Electrochemical Oxidation of Troglitazone: Identification and

Sep 13, 2008 - Steen H. Hansen,† and Jørgen Olsen|. Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, UniVersi...
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Chem. Res. Toxicol. 2008, 21, 2035–2041

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Electrochemical Oxidation of Troglitazone: Identification and Characterization of the Major Reactive Metabolite in Liver Microsomes Kim G. Madsen,*,† Gunnar Gro¨nberg,‡ Christian Skonberg,† Ulrik Jurva,§ Steen H. Hansen,† and Jørgen Olsen| Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, UniVersity of Copenhagen, Denmark, Medicinal Chemistry and DMPK & Physical Chemistry, Lead Generation, AstraZeneca R&D, Mo¨lndal, Sweden, and ADME & Assay Technology, NoVo Nordisk A/S, DK-2760 MåløV, Denmark ReceiVed June 18, 2008

Troglitazone (TGZ) was developed for the treatment of type 2 diabetes but was withdrawn from the market due to hepatotoxicity. The formation of reactive metabolites has been associated with the observed hepatotoxicity. Such reactive metabolites have been proposed to be formed via three different mechanisms. One of the proposed mechanisms involves the oxidation of the chromane moiety of TGZ to a reactive o-quinone methide. The two other mechanisms involve metabolic activation of the thiazolidinedione moiety of TGZ. In the present study, it is shown that electrochemical oxidations can be used to generate a reactive metabolite of TGZ, which can be trapped by GSH or N-acetylcysteine. From incubations of TGZ with rat and human liver microsomes in the presence of either GSH or N-acetylcysteine, it was shown that similar conjugates were formed in vitro as formed from electrochemical oxidations of TGZ. One- and two-dimensional NMR studies of the troglitazone-S-(N-acetyl)cysteine conjugate revealed that N-acetylcysteine was attached to a benzylic carbon in the chromane moiety, showing that the conjugate was formed via a reaction between the o-quinone methide of TGZ and N-acetylcysteine. From electrochemical oxidations of rosiglitazone, pioglitazone, and ciglitazone in the presence of GSH, no GSH conjugates could be identified. These three compounds all contain a thiazolidinedione moiety. In conclusion, it has been shown that the primary reactive metabolite of TGZ formed from electrochemical oxidation was the o-quinone methide, and this metabolite was similar to what was observed to be the primary reaction product in human and rat liver microsomes. Introduction 1

Troglitazone (TGZ) belongs to the thiazolidinedione (TZD) structural class of compounds, developed for the treatment of type 2 diabetes. The use of TGZ has been associated with hepatotoxicity, and it has therefore been withdrawn from the market (1). At present, there are different theories about the cause of the toxicity, one being that reactive metabolites might be involved (1). Kassahun et al. identified five different glutathione (GSH) conjugates from incubations of TGZ with human liver microsomes. One of these GSH conjugates was proposed to be formed via a reactive o-quinone methide, formed from oxidation of the chromane moiety of TGZ, which was supported by 1H NMR studies (Scheme 1) (2). In contrast, Tettey et al. identified a GSH conjugate with an identical m/z value but with another retention time (3), rather than the conjugate proposed by Kassahun et al. Tettey et al. proposed that the GSH * To whom correspondence should be addressed. † University of Copenhagen. ‡ Medicinal Chemistry, AstraZeneca R&D. § DMPK & Physical Chemistry, Lead Generation, AstraZeneca R&D. | Novo Nordisk A/S. 1 Abbreviations: CGZ, ciglitazone; COSY, correlation spectroscopy; GSH, glutathione; HMBC, heteronuclear multiple bond correlation; HSQC, heteronuclear single quantum coherence; NOESY, nuclear Overhauser effect spectroscopy; PGZ, pioglitazone; RGZ, rosiglitazone; ROESY, rotational frame nuclear Overhauser spectroscopy; SRM, selected reaction monitoring; TGZ, troglitazone; TGZ-NAC, troglitazone-S-(N-acetyl)cysteine thioether conjugate; TGZQ, troglitazone quinone; TOCSY, total correlation spectroscopy; TZD, thiazolidinedione.

