Cytochrome P450 Oxidation of the Thiophene-Containing

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Cytochrome P450 Oxidation of the Thiophene-Containing Anticancer Drug 3-[(Quinolin-4-ylmethyl)-amino]-thiophene2-carboxylic Acid (4-Trifluoromethoxy-phenyl)-amide to an Electrophilic Intermediate Christine Medower,‡ Lian Wen,† and William W. Johnson* Drug Metabolism and Pharmacokinetics, OSI Pharmaceuticals, Boulder, Colorado 80301 ReceiVed December 7, 2007

Compounds that are enzymatically transformed to reactive intermediates are common in nature. Some drugs and many phytochemicals that contain a thiophene ring are oxidized by cytochrome P450 to biological reactive intermediates (BRI) that can covalently bind to thiol nucleophiles. The investigational anticancer agent 3-[(quinolin-4-ylmethyl)-amino]-thiophene-2-carboxylic acid (4-trifluoromethoxy-phenyl)amide (OSI-930) contains a thiophene moiety that can be oxidized by P450s to an apparent sulfoxide, which can react via Michael-addition to the 5-position of the thiophene ring, as demonstrated by mass spectral characterization of several thioether conjugates of the presumed thiophene S-oxide. Furthermore, a stable deuterium isotope retention experiment in which solvent deuterium was incorporated into the thiophene verifies the sulfoxide pathway. Various thiol nucleophiles are shown by tandem mass spectra to bind with this BRI, which is activated by P450 3A4 and to a slight degree, P450 2D6. Yet various safe drugs, phytochemicals, and endogenous molecules, all noted for their activation to BRI, are not toxic at a normal dose. Thus, multiple features determine any consequence of a BRI, with these complexities determining why one BRI is benign while another is not. The retention of covalent protein adducts of radio-labeled intermediate rat tissue has a half-life of about 1-1.5 days; hence, modified protein is cleared and replaced relatively quickly. Introduction Thiophene derivatives are ubiquitous in nature, with some produced by the cooking process and others by the combustion of fossil fuels (1, 2). Polythiophenes are commonly used in materials chemistry, while thiophene rings occur in many drugs (3). Thiophene compounds have been reported to be activated to electrophilic intermediates by cytochrome P450-mediated oxidation. The resulting sulfoxides can then be covalently modified by glutathione and other thiol-containing compounds. Several publications describe evidence for the formation of thiophene sulfoxides as a primary intermediate in the oxidative metabolism of two thiophene derivatives (4–7). These sulfoxides react rapidly with various nucleophiles by a Michael-type addition at position 5 of the thiophene ring; reactions with nucleophilic residues of proteins result in covalent binding to proteins (6). Many xenobiotics are oxidized by cytochrome P450 enzymes in the liver to become electrophilic metabolites, such as quinoids and epoxides, which have the potential to covalently modify cellular macromolecules and cause cytotoxicity. After formation in ViVo, these alkylating agents meet one of several fates: they (1) covalently bind to the active site of the enzyme in which they were formed; (2) undergo hydrolysis; or (3) are released from the enzyme followed by alkylation of another biological nucleophile such as glutathione (GSH). Therefore, sequestration of reactive metabolites by GSH and other thiols was used in the present study to * To whom correspondence should be addressed. Drug Metabolism and Pharmacokinetics, OSI Pharmaceuticals, Inc., 2860 Wilderness Place, Boulder, CO 80301. E-mail: [email protected]. † Present address: DMPK and Bioanalytical Services, ABC Laboratories, Inc., 7200 E. ABC Lane, Columbia, MO 65202. ‡ Present address: Tucson, AZ.

characterize the chemical pathway and intermediates. Any subsequent protein modification by biological reactive intermediates in ViVo, and potential toxicity therefrom, is dependent on many variables, including protein target (Critical Protein theory), effect on the target protein, state of oxidative stress and oxidative defenses, concentration (of intermediate and parent), extent of modification (reactivity), and detoxication routes such as epoxide hydrolase, GSH S-transferase, UDP-glucuronosyl transferase, sulfotransferase, aldehyde dehydrogenase, superoxide dismutase, and even other P450-mediated products (8–13). Other factors cause variability in the clinical outcome, such as diet, infection, enzyme induction, polymorphisms, cytokines, and immune responsiveness. Consequently, although many drugs are metabolized to reactive intermediates, they are commonly not toxic at efficacious doses. Such drugs currently marketed include clozapine (14), acetaminophen (15), diclofenac (16, 17) phenytoin (18), and estrogens (19). Indeed, covalent binding can be a detoxification step for reactive intermediates. Notably, some drugs depend on covalent bonding to proteins for efficacy (e.g., penicillins, aspirin, and omeprazole). Moreover, nutrients and endogenous compounds can form reactive intermediates (20–25). Therefore, it is critical to understand the metabolic mechanism when considering any toxicity relevance. OSI-930 (3-[(quinolin4-ylmethyl)-amino]-thiophene-2-carboxylic acid (4-trifluoromethoxy-phenyl)-amide) is an investigational anticancer agent that contains a thiophene moiety (26, 27). The results herein describe the P450-mediated biotransformation of the thiophene moiety in OSI-930 to a sulfoxide and the subsequent covalent reaction with thiols such as GSH.1 Although the compound 1

Abbreviations: BRI, biological reactive intermediate; GSH, glutathione.

