Biotransformation Reactions of Five-Membered Aromatic Heterocyclic

MARCH 2002 VOLUME 15, NUMBER 3 © Copyright 2002 by the American Chemical Society

Invited Review Biotransformation Reactions of Five-Membered Aromatic Heterocyclic Rings Deepak K. Dalvie,*,† Amit S. Kalgutkar,† S. Cyrus Khojasteh-Bakht,‡ R. Scott Obach,† and John P. O’Donnell† Pharmacokinetics, Dynamics and Drug Metabolism, Pfizer Global Research and Development, Eastern Point Road, Groton, Connecticut 06340, and Small Molecule Pharmacology, Genentech Inc., 1 DNA Way South, San Francisco, California 94080-4990 Received October 16, 2001

Contents 1. Introduction 2. Heterocycles with One Heteroatom 2.1. Pyrroles 2.2. Indoles 2.3. Furans 2.4. Thiophenes 3. Heterocycles with Two Heteroatoms 3.1. Imidazoles 3.2. Oxazoles 3.3. Thiazoles 3.4. Pyrazoles 3.5. Isoxazoles 3.6. Isothiazoles 4. Heterocycles with Three or More Heteroatoms 4.1. Triazoles 4.2. Oxadiazoles 4.3. Thiadiazoles 4.4. Tetrazoles 5. Conclusions

1. Introduction 269 270 270 272 275 278 281 282 284 285 287 288 289 289 290 291 292 292 293

* To whom correspondence should be addressed. Phone: (860) 441-4641. Fax: (860) 715-7888. E-mail: Deepak•k•Dalvie@ groton.pfizer.com. † Pfizer Global Research and Development, Groton, CT. ‡ Genentech Inc. South San Francisco, CA.

Organic compounds containing five-membered aromatic heterocyclic rings are widely distributed in nature and often play an important role in various biochemical processes. Heteroaromatic rings also serve as bioisosteres of several substituents including phenyl rings or carboxylic acid and its ester analogues and oftentimes render greater pharmacological activity to the resulting compounds. As a result, they are commonly incorporated into new chemical entities by medicinal chemists (1, 2). Structure-metabolism relationship (SMR)1 studies often reveal that incorporation of one or more heteroatoms in an aromatic ring influences the chemical and biochemical reactivity of these compounds and therefore alter their metabolism. In some instances, however, these rings can serve as alternative sites for metabolic attack and at times may have the potential of undergoing unusual metabolic transformations that can result in toxic events (3, 4). Several therapeutic agents containing aromatic heterocyclic rings have therefore been withdrawn from the clinic due to specific organ toxicities and related idiosyncratic autoimmune reactions. The volume of literature on five-membered heteroaromatic rings has experienced an enormous expansion over the past few decades (5). Thus, there exists a need to 1Abbreviations: SMR, structure-metabolism relationship; SAR, structure-activity relationship; P450, cytochrome P450; GSH, reduced glutathione; UGT, glucuronosyl transferase.

10.1021/tx015574b CCC: $22.00 © 2002 American Chemical Society Published on Web 02/07/2002

270

Chem. Res. Toxicol., Vol. 15, No. 3, 2002

Dalvie et al.

Chart 1. Chemical Structures of Five-Membered Aromatic Heterocyclic Rings

review the many interesting biotransformation pathways of heteroaromatic ring systems. The structural diversity of the heteroaromatic rings makes it extremely difficult to review all the ring systems. Therefore, the major theme is limited to the metabolism of five-membered heteroaromatic rings (Chart 1). An attempt has been made to correlate the effect of physicochemical properties such as the electronic effects of heteroatoms, aromaticity, and acidity/basicity (pKa) of the rings on their biotransformation. As available data permits, the generation of reactive metabolites following oxidation or reduction of these heterocycles and the subsequent toxicological consequences are also discussed.

2. Heterocycles with One Heteroatom Pyrrole, furan, thiophene, and their benzo-fused derivatives such as indoles, benzofurans, and benzothiophenes belong to this group. These rings constitute an important group of five-membered aromatic heterocycles that are substructures of many drug molecules. These heteroaromatic rings are considered to be electron rich systems and have an excess of π-electrons (six electrons are distributed over five atoms). The aromaticity of pyrrole and thiophene is comparable and both rings are often compared to the nucleophilic benzenoid rings such as aniline and phenol (6). In contrast, furan has low resonance energy associated with it and often mimics conjugated diene systems containing an appropriate heteroatom at the C1 position (7, 8). Despite the differences in their electronic properties, these rings often display broad similarities in their metabolic fate and are sometimes bioactivated to products that can cause liver, lung, and/or kidney necrosis (3).

2.1. Pyrroles There is considerable evidence that pyrroles are metabolized by cytochrome P450. For instance, 1,3,4-trisubstituted pyrrole analogues such as 1 are bioactivated by P450 in the presence of NADPH to a reactive iminium intermediate 3, via the primary alcohol 2 (Scheme 1) (9). Although the pyrrole ring is not oxidized in this bioactivation process, the formation of 3 is facilitated by the

Scheme 1. Bioactivation of 1,3,4-Trimethylpyrrole (1)

lone pair of electrons on the nitrogen atom of pyrrole. Examples of P450-catalyzed oxidative metabolism of pyrrole rings in xenobiotics are also well-known. Oxidations occur predominantly at the carbon atoms adjacent to the pyrrolyl nitrogen, in analogues such as 4, generating the corresponding 3- or 4-pyrrolin-2-ones, 6a and 6b, presumably via an initial epoxide intermediate 5 (Scheme 2). The pyrrolinone tautomers are in equilibrium with 2-hydroxypyrrole (6c), the corresponding enol form of 6a and 6b. The reaction sequence is analogous to the oxidation of benzenoid rings to phenols via arene-oxide intermediates (10). For example, the anticonvulsant methyl-4-p-chlorophenyl-pyrrole-3-morpholino-2-carboxylate (8) is primarily metabolized by P450 to hydroxypyrrole (9) (Scheme 2) (11). Similarly, the antianxiety agent, premazepam (10) with a fused pyrrole ring system, is converted to pyrrolinone 11 (12). However, this oxidation is preceded by an initial diazepine ring cleavage. The pyrrolinone metabolites 6a and 6b often are susceptible to further oxidation yielding the corresponding hydroxypyrrolinone derivatives 7 (see Scheme 2). For instance, pyrrolonitrin (12), a broad-spectrum antifungal agent, is oxidized to 4-chloro-3-(3-chloro-2-nitrophenyl)-3-pyrrolin2-one (13) and 4-chloro-3-(3-chloro-2-nitrophenyl)-5-hydroxy-3-pyrrolin-2-one (14) by rat liver microsomes in the presence of NADPH (13). Subsequent oxidation of 13 yields 3-(3-chloro-2-nitrophenyl)-maleimide (15), which is further reduced to 4-chloro-3-(3-chloro-2-nitrophenyl)succinimide (16) as illustrated in Scheme 2. Castagnoli and co-workers have extensively characterized the metabolic fate of β-nicotyrine, (17), a minor tobacco alkaloid and a pyrrolyl analogue of (S)-nicotine (14). Like other pyrrole-containing compounds, 17 is primarily oxidized to 3- and 4-pyrrolin-2-ones (18a and

Invited Review

Chem. Res. Toxicol., Vol. 15, No. 3, 2002 271

Scheme 2. P450-Catalyzed Biotransformation Reactions on the Pyrrole Ring System

18b) and 5-hydroxy-3-pyrrolin-2-one (20) upon incubation with NADPH-supplemented rabbit lung and liver microsomes (Scheme 3) (15). The conversion of the 4-pyrrolin-2-one derivative 18a to the corresponding 5-hydroxy3-pyrrolin-2-one (20) metabolite is thought to proceed by

autoxidation of the tautomeric hydroxypyrrole 18c, via a resonance stabilized carbon-centered radical 19, followed by C5 hydroxylation or a sequential second electron oxidation followed addition of water. In addition to 18 and 20, 5-hydroxycotinine (21), formed presumably via hydration of 18a, is also detected in these microsomal incubation mixtures. Interestingly, the isomeric cis-3′hydroxycotinine (24) has been identified as the major urinary metabolite in rabbits treated with 17 (see Scheme 3) (16). The pathway leading to 24 is thought to involve the initial two-electron oxidation of 18 to the corresponding iminium species 22, hydration of which generates 3-hydroxypyrrolinone (23), that is reduced to 24. The lack of formation of 24 in the microsomal incubations suggests the possibility of involvement of an extrahepatic pathway. Egger and co-workers have reported the formation of unusual oxidative metabolites during biotransformation studies on an investigational antiinflammatory agent prinomide (25) (17). A novel bicyclic metabolite 29 has been identified in urine samples collected from preclinical species treated with radiolabeled and unlabeled 25, in addition to the corresponding pyrrolin-2-one (26) and phenol (27) metabolites (Scheme 4). Pyrrolin-2-one 26 undergoes autooxidation to afford the corresponding hydroxypyrrolin-2-one 28, which undergoes spontaneous intramolecular cyclization to yield 29. In addition, the de novo fused succinimide metabolite 30 was also detected in the urine. Spectroscopic analysis and X-ray crystal structure analysis of the isolated metabolite unequivocally confirmed the structure of 30. Although the mechanism for the formation of 30 is unclear, it is proposed that the intermediate that leads to the formation of 29 also forms 30. Assandri and co-workers have also reported interesting biotransformation reactions involving the oxidative pyrrole ring opening, in mopidralazine (31), an antihypertensive agent (Scheme 5) (18-20). Wistar rats treated

Scheme 3. P450-Catalyzed Metabolism of β-Nicotyrine (17)

272


Dalvie et al.

Scheme 4. Metabolism of Prinomide (25) in Preclinical Species

Scheme 5. Metabolism of Mopidralazine (31) in Rats

with [14C]- or [13C]31 transformed the pyrrole moiety of the pyrrolylpyridazinamine primarily to a six-membered sydnone-like metabolite (34). Several other interesting metabolites such as the dione 35, triazine 36, and the triazolopyridazine 38 also were detected in rat urine. A peroxidative mechanism, rather than a simple P450catalyzed mono-oxygenation, has been proposed for this transformation (18, 20). It is thought that 31 undergoes an addition of a dioxygen across the pyrrolyl 2′-3′-double bond yielding the corresponding endoperoxide 32. Ring opening of 32 generates intermediates 33 or 37 followed by a series of chemical rearrangements resulting in 3436 and 38, respectively (Scheme 5).

2.2. Indoles The indole ring is also subjected to extensive P450catalyzed oxidative metabolism. Primary sites of metabolism of an indole ring are the aromatic ring, the pyrrolyl moiety and the nitrogen atom. Hydroxylation of the aromatic ring is by far the most common site of metabolism on the indole ring (Scheme 6) (21-25). In most cases, aromatic hydroxylation occurs on the C-5 and/or C-6 position of the indole ring; however, oxidations at the C-4

and C-7 positions have also been observed. The hydroxylated metabolites typically undergo phase II conjugation leading to the sulfate and glucuronide conjugates and are eliminated as such. There is evidence that the site of hydroxylation is influenced by the type of substitutents on the ring as predicted by theoretical calculations of aromatic electrophilic substitution patterns (26). King and co-workers have studied the metabolism of radiolabeled indole in rats and in NADPH-supplemented rat liver microsomes (Scheme 6) (27). Unlike pyrroles, oxidation of indole primarily yields 3-hydroxyindole (39) (C-3 is the site of highest electron density) and 2-hydroxyindole (40), to a lesser extent. Formation of 39 and 40, presumably proceeds via the epoxide intermediate as discussed with pyrroles. Like pyrroles, both 39 and 40 are in equilibrium with their respective tautomers, 39a and oxindole (40a). Recent studies have indicated that human CYP2A6 is the main isoform that catalyzes the conversion of indole to 3-hydroxyindole (39), whereas CYP2A6, 2C19, and 2E1 oxidize indole to oxindole (40) (28). The differing product ratios and/or products formed from different isoforms suggest that electronic effects are not the only factor that determines the sites of oxidation by P450.

Invited Review

Chem. Res. Toxicol., Vol. 15, No. 3, 2002 273 Scheme 6. Biotransformation Pathways of Indole

Besides 39 and 40, isatin (41), N-formyl anthranilic acid (42) and anthranilic acid (43) (Scheme 6) have also been detected in urine of rats and in the microsomal incubation mixtures. These observations suggest that hydroxyindoles are prone to further oxidation and/or undergo oxidative ring scission (27). The observation that anthranilic acid (43) is detected in rat urine after administration of 3-hydroxyindole (39) suggests that 39 is a common intermediate that undergoes cleavage to 42 and 43 (27). More recently, Gillam and co-workers have shown that indoles undergo P450-mediated oxidative coupling reactions that lead to the formation of indigoid pigments (see Scheme 6) (28). The mechanism for formation of these dyes has been described in detail by Guengerich (29). Overall, these observations confirm the previous results by King and co-workers who have demonstrated the formation of complex dyes such as indigo (44) and indirubin (45) upon incubation of indole with NADPH rat liver microsomes (27). In addition to these two dyes, 6H-oxazolo-[3,2-a-4,5-b′]-diindole (46), a compound formed by oxidative coupling of 39a and indole has also been identified in incubations of indole with NADPH supplemented human liver microsomes (28, 29). Other examples where the indole ring undergoes oxidative ring cleavage have been highlighted in the biotransformation studies of the dopamine receptor agonist, proterguride (47) (30), and the β-blocker, pindolol (50) (31, 32) (Chart 2). Both of the compounds undergo initial oxidation to yield the oxo-derivatives 48 and 51, respectively, followed by ring cleavage leading to 49 and the anthranilic acid derivative 52, respectively. Kiechel and co-workers have also detected the indigotin analogue 53 resulting from oxidative coupling of pindolol (50) (32). Similar oxidative ring opening has also been demonstrated in the metabolism studies of a 5,6-fused pyrrolo[2,3-d]pyrimidine, LY231514 (54) (Chart 2) (33). LY231514 is a novel antifolate agent that is primarily excreted

unchanged in the urine of mice and dogs. However, the compound undergoes biotransformation to a ring cleaved product 55 in addition to oxidation on the pyrrole nucleus leading to 56. Although the role of P450 in the oxidative ring opening of indole and indole-containing compounds is widely accepted, a peroxidative pathway similar to the one demonstrated by Assandri and co-workers in the metabolism studies of 31 cannot be ruled out (see Scheme 5). Enzymes such as indoleamine-2,3-dioxygenase or tryptophan-2,3-dioxygenase that have been known to oxidize tryptophan or indoleamines using a superoxide anion as an oxygen source can possibly play a role in these reactions as well (33-35). A mechanism involving direct addition of dioxygen across the 2,3-double bond in indole (or via the hydroperoxide 57) followed by ring opening of the corresponding endoperoxide intermediate 58 to ring opened metabolites also can be proposed in the formation of the aniline derivatives such as 59 (Scheme 7). Another example where the indole nucleus undergoes P450-mediated oxidation is illustrated in the biotransformation studies on the pneumotoxin, 3-methylindole (60), a degradation product of tryptophan synthesis (36). 3-Methylindole (60) is primarily metabolized to 3-methyloxindole (63), carbinol 66, and 3-methyleneindolenine (70) upon incubation with NADPH-supplemented vaccinia-expressed human P450s (37). It is excreted predominantly as unconjugated or glucuronidated 3-hydroxy3-methyloxindole (65) and indole-3-carboxylic acid (68) metabolites in the urine of mouse and goats (Scheme 8) (38, 39). Additionally, a mercapturic acid metabolite, 3-[(N-acetyl-L-cystein-S-yl)methyl]indole (71) and sulfate conjugates of 4-, 5-, 6-, or 7-hydroxy-3-methyloxindole (64) have also been detected in urine of both species. Three reactive intermediates, 3-methyleneindolenine (70), 2,3-epoxy-3-methylindoline (61), and 3-hydroxy-3-

274


Dalvie et al.

Chart 2. Structures of Proterguride (47), Pinodolol (50), LY231514 (54) and Their Metabolites

Scheme 7. Plausible Mechanism for the C2-C3 Double Bond Cleavage in Indole Derivatives

Scheme 8. P450-Catalyzed Bioactivation of the Pneumotoxin 3-Methylindole (60)

methylindolenine (62), that can form covalent adducts with cysteine, glutathione (GSH), and DNA have been implicated as electrophiles of 3-methylindole (Scheme 8) (40-45). It has been established that 3-methyleneindo-

lenine (70) is the major ultimate toxin and that P450mediated dehydrogenation of 60 is the predominant route leading to this metabolite in goat lung microsomes (46). The reaction is thought to proceed by hydrogen atom

Invited Review


Chart 3. Examples of Metabolism on the Indole Nitrogen

abstraction followed by a one-electron oxidation step (see Scheme 8). Studies with vaccinia virus-expressed P450s have suggested that P4502F enzymes (human CYP2F1 and goat CYP2F3) are responsible for the generation of 70 (47-49). Apart from the above-mentioned biotransformation pathways of the indole nucleus, several interesting modifications of the indole nitrogen atom have also been reported. For instance, the indole nitrogen of delavirdine (72) and carvedilol (74) undergoes glucuronidation, yielding N-glucuronides (73 and 75), respectively, in preclinical species (Chart 3) (50, 51). Similarly, N-hydroxylation of 2-phenylindole (76) resulting in a stable N-hydroxy metabolite (77) has been reported upon incubation of 76 with liver tissue of the rabbit and guinea pig pretreated with 3-methylcholanthrene (Chart 3) (52). This result suggests that N-hydroxylation may also occur in other ring systems at the pyrrolyl nitrogen, but the resulting unstable water-soluble metabolites are often undetectable. Besides conjugation and/or oxidation reactions, simple N-dealkylation of N-alkylindole derivatives have been characterized, as exemplified by N-demethylation of zafirlukast (78) to 79 (Chart 3) (53).

