Toxic Metabolite Formation from Troglitazone (TGZ): New Insights from

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Toxic Metabolite Formation from Troglitazone (TGZ): New Insights from a DFT Study Vaibhav A. Dixit and Prasad V. Bharatam* Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), S. A. S. Nagar, Punjab-160062, India

bS Supporting Information ABSTRACT: The hepatotoxicity of Troglitazone (TGZ) has been ascribed to the formation of reactive metabolites, and the primary reactive metabolite of TGZ has been confirmed to be an o-quinone methide. Oxidation of the chromane moiety is also known to produce quinone containing metabolites. Quantum chemical studies have been performed to analyze the possible reaction pathways for the metabolism of the TGZ side chain, 6-hydroxy-2,2,5,7,8-pentamethylchromane (HPMC). From this analysis, a new pathway including oxidation at the C13 and C14 atoms of HPMC has been proposed for the formation of o-quinone methide (M2), while oxidation at the hydroxyl group leads to the formation of the quinone metabolite (M7). o-Quinone methide reactive metabolites have been shown to be more electrophilic at the reactive methylene center using quantum chemically estimated parameters.

1. INTRODUCTION Sohda et al. in 1982 identified the thiazolidinedione (TZD) class of compounds as one of the potential antidiabetics.1 Troglitazone (TGZ) was introduced in 1997, as the first TZD based oral antidiabetic drug after a gap of 30 years. Developed as an antioxidant and antihyperlipidemic agent with a TZD ring and a chromane ring of vitamin E, TGZ was found to lower plasma glucose levels by increasing insulin sensitivity.2 Rosiglitazone (RGZ) and Pioglitazone (PGZ) soon followed (1999), and PGZ still continues to be widely used for Type II diabetes. Reports of liver failure forced the withdrawal of TGZ from the market in February 2000 by the USFDA.3 Except for rare reports,4 RGZ and PGZ have not shown such liver damage or failures over the past decade and are considered relatively safe antidiabetic drugs.5,6 TZDs act as agonists at peroxisome proliferator-activated receptor-γ (PPARγ) and tend to rectify errors in glucose and lipid metabolism relieving hyperglycemic and hyperlipidimic states.7 As a result, despite the withdrawal of TGZ,8 PGZ still continues to be in the market as effective treatment against Type II diabetes. It is only recently that reports of cardiotoxicity of RGZ have forced regulatory agencies to recommend the withdrawal of RGZ in Europe and Asia, but the drug continues to be in the market in the US with warning labels. The major reason for the withdrawal of TGZ was hepatotoxicity, and mechanisms of such toxicity have been amply studied.9,10 Reasons such as (i) high dose (200600 mg/day) requirement, (ii) induction of CYP3A4 and inhibition of CYP2C8, CYP2C9, and CYP2C19, (iii) accumulation in the liver through enterohepatic circulation, and (iv) extensive binding (99%) to albumin added to the hepatotoxicity of this chromane ring containing ex-drug.11,12 r 2011 American Chemical Society

Although designed to act as an antioxidant and to neutralize free radicals generated during oxidative stress,13 the chromane moiety has equal potential to lead to the formation of such free radicals through P450 mediated hydrogen abstraction and quinone and oquinone methide formation.1416 These reactive metabolites are also known to form adducts with nucleotides which serve as reservoirs for these reactive species.16 They also form quinone epoxide,17 sulfate conjugates, GSH adducts, and quinone metabolites leading to cytotoxicity.14,18 Figure 1 shows a schematic diagram explaining the reaction path involved in the metabolic profile of TGZ. This cytotoxicity has prompted many research groups to design compounds with the o-quinone methide framework as anticancer and antibiotic drugs.1922 o-Quinone methides show electrophilic character and are known to react as Michael acceptors and add to nucleophiles at the exocyclic methylene group.2325 A study for identifying reactive metabolites of TGZ, RGZ, and PGZ utilizing electrochemical oxidation and incubation with liver microsomes has confirmed these facts.26 Mechanisms for TGZ induced hepatotoxicity have been classified mainly as (i) metabolic and (ii) nonmetabolic and include complex events at the cellular and molecular levels in addition to the variable patient condition/response, and have kept researchers speculating about the exact nature of the cause.10 Although many factors have been proposed to contribute to the mechanisms underlying the idiosyncratic toxicity of TGZ, rigorous efforts over the years have highlighted that the class effect of TZDs is more or Received: March 11, 2011 Published: June 09, 2011 1113

dx.doi.org/10.1021/tx200110h | Chem. Res. Toxicol. 2011, 24, 1113–1122

Chemical Research in Toxicology

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Figure 1. Metabolic products generated from CYP2C8 and 3A4 mediated oxidation of TGZ.