conjugate was formed from metabolic activation of the TZD moiety (3), which was corroborated by He et al., who supported the hypothesis with 1H NMR data and two-dimensional (2D) total correlation spectroscopy (TOCSY) data (Scheme 1) (4). Rosiglitazone (RGZ) and pioglitazone (PGZ) are two other TZDs in clinical use. The use of RGZ or PGZ is rarely, if ever, associated with hepatotoxicity (5). Alvarez-Sa´nchez et al. have performed a detailed mechanistic study of the bioactivation of TZD-containing compounds by synthesizing TGZ, RGZ, and PGZ with four labeled atoms incorporated into the TZD moiety. A range of GSH conjugates of all three compounds were identified from incubations with human and rat liver microsomes. For RGZ and PGZ, some of the labeled atoms were lost during bioactivation indicating that the TZD moiety underwent some kind of metabolic activation at a substrate concentration of 100 µM, but at concentrations of 10 µM, only traces of GSH adducts could be identified. However, from incubations of TGZ in liver microsomes, the labeling was not seen to be lost in the most dominant GSH adduct, which could be explained by activation of the chromane moiety instead of the TZD moiety. This GSH adduct was proposed to have originated from the reaction between the o-quinone methide and GSH, as observed by Kassahun et al. (2), and it was concluded that TZD ring scission did not contribute significantly to the GSH adduct formation (6). It has previously been shown that it is possible to use electrochemical oxidations for mimicking many oxidative metabolic reactions, including the formation of reactive me-

10.1021/tx8002214 CCC: $40.75  2008 American Chemical Society Published on Web 09/13/2008

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Scheme 1. Proposed Mechanisms for the Bioactivation of TGZ to Reactive Metabolites (2-4)

Scheme 2. Four Compounds Included in the Electrochemical Study of Glitazones

tabolites (7-18). For example, it has been shown that TGZ can be oxidized to the troglitazone quinone (TGZQ) by electrochemical oxidation (19). In the present study, the electrochemical oxidation of TGZ to a reactive intermediate, which could be trapped with GSH or N-acetylcysteine, has been studied. The trapped N-acetylcysteine conjugate was synthesized electrochemically, purified by preparative HPLC, and characterized by MS. The full elucidation of the structure was performed using one-dimensional (1D) and 2D NMR. The conjugation products had identical retention times, m/z values, and fragmentation patterns as the conjugates formed from incubation of TGZ with GSH or N-acetylcysteine, respectively, in liver microsomes. For comparison, RGZ, PGZ, and ciglitazone (CGZ), all containing the TZD moiety, were also subjected to electrochemical oxidation and incubation with liver microsomes, but no significant GSH conjugates were detected. The chemical structures of the compounds included in these studies are shown in Scheme 2.

Experimental Procedures Chemicals and Reagents. TGZ was obtained from Cayman Chemical Co. (Tallinn, Estonia). CGZ was obtained from Calbiochem (San Diego, CA). PGZ and RGZ were kindly provided by Novo Nordisk A/S (Måløv, Denmark). Pooled human liver microsomes (27 individuals) and pooled rat liver microsomes (male, Sprague-Dawley) were obtained from BD Biosciences (St. Woburn, MA). DMSO-d6 was obtained from Armar Chemicals (Do¨ttingen, Switzerland).