10.1021/tx700430n CCC: $40.75  2008 American Chemical Society Published on Web 08/02/2008

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Table 1 adduct precursor ion (M + H+)

adduct minus 129 product ion (m/z)

749

620

plus 16 ethyl

765 777

636 648

ethyl 16

793

664

Adduct Molecule OSI-930 MW 443

glutathione glutathione glutathione ester glutathione ester plus

forms BRI and also goes on to form an adduct with a surrogate reagent, neither these entities nor these processes provide any evidence of a toxic phenomenon. Indeed, despite the formation of BRI (and an adduct), OSI-930 was well tolerated at efficacious doses in preclinical models and is well tolerated in normal healthy volunteers.

Materials and Methods Materials. Pooled human liver microsomes were purchased from Xenotech (Lenexa, KS) and In Vitro Technologies (Baltimore, MD). Recombinant cytochrome P450s (Bactosomes) were purchased from Xenotech (Lenexa, KS). β-Nicotinamide adenine dinucleotide phosphate, glutathione, glutathione ethyl ester, and N-acetyl-Lcysteine were purchased from Sigma-Aldrich (St. Louis, MO). ACS grade formic acid and HPLC grade acetonitrile and methanol were purchased from VWR (West Chester, PA). Microsomal Incubations. Incubations were conducted at 37 °C for 60 min. Final reaction mixtures contained 1 mg/mL human liver microsomal protein, 2 mM NADPH, 20 µM test compound, and a thiol-containing trapping agent. Trapping agents used were GSH, GSH ethyl ester (GSHEE), and N-acetylcysteine (NAC) at final concentrations of 5, 5, and 1 mM, respectively. Control assays with no NADPH, no trapping agent, and no test compound were performed. Incubation reaction mixtures were brought to a final volume of 300 µL with the addition of 50 mM potassium phosphate buffer (pH 7.4). Acetonitrile (0.6 mL) was used to terminate incubations. Reaction mixtures were centrifuged for 15 min at 17,000g. Supernatants were then transferred to another tube and stored at -20 °C until analysis. Recombinant Cytochrome P450 Incubations. Conditions were equivalent to the microsomal incubations except for the following: final reaction mixtures contained 1 mM NADPH, 2 mM GSHEE, and P450 concentrations varying from 8-13 pmol/mL. LC-MS/MS. Supernatant (200 µL) was transferred to a 96-well plate and evaporated to dryness under a nitrogen stream at 35 °C. Samples were resuspended in 100 µL water/methanol, 95%/5%, v/v matching the initial chromatographic conditions. A CTC HTC PAL autosampler (LEAP Technologies, Carrboro, NC) was used to inject 20 µL of sample onto a Phenomenex (Torrance, CA) Synergi 4 µ Hydro-RP 80A HPLC column (250 mm × 3.00 mm, 4 µM column). Using an Agilent 1100 HPLC system (Santa Clara, CA), the mobile phase began at 300 µL/min, 95%A, 5%B v/v (where A ) water, 0.1% formic acid, and B ) methanol, 0.1% formic acid). From 1 to 25 min, a linear gradient brought the mobile phase to 5% A, 95% B v/v, where conditions were held for an additional 5 min. Re-equilibration at the mobile phase starting conditions occurred for 10 min prior to the next injection. Mass spectrometric analysis was conducted on an Applied Biosystems 4000 Q-trap (Foster City, CA) using positive ion electrospray ionization. Two techniques were employed in the screening for GSH and GSHEE conjugated test compounds: (1) a neutral loss 129 scan and (2) a selected reaction monitoring (SRM) technique (28). The latter technique involved predicting the precursor ion/product ion transitions of potential adducts. An extensive list of transitions was created, taking into account a variety of bioactivation mechanisms (oxidations, hydrations, etc.). The list comprised calculated theoretical precursor ions (i.e., the protonated adduct molecule as the test compound plus the thiol trapping agent or the test compound plus the thiol trapping agent plus 16, 17, or 18 amu) and product ions based on the typical/

observed fragmentation of the trapping agent (the characteristic loss of 129 amu from GSH and GSHEE, the loss of 75 amu from GSH, and the loss of 103 amu from GSHEE). Screening for NAC conjugates was accomplished exclusively via the SRM method, and product ions were based on assumed losses of the NAC portion of the adduct molecule: minus the ketone (42 amu), minus NAC except sulfur (129 amu), or minus the whole NAC molecule (163 amu) (Table 1).

