ARTICLE pubs.acs.org/crt
Bioactivation of Substituted Thiophenes Including r-Chlorothiophene-Containing Compounds in Human Liver Microsomes Weiqi Chen, Janet Caceres-Cortes, Haiying Zhang, Donglu Zhang, W. Griffith Humphreys, and Jinping Gan* Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Research and Development, Princeton, New Jersey 08543, United States
bS Supporting Information ABSTRACT: The thiophene moiety has been recognized as a toxicophore because of the potential of oxidative bioactivation leading to electrophilic species. The introduction of bulky or electron-withdrawing groups at the R-carbon to the sulfur atom has the potential to reduce or eliminate bioactivation. In this article, we describe the bioactivation of a variety of substituted thiophenes. These compounds were incubated in NADPH-fortified human liver microsomes with or without the addition of reduced glutathione (GSH) as a trapping agent. The resulting GSH adducts were characterized by high performance liquid chromatography/high resolution mass spectrometry with the aid of a background subtraction methodology. Four of the five R-chlorothiophenes tested formed NADPH-dependent GSH adducts. Most adducts had masses consistent with the nominal substitution of chlorine by GSH. LC/MS/MS and proton NMR of the major GSH adduct of 1-(5-chlorothiophen-2-yl)ethanone (1a) confirmed that GSH displaced chlorine. To further explore the effect of different substitutions on the bioactivation potential, a series of 2-acetylthiophenes substituted at the C4 or C5 positions were tested in a quantitative thiol-trapping assay using dansyl glutathione. Substitutions at the C4 or C5 positions gave adduct levels that decreased in the following order: 4-H, 5-H (no substitution) > 4-Br ∼ 4-Cl > 5-Cl > 5-CN > 4-CH3 > 5-Br > 5-CH3 (no adduct detected). In conclusion, bioactivation was detected in a series of substituted thiophenes. Although substitutions on the thiophene ring can reduce the formation of reactive metabolites, the degree of reduction is dependent on the substitution position and substituent.
’ INTRODUCTION The thiophene moiety is a known structural alert due to a high potential for bioactivation which in some cases has been linked to toxicity.15 Medicinal chemistry approaches to minimize the bioactivation potential of thiophenes often include replacement of the thiophene with other heterocycles or introduction of substituents at the R-carbon position to provide steric hindrance and a potential site for metabolism or to serve as an electron withdrawing group. One such thiophene derivative that is often used in medicinal chemistry is R-chlorothiophene, and there are several examples of marketed drugs that containing this moiety such as tioconazole, lornoxicam, and rivaroxaban. However, there is no literature on the effectiveness of such substitution in reducing the bioactivation potential of the thiophene moiety. It is well established that bioactivation of thiophene-containing compounds can be initiated by both S-oxidation and epoxidation57 to form reactive intermediates. These intermediates effectively react with thiol trapping agents leading to the formation of multiple products, including S-oxide thiol adducts and thiol adducts with apparent direct thiol addition to the thiophene ring. The direct addition products likely arise from addition of thiol to an epoxide followed by the loss of water or from thiol addition to a S-oxide followed by reduction of the S-oxide by excess thiols in the incubations. Additionally, r 2011 American Chemical Society
thiophene S-oxides are also known to form dimers via DielsAlder reactions.7 Glutathione (GSH)-trapped reactive metabolites can be detected using a number of liquid chromatography/mass spectrometry (LC/MS)-based techniques.8,9 Some of these techniques include neutral loss scanning of the glutamate residue (129 Da), precursor ion scanning of an anion fragment at m/z 272 in the negative ionization mode, the use of stable-isotope labeled glutathione to improve analytical efficiency, and the use of high resolution mass spectrometry-based mass defect filtering to facilitate detection. Most of the techniques target a specific behavior or property of GSH adducts, although not all GSH adducts behave the same way or follow the same fragmentation pattern. Recently, a high resolution mass spectrometry-based background subtraction technique has been reported, which seems promising to discern drug metabolite peaks in complex biological samples without the need for predicting their masses or fragmentation patterns.1012 The utility of this background subtraction technique for screening GSH-trapped reactive metabolites has been demonstrated.10,11 Received: November 3, 2010 Published: March 22, 2011 663
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Figure 1. Chemical Structures of R-chlorothiophenes tested (1ae) for GSH adduct formation and R-acetylthiophenes tested (2a2g and 1a) for dGSH adduct formation.