2.3. Furans As observed with pyrroles, P450 is also primarily responsible for the oxidative metabolism of furans. Most furan-containing compounds are hepatotoxic and hepatocarcinogenic in preclinical species and humans. For example, furan itself, (see Chart 1) which is used both as an industrial chemical and present in many foods and beverages, is a liver toxicant and genotoxic in rats and mice (54, 55). Likewise, 3-methylfuran has been shown to be a potent pneumotoxin in animals. Various groups have proposed that the toxicity of furans is a result of metabolic activation, mediated by P450. The importance of P450 in the bioactivation of the furan ring has been further substantiated by dosing furan or furan-containing compounds to rats pretreated with inhibitors or inducers that either alleviate or augment, respectively, the toxic effects of these compounds (66). Studies on disposition of [2,5-14C]furan in rats have illustrated that [14C]carbon

dioxide is the primary metabolite in furan metabolism and suggests that ring scission followed by complete oxidation of at least one of the labeled carbons constitutes its major metabolic fate (57). Furthermore, approximately 80% of the radioactivity in the liver is not extractable by organic solvents and is associated with proteins. A more or less linear increase in the covalent binding to proteins is observed upon administration of multiple doses of [14C]furan. The predominant biotransformation pathway of furan involves its ring scission to R,β-unsaturated dicarbonyl metabolites. For example, furan, 2-methylfuran (82) and 3-methylfuran (83) generate cis-butene-1,4-dial (84), 4-oxo-2-pentenal (85), and 2-methylbutene-1,4-dial (86), respectively, upon incubation with liver microsomes (Scheme 9) (58-60). These electrophilic R,β-unsaturated dicarbonyl metabolites react with biological macromolecules via a 1,4-Michael addition across the R,β-unsaturated dicarbonyl moiety or via nucleophilic 1,2-addition to the aldehyde. As a consequence, these unsaturated metabolites have been implicated in the covalent binding of furan and furan-containing compounds to proteins and DNA resulting in toxicity. Nucleophiles such as GSH, semicarbazide, or methoxylamine can react with these R,β-unsaturated carbonyl compounds and thereby prevent irreversible binding to proteins. For reasons that remain unclear, N-acetylcysteine enhances covalent protein binding of these intermediates (61). bis-Semicarbazones (87-89) have been detected upon incubation of furan, 82 and 83, respectively, with microsomes in the presence of semicarbazide (58, 59). Studies on furan bioactivation in liver microsomes containing GSH or N-acetyllysine have demonstrated that the cis-butene1,4-dial formed in the incubation mixture reacts rapidly with the nucleophiles forming cross-links and yielding cyclic or acyclic adducts (90-93b) (Scheme 9) (62). The type and the extent of adduct formation is dependent on the concentration of nucleophile(s) in the incubation mixture. Several furan-containing compounds such as furosemide (80) and ipomeanol (81) (Chart 4) also cause hepatic and renal necrosis in mouse and humans or develop potentially lethal pulmonary lesions in rat (6370). Oxidative ring opening of furan and irreversible protein binding of the corresponding substituted γ-ketoenals has been reported during metabolism studies on many furan-containing biologically active compounds. Studies with the lipooxygenase inhibitor L-739,010 (94) and an experimental anti HIV drug, L-754,394 (95) have demonstrated the formation of O-methyloxime derivatives 96 and 97, respectively, (Chart 4), when incubated with human liver microsomes containing methoxylamine (71, 72). In addition, the S-linked conjugates of GSH or N-acetylcysteine, 98 and 99, respectively, have been identified in the metabolism studies of L-754,394 upon inclusion of these trapping agents in the incubation mixtures (72). In contrast, diclofurmine (100), furfenorex (101), or the furanocoumarin, methoxsalen (102), are primarily converted in preclinical species to γ-ketobutyric acid and acetic acid metabolites 103, 104, 105, respectively, following oxidation of the furan moiety in the parent compounds (Chart 4) (73-75). Studies with menthofuran (107), a proximate toxin responsible for the hepatotoxicity mediated by the P450-catalyzed oxidation of pulegone (106), have also demonstrated that the furan ring in 107 undergoes further oxidiation to 110 (Scheme

276


Dalvie et al.

Scheme 9. Metabolic Pathways of Substituted Furan and Furan Derivatives 82 and 83

Chart 4. Examples of Furan-based Xenobiotics and Their Respective Metabolites

10) (77-83). Several other novel metabolites including 2-hydroxyfuran (108), which exists in equilibrium with its lactone tautomer 109, have been identified as oxida-

tive metabolites of 107. Co-incubation of 107 with phenobarbital-induced rat liver microsomes containing NADPH and semicarbazide results in the formation of a

Invited Review Scheme 10. P450-Catalyzed Metabolism of Pulegone (106) and Menthofuran (107)

cinnoline derivative 111, presumably via the γ-ketoenal 110 intermediate. It has been well established that this γ-ketoenal 110 is the ultimate toxin responsible for the toxicity of pulegone (106) (79-83). The mechanism of oxidative opening of furan rings to the corresponding R,β-unsaturated dicarbonyl or γketoacid metabolites is thought to proceed via the initial formation of furan-2,3-epoxide (112) as shown in Scheme 11. This 2,3-epoxide directly rearranges to hydroxyfuran, which predominantly exists as the lactone tautomer. Hydrolysis of the lactone affords the γ-ketocarboxylic acid as shown in pathway a (Scheme 11) (73, 75). Alternately,


ring opening of the furanoyl epoxide can also lead to the formation of γ-ketoenal (Scheme 11, pathway b) (83). Kobayashi and co-workers have proposed an alternate mechanism for ring opening using deuterated TA-1801 (113), a hypolipidemic agent, as a probe (Scheme 12). Their proposal suggests that the C-O bond in the furan ring is directly cleaved following transfer of oxygen from P450 to the substrate (Scheme 12) and is based on the observation that 113 is converted to the corresponding deuterated γ-keto alcohol 115 and nondeuterated γketoacid 116 metabolites (84). These results have been explained by a pathway in which 113 undergoes an oxidative ring opening to deuterated R,β-unsaturated aldehyde 114 followed by enzyme-assisted reduction to 115 (Scheme 12). Oxidation of the alcohol 115 results in the loss of the deuterium atom leading to the corresponding carboxylic acid 116. Although this mechanism argues against the epoxide intermediate, evidence for the formation of the furan epoxide intermediate has been shown in the metabolism studies of R-(+)-pulegone and R-(+)menthofuran by human liver P450s using 18O2 and H218O and C-2 deuterium-labeled menthofuran (83). Indirect evidence for an epoxidation pathway has also been reported for furosemide (80) (see Chart 4). Incubation of 80 with mouse liver microsomes in the presence of epoxide hydrolase inhibitor, 1,2-epoxy-3,3,3,-trichloropropane, results in a 2-fold increase in covalent binding (85). This increase in reactivity is attributed to the higher

Scheme 11. Plausible Mechanism of P450-Catalyzed Oxidative Ring Opening in Substituted Furans and Thiophenes

Scheme 12. Plausible Mechanism of P450-Catalyzed Oxidative Ring Opening in the Substituted Furan, TA-1801 (113)

278


Dalvie et al.

Scheme 13. Reductive Metabolism of Furazolidone (117)

Chart 5. Structures of Nitrofurans, Nitrofurazone (118), NFTA (126), and 7-methoxy-2-nitronaphtho[2,1-b]furan (128) and Their Metabolites

epoxide concentrations that result from inhibition of epoxide hydrolase activity in the microsomes. Furthermore, the presence of GSH or N-acetylcysteine in microsomal incubations significantly diminishes the extent of covalent binding to proteins. Unlike unsubstituted furans, nitrofurans such as furazolidone (117) (Scheme 13) and nitrofurazone (118) (Chart 5) are activated by reduction under anaerobic condition (86). For instance, furazolidone (117) undergoes a two-electron reduction to the hydroxylamine metabolite 119 upon incubation with rat liver microsomes. Two other metabolites, the amine 120 and the nitrile 121, have also been detected in urine of rabbits dosed with 117 (8789). It is proposed that a second two-electron reduction of 119 generates 120. Alternatively, the hydroxylamine intermediate can dehydrate to a nitrile 119b (as shown in Scheme 13) followed by reduction of the double bond to yield 121. Additional metabolites, such as the carboxylic acid 122, R-ketoglutaric acid 123 and the dimer 124 have also been detected in in vitro and in vivo studies. Hydrolysis of the cyano group of 121 can generate the acid 122, further degradation of which can account for 123. The dimer 124 is perhaps a result of the coupling of hydroxylamine 119 with furazolidone

(117), since addition of GSH to the incubation mixture attenuates the formation of both 121 and 124. Phenotyping studies have demonstrated involvement of xanthine oxidase, NADPH-cytochrome P450 reductase, cytochrome c reductase or aldehyde oxidase in the reductive cleavage of the nitrofuran ring (90-94). The nitrenium intermediate 125 formed from 119 has been implicated as the reactive intermediate in the toxicity of nitrofurans and forms covalent adducts with macromolecules (95). Similar NADPH:cytochrome P450 reductase catalyzed reductive ring opening resulting in the formation of a nitrile 127 has been reported in the biotransformation of NFTA (126) (Chart 5) (96). In contrast, Maurizis and co-workers have shown the formation of a primary amine 130 in the metabolism studies of 7-methoxy-2-nitronaphtho[2,1-b]furan (128) in rats (97). It is proposed that formation of the primary amine involves a ring cleavage to a nitrile intermediate 129, which is further reduced to 130.

2.4. Thiophenes Thiophene rings (Chart 1) have proven to be an attractive isosteric replacement in the quest for improved potency and selective SAR (1). However, few reports on the metabolism of this heteroaromatic ring have been published (98). Recently, removal of some of the thiophenecontaining therapeutic agents from use due to hepatotoxicities has led to an increased effort in understanding the cause of these toxic events particularly in relation to their bioactivation potential. As observed with pyrroles and furans, the metabolism of thiophenes is principally mediated by P450. Like pyrroles, thiophenes undergo hydroxylation at the carbon atoms adjacent to the sulfur atom and are converted to 2 or 5-hydroxythiophenes. Earlier studies by Bray and co-workers provided evidence that thiophene was converted to N-acetyl-S-thienylcysteine (131) and premercapturic acid (132) in rats and rabbits (Chart 6). Thiophene-2,3-epoxide has been postulated to be the obligatory intermediate in the formation of 131 and 132 (99). Most drugs containing thiophene rings are hydroxylated on the carbon atoms adjacent to sulfur (Chart 6). For example, the diuretic, tienilic acid (133) (100-102), nonsteroidal antiinflammatory agents, such as suprofen (134) (103, 104), S-2-[4-(3-methyl-2-

Invited Review


Chart 6. Structures of Thiophene-based Xenobiotics and the Site of Hydroxylation of the Thiophene Moiety

thienyl)phenyl]propionic acid (135) (105), tenoxicam (136) (106), and others like tiquizium bromide (137) (107), morantel (138) (108), and ticlopidine (139) (109) represent some drugs that are predominantly metabolized to 5-hydroxythiophene derivatives in humans or other preclinical species. Cytochrome P4502C9 has been implicated as the main P450 isoform responsible for the hydroxylation of the thiophene ring in tienilic acid (133) and S-2-[4-(3-methyl-2-thienyl)phenyl]propionic acid (135) (110-113). An antirheumatic drug tenidap (140) is also metabolized to 5-hydroxytenidap (141) in rats, monkeys, and humans, and is primarily excreted as a glucuronide conjugate of 5-hydroxytenidap (142) in the bile of all species (Scheme 14) (114). A novel thiolactone metabolite 144 has been detected in the fecal extracts and plasma of preclinical species and in humans treated with radiolabeled tenidap. It is postulated that the glucuronide conjugate of 5-hydroxytenidap is excreted into the gastrointestinal tract via the bile and is hydrolyzed by the gut microflora. The resulting 5-hydroxytenidap (141) that exits as the R,β-unsaturated thiolactone derivative 143 is reduced anaerobically in the gut to yield the corresponding thiolactone (144) (114).

Thiophenes can also undergo a P450-catalyzed oxidation to unstable and reactive thiophene-S-oxide metabolites 145 (Scheme 15) (115, 116). The detection and isolation of diastereoisomeric thiophene-S-oxide dimers (146a and 146b) provided evidence for formation of thiophene-S-oxide 145 in in vitro and in vivo studies (Scheme 15) (117). The structure of the dimers was further confirmed by an independent synthesis. A mercapturic acid adduct 148 of dihydrothiophene-S-oxide was also identified in urine of rats treated with radiolabeled thiophene. This adduct was a result of a 1,4-Michael-type addition of GSH on 145 leading to 147, followed by the usual transformation of GSH adducts to the corresponding mercapturate 148. Studies with the 3-aroylthiophene analogue 149 of tienilic acid have also shown that 149 is also metabolized to a sulfoxide (150) by NADPH supplemented rat liver microsomes. Incubation in the presence of mercaptoethanol as a trapping agent has led to the isolation of mercaptoethanol adducts (151-154) (Scheme 16, pathway a) (117, 118). It has been proposed that tienilic acid undergoes sulfoxidation to a reactive electrophilic, tienilic acid-Soxide (155) by human P4502C9, which inactivates the enzyme (119). Tienilic acid specific autoantibodies, called anti-LKM2, directed against P4502C9 have been detected in patients treated with tienilic acid and suffering with tienilic acid-induced hepatitis (120). The tienilic acid-Soxide (155) is also thought to be responsible for the formation 5-hydroxytienilic acid (156), in addition to inactivating the enzyme. According to this mechanism (Scheme 16B) 155 reacts with various nucleophiles in the active site of the enzyme by a Michael type 1,4-addition resulting in specific covalent binding to the active site. Alternatively, reaction of 155 with water leads to the formation of 5-hydroxytienilic acid (156) (110, 118, 121, 122). The hypothesis for the formation of 5-hydroxytienilic acid metabolites via the thiophene sulfoxide intermediate has been recently investigated by studies with stable isotopes (123). Microsomal incubation of tienilic acid (133) and its analogue 149 in the presence of NADPH and under 18O2 atmosphere results in the incorporation of 18O in 5-hydroxythiophene metabolites. This suggests that the oxygen in hydroxythiophene metabolites is almost exclusively derived from molecular oxygen and not from water as was proposed previously. Similar results have been observed upon incubation of a probe substrate 2-(4-methoxybenzoyl)thiophene (157) (see Scheme 17) with 18O2 in our laboratories (J. P. O’Donnell, unpublished results). Overall, these results provide indirect evidence that 5-hydroxythiophene metabolites also can be formed via an epoxide pathway in addition to the sulfoxide pathway, unless the oxygen of thiophene sulfoxide rearranges to the give hydroxythiophene. It has been shown that the position of the substituents on the thiophene ring can also alter the reactivity of the sulfoxide intermediates formed upon oxidation of the ring

Scheme 14. Metabolism of Tenidap (140) in Rat, Monkey and Humans

280


Scheme 15. In Vitro and in Vivo Metabolism of Thiophene to Thiophene-S-oxide Metabolites

by P450 (121, 124). For example, the different behavior of tienilic acid (133) and its analogue (149) has been

Dalvie et al.

explained by the very different reactivity of their sulfoxide intermediates. It has been shown that 149 is hepatotoxic in laboratory animals but does not inactivate P4502C9 like tienilic acid. Furthermore, metabolism of 149 results in covalent binding to microsomal proteins which is reduced upon incubation in the presence of GSH. On the contrary, tienilic acid is not hepatotoxic in laboratory animals and inactivation of P4502C9 cannot be inhibited by addition of GSH. Like furans, several drugs such as S-2-[4-(3-methyl2-thienyl)phenyl]propionic acid (135) (105), morantel (138) (108), ticlopidine (139) (109), and tenidap (140) (H. G. Fouda, unpublished results) (Chart 6) have also demonstrated the transformation of thiophene ring to the γ-ketocarboxylic acid metabolite, in addition to the

Scheme 16. Reactivity of the Sulfoxide Metabolite Derived from Tienilic Acid (133) and Its Positional Isomer (149)

Scheme 17. P450-Catalyzed Metabolism of 2-(4-Methoxybenzoyl)thiophene (157)

Invited Review Chart 7. Metabolic Ring Opening in 2-(substituted)Thiophene Analogs

Chem. Res. Toxicol., Vol. 15, No. 3, 2002 281 Scheme 18. Peroxidase-Catalyzed Metabolism of Ticlopidine (139)

Chart 8. Examples of Benzothiophene-based Xenobiotics and Their Respective Metabolites

formation of 5-hydroxythiophene derivative, upon administration to preclinical species and humans. As observed with furans, the 5-hydroxythiophene metabolite is presumably the precursor in the formation of γketoacids. It is thought that R,β-unsaturated thiolactone, a tautomer of 5-hydroxythiophene, is hydrolyzed to the γ-thionoscarboxylic acid metabolite. The corresponding thionoacid is then converted to the γ-ketoacid metabolite upon loss of hydrogen sulfide (see Scheme 11). A similar pathway has also been proposed in the microbial metabolism of thiophene-2-carboxylic acid (158) to the diacid 159 (Chart 7) (125). Recently, studies with a radiolabeled arylthiophene (160) in our laboratory have shown that 60% of the radiolabeled dose is excreted as a taurine conjugate 162 of γ-ketoacid (161) in urine of rats treated with 160 (126). Recent studies on the mechanism of the ring opening of thiophene rings using 2-(4-methoxybenzoyl)thiophene (157) as a probe substrate have suggested that thiophenes undergo a ring opening to R,βunsaturated aldehydes (J. P. O’Donnell, unpublished results) (Scheme 17). Incubation of 157 with human liver microsomes and baculovirus expressed recombinant human CYP2C9 in the presence of semicarbazide, resulted in a stable pyridazine derivative (166) as well as the 5-hydroxythiophene and the phenol metabolites, 163 and 167, respectively. The formation of 166 indicates that the R,β-unsaturated carbonyl metabolites (164 or 165) are obligatory intermediates in the metabolism of 157, as observed in furans. Furthermore, the presence of 163 along with 166 suggests that the epoxide 157a is presumably a key intermediate in the formation of both 166 and 163. The metabolism of compounds containing a benzothiophene ring has also been reported. Like indoles, benzothiophene rings are mainly hydroxylated on the phenyl rings but also undergo sulfoxidation to benzothiophene-S-oxides. For instance, oxidation of benzothiophene by rat liver microsomes in the presence of NADPH led to the sulfoxide 168 (Chart 8) (116). Incubation in the presence of mercaptoethanol and at pH 8.5 gave the mercaptoethanol adduct (169) of the sulfoxide 168. Similarly, metabolism studies with the 5-lipoxygenase inhibitor, zileuton (170), and its reduced metabolite

172 have demonstrated the formation of S-oxides 171 and 173, respectively (Chart 8) (127). More recently, studies by Liu and Uterecht have shown that the thiophene ring of ticlopidine (139) is metabolized by activated neutrophils resulting in a reactive metabolite (Scheme 18) (128). The potential formation of the reactive metabolite has been proposed to be the cause for the incidence of agranulocytosis observed in patients treated with ticlopidine. Incubation of ticlopidine by activated neutrophils and myeloperoxidase, a major enzyme found in the neutrophils, results in complete transformation of 139 to chloroticlopidine (175) and dehydrogenated ticlopidine (176). The neutrophil-derived metabolite of ticlopidine has also been trapped with GSH to yield a GSH adduct 177. The formation of 176 can be explained by a mechanism in which the sulfur is chlorinated to a reactive metabolite thiophene-S-chloride (174), which upon loss of HCl is converted to the proposed 176 (128).