Results Oxidation of OSI-930 by Liver Microsomes in the Presence or Absence of Glutathione. Glutathione (GSH) and other thiol-containing compounds were used to covalently bind with any incidental soft electrophiles produced by the P450mediated oxidation of the thiophene containing OSI-930. The complete microsomal reaction incubation, including the cofactor NADPH and GSH, produced an oxidized thiophene that then was covalently modified by the sulfhydryl containing GSH. Figure 1 shows the resultant analytical HPLC peak to be more hydrophilic than OSI-930 when NADPH is added to the incubation, while the subsequent MS spectrum analysis of this analyte, (Figure 2), reveals a parent mass to charge ratio equivalent to the protonated GSH conjugate of OSI-930 plus the oxidation of the thiophene. The incremental gain of 18 amu indicates an addition product of GSH to the presumed thiophene sulfoxide. Further examination of the spectrum shows the typical GSH fragment loss of the elements of pyroglutamic acid (129 amu) from the parent (MH+) ion and many characteristic peaks of the OSI-930 fragmentation pattern. The structure illustrated for this GSH adduct is in agreement with the results of the MS spectrum (i.e., loss of 18 corresponding to H2O and other diagnostic transitions). Analysis of the Product Conjugate. Comparison of the MS spectrum of OSI-930 (Figure 3) with that of the novel 767 m/z ion shows that the peaks at m/z 206, 239, 265, 317, and 442 occur in similar relative intensities in the spectrum of the reaction product. (Energy-induced cleavage at the C-S bond may result in a carbocation to give an m/z value that is 2 amu less than the signals at m/z 444 and 267, characteristic of OSI930.) Notably, the product ion of m/z 749 is consistent with typical thiophene sulfoxide loss of water (although GSH adducts can also dehydrate). The trans-dihydrodiols and glutathione adducts are labile, especially in acidic conditions and rearomatize with loss of water to phenols and aromatic glutathione adducts, respectively. Because 18 amu is lost from several dominant ions, these fragments likely contain the thiophene sulfoxide. Several other likely fragment ions of the OSI-930GSH conjugate are discernible (Figure 2), e.g., the diagnostic ions at m/z 572 and 620 (Figure 3 shows the MS spectrum of OSI-930). The substructure with an m/z 442 peak is the likely result from the 307 amu loss due to cleavage at the glutathionyl sulfur (rather than 129 from GSH with the loss of the NH3 benzene-OCF3). The ethylester of GSH (GSHEE) is a GSH analogue shown to have superior detection efficiency and, hence, better sensitivity (28). The OSI-930-GSHEE spectrum using the selected reaction monitoring (SRM) method (Figure 4) shows the indicative fragmentation peaks at m/z 442, 471, 600, 648, and 666 and the characteristic water loss at m/z 777. The OSI-930-GSHEE spectrum showed an m/z 471 fragment (Figure 5), indicating the subsequent loss of both 129 and NH-benzene-OCF3; an m/z 442 fragment indicates the loss of 335 (GSHEE). These were considered to be odd-electron ions (i.e., radical cations) because the m/z values do not correspond to protonated ions. The peak at 666 m/z results

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Figure 1. LC/MS spectrum. Panel A: TIC result from microsomal reaction incubations with OSI-930 and without NADPH. Panel B: result when all components, cofactors, and GSH are present. The new peak introduced at 24.3 min is labeled. The x-axis is in time, and the y-axis is intensity in counts per second (cps).

Figure 2. Enhanced product ion spectra of the GSH conjugate with the thiopene sulfoxide. The typical 129 amu neutral loss from 767 and 749 m/z to 638 and 620 m/z, respectively, along with several other characteristic ions are shown and related to the molecular scheme (including loss of H2O). Products ions noted include 749, 638, 620, 572, and 442 m/z. Product ions are illustrated as corresponding to the molecular substructures in the inset. The x-axis is in m/z, and the y-axis is intensity in counts per second (cps).

from the characteristic 129 amu loss for GSH (or GSHEE) adducts, with the m/z 648 peak at one H2O less. The NAC thiol-containing nucleophile also covalently modified OSI930 to form a similar adduct (Figure 6). The parent mass (m/z 623) and the loss of H2O is in agreement with the OSI930-NAC structure (Figure 6). The water loss results in a

fragment ion at m/z 605 from the parent ion of 623. Other typical fragments of the thiol conjugate were prominent at m/z 239, 317, 428, and 442. From the OSI-930-NAC adduct, the 239 m/z represents the quinoline and thiophene; m/z 267 represents the previous substructure plus a carbonyl; and, with a sulfur added, the m/z is at 299; an addition of H2O to


Figure 3. Product ion spectra of OSI-930 molecular ion at 444 (MH+) and product ions at 267, 239, and 142 m/z. The x-axis is in m/z, and the y-axis is intensity in counts per second (cps).

Figure 4. LC/MS spectrum shows the TIC result from microsomal reaction incubations with OSI-930 and when all components, cofactors, and GSHEE are present. The new peak introduced at 25.9 min is labeled. The x-axis is in m/z, and the y-axis is intensity in counts per second (cps).