In this study, we describe the screening of a series of substituted thiophene compounds (Figure 1) for bioactivation potential after incubation in human liver microsomes (HLM) in the presence of NADPH and GSH. Initially, a series of 2-chloro5-substituted thiophenes was examined followed by an experiment to determine the structurebioactivation potential relationship of a series of 2-acetylthiophene analogues with substitutions on the C5- and C4-positions (R1 and R2, respectively). In order to develop the structurebioactivation relationship, the study was done with dansyl glutathione (dGSH) in HLM to allow a more quantitative estimate of adduct levels.13 The adduct masses were determined by linear ion-trap mass spectrometry. Nuclear magnetic resonance (NMR) spectra were utilized to characterize a representative GSH adduct, and a mechanism was proposed for the formation of this adduct.
reaction was terminated by the addition of 1 volume of acetonitrile. The tubes were vortexed and centrifuged, and the resulting supernatants were stored at 80 °C before analysis.
In Vitro Incubations for the Quantitation of dGSH Adducts. Test compounds (30 μM) were preincubated in 0.1 M potassium phosphate buffer (pH 7.4) with 1 mg/mL HLM and 1 mM dGSH for 5 min at 37 °C. NADPH (1 mM final concentration) was then added to initiate the reaction. The final incubation volume was 1 mL. After 30 min, the reaction was terminated by the addition of 2 volumes of ice-cold methanol with 5 mM thiothreitol (DTT). The tubes were vortexed and centrifuged, and the resulting supernatants were stored at 80 °C before analysis. LC/MS Method for the Identification of GSH Adducts. LC/ MS data were acquired using a Thermo Surveyor HPLC and a Thermo LTQ-Orbitrap Discovery (Thermo Fisher, CA). Adduct separation was carried out on a Waters Atlantis C18 column (150 2.1 mm, 5 μm, Waters, Milford, MA) using 0.1% formic acid as mobile phase A and acetonitrile as mobile phase B at a flow rate of 0.25 mL/min. The gradient was maintained at 10% B for 2 min and increased to 50% B in 18 min, followed by 50% B to 90% B in 1 min, and was maintained at 90% B for 4 min prior to column re-equilibration. The HPLC eluent was introduced into an electrospray ionization source in positive ionization mode with a source voltage of 5 Kev and a source temperature of 300 °C. Full scan spectra were acquired at an m/z range of 200 to 900 with data-dependent acquisition of MS2 for the most intense m/z peaks in the spectrum. The adduct peaks were identified by visually inspecting the base peak chromatograms in comparison with their respective controls in the absence of NADPH. The full scan mass chromatograms were further interrogated for adduct peaks by using an in-house background subtraction program to subtract background ions from incubations either without NADPH or without the study compound. The MS2 and MS3 spectra of the resulting adduct peaks were used to aid the positive identification of GSH adducts formed.
’ EXPERIMENTAL PROCEDURES Materials. Pooled human liver microsomes (HLM) and human liver S9 fractions were obtained from BD Gentest (Woburn, MA), and dansylated reduced glutathione (dansyl glutathione, dGSH) was synthesized and purified in house according to published procedures.13 Reduced glutathione (GSH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), 3,4-dichloronitrobenzene, 1-(5-chlorothiophen-2-yl)ethanone (1a), 1-(thiophen-2-yl)ethanone (2a), 1-(5methylthiophen-2-yl)ethanone (2b), 1-(5-bromothiophen-2-yl)ethanone (2c), and 1-(4-methylthiophen-2-yl)ethanone (2e) were purchased from Sigma-Aldrich (Milwaukee, WI); 2-chloro-5-methylthiophene (1d), 5-acetylthiophene-2-carbonitrile (2d), and 1-(4-chlorothiophen2-yl)ethanone (2f) were from Alfa Aesar (Ward Hill, MA); 1-(4bromothiophen-2-yl)ethanone (2g) was from Biogene Organics (The Woodlands, TX); 5-chlorothiophene-2-carboxylic acid (1b) was from Lancaster Synthesis (Windham, NH); 5-chloro-2-thienyphenyl methanone (1c) was from Ryan Scientific Inc. (Mt. Pleasant, SC); and N-(4acetylphenyl)-5-chlorothiophene-2-carboxamide (1e) was from Matrix Scientific (Columbia, SC). All other reagents and solvents were of analytical reagent grade or better.