3. Heterocycles with Two Heteroatoms Five-membered aromatic heterocycles containing two heteroatoms represent a large and structurally diverse group. Most of these ring systems are formally derived from pyrrole, furan and thiophene by replacement of one of the methine (-CH group) by an sp2-hybridized nitrogen atom (Chart 1). It is the variation in the position of additional nitrogen that leads to the structural diversity of this group of heterocycles. Replacement of an additional methine in pyrrole, thiophene, and furan with a nitrogen atom has important effects on the properties of the resulting rings. The additional nitrogen atom causes a decrease in the energy levels of the π-orbitals so that the heterocycles are less “π-electron rich” (129). Second, the lone pair of electrons of the nitrogen atom provides a site for protonation and alters the acidity and basicity of these heterocycles (Table 1) (130-133). Both of these properties can affect the metabolism of these heterocyclic rings. Additionally, the position of the second heteroatom and its electronegativity also can affect the biotransformation pathways of the heteroaromatic rings. For example, imidazole, oxazole, and thiazole rings are susceptible to oxidation reactions whereas, isoxazole or isothiazole rings undergo reduction. In contrast, the pyrazole

282


Table 1. Acid-Base Properties (pKa) of Five-Membered Nitrogen-Containing Heterocycles in Solutions and in Gas Phase heterocycle

Het h Het- + H+

pyrrole imidazole 1,2,3-triazole 1,2,4-triazole tetrazole benzimidazole

16.2 14.5 10.0 9.4 4.8 13.2

heterocycle

Het H+ h Het + H+

pyrrole imidazole thiazole oxazole benzimidazole pyrazole isothiazole isoxazole benzisoxazole 1,2,4-triazole 1,2,3-triazole tetrazole

-3.8 7.0 2.53 0.8 5.5 2.5 -0.51 -2.97 -4.7 2.2 1.2 -3.0

Dalvie et al. Chart 10. Examples of Imidazole-based Xenobiotics and Their Respective Metabolites

Chart 9. Examples of Antifungal Agents, Ketoconazole (178), Its Metabolites and Clotrimazole (179)

Scheme 19. P450-Catalyzed Metabolism of Imidazole and Histidine (187)

ring is relatively stable to metabolism, and very few reports of oxidation of this ring system have been published. It is important to note that, unlike isoxazoles and isothiazoles, this ring has not been shown to undergo reductive metabolism.

3.1. Imidazoles The incorporation of an imidazole ring in medicinal chemistry has grown considerably as judged from the large number of therapeutically useful imidazole-based compounds including antifungals, anticonvulsants, thromboxane synthase-, aromatase-, and phosphodiesterase III inhibitors (134). Imidazole-containing compounds are often strong ligands of P450 heme and bind to the oxidized (or even reduced) form of the enzyme. As a result, most imidazole-containing compounds are potent inhibitors of P450. For example, ketoconazole (178) and clotrimazole (179) (Chart 9) are potent inhibitors of human CYP3A4 (135-137). The mechanism of inhibition relates to the coordination of the nitrogen lone pair electrons with the prosthetic heme iron in P450. The process shifts the ferric iron from a high-to-low spin state, making its reduction by P450 oxidoreductase more difficult, and consequently results in inhibition of enzyme activity (138, 139). Introduction of alkyl substituents at the C-2 position on the imidazole ring sterically hinders interaction of the imidazole nitrogens with the heme iron and attenuates P450 inhibition (140, 141).

Irrespective of their inhibitory properties, imidazole and imidazole-containing compounds are primarily metabolized by P450. Ring oxidation mainly results in C-2, C-4, and/or C-5 hydroxylation, and these hydroxylated metabolites generally undergo further ring oxidation or ring scission resulting in hydantoin derivatives or substituted ureas. In some instances the hydroxylated imidazoles are eliminated as glucuronide conjugates. For example imiloxan (180), a 2-substituted imidazolecontaining compound, is primarily hydroxylated at the C4 or C5 position of the imidazole ring resulting in the corresponding hydroxyimidazole metabolite 181, in humans (Chart 10) (142). The hydroxylated metabolite is excreted as an O-glucuronide conjugate in urine or is further oxidized to metabolites 182 and 183. Imidazole itself also has been shown to undergo oxidation to the 2-imidazolone metabolite 184, which upon further oxidation and ring cleavage yields the hydantoin (185) and N-(carboxymethyl)urea (186) (Scheme 19) (143). Similarly, (5-carboxyethyl)hydantoin (188) and glutamic acid derivative 189 are the predominant metabolites (after

Invited Review

Chem. Res. Toxicol., Vol. 15, No. 3, 2002 283 Scheme 20. Metabolism of Croconazole (201) in Rats and Rabbits

deamination) of the imidazole-based amino acid, histidine (187) (144, 145). Cytochrome P450-mediated oxidation of the imidazole ring to the hydantoin has also been observed in the biotransformation studies of an anticonvulsant nafimidone (190) (146, 147) and an antifungal agent, econazole (191) (Chart 10) (148). The major urinary metabolite of nafimidone (190) in man is the secondary alcohol 192, which is further converted to the hydantoin derivative 193 (Chart 10). The fungicide prochloraz (194) undergoes ring cleavage to afford urea derivatives (195 and 196) (Chart 10) (149, 150). Another example of hydroxylation followed by opening of the imidazole ring has been illustrated in the metabolism studies with ketoconazole (178) (Chart 9) (151). In mouse, ketoconazole (178) undergoes a P450-catalyzed oxidation to carbinolamine 197 that exists in equilibrium with the ring opened formamide derivative 198 in addition to N-oxide (199) formation. Like most other N-alkyl and N-arylamines (152), N-alkyl or N-arylimidazoles also are prone to N-dealkylation or lose the aromatic ring, respectively. NDeethylation has been observed in the biotransformation studies of xanthine-related nootropic drug, ethimizol (200) (Chart 10) (153). An interesting example of a loss of the imidazole ring has been demonstrated in the biotransformation studies of an antifungal agent croconazole (201) (Scheme 20). The primary alcohol 204 was detected in the urine of rats and rabbits treated with croconazole (154, 155). The loss of the imidazole ring was investigated by incubating 201 with rabbit liver microsomes in the presence of NADPH and 18O2. Mass spectrometric analysis of the incubate indicated that one 18O atom was incorporated into the metabolite 204 (155) suggesting that the formation of 204 possibly involved P450-generated epoxide intermediate 202, which spontaneously hydrolyzed to the diol 203 followed by elimination of imidazole to yield 204. The detection of imidazole in organic extracts from the microsomal incubations further confirmed the hypothesis. Besides P450-mediated oxidative metabolism and ring scission, the imidazole moiety is also susceptible to glucuronidation. For instance, incubation of the model compound 1-phenylimidazole (205) with alamethacinactivated human liver microsomes generated quaternary glucuronide 206 (Chart 11), the structure of which was confirmed by comparison of its spectral properties to that of its synthetic standard (156). The formation of 206 was catalyzed by cDNA-expressed glucuronsyl transferase isozymes, UGT1A3 and UGT1A4, respectively. Huskey and co-workers have characterized the relative substrate specificities of 2-(methylbiphenyl)imidazole (207), 4-(methylbiphenyl)imidazole (208) and several other analogous five-membered heterocycles toward glucuronidation reactions (157, 158). In general, the nitrogen atom adjacent to the substituted carbon is the least susceptible to

Chart 11. Glucuronidation of Imidazole-based Xenobiotics

Chart 12. Methimazole (209), and Its Metabolites

glucuronidation. For instance, the 2-substituted imidazole derivative 207 was stable toward glucuronidation whereas 4-substituted imidazole analogue 208 underwent glucuronidation at the N1 position of the imidazole moiety. N-Glucuronidation of the imidazole ring has also been detected in the metabolism studies of imiloxan (180) and the alcohol metabolite (192) of nafimidone (142, 146). Bioactivation of the imidazole ring leading to a reactive metabolite that covalently reacts with cellular proteins and DNA has also been reported in the literature. For example, methimazole (209) (Chart 12), a drug widely used for hyperthyroidism, is thought to be metabolically activated to metabolites that cause hepatoxicity and bone marrow suppression in mice and humans (159-163). Structure-activity relationship (SAR) studies have indicated that the imidazole moiety and the free thiol group in 209 are essential for hepatotoxicity since the saturated imidazoline derivative 210 and the S-methyl ether analogue 211 are nontoxic in animal models (Chart 12) (164). Additional insight into the mechanism of bioactivation was obtained upon identification of metabolites of 209 in preclinical species. These studies revealed the presence of glyoxal (212) and N-methylthiourea (213) in significant quantities in these species (165, 166). Administration of 213 to mice caused a marked increase in hepatotoxic injury, suggesting that 213 may be the ultimate toxicant derived from 209 (167-169). Although the sequence of reactions leading to the formation of 212 and 213 has not been fully characterized, the cleavage of the imidazole ring possibly takes place via the epoxide/ diol pathway as illustrated in the general scheme for oxidative ring opening of azoles (Scheme 21). Thus, epoxidation of the double bond between the 4,5-position followed by hydrolysis of the corresponding epoxide yields the dihydrodiol derivative. Subsequent rearrangement of

284


Dalvie et al.

Scheme 21. General Mechanism for Oxidative Ring Opening of Azoles

Scheme 22. Bioactivation of 2-Methylimidazole (214) to Nucleophilic Reactive Intermediates

the diol intermediate affords the corresponding R-dicarbonyl and the amide metabolites, respectively. Metabolism of imidazole to nucleophilic intermediates that covalently react with biological electrophiles has also been investigated (170-172). Studies have shown that significant amounts of imidazole-based drugs are retained in the connective tissue, upon administration to laboratory animals. For instance, intravenous administration of [2-14C]imidazole or 2-methyl-[2-14C]imidazole (214) in rats reveal considerable accumulation of irreversibly bound radioactivity to elastin within the connective tissue of the aorta (170). The observation that P450 inhibitors do not prevent this binding to elastin suggests that non-P450 enzymes may catalyze the bioactivation process. Analysis of urinary metabolites indicates the presence of 2-methylimidazolone (215) as the principal metabolite of 214 (Scheme 22) (173). The facile aldol condensation of 215 with acetaldehyde (216) leading to 217 under physiological conditions suggests that 215 must possess sufficient nucleophilicity to react with aldehyde-based peptidyl residues in elastin such as allysine and hydroxyallysine generating covalent adducts such as 218 (Scheme 22) (173). Formation of such covalent linkages could disrupt the normal physiological role of these aldehyde groups including their involvement as intermediates of covalent cross linkages that contribute to elasticity or tensile strength for the respective fibers (174). The metabolism of benzimidazoles has also been studied in mammalian systems (175-177). Metabolism of [14C]benzimidazole (219) in rats has shown that like other benzo-fused heterocycles, the compound is primarily metabolized to 5-hydroxybenzimidazole (220) (Chart 13) (178). Several other studies with compounds such as 1-benzyl-2-aminobenzimidazole (221) and an omeprazole derivative, H259/31 (222) have indicated that hydroxylation of the phenyl moiety is the major metabolic pathway of benzimidazole-containing drugs (179, 180). Unlike indole, benzothiophene, and benzofurans, no

Chart 13. Examples of Benzimidazole- and Benzoxazole-based Xenobiotics and Their Metabolites

reports of the modification of the imidazole moiety of benzimidazole have been reported.

3.2. Oxazoles Given the similarity in the electronic properties of oxazole, imidazole, and thiazole ring systems, it is not surprising that oxazoles are primarily metabolized to ring opened products as observed in the biotransformation of imidazoles. A few cases of oxazole ring hydroxylation have also been reported, but most often the hydroxylated metabolite is hydrolyzed to the open chain carboxylic acid metabolite. Ring opening of benzoxazole and 2-(substituted)benzoxazoles such as 223 to o-aminophenol metabolites such as 224 was reported in the early 1950s by Bray and co-workers (see Chart 13) (181). The metabolite was eliminated as an acetyl, sulfate, or a glucuronide conjugate in urine. Oxidative metabolism and ring cleavage of the oxazole ring has been observed in the metabolism studies of the platelet aggregation inhibitor, ditazole (225) (Scheme 23) (182, 183). Ditazole is primarily metabolized to oxazolone (228) in rabbits but is apparently cleaved to benzil (229) in humans. The metabolite 228 is formed by sequential N-dealkylation to secondary and primary aminooxazole metabolites 226 and 227, respectively, followed by hydrolysis of aminooxazole (227) (via the imine tautomer) to the oxazolone derivative 228. Detection of aminooxazoles 226 and 227 provided strong evidence for this pathway. A similar oxidative deamination pathway resulting in chlorzoxazone (231) has been observed in the biotransformation studies of a myorelaxant and uricosuric agent zoxazolamine (230) in the rat (Chart 13)

Invited Review

Chem. Res. Toxicol., Vol. 15, No. 3, 2002 285 Scheme 23. Metabolism of Ditazole (225) in Rabbit and Man

Scheme 24. Metabolism of Isomoxole (232) in Rats and Guinea Pigs

(184). The benzil formation from ditazole (225) in humans, on the other hand, can be rationalized by epoxide/ diol pathway illustrated in Scheme 21. Similarly, rats and guinea pigs treated with radiolabeled isomoxole (232) result in formation of methylglyoxal (233) and N-substituted urea (234) (Scheme 24) (185). The urea derivatives 236 and 238 are presumably the hydrolysis products of oxazolones 235 and 237, respectively. In addition, an unusual hydantoin derivative, 239, resulting from cyclization of the urea 238 or hydrolysis and subsequent cyclization of 236, has been detected as a metabolite of isomoxole.

3.3 Thiazoles Like oxazoles, thiazoles (as well as benzothiazoles) predominantly undergo P450-catalyzed oxidative ring opening. In recent years, Mizutami and Suzuki have demonstrated that ring cleavage of substituted thiazoles such as 240 results in the formation of the corresponding R-dicarbonyl metabolites 241 and thioamide derivatives 242 (Chart 14) (186, 187). The mechanism of the ring scission is thought to be similar to that shown with

oxazole and imidazole rings (Scheme 21). The wellestablished toxic properties of thioamides and thioureas have led to the speculation that the toxicity of thiazoles is attributed to the P450-catalyzed bioactivation of the thiazole rings to the corresponding thioamide metabolites. Treatment with P450 inhibitors results in the attenuation of the toxic effects caused by these compounds (188, 189). Recent studies on metabolism and toxicity of thiabendazole (243), a compound that is widely used as an agricultural fungicide and an antihelmintic, have demonstrated that the compound is also oxidatively metabolized to thioformamide and the benzimidazol-2ylglyoxal (244) metabolites in preclinical species and humans (Chart 14) (190). The thioformamide thus formed is also believed to be the proximate toxicant derived from thiabendazole (191). Thiazole ring opening has also been observed in metabolism studies with the immunomodulatory agent, SM-8849 (245) (192), antiinflammatory 2-acetylaminothiazoles (246-248) (193), and the hepatoprotective agent YH-439 (249) (194) (Chart 14). SM-8849 (245) is primarily metabolized to N-methylthiourea (250) follow-

286


Dalvie et al.

Chart 14. Structures of Thiazole-based Xenobiotics and Their Respective Metabolites

Scheme 25. Metabolism of Sudoxicam (254) and Meloxicam (255) in Rats

ing scission of its methylaminothiazole ring. Likewise, acetylaminothiazoles (246-248) and YH-439 (249) produce acetylthiourea and thiohydantoic acids (251-253) as minor and major metabolites, respectively. Metabolic switching and resistance to ring opening has been observed upon incorporation of substituents in the 5-position of the thiazole ring. A comparison of the biotransformation pathways of the two oxicam derivatives, sudoxicam (254) and meloxicam (255) indicates that sudoxicam is metabolized to the thiohydantoic acid 257 and thiourea derivative 258, respectively, in rats (Scheme 25) (195). The thiazole ring opening in 254 most likely occurs via hydrolysis of the thiazolone intermediate 256. In contrast, incorporation of a methyl group at the 5-position of the thiazole ring in meloxicam (255) results in extensive oxidation of the methyl group to form an alcohol 259 that undergoes further oxidation to carboxylic acid analogue 260. Little to no oxidative ring opening of the thiazole moiety in 255 is observed (196). These results also provide evidence that the 5-position of the thiazole ring is more susceptible to hydroxylation and the formation of thiohydantoic acids, as observed in the metabolism of oxazoles. Ring opening of the thiazole moiety has also been observed in benzothiazoles. For instance, benzothiazole itself is converted to S-methylmercaptoaniline (262), (Scheme 26) which undergoes further oxidation at the sulfur and nitrogen resulting in sulfinyl (263), sulfonyl (264), and hydroxylamine derivatives (265 and 266), respectively (197). The pathway is similar to the previously described biotransformation of benzoxazoles, and

presumably takes place by hydroxylation followed by hydrolysis and decarboxylation of the corresponding 2-oxobenzothiazole (261) (see Scheme 26). S-Methyltransferase is presumably responsible for methylation of the thiophenol metabolite resulting in 262. In some instances, hydroxylated thiazoles or the Soxides of thiazoles have been detected in the metabolism studies with thiazole-containing compounds. For example, chlormethiazole (267) (Chart 15) is hydroxylated at the 2-position of the thiazole ring (268) (198, 199) in addition to the formation of the “dioxide” metabolite, 4,5dimethylthiazole-N-oxide-S-oxide (269) (200). A unique metabolite of chlormethiazole, the 3-mercapto-2,4-pentadione metabolite (270), has also been identified in urine of humans administered with 267 (201). The mechanism for the formation of 270 is unknown. The authors have speculated that chlormethiazole undergoes degradation of the chloroethyl side chain followed by oxidative cleavage of the thiazole ring to yield 270 (201). Likewise, incubation of L-766,112 (271), a potent and selective inhibitor of human COX-2, with rat and rhesus monkey hepatic microsomal fractions under oxidative conditions generates the electrophilic thiazole-S-oxide metabolite 272. The corresponding S-oxide has been trapped with GSH to a give the GSH conjugate 273 (Chart 15) (202). In other examples, nucleophilic displacement of groups substituted at the C-2 position of the benzothiazole has been reported (203-205). For instance, administration of benzothiazole-2-sulfonamides (274, 275, and 276) and 2-thiobenzothiazole (277) to rats yields the 2-mercapturic acid metabolite 278.