this gives m/z 317; the m/z 428 daughter corresponds to the 267 ion plus N-acetylcysteine. Results with the Thiophene-Containing Substructure. A thiophene-containing substructure OSI-458895, the basis for the OSI-930 molecule, may be similarly activated to an electrophilic sulfoxide. OSI-458895 was also shown to form a sulfoxide product (Figure 7). The structure illustrates a covalently modified OSI-458895 with the GSHEE acting as the thiol nucleophile in this case. The peaks at m/z 654, 636, 525, 507, 459, and 330 represent the characteristic fragment ions indicated in the figure. The adduct forms both on this subfragment of OSI-930 and on the 267 fragment of OSI-930 itself, with the only possibility for activation occurring at the thiophene. Individual P450s Involved. Various P450s were tested for the capacity to form the reactive intermediate that could result in the formation of the conjugate. Incubations were performed in the presence of P450 3A4, P450 2D6, P450 2E1, P450 1A2, P450 2B6, P450 2C19, or P450 2A6. The LC-MS/MS analysis based on the SRM revealed a significant peak from the P450 3A4 reactions and a detectable peak from the P450 2D6 reaction, suggesting that this bioactivation is catalyzed by P450 and that the P450 3A4 is the enzyme predominantly responsible. There was no product detected from the other isoforms. Isotopic Retention. To discern the pathway of the reaction (i.e., sulfoxide versus oxirane) and the operative reactive intermediate that leads to the covalent adduct, the incorporation and retention


of solvent water proton (or deuterium) was examined. Performing the reactive-intermediate trapping experiment in the presence of the deuterated water solution (D2O) allows the insertion of a deuterium atom to be measured by a single amu mass shift in the product (Scheme 1). The pathway via the sulfoxide has a solvent proton (or deuterium) incorporated into the ring, and it is largely retained through the dehydration/rearomatization due to a partial isotope effect. The considered pathway via the epoxide, however, does not incorporate solvent protons after the loss of H2O. Consequently, the deuterium incorporation experiment resulted in a GSH conjugate that is shifted by 1 amu when compared to the same conjugate produced in water (Figure 6). The spectrum also showed a 1 amu increase in many of the distinguishing CID peaks that represent thiophene-containing moieties (Figure 8). The proportion of deuterium retention to total area of both conjugate peaks (the other analyte peak having incorporated hydrogen) was ∼70%, which indicates some hydrogen incorporation may result from the small amount of contaminating H2O. Because the deuterium is predominantly incorporated, the sulfoxide pathway is imperative. In ViWo Protein Binding Quantification. The radiolabeled OSI-930 was administered to rats to quantify any protein adduct formation in the liver, kidney, and intestines. The reactive intermediate produced by oxidative metabolism in ViVo covalently bound to protein residues within these tissues. Samples collected on days 2, 4, and 7 after dose administration were analyzed. After multiple extraction of the tissue homogenate, the retained protein was measured for radioactivity (DPM) and normalized to total protein. The results are plotted and shown in Figure S1 (Supporting Information). The decrease in labeled protein has an adducted protein clearance half-life of less than 2 days in liver tissue. Since the Tmax of orally administered OSI-930 is about 8 h and the parent compound is still at significant concentrations thereafter, it is implicit that the transient reactive intermediate is maximally produced during this time. Hence, the exposure to the activated intermediate and the maximum protein binding is subsequent to 8 h postdose. Consequently, the real half-life would be about 8-10 h less than the apparent or observed half-life. The clearance from liver of the covalently modified protein is relatively quick at ≈1-1.5 days. Therefore, the adducted residues are not intransigent and could be replaced de noVo at a pace that is typical of some P450s (29). The quantity of adducts is comparatively low at about 14 pmol (drug equivlnt/mg total protein) in the liver at 2 days postdose. The adduct levels were lower in the kidney, yet the 2-day time point in the intestine is similar to that of the liver until day 4, following a precipitous decline due, most probably, to natural enterocyte sloughing.

Discussion The adduct clearance half-life of less than 2 days indicates that the modified proteins are not intransigent and are removed from the cell comparatively rapidly. This rate of clearance is similar to the turnover of membrane-associated endoplasmic reticulum proteins such as cytochrome P450, cytochrome P450 reductase, cytochrome b5, epoxide hydrolase, and others (29). Some P450s have turnover half-lives of 1 or 2 days. This further supports a purported safety since affected proteins are not retained over significant durations. The quantity of adducts at the 2 day time point is 14 pmol drug equivalent/mg total protein. This is a comparatively very low amount and is significantly less than half of the reference guideline of 50 pmol(drug equivalent)/mg total protein proposed recently based on studies of hepatotoxic and well-tolerated drugs known to form BRI (30). Unlike benzothiophene S-oxides, thiophene S-oxides are very reactive species. Although few of them have been described

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Figure 5. Enhanced product ion spectra of the GSHEE conjugate with the thiopene sulfoxide. The typical 129 amu neutral loss from 795 and 777 m/z to 666 and 648 m/z, respectively, along with several other characteristic ions are shown and related to the molecular scheme (including loss of H2O). Products ions noted include 777, 759, 666, 648, and 442 m/z. Product ions are illustrated as corresponding to the molecular substructures in the inset. The x-axis is in m/z, and the y-axis is intensity in counts per second (cps).