Water for mobile phases and solutions for the electrochemical oxidation were purified with a Milli-Q deionization unit Millipore (Molsheim, France). All solvents used for mobile phases and electrochemical oxidation were of HPLC-grade. Instrumentation. LC-MS and LC-MS/MS were performed using a Thermo Finnigan Surveyor LC system coupled to a Thermo Finnigan TSQ Quantum Ultra AM triple quadrupole mass spectrometer with an ESI interface from Thermo Finnigan (San Jose, CA). An Agilent 1100 Series LC coupled to an Agilent single-quadrupole mass spectrometer from Agilent Technologies (Waldborn, Germany) was used for the analyses of the intrinsic clearance. Both mass spectrometers were operated in positive and negative ionization mode. Preparative chromatography was performed using an Agilent 1100 Series LC system equipped with a fraction collector. NMR spectroscopic experiments [1H NMR, 13C NMR, correlation spectroscopy (COSY), heteronuclear single quantum coherence (HSQC), heteronuclear multiple bond correlation (HMBC), nuclear Overhauser effect spectroscopy (NOESY), and rotational frame nuclear Overhauser spectroscopy (ROESY)] were performed on a Bruker 600 MHz instrument equipped with a cryo probe using standard Bruker pulse sequences. The sample was dissolved in 120 µL of DMSO-d6 and transferred to a 2.5 mm NMR tube. The electrochemical oxidations were performed in an ESA 5011 analytical electrochemical cell equipped with a porous graphite working electrode, a Pd counter electrode, and a Pd/ H2 reference electrode. The cell was controlled by an ESA Coulochem potentiostat (model 5100A, ESA Bioscience Inc., Chelmsford, MA). Chromatography. All separations were performed with a Luna C18(2), 3 µm, 100 mm × 2 mm column from Phenomenex (Torrance, CA) using a gradient elution with mobile phase A consisting of 5% acetonitrile and 0.2% formic acid in water and mobile phase B consisting of acetonitrile containing 0.2% formic acid. The gradient was linear from 0 to 95% B over 10 min, which was held for 2 min. Finally, the column was allowed to re-equilibrate for 6 min. The chromatography was performed at 40 °C with a flow rate of 250 µL/min. Preparative chromatography was performed on a Luna C18(2), 3 µm, 150 mm × 4.6 mm column from Phenomenex (Torrance, CA). The chromatography was performed with a mobile phase consisting of 50% acetonitrile and 0.2% formic acid, using a flow rate of 1 mL/min at 40 °C. The compounds were detected by UV spectrophotometry at 235 and 275 nm. Electrochemical Oxidation of TGZ, RGZ, PGZ, and CGZ. Twenty-five micromolar solutions of TGZ, RGZ, PGZ, or CGZ in a 0.1 M phosphate buffer (pH 7.4)/acetonitrile (3:1

Electrochemical Oxidation of Troglitazone

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Scheme 3. Proposed Mechanism for the Electrochemical Formation of TGZQ at +200 mV

v/v) were oxidized in intervals of 100 mV from 0 to +1000 mV vs the Pd/H2 reference electrode. GSH (final concentration, 5 mM) was added to the sample before the oxidation was performed. The solutions were pumped through the electrochemical cell using a syringe pump (50 µL/min), and the samples were collected in vials and analyzed by LC-MS and LC-MS/MS. Electrochemical Synthesis of Troglitazone-S-(N-acetyl)cysteine thioether conjugate (TGZ-NAC). A 100 µM solution of TGZ was oxidized in the presence of N-acetylcysteine (5 mM) in a 0.1 M phosphate buffer (pH 7.4)/acetonitrile (3:1 v/v) at +300 mV. The oxidized sample was collected on ice. The sample was lyophilized, and the remaining solid was redissolved in water/acetonitrile (2:1 v/v). The solution was purified by solid phase extraction (SPE) using an Oasis HLB (3 cc, 50 mg) SPE cartridge from Waters (Milford, MA) to remove the buffer salts and the excess of N-acetylcysteine. The adsorbed compounds were eluted with methanol. Methanol was removed under a stream of nitrogen gas. Finally, the TGZ-NAC was purified by preparative chromatography. Incubations in Human and Rat Liver Microsomes. TGZ, RGZ, PGZ, or CGZ (final concentration 10 µM) were incubated in human and rat liver microsomes (0.5 mg protein/mL). All incubations were performed in a 0.1 M phosphate buffer, pH 7.4, at 37 °C for 30 min. The final concentration of GSH or N-acetylcysteine was 5 mM, and the final concentration of NADPH was 1 mM. The incubations were terminated by transferring the incubation mixture to an equal amount of cold acetonitrile containing 0.2% formic acid. The samples were centrifuged (Eppendorf Centrifuge 5804 R) for 10 min at 4 °C at 4500g. The supernatant was diluted with an equal amount of water and analyzed by LC-MS and LC-MS/MS. Intrinsic Clearance of TGZ, PGZ, RGZ, and CGZ. TGZ, RGZ, PGZ, or CGZ (final concentration 10 µM) were incubated with human and rat liver microsomes (0.5 mg protein/mL). The NADPH concentration was 1 mM. All incubations were performed in a 0.1 M phosphate buffer, pH 7.4, at 37 °C (n ) 2, each compound). The experiments were performed with and without the addition of 5 mM GSH. The incubation was terminated at selected time-points (0, 5, 15, 30, 45, and 60 min) by transferring 150 µL of the mixture to 150 µL of cold acetonitrile containing 0.2% formic acid. The samples were centrifuged, and the supernatant was diluted with an equal amount of water. The samples were analyzed by LCMS.