HPLC/UV/Fluorescence Method for the Quantitatation of dGSH Adducts. All incubation samples were injected onto a Shimadzu LC-10Avp HPLC system comprising binary pumps, a well-plate autoinjector (chilled), and a fluorescence detector. Analyte separation was accomplished using a reverse phase HPLC column (Phenomenex Prodigy ODS2, 4.6 mm 150 mm). A shallow mobile phase gradient (3 min at 20% acetonitrile in 0.1% formic acid, ramping up to 50% acetonitrile in 20 min, followed by another ramp to 90% in 10 min) was used to ensure adequate separation of adduct peaks.13 The mobile phases contained 0.1% formic acid in water and acetonitrile.
In Vitro Incubations with HLM for GSH Adduct Screening. Test compounds (30 μM) were preincubated in 0.1 M potassium phosphate buffer (pH 7.4) with 2 mg/mL HLM and 5 mM GSH for 5 min at 37 °C. NADPH (2 mM final concentration) was then added to initiate the reaction. The final incubation volume was 1 mL. After 1 h, the 664
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Figure 2. Example base peak chromatograms of 1d coincubated with NADPH and GSH in HLM over the full scan mass range of m/z 200900 before (A) and after (B) background subtraction. The mass spectra of the GSH adduct at retention time 6.9 min are shown before (C) and after (D) background subtraction. For the determination of dGSH adduct concentration, dansyl-related fluorescence was monitored at λex of 340 nm and λem of 525 nm. The adduct peaks were identified by visual comparison of chromatograms from the incubated samples and their relevant controls. An external standard curve of dGSH (concentration range: 0.068 to 16.67 μM) was used for the quantitative determination of dGSH adduct concentrations. LC/MS for dGSH Adduct Mass Determination. LC/MS data were acquired using an Agilent 1100 HPLC binary pump, an Agilent well-plate autoinjector, and a Thermo LTQ XL linear ion trap (Thermo Fisher, CA). For adduct separation, a Waters Atlantis C18 column (150 2.1 mm, 5 μm, Waters, Milford, MA) was used with 0.1% formic acid as mobile phase A and acetonitrile as mobile phase B at a flow rate of 0.25 mL/min. The gradient started by increasing from 10% B to 60% B in 30 min, followed by 60% B to 95% B in 1 min and was maintained at 100% B for 3 min prior to column re-equilibration. The HPLC eluent was introduced to an electrospray ion source with alternating polarity. dGSH was used as the tuning standard for the optimization of source parameters. Nuclear Magnetic Resonance. Compound 1a and its major GSH adduct were each dissolved in 180 μL of DMSO-d6 and placed in 3 mm NMR tubes. The samples were analyzed using a Bruker Avance 600 MHz NMR spectrometer equipped with a 5-mm TCI triple
resonance cryoprobe (Bruker, Billerica, MA) at 27 °C. The 1H and C chemical shifts are reported on the δ scale (ppm) downfield from tetramethylsilane. The peak assignments were based on 1H, 1H-1Hgradient correlation spectroscopy, 1H-13C- heteronuclear single quantum coherence, and multiple-bond correlation spectroscopy (HSQC and HMBC) analyses. Full assignment of all of the protons in the spectra of the GSH adduct was not completed due to limited sample amounts and spectral overlap. 1a: 1H NMR (600 MHz, DMSO-d6): 7.84 (d, J = 4.1 Hz 1H), 7.28 (d, J = 4.1 Hz, 1H) 2.49 (s, 3H). 13C NMR: 190.4, 143.2, 137.5, 133.8, 128.7, 25.6. GSH adduct of 1a (partial assignments): 1H NMR (600 MHz, DMSO-d6): 7.83 (d, J = 3.3 Hz 1H), 7.21 (d, J = 3.3 Hz, 1H), 4.41 (overlap), 3.40 (overlap), 3.13 (overlap), 2.48 (s, 3H). 13C NMR: 189.9, 144.6, 134.5, 130.9, 26.0. 13
’ RESULTS AND DISCUSSION In order to develop a better understanding of the potential for the bioactivation of substituted thiophenes, five R-chlorothiophene-containing compounds were tested for glutathione adduct 665
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Table 1. Accurate Masses, MS Fragments, and the Proposed Chemical Compositions of GSH Adducts from Incubations of Various r-Chlorothiophene Compounds with HLM in the Presence of NADPH and GSH
a
P: parent mass. b Doubly charged ions. ND: not detected. NA: not applicable.