Invited Review

Chem. Res. Toxicol., Vol. 15, No. 3, 2002 287 Scheme 26. Metabolic Ring Opening of Benzothiazoles and Benzoxazoles

Chart 15. Structures of Thiazole and Benzothiazole-based Xenobiotics and Their Metabolites

Chart 16. Structures of Metabolites of Pyrazole

Chart 17. Examples of Pyrazole-containing Drugs

3.4. Pyrazoles Several drugs with a pyrazole, fused pyrazole, and indazole (a benz-fused pyrazole) have been examined with regard to mammalian biotransformation pathways. As mentioned before the presence of the heteroatom decreases the basicity of the pyrazole ring (pKa of 2.02.5, Table 1). The greatest electron density resides on the N1 nitrogen, followed by N2, C4, C5, and C3 positions, respectively. Unlike the imidazole, thiazole, and oxazole heterocycles, which undergo oxidative cleavage to electrophilic fragments, pyrazole-containing compounds are relatively more stable to oxidation by oxygenases including P450. It is probably the strongly acidic nature of these rings that makes these heterocycles less susceptible to

oxidative metabolism. However, oxidation of the pyrazole ring has been reported for some compounds. Incubation of pyrazole with rat liver microsomes and hepatocytes or recombinant human P4502E1 have revealed the formation of 4-hydroxypyrazole (279) (Chart 16) (206208). Incubations with rat liver microsomes isolated from animals treated with P4502E1 inducers (such as pyrazole itself) increase the rate and extent of formation of 279 (209). Administration of [3,4-14C]pyrazole to rats also results in the urinary excretion of 279 and its corresponding glucuronide and sulfate conjugate as major metabolites (210). Other metabolites such as 3-hydroxy-

288


Dalvie et al.

Scheme 27. Metabolism of Tracazolate (286) to a Vinyl Metabolite (293)

Scheme 28. Metabolic Cleavage of Mepirizole (295) in Rats and Rabbits

mepirizole with hydrogen peroxide. An N-oxide metabolite 296, resulting from oxidation of 295, is presumably the intermediate in the formation of 297, as shown in Scheme 28.

3.5. Isoxazoles

pyrazole (280a) (which exists in equilibrium with the keto tautomer 280b), 3,4-dihydroxypyrazole (281), and 1,3,4trihydroxypyrazole (282), respectively, have also been detected in mice treated with pyrazole (Chart 16) (211). In addition to oxidative metabolism, N-glucuronidation (283) and N-ribosylation (284, an unusual pathway of xenobiotic conjugation) have also been identified as metabolic pathways involved in the elimination of pyrazole in vivo. Drugs containing N-substituted pyrazoles as part of the substructure often lose the substituent on the pyrazole ring as a part of their biotransformation. For instance, zolazepam (285) (212), tracazolate (286) (213, 214), cartazolate (287) (215), benzydamine (288) (216219), and granisetron (289) (220) (Chart 17) yield Ndealkylated metabolites. Similarly, hydrolysis of the sulfonamide moiety on the pyrazole ring in zanoterone (290) results in the formation of a desmethyl sulfone metabolite 291 in rat (221). Interestingly, tracazolate (286) is also converted to an N-ethenyl metabolite 293 (213, 214), in addition to the usual N-deethyl metabolite 294. This unusual metabolite 293 was proposed to arise via dehydration of the hydroxylated metabolite 292 (Scheme 27) (213). However, direct CYP-mediated dehydrogenation of 286 to 293, where alcohol is not an intermediate, cannot be ruled out as an alternate pathway. An interesting metabolite resulting from a reaction that is analogous to a simple N-dearylation reaction has been observed in the metabolism studies of mepirizole (295) in rats and rabbits (222, 223). These studies have shown that 295 is transformed to a unique 4-methoxy6-methyl-2-pyrimidone metabolite 297 with the loss of 1-hydroxy-3-methyl-5-methoxypyrazole (298) (Scheme 28). The metabolite is also generated by treatment of

Unlike the metabolism of pyrazole rings, biotransformation of isoxazole rings results in extensive ring opening. However, in contrast to the oxidative cleavage of thiazoles and oxazoles, isoxazole rings undergo a reductive ring cleavage. It can be speculated that the greater susceptibility of the isoxazole moiety compared to pyrazoles toward reductive ring cleavage is attributed to the greater electronegativity of the oxygen atom adjacent to the nitrogen in the isoxazole ring. Furthermore, compounds containing the benzisoxazole moiety are reductively cleaved to phenols. The most extensively studied isoxazole-containing drug with regard to biotransformation is the anticonvulsant drug zonisamide (299). In rats, zonisamide is metabolized to 2-(sulfamoylacetyl)phenol (301) and is eliminated as a glucuronide conjugate in urine (Scheme 29) (224). The mechanism for the formation of the phenol metabolite 301 involves a two electron reductive cleavage of the N-O bond to an imine intermediate 300 that is hydrolyzed to 301. Initial metabolism studies of zonisamide using rat and human liver tissue have suggested the involvement of P450 in this metabolic pathway (225, 226). The reduction is inhibited by carbon monoxide, ketoconazole, troleandomycin, or cimetidine and is faster in microsomes obtained from phenobarbital or dexamethasone pretreated animals (225, 227-229). Interestingly, SKF-525A, a nonspecific inhibitor of P450, stimulated the reduction of zonisamide in rats. Besides P450, evidence that aldehyde oxidase is also capable of reducing 299 to 301 was demonstrated by incubating 299 with cytosolic preparations under anaerobic conditions and by inhibiting the reaction with menadione, a potent, selective inhibitor of aldehyde oxidase activity. On the other hand, the reduction was stimulated by addition of Scheme 29. Reductive Metabolism of Zonisamide (289)

Invited Review


Chart 18. Structures Risperidone (303), Iloperidone (304), and Leflunomide (311) and Its Cyano Metabolite (312)

amine metabolite (307). The formation of 310 possibly involves further oxidation of the cyano metabolite (309). The formation of a cyano metabolite (312) has also been demonstrated in biotransformation studies with leflunomide, 311 (Chart 18) (235). The reaction is thought to occur in the gut wall, plasma, and liver (both cytosolic and microsomal fractions). In case of leflunomide, this represents an important biotransformation pathway since the resulting 312 is partly responsible for the pharmacological activity of 311.

3.6. Isothiazoles

electron donors utilized by aldehyde oxidase including, 2-hydroxypyrimidine, N′-methylnicotinamide, and benzaldehyde (230). Finally, purified rat and rabbit aldehyde oxidase was also capable of carrying out this transformation. In addition to 301, a unique N-glucuronide conjugate 302 of 299 was also detected as a metabolite in rats (Scheme 29). The antipsychotic agents, risperidone (303) and iloperidone (304) (Chart 18), are also metabolized to corresponding phenol metabolites by reduction of the benzisoxazole rings in preclinical species and man (231-233). It was proposed that reductive metabolism of 303 was catalyzed by the gut microflora since the phenol was also observed in incubations of 303 with intestinal contents. Further evidence for the involvement of gut microflora was provided by the observations that the reductive metabolites were present only in the feces and not in bile of these animals. In contrast, Mutlib and co-workers have shown that reduction of iloperidone (304) in rat, dog, and humans is primarily catalyzed by hepatic enzymes and not by gut microflora (233). However, the enzymes responsible for the reduction have not been identified. An interesting example in which the isoxazole ring undergoes ring opening to a unique cyano derivative has also been reported (234). Biotransformation studies with radiolabeled danazol (305) have demonstrated that the isoxazole ring of 305 is metabolized to the corresponding amino (307), cyano (309), and dicarbonyl (310) metabolites in the rat, monkey, and human volunteers (Scheme 30). The mechanism for formation of these metabolites is unclear. However, a key intermediate is presumably the imine 306, which is formed by a two-electron reduction of the isoxazole ring. The imine (306) can undergo dehydration to a cyano metabolite 309, via the hydroxylamine intermediate (308) or is reduced to a primary

Isothiazoles and benzisothiazole rings, in which the nitrogen atom is adjacent to the sulfur, possess similar electronic properties as that observed with isoxazoles. Although several drugs containing isothiazole and benzoisothiazole ring systems are known, very little information regarding their metabolism and toxicity has been reported. Prakash and co-workers have shown that the benzoisothiazole ring in ziprasidone (313) undergoes extensive reductive and oxidative biotransformation reactions when administered to rats, monkeys, and humans (Scheme 31) (236). The sulfur atom in the benzisothiazole ring undergoes a CYP3A4-catalyzed oxidation to the sulfoxide and sulfone metabolites, 314 and 315, respectively. Furthermore, the S-methyldihydroziprasidone (317) resulting from reductive cleavage of the benzisothiazole ring has been detected in the biological samples of preclinical species and humans. The mechanism for the formation of 317 involves reduction of the benzisothiazole ring in 313 to the corresponding thiophenol derivative 316 that undergoes S-methylation to afford 317 (237). In vitro incubations with human cytosol and S-9 fractions using menadione as an inhibitor have suggested that this reduction is catalyzed by aldehyde oxidase (Kamel, unpublished results). The sulfinylamide metabolite 318 has also been detected in the urine and bile of rats treated with ziprasidone (Scheme 31) (238). However, the mechanism for the formation of this metabolite remains unclear. In contrast to the metabolism of ziprasidone, in vitro and in vivo metabolism studies of tiospirone (319) (Scheme 31) have demonstrated the formation of benzisothiazole sulfoxide and the sulfone metabolites, which are analogous to 314 and 315 (239, 240). No reduced metabolites such as 316 or 317 were detected in the urine or plasma of any species.

4. Heterocycles with Three or More Heteroatoms Heteroaromatic rings such as triazoles, oxadiazoles, thiadiazoles and tetrazoles belong to this class of het-

Scheme 30. Metabolism of Danazol (305) in Preclinical Species

290


Dalvie et al.

Scheme 31. Metabolism of Ziprasidone (313) in Preclinical Species and Man

Chart 19. Structures of Drugs Containing Triazoles and Thiadiazoles

erocycles (Chart 1). Many biologically active compounds including drugs as well as pesticides contain these ring systems as a part of their structure. The most common among them are the triazole and tetrazole rings. For example, triazoles are frequently found in herbicides such as amitrole (320) and in several antimycotic agents such as itraconazole (321) or fluconazole (322) (Chart 19). Examples of drugs containing thiadiazole rings include timolol (323), which is used in the treatment of glaucoma and as a protective drug for patients who have suffered heart attacks, and the diuretic, acetazolamide (324). Tetrazoles and N-substituted tetrazoles are commonly used in medicinal chemistry as bioisosteric replacements of carboxylic acid groups (1). This is because the pKa of tetrazole is similar to that of the carboxylic acid. Also, the tetrazole is a planar delocalized ring system with approximately the same spatial requirement as the carboxy group. An increasing number of electronegative nitrogen atoms in these heteroaromatic rings have a considerable effect on their acidity and basicity (Table 1) as well as the electron density. Thus, the electrophilic substitutions and oxidations are very rare in these ring systems. Although the chemistry of these ring systems has been studied extensively, details concerning their metabolism are less known mainly because these rings are quite inert to biotransformation reactions.

Scheme 32. Proposed Mechanism for Inactivation of P450 by 1-Aminobenzotriazole (325)

4.1. Triazoles Triazole rings contain three nitrogen atoms and generally exist as two isomers namely, the 1,2,3-triazole and the 1,2,4-triazole (Chart 1). Most compounds containing these rings are potent P450 inhibitors. For example, itraconazole is a potent inhibitor of human CYP3A4 whereas fluconazole is known to inhibit human CYP2C9 and CYP2C19 isoforms (241, 242). Like imidazoles, these compounds can inhibit P450 by binding to the heme iron. Benzotriazole derivatives such as 1-aminobenzotriazole (325) and N-substituted aminobenzotriazoles (326-329) have been extensively studied as mechanism-based inactivators of P450 as well as peroxidases in vitro and in vivo (Scheme 32) (243-249). 1-Aminobenzotriazole (325) is a nonselective mechanism-based inhibitor of P450 and inactivates P450 via alkylation of the prosthetic heme moiety. This has been demonstrated by isolation of a N,Nbridgedphenylene-protoporphyrin IX adduct from livers of rats treated with 325 (243). The proposed mechanism of inactivation involves oxidation of 325 to a benzyne (330) with a concomitant loss of nitrogen in the presence of NADPH (Scheme 32) (250). Recently, the metabolism and covalent binding of N-benzylaminobenzotriazole (326) have been examined in hepatic or pulmonary microsomes from untreated and phenobarbital or naphthoflavone induced guinea pigs. Results from this study have suggested that, unlike 325, the mechanism of inactiva-

Invited Review

Chem. Res. Toxicol., Vol. 15, No. 3, 2002 291 Chart 20. Examples of Drugs Containing the 1,2,3- or 1,2,4-Triazole Ring System

tion by 326 probably involves the modification of an active site residue rather than heme alkylation (250). Most triazole-containing compounds are primarily eliminated as N-glucuronides. Studies by Huskey and coworkers have suggested that, like imidazoles, the nitrogen atoms in close proximity to the substituted carbon atoms in the triazole ring are less prone to glucuronidation (157, 158). For example, the nitrogen atom in the 3-position of methylbiphenyl-1,2,3-triazole (331) and the 2 and 4-positions of methylbiphenyl-1,2,4-triazole (332) (Chart 20) were relatively less reactive toward N-glucuronidation. Also, some regioselective differences in Nglucuronidation of triazoles are exhibited by glucuronosyltransferases in human liver microsomes when compared to rats and monkeys. For instance, the 1,2,4-triazole derivative (332) is glucuronidated at the N1 position when incubated with human liver microsomes, in contrast to the N2 position in rat liver microsomes. Similarly, glucuronidation in the 1,2,3-triazole derivative (331) occurred predominantly at the N1 position in monkey liver microsomes, whereas the N2 position in 331 was the major site of glucuronidation upon incubation with microsomes from rats and humans. Although oxidation of triazoles is very rare, metabolism studies of estazolam (333), where the triazole ring is fused to a benzodiazepine moiety, have revealed the formation of the corresponding triazolone metabolite (334) as a minor metabolite in dogs and humans (see Chart 20) (251). In another report, metabolism studies with 1,2,4-triazolyl derivative, triazophos (335) (Chart 20) in rats have demonstrated the formation of novel triazole ring opened metabolites namely 1-phenylsemicarbazide (336), semicarbazide (337), and (338), respectively, which are excreted as N-glucuronides in the urine of rats in addition to 1-phenyl-1,2,4-triazol-3-ol glucuronide (339) (see Chart 20) (252). The mechanism of triazole ring opening leading to semicarbazide metabolites is unknown.

4.2. Oxadiazoles Oxadiazoles are analogues of isoxazole in which one of the methine groups is replaced with an additional nitrogen atom. There are four isomeric types of oxadiazoles (Chart 1). These heterocyclic ring systems are considered to be bioisoteres of ester groups and are frequently incorporated in lieu of the ester group to generate the compounds resistant to hydrolysis by esterases (253-260). Although, examples of all oxadiazole

Scheme 33. Reductive Metabolism of the Oxadiazole Analogue SQ-18506 (340)

Chart 21. Structures of U-78875 (345) and Sirdalud (350) and Their Metabolites

isomers are known, only the biotransformation pathways of compounds containing 1,2,4-oxdiazole rings have been reported so far. Like isoxazoles, the 1,2,4-oxadiazole rings have a potential to undergo ring opening as observed in the biotransformation of SQ-18506 (340) (Scheme 33) (261). The oxadiazole moiety of SQ-18506 undergoes a reductive N-O bond cleavage to generate the ring-opened products such as the amidine, amide and carboxylic acid derivatives (341-344) (Scheme 33). The reduction is NADPH dependent although, xanthine oxidase also has been shown to be involved in the reduction process. A similar metabolic transformation has been reported for U-78885 (345), a nonbenzodiazepine anxiolytic agent that yields the urea derivative 346 (Chart 21) (262). In contrast to the reductive ring opening, an interesting biotransformation pathway involving an unusual cleavage of the 1,2,4-oxadiazole ring has been reported in the metabolism studies with SM-6586 (347) (Scheme 34) (263). A novel nitrile derivative 349 is detected in the

292


Dalvie et al.

Scheme 34. Metabolism of SM-6586 (347) in Rats

bile of rats treated with 337. Hydroxylation of the benzylic methylene of 347 and spontaneous rearrangement of the corresponding hydroxylated metabolite 348 can be speculated as one of the pathways for the formation of 349.

Scheme 36. P450 Metabolism of 1,2,3-Thiadiazole Derivatives

4.3. Thiadiazoles The thiadiazole ring, like oxadiazoles, also exists in four isomeric forms but contain a sulfur atom instead of oxygen (Chart 1). Like oxadiazoles, very few reports on the metabolism of this ring system have been published. One example of biotransformation of the thiadiazole ring is observed in the metabolism studies of a muscle relaxant, sirdalud (350) (Chart 21) (264). In addition to hydroxylation of the phenyl ring, which represents one of the major metabolites of sirdalud, the benzothiadiazole portion of 350 undergoes some unique transformations leading to the cyclic sulfonylurea derivative 351 (Chart 21). This metabolite has been detected in several species and constitutes 98% of the circulating drug-related material in the rat. An interesting example of a biotransformation reaction involving the thiadiazole ring is that of the fungicidal agent etridazole (352) (Scheme 35) (265). Although the thiadiazole ring of etridazole is not modified, the trichloromethyl group on the ring is converted to the carboxylic acid 355. This overall conversion possibly occurs (either by hepatic enzymes or gut floral metabolism) via a dichloromethyl intermediate 353 that undergoes hydroxylation and rearrangement to an acid chloride 354 followed by hydrolysis to 355 (Scheme 35). Although this biotransformation pathway does not constitute an example of a thiadiazole ring modification, it is tempting to speculate that the thiadiazole ring participates in this unique reaction. Scheme 35. Metabolism of the Fungicidal Agent Etridazole (352)

Babu and Vaz have also studied the metabolism of 1,2,3-thiadiazole ring systems in the course of investigation of these rings as heme ligands and mechanism-based inactivators of P450 (266). These studies have demonstrated that the monocyclic 1,2,3-thiadiazoles (356-358) undergo S-oxidation and rearrangement to acetylene derivatives (359-361) via extrusion of heteroatoms in the form of SO and nitrogen but do not inactivate P450 (Scheme 36). In contrast, the fused bicyclic thiadiazole

such as 362 inactivates the P4502E1 and 2B4 in a mechanism-based manner (266).