less a physiological and heamodynamical effect resulting in weight gain and fluid retention.2729 Many old and recent reports point to the fact that hepatotoxicity of TGZ is most probably a result of the metabolism of the side chain chromane moiety to reactive metabolites o-quinone methide and quinone.9,14,28,30 TZD ring based reactive metabolites have also been invoked in the hepatotoxicity, but the absence of similar circumstantial evidence against RGZ and PGZ weakens the case.31,32 Moreover, TGZ is metabolized by both CYP3A4 and 2C8 and is a known inducer of CYP3A4, which catalyzes side chain oxidation;33 this P450 induction increases the potential of adverse drug interactions with molecules such as acetaminophen (APAP).3436 Thus, it is worth exploring the molecular level details which trigger the hepatotoxic influence of TGZ. Although, many studies proposed the reaction pathways for the TGZ toxicity, a thorough analysis of the possible reaction pathways and product stabilities has not been taken up. Both quinone and o-quinone methides are reported to be generated during the metabolism of TGZ; however, the toxicity is mostly attributed to the quinone methide reactive metabolites.14 The reasons for the preferential generation of o-quinone methides have not been explored. The reaction pathway leading to the quinone and o-quinone methide reactive metabolites had been presumed to be the same, and the reaction mechanism leading to the formation of two reactive metabolites have not been distinguished. The relative stabilities of the reactive metabolite and relative reactivity have not been explored. It is worth establishing the pathways leading to the formation of the

reactive metabolite, their relative stabilities, and reactivities. Density functional studies have been shown to be quite useful in exploring the reaction mechanism of metabolite formation and associated toxicity of drugs.3746 This article presents a DFT study on the relative energetics of the oxidation pathways of the TGZ side chain, i.e., HPMC (6-hydroxy-2,2,5,7,8-pentamethylchromane). Hydrogen abstraction energy, relative product stabilities, and enthalpies of Cpd I mediated oxidation have been calculated to analyze the differences in the metabolism of TGZ. The reactivity of reactive metabolites has been studied using global and local electrophilicities and Fukui functions. Further, a study on the potential energy surface (PES) for the nucleophilic attack of methanethiol on o-quinone methides is presented to analyze the reactivity patterns, intermediates, transition states, and product stabilities. A comparative study of the reactive metabolite formation from RGZ and PGZ is also reported using the model systems 2-DMAP (2-(N,N-dimethylaminopyridine) and EMP (5-ethyl-2-methylpyridine). Enthalpies of Cpd I mediated oxidation have been estimated which further support the conclusions.

2. EXPERIMENTAL PROCEDURES 2.1. Methodology. Ab initio calculations for all systems under consideration have been carried out using the Gaussian03 suite of programs.47 Geometry optimizations were performed using DFT theory at the B3LYP/ 6-31+G(d) level. Analytical frequencies have been estimated by carrying out frequency calculations at the B3LYP/6-31+G(d) level to characterize the 1114

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Figure 2. Free radical hydrogen 3 abstraction pathways (A, B, and C) for OH bond and (D and E) for CH bond homolytic cleavage leading to the formation of the quinone and o-quinone methide intermediate from the chromane side chain model (HPMC). See Table 1 for the energies associated with the depicted reaction paths. optimized structures as minima or transition states (one negative frequency) on the potential energy surface. Single point energies were determined at B3LYP/6-311++G(2df,3pd) using the B3LYP/6-31+G(d) geometries. The zero point vibrational energy (ZPVE) values have been scaled by 0.9806.48 Partial atomic charges were estimated by performing natural bond orbital (NBO) analysis.49 The LAN2DZ pseudopotential was used on the Fe atom in Cpd I and Cpd IH, while the rest of the molecule was treated with the 6-31+G(d) basis set. Both double and quartet spin states of Cpd I were considered during calculations. Solvent phase calculations were performed at B3LYP/6-311++G(2df,3pd)//B3LYP/6-31+G(d) using the IEF-PCM model with water as solvent.