Results and Discussion Electrochemical and Liver Microsomal Oxidation of TGZ. TGZ was oxidized in the electrochemical cell with GSH

added to the sample prior to the electrochemical oxidation. The applied potential was increased from 0 to +1000 mV in steps of 100 mV, and a sample was collected at each potential. The amount of TGZ in the samples from the electrochemical oxidation rapidly decreased when the applied potential was increased. At +300 mV, it was not possible to detect any TGZ left in the oxidized samples. Two major oxidation products were detected by LC-MS. The first product formed from the electrochemical oxidation of TGZ was detected at m/z 455.9 ([TGZQ - H]-) (tR ) 9.8 min), which corresponded to TGZQ. It has previously been reported by Tahara et al. that TGZQ could be formed from electrochemical oxidation of TGZ (19). The product was formed at +200 mV, and the amount of TGZQ did not increase when the applied potential was increased stepwise from +300 to +1000 mV. On the basis of the MS response of TGZ and TGZQ in negative ESI mode, the conversion efficiency of the method was estimated. At +200 mV, approximately 1% of TGZ was left in the oxidized sample, indicating that the electrochemical cell had a conversion efficiency close to 100%. Assuming that TGZ and TGZQ are ionized to the same degree in the ESI source, approximately 90% of TGZ was converted to TGZQ. The proposed mechanism for the electrochemical formation of TGZQ is shown in Scheme 3. The initial step involves an electron abstraction, leading to oxygen-centered radical cation, which undergoes deprotonation. The oxygen-centered radical rearranges and undergoes a second electron abstraction, leading to a carbocation. A water molecule reacts with the carbocation, and by rearrangement, TGZQ is formed. In human liver microsomes, the formation of TGZQ has been shown to be catalyzed by CYP2C8 and CYP3A4 (20). A metabolite with the same retention time and fragmentation pattern was observed from incubation of TGZ with human liver microsomes, GSH, and NADPH (chromatograms and MS/MS spectra are shown in the Supporting Information, Figure 1). In this context, Yamamoto et al. have identified an epoxide of TGZQ formed in HepG2 cells and human hepatocytes, showing that TGZQ can undergo further oxygenation (21). A product corresponding to the epoxide of TGZQ was not identified from the electrochemical oxidations of TGZ, but it has to our knowledge never been shown that electrochemical oxidations can be used for generating epoxides, which of course is a general limitation of the electrochemical method. It was not possible in the present experiment to identify a product with an m/z value corresponding to the epoxide of TGZQ from the liver microsomal incubations. In addition to the observed TGZQ, a GSH conjugate was detected at m/z 747.2 ([TGZ-SG + H]+) (tR ) 7.5 min) when the sample was oxidized at +200 mV, which corresponds to