4.4. Tetrazoles Tetrazoles are five-membered aromatic heterocycles with four nitrogens in the ring (Chart 1). The past two decades have seen frequent incorporation of tetrazole moieties as bioisoteres of carboxylic acids in various drug molecules (1, 267). A review of the literature of tetrazolecontaining compounds indicates that, like triazoles, these heterocycles are resistant to oxidative metabolism. NGlucuronidation has been shown to be an important clearance pathway of tetrazole-containing compounds. Nohara and co-workers were the first to identify a tetrazole-N-1-glucuronide (364) in urine of animals dosed with 6-ethyl-3-(1H-tetrazol-5-yl)chromone (363) (Chart 22) (268). Since then several N2-glucuronide conjugates of tetrazoles have been reported as metabolites in metabolism studies of the tetrazole-containing angiotensin II receptor antagonist losartan (365), L-158,338 (366), L-158,809 (367), irbesartan (368), and the model biphenyltetrazole (369) (Chart 22) (269-273). Recent metabolism studies with RG-12525 (371), a new chemical entity for Type II diabetes, have suggested that glucuronosyl transferase isoforms 1A1 and 1A3 display the highest rate of N2-glucuronidation of tetrazoles (274). N-Glucuronides of tetrazoles have also been detected in bile or urine following administration of irbesartan (368) or DMP 811 (370), to preclinical species or humans (275277). A few reports describing the species differences in N-glucuronidation of tetrazoles have been published. Comparison of N-glucuronidation in rat, monkey and human hepatic microsomes have indicated that like triazoles, monkeys glucuronidate L-158,809 (367) with a relatively higher catalytic efficiency than rat and humans (11.0 and 2.6-fold higher, respectively) (273). The

Invited Review

Chem. Res. Toxicol., Vol. 15, No. 3, 2002 293 Chart 22. Glucuronidation of Tetrazolyl Derivatives

optimal pH value for the transformation of tetrazoles to their respective N2-glucuronide conjugates correlates very well with the reported pKa of tetrazole (pKa ) 4.9) (273). In vivo and in vitro studies with losartan (365) by Krieter and co-workers have shown that intestine is also the site of glucuronidation of tetrazoles (278). It is observed that approximately 12 to 20% of losartan transported across the duodenum and jejunum is glucuronidated, whereas no conjugate is formed in the ileum or colon.

5. Conclusions In summary, an attempt has been made to compile the important and unique biotransformation pathways of most commonly used five-membered aromatic heterocycles. Wherever possible, the focus of the review has also been to understand the mechanisms of formation of these unique metabolites. To our knowledge, the review covers most of the biotransformation reactions of five-membered aromatic heterocycles. It is clear from the overview that the most of the fivemembered heteroaromatic rings are metabolized by P450. However, other oxidizing enzymes sometimes play a role in their biotransformation. The number of heteroatoms in a ring as well as the electronegativity and the position of these heteroatoms clearly play a major role in the metabolism of these rings. One other common factor among most of the rings is that the oxidation or reduction of the heteroaromatic rings often leads to bioactivation that generates electrophilic intermediates capable of reacting with biomacromolecules and resulting in toxicological consequences. Incorporation of appropriate substituents in these ring systems, however, provides a facile methodology to circumvent potential bioactivation issues and improve the metabolic stability of these ring systems. It is our hope that summarizing the metabolism of these important heteroaromatic ring systems will not only aid medicinal chemists in their quest for the design of new pharmacophores containing these ring systems but will also guide drug metabolism scientists in rationalizing the metabolic pathways of these compounds.

References (1) Patani, G. A., and LaVoie, E. J. (1996) Bioisosterism: A rational approach in drug design. Chem. Rev. 96, 3147-3176. (2) Lipinski, C. A. (1986) Bioisosterism in drug design. Ann. Rep. Med. Chem. 21, 283-291. (3) Nelson, S. D. (1982) Metabolic activation and drug toxicity. J. Med. Chem. 25, 753-765. (4) Ashby, J., and Elliott, B. M. (1984) Toxicity of heterocycles. In Comprehensive Heterocyclic Chemistry (Katritzky, A. R., and Rees, C. W., Eds.) Vol. 1, pp 111-141, Pergamon press, New York. (5) Damani, L. A., and Case, D. E. (1984) Metabolism of heterocycles. In Comprehensive Heterocyclic Chemistry: The Structure, Reactions, Synthesis and Uses of Heterocyclic Compounds (Katritzky, A. R., and Rees, C. W., Eds.) Vol. 1, pp. 111-141, Pergamon Press, New York. (6) Jackson, A. H. (1979) Pyrroles. In Comprehensive Organic Chemistry: The Synthesis and Reactions of Organic Compounds (Barton, D., and Ollis, W. D., Eds.) Vol. 4, pp 275-330, Pregamon Press, New York. (7) Gilchrist, T. L. (1987) Five membered ring compounds with one heteroatom. In Heterocyclic Chemistry, pp 126-185, Wiley Inc., New York. (8) Campos-Vallette, M. M., and Clavijo, C. R. E. (1985) Molecular vibrational constants and chemical bonding in five-membered heterocyclic compounds. Spectrosc. Lett. 18, 759-66. (9) Guengerich, F. P., and Mitchell, M. B. (1980) Metabolic activation of model pyrroles by cytochrome P450. Drug Metab. Dispos. 8, 34-38. (10) Testa, B., and Caldwell, J. Carbon oxidations catalyzed by cytochromes P-450. The Metabolism of Drugs and Other Xenobiotics, pp 122-163, Academic Press, New York. (11) Langner, A., Ra¨tzer, J., Rickert, H., Nerlich, C., and Franke, P. (1993) Untersuchungen zur biotransformation des antikonvulsivusms 4-p-chlorophenylpyrrol-3-morpholino-2-carbonsauremethylester (AWD 140-076) Pharmazie 48, 849-853. (12) Assandri, A., Barone, D., Ferrari, P., Perazzi, A., Ripamonti, A., Tuna, G., and Zerrilli, L. F. (1984) Metabolic fate of premazepam, a new antianxiety drug, in the rat and the dog. Drug Metab. Dispos. 12, 257-263. (13) Murphy, P. A., and Williams, T. L. (1972) Biological inactivation of pyrrolnitrin. Identification and synthesis of pyrrolnitrin metabolites. J. Med. Chem. 15, 137-139. (14) Shigenaga, M. K., Kim, B. H., Caldera-Munoz, P., Cairns, T., Jacob, P., III, Trevor, A. J., and Castagnoli, N., Jr. (1989) Liver and lung microsomal metabolism of the tobacco alkaloid β-nicotyrine. Chem. Res. Toxicol. 2, 282-287. (15) Liu, X., Zang, L., Van Der Schyf, C. J., Igarashi, K., Castagnoli, K., and Castagnoli, N., Jr. (1999) Studies on the pyrrolinone metabolites derived from tobacco alkaloid 1-methyl-2-(3-pyridinyl)pyrrole (β-Nicotyrine). Chem. Res. Toxicol. 12, 508-512.

294


(16) Liu, X., Castagnoli, K., Van Der Schyf, C. J., and Castagnoli, N., Jr. (2000) Studies on the in vivo biotransformation of the tobacco alkaloid β-nicotyrine. Chem. Res. Toxicol. 13, 336-341. (17) Egger, H., Itterly, W., John, V., Rodebaugh, R., Shimanskas, C., Stancato, F., and Kapoor, A. (1988) Disposition and metabolism of prinomide in laboratory animals. Drug Metab. Dispos. 16, 568575. (18) Assandri, A., Perrazi, A., Baldoli, E., Ferrari, P., Ripamonti, A., Bellasio, E., Tuan, G., Zerilli, L. F., and Tarzia, G. (1985) Metabolic pathways of the anti-hypertensive agent, N-(2,5dimethyl-1H-pyrrol-1-yl)-6-(4-morpholinyl)-3-pyridazinamine hydrochloride. I. Studies in the rat. Xenobiotica 15, 1069-1087. (19) Assandri, A., Perrazi, A., Baldoli, E., Ferrari, P., Ripamonti, A., Bellasio, E., Tuan, G., Zerilli, L. F., and Tarzia, G. (1985) Metabolic pathways of the anti-hypertensive agent, N-(2,5dimethyl-1H-pyrrol-1-yl)-6-(4-morpholinyl)-3-pyridazinamine hydrochloride. II. Studies in the dog. Xenobiotica 15, 1089-1102. (20) Assandri, A., Tarzia, G., Bellasio, E., Ciabatti, R., Tuan, G., Ferrari, P., and Zerilli, L. (1987) Metabolic oxidation of the pyrrole ring: Structure and origin of some urinary metabolites of the antihypertensive pyrrolylpridazinamine, mopidralazine. III: Studies with 13C-labelled drug. Xenobiotica 17, 559-573. (21) Tse, F. L. S., Orwig, B. A., Jaffe, J. M., and Dain, J. G. (1987) Pharmacokinetics and metabolism of R-[(dimethylamino)methyl]2-(3-ethyl-5-methyl-4-isoxazolyl)-1H-indole-3-methanol, a hypoglycemic agent in man. Xenobiotica 17, 1259-1267. (22) Dain, J. G., Fu, E., Gorski, J., Nicoletti, J., and Scallen, T. J. (1993) Biotransformation of fluvastatin sodium in humans. Drug Metab. Dispos. 21, 567-572. (23) Fischer, V., Baldeck, J.-P., and Tse, F. L. S. (1992) Pharmacokinetics and metabolism of the 5-hydroxytryptamine antagonist tropisetron after single oral doses in humans. Drug Metab. Dispos. 20, 603-607. (24) Dixon, C. M., Colthup, P. V., Serabjit-singh, C. J., Kerr, B. M., Boehlert, C. C., Park, G. R., and Tarbit, M. H. (1995) Multiple forms of cytochrome P 450 are involved in the metabolism of ondansetron in humans. Drug Metab. Dispos. 23, 1225-1230. (25) Pierce, D. M. (1990) A review of the clinical pharmacokinetics and metabolism of the R1-adrenorecptor antagonist indoramin. Xenobiotica 20, 1357-1367. (26) Chang, M., Sood, V. K., Wilson, G. J., Kloosterman, D. A., Sanders, P. E., Schuette, M. R., Judy, R. W., Voorman, R. L., Maio, S. M., and Slatter, J. G. (1998) Absortion, distribution, metabolism, and excretion of atervirdine in the rat. Drug Metab. Dispos. 26, 10081018. (27) King, L. J., Parke, D. V., and Williams, R. T. (1966) The metabolism of [2-14C]indole in the rat. Biochem. J. 98, 266-277. (28) Gillam, E. M. J., Notley, L. M., Cai, H., De Voss, J. J., and Guengerich, F. P. (2000) Oxidation of indole by cytochrome P 450 enzymes. Biochemistry 39, 13817-13824. (29) Guengerich, F. P. (2001) Common and uncommon cytochrome P 450 reactions related to metabolism and chemical toxicity. Chem. Res. Toxicol. 14, 611-650. (30) Krause, W., Du¨sterberg, B., Jakobs, U., and Hoyer, G.-A., (1993) Biotransformation of proterguride in the perfused rat liver. Drug Metab. Dispos. 21, 203-208. (31) Paine, A. J., Maurer, G., and von Wartburg, B. R., (1984) The application of hepatocyte culture to the identification of pathways of drug metabolism: Studies with pindolol and fluperlapine. Biochem. Pharmacol. 33, 3111-3114. (32) Kiechel, J. R., Niklaus, P., Schreier, E., and Wagner, H. (1975) Metabolites of pindolol in different animal species. Xenobiotica 5, 741-754. (33) Woodland, J. M., Barnett, C. J., Dorman, D. E., Gruber, J. M., Shih, C., Spangle, L. A., Wilson, T. M., and Ehlhardt, W. J. (1997) Metabolism and disposition of the antifolate LY231514 in mice and dogs. Drug Metab. Dispos. 25, 693-700. (34) Sun, Y. (1989) Indoleamine 2,3-dioxygenase - a new antioxidant enzyme. Mater. Med. Pol. 21, 244-250. (35) D’Ischia, M., and Prota, G. (1986) Photo-oxidation of 5,6-dihydroxyindoles. Gaz. Chim. Ital. 116, 407-410. (36) Carlson, J. R., and Yost, G. S. (1989) 3-Methylindole-induced acute lung injury resulting from ruminal fermentation of tryptophan. In Toxicants of Plant Origin (Cheeks, P. R., Ed.) pp 108-123, CRC Press, Boca Raton, FL. (37) Thronton-Manning, J., Appleton, M. L., Gonzalez, F. J., and Yost, G. S. (1996) Metabolism of 3-methylindole by vaccinia-expressed P450 enzymes: correlation of 3-methyleneindolenine formation and protein binding. J. Pharmacol. Exp. Ther. 276, 21-29. (38) Smith, D. J., Skiles, G. L., Appleton, M. L., Carlson, J. R., and Yost, G. S. (1993) Identification of goat and mouse urinary metabolites of the puemotoxin, 3-methylindole. Xenobiotica 23, 1025-1044.

Dalvie et al. (39) Skiles, G. L., Adams, J. D., Jr., and Yost, G. S. (1989) Isolation and identification of 3-hydroxy-3-methyloxindole, the major murine metabolite of 3-methylindole. Chem. Res. Toxicol. 2, 254259. (40) Diaz, G. J., Skordos, K. W., Yost, G. S., and Squires, E. J. (1999) Identification of phase I metabolites of 3-methylindole produced by pig liver microsomes. Drug Metab. Disp. 27, 1150-1156. (41) Skordos, K. W., Skiles, G. L., Laycock, J. D., Lanza, D. L., and Yost, G. S. (1998) Evidence supporting the formation of 2,3-epoxy3-methylindoline: a reactive intermediate of the puemotoxin 3-methylindole. Chem. Res. Toxicol. 11, 741-749. (42) Skordos, K. W., Laycock, J. D., and Yost, G. S. (1998) Thioether adducts of a new imine reactive intermediate of the puemotoxin 3-methylindole. Chem. Res. Toxicol. 11, 1326-1331. (43) Ruangyuttrkarn, W., Skiles, G. L., and Yost, G. S. (1992) Identification of a cysteinyl adduct of oxidized 3-methylindole from goat lung and human liver microsomal proteins. Chem. Res. Toxicol. 5, 713-719. (44) Skiles, G. L., Smith D. J., Appleton, M. L., Carlson, J. R., and Yost, G. S. (1991) Isolation of a mercapturate adduct produced by susequent glutathione conjugation to bioactivated 3-methylindole. Toxicol. Appl. Pharmacol. 108, 531-537. (45) Regal, K. A., Laws, G. M., Yuan, C., Yost, G. S., and Skiles, G. L. (2001). Detection and characterization of DNA adducts of 3-methylindole. Chem. Res. Toxicol. 14, 1014-1024. (46) Skiles, G. L., and Yost, G. S. (1996) Mechanistic studies on the cytochrome P450 catalyzed dehydrogenation of 3-methylindole. Chem. Res. Toxicol. 9, 291-297. (47) Wang, H., Lanza, D. L., and Yost, G. S. (1998) Cloning and expression of CYP2F3, a cytochrome P450 that bioactivates the selective puemotoxins 3-methylindole and naphthalene. Arch. Biochem. Biophys. 349, 329-340. (48) Lanza, D. L., Code, E., Crespi, C. L., Gonzalez, F. J., and Yost, G. S. (1999) Specific dehydrogenation of 3-methylindole and epoxidation of naphthalene by recombinant human CYP2F1 expressed in lymphoblastoid cells. Drug Metab. Disp. 27, 798803. (49) Lanza, D. L., and Yost, G. S. (2001) Selective dehydrogenation/ oxygenation of 3-methylindole by cytochrome P450 enzymes. Drug Metab. Disp. 29, 950-953. (50) Chang, M., Sood, V. K., Klosterman, D. A., Haurer, M. J., Fagerness, P. E., Sanders, P. E., and Vrbanac, J. J. (1997) Identification of the metabolites of the HIV-1 reverse transcriptase inhibitor delavirdine in monkeys. Drug Metab. Dispos. 25, 814-827. (51) Schaefer, W. H., Goalwin, A., Dixon, F., Hwang, B., Killmer, L., and Kuo, G. (1992) Structural determination of glucuronide conjugates and a carbamoyl glucuronide conjugate of carvedilol: Use of acetylation reactions as an aid to determine positions of glucuronidation. Biol. Mass Spectrom. 21, 179-188. (52) Jaccarini, A., and Felice, M. A. (1978) The metabolism of pyrroles and indoles. Ring nitrogen oxidation. In The Biological Oxidation of Nitrogen (Gorrod, J. W., Ed.) pp 169-175, Elsevier/NorthHolland Biomedical Press, Amsterdam. (53) Savidge, R. D., Bui, K. H., Birmingham, B. K., Morse, J. L., and Spreen, R. C. (1998) Metabolism and excretion of zafirlukast in dogs, rats and mice. Drug Metab. Dispos. 26, 1069-1076. (54) Sirica, A. E. (1996) Biliary proliferation and adaptation in furaninduced rat liver injury and carcinogenesis. Toxicol. Pathol. 24, 90-99. (55) Peterson, L. A., Naruko, K. C., and Predecki, D. P. (2000) A reactive metabolite of furan, cis-2-butene-1,4-dial, is mutagenic in the Ames assay. Chem. Res. Toxicol. 13, 531-534. (56) Kedderis, G. L., Carfagna, M. A., Held, S. D., Batra, R., Murphy, J. E., and Gargas, M. L. (1993) Kinetic analysis of furan biotransformation by F-344 rats in vivo and in vitro. Toxicol. Appl. Pharmacol. 123, 274-82. (57) Burka, L. T., Washburn, K. D., and Irwin, R. D. (1991) Disposition of [14C]furan in the male F344 rat. J. Toxicol. Environ. Health 34, 245-57. (58) Chen, L.-J., Hecht, S. S., and Peterson, L. A. (1995) Identification of cis-2-butene-1,4-dial as a microsomal metabolite of furan. Chem. Res. Toxicol. 8, 903-906. (59) Ravindranath, V., Burka, L. T., and Boyd, M. R. (1984) Reactive metabolites from the bioactivation of toxic methylfurans. Science 224, 884-886. (60) Ravindranath, V., and Boyd, M. R. (1985) Metabolic activation of 2-methylfuran by rat microsomal systems. Toxicol. Appl. Pharmacol. 78, 370-376. (61) Parmar, D., and Burka, L. T. (1993) Studies on the interaction of furan with hepatic cytochrome P450. J. Biochem. Toxicol. 8, 1-9.