Figure 6. Enhanced product ion spectra of the N-acetyl-cysteine conjugate with thiopene sulfoxide. The expected molecular ion at 623 m/z and typical characteristic ions are shown and related to the molecular scheme. Products ions noted include 605, 442, 428, and 299 m/z. Product ions are illustrated as corresponding to the molecular substructures in the inset. The x-axis is in m/z, and the y-axis is in counts per second (cps).

thus far (31–34), recent results show that oxidation of thiophene leads to adducts from a Michael-type addition of a thiolcontaining nucleophilic reagent to a thiophene S-oxide intermediate (5). Tienilic acid (TA) is an uricosuric diuretic drug that has been found to cause immunoallergic hepatitis in about 1/10,000 patients treated (35). Covalent binding of TA and a TA isomer is cytochrome P450-dependent and is almost completely inhibited in the presence of sulfur-containing nucleophiles such as glutathione (34). A reactive metabolite thiophene S-oxide was stable enough to be characterized with 1 H NMR, which showed only one vinylic proton coupled to two geminal protons (H5), a singlet for H2, and other spectrum signals indicating the structure as the S-oxide shown (5), rather than epoxide. The UV spectrum, pH-dependent changes (H2O loss), and MS spectrum also support the conclusion that the pathway is via sulfoxide.

The reactive intermediate formed from TA enables mechanism-based inactivation of P450 2C9 and P450 2C10 enzymes without protection by exogenous GSH; oxidation also produces a concomitant 5-hydroxytienilic acid (5-OHTA) product (36). The proposed mechanism involves the formation of intermediate thiophene sulfoxide, which may react at the strongly electrophilic position 5 of its thiophene ring either with H2O, to give 5-OHTA, or with a nucleophilic group of an amino acid residue of the P450 active site, to result in covalent binding to the P450 protein. The partition ratio of about 11-12 and the significantly higher microsomal protein binding in the absence of GSH show that TA can diffuse out of the P450 active site and react with other proteins or GSH. Although performing the reaction incubation in 70% H218O resulted in no 18O incorporation, the presence of ∼90% 18O2 showed about 95% incorporation of one 18O atom, results that confirm an S-oxide mechanism (37).



Figure 7. Enhanced product ion spectra of the GSH conjugate with the thiopene sulfoxide of the subfragment of OSI-930. The typical 129 amu neutral loss from 654 and 636 m/z to 525 and 507 m/z, respectively, along with several other characteristic ions are shown and related to the molecular scheme (including loss of H2O). Products ions noted include 636, 525, 507, 459, and 330 m/z. Product ions are illustrated as corresponding to the molecular substructures in the inset. The x-axis is in m/z, and the y-axis is in counts per second (cps).

Scheme 1

Moreover, characterization of the adduct with the P450 protein demonstrates that the mass shifts include 16 additional amu over TA alone (38). Studies involving the antirheumatic drug Tenidap, which also contains a thiophene, however, indicate that a major route of metabolism is hydroxylation of that ring with no reported apparent sulfoxide (39). A similar pathway is operative for the anti-inflammatory suprofen (40) and the anticholinergic tiquizium bromide (41). The spectroscopic/spectrometric (NMR, IR, UV, and MS) data of an in ViVo conjugate of thiophene itself are consistent with the dihydrothiophene sulfoxide structure and not the epoxide, as the 1H NMR signals for the protons using doubleirradiation and 2D COSY experiments demonstrate; the 13C NMR signals are consistent with this structure (4). Hence, they suggest a mechanism of S-oxidation, Michael-type GSH addition at position 2, protonation at position 5, and physiological transformation to N-acetylcysteine conjugates. A more comprehensive study of 3-aroylthiophene that added MS to the

analysis shows clear evidence for the sulfoxide intermediate via metabolic oxidation in Vitro and in ViVo (6), with the geminal H5 protons detected by 1H NMR spectroscopy characteristic of the sulfoxide. In addition to its reaction with nucleophiles such as glutathione, the thiophene S-oxide can dimerize via a Diels-Alder reaction (7). X-ray crystal diffraction determination of the structures of the two diastereoisomeric thiophene S-oxide dimers, reported for thiophene (7), provides direct evidence for the transient thiophene S-oxide reactive intermediate in the metabolism of thiophene. The thiophene S-oxide dimers can also form in ViVo in rats (7). The thiophene-containing drug ticlopidine is oxidized by P450 2C19 to the dimer of the sulfoxide via a Diels-Alder reaction (42), with this oxidation alternatively resulting in P450 2C19 inactivation by apparent alkylation of the active site. Oxidation of thiophene with peracids also resulted in the formation of dimers and was rationalized, as well, by the initial oxidation at the sulfur atom to yield

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interactions and entities underscores the fact that the presence of a BRI is not an accurate indicator of human toxicity. Rather, any consequence of a BRI depends on the complex relationships among many factors, all of which must be examined in the determination of why one BRI is toxic and another is benign. Acknowledgment. We are very grateful to A. L. Stewart for editorial assistance. We thank a reviewer who provided valuable critiques and suggestions that significantly enhanced the manuscript. Supporting Information Available: Results of in ViVo protein covalent bonding of radio-labeled drug equivalent and clearance over a week. This material is available free of charge via the Internet at http://pubs.acs.org.