3. RESULTS AND DISCUSSION 3.1. Reactive Metabolite Formation from HPMC. Kassahun et al. proposed a reaction mechanism involving radical formation leading to the reactive metabolites of TGZ.14 Alvarez-Sanchez et al. performed a detailed mechanistic study and supported the proposal that the chromane moiety gets preferentially metabolized in TGZ.50 Madsen et al. performed electrochemical oxidation of TGZ and compared the results with liver microsomal oxidation and showed that the primary reactive metabolite of

TGZ is o-quinone methide.26 To study the details of these mechanisms, in this work three different sections are considered to explore the atomic level details of oxidation of HPMC. The first one involves the study of radical generation from HPMC by the hydrogen abstraction process and the possible consequences experienced by the radicals generated. The second section involves the study of the hydrogen abstraction process in the presence of model oxidizing agent hydrogen peroxide. The third section involves the Cpd I mediated formation of radicals. The results obtained are presented in the following three sections. 3.1.1. Radical Generation from HPMC. For the generation of radicals, hydrogen abstraction is the first step; the proposed mechanism involves the abstraction of hydrogen from the hydroxyl group of the chromane moiety. Alternative paths can also be suggested, which involve hydrogen abstraction from the methyl groups of the chromane moiety; all of the pathways are depicted in Figure 2. Hydrogen atom abstraction from the hydroxyl group of HPMC (a model of chromane moiety) leads to the formation of the radical R1. R1a to R1d are resonance representations of the radical R1. R1b can lead to the formation of M1, a quinone derivative after an additional H atom abstraction (path A). R1c can similarly lead to M2, an o-quinone methide (path B), whereas R1d can lead to M3 1115



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Table 1. Reaction Enthalpies (ΔH) on Paths A to E (Figure 2) Estimated Using the B3LYP/6-311++G(2df,3pd)//B3LYP/ 6-31+G(d) Level in Gas Phase and in Solvent Medium [IEF-PCM (Water)] energies (kcal/mol) path A/B/C A A B C D E

reaction HPMC f R1 + H 3 R1 f R10 + H 3

R1 f M1 + H 3 R1 f M2 + H 3

R1 f M3 + H 3 HPMC f R2 + H 3

HPMC f R3 + H 3 M6 f 13Crad-M6 + H 3

gas phase

solvent phase

BDE

71.52

70.95

BDE

27.57

29.16

BDE

65.41

66.41

BDE

64.29

65.29

BDE

67.42

67.73

BDE

83.23

83.02

BDE BDE

84.30 83.97

84.00 82.84

(path C), an isomer of M2. Alternatively, the metabolites M2 and M3 may be formed in a cyctochrome mediated oxidation pathway involving a rebound mechanism,51 initiated by hydrogen radical abstraction from the methyl groups (paths D and E). Quantitative estimation of the hydrogen abstraction energies (in terms of bond dissociation energy (BDE)) are required to distinguish various possible pathways. They have been successfully used for the prediction of the site of metabolism (SOM), and the use of such protocols in combination with QSAR, molecular docking, have become a routine procedure to predict SOM in a drug discovery setup.52 The CH and OH BDEs for a variety of substituted phenols,5355 semiquinones,56 tocopherols, and chromanes57 have also been calculated and compared against experimental values. The ability of the DFT based methods, especially the B3LYP functional, in satisfactorily predicting BDEs has been highlighted in all these studies, and this prompted us to use the same for the current study also.5357 BDE for the formation of R1 is 71.52 kcal/mol (Table 1), whereas the BDE for the formation of radicals R2 and R3 are, respectively, 83.23 and 84.30 kcal/mol. Under gas phase conditions, R1 formation appears to be relatively more favorable; however, the cytochrome mediated path may favor the formation of R2/R3. The resonance stabilization of radical R1 is clear from the estimated geometric parameters as well as its spin densities, and its characteristics can be explained with the help of conjugation, hyperconjugation concepts, and measurement of unpaired spin density.58,59 The chromane nucleus of HPMC is a special kind of phenol with an oxygen atom at the para position to a hydroxyl group; this leads to extensive delocalization of electron spin density in the radical and shortening of the C6O6 bond to 1.26 Å. Thus, this radical is essentially a carbon center radical with spin density distribution at O6 (0.351), C5 (0.308), C7 (0.230), and C9 (0.311), supporting the resonance stabilization of radical R1. However, the spin densities of radicals R2 and R3 are localized on the carbon atom at which the radical is generated (C13 (0.737) in R1 and C14 (0.771) in R3). This indicates that the greater stabilization of R1 over R2/R3 (by ∼11.17 kcal/mol) is due to the resonance effect. Generation of R10 from R1 (see Figure 2) is an energetically uphill task (ΔE: 27.57 kcal/mol), and hence, the formation of M1 as in path A cannot be expected; experimentally no report suggested the formation of M1. Similarly, the formation of M2 and M3 along paths B and C are uphill tasks because these paths involve the abstraction of two hydrogen radicals which need additional energy (∼6468 kcal/mol, Table 1). Formation of