Invited Review (62) Chen, L.-J., Hecht, S. S., and Peterson, L. A. (1997) Characterization of amino acid and glutathione adducts of cis-2-butene-1,4dial, a reactive metabolite of furan. Chem. Res. Toxicol. 10, 866874. (63) McCurtry, R. J., and Mitchell, J. R. (1977) Renal and hepatic necrosis after metabolic activation of 2-substituted furans and thiophenes, including furosemide and cephalosporine. Toxicol. Appl. Pharmcol. 42, 285-300. (64) Mitchell, J. R., Potter, W. Z., Hinson, J. A., and Jollow, D. J. (1974) Hepatic necrosis caused by furosemide. Nature (London) 251, 508-511. (65) Walker, R. M., and McElligott, T. F. (1981) Furosemide induced hepatotoxicity. J. Pathol. 135, 301-314. (66) Boyd, M. R., Burka, L. T., Neal, R. A., Wilson, B. J., and Holscher, M. M. (1974) Role of covalent binding in the toxic mechanism of the lung-edemagenic agent, 4-ipomeanol. Fed. Proc. 33, 234. (67) Boyd, M. R. (1976) Role of metabolic activation in the pathogenesis of chemically induced pulmonary disease: Mechanism of action of furan lung-toxic 4-ipomeanol. Environ. Health Prospect. 16, 127-138. (68) Mitchell, J. R., Nelson, W. L., Potter, W. Z., Sasame, H. A., and Jollow, D. J. (1976) Metabolic activation of furosemide to a chemically reactive, hepatotoxic metabolite. J. Pharmacol. Exp. Ther. 199, 41-52. (69) Boyd, M. R. (1975) Hepatotoxicity produced by the lung-toxic furanoterpenoid, 4-ipomeanol, after mixed function oxidase induction: Reversal of target organ for toxicity and covalent binding in rats pretreated with 3-methylchlolanthrene. Toxicol. Appl. Pharmcol. 33, 132. (70) Boyd, M. R., Burka, L. T., Wilson, B. J., and Sasame, H. A. (1978) In vitro studies on the metabolic activation of the pulmonary toxin, 4-ipomeanol, by rat lung and liver microsomes. J. Pharmacol. Exp. Ther. 207, 677-686. (71) Zhang, K. E., Naue, J. A., Arison, B., and Vyas, K. P. (1996) Microsomal metabolism of the 5-lipoxygenase inhibitor L-739,010: Evidence for furan bioactivation. Chem. Res. Toxicol. 9, 547-554. (72) Sahali-Sahly, Y., Balani, S. K., Lin, J. H., and Baillie, T. A. (1996) In vitro studies on the metabolic activation of the furanopyridine L-754,394, a highly potent and selective mechanism-based inhibitor of cytochrome P450 3A4. Chem. Res. Toxicol. 9, 1007-1012. (73) Le Fur, J. M., and La Baune, J. P. (1985) Metabolic pathway by cleavage of furan. Xenobiotica 15, 567-577. (74) Inoue, T., Yasuda, T., Kishi, T., and Niwaguchi, T. (1986) The metabolism of 1-phenyl-2-(N-methyl-N-furfurylamino)propane (furfenorex) in the rat in vivo and in vitro. Xenobiotica 16, 109121. (75) Kollis, S. J., Williams, T. H., Postma, E. J., Sasso, G. J., Confalone, P. N., and Schwartz, M. A. (1979) The metabolism of 14Cmethoxsalen by dog. Drug Metab. Dispos. 7, 220-225. (76) Mays, D. C., Rogers, S. L., Guiler, R. C., Sharp, D. E., Hecht, S. G., Staubus, A. E., and Gerber, N. (1986) Disposition of 8-methoxypsoralen in the rat: Methodology for measurement, dosedependent pharmcokinetics, tissue distribution and identification of metabolites. J. Pharmacol. Exp. Ther. 236, 364-373. (77) Gordon, W. P., Huitric, A. C., Seth, C. L., McClanahan, R. H., and Nelson, S. D. (1987) The metabolism of the abortifacient terpene R-(+)-pulegone, to a proximate toxin, menthofuran. Drug Metab. Dispos. 15, 589-594. (78) Moorthy, B., Madyastha, P., and Madyastha, K. M. (1989) Metabolism of a monoterpene ketone, R-(+)-pulegone a hepatotoxin in rat. Xenobiotica 19, 217-224. (79) McClanahan, R. H., Thomassen, D., Slattery, J. T., and Nelson, S. D. (1989) Metabolic activation of R-(+)-pulegone to a reactive enonal that covalently binds the mouse liver proteins. Chem. Res. Toxicol. 2, 349-355. (80) Madyastha, K. M., and Paul Raj, C. (1992) Metabolic fate of menthofuran in rats: Novel oxidative pathways. Drug Metab. Dispos. 20, 295-301. (81) Madyastha, K. M., and Paul Raj, C. (1990) Biotransformations of R-(+)-pulegone and menthofuran in vitro. Chemical basis for toxicity. Biochem. Biophys. Res. Commun. 173, 1086-1090. (82) Thomassen, D., Knebel, N., Slattery, J. T., McClanahan, R. H., and Nelson, S. D. (1992) Reactive intermediates in the oxidation of menthofuran by cytochrome P450. Chem. Res. Toxicol. 5, 123130. (83) Khojasteh-Bakht, S. C., Chen, W., Koenigs, L. L., Peter, R. M., and Nelson, S. D. (1999) Metabolism of R-(+)-pulegone and R-(+)menthofuran by human liver cytochrome P450s: Evidence for formation of a furan epoxide. Drug Metab. Dispos. 27, 574-580. (84) Kobayashi, T., Sugihara, J., and Harigaya, S. (1987) Mechanism of metabolic cleavage of a furan ring. Drug Metab. Dispos. 15, 877-881.

Chem. Res. Toxicol., Vol. 15, No. 3, 2002 295 (85) Wirth, P. J., Bettis, C. J., Nelson, W. L. (1976) Microsomal metabolism of furosemide: Evidence for the nature of the reactive intermediate involved in covalent binding. Mol. Pharmacol. 12, 759-768. (86) Kedderis, G. L., and Miwa, G. T. (1988) The metabolic activation of nitroheterocyclic therapeutic agents. Drug Metab. Rev. 19, 3362. (87) Tatsumi, K., Nakabeppu, H., Takahashi, Y., and Kitamura, S. (1984) Metabolism in vivo of furazolidone: Evidence for formation of open chain carboxylic acid and R-ketoglutaric acid from the nitrofuran in rats. Arch. Biochem. Biophys. 234, 112-116. (88) Tatsumi, K., Ou, T., Yamada, H., Yoshimura, H., Koga, H., and Horiuchi, T. (1978) Isolation and identification of the metabolite of N-(5-nitro-2-furfurylidene)-3-amino-2-oxazolidone (furazolidone). J. Pharmacobio-Dyn. 1, 256-261. (89) Voormen, L. H. M., van Ommen, B., and van Bladeren, P. J. (1987) Quantitative studies of the metabolism of furazolidone by rat liver microsomes. Toxicol. in Vitro 1, 97-101. (90) Taylor, J. D., Paul, H. E., and Paul, M. F. (1951) Metabolism of the nitrofurans. III. Xanthine oxidase in vitro. J. Biol. Chem. 191, 223-231. (91) Morita, M., Feller, D. R., and Gillette, J. R. (1971) Reduction of niridazole by rat liver xanthine oxidase. Biochem. Pharmacol. 20, 217-226. (92) Wang, C. Y., Behrens, B. C., Ichikawa, M., and Bryan, G. T. (1974) Nitroreduction of 5-nitrofuran derivatives by rat liver xanthine oxidase and reduced nicotinamide adenine dinucleotide phosphatecytochrome c reductase. Biochem. Pharmacol. 23, 3395-3404. (93) Tatsumi, K., Kitamura, S., and Yoshimura, H. (1976) Reduction of nitrofuran derivatives by xanthine oxidase and microsomes. Isolation and identification of reduction products. Arch. Biochem. Biophys. 175, 131-137. (94) Wolpert, M. K., Althaus, J. R., and Johns, D. G. (1973) Nitroreductase activity of mammalian liver aldehyde oxidase. J. Pharmacol. Exp. Ther. 185, 202-213. (95) Streeter, A. J., and Hoener, B. (1988) Evidence for the involvement of nitrenium ion in the covalent binding of nitrofurazone to DNA. Pharm. Res. 5, 434-436. (96) Chatfield, D. H. (1976) Disposition and metabolism of some nitrofurylthiazoles possessing antiparasitic activity. Xenobiotica 6, 509-520. (97) Maurizis, J. C., Madelmont, J. C., and Parry, D. (1986) Main excretion metabolites of 7-methoxy-2-nitronaphtho[2,1-b]furan (R7000) in rats. Xenobiotica 16, 635-643. (98) Rance, D. J. (1989) in Sulfur-containing drugs and related organic compounds, chemistry, biochemistry and toxicology: Metabolism of sulfur functional groups (Damani, L. A., Ed.) Vol. 1, part B, pp 217-268, Ellis Horwood Ltd., Chichester. (99) Bray, H. G., Carpanini, M. B., and Water, B. D. (1971) The metabolism of thiophene in the rabbit and the rat. Xenobiotica 1, 157-168. (100) Neau, E., Dansette, P. M., Andronik, V., and Mansuy, D. (1990) Hydroxylation of the thiophene ring by hepatic monooxygenases: Evidence for 5-hydroxylation of 2-aroylthiophenes as a general metabolic pathway using a simple UV-visible assay. Biochem. Pharmacol. 39, 1101-1107. (101) Mansuy, D., Dansette, P. M., Foures, C., Jaouen, M., Moinet, G., and Bayer, N. (1984) Metabolic hydroxylation of the thiophene ring: Isolation of 5-hydroxy-tienilic acid as the major urinary metabolite of tienilic acid in man and rat. Biochem Pharmacol. 33, 1429-1435. (102) Dansette, P. M., Amar, C., Smith, C. B., Neau, E., Pons, C., and Mansuy, D. (1989) Hydroxylation of the thiophene ring: In vitro the 5-hydroxylation of tienilic acid is catalyzed by cytochrome P 450 in rat and human microsomes. In Cytochromes P-450 Biochemistry and Biophysics (Schuster, Ed.) pp 751-754, Taylor and Francis, London. (103) Mori, Y., Sakai, Y., Kuroda, N., Yokoya, F., Toyoshi, K., Horie, M., and Baba, S. (1984) Further structural analysis of urinary metabolites of suprofen in rats. Drug Metab. Dispos. 12, 767771. (104) 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, 239245. (105) Konishi, T., Okada, S., Nishikawa, H., Esumi, Y., Kitamura, S., and Kiyoshi, T. (1998) Disposition of a new nonsteroidal antiinflammatory agent, S-2-[4-(3-methyl-2-thienyl)phenyl]propionic acid in rats, dogs, and monkeys. Arzneim.-Forsch./Drug Res. 48, 266-274. (106) Ichihara, S., Tsuyuki, Y., Tomisawa, H., Fukazawa, N., Nakayama, N., Tateishi, M., and Joly, R. (1984) Metabolism of tenoxicam in rats. Xenobiotica 14, 727-739.

296


(107) 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. (108) Lynch, M. J., Mosher, F. R., Levesque, W. R., and Newby, T. J. (1987) The in vitro and in vivo metabolism of morantel in cattle and toxicology species. Drug Metab. Rev. 18, 253-288. (109) Picard-Fraire, C. (1984) Pharmacokinetics and metabolic characteristics of ticlopidine in relation to its inhibitory properties on platelet function. Agents and Actions (suppl.) 15, 68-75. (110) Jean, P., Lopez-Garcia, P., Dansette, P., Mansuy, D., and Goldstein, J. L. (1996) Oxidation of tienilic acid by human yeastexpressed cytochromes P-450 2C8, 2C9, 2C18, and 2C19. Evidence that this drug is a mechanism-based inhibitor specific for cytochrome P-450 2C9. Eur. J. Biochem. 241, 797-804. (111) 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. Biochem. Pharmacol. 41, 553-560. (112) Lopez-Garcia, M. P., Dansette, P. M., Valadon, P., Amar, C., Beaune, P. H., Guengerich, F. P., and Mansuy, D. (1993) Human liver cytochromes P450 expressed in yeast as tools for reactive metabolite formation studies. Eur. J. Biochem. 213, 223-232. (113) Taguchi, K., Konishi, T., Nishikawa, H., and Kitamura, S. (1999) Identification of human cytochrome P450 isoforms involved in the metabolism of S-2-[4-(3-methyl-2-thienyl)phenyl]propionic acid. Xenobiotica 29, 899-907. (114) Fouda, H. G., Avery, M. J., Dalvie, D. K., Falkner, F. C., Lawrence, M. S., and Ronfeld, R. A. (1997) Disposition and metabolism of tenidap in the rat. Drug Metab. Dispos. 25, 140148. (115) 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. (116) 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 P450 dependent oxidation and reaction with nucleophiles. J. Am. Chem. Soc. 113, 7825-7826. (117) 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 as an intermediate in thiophene metabolism in vivo and in vitro. J. Am. Chem. Soc. 119, 1565-1571. (118) Valadon, P., Dansette, P. M., Girault, J.-P., Amar, C., and Mansuy, D. (1996) Thiophene-S-oxides 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. (119) Lopez-Garcia, M. P., Dansette, P. M., and Mansuy, D. (1994) Thiophene derivatives as new mechanism based inhibitors of cytochromes P450: Inactivation of yeast expressed human liver cytochrome P450 2C9 by tienilic acid. Biochemistry 33, 166175. (120) Lecoeur, S., Andre, C., and Beaune, P. H. (1996) Tienilic acid induced autoimmune hepatitis: Anti-liver and -kidney microsomal type 2 autoantibodies recognize a three-site conformational epitope on cytochrome P4502C9. Mol. Pharmacol. 50, 326-333. (121) Mansuy, D. (1997) Molecular structure and hepatotoxicity: compared data about two closely related thiophene compounds. J. Hepatol. 26, (Suppl. 2) 22-25. (122) Koenigs, L. L., Raimund, P. M., Hunter, A. P., Haining, R. L., Rettie, A. E., Friedberg, T., Pritchard, M., 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, 23122319. (123) Belghazi, M., Jean, P., Poli, S., Schmitter, J.-M., Mansuy, D., and Dansette, P. M. (2000) Use of isotopes and LC-MS-ESI-TOF for mechanistic studies of tienilic acid and tienilic acid isomer mechanistic activation. Drug Metab. Rev. 32, 42. (124) 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 anti-tienilic acid-protein adduct antibodies and the possible relationship with different hepatotoxic effects of the two compounds. Chem. Res. Toxicol. 12, 286-296. (125) Cripps, R. (1973) The microbial metabolism of thiophen-2carboxylate. Biochem. J. 134, 353-366.

Dalvie et al. (126) O’Donnell, J. P., and Khojasteh-Bakht, S. C. (1999) Oxidative fate of an aryl thiophene: In vivo and in vitro investigation of a possible non-P450 mediated ring opening mechanism. ISSX Proc. 15, 115. (127) Machinist, J. M., Mayer, M. D., Manjunath, S. S., Ferrero, J. L., and Rodrigues, A. D. (1995) Identification of the human liver cytochrome P450 enzymes involved in the metabolism of zileuton (ABT-077) and its N-dehydroxylated metabolite, Abbot-66193. Drug Metab. Dispos. 23, 1163-1174. (128) Liu, Z. C., and Utrecht, J. P. (2000) Metabolism of ticlopidine by activated neutrophils: Implications for ticlopidine-induced agranulocytosis. Drug Metab. Disp. 28, 726-730. (129) Gilchrist, T. L. (1985) Five membered ring compounds with two or more heteroatoms. Heterocyclic Chemistry, pp 186-238, Longman Scientific and Technical, England. (130) Sokolova, M. M., Ostrovskii, V. A., Koldobskii, G. I., Mel’nikow, V. V., and Gidaspov, B. V. (1974) Protonation of tetrazole. Zh. Org. Khim. 10, 1085-1088. (131) Zoltewicz, J. A., and Deady, L. W. (1978) Quaterniztion of heteroaromatic compounds. Quantitative aspects. Adv. Heterocycl. Chem. 22, 71-121. (132) Catalan, J., Menendez, M., and Elguero, J. (1985) On the relationships between basicity and acidity in azoles. Bull. Soc. Chim. Fr. 1, 30-33. (133) Catalan, J., and Elguero, J. (1984) Basicity of azoles. IV. Empirical relationships between basicity and ionization potential for aromatic five membered rings containing nitrogen and oxygen. J. Heterocycl. Chem. 21, 269-270. (134) Matuszak, C. A., and Matuszak, A. J. (1976) Imidazole-versatile today, prominent tomorrow. J. Chem. Educ. 53, 280-284. (135) Wilkinson, C. F., Htnarski, K., Cantwell, G. P., and Di Carlo, F. J. (1974) Structure-activity relationships in the effects of 1-alkylimidazoles on microsomal oxidation in vitro and in vivo. Biochem. Pharmacol. 23, 2377-2386. (136) Ahmed, S., Smith, J. H., Nicholls, P. J., Whomsley, R., and Cariuk, P. (1995) Synthesis and biological evaluation of imidazole based compounds as cytochrome P-450 inhibitors. Drug Dis. Discov. 13, 27-41. (137) Halpert, J. R. (1995) Structural basis of selective cytochrome P450 inhibition. Annu. Rev. Pharmacol. Toxicol. 35, 29-53. (138) Schenkman, J. B., Sligar, S. G., and Cinti, D. L. (1982) Substrate interaction with cytochrome P450. In hepatic cytochrome P-450 monooxygenase system (Schenkman, J. B., and Kupfer, D., Ed.) pp 587-615, Pergamon Press, New York. (139) Ortiz de Montellano, P. R., and Correia, M. A. (1995) Inhibition of cytochrome P-450 enzymes. In Cytochrome P-450: Structure, mechanism and biochemistry (Ortiz de Montellano, P. R., Ed.) pp 305-364, Plenum Press, New York. (140) Rogerson, T. D., Wilkinson, C. F., and Hetarski, K. (1977) Steric factors in the inhibitory interaction of imidazoles with microsomal enzymes. Biochem. Pharmacol. 26, 1039-1042. (141) Tang, C., Chiba, M., Nishime, J., Hochman, J. H., Chen, I.-W., Williams, T. M., and Lin, J. H. (2000) Comparison of imidazoleand 2-methylimidazole-containing farnesyl protein transferase inhibitors: interaction with and metabolism by rat hepatic cytochrome P450s. Drug Metab. Dispos. 28, 680-686. (142) Rush, W. R., Hall, D. J., Graham, D. J. M., and Selby, I. A. (1992) The metabolism of imiloxan hydrochloride in healthy male volunteers. Xenobiotica 22, 237-246. (143) Mariaggi, N., Cornu, A., and Rinaldi, R. (1973) Investigation and identification of imidazole metabolites in the rat, using thinlayer chromatography, infrared spectroscopy, and mass spectrometry. Analysis 2, 485-488. (144) Brown, D. D., and Kies, M. W. (1959) Mammalian metabolism of L-histidine. II. Enzymatic formation, stabilization, purification, and properties of 4(5)-imidazolone-5(4)-propionic acid, the product of urocanase activity. J. Biol. Chem. 234, 3188-3191. (145) Cooper, T. A., Brinigar, W. S., and Wang, J. H. (1968) Characterization of the reaction intermediates in a model oxidative phosphorylation reaction. J. Biol. Chem. 243, 5854-5458. (146) Rush, W. R., Alexander, O. F., Hall, D. J., Dow, R. J., Tokes, L., Kurz, L., and Graham, D. J. M. (1990) The metabolism of nafimidone hydrochloride in the dog, primates, and man. Xenobiotica 20, 123-132. (147) Graham, D. J. M., Hama, K. M., Smith, S. A., Kurz, L., Chaplin, M. D., and Hall, D. J. (1987) Disposition of nafimidone in rats. Drug Metab. Dispos. 15, 565-570. (148) Midgley, I., Biggs, S. R., Hawkins, D. R., Chasseaud, L. F., Darragh, A., Brodie, R. R., and Walmsley, L. M. (1981) The metabolic fate of [3H]-econazole in man. Xenobiotica 11, 595608.

