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Remoxipride is an atypical antipsychotic displaying selective binding to the dopamine D2 receptor. Several cases of aplastic anemia led to the withdra...
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Chem. Res. Toxicol. 2004, 17, 564-571

Characterization of Glutathione Conjugates of the Remoxipride Hydroquinone Metabolite NCQ-344 Formed in Vitro and Detection following Oxidation by Human Neutrophils John C. L. Erve,*,† Mats A. Svensson,‡ Hans von Euler-Chelpin,† and Eva Klasson-Wehler† Departments of DMPK & Bioanalytical Chemistry and Computational Chemistry, AstraZeneca R&D, S-151 85 So¨ derta¨ lje, Sweden Received November 19, 2003

Remoxipride is an atypical antipsychotic displaying selective binding to the dopamine D2 receptor. Several cases of aplastic anemia led to the withdrawal of remoxipride from the market in December 1993. The remoxipride metabolite NCQ-344 is a hydroquinone while the structural isomer NCQ-436 is a catechol, both of which have been suggested to be capable of forming a reactive para- and ortho-quinone, respectively. Recently, these two remoxipride metabolites were shown to induce apoptosis in human bone marrow progenitor cells. Furthermore, NCQ344 also caused necrosis of these cells unlike NCQ-436. Although NCQ-344 has been detected in plasma of humans dosed with remoxipride, to date, no experimental evidence for the formation of the corresponding para-quinone has been obtained. Here, we report the detection of three glutathione (GSH) conjugates of NCQ-344 in vitro that were formed following a chemical reaction and characterized by tandem mass spectrometry and for a cyclized conjugate additionally with derivatization and deuterium exchange. In contrast, NCQ-436 did not form a GSH conjugate. Hypochlorous acid oxidized NCQ-344 to the para-quinone while NCQ-436 was resistant to oxidation. Upon incubation with NCQ-344, stimulated human neutrophils produced from 2- to 5-fold greater amounts of glutathione conjugates than unstimulated neutrophils. Ab initio calculations on these remoxipride metabolites indicated that the reaction leading to the respective quinone was spontaneous for the para-quinone (e.g., from NCQ-344) while ortho-quinone (e.g., from NCQ-436) formation was not. These results demonstrate that NCQ-344 is capable of facile formation of a reactive para-quinone capable of reacting with GSH and may rationalize previous findings regarding the biological effects observed in vitro with these two remoxipride metabolites.

Introduction Remoxipride (Roxiam), (S)-(-)-3-bromo-N-[1-ethyl-2pyrrrolidinyl)methyl]-2,6-dimethoxybenzamide, is an atypical neuroleptic drug displaying selective antagonism for dopamine D2 receptors (1). Clinical trials demonstrated that remoxipride produced antischizophrenic effects with few adverse side effects such as extrapyramidal symptoms (2) that limit the usefulness of other drugs in this class. Remoxipride was marketed in several European countries during the early 1990s. During the fall of 1993, eight cases of aplastic anemia were reported in patients taking remoxipride, representing a strong epidemiological connection of 1 in 50 000 vs 1 in ≈500 000 spontaneous incidence (3), and it was decided in December of that year to withdraw remoxipride from the market (4). The apparent idiosyncratic toxicity that remoxipride displayed has raised the suggestion that reactive metabolite(s) derived from remoxipride were involved in causing the aplastic anemia although to date no such metabolites have been characterized. * To whom correspondence should be addressed. † Department of DMPK & Bioanalytical Chemistry. ‡ Department of Computational Chemistry.

Remoxipride (Scheme 1) (1) undergoes extensive metabolism in vivo with the predominant production of pyrrolidine ring oxidation products in humans and dogs and aromatic hydroxylation products in rodents (5) although all species produce the same metabolites to different degrees. Remoxipride is metabolized in part by the polymorphic enzyme P450 2D6, and some of the pyrrolidine ring metabolites possess pharmacologic activity (6). A number of polyphenolic metabolites arise due to sequential O-demethylation and aromatic hydroxylation, and these metabolites have received particular attention due to their potential to form reactive quinones (7). Two such metabolites, NCQ-344 (2),1 a hydroquinone, and the structural isomer NCQ-436 (4), a catechol, were shown to be capable of inducing apoptosis in the human promyeolocytic cell line HL-60 (8) with the apoptosis accompanied by phosphatidyl serine exposure, caspase activation, and DNA cleavage (9). Unique to NCQ-344 was its biphasic ability to cause necrosis in addition to 1 Abbreviations: ACN, acetonitrile; DMSO, dimethyl sulfoxide; GSH, glutathione; MPO, myeloperoxidase; NCQ-344, [(-)-S-3-bromoN-[(1-ethyl-2-pyrrolidinyl)methyl]-5-hydroxy-6-methoxysalicylamide]; NCQ-436, [(-)-S-3-bromo-N-[(1-ethyl-2-pyrrolidinyl)methyl]5,6-dihydroxy-2-methoxybenzamide]; PBS, phosphate-buffered saline; PMA, phorbol 12-myristate 13-acetate; TFA, trifluoroacetic acid.