formation. Supernatants from microsomal incubation mixtures were directly injected onto an LC/LTQ-Orbitrap. The full scan LC/MS data were processed with an in-house developed background subtraction methodology.11 This method relies upon the subtraction of accurate masses that are shared by the incubation sample and its controls (incubations either without compound or without GSH) with a mass tolerance of (5 ppm, a retention time tolerance of (0.3 min, and an intensity amplification factor of 50 to ensure adequate subtraction. An intensity value of zero is assigned if the subtraction result is negative. The resulting peaks in the processed mass chromatograms were inspected to determine the molecular ions of the glutathione adducts and to obtain their respective MS2 fragmentation data to confirm GSH adduct formation. Shown in Figure 2 are examples of chromatograms and mass spectra at the retention time of the GSH adduct from injected samples involving incubations with 1d before and after background subtraction. Of the five R-chlorothiophene-containing compounds incubated in HLM with NADPH and GSH, four of them formed
GSH adducts. We checked the turnover of 1b in the incubation mixture by LC/negative electrospray ionization mass spectrometry and found that 1b was stable under the experimental conditions described. For the four compounds with detectable GSH adducts, the accurate masses recorded for the GSH adducts and their proposed chemical compositions are listed in Table 1. The observed accurate masses are all within 0.0005 Da from the predicted accurate masses based on the proposed chemical composition. The majority of the adduct masses are consistent with the apparent substitution of the chlorine atom with a glutathionyl moiety. We obtained the MS2 and MS3 fragmentation spectra of adducts of interest (Supporting Information), and the typical neutral loss of 129 mass units verified the formation of GSH adducts. Compound 1a was selected for further structural determination, and enough 1a GSH adduct was isolated to allow NMR analysis. The proton NMR spectrum of 1a and its GSH adduct are shown in Figure 3. The protons of the 2-acetyl-5chloro-thiophene moiety (H3, H4, and H9) are present in both 666
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spectra. Chemical shift changes of 0.01 ppm were observed for H3 and H9, while a 0.07 ppm change was observed for H4 between 1a and its GSH adduct. The relatively small changes in chemical shifts observed for the protons of 1a suggest that the addition of glutathione does not displace any one of the original protons; therefore, the most plausible structure of the 1a glutathione adduct is as shown in Figure 3B where the glutathionyl group replaced the chlorine at the C5 position. The adduct formation was NADPH-dependent; therefore, it is unlikely a product of microsomal glutathione S-transferase (GST) mediated substitution of chlorine. To confirm the lack of GST involvement in the adduct formation with 1a, additional experiments were conducted with human liver S9 in the absence of NADPH. A glutathione adduct of 1a was not formed under conditions where GST-mediated glutathione replacement of chlorine was observed with a positive control compound, 3,4dichloronitrobenzene (data not shown). Because liver S9
contains both microsomal and cytosolic GST enzymes, we concluded that oxidative enzymes rather than GST are involved in the formation of the 1a glutathione adduct. The nominal substitution of chlorine with glutathione in 1a may be a result from the sequential reaction of bioactivated intermediates with glutathione as shown in Scheme 1. One possible mechanism is adapted from earlier studies by Valadon et al.5 with 3-aroylthiophenes. Briefly, microsomal oxidation of chlorothiophene results in a reactive sulfoxide. This intermediate then reacts with glutathione with opening of the ring leading to a glutathione substituted sulfenic acid. Subsequently, two molecules of glutathione are used in the reduction of sulfenic acid, leading to the formation of a sulfhydryl group. Finally, ring closure leads to the final GSH adduct with the loss of the elements of hydrochloric acid. Alternatively, GSH can displace the chlorine from the sulfoxide intermediate, followed