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Figure 1. Chromatograms and MS/MS spectra of TGZ-SG ([TGZ-SG + H]+ at m/z 747.2) generated from (A) electrochemical oxidation of TGZ in the presence of GSH in a 0.1 M phosphate buffer (pH 7.4)/ acetonitrile (3:1) at +200 mV; (B) incubation of TGZ with human liver microsomes, NADPH, and GSH in a 0.1 M phosphate buffer (pH 7.4) at 37 °C; and (C) incubation of TGZ with rat liver microsomes, NADPH, and GSH in a 0.1 M phosphate buffer (pH 7.4) at 37 °C.

the addition of GSH to TGZ. The amount of GSH conjugate formed decreased at +300 mV, as compared to +200 mV, and the GSH conjugate was not detected when the applied potential was increased to +400 mV. By MS/MS fragmentation of m/z 747.2 ([TGZ-SG + H]+), fragment ions were detected as follows: m/z 440.1 ([TGZ-SG - GSH - 2H + H]+); m/z 308.0 ([TGZ-SG - TGZ + H]+); m/z 178.9 ([TGZ-SG - GSH C3HNO2S (TZD moiety) - C10H11O]+) (Figure 1). Following incubation of TGZ with human or rat liver microsomes in the presence of GSH and NADPH, a GSH conjugate with the same

Madsen et al.

retention time, m/z value, and fragmentation pattern, as the GSH conjugate formed from electrochemical oxidation of TGZ in the presence of GSH, was identified (chromatograms and MS/ MS data are shown Figure 1). These results show that the same reactive intermediate was formed from both electrochemical oxidations of TGZ and from liver microsomal incubations of TGZ. Approximately 8% of TGZ was oxidized to the o-quinone methide based on the formed amount of TGZ-SG, assuming that the formed o-quinone methide reacts quantitatively with GSH. When a potential from +300 to +600 mV was applied, another GSH conjugate appeared at m/z 763.2 ([T1 + H]+) (tR ) 6.9 min), which corresponds to an addition of 16 Da to the TGZ-SG conjugate. By MS/MS fragmentation of m/z 763.2, fragment ions were detected as follows: m/z 745.1 ([T1 - H2O + H]+); m/z 688.1 ([T1 - glycine + H]+); m/z 634.1 ([T1 glutamic acid + H]+); m/z 616.1 ([T1 - glutamic acid - H2O + H]+); m/z 531.2 ([T1 - glutamic acid - glycine - CO + H]+); m/z 470.2 ([T1 - glutamic acid - glycine - CO CONH (TZD moiety) - H2O + H]+); m/z 393.0 ([T1 glutamic acid - glycine - CO - CONH (TZD moiety) - CO - SH2 - HN3 - H2O]+); m/z 231.2 ([T1 - TGZ-(OH) glycine + H]+); m/z 215.2 ([T1 - TGZQ - glycine - H2O + H]+) (MS/MS spectrum is shown in the Supporting Information, Figure 2). This GSH conjugate was only identified in small amounts in human and rat liver microsomes by a sensitive LCMS method using selected reaction monitoring (SRM) (data not shown). A conjugate with the same m/z value has previously been reported by Kassahun et al., who proposed that the conjugate was obtained from reaction of GSH with a reactive isocyanate of TGZ formed from metabolic ring opening of the TZD moiety (2). From the electrochemical oxidations of RGZ, PGZ, and CGZ (see later section), no GSH conjugates were observed, indicating that a reactive isocyanate intermediate of the TZD moiety may not be formed electrochemically. In the present study, the conjugate observed may also be formed from electrochemical oxidation of the TGZ-SG conjugate. When the potential was further increased to +800 mV, a third GSH conjugate appeared at m/z 781.2 (tR ) 6.2 min). The conjugate observed at m/z 781.2 ([T2 + H]+) corresponded to an addition of 34 Da to the TGZ-SG conjugate. By MS/MS fragmentation of m/z 781.2, fragment ions were detected as follows: m/z 763.1 ([T2 - H2O + H]+); m/z 706.3 ([T2 glycine + H]+); m/z 652.2 ([T2 - glutamic acid + H]+); m/z 634.0 ([T2 - glutamic acid - H2O + H]+); m/z 616.5 ([T2 glutamic acid - 2H2O + H]+); m/z 540.1 ([T2 - TZD moiety - C7H6O]+); m/z 458.2 ([T2 - GSH + H]+); m/z 411.1 ([T2 - TZD moiety - glutamic acid - C7H6O]+) (the MS/MS spectrum is shown in the Supporting Information, Figure 3). This GSH conjugate was only identified in small amounts in human and rat liver microsomes by a sensitive LC-MS method using SRM (data not shown). A conjugate with the same m/z value has previously been reported by Kassahun et al., who proposed that the conjugate was obtained from reaction of GSH with a reactive isocyanate metabolite of TGZ formed from metabolic ring opening of the TZD moiety (2). It is proposed that this conjugate may be formed from further electrochemical oxidation of the TGZ-SG conjugate, as some of the fragment ions of this GSH conjugate indicate that it is a GSH conjugate of TGZQ. Electrochemical Synthesis and Structure Elucidation of the TGZ-NAC. To obtain more information on the mechanism underlying the reactive metabolite formation of TGZ, TGZNAC was synthesized electrochemically. N-Acetylcysteine was