10.1021/tx034238n CCC: $27.50 © 2004 American Chemical Society Published on Web 03/30/2004

GSH Conjugates of the Remoxipride Metabolite NCQ-344 Scheme 1. Metabolism of Remoxipride

apoptosis with necrosis predominating at higher concentrations, an effect not observed with NCQ-436. The promyelocytic cell line HL-60 contains high MPO and low NAD(P)H quinone oxidoreductase activities (10), a situation that makes HL-60 cells more sensitive to the toxic effects of the polyhydroxy metabolites of benzene (11). A similar pattern of apoptosis induced by both benzene and remoxipride is consistent with formation of common quinone metabolites. P450 in hepatocytes is responsible for much drug oxidation, but recent work has found evidence for mRNA from five P450 enzymes including P450 2D6 in human bone marrow, bone marrow-derived macrophages, and other human hemopoietic cell lines (12) raising the possibility that drugs such as remoxipride may be metabolized to reactive metabolites directly in the target cell. A major source of extrahepatic drug oxidation are neutrophils (13) via the MPO-generated oxidant hypochlorous acid. The MPO system in neutrophils can cause oxidation of drugs and metabolites to quinone imines or quinones as has been demonstrated for amodiaquine (14) and a metabolite of prinomide (15), respectively. The purpose of the work described here was to investigate the potential for GSH conjugate formation caused by the chemical reactivity of the remoxipride metabolites NCQ-344 and NCQ-436. In addition, hypochlorous acid and activated human neutrophils were employed to investigate their ability to activate NCQ-344 to a reactive para-quinone metabolite. To this end, a combination of MS techniques in conjunction with various chemical derivatization strategies was employed to detect and characterize the resulting GSH conjugates.