by reduction by either GSH (or possibly NADPH) of the sulfoxide, leading to the same product. The GSH substituted sulfoxide is still activated, and one additional GSH could add to the ring resulting in a bis-glutathionyl sulfoxide, and its subsequent reduction would generate the bis-glutathionyl adduct (observed with 1a and 1c). While there was clearly an adduct with mass consistent with a bis-glutathionyl adduct, the adduct was extremely unstable, and multiple attempts to isolate and perform further structural characterization failed. Mechanisms involving epoxide formation are unlikely for 1a adduct formation. However, epoxide mechanisms could be possible for the adduct formation from 1d, 1e, 2a, 2d, and 2e. Definitive structure elucidation of these adducts may aid the elucidation of bioactivation mechanisms. As a follow-up study, we incubated a series of 2-acetylthiophenes with substitutions on the C4- and C5-positions of the thiophene ring. The substitutions included chlorine, bromine, nitrile, and methyl groups. The objective was to explore a structurebioactivation relationship using dGSH adduct levels as a quantitative readout. Similar to the GSH adduct masses of the chlorothiophenes tested, the masses of the major dGSH adducts from incubations with halogenated 2-acetylthiophenes are consistent with the nominal displacement of halogens with dGSH. The dGSH adducts of 4-methyl and 5-nitrile substituted 2-acetylthiophenes have masses consistent with the oxidative addition of dGSH (parent mass þ dGSH 2H). The two major adducts of unsubstituted 2-acetylthiophene have masses of either
Figure 3. Proton NMR spectra of 1a (A) and its GSH adduct (B). The structures are displayed with atom numbering, and the spectra are labeled accordingly. The intensity of some peaks of the GSH adduct in spectrum B are reduced due to solvent suppression. Impurity peaks present in spectrum B prevented the assignment of some of the GSH protons.
Scheme 1. Possible Mechanisms for the in Vitro Formation of Deschloro-glutathione Adducts from 1aa
a
The steps with two arrows are reduction steps currently not well understood. 667
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Table 2. Extent of dGSH Adduct Formation and Substrate Turnover (As % of Initial Parent Concentrations) and the Mass Assignment of Major Adducts for All Compounds with Detectable dGSH Adduct Formation
compounds
a
R1a
R2a
formation of GSH (% of parent)
number of dGSH adducts
proposed chemical composition of major adduct
2a
H
H
4.14
2
2(PþO)þdGSH-4H and PþdGSH-S
2g
H
Br
2.68
4
PþdGSH-HBr
2f
H
Cl
2.37
3
PþdGSH-HCl
1a
Cl
H
1.67
3
PþdGSH-HCl
2d 2e
CN H
H CH3
0.81 0.62
2 1
PþdGSH-2H PþdGSH-2H
2c
Br
H
0.50
1
PþdGSH-HBr
2b
CH3
H
ND
NA
NA
R1, substituents at position C5; R2, substituents at position C4. ND: not detected. NA: not applicable.
apparent dimerization of sulfoxide plus dGSH or an addition of dGSH with loss of a sulfur atom. The dimerization of thiophene sulfoxides has been described,6 but the loss of a sulfur atom in the oxidation of 2-acetylthiophene has not been reported and deserves further investigation. The quantitative results of the total adduct amounts are summarized in Table 2. Overall, the adduct levels decrease in the order of no substitution (4-H, 5-H) > 4-Br ∼ 4-Cl > 5-Cl > 5-CN > 4-CH3 > 5-Br > 5-CH3. It is clear that substitutions on the C5 position result in greater reduction of bioactivation than the respective substitutions on the C4 position. Methyl substitutions were better than halogens in the reduction of bioactivation as well, with the 5-methyl substitution completely abolishing bioactivation. In summary, a study on the bioactivation of R-chlorothiophene-containing compounds is described. While the R-chloro substitution of thiophenes does seem to reduce bioactivation, it does not completely block bioactivation as evidenced by the formation of glutathione adducts from the majority of the R-chlorothiophenes tested. In addition, a structurebioactivation relationship was explored with a series of C4- and C5-substituted 2-acetylthiophenes. It is concluded that the introduction of alkyl substitutions on the C5 position may be optimal in order to reduce the bioactivation potential of acetylthiophene-containing compounds.