(M2 and M3) follows an alternative P450 mediated rebound mechanism path involving the formation of hydroxylated metabolites M4 (and M5), respectively, paths D (and E). Water elimination from the species leads to the formation of o-quinone methides. The water elimination reaction from M4 to give M2 is more facile compared to the known water elimination reaction from 2-(hydroxymethyl)phenol.24 This is supported by the fact that o-quinone methide formation from M4 requires only 10.55 kcal/ mol, whereas water elimination from 2-(hydroxymethyl)phenol requires 18.68 kcal/mol in gas phase (see Table 3). The barrier for water elimination is 29.33 kcal/mol for M4 and 32.80 kcal/mol in 2-(hydroxymethyl)phenol in gas phase. The presence of oxygen at the para position to the hydroxyl group is facilitating the water elimination. The driving force in the greater ease of formation of oquinone methides should be the resonance stabilization in o-quinone methides. NBO analysis shows that the positive charge at C13 (0.131) in M2 is less than that of the corresponding carbon in unsubstituted o-quinone methide (0.195). Overall, section 3.1.1 suggests that the formation of radical R1 is easier in comparison to that of R2 and R3, but the formation of reactive metabolites from R1 are not expected on paths A, B, and C because these involve higher energy paths, i.e., abstraction of two hydrogen radicals. 3.1.2. Radical Generation with the Help of Model Oxidizing Agent Hydrogen Peroxide. To further analyze the importance of paths B vs D in the generation of M2, the oxidation of HPMC by the model oxidizing agent hydrogen peroxide was studied using quantum chemical methods (Figure 3). This study revealed that the quinone (M7) can be generated by the oxidizing agents in a pathway involving radical R1 (path F). However, the formation of o-quinone methide (M2) can happen only on a pathway involving radical R2 (paths G and H). The energetics associated with these paths is given in Table 2. Formation of radical R1 is found to be a favorable process on path F, requiring only about 0.66 kcal/mol (in fact, in water medium, this reaction was found to be exothermic by about 3 kcal/mol). The hydroxyl radical attack on R1 leads to the formation of the metabolite M6 in an exothermic process by about 47 kcal/mol. The quinone metabolite M7 is more stable than M6 by about 8 kcal/mol, and the conversion of M6 to M7 requires a tautomeric process with a barrier of 32.90 kcal/mol (34.55 kcal/mol in water), which can be readily available in the reaction due to the large exothermicity in the formation of M6. Hence, the formation of the quinone metabolite (M7) on path F is a thermodynamically favorable process. M2 was found to be thermodynamically more stable than M6 by 3.48 kcal/mol in gas phase and 9.51 kcal/mol in water medium, but no concerted or conjugative water elimination path is feasible from M6 to M2 because of the large distance (4.75 Å) between the corresponding hydroxyl group and methyl hydrogen in M6. Also, any P450 mediated oxidation of M6 would lead to radical character at the methyl group precluding the possibility of any path leading to M2. Further uncatalyzed free radical oxidation of M6 at the methyl group requires at least 83.97 kcal/mol in gas phase (82.84 kcal/mol in aqueous medium). Hence, M6 is expected to form M7 rather than M2. The formation of R2 in path G is an endothermic process requiring about 12.4 kcal/mol. However, the formation of reactive metabolite M4 is a highly exothermic process (ΔH = 60.6 kcal/ mol), and its formation via radical R2 should be highly feasible even though R2 generation is slightly endothermic. M4 can release water to give M2 as discussed in the previous section. The energy profile for the formation of M3 on path H is quite comparable to that of M2 on path G. 1116



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Figure 3. Possible reaction paths in the generation of reactive metabolites from HPMC, using model oxidizing agent hydrogen peroxide. See Table 2 for the energies associated with the depicted reaction paths.