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Materials and Methods Chemicals. NCQ-344 and NCQ-436 were synthesized by AstraZeneca (So¨derta¨lje, Sweden) as described previously (16, 17). GSH was obtained from Fluka Chemie AG (Buchs, Switzerland). Sodium hypochlorite was from Arcos (Glee, Belgium). Acetic anhydride, acetyl chloride, and DMSO were from Aldrich (St. Louis, MO). Water of 18 MΩ quality was prepared by a Milli-Q system (Millipore, Bedford, MA), and deuterium oxide (99.9% purity) was from Cambridge Isotope Laboratories (Andover, MA). Acetonitrile (ACN), methanol, and trifluoroacetic acid (TFA) were HPLC grade obtained from Merck KGaA (Darmstadt, Germany) while 2-propanol was from RiedeldeHaen (Seelze, Germany). S-(p-Nitrobenzyl)-GSH, phorbol 12myristate 13-acetate (PMA), Trypan blue, and Ficoll-Paque 1077 and 1119 were from Sigma. Hanks balanced salt without phenol red and PBS were both purchased from Invitrogen Corp. (U.K.). Reaction Conditions. NCQ-344 or NCQ-436 solutions (5 µL, 1 mM in ACN) were added to GSH (25 µL, 5 mM in ammonium bicarbonate buffer, pH 7.4-7.6) and allowed to react at T ) 37 °C. The reaction mixture was sampled at times ranging from 5 min to 24 h by withdrawing 2 µL and adding to 1 mL of MS solvent (50:50 ACN/H2O with 0.1% formic acid). For oxidation experiments, 1 µL of NCQ-344 or NCQ-436 stock solutions was added to 1 µL of NaOCl solution (50 mM in 1% acetic acid) with 5 µL of ammonium bicarbonate (50 mM, pH 7.2-7.6). Incubations were sampled at 0, 5, and 10 min by withdrawing 1 µL and diluting with 400 µL of MS solvent. Acetylation of the bis- and cyclized conjugates was performed by adding 40 µL of acetylation reagent (100 µL of acetic anhydride + 300 µL of methanol) and incubating for 30 min at T ) 37 °C. Carboxy methyl esterfication was accomplished by preparing an esterfication reagent (160 µL of acetyl chloride added dropwise with constant stirring to 1 mL of methanol). To a sample containing both bis- and cyclized conjugates, 40 µL of esterfication reagent was added and incubated for 2 h at T ) 37 °C. Reagent was removed by rotoevaporation before MS analysis. Reactions in deuterium oxide were performed by preparing GSH and ammonium bicarbonate buffer in deuterium oxide and reacting as described above. Neutrophil Isolation. A density gradient was prepared by layering 12.5 mL of Ficoll-Paque 1119 on top of an equal volume of Ficoll-Paque 1077 in a 50 mL Falcon tube. Twenty-five milliliters of fresh human blood (collected by venipuncture from healthy volunteers) was layered on top of the Ficoll-Paque and centrifuged at 700g (25 °C) for 30 min. The neutrophil layer was removed and washed with 45 mL of Hanks buffer followed by centrifugation at 200g for 10 min. To the resulting pellet was added 5 mL of deionized water followed by brief vortexing (5 s) to lyse contaminating erythrocytes. Following a second wash, the neutrophil pellet was resuspended in 1 mL of 1× PBS. The procedure was found to allow isolation of neutrophils at a concentration of approximately (1-2) × 107 cells/mL with a viability greater than 90% as judged microscopically by the Trypan blue (0.1% w/v) exclusion test. Neutrophil-Mediated Metabolism of GSH. To 375 µL of neutrophils in 1× PBS was added 15 µL of 10 mM GSH in 10× PBS. Activation of neutrophils was accomplished with the addition of 0.5 µL of PMA (0.05 mg/mL) in DMSO (0.5 µL of DMSO to control). Duplicate incubations were placed in an incubator at T ) 37 °C with constant shaking. Sampling was performed at T ) 0, 15, 45, 90, and 120 min by withdrawing 65 µL and adding to an equal volume of 2% acetic acid containing 5 µM S-(p-nitrobenzyl)-GSH as an internal standard. Samples were processed by ultrafiltration using a centrifugal filter device with a 10 kDa exclusion limit (Millipore) by centrifugation at 14 000 rpm for 30 min. The ultrafiltrate was stored at -25 °C until LC/MS/MS analysis. Neutrophil-Mediated Metabolism of GSH and NCQ-344. As above except after 15 min of stimulation, NCQ-344 was added to both stimulated and control neutrophils (in duplicate, 13 µM final concentration) and samples were taken at T ) 0, 15, 45, 90, and 120 min after addition of NCQ-344.

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Table 1. Relative Energiesa ∆E (in kcal/mol) for the Remoxiprideb Metabolites and Effect of Substituents

a b c

R1

R2

R3

∆E I f III

∆E II f IV

∆(∆E)

H H C(O)NH

H H OCH3

H Br Br

-13.8 -13.8 -2.0

-4.5 -3.9 +7.1

9.4 9.9 9.1

a The relative energy (∆E) is defined from the reaction HQ + 1/2O2 + ∆E f Q + H2O. b For simplicity, the ethyl-pyrrolidine group was not included in the structures.