’ REFERENCES (1) Nelson, S. D. (2001) Structure toxicity relationshipshow useful are they in predicting toxicities of new drugs? Adv. Exp. Med. Biol. 500, 33–43. (2) Dalvie, D. K., Kalgutkar, A. S., Khojasteh-Bakht, S. C., Obach, R. S., and O’Donnell, J. P. (2002) Biotransformation reactions of fivemembered aromatic heterocyclic rings. Chem. Res. Toxicol. 15, 269–299. (3) Joshi, E. M., Heasley, B. H., Chordia, M. D., and Macdonald, T. L. (2004) In vitro metabolism of 2-acetylbenzothiophene: relevance to zileuton hepatotoxicity. Chem. Res. Toxicol. 17, 137–143. (4) Graham, E. E., Walsh, R. J., Hirst, C. M., Maggs, J. L., Martin, S., Wild, M. J., Wilson, I. D., Harding, J. R., Kenna, J. G., Peter, R. M., Williams, D. P., and Park, B. K. (2008) Identification of the thiophene ring of methapyrilene as a novel bioactivation-dependent hepatic toxicophore. J. Pharmacol. Exp. Ther. 326, 657–671. (5) Valadon, P., Dansette, P. M., Girault, J.-P., Amar, C., and Mansuy, D. (1996) Thiophene sulfoxides as reactive metabolites: formation upon microsomal oxidation of a 3-aroylthiophene and fate in the presence of nucleophiles in vitro and in vivo. Chem. Res. Toxicol. 9, 1403–1413. (6) Dansette, P. M., Bertho, G., and Mansuy, D. (2005) First evidence that cytochrome P450 may catalyze both S-oxidation and epoxidation of thiophene derivatives. Biochem. Biophys. Res. Commun. 338, 450–455. (7) Treiber, A., Dansette, P. M., El Amri, H., Girault, J.-P., Ginderow, D., Mornon, J.-P., and Mansuy, D. (1997) Chemical and biological oxidation of thiophene: preparation and complete characterization of thiophene S-oxide dimers and evidence for thiophene s-oxide as an intermediate in thiophene metabolism in vivo and in vitro. J. Am. Chem. Soc. 119, 1565–1571. (8) Ma, S., and Subramanian, R. (2006) Detecting and characterizing reactive metabolites by liquid chromatography/tandem mass spectrometry. J. Mass Spectrom. 41, 1121–1139. (9) Ma, S., and Zhu, M. (2009) Recent advances in applications of liquid chromatography-tandem mass spectrometry to the analysis of reactive drug metabolites. Chem.-Biol. Interact. 179, 25–37. (10) Zhang, H., Ma, L., He, K., and Zhu, M. (2008) An algorithm for thorough background subtraction from high-resolution LC/MS data: application to the detection of troglitazone metabolites in rat plasma, bile, and urine. J. Mass Spectrom. 43, 1191–1200. (11) Zhang, H., and Yang, Y. (2008) An algorithm for thorough background subtraction from high-resolution LC/MS data: application for detection of glutathione-trapped reactive metabolites. J. Mass Spectrom. 43, 1181–1190.
’ ASSOCIATED CONTENT
bS
Supporting Information. MS2 spectra of GSH adducts from 1a and 1d. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Phone: (609) 252-7885. E-mail:
[email protected].
’ ABBREVIATIONS GSH, reduced glutathione; LC/MS, liquid chromatography/ mass spectrometry; dGSH, dansyl glutathione; NMR, nuclear magnetic resonance; HLM, human liver microsomes; GST, glutathione S-transferase. 668
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(12) Zhu, P., Ding, W., Tong, W., Ghosal, A., Alton, K., and Chowdhury, S. (2009) A retention-time-shift-tolerant background subtraction and noise reduction algorithm (BgS-NoRA) for extraction of drug metabolites in liquid chromatography/mass spectrometry data from biological matrices. Rapid Commun. Mass Spectrom. 23, 1563–1572. (13) Gan, J., Harper, T. W., Hsueh, M. M., Qu, Q., and Humphreys, W. G. (2005) Dansyl glutathione as a trapping agent for the quantitative estimation and identification of reactive metabolites. Chem. Res. Toxicol. 18, 896–903.
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