Electrochemical Oxidation of Troglitazone

Chem. Res. Toxicol., Vol. 21, No. 10, 2008 2039 Table 1. Proton and Carbon Chemical Shifts Observed for the TGZ-NAC and TGZ in DMSO-d6a TGZ-NAC

Figure 2. 1H NMR spectrum of the TGZ-NAC conjugate (in DMSOd6), generated by electrochemical oxidation of TGZ in the presence of N-acetylcysteine at +200 mV.

used as trapping agent since NMR spectra of N-acetylcysteine conjugates are simpler to interpret than NMR spectra of GSH conjugates (less protons to assign). A solution of TGZ (100 µM) and N-acetylcysteine (5 mM) in a 0.1 M phosphate buffer (pH 7.4)/acetonitrile (3:1) was pumped through the electrochemical cell at a potential of +200 mV. As shown previously, a product that corresponded to TGZQ was formed at this potential, but the highest yield of TGZ-NAC was obtained at this potential. TGZ-NAC was isolated, purified by preparative LC, and characterized by MS, MS/MS, and NMR. The TGZ-NAC conjugate was observed at m/z 601.2 ([TGZNAC - H]-) (tR ) 9.2 min). By MS/MS fragmentation of m/z 601.2, fragment ions were detected as follows: m/z 437.9 ([TGZNAC - NAC - 2H - H]-); m/z 395.0 ([TGZ-NAC - NAC - 2H - CONH - H]-); m/z 351.0 ([TGZ-NAC - NAC - 2H - CONH - CO - H2O - H]-); m/z 161.7 ([TGZ-NAC TGZ - H]-). From incubation of TGZ with N-acetylcysteine and NADPH with rat or human liver microsomes, a TGZ-NAC conjugate was identified with an identical retention time, m/z value, and fragmentation pattern (chromatograms and MS/MS spectra are shown in the Supporting Information, Figure 4). The structure of purified TGZ-NAC was elucidated with 1D and 2D NMR experiments (1H NMR, 13C NMR, COSY, NOESY, ROESY, HSQC, and HMBC), and chemical shifts and correlations were compared to a reference sample of TGZ. The 1 H NMR spectrum of TGZ-NAC along with the structure is shown in Figure 2. The protons are numbered according to the carbon to which they are attached. The 13C NMR spectrum of TGZ-NAC is shown in the Supporting Information (Figure 5), and the chemical shifts for the protons and carbons in TGZNAC and TGZ are summarized in Table 1. Reference spectra of TGZ in DMSO-d6 are shown in the Supporting Information (Figures 6 and 7). From the COSY experiment, the coupling path between the protons was established (the COSY spectrum is shown in the Supporting Information, Figure 8). This information together with connections through space derived from a ROESY experiment (the ROESY spectrum is shown in Supporting Information, Figure 9) allowed assignment of all proton resonances. The edited HSQC experiment was used to obtain one bond proton-carbon correlations with different signs for CH2 groups relative to CH and CH3 groups (the HSQC spectrum is shown in the Supporting Information, Figure 10). From the HMBC experiment, long-range proton-carbon couplings were obtained (the HMBC spectrum is shown in the Supporting Information, Figure 11).