Instrumentation. Flow infusion analysis was performed on either a Quattro Micro triple quadrupole MS or Q-TOF2 orthogonal time-of-flight MS (Micromass, Manchester, U.K.) equipped with an electrospray ion source operated in the positive ion mode. Samples were infused at a rate of 10-15 µL /min into the ion source. The spray voltage was set at 3.25 kV, and the cone voltage was set from 45 to 50 V. The desolvation gas flow was 600 L/h. The ion source and desolvation temperatures were 80 and 100 °C, respectively. Collision-induced dissociation was performed with collision energies ranging from 30 to 50 eV (laboratory frame of reference) with argon as a collision gas (∼3.5 × 10-3 Torr). LC/MS/MS was performed with a HewlettPackard 1100 gradient pump equipped with an autosampler (Hewlett-Packard, San Jose, CA), a Shimadzu LC-9A isocratic pump for sample loading (Shimadzu Deutschland, GmbH, Duisburg, Germany) onto a Hypercarb trap column (Thermo Hypersil-Keystone, Bellefonte, PA), and a Quattro Micro triple quadrupole MS. Chromatographic separation was accomplished with an analytical Hypercarb column (1 mm × 100 mm; 5 µm particle size) employing a binary solvent system: solvent A, 0.05% TFA; solvent B, 75% ACN, 25% 2-propanol containing 0.05% TFA at a flow rate of 0.05 mL/min. Samples were first loaded onto the trap column at a flow rate of 0.2 mL/min using solvent A followed by elution onto the analytical column. The separation gradient began with 2 min at 100% A with a linear increase to 80% B in 6 min. Following a hold at 80% B for 0.5 min, the starting conditions were reinstated and held for 10 min before the next injection. Multiple reaction monitoring of GSH sulfonamide (338 f 262), NCQ-344 (373 f 112), monoconjugate (600 f 98), bis-conjugate (905.3 f 775.3 + 98), cyclized conjugate (871.3 f 742 + 98), and the internal standard S-(pnitrobenzyl)-GSH (443 f 136) was performed with a dwell time of 0.1 s and delay of 0.1 s employing cone voltages and collision energies optimized for each compound monitored. Peak areas were normalized by dividing the peak area of the compound of interest by the area of internal standard divided by 1000. Ab Initio Calculations. Geometries (bond lengths, bond angles, and torsional angles) of the structures shown in Table 1 were fully optimized in the gas phase and for some structures also in water. For simplicity, the ethyl-pyrrolidine group was not included in the structures of the metabolites. Relative electronic energies of the optimized structures were calculated using the gradient corrected hybrid density functional method B3LYP (18, 19) and the 6-31G** basis set as implemented in the Jaguar program (Version 4.1, Schro¨dinger Inc., Portland OR). For bromine, a basis set including an effective core potential for the inner shell electrons was used (20).

Results Characterization GSH Conjugates from NCQ-344 by MS. NCQ-344 and NCQ-436 were evaluated for their ability to produce GSH conjugates in vitro. The reactions were conducted in an aqueous buffer containing up to 16% ACN. When subjected to tandem MS, NCQ-344

Figure 1. Tandem mass spectrum of NCQ-344. The amide bond cleaves to release the aromatic nucleus at m/z 244. The pyrrolidine ring generates the lower mass fragments that are diagnostic for NCQ-344.

Figure 2. Tandem mass spectrum of the monoconjugate with m/z 600 formed by reaction between NCQ-344 and GSH after 10 min. The reaction is between the quinone form of NCQ-344 and GSH resulting in displacement of bromine. Insert: The molecular ion at m/z 598 represents the quinone form of the conjugate (Scheme 2; ref 7), which is still reactive.

produced the product ion spectrum with the proposed fragment ions shown in Figure 1. NCQ-436 produced a product ion spectrum with the identical fragment ions (data not shown). Upon reaction of NCQ-344 with GSH, a molecular ion with m/z 600 could be detected after 10 min corresponding to a monoconjugate formed most probably by Michael addition of GSH to the para-quinone form of NCQ-344 with subsequent loss of bromine. Upon fragmentation of the monoconjugate, the daughter ion spectrum shown in Figure 2 was produced, which displayed the diagnostic fragment ions (m/z values 98, 112, and 129) derived from NCQ-344 as observed in Figure 1. Upon close examination of the molecular ion, the presence of m/z 598 could be observed representing the quinone form of the monoconjugate (Figure 2, insert). After 20 min, the monoconjugate had disappeared while a new ion at m/z 905 appeared consistent with incorporation of an additional molecule of GSH producing a bisconjugate. Concurrently, a peak 34 Da less in mass than

GSH Conjugates of the Remoxipride Metabolite NCQ-344

Figure 3. (A) Tandem MS spectrum of the product formed by addition of GSH to the monoconjugate to produce a bis-conjugate at m/z 905. (B) Tandem MS spectrum of the product derived from the bis-conjugate following loss of 34 Da to produce the cyclized conjugate at m/z 871. Note the low mass fragment ions characteristic of NCQ-344.