Table 2. Reaction Enthalpies and Activation Energies on Paths F to G (Figure 3) Estimated Using the B3LYP/6-311+ +G(2df,3pd)//B3LYP/6-31+G(d) Level in Gas Phase and in Solvent Medium [IEF-PCM (Water)]

Table 3. Enthalpies for Cpd I Mediated Oxidation of HPMC at the B3LYP/6-31+G(d) Level in kcal/mol ΔH

energies (kcal/mol) path

reaction

ΔH/ Ea

gas phase

solvent phase

F F F F F G G G G H H H H

HPMC + H2O2 f R1 + 3 OH + H2O HPMC + H2O2 f R10 + 3 OH + H2O HPMC + H2O2 f M6 + H2O HPMC + H2O2 f M7 + H2O M6 f M7 HPMC + H2O2 f R2 + 3 OH + H2O HPMC + H2O2 f M4 + H2O M4 f M2 + H2O M4 f M2 + H2O HPMC + H2O2 f R3 + 3 OH + H2O HPMC + H2O2 f M5 + H2O M5 f M3 + H2O M5 f M3 + H2O M6 f M2 + H2O

ΔH ΔH ΔH ΔH Ea ΔH ΔH ΔH Ea ΔH ΔH ΔH Ea ΔH

0.66 28.23 46.54 54.88 32.92 12.37 60.57 10.55 29.33 13.44 60.82 13.92 30.71 3.48

2.90 26.26 43.83 54.71 34.55 9.16 60.33 6.99 28.31 10.14 60.24 9.34 29.08 9.51

Reversibility and reactivity of o-quinone methides have been studied extensively.60,61 Reversible o-quinone methide formation

a

entry

reaction

doublet

quartet

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

HPMC + Cpd I f R1 + Cpd IH HPMC + Cpd I f R2 + Cpd IH HPMC + Cpd I f R3 + Cpd IH R1 + Cpd I f M1 + Cpd IH R1 + Cpd I f M2 + Cpd IH R1 + Cpd I f M3 + Cpd IH R2 + Cpd I f M2 + Cpd IH R3 + Cpd I f M3 + Cpd IH HPMC + 2Cpd I f M1 + 2Cpd IH HPMC + 2Cpd I f M2 + 2Cpd IH HPMC + 2Cpd I f M3 + 2Cpd IH HPMC + Cpd I f M4 + PorFe(III)a HPMC + Cpd I f M5 + PorFe(III) HPMC + Cpd I f M7 + PorFe(III) HPMC + Cpd I f M1 + H2O + PorFe(III) HPMC + Cpd I f M2 + H2O + PorFe(III) HPMC + Cpd I f M3 + H2O + PorFe(III)

04.42 12.65 13.55 04.66 07.53 04.32 19.85 17.49 09.08 07.20 03.94 68.13 68.41 59.67 49.88 52.74 49.54

04.49 12.55 13.48 04.73 07.59 04.39 19.92 17.56 09.22 07.37 04.08 67.96 68.24 59.50 49.71 52.57 49.37

PorFe(III) is the resting state model of P450.51

from o-hydroxybenzyl alcohols has been suggested by Chiang et al.24 The relative stability of M2 and M4 was found to be 1117



Figure 4. Model reactions for compound I (Cpd I) mediated oxidation of HPMC.