Invited Review (149) Needham, D., and Challis, I. R. (1991) The metabolism and excretion of prochloraz, an imidazole-based fungicide, in the rat. Xenobiotica 21, 1473-1482. (150) Laignelet, L., Riviere, J.-L., and Lhuguenot, J.-C. (1992) Metabolism of an imidazole fungicide (prochloraz) in the rat after oral administration. Food Chem. Toxicol. 30, 575-583. (151) Whitehouse, L. W., Menzie, A., Dawson, B., Cyr, T. D., By, A. W., Black, D. B., and Zamecnik, J. (1994) Mouse hepatic metabolites of ketoconazole: isolation and structure elucidation. J. Pharm. Biomed. Anal. 12, 1425-1441. (152) Testa, B. (1995) Mono-oxygenase catalyzed N-C cleavage. In The Metabolism of Drugs and Other Xenobiotics (Testa, B., and Caldwell, J., Eds.) pp 201-234, Academic Press, NY. (153) Bezek, S., Kukan, M., Kallay, Z., Trnovec, T., Stefek, M., and Piotrovskiy, L. B. (1990) Disposition of ethimizol, a xanthinerelated nootropic drug, in perfused rat liver and isolated hepatocytes. Drug Metab. Dispos. 18, 88-95. (154) Nakano, M., Takeuchi, M., Kawahara, S., and Mizojiri, K. (1990) Croconazole metabolism in rats. Xenobiotica 20, 385-393. (155) Nakano, M., Nakajima, Y., Iwatani, K., Ikenishi, Y., Nakagawa, Y., and Sugeno, K. (1989) Metabolism of the antimycotic agent, croconazole, in rabbits. Drug Metab. Dispos. 17, 323-329. (156) Vashishtha, S. C., Hawes, E. M., McKay, G., and McCann, D. J. (2000) Synthesis and identification of the quaternary ammonium-linked glucuronide of 1-phenylimidazole in human liver microsomes and investigation of the human UDP-glucuronosyltransferases involved. Drug Metab. Dispos. 28, 1009-1013. (157) Huskey, S. W., Doss, G. A., Miller, R. R., Schoen, W. R., and Chiu, S.-H. L. (1994) N-Glucuronidation reactions. II. Relative N-glucuronidation reactivity of methylbiphenyl tetrazole, methylbiphenyl triazole, and methylbiphenyl imidazole in rat, monkey, and human hepatic microsomes. Drug Metab. Dispos. 22, 651-658. (158) Huskey, S. W., Magdalou, J., Ouzzine, M., Siest, G., and Chiu, S.-H. L. (1994) N-Glucuronidation reactions. III. Regioselectivity of N-glucuronidation of methylbiphenyl tetrazole, methylbiphenyl triazole, and methylbiphenyl imidazole using human and rat recombinant UDP-glucuronosyltransferases stably expressed in V79 cells. Drug Metab. Dispos. 1994, 22, 659-662. (159) Wing, S., and Fantus, I. (1987) Adverse immunologic effects of antithyroid drugs. Can. Med. Assoc. J. 136, 121-127. (160) Bartalena, L., Bogazzi, F., and Martino, E. (1996) Adverse effects of thyroid hormone preparations and antithyroid drugs. Drug Safety 15, 53-63. (161) Martinez-Lopez, J. I., Greenberg, S. E., and Kling, R. R. (1962) Drug-induced hepatic injury during methimazole therapy. Gastroenterology 43, 84-87. (162) Vitug, A. C., and Goldman, J. M. (1985) Hepatotoxicity from antithyroid drugs. Horm. Res. 21, 229-234. (163) Mizutani, T., Murakami, M., Shirai, M., Tanaka, M., and Nakanishi, K. (1999) Metabolism-dependent hepatotoxicity of methimazole in mice depleted of glutathione. J. Appl. Toxicol. 19, 193-198. (164) Mizutani, T., Yoshida, K., Murakami, M., Shirai, M., Kawazoe, S. (2000) Evidence for the involvement of N-methylthiourea, a ring cleavage metabolite, in the hepatotoxicity of methimazole in glutathione-depleted mice: structure-toxicity and metabolic studies. Chem. Res. Toxicol. 13, 170-176. (165) Skellern, G. G., and Steer, S. T. (1981) The metabolism of [2-14C]methimazole in the rat. Xenobiotica 11, 627-634. (166) Kedderis, G. L., and Rickert, D. E. (1985) Loss of rat liver microsomal cytochrome P-450 during methimazole metabolism. Role of flavin-containing monooxygenase. Drug Metab. Dispos. 13, 58-61. (167) Neal, R. A., and Halpert, J. (1982) Toxicology of thiono-sulfur compounds. Annu. Rev. Pharmacol. Toxicol. 22, 321-339. (168) Poulsen, L. L., Hyslop, R. M., and Ziegler, D. M. (1979) S-Oxygenation of N-substituted thioureas catalyzed by the pig liver microsomal FAD-containing monooxygenase. Arch. Biochem. Biophys. 198, 78-88. (169) Cashman, J. R. (1995) Structural and catalytic properties of the mammalian flavin-containing monooxygenase. Chem. Res. Toxicol. 8, 165-181. (170) Ohta, K., Yamaguchi, J.-I., Akimoto, M., Fukushima, K., Suwa, T., and Awazu, S. (1996) Retention mechanism of imidazoles in connective tissue. I. Binding to elastin. Drug Metab. Dispos. 24, 1291-1297. (171) Ohta, K., Fukasawa, Y., Yamaguchi, J.-I., Akimoto, M., Kohno, Y., Fukushima, K., Suwa, T., and Awazu, S. (1998) Retention mechanism of imidazoles in connective tissue. III. Aldehyde adduct formation of a 4(5H) (or 5(4H))-imidazolone product in vitro. Biol. Pharm. Bull. 21, 958-963.

Chem. Res. Toxicol., Vol. 15, No. 3, 2002 297 (172) Ohta, K., Fukasawa, Y., Yamaguchi, J.-I., Kohno, Y., Fukushima, K., Suwa, T., and Awazu, S. (1998) Retention mechanism of imidazoles in connective tissue. IV. Identification of a nucleophilic imidazolone metabolite in rats. Biol. Pharm. Bull. 21, 1334-1337. (173) Yamagami, S., Kawasaki, E., and Egawa, A. (1993) Studies on the disposition of fadrazole hydrochloride (CGS 16949A). II. Absorption, distribution, metabolism and excretion after repeated oral administration to rats. Xenobiot. Metab. Dispos. 8, 1129-1146. (174) Jacqmin, P., Herin, M., Cavalier, R., and Lesne, M. (1993) Irreversible binding to proteins after single and repeated daily oral administration of 4-(2,2-diphenylethyl)imidazole (SC-46264) to the cynomolgus monkey. Eur. J. Drug Metab. Pharmacokinet. 18, 299-307. (175) Dicullo, C. J., Miller, J. A., Mendelson, W. L., and Pagano, J. F. (1974) Metabolic and tissue residue studies on parbendazole in sheep. J. Agri. Food Chem. 22, 948-953. (176) Douch, P. G. (1973) The metabolism of benomyl fungicide in mammals. Xenobiotica 3, 367-380. (177) Tocco, D. J., Roseblum, C., Martin, C. M., and Robinson, H. J. (1966) Toxicol. Appl. Pharmacol. 9, 31-39. (178) Tyler, T. L., Lee, J. T., Flynn, H., and Vandenheuvel, W. J. A. (1976) Identification of 5-hydroxybenzimidazole as a major urinary metabolite of benzimidazole in the rat. Drug Metab. Dispos. 4, 177-179. (179) Ogunbiyi, O., Kabaf, M., Lamb, J. H., Jahanshahi, M., Gorrod, J. W., and Naylor, S. (1995) Characterization of 2-amino-1benzylimidazole and its metabolites using tandem mass spectrometry. Toxicol. Lett. 78, 25-33. (180) A ¨ belo¨, A., Andersson, T. B., Bredberg, U., Skåsnberg, I., and Weidolf, L. (2000) Stereoselective metabolism by human liver CYP enzymes of a substituted benzimidazole. Drug. Metab. Dispos. 28, 58-64. (181) Bray, H. G., Clowes, R. C., and Thorpe, W. V. (1952) Metabolism of aminophenols, o-formamidophenol, benzoxazole, 2-methyl- and 2-phenylbenzoxazoles, and benzoxazolone in the rabbit. Biochem. J. 51, 70-78. (182) Marchetti, E., Mattalia, G., and Bergesi, G. (1973) Ditazol metabolism in rat, rabbit, and man. Arzneim-Forsch. (Drug Res.) 23, 1291-1295. (183) Maurer, H., and Kleff, I. (1988) On the metabolism of ditazole in man. Arzneim-Forsch. (Drug Res.) 38, 1843-1845. (184) Van Der Graaff, M., Vermeulen, N. P. E., Crul, I. E. P., and Breimer, D. D. (1986) Dose dependent pharmacokinetics of zoxazolamine in the rat. Drug Metab. Dispos. 14, 331-335. (185) Chatfield, D. H., Green, J. N., Kao, J. C., Ross, W. J., and Woodage, T. J. (1979) The metabolism of isomoxole in the rat and guinea pig. Xenobiotica 9, 585-594. (186) Mizutani, T., and Suzuki, K. (1996) Realtive hepatotoxicity of 2-(substituted phenyl)thiazoles and substituted thiobenzamides in mice: evidence for the involvement of thiobenzamides as ring cleavage metabolites in the hepatotoxicity of 2-phenylthiazoles. Toxicol. Lett. 85, 101-105. (187) Mizutani, T., Yoshida, K., and Kawazoe, S. (1994) Formation of toxic metabolites from thiabendazole and other thiazoles in mice: Identification of thioamides as ring cleavage products. Drug Metab. Dispos. 22, 750-755. (188) Mizutani, T., Ito, K., Nomura, H., and Nakanishi, K. (1990) Nephrotoxicity of thiabendazole in mice depleted of glutathione by treatment with DL-buthionine sulfoximine. Food Chem. Toxicol. 28, 169-177. (189) Mizutani, T., Yoshida, K., and Ito, K. (1992) Nephrotoxicity of thiazoles strucutrally related to thiabendazole in mice depleted of glutathione by treatment with buthionine sulfoxoimine. Res. Commun. Chem. Pathol. Pharmacol. 75, 29-38. (190) Mizutani, T., Yoshida, K., and Kawazoe, S. (1993) Possible role of thioformamide as a proximate toxicant in the nephrotoxicity of thiabendazole and related thiazoles in glutathione-depleted mice: Structure-toxicity and metabolic studies. Chem. Res. Toxicol. 6, 174-179. (191) Coulet, M., Eeckhoutte, C., Larrieu, G., Sutrra, J.-F., Alvinerie, M., Mace, K., Pfiefer, A., Zucco, F., Stammati, A. L., De Angelis, I., Vignoli, A. L., and Galtier, P. (2000) Evidence for cytochrome P4501A2-mediated protein covalent binding of thiabendazole and for its passive intestinal transport: use of human and rabbit derived cells. Chem.-Biol. Interact. 127, 109-124. (192) Yabuki, M., Shimakura, J., Ito, M., Kanamura, H., Iba, K., and Nakatasuka, I. (1997) Metabolism of 4-[1-(2-fluoro-4-biphenyl)ethyl]-2-methylaminothiazole (SM-8849), in rats. Eur. J. Drug Metab. Dispos. Pharmacokinet. 22, 25-33.

298


(193) Chatfield, D. H., and Hunter, W. H. (1973) The metabolism of acetamidothiazoles in the rat: 2-acetamido-, 2-acatamido-4methyl- and 2-acetamido-4-phenylthiazole. Biochem. J. 134, 869-878. (194) Yoon, W. H., Yoo, J. K., Lee, J. W., Shim, C.-K., and Lee, M. G. (1998) Species differences in pharmacokinetics of a hepatoprotective agent, YH-439, and its metabolites M4, M5, and M7, after intravenous and oral administration to rats, rabbits and dogs. Drug Metab. Dispos. 26, 152-163. (195) Hobbs, D. C., and Twomey, T. M. (1977) Metabolism of sudoxicam by the rat, dog and monkey. Drug Metab. Dispos. 5, 7581. (196) Schmid, J., Busch, U., Trummlitz, G., Prox, A., Kaschke, S., and Wachsmuth, H. (1995) Meloxicam: Metabolic profile and biotransformation products in the rat. Xenobiotica 25, 1219-1236. (197) Wilson, K., Chissick, H., Fowler, A. M., Frearson, F. J., Gittins, M., and Swinbourne, F. J. (1991) Metabolism of benzothiazole. I. Identification of ring cleavage products. Xenobiotica 21, 11791183. (198) Herbertz, G., and Reinauer, H. (1971) Metabolism of chlormethiazol in the rat. Nanuyn-Schmiedebergs Arch. Pharmakol. 270, 192-202. (199) Herbertz, G., Metz, T., Reinauer, H., and Staib, W. (1973) Metabolism of chlormethiazol in the perfused liver of the rat. Biochem. Pharmacol. 22, 1541-1546. (200) Offen, C. P., Frearson, M. J., Wilson, K., and Burnett, D. (1985) 4,5-dimethylthiazole-N-oxide-S-oxide: a metabolite of chlormethiazole in man. Xenobiotica 15, 503-511. (201) Grupe, A., and Spiteller, G. (1982) Unexpected metabolites produced from chlormethiazole. J. Chromatogr. 230, 335-344. (202) Trimble, L. A., Chauret, N., Silva, J. M., Nicoll-Griffith, D. A., Li, C.-S., and Yergey, J. A. (1996) Characterization of the in vitro oxidative metabolites of the COX-2 selective inhibitor L-766,112. Bioorg. Med. Chem. Lett. 7, 53-56. (203) Colucci, D. F., and Buyske, D. A. (1965) The biotransformation of a sulphonamide to a mercaptan and to a mercapturic acid and glucuronide conjugates. Biochem. Pharmacol. 14, 457-466. (204) Conroy, C. W., Schwann, H., and Maren, T. W. (1984) The nonenzymatic displacement of the sulfamoyl group from different classes of aromatic compounds by glutathione and cysteine. Drug Metab. Dispos. 12, 614-618. (205) Fukuoka, M., Satoh, M., and Tanaka, A. (1995) Metabolism of 2-thiobenzothiazoles in the rat. Arch. Toxicol. 70, 1-7. (206) Clejan, L. A., and Cederbaum, A. I. (1990) Comparison of the interaction of pyrazole and its metabolite 4-hydroxypyrazole with rat liver microsomes. Drug Metab. Dispos. 18, 393-397. (207) Clejan, L. A., and Cederbaum, A. I. (1990) Oxidation of pyrazole by reconstituted systems containing cytochrome P450 2E1. Biochim. Biophys. Acta 1034, 233-237. (208) Puntarulo, S., and Cederbaum, A. I. (1988) Oxidation of pyrazole to 4-hydroxypyrazole in intact rat hepatocytes. Biochem. Pharmacol. 37, 1555-1561. (209) Feierman, D. E., and Cederbaum, A. I. (1987) Oxidation of the alcohol dehydrogenase inhibitor pyrazole to 4-hydroxypyrazole by microsomes. Effect of cytochrome P-450 inducing agents. Drug Metab. Dispos. 15, 634-639. (210) Deis, F. H., Grace, W., and Lester, D. (1977) Metabolism of pyrazole in rat. In Alcohol Aldehyde Metabolizing Systems (Thuman, R. G., Ed.) 3, 399-405. (211) Clay, K. L., Watkins, W. D., and Murphy, R. C. (1977) Metabolism of pyrazole. Structure elucidation of urinary metabolites. Drug Metab. Dispos. 5, 149-156. (212) Baukema, J., Okerholm, R. A., and Glazko, A. J. (1975) Comparative metabolism of zolazepam in rat, dog, and monkey. Res. Comm. Chem. Path. Pharmacol. 10, 227-238. (213) Zuleski, F. R., Kirkland, K. M., Melgar, M. D., and Malbica, J. O. (1985) Tracazolate metabolites in rat tissue. Drug Metab. Dispos. 13, 139-147. (214) Heald, A. F., Dizio, D. P., Kirkland, K. M., Loftus, P., and Malbica, J. O. (1986) Metabolism of tracazolate. Metabolites in dog and rat urine. Drug Metab. Dispos. 14, 631-636. (215) Kripalani, K. J., Dreyfuss, J., Nerhec, J., Cohen, A. I., Meeker, F., and Egli, P. (1981) Biotransformation in the monkey of cartazolate (SQ 65,396), a substituted pyrazolopyridine having anxiolytic activity. Xenobiotica 11, 481-488. (216) Kataoka, S., Tiara, K., and Takabatake, E. (1971) Metabolism of benzydamine hydrochloride. Chem. Pharm. Bull. 19, 15111513. (217) Kataoka, S., Tiara, K., Ariyoshi, T., and Takabatake, E. (1973) Metabolism of benzydamine hydrochloride. Species differences and the identification of unconjugated metabolites in rabbit urine. Chem. Pharm. Bull. 21, 358-365.