the bis-conjugate was also observed. With time, this peak at m/z 871 became more prominent such that by 24 h the peak at m/z 905 had disappeared while the peak at m/z 871 remained (data not shown). The daughter ion spectra produced from collision-induced dissociation of m/z 905 and m/z 871 are displayed in Figure 3A,B, respectively. The spectra display similarities such as the low mass diagnostic fragment ions derived from NCQ344 and the loss of 129 Da from the respective molecular ion. However, each molecular ion produced a unique pattern of fragment ions in the mid range of the mass spectrum. When NCQ-436 was reacted under identical conditions as used with NCQ-344, no GSH conjugates were detected. Characterization of the Bis- and Cyclized Conjugates of NCQ-344 with Chemical Derivatization and Deuterium Exchange. The reaction between GSH and NCQ-344 was repeated in deuterated buffer to determine the number of exchangeable hydrogens present in the bis- and cyclized conjugates as an aid to structure elucidation, particularly of the cyclized conjugate. Following reaction in deuterated buffer, the molecular ions of interest were observed at m/z 884 and m/z 921, rather than at m/z 871 and m/z 905, respectively. The observed mass shift indicated 12 and 15 exchangeable hydrogens for the cyclized and bis-conjugate, respectively. To determine the number of amino groups that could be acetylated in each conjugate, an aliquot of a reaction mixture containing both conjugates was acetylated as described in the Materials and Methods. The resulting products were mass measured to reveal that the mass increments for m/z 871 and m/z 905 were 42 and 84 Da, corresponding to one and two free amino groups, respectively. To determine the number of carboxyl groups present, an aliquot of a reaction mixture containing both conjugates was subject to methyl esterfication as described in the Materials and Methods. The resulting products were mass measured to reveal that the greatest mass increment for these conjugates was 56 Da indicating the presence of four carboxylate groups on each of the conjugates. The results of these experiments support the postulated structures 11 and 12 (Scheme 2). Reaction of NCQ-344 and NCQ-436 with the Oxidant HOCl. The oxidant hypochlorous acid (HOCl) was

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Figure 4. (A) Full scan spectra of NCQ-344 before and after addition of the oxidant HOCl. The isotopic pattern consisting of peaks at m/z 373 and m/z 375 is due to bromine. Immediately after mixing, the pattern shifted to produce the isotopic pattern with m/z 371 and m/z 373 as the major peaks indicating loss of 2 Da. (B) NCQ-436 was not oxidized as indicated by the identical isotope pattern before and after 5 min with hypochlorous acid.

added as a solution of NaOCl in 1% acetic acid to a separate buffered solution of either NCQ-344 or NCQ436. After addition of HOCl to NCQ-344, a change in the isotope pattern of the molecular ion was immediately apparent by MS analysis as depicted in Figure 4A. Mass measurement revealed a 2 Da decrease in mass of the molecular ion based on the shift in the isotope cluster from m/z 373/375 to m/z 371/373. This is consistent with the formation of a para-quinone from the hydroquinone. In contrast, an identical incubation of NCQ-436 with HOCl failed to produce an analogous change in the mass spectrum even after 5 min as shown in Figure 4B. Neutrophil-Mediated Bioactivation of NCQ-344. Stimulated neutrophils, which produce HOCl, were investigated for their ability to oxidize NCQ-344 to the para-quinone. GSH sulfonamide, proposed as a biomarker of MPO-derived hypochlorous acid (21), was detected by LC/MS/MS with a retention time of 10.6 min in neutrophils stimulated with PMA after 15 min. Levels were still increasing as judged by peak area after 120 min, the last time point sampled (data not shown). By 45 min, approximately four times more GSH sulfonamide was present than at the start. GSH sulfonamide could not be detected in unstimulated neutrophils. In addition to detection of GSH sulfonamide, stimulated neutrophils could be distinguished visually by a yellow tinge in contrast to the pink tinge of unstimulated neutrophils. On the basis of the results of this experiment, it was decided to stimulate the neutrophils for 15 min prior to addition of NCQ-344. NCQ-344 was added to control (unstimulated) neutrophils or neutrophils that had been stimulated with PMA. GSH sulfonamide, NCQ-344, and the mono-, bis-, GSH, and cyclized conjugates in incubations from both unstimulated and stimulated neutrophils were monitored by LC/MS/MS. As in incubations without NCQ-344, GSH sulfonamide was only detected in stimulated neutrophils (Figure 5A). Retention times for NCQ-344, mono-, bis-, and cyclized GSH conjugates were 12.5, 11.8, 11.7, and 11.7 min, respectively. The initial concentration of NCQ344 (13 µM) did not decrease substantially during the sampling period for unstimulated neutrophils but did decrease to less that 1 µM by 120 min in stimulated neutrophils as judged by peak areas (Figure 5B). The

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Figure 5. Comparison of GSH sulfonamide (A), NCQ-344 (B), monoconjugate (C), and bis-conjugate (D) in PMA stimulated vs unstimulated human neutrophils. The y-axis represents the ratio of the compound of interest with the internal standard (IS) S-(pnitrobenzyl)-GSH. Peak area of the internal standard was divided by 1000 before forming the ratio. Error bars represent ( one standard deviation of duplicate measurements.