dependent on the solvent environment due to the considerable contribution of the dipolar form in the structure of M2. Such a dipolar form is more stabilized in water bulk, and the difference drops to ∼7 kcal/mol in comparison to the gas phase value of 10.6 kcal/mol. These results are in the range suggested earlier form experimental kinetic studies on o-quinone methides.24 Though the o-quinone methide formation step is endothermic (i.e., M4 to M2), the formation of M4 from HPMC is highly exothermic, and thus, o-quinone methides are expected in this reaction (path G); these are trapped by glutathione in the metabolism studies. The moderate endothermicity of water elimination from M4 and M5 indicates that formation of M2 and M3 is equally likely. Thus, the paths F, G, and H suggested in Figure 3 seem equally likely given the kinetic and thermodynamic considerations and the fact that P450s show diverse regio- and chemoselectivity patterns.51 Path F can lead to the formation of the quinone reactive metabolite only. Paths G and H are the paths which preferentially lead to o-quinone methides. This implies that o-quinone methides can form only due to the activation of the methyl group in TGZ by the catalytic action of P450 but not by hydrogen abstraction from the hydroxyl group. 3.1.3. Enthalpy Estimation in the Presence of Cpd I. Under the in vivo conditions, P450 oxidizes the drugs to produce reactive metabolites. In the catalytic cycle of P450, compound I, an important reactive species, is considered to be performing the actual oxidation.51,62 The oxidation reactions involving cytochromes can be modeled using the model Cpd I (Figure 4) to estimate the energy profile and to elucidate the reaction mechanisms.51 However, tracing the entire path using Cpd I is computationally prohibitive. Enthalpy estimates can be performed using equations in Figure 4 and Table 3 to obtain clues regarding the preferential formation of radicals and reactive metabolites. B3LYP/6-31+G(d) calculations (employing the LAN2DZ basis set for Fe) were performed to estimate the energies associated with the reactions in Figure 4. Table 3 lists the energies associated with these reactions. The formation of radical R1 is an exothermic process by about 45 kcal/mol, whereas the formation of radicals R2/R3 are endothermic by 1214 kcal/mol on this path. These facts indicate that reaction paths involving radical R1 probably are more feasible. R1 can give rise to metabolites M1, M2, or M3 only when another hydrogen radical is abstracted. This would require another catalytic cycle of P450 oxidation. The enthalpies of hydrogen radical abstraction during the second cycle of P450 mediated oxidation from R1 are 4.66 kcal/mol (M1 formation), 7.53 kcal/mol (M2 formation), and 4.32 kcal/mol (M3 formation) (entries 4, 5, and 6 in Table 3). However, the formation of M2 from R2 through the

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second cycle is exothermic by 19.85 kcal/mol (entry 7, Table 3). Similarly, the formation of M3 is also an exothermic process by 17.49 kcal/mol. When the enthalpies of M1, M2, and M3 are directly estimated from HPMC, employing two cycles of P450 oxdiation, the reactions are found to be exothermic by 9.08, 7.20, and 3.94 kcal/mol (entries 9, 10, and 11 in Table 3). Hence, if the hypothesis based on two cycles of P450 oxidation is acceptable, the reactions for the formation of all three metabolites M1, M2, and M3 turn out to be exothermic. To the best of our knowledge, such reactions, involving two cycles of P450 have not been invoked previously. Hence, alternative paths were considered which involve the rebound mechanism. R1b can undergo CO bond cleavage to give R10 (Figure 3, Path A), which can get rebound to P450 and eventually lead to the formation of M7; this reaction is exothermic by 59.67 kcal/mol (entry 14, Table 3). Similarly, radicals R2 and R3 can lead to metabolites M4 and M5; the reactions are exothermic by 68.13 and 68.41 kcal/mol (entries 12, 13; Table 3). These reactions follow the rebound mechanism which has been proven to be true using both theoretical and experimental methods.62 The overall reactions leading to reactive metabolites M1, M2, and M3 from HPMC through the rebound mechanism are exothermic by 49.88, 52.74, and 49.54 kcal/mol (entries 15, 16, and 17 in Table 3). This data clearly indicates that the formation of reactive o-quinone methide metabolite M2 on a path involving R2 followed by rebounding to give M4 and consequent loss of water is a favorable pathway in comparison to other considered pathways. Taken together (Figures 24), the results from three sections indicate that the formation of radical R1 is energetically more favorable than the formation of radical R2/R3. Radical R1 should either release the hydrogen radical to give the metabolite M2/ M3 or should react with the hydroxyl radical to lead to the metabolite M6 and eventually metabolite M7. Pathways involving second hydrogen radical loss are highly endothermic, while those with two catalytic cycles of P450 oxidation are much less exothermic and hence appear to be hypothetical under the reaction conditions. Radical R1 may react with water to produce M6 and M7; however, radicals R2 and R3 can lead to metabolites M4 and M5 through the rebound mechanism. It should be noted that metabolite M7 is less stable than metabolite M4 (and M5) by ∼5.7 kcal/mol. The pathways involving radical R2 (and R3) formation is most likely the pathway for the generation of o-quinone methides. Hence, the pathway involving radical R1 shall lead to the relatively less stable quinone metabolite M7, whereas the pathway involving radicals R2/R3 leads to the relatively more stable metabolite M4/ M5, which is in equilibrium with the corresponding reactive oquinone methides. Thus, the thermodynamic consideration of the formation of reactive metabolites explains the experimental observation that o-quinone methide based metabolites as well as the quinone based metabolites are identified in comparable amounts, o-quinone methide formation being marginally larger. The electrophilic center in o-quinone methide explains its greater involvement in toxicity as explained in the next section. 3.2. Reactivity of Reactive Metabolites. The reactive metabolites M2, M3, and M7 are important in determining the toxicity profiles arising from the chromane side chain of TGZ. To establish their relative reactivities, the electrophilicity parameters have been estimated. The global electrophilicity index (ω), local electrophilicity (ωc+), and local Fukui Function (fc+) can be estimated (see Supporting Information) using standard equations.39,63,64 Table 4 lists these values for M2, M3, and M7. ω represents the global electrophilicity of any species; the greater its positive value, the higher is the overall electrophilicity of the reactive metabolite. 1118