References

Figure 8. (a) Enhanced product ion spectra of the GSHEE conjugate with the thiopene sulfoxide. The typical 129 amu nuetral loss from 795 and 777 m/z to 666 and 648 m/z, respectively, along with several other characteristic ions are shown and related to the molecular scheme (including loss of H2O). Products ions noted include 777, 759, 666, 648, and 442 m/z. Product ions are illustrated as corresponding to the molecular substructures in the inset. The x-axis is in m/z, and the y-axis is intensity in counts per second (cps). (b) Enhanced product ion spectra of the GSHEE conjugate with the thiopene sulfoxide when the activation biotransformation reaction was performed in D2O. The typical 129 amu nuetral loss from 796 and 778 m/z to 667 and 649 m/z, respectively, along with several other characteristic ions are shown and related to the molecular scheme (including loss of H2O). Products ions are 1 amu greater than the corresponding experiment in H2O and include 778, 760, 667, 648, and 443 m/z. Product ions are illustrated as corresponding to the molecular substructures in the inset. The x-axis is in m/z, and the y-axis is intensity in counts per second (cps).

thiophene-S-oxide followed by a Diels-Alder-type dimerization (43). We looked for, but did not find, a sulfoxide dimer. Oxidation of 2-phenylthiophene by microsomal incubations produced the sulfoxide dimers by a Diels-Alder dimerization, the GSH adduct of the sulfoxide, and, interestingly, some areneoxide of the thiophene ring (44), thus providing the first evidence that P450 may catalyze both S-oxidation and epoxidation of thiophene derivatives. Additionally, experimental results of P450 2C9 inactivation by suprofen are consistent with both the sulfoxide and epoxide intermediates (45). Our results demonstrate that the thiophene in OSI-930 undergoes a P450-mediated biotransformation to a sulfoxide, and a subsequent covalent reaction with thiols such as GSH. We have not confirmed the exact site of adduction, and there is a conceivable pathway via concerted epoxide and thiophene ring-opening to produce a γ-aldo thiocarbonyl that could incorporate a deuterium. Yet neither these processes nor these products necessarily cause toxicity (46). Indeed, the complex nature of these many

(1) Ebermann, R., Alth, G., Kreitner, M., and Kubin, A. (1996) Natural products derived from plants as potential drugs for the photodynamic destruction of tumor cells. J. Photochem. Photobiol. B 36 (2), 95–97. (2) Dyreborg, S., Arvin, E., and Broholm, K. (1996) Effects of creosote compounds on the aerobic bio-degradation of benzene. Biodegradation 7 (3), 191–201. (3) Dalvie, D. K., Kalgutkar, A. S., Khojasteh-Bakht, S. C., Obach, R. S., and O’Donnell, J. P. (2002) Biotransformation reactions of fivemembered aromatic heterocyclic rings. Chem. Res. Toxicol. 15 (3), 269–299. (4) Dansette, P. M., Thang, D. C., el Amri, H., and Mansuy, D. (1992) Evidence for thiophene-S-oxide as a primary reactive metabolite of thiophene in vivo: Formation of a dihydrothiophene sulfoxide mercapturic acid. Biochem. Biophys. Res. Commun. 186, 1624–1630. (5) Mansuy, D., Valadon, P., Erdelmeier, I., Lopez-Garcia, P., Amar, C., Girault, J. P., and Dansette, P. M. (1991) Thiophene S-oxides as new reactive metabolites: Formation by cytochrome P-450 dependent oxidation and reaction with nucleophiles. J. Am. Chem. Soc. 113, 7825–7826. (6) Valadon, P., Dansette, P. M., Girault, J. P., Amar, C., and Mansuy, D. (1996) Thiophene sulfoxides as reactive metabolites: Formation upon microsomal oxidation of a 3-aroylthiophene and fate in the presence of nucleophiles in vitro and in vivo. Chem. Res. Toxicol. 9, 1403–1413. (7) Treiber, A., Dansette, P. M., El Amri, H., Girault, J. P., Ginderow, D., Mornon, J.-P., and Mansuy, D. (1997) Chemical and biological oxidation of thiophene: Preparation and complete characterization of thiophene S-oxide dimers and evidence for thiophene S-oxide as an intermediate in thiophene metabolism in ViVo and in Vitro. J. Am. Chem. Soc. 119, 1565–1571. (8) Guengerich, F. P. (2003) Cytochromes P450, drugs, and diseases. Mol. InterV. 3 (4), 194–204. (9) Guengerich, F. P. (2005) Generation of reactive intermediates. J. Biochem. Mol. Toxicol. 19 (3), 173–174. (10) Guengerich, F. P. (2006) Cytochrome P450s and other enzymes in drug metabolism and toxicity. AAPS J. 8 (1), E101–E111. (11) Guengerich, F. P., and Macdonald, J. S. (2007) Applying mechanisms of chemical toxicity to predict drug safety. Chem. Res. Toxicol. 20 (3), 344–369. (12) Park, B. K., Kitteringham, N. R., Maggs, J. L., Pirmohamed, M., and Williams, D. P. (2005) The role of metabolic activation in drug-induced hepatotoxicity. Annu. ReV. Pharmacol. Toxicol. 45, 177–202. (13) Kaplowitz, N. (2005) Idiosyncratic drug hepatotoxicity. Nat. ReV. Drug DiscoVery 4 (6), 489–499. (14) Williams, D. P., O’Donnell, C. J., Maggs, J. L., Leeder, J. S., Uetrecht, J., Pirmohamed, M., and Park, B. K. (2003) Bioactivation of clozapine by murine cardiac tissue in vivo and in vitro. Chem. Res. Toxicol. 16 (10), 1359–1364. (15) Hinson, J. A., Pumford, N. R., and Roberts, D. W. (1995) Mechanisms of acetaminophen toxicity: immunochemical detection of drug-protein adducts. Drug Metab. ReV. 27 (1-2), 73–92. (16) Kretz-Rommel, A., and Boelsterli, U. A. (1994) Selective protein adducts to membrane proteins in cultured rat hepatocytes exposed to diclofenac: radiochemical and immunochemical analysis. Mol. Pharmacol. 45 (2), 237–244. (17) Pumford, N. R., Myers, T. G., Davila, J. C., Highet, R. J., and Pohl, L. R. (1993) Immunochemical detection of liver protein adducts of the nonsteroidal antiinflammatory drug diclofenac. Chem. Res. Toxicol. 6 (2), 147–150. (18) Munns, A. J., De Voss, J. J., Hooper, W. D., Dickinson, R. G., and Gillam, E. M. (1997) Bioactivation of phenytoin by human cytochrome