δ 1H NMR (ppm)

δ

13

1.29 1.84 1.79 1.96 1.97

21.5 22.8 28.0

2.05 2.48 2.66 2.71 2.79 2.84 3.29 3.64 3.89 3.94 4.33 4.49 6.89 7.12 7.68 ND 8.29 ND

12.9 33.5

C NMR (ppm)

TGZ δ 1H NMR (ppm)

δ

13

C NMR (ppm)

assignment

1.29

21.8

1.80 1.97 1.95 2.03 2.05

28.3 11.9 11.9 12.8

24 27 (TGZ) 25 31

18.6

2.56

19.8

16

37.8

3.04 3.29

36.3

6

12.0

26.3 72.0 53.5 55.9 114.5 130.1

27 (TGZ-NAC) 3.93

72.4

4.85 6.90 7.13

53.1 114.6 130.4

7.45 164.7 12.02 74.0 116.8 121.3 123.1 124.3 130.5 144.4 146.7 157.6 169.4 173.4 ND ND

14 39 15

73.8 116.7 120.4 121.1 122.8 128.8 145.4 144.1 157.9 171.8 175.8

12 32 4 9 8 36 (NH) 21 (OH) NA 1 (NH) 13 17 20 23 22 7 18 21 10 37 33 2 5

a See Figure 2 and the Supporting Information, Figures 5-7 for the spectra and numbers used in the assignment. Chemical shifts are measured at 25 °C relative to the internal residual trace of DMSO set to 2.5 ppm (1H), respectively, 39.5 ppm (13C). NA, not assigned; ND, not detected.

It was noted that only four methyl signals were present in TGZ-NAC, whereas an extra CH2 group was found at δC:δH (26.3 ppm:3.64/3.89 ppm). Two methyl signals at 21.6 and 22.9 ppm were identified as C14 and C39, respectively. This indicated that one of the methyl groups, C24, C25, or C27, was missing. HMBC long-range correlations from these CH2 protons at 3.64/3.89 ppm to C31 as well as C17, C20, and C21 (Figure 3) indicated that the N-acetylcysteine group was attached to C27. From a ROESY experiment, it was observed that the protons at position C27 correlated with the protons at C16 and C31, indicating that these protons are close to each other in space, which further support this assignment (Figure 4). The signals from C4 were observed at δC:δH (55.9 ppm: 4.49 ppm), which would not be the case if a thioether conjugate was formed according to the mechanism proposed by Tettey et al. (3) and He et al. (4). From the performed NMR experiments, it was concluded that N-acetylcysteine was attached to C27 and most likely formed via a reaction between the N-acetylcysteine and the o-quinone methide of TGZ as proposed by Kassahun et al. (2). The 13C chemical shifts of C2 and C5 of the TZD ring were not readily observed. C4 and C6 showed the expected COSY, HSQC, and HMBC correlations and connections between each

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Figure 3. Section of the HMBC spectrum of the TGZ-NAC conjugate, which shows the coupling between protons at C27 and C17, C20, C21, and C31.