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Table 4. Electrophilicity Index (ω, Electronvolts), Local Fukui Function (fc+), and Local Electrophilicity (ωc+) Using the B3LYP/6-311++G(2df,3pd)//B3LYP/6-31+G(d) Method in Gas Phase molecule

reactive centre

ω

fc+

ωc+

o-QM

C7

3.990

0.253

0.858

M2

C13

2.684

0.237

0.636

M3

C14

2.844

0.208

0.591

M7

C1

3.535

0.092

0.325

C2

0.078

0.276

C3

0.041

0.145

C4 C5

0.094 0.058

0.332 0.205

C6

0.059

0.209

The ω value for M7 is 3.54, which is much larger than that of M2 (2.68) and M3 (2.84); this makes the quinone based reactive metabolite more electrophilic. However, the local electrophilicity indicators (ωc+ and fc+) are more pronounced at the electrophilic carbons C13 and C14 in M2 (0.636) and M3 (0.591), respectively, in relation to any center in M7. This establishes the fact that the overall toxic effect of the chromane side chain in TGZ arises from the local electrophilicity experienced by the methylene reactive center in o-quinone methides. On a relative scale, the C13 center in the oquinone methide (M2) is more electrophilic than the C14 center in M3. This explains the observed glutathione adduct formation from M2. However, it would be worth taking up experimental metabolism studies to identify the metabolic adducts arising from the C14 atom of HPMC and corresponding center in TGZ. 3.2.1. Nucleophilic Attack of Methanethiol on M2 and M3. The o-quinone methides M2/M3 generated from HPMC are reactive intermediates, they can easily react with S nucleophiles, and get eliminated. This is exemplified by the fact that GSH adducts of M2 are experimentally observed using mass spectrometric methods.14,26,31 To model the reactivity of o-quinone methides toward thiols, the energy profile of M2 and M3 vs methanethiol (MeSH) has been explored using the B3LYP/ 6-311++G(2df,3pd)//B3LYP/6-31+G(d) method. Figure 5 shows the PES for the formation of adducts in the gas phase and in a water medium. The adduct formation from M2 is exothermic by 16.5 kcal/mol, and the reaction required an activation energy of 15.85 kcal/mol in the water medium. This clearly establishes the high reactivity of o-quinone methides toward S nucleophiles. Figure 5 shows the comparative energy profiles of a simple o-quinone methide and M2 and M3. It revealed that M2 and M3 are less reactive toward methanethiol in comparison to simple o-quinone methide. This can be understood in terms of the reduced electrophilic character of M2 and M3 in relation to unsubstituted o-quinone methide (Table 4). Because of the presence of the electron releasing oxygen at the para position, M2 and M3 are less electrophilic. In polar media, the reaction of o-quinone methides with thiols appears to be less favorable, as shown in Figure 5. These o-quinone methides may react with N and O based nucleophiles also, but the estimated barriers for these reactions are high. Thus, it can be further inferred that the high reactivity of oquinone methides toward thiol nucleophiles leads to the depletion of glutathione concentration and causes the toxicity associated with o-quinone methides as well as drugdrug interactions.

Figure 5. Potential energy surface (PES) for the reaction of MeSH with o-quinone methide, M2, and M3. o-QM (o-quinone methides), TS (transition states), and P (methanethiol adducts). Values indicate TS barriers in kcal/mol.

