P450 catalysed oxidation reactions,45 for instance it is universally accepted that the bioactivation of APAP to NAPQI is a P450-dependent (primarily CYP2E1) mechanism.46 In the present study, pre-incubation of microsomes with the irreversible P450 inhibitor ABT, reduced the formation of GSH-EE trapped conjugates in NADPH supplemented human liver microsomes suggesting that the bioactivation of aminopyrazoles to iminopyrazole reactive intermediates is primarily mediated via NADPH-dependent P450 enzymes. Flavin monooxygenases (FMO),47 monoamine oxidases (MAO)48 and heme-containing peroxidases49 as well as autoxidation50 have also being shown to play a role in oxidation-mediated bioactivation of drugs. For instance, fluoro-Nmethylaniline forms a quinoneimine reactive intermediate via FMO1 mediated oxidation and defluorination,47 haloperidol, a clinically used neuroleptic agent, has being shown to undergo MAO-B induced bioactivation to a pyridinium metabolite48 while

amodiaquine,

an

antimalarial

associated

with

agranulocytosis

and

hepatotoxicity in man, forms a chemically reactive quinoneimine in neutral solution under air as well as via peroixdases and P450 mechanisms.50 In the present study, certain aminopyrazole derivatives appeared to form covalent adducts with GSH-EE directly in liver microsomes in the absence of NADPH (albeit with an LC/MS signal much lower than that observed in the presence of NADPH) suggesting an additional


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P450 independent enzymatic or chemical mechanism. Since no adducts were formed in the absence of liver microsomes, thus eliminating the possibility of autooxidation, the formation of non-NADPH mediated iminopyrazole reactive intermediates most likely involves other non-P450 enzymes, further investigation is required to determine the exact mechanism of this metabolic pathway. Overall, we present evidence for the in vitro bioactivation of unsubstituted 3and 4-aminopyrazoles which appeared to proceed via oxidation to form a conjugated imine intermediate. We have shown that the reactivity of this class of compounds may be attenuated by hindering the formation of the imine resonance structure by blocking the addition of GSH-EE by methyl substitution on the carbons beta to the amine on 4aminopyrazole or by N-methylation. However, N-dealkylation of pyrazoles is a major metabolic pathway and the resultant metabolite would potentially be able to form the reactive iminopyrazole intermediate.

Funding Source The authors would like to thank the Australian government for funding through the Cooperative Reasearch Ceters (CRC) and the Centre for Drug Candidate Optimisation.

Acknowledgment The authors would like to acknowledge Dr Scott Walker and Dr Paul Stupple for their contribution with the preparation of this manuscript.



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Supporting Information Available Synthesis and characterisation of the aminopyrazole compounds. LC/MS (Figure S1) and MS/MS data (Figure S2) for observed GSH-EE adducts. This material is available free of charge via the internet at http://pub.acs.org. Abbreviations ABT: 1-Aminobenzotriazole, GSH-EE: Glutathione Ethyl Ester, MS/MS: Mass Spectrometry / Mass Spectrometry, NAPQI: N-Acetyl-p-benzoquinone-imine, RMs: Reactive Metabolites, UPLC: Ultra Performance Liquid Chromatography.

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Table 1. Detection of aminopyrazole trapped reactive metabolites in NADPHsupplemented HLMs employing GSH-EE as a trapping agent. GSH-EE Adduct Detected

MH+ Adduct (theoretical)

MH+ Adduct (observed)

1

Yes

524.2040

524.2015 -0.0025

0.060

2

No

538.2197

-

-

3

Yes

558.1650

558.1600 -0.0050

4

No

572.1807

-

5

Yes

592.1914

592.1910 -0.0004

0.026

6

No

606.2071

-

-

7

Yes

606.2071

606.2005 -0.0066

0.005

8

No

620.2227

-

-

-

9

No

620.2227

-

-

-

10

No

634.2384

-

-

-

11

Yes

592.1914

592.1910 -0.0004

0.029

12

No

606.2071

-

-

13

Yes b

592.1914

592.1811 -0.0103

0.003

14

No

606.2071

-

-

-

15

No

606.2071

-

-

-

Compound

Structure

a

∆ MH+ (theoretical – observed)

-

Relative adduct formationa

0.093

-

-

-

Relative adduct formation is based on peak area of the GSH-EE conjugate (at the end of the 2 hour incubation) over the peak area of the parent (at the start of the incubation) observed when the parent compound is incubated with NADPHsupplemented human liver microsomes in the presence of GSH-EE. b Two GSH-EE conjugates were detected for compound 13.



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Figure 1 Figure Legends Figure 1. Proposed bioactivation mechanism of aminopyrazoles. Mechanism involves oxidation to a transient reactive iminopyrazole which undergoes nucleophile attack from GSH-EE.


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Evidence for the in Vitro Bioactivation of Aminopyrazole Derivatives

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