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Electrochemical Oxidation and Liver Microsomal Incubations of CGZ, PGZ, and RGZ. To support the hypothesis that the TZD moieties do not undergo activation to a reactive intermediate by electrochemical oxidation, three other compounds, which contain a TZD moiety, were included in the electrochemical studies. These three compounds were CGZ, PGZ, and RGZ. All three compounds were subjected to electrochemical oxidation under the same conditions as described for the oxidation of TGZ. The applied potential was varied between 0 and +1000 mV in intervals of 100 mV. Furthermore, RGZ, PGZ, and CGZ were incubated with human and rat liver microsomes. The electrochemical disappearances of RGZ, PGZ, and CGZ were investigated in the presence of GSH. The concentrations of RGZ, PGZ, and CGZ were almost constant in samples from electrochemical oxidation from 0 to +1000 mV, and it was not possible to identify any GSH conjugates of the three tested compounds from the electrochemical oxidations by LC-MS. However, small amounts of other products corresponding to hydroxylations and N-dealkylations were observed. No GSH conjugates were observed from the liver microsomal incubations of RGZ, PGZ, and CGZ in the presence of GSH, which is consistent with the results on TGZ, showing that the bioactivation took place in the chromane moiety. The intrinsic clearance in human and rat liver microsomes was determined for the three glitazones aiming at showing that the compounds were metabolized, although not to reactive metabolites. The three tested compounds were metabolized in human and rat liver microsomes, as determined from measurements of their intrinsic clearance. The intrinsic clearance was determined to be 26 and 75 µL/min/mg protein (human/rat liver microsomes) for TGZ, 9 and 18 µL/min/mg protein (human/rat liver microsomes) for PGZ, 14 and 58 µL/min/mg protein (human/rat liver microsomes) for RGZ, and 60 and 35 µL/min/ mg protein (human/rat liver microsomes) for CGZ. The intrinsic clearances of the four tested compounds were not influenced by adding GSH to the liver microsomal incubations. Only metabolites that previously have been identified (22-25) were detected.

Conclusion

Figure 4. Section of the ROESY spectrum of the TGZ-NAC conjugate, which shows the correlation between the protons at C27 and the protons at C16 and C31.

other and to the phenoxy ring, but C2 and C5 could not be detected in the 13C spectrum. A correlation was seen in the HMBC spectrum from C4 and one of the C6 protons to 182.5 ppm and an extra CH signal at 164.7 ppm, with a C-H coupling constant of 210 Hz, which was present in the TGZ-NAC spectra but could not be correlated to the rest of the molecule. This signal might be derived from residual formic acid but was not confirmed. The 13C chemical shift changes of C4 from δC:δH (53.1 ppm:4.85 ppm) in TGZ to δC:δH (55.9 ppm:4.49 ppm) in TGZ-NAC also indicated that a change occurred in the TZD ring in the NMR sample. In conclusion, it was clearly shown that the sulfur of the N-acetylcysteine moiety is attached to C27 in TGZ-NAC.

In this study, it has been shown that electrochemical oxidations could be used to generate the reactive metabolite of TGZ, which was trapped with GSH and N-acetylcysteine. Electrochemical oxidations were used to discriminate between the three proposed mechanisms for formation of reactive metabolites of TGZ and compared to results obtained from incubation of TGZ in microsomes. From these studies, it is reasonable to suggest that the major reactive metabolite of TGZ in liver microsomes is formed via metabolic oxidation of the chromane moiety in TGZ to a reactive o-quinone methide. In addition, the contribution of bioactivation of the TZD moiety appears to be limited based on the data presented in our studies. These results illustrate the benefits of using electrochemical oxidations in combination with NMR for small scale synthesis of metabolites for structural characterization and thus obtaining information on the mechanism involved in the formation of reactive metabolites. Supporting Information Available: Chromatograms and MS/MS spectra of TGZQ, MS/MS spectra of the GSH conjugate, chromatograms and MS/MS spectra of TGZ-NAC thioether conjugate, 13C NMR spectrum of the TGZ-NAC conjugate, 1H NMR and 13C NMR spectra of TGZ, and COSY, ROESY,

Electrochemical Oxidation of Troglitazone

HSQC, and HMBC spectra of TGZ-NAC. This material is available free of charge via the Internet at http://pubs.acs.org.

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