3.3. Comparison with RGZ and PGZ. Hydrogen abstraction from the methyl group of NMe2 in RGZ requires 85.98 kcal/mol in gas phase conditions and 16.38 kcal/mol with the help of Cpd I (see Supporting Information). Similarly, the hydrogen abstraction energy from the ethyl group of PGZ requires 82.10 and 11.68 kcal/mol, respectively. These are quite comparable to the energies associated with the generation of radicals R2 and R3; thus, hydrogen abstraction energies do not distinguish RGZ/PGZ and TGZ. The radicals from RGZ lead to the formation of stable oxidized metabolites (Figure S1, Supporting Information). The radicals from PGZ also lead to the formation of stable oxidized metabolites (Figure S2, Supporting Information). These metabolites are not reactive metabolites unlike M2 and M3. While M4 and M5 are in equilibrium with o-quinone methides, the metabolites from RGZ and PGZ are stable and eliminated as glucoronides. Thus, it is clearly established that reactive o-quinone methide formation in TGZ leads to toxicity but not the initial step of hydrogen abstraction.

4. CONCLUSIONS Quantum chemical studies have been performed on the model species 6-hydroxy-2,2,5,7,8-pentamethylchromane (HPMC) to explore the reaction mechanism of the generation of reactive metabolites from Troglitazone (TGZ). B3LYP/6-311++G(2df,3pd)//B3LYP/6-31+G(d) calculations have been performed to estimate the radical generation from HPMC. To understand the reaction mechanism, analysis was carried out at three stages. The first stage involves the estimation of hydrogen abstraction energies. The results from this stage indicate that hydrogen abstraction from the hydroxyl group is favorable in comparison to that of other alternative paths. In the second stage, the reaction is explored with the model oxidizing agent hydrogen peroxide. This study revealed that quinones may be preferentially formed in a path involving radical abstraction from a hydroxyl group, whereas o-quinone methides can be formed only on a path involving hydrogen abstraction from a methyl group. The third stage analysis involved the estimation of the enthalpies of radical formation using Cpd I. The combination of the analysis from these three stages helped in distinguishing the reaction mechanisms leading to the formation of quinones and o-quinone methides. A reaction path involving hydrogen abstraction from the hydroxyl group of HPMC has been shown to lead to the formation of the quinone reactive metabolite, whereas hydrogen abstraction from the methyl group of HPMC has been shown to 1119


Chemical Research in Toxicology yield an o-quinone methide reactive metabolite. 13-HydroxyHPMC (M4), 14-hydroxy-HPMC (M5), and quinone metabolite (M7) are stable reactive metabolites, with the quinone metabolite being less stable by about 5.7 kcal/mol. The formation of M4 and M5 are exothermic by ∼60 kcal/mol on a path involving hydrogen peroxide. The hydroxyl-HPMC reactive metabolites are in equilibrium with o-quinone methide reactive metabolites (Ea ∼79 kcal/mol), which react with nucleophiles leading to toxicity. The activation energy for the reaction between M2/M3 and methanethiol is estimated to be small (11.21 and 9.46 kcal/mol, respectively, in gas phase) establishing the high reactivity of these reactive metabolites with nucleophiles. The radical generation from the side chains of TGZ, RGZ, and PGZ have been found to be quite comparable. Whereas the reactive metabolites from TGZ (M4 and M5) are in equilibrium with highly reactive electrophilic reactive metabolites (i.e., o-quinone methides), the reactive metabolites from RGZ and PGZ do not carry such a possibility, thus explaining the observed toxicity of TGZ but not of RGZ and PGZ. This detailed mechanistic pathway provides atomic level clues regarding the formation of reactive metabolites and offers new insights into the toxicity of TGZ.

’ ASSOCIATED CONTENT

bS

Supporting Information. Absolute energies of coordinates for the reactants, transition states, reactive metabolites, and products; data on electronegativity (χ), hardness (η), and electrophilicity index (ω); data on local electrophilicity (ωc+) and local Fukui functions (fc+); NBO charges on reactive metabolites; spin densities on atoms in reactive metabolites; hydrogen abstraction and hydrogen peroxide mediated oxidation paths for RGZ; hydrogen abstraction and hydrogen peroxide mediated oxidation paths for PGZ; and the coordinates for all the molecular species. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Tel: +91 172 2292018. Fax: +91 172 2214692. E-mail: pvbharatam@ niper.ac.in. Funding Sources

We thank Department of Science and Technology (DST), New Delhi, for financial support.

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Toxic Metabolite Formation from Troglitazone (TGZ): New Insights from

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