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(21)

(22) (23)

(24) (25)

(26)

(27)

(28)

(29) (30)

(31) (32)

P450: characterization of the mechanism and targets of covalent adduct formation. Chem. Res. Toxicol. 10 (9), 1049–1058. Maggs, J. L., Grabowski, P. S., and Park, B. K. (1983) Drug protein conjugates-III. Inhibition of the irreversible binding of ethinylestradiol to rat liver microsomal protein by mixed-function oxidase inhibitors, ascorbic acid and thiols. J. Steroid Biochem. 19 (3), 1273–1278. Chaudhary, A. K., Nokubo, M., Reddy, G. R., Yeola, S. N., Morrow, J. D., Blair, I. A., and Marnett, L. J. (1994) Detection of endogenous malondialdehyde-deoxyguanosine adducts in human liver. Science 265, 1580–1582. Plastaras, J. P., Guengerich, F. P., Nebert, D. W., and Marnett, L. J. (2000) Xenobiotic-metabolizing cytochromes P450 convert prostaglandin endoperoxide to hydroxyheptadecatrienoic acid and the mutagen, malondialdehyde. J. Biol. Chem. 275, 11784–11790. West, J. D., and Marnett, L. J. (2006) Endogenous reactive intermediates as modulators of cell signaling and cell death. Chem. Res. Toxicol. 19, 173–194. Alderson, N. L., Wang, Y., Blatnik, M., Frizzell, N., Walla, M. D., Lyons, T. J., Alt, N., Carson, J. A., Nagai, R., Thorpe, S. R., and Baynes, J. W. (2006) S-(2-Succinyl) cysteine: A novel chemical modification of tissue proteins by a Krebs cycle intermediate. Arch. Biochem. Biophys. 450, 1–8. Januszewski, A. S., Alderson, N. L., Jenkins, A. J., Thorpe, S. R., and Baynes, J. W. (2005) Chemical modification of proteins during peroxidation of phospholipids. J. Lipid Res. 46, 1440–1449. Skordos, K. W., Skiles, G. L., Laycock, J. D., Lanza, D. L., and Yost, G. S. (1998) Evidence supporting the formation of 2,3-epoxy-3methylindoline: a reactive intermediate of the pneumotoxin 3-methylindole. Chem. Res. Toxicol. 11 (7), 741–749. Petti, F., Thelemann, A., Kahler, J., McCormack, S., Castaldo, L., Hunt, T., Nuwaysir, L., Zeiske, L., Haack, H., Sullivan, L., Garton, A., and Haley, J. D. (2005) Temporal quantitation of mutant Kit tyrosine kinase signaling attenuated by a novel thiophene kinase inhibitor OSI-930. Mol. Cancer Ther. 4, 1186–1197. Garton, A. J., Crew, A. P., Franklin, M., Cooke, A. R., Wynne, G. M., Castaldo, L., Kahler, J., Winski, S. L., Franks, A., Brown, E. N., Bittner, M. A., Keily, J. F., Briner, P., Hidden, C., Srebernak, M. C., Pirrit, C., O’Connor, M., Chan, A., Vulevic, B., Henninger, D., Hart, K., Sennello, R., Li, A. H., Zhang, T., Richardson, F., Emerson, D. L., Castelhano, A. L., Arnold, L. D., and Gibson, N. W. (2006) OSI-930: A novel selective inhibitor of Kit and kinase insert domain receptor tyrosine kinases with antitumor activity in mouse xenograft models. Cancer Res. 66, 1015–1024. Soglia, J. R., Harriman, S. P., Zhao, S., Barberia, J., Cole, M. J., Boyd, J. G., and Contillo, L. G. (2004) The development of a higher throughput reactive intermediate screening assay incorporating microbore liquid chromatography-micro-electrospray ionization-tandem mass spectrometry and glutathione ethyl ester as an in vitro conjugating agent. J. Pharm. Biomed. Anal. 36, 105–116. Correia, M. A. (2003) Hepatic cytochrome P450 degradation: mechanistic diversity of the cellular sanitation brigade. Drug Metab. ReV. 35, 107–143. Evans, D. C., Watt, A. P., Nicoll-Griffith, D. A., and Baillie, T. A. (2004) Drug-protein adducts: An industry perspective on minimizing the potential for drug bioactivation in drug discovery and development. Chem. Res. Toxicol. 17, 3–16. Mansuy, D. (1997) Molecular structure and hepatotoxicity: compared data about two closely related thiophene compounds. J. Hepatol. 26 (Suppl. 2), 22–25. Dansette, P. M., Amar, C., Smith, C., Pons, C., and Mansuy, D. (1990) Oxidative activation of the thiophene ring by hepatic enzymes. Hydroxylation and formation of electrophilic metabolites during