monoconjugate was detected in unstimulated neutrophils at all time points, and levels did not change substantially (Figure 5C). The bis-conjugate was also detected at all time points and increased slightly during the course of the experiment (Figure 5D). In stimulated neutrophils, the monoconjugate was detected at all time points and had approximately doubled after 15 min following which levels remained constant for the rest of the experiment. Levels of bis-conjugate increased throughout the experiment and were over 5-fold higher at 120 min as compared to initial levels. The bis-conjugate was also approximately 5-fold higher than in unstimulated neutrophils at 45 min. The cyclized conjugate had a retention time almost identical to the bis-conjugate and was detected in some but not all experiments. When detected, amounts paralleled that of the bis-conjugate but at lower levels. In the presence of NCQ-344, no visual distinction between stimulated and unstimulated neutrophils was possible. Ab Initio Calculations. The relative electronic energies (∆E) defined by the oxidation reaction of the hydroquinone to para-quinone and catechol to ortho-quinone (HQ + 1/2O2 + ∆E f Q + H2O) and the corresponding energies for NCQ-344 and NCQ-436 leading to the respective quinones and water were calculated using density functional theory. For the remoxipride metabolites, to identify key contributions to the thermodynamics of the reaction by the various chemical substituents, calculations were also performed without the amide and methoxy groups. The relative electronic energies of the reactions of the hydroquinone and catechol leading to the para- or ortho-quinones were -13.8 and -4.5 kcal/mol, respectively (Table 1, entry a, I f III; II f IV). Including the bromine substituent decreased the exothermicity (as compared to the respective unsubstituted hydroquinone or catechol) by 0.6 kcal/mol (Table 1, entry b, I f III) for the ortho-quinone and had no effect on the para-quinone. The effects of the amide and methoxy substituents on the exothermicity for the para- and ortho-quinones were

11.6 and 11.8 kcal/mol, respectively. The effect of water on the relative energies was also evaluated. In an aqueous phase (water), the relative energy difference between the formation of the ortho- and the para-quinone was 7.9 kcal/mol. Thus, the effect of water was relatively small (1.2 kcal/mol) and changed the magnitude of the gas phase calculations only slightly. These results indicate that oxidation of NCQ-344 to the para-quinone is spontaneous while oxidation of NCQ-436 to the orthoquinone is not.

Discussion Although postulated to be involved in inducing apoptosis (8, 9), the respective para- and ortho-quinones of NCQ-344 (3) (Scheme 1) and NCQ-436 (5) have not yet been detected. In experiments described here, NCQ-344 incubated in vitro with GSH was shown to be capable of facile reaction to produce three distinct conjugates (Scheme 2): a mono- (6) and bis-GSH (8) conjugate and a cyclized conjugate existing as tautomers (11, 12). The cyclized conjugate formed from the bis-GSH conjugate following intramolecular cyclization with displacement of the methoxy substituent by the R-amino group of the γ-glutamic acid of GSH. These findings are indeed consistent with para-quinone formation arising from the hydroquinone NCQ-344. In contrast, no GSH conjugates were detected with NCQ-436. Work by Ross and co-workers demonstrated that both NCQ-344 and NCQ-436 induced apoptosis and that at higher concentrations NCQ-344 caused necrosis (8). No necrosis was observed for NCQ-436. It was proposed that these effects were due to formation of reactive quinone metabolites. Our work has demonstrated that only NCQ344 has the ability to oxidize spontaneously to the paraquinone whereas due to structural factors NCQ-436 does not form an ortho-quinone. Thus, the ability to induce apoptosis may be a property inherent to NCQ-344 and

GSH Conjugates of the Remoxipride Metabolite NCQ-344

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Scheme 2. Proposed Pathway for Formation of GSH Conjugates of NCQ-344