Dalvie et al. (218) Kataoka, S., Nishimura, K., and Naito, T. (1979) In vivo metabolism and antiinflammatory activity of benzydamine hydrochloride in rats treated with carrageenin. Chem. Pharm. Bull. 27, 2890-2903. (219) Koppel, C., and Tenczer, J. (1985) Metabolism of benzydamine Arzneim. Forsch. 35, 634-635. (220) Clarke, S. E., Austin, N. E., Bloomer, J. C., Haddock, R. E., Higham, F. C., Hollis, F. J., Nash, M., Shardlow, P. C., Tasker, T. C., Woods, F. R., and Allen, E. D. (1994) Metabolism and disposition of 14C-granisetron in rat, dog and man after intravenous and oral dosing. Xenobiotica 24, 1119-1131. (221) Descotes, G., Osman, H., Gallas, J. F., Penacchio, E., and Moreau, C. (1996) Functional, biochemical, and morphological hepatobiliary effects in rats chronically exposed to a steroidal antiandrogen. Toxicol. Appl. Pharmacol. 137, 23-33. (222) Kodama, R., and Takabatake, E. (1972) Metabolism of mepirizole. Isolation of a new metabolite from dog urine and species differences in urinary metabolites. Xenobiotica 2, 479-488. (223) Takabatake, E., Kodama, R., Tanaka, Y., Dohmori, R., Tachizawa, H., and Naito, T. (1970) Metabolic fate of 1-(4-methoxy6-methyl-2-pyrimidinyl)-3-methyl-5-methoxypyrazole (mepirizole, DA-398) in rats and rabbits. Chem. Pharm. Bull. 18, 19001907. (224) Stiff, D. D., and Zemaitis, M. A. (1990) Metabolism of the anticonvulsant agent zonisamide in the rat. Drug Metab. Dispos. 18, 888-894. (225) Stiff, D. D., Robicheau, J. T., and Zemaitis, M. A. (1992) Reductive metabolism of the anticonvulsant agent zonisamide, a 1,2-benzisoxazole derivative. Xenobiotica 22, 1-11. (226) Nakasa, H., Komiya, M., Ohmori, S., Rikihisa, T., Kiuchi, M., and Kitada, M. (1993) Characterization of human liver microsomal cytochrome P450 involved in the reductive metabolism of zonisamide. Mol. Pharmacol. 44 216-220. (227) Nakasa, H., Komiya, M., Ohmori, S., Kitada, M., Rikihisa, T., and Kanakubo, Y. (1992) Formation of reductive metabolite, 2-sulfamoylacetylphenol, from zonisamide in rat liver microsomes. Res. Comm. Chem. Path. Pharmacol. 77, 31-41. (228) Nakasa, H., Komiya, M., Ohmori, S., Rikihisa, T., and Kitada, M. (1993) Rat liver microsomal cytochrome P-450 responsible for reductive metabolism of zonisamide. Drug Metab. Dispos. 21, 777-781. (229) Nakasa, H., Ohmori, S., and Kitada, M. (1996) Formation of 2-sulfamoylacetylphenol from zonisamide under aerobic conditions in rat liver microsomes. Xenobiotica 26, 495-501. (230) Sugihara, K., Katamuram S., and Tatsumi, K. (1996) Involvement of mammalian liver cytosols and aldehyde oxidase in reductive metabolism of zonisamide Drug Metab. Dispos. 24, 199-202. (231) Mannens, G., Huang, M.-L., Meudermans, W., Hendrickx, J., Woestenborghs, R., and Heykants, J. (1993) Absorption, metabolism, and excretion of risperidone in humans. Drug Metab. Dispos. 21, 1134-1141. (232) Meudermans, W., Hendricks, J., Manneris, G., Lavnjsen, K., Janssen, C., Bracke, J., LeJeune, L., Lauwers, W., and Heykants, J. (1994) The metabolism and excretion of risperidone after oral administration in rats and dogs. Drug Metab. Dispos. 22, 129138. (233) Mutlib, A. E., Strupczewski, J. T., and Chesson, S. M. (1995) Application of hyphenated LC/NMR and LC/MS techniques in rapid identification of in vitro and in vivo metabolites of iloperidone. Drug Metab. Dispos. 23, 951-964. (234) Davison, C., Banks, W., and Fritz, A. (1976) The absorption, distribution and metabolic fate of Danazol in rats, monkeys and human volunteers. Arch. Int. Pharmacodyn. 221, 294-310. (235) Prakash, A., and Jarvis, B. (1999) Leflunomide: a review of its use in active rheumatoid arthritis. Drugs 58, 1137-1164. (236) Prakash, C., Kamel, A., Gummerus, J., and Wilner, K. (1997) Metabolism and excretion of a new antipsychotic drug, ziprasidone, in humans. Drug Metab. Dispos. 25, 863-872. (237) Prakash, C., Kamel, A., and Cui, D. (1997) Characterization of the novel benzisothiazole ring-cleaved products of the antipsychotic drug ziprasidone. Drug Metab. Dispos. 25, 897-901. (238) Prakash, C., Kamel, A., Anderson, W., and Howard, H. (1997) Metabolism and excretion of the novel antipsychotic drug ziprasidone in rats after oral administration of a mixture of 14Cand 3H-labeled ziprasidone. Drug Metab. Dispos. 25, 206-218. (239) Jajoo, H. K., Blair, I. A., Klunk, L. J., and Mayol, R. F. (1990) Characterization of in vitro metabolites of the antipsychotic drug tiospirone by mass spectrometry. Biomed. Environ. Mass Spec. 19, 281-285. (240) Mayol, R. F., Jajoo, H. K., Klunk, L. J., and Blair, I. A. (1991) Metabolism of the antipsychotic drug tiospirone in humans. Drug Metab. Dispos. 19, 394-399.

Invited Review (241) Venkatakrishnan, K., von Moltke, L. L., and Greenblatt, D. J. (2000) Effects of the antifungal agents on oxidative drug metabolism: clinical relevance. Clin. Pharmacokinet. 38, 111180. (242) Wienkers, L. C., Wurden, C. J., Storch, E., Kunze, K. L., Rettie, A. E., and Trager, W. F. (1996) Formation of (R)-8-hydroxywarfarin in human liver microsomes. A new metabolic marker for the (S)-mephenytoin hydroxylase, P4502C19. Drug Metab. Dispos. 24, 610-614. (243) Ortiz de Montellano, P. R., and Mathews, J. M. (1981) Autocatalytic alkylation of the cytochrome P450 prosthetic haem group by 1-aminobenzotriazole. Biochem. J. 195, 761-764. (244) Ortiz de Montellano, P. R., Mico, B. A., Mathews, J. M., Kunze, K. L., Miwa, G. T., and Lu, A. Y. H. (1981) Selective inactivation of cytochrome P450 isozymes by suicide substrates. Arch. Biochem. Biophys. 210, 717-728. (245) Mathews, J. M., Dostal, L. A., and Bend, J. R. (1985) Inactivation of rabbit pulmonary cytochrome P450 in microsomes and isolated perfused lungs by the suicide substrate 1-aminobenzotriazole. J. Pharmacol. Exp. Ther. 235, 186-190. (246) Woodcroft, K. J., Szczepan, E. W., Knickle, L. C., and Bend, J. R. (1990) Three N-aralkylated derivatives of 1-aminobenzotriazole as potent and isozyme-selective, mechanism-based inhibitors of guinea pig pulmonary cytohrome P450 in vitro. Drug Metab. Dispos. 18, 1031-1037. (247) Mathews, J. M., and Bend, J. R. (1993) N-Araalkyl derivatives of 1-aminobenzotriazole as potent isozyme-selective mechanism based inhibitors of rabbit pulmonary cytochrome P450 in vivo. J. Pharmacol. Exp. Ther. 265, 281-285. (248) Ortiz de Montellano, P. R., Mathews, J. M., and Langry, K. C. (1984) Autocatalytic inactivation of cytochrome P450 and chloroperoxidase by 1-aminobenzotriazole and other aryne precursors. Tetrahedron 40, 511-519. (249) Mathews, J. M., and Bend, J. R. (1986) N-Alkylaminobenzotriazoles as isozyme-selective suicide inhibitors of rabbit pulmonary microsomal cytochrome P450. Mol. pharmacol. 30, 25-32. (250) Woodcroft, K. J., Webb, C. D., Yao, M., Weedon, A. C., and Bend, J. R. (1997) Metabolism of the cytochrome P450 mechanism based inhibitor N-benzyl-1-aminobenzotriazole to products that covalently bind with protein in the guinea pig liver and lung microsomes: Comparative study with 1-aminobenzotriazole. Chem. Res. Toxicol. 10, 589-599. (251) Machinist, J. M., Bopp, B. A., Anderson, D. J., Granneman, G. R., Sonders, R. C., Tolman, K., Buchi, K., and Rollins, D. (1986) Metabolism of 14C-estazolam in dogs and humans. Xenobiotica 16, 11-20. (252) Rudolf, B., and Werner, T. (1976) Metabolism and fate of triazphos in rats. Pestic. Sci. 7, 307-314. (253) Saunders, J., Cassidy, M., Freedman, S. B., Harley, E. A., Iversen, L. L., Kneen, C., MacLeod, A. M., Merchant, K. J., Snow, R. J., and Baker, R. (1990) Novel quinuclidine-based ligands for the muscarinic cholinergic receptor. J. Med. Chem. 33, 11281138. (254) Swain, C. J., Baker, R., Kneen, C., Moseley, J., Saunders, J., Seward, E. M., Stevenson, G., Beer, M., Stanton, J., and Watling, K. (1991) Novel 5-HT3 antagonists. Indole oxadiazoles. J. Med. Chem. 34, 140-151. (255) Lipinski, C. A., Fiese, E. F., and Korst, R. J. (1991) pKa, Log P and MedChem CLOGP fragment values of acidic heterocyclic potential bioisosteres. Quant. Struct-Act. Relat. 10, 109-117. (256) Diana, G. D., Rudewicz, P., Pevear, D. C., Nitz, T. J., Aldous, S. C., Aldous, D. J., Robinson, D. T., Draper, T. and Dutko, F. J. (1995) Picornavirus inhibitors: trifluoromethyl substitution provides a global protective effect against hepatic metabolism. J. Med. Chem. 38, 1355-1371. (257) Harfenist, M., Heuser, D. J., Joyner, C. T., Batchelor, J. F., and White, H. L. (1996) Selective inhibitors of monoamine oxidase. 3. Structure-activity relationship of tricyclics bearing imidazoline, oxadiazole, or tetrazole groups. J. Med. Chem. 39, 18571863. (258) Gaster, L. M., Blaney, F. E., Duckworth, D. M., Ham, P., Jenkins, S., Jennings, A. J., Joiner, G. F., King, F. D., Mulholland, K. R., Starr, S., Wyman, P. A., Hagan, J. J., Hatcher, J., Jones, B. J., Middlemiss, D. N., Price, G. W., Riley, G., Roberts, C., Routledge, C., Selkirk, J., and Slade, P. D. (1998) The selective 5-HT1B receptor inverse agonist 1′-methyl-5-[[2′-methyl-4′- (5-methyl1,2,4-oxadiazol-3-yl)biphenyl-4-yl]carbonyl]-2,3,6,7-tetrahydrospiro[furo[2,3-f]indole-3,4′-piperidine] (SB-224289) potently blocks terminal 5-HT autoreceptor function both in vitro and in vivo. J. Med. Chem. 41, 1218-1235. (259) Cerecetto, H., Di Maio, R., Gonzalez, M., Risso, M., Saenz, P., Seoane, G., Denicola, A., Peluffo, G., Quijano, C., and Olea-Azar, C. (1999) 1,2,5-Oxadiazole N-oxide derivatives and related


(260)

(261)

(262)

(263)

(264) (265)

(266)

(267)

(268)

(269)

(270)

(271)

(272)

(273)

(274)

(275)

(276)

(277)

(278)

compounds as potential antitrypanosomal drugs: structureactivity relationships. J. Med. Chem. 42, 1941-1950. Allen, J. G., Blackburn, M. J., and Caldwell, S. M. (1971) Metabolism by man of an anticonvulsant oxadiazole. Unusual metabolic route for tertiary amides. Xenobiotica 1, 3-12. Lan, S. J., Weliky, I., and Schreiber, E. C. (1973) Metabolic studies with trans-5-amino-3-[2-(5-nitro-2-furyl)vinyl]-1,2,4[514C]oxadiazole (SQ 18,506). 1. Reductive cleavage of the 1,2,4oxadiazole ring. Xenobiotica 3, 97-102. Speed, W., Parton, A. H., Martin, I. J., and Howard, M. R. (1994) The use of liquid chromatography/thermospray mass spectrometry with online ultraviolet diode array and radiochemical detection: characterization of the putative metabolites of U-78875 in female rat feces. Biol. Mass Spectrom. 23, 1-5. Yabuki, M., Shono, F., Nakatsuka, I., and Yoshitake, A. (1993) Novel cleavage of the 1,2,4-oxadiazole ring in rat metabolism of SM-6586, a dihydropyridine calcium antagonist. Drug Metab. Dispos. 21, 1167-1169. Koch, P., Hirst, D. R., and von Wartburg, B. R. (1989) Biological fate of sirdalud in animals and man. Xenobiotica 19, 1255-1265. Van Welie, R. T. H., Mensert, R., Van Duyn, P., and Vermeulen, N. P. E. (1991) Identification and quantitative determination of a carboxylic and a mercapturic acid metabolite of etridiazole in urine of rat and man. Potential tools for biological monitoring. Arch. Toxicol. 65, 625-632s. Babu, R. G., and Vaz, A. D. N. (1997) 1,2,3-Thiadiazoles: A novel heterocyclic heme ligand for the design of cytochrome P450 inhibitors. Biochemistry 36, 7209-7216. Butler, R. N. (1984) Tetrazoles. In Comprehensive Heterocyclic Chemistry: The Structure, Reactions, Synthesis, and Uses of Heterocyclic Compounds (Katritzky, A. R., and Rees, C. W., Eds.) Vol. 5, 791-838. Nohara, A. (1980) Clinically effective 6-ethyl-3-(1H-tetrazol-5yl)chromone, (AA-344). American Chemical Society Symposium Series 118, Drugs Affecting Respiratory Systems, pp 125-144, American Chemical Society (ACS), Washington, DC. Stearns, R. A., Doss, G. A., Miller, R. R., and Chiu, S.-H. L. (1991) Synthesis and identification of a novel tetrazole metabolite of the angiotensin II receptor antagonist DuP 753. Drug Metab. Dispos. 19, 1160-1162. Stearns, R. A., Miller, R. R., Doss, G. A., Chakravarty, P. K., Rosegay, A., Gatto, G. J., and Chiu, S. H. (1992) The metabolism of DuP 753, a nonpeptide angiotensin II receptor antagonist by rat, monkey, and human liver slices. Drug Metab. Dispos. 20, 281-287. Rios, G. R., Green, M. D., Schuetz, E., and Tephly, T. R. (1998) Metabolism of losartan by polymorphic human UDP-glucuronosyltransferase (UGT)2B7. FASEB J. 5573, A962. Perrier, L., Bourrie´, M., Marti, E., Trounquet, C., Masse´, D., Berger, Y., Magdalou, J., and Fabre, G. (1994) In vitro N-glucuronidation of SR-47436 (BMS 186295), a new AT1 nonpeptide angiotensin II receptor antagonist, by rat, monkey and human microsomal fractions. J. Pharmacol. Exp. Ther. 271, 91-99. Huskey, S.-E., Miller, R. R., and Chiu, S.-H. L. (1993) NGlucuronidation reactions. I. Tetrazole N-glucuronidation of selected angiotensin II receptor antagonists in hepatic microsomes from rats, dogs, monkeys and humans. Drug. Metab. Dispos. 21, 792-799. Stevens, J. C., Fayer, J. L., and Cassidy, K. C. (2001) Characterization of 2-[[4-[[2-(1H-tetrazol-5-ylmethyl)phenyl]-methoxy]phenoxy]methyl]quinoline N-glucuronidation by in vitro and in vivo approaches. Drug Metab. Dispos. 29, 289-295. Davi, H., Tronquet, C., Miscoria, G., Perrier, L., DuPont, P., Caix, J., Simiand, J., and Berger, Y. (2000) Disposition of irbesartan, an angiotensin II AT1-receptor antagonist in mice, rats, rabbits, and macaques. Drug Metab. Dispos. 28, 79-88. Chando, T. J., Everett, D. W., Kahle, A. D., Starrett, A. M., Vachharajani, N., Shyu, W. C., Kripalani, K. J., and Barbhaiya, R. H. (1998) Biotransformation of ibesartan in man. Drug Metab. Dispos. 26, 408-417. Zhang, K. E., and Vyas, K. P. (1995) Disposition of DMP 811 (L-708,404), a potent angiotensin II receptor antagonist, in the rat, monkey, and chimpanzees. Drug Metab. Dispos. 23, 11041109. Krieter, P. A., Colletti, A. E., Miller, R. R., and Stearns, R. A. (1995) Absorption and glucuronidation of the angiotensin II receptor antagonist losartan by the rat intestine. J. Pharmacol. Exp. Ther. 273, 816-822.

TX015574B

Biotransformation Reactions of Five-Membered Aromatic Heterocyclic

Recommend Documents