(33)

(34)

(35) (36)

(37)

(38)

(39) (40) (41)

(42)

(43) (44)

(45)

(46)

metabolism of tienilic acid and its isomer by rat liver microsomes. Biochem. Pharmacol. 39 (5), 911–918. Bonierbale, E., Valadon, P., Pons, C., Desfosses, B., Dansette, P. M., and Mansuy, D. (1999) Opposite behaviors of reactive metabolites of tienilic acid and its isomer toward liver proteins: Use of specific antitienilic acid-protein adduct antibodies and the possible relationship with different hepatotoxic effects of the two compounds. Chem. Res. Toxicol. 12, 286–296. Dansette, P. M., Amar, C., Valadon, P., Pons, C., Beaune, P. H., and Mansuy, D. (1991) Hydroxylation and formation of electrophilic metabolites of tienilic acid and its isomer by human liver microsomes. Catalysis by a cytochrome P450 IIC different from that responsible for mephenytoin hydroxylation. Biochem. Pharmacol. 41, 553–560. Homberg, J. C., Andre, C., and Abuaf, N. (1984) A new anti-liverkidney microsome antibody (anti-LKM2) in tienilic acid-induced hepatitis. Clin. Exp. Immunol. 55, 561–570. Lopez-Garcia, M. P., Dansette, P. M., and Mansuy, D. (1994) Thiophene derivatives as new mechanism-based inhibitors of cytochromes P-450: Inactivation of yeast-expressed human liver cytochrome P-450 2C9 by tienilic acid. Biochemistry. 33, 166–175. Belghazi, M., Jean, P., Poli, S., Schmitter, J.-M., Mansuy, D., and Dansette, P. M. (2001) Use of Isotopes and LC-MS-TOF for Mechanistic Studies of Tienilic Acid Metabolic Activation. In Biological ReactiVe Intermediates VI (Dansette, P. M. et al., Eds.) Plenum Publishers, New York. Koenigs, L. L., Peter, R. M., Hunter, A. P., Haining, R. L., Rettie, A. E., Friedberg, T., Pritchard, M. P., Shou, M., Rushmore, T. H., and Trager, W. F. (1999) Electrospray ionization mass spectrometric analysis of intact cytochrome P450: Identification of tienilic acid adducts to P450 2C9. Biochemistry 38, 2312–2319. Fouda, H. G., Avery, M. J., Dalvie, D., Falkner, F. C., Melvin, L. S., and Ronfeld, R. A. (1997) Disposition and metabolism of tenidap in the rat. Drug Metab. Dispos. 25, 140–148. Mori, Y., Kuroda, N., Sakai, Y., Yokoya, F., Toyoshi, K., and Baba, S. (1985) Species differences in the metabolism of suprofen in laboratory animals and man. Drug Metab. Dispos. 13, 239–245. Nishikawa, T., Nagata, O., Tanbo, K., Yamada, T., Takahara, Y., Kato, H., and Yamamoto, Y. (1985) Absorption, excretion and metabolism of tiquizium bromide in dogs, and relationship between pharmacological effect and plasma levels of unchanged drug. Xenobiotica 15, 1053– 1060. Ha-Duong, N. T., Dijols, S., Macherey, A. C., Goldstein, J. A., Dansette, P. M., and Mansuy, D. (2001) Ticlopidine as a selective mechanism-based inhibitor of human cytochrome P450 2C19. Biochemistry 40, 12112–12122. Treiber, A., Dansette, P. M., and Mansuy, D. (2002) Mechanism of the aromatic hydroxylation of thiophene by acid-catalyzed peracid oxidation. J. Org. Chem. 67, 7261–7266. Dansette, P. M., Bertho, G., and Mansuy, D. (2005) First evidence that cytochrome P450 may catalyze both S-oxidation and epoxidation of thiophene derivatives. Biochem. Biophys. Res. Commun. 338, 450– 455. O’Donnell, J. P., Dalvie, D. K., Kalgutkar, A. S., and Obach, R. S. (2003) Mechanism-based inactivation of human recombinant P450 2C9 by the nonsteroidal anti-inflammatory drug suprofen. Drug Metab. Dispos. 31, 1369–1377. Johnson, W. W. (2008) Many drugs and phytochemicals can be activated to biological reactive intermediates. Curr. Drug Metab. 9, 344–351.

TX700430N

Cytochrome P450 Oxidation of the Thiophene-Containing

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