NCQ-436 themselves and not dependent on reactive metabolite formation. The ability of NCQ-344 to cause necrosis is probably a reflection of its ability to form a reactive metabolite. Taken together, the picture that emerges is that subtle differences in molecular structure can influence a drug’s (or metabolite’s) ability to cause apoptosis, necrosis, or both. Indeed, examples of compounds exist, such as the redox cycling quinone 2,3dimethoxy-1,4-naphthoquinone, which depending on concentration and exposure duration stimulate growth, induce apoptosis or cause necrosis of pancreatic RINm5F cells (22). The remoxipride metabolite NCQ-344, which is detectable in human plasma (23), was shown here to react with GSH at physiologic pH and temperature consistent with the presence of a fraction of NCQ-344 as the paraquinone. This para-quinone reacted with the nucleophile GSH via a Michael addition to form a monoconjugate resulting in bromine elimination. Of comparative interest is the formation of two mono-GSH conjugates of the chloroaromatic drug apraclonidine, where elimination of chlorine also occurs although to a lesser degree than the alternative aromatic substitution without chlorine displacement (24). Apparently, the hydroquinone structure of the monoconjugate could reoxidize, to reform a para-quinone that reacted further with GSH to form a bis-conjugate. The presence of the para-quinone was indicated by a small peak in the mass spectrum at m/z 598, 2 Da less than the monoconjugate at m/z 600 (see Figure 2, insert). It has been demonstrated for certain compounds capable of ortho-quinone formation that once the thioether conjugate is formed, the resulting conjugate is more prone to oxidation than the parent compound and thus readily undergoes further chemical reactions. Examples of molecules displaying such chemistry include bromobenzene (25) and salsinolol (26) in which the initial cysteine thioether conjugate undergoes intramolecular cyclization via a condensation of the R-amino group of γ-glutamic acid of GSH with the carbonyl group of the ortho-quinone to generate a 1,4-benzothiazine structure. A similar condensation between the para-quinone carbonyl and the

R-amino group in the bis-conjugate would produce a cyclized quinone diimine (Scheme 2; 10) although this chemical transformation product was not observed. An alternative transformation of the bis-conjugate proposed here requires the R-amino group of the γ-glutamic acid of GSH to displace the methoxy group (see Scheme 2) in an intramolecular cyclization reaction producing a cyclized conjugate. As observed in the MS spectrum of the monoconjugate, close examination of the molecular ion corresponding to the bis-conjugate at m/z 905 revealed the presence of m/z 903 representing the quinone form (data not shown). Cyclization is made possible by the fact that the methoxy group is “doubly” activated by the presence of the conjugated system and amide group making it highly susceptible to displacement. Measured experimental data that support the cyclic tautomeric structures (Scheme 2; 11 and 12) in addition to molecular weight include the 12 exchangeable hydrogens, one free amino group, and four carboxylate groups. Intramolecular cyclization can be viewed as a detoxification pathway as has been suggested on the basis of detailed studies of several quinone/quinol thioethers (27). The conversion of the bis-conjugate to the cyclized conjugate may also be viewed as a detoxification event as it prevents the bisconjugate with its continued electrophilic character from reacting further with more critical cellular nucleophiles or to undergo redox cycling with generation of reactive oxygen species. However, GSH conjugation can sometimes allow transport of a reactive metabolite from its site of formation to distal sites (28). For example, methylisocyanate can be transported as GSH conjugates from the liver (29) and a primary metabolite of Felbamate can cyclize to an oxazolidinone for stable transport from the liver before conversion to a reactive aldehyde (30). Further investigations would be needed to explore this possibility with regards to the potential toxicity of this GSH conjugate of NCQ-344. Experiments were performed with human neutrophils based on the hypothesis that stimulated neutrophils by virtue of hypochlorous acid generation would produce more NCQ-344 GSH conjugates than unstimulated neutrophils. Measurement of the proposed biomarker of MPO

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activity, GSH-sulfonamide, provided evidence that PMA treatment stimulated neutrophils and served as a control to these experiments. Stimulated human neutrophils consumed NCQ-344 to a far greater extent than unstimulated neutrophils, consistent with greater levels of para-quinone metabolite due to oxidation by hypochlorous acid. The finding of higher amounts of GSH conjugates (from 2- to 5-fold) in stimulated as compared to unstimulated neutrophils is consistent with our hypothesis. It is difficult to compare quantitatively by MS alone whether the amount of GSH conjugates formed accounts for the amount of NCQ-344 consumed. Such a quantitative endeavor is made more difficult as it is likely that the para-quinone of NCQ-344 is reacting with proteins such as membrane proteins, to form adducts in addition to GSH conjugates. Finally, the variability in formation of the cyclized conjugate may reflect differences between the human neutrophils used in these experiments, which can add factors that are difficult to control. Catechols are known to be capable of generating reactive ortho-quinones such as occurs in the metabolism of benzene (31). NCQ-436 did not form any GSH conjugates ostensibly due to its inability to oxidize to a reactive ortho-quinone because unique electronic factors created by the aromatic ring substituents (discussed below) that made oxidation of NCQ-436 energetically unfavorable. Supporting this hypothesis are ab initio calculations indicating that formation of the ortho-quinone from NCQ436 was not energetically favored with a reaction energy of +7.1 kcal/mol (Table 1, entry c, II f IV). The large positive value may also explain the experimental observation that even the oxidant hypochlorous acid was unable to oxidize NCQ-436 to the ortho-quinone. In contrast, para-quinone formation from NCQ-344 was energetically favored by 9.1 kcal/mol in comparison to ortho-quinone formation from NCQ-436 with a reaction energy of -2.0 kcal/mol (Table 1, entry c, I f III) and thus proceeded spontaneously under physiologic conditions and was accelerated in the presence of the oxidant hypochlorous acid. Our analysis employing ab initio calculations indicates that the key determinant in influencing the electronic energy for both NCQ-344 and NCQ-436 was the combination of the methoxy and amide carbonyl groups. orthoQuinone formation from catechol is favorable by -4.5 kcal/mol (Table 1, entry a, II f IV). Examination of energy-minimized structures of both NCQ-344 and NCQ436 and their respective quinones (available as Supporting Information) enables speculation as to the reasons behind their different properties. The catechol NCQ-436 is stabilized by three hydrogen bonding interactions: between the amide carbonyl oxygen and the aromatic hydroxyl, between the methoxy oxygen and the amide hydrogen, and between the two adjacent aromatic hydroxyl groups. In the ortho-quinone structure, a repulsive interaction also exists between the methoxy and the amide groups that destabilize the quinone. With the hydroquinone NCQ-436, only two of these stabilizing hydrogen bonding interactions exist (no adjacent aromatic hydroxyls), and there is no destabilizing interaction between the methoxy and the amide groups such that the net stabilization is not enough to offset the greater intrinsic exothermicity of para-quinone formation of -13.8 kcal/mol (Table 1, entry a, I f III). Interestingly, bromine did not have a significant impact on the relative

Erve et al.

energies of these metabolites (Table 1, entry b, I f III, II f IV). The apparent association between aplastic anemia and remoxipride suggests that the toxicity was idiosyncratic as hemotoxicity was not detected during preclinical trials preceding release to market (32, 33). Aplastic anemia is characterized as a pancytopenia with nonfunctioning bone marrow (34). Although the etiology is poorly understood, the highly proliferative nature of hematopoietic cells may make them more susceptible with stromal cells being suspected targets for some time (35). Direct cytotoxicity to bone marrow cells or their precursors is a possible mechanism for aplastic anemia, but a more probable mechanistic explanation with respect to aplastic anemia of an idiosyncratic nature involves development of an immune response targeting critical cell types. Oxidative bioactivation to reactive intermediates with subsequent protein binding has been proposed as a common mechanism for drugs to cause idiosyncratic toxicity (36, 37). Although the exact mechanisms by which reactive metabolites lead to drug-induced dyscrasias such as aplastic anemia are not known in detail, most drugs associated with a high incidence of idiosyncratic hemotoxicity are oxidized to reactive intermediates (38). Reactive metabolites derived from a number of drugs causing idiosyncratic toxicity have been identified, including those causing aplastic anemia, such as trimethoprim (39) and carbamazepine (40). With respect to NCQ-344, one may also find covalent modification of protein thiols resulting in haptenation. What mechanistic role the reactive metabolite derived from NCQ-344 may have, if any, in causing aplastic anemia in humans via formation of protein conjugates remains to be elucidated.

Conclusion The remoxipride metabolite, NCQ-344, oxidizes spontaneously to a para-quinone that can react with GSH forming conjugates whereas the related structural isomer NCQ-436 does not oxidize to an ortho-quinone. These results allow rationalization of previous findings that only NCQ-344 had the ability to induce both apoptosis and necrosis (8). Necrosis may reflect the ability of the para-quinone of NCQ-344 to deplete GSH and/or modify critical protein thiol residues. Our findings demonstrate how minor modifications in structure can have a major impact on physicochemical properties, which in turn are manifested in critical biological events involved in toxic pathways.

Acknowledgment. We thank Dr. Anna Bogstedt for assistance in the isolation of neutrophils and Dr. Staffan Schmidt and Ms. Therese Ekelin for assistance with setting up the GSH sulfonamide LC/MS/MS method. The critical reviews by Drs. Magnus Halldin and Gerry Kenna are gratefully acknowledged. Supporting Information Available: Energy-minimized molecular structures. This material is available free of charge via the Internet at http://pubs.acs.org.

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