Bioactivation potential of thiophene-containing drugs.

Thiophene is a five-membered, sulfur-containing heteroaromatic ring commonly used as a building block in drugs. It is considered to be a structural alert, as its metabolism can lead to the formation of reactive metabolites. Thiophene S-oxides and thiophene epoxides are highly reactive electrophilic thiophene metabolites whose formation is cytochrome P450-dependent. These reactive thiophene-based metabolites are quite often responsible for drug-induced hepatotoxicity. Tienilic acid is an example of a thiophene-based drug that was withdrawn from the market after only a few months of use, due to severe cases of immune hepatitis. However, inclusion of the thiophene moiety in drugs does not necessarily result in toxic effects. The presence of other, less toxic metabolic pathways, as well as an effective detoxification system in our body, protects us from the bioactivation potential of the thiophene ring. Thus, the presence of a structural alert itself is insufficient to predict a compound's toxicity. The question therefore arises as to which factors significantly influence the toxicity of thiophene-containing drugs. There is no easy way to answer this question. However, the findings presented here indicate that, for a number of reasons, daily dose and alternative metabolic pathways are important factors when predicting toxicity and will therefore be discussed together with examples.

Differential oxidation of two thiophene-containing regioisomers to reactive metabolites by cytochrome P450 2C9.

The uricosuric diuretic agent tienilic acid (TA) is a thiophene-containing compound that is metabolized by P450 2C9 to 5-OH-TA. A reactive metabolite of TA also forms a covalent adduct to P450 2C9 that inactivates the enzyme and initiates immune-mediated hepatic injury in humans, purportedly through a thiophene-S-oxide intermediate. The 3-thenoyl regioisomer of TA, tienilic acid isomer (TAI), is chemically very similar and is reported to be oxidized by P450 2C9 to a thiophene-S-oxide, yet it is not a mechanism-based inactivator (MBI) of P450 2C9 and is reported to be an intrinsic hepatotoxin in rats. The goal of the work presented in this article was to identify the reactive metabolites of TA and TAI by the characterization of products derived from P450 2C9-mediated oxidation. In addition, in silico approaches were used to better understand both the mechanisms of oxidation of TA and TAI and/or the structural rearrangements of oxidized thiophene compounds. Incubation of TA with P450 2C9 and NADPH yielded the well-characterized 5-OH-TA metabolite as the major product. However, contrary to previous reports, it was found that TAI was oxidized to two different types of reactive intermediates that ultimately lead to two types of products, a pair of hydroxythiophene/thiolactone tautomers and an S-oxide dimer. Both TA and TAI incorporated ¹8O from ¹8O2 into their respective hydroxythiophene/thiolactone metabolites indicating that these products are derived from an arene oxide pathway. Intrinsic reaction coordinate calculations of the rearrangement reactions of the model compound 2-acetylthiophene-S-oxide showed that a 1,5-oxygen migration mechanism is energetically unfavorable and does not yield the 5-OH product but instead yields a six-membered oxathiine ring. Therefore, arene oxide formation and subsequent NIH-shift rearrangement remains the favored mechanism for formation of 5-OH-TA. This also implicates the arene oxide as the initiating factor in TA induced liver injury via covalent modification of P450 2C9. Finally, in silico modeling of P450 2C9 active site ligand interactions with TA using the catalytically active iron-oxo species revealed significant differences in the orientations of TA and TAI in the active site, which correlated well with experimental results showing that TA was oxidized only to a ring carbon hydroxylated product, whereas TAI formed both ring carbon hydroxylated products and an S-oxide.

Comparative NMR-based metabonomic investigation of the metabolic phenotype associated with tienilic acid and tienilic acid isomer.

An NMR-based metabonomic approach was applied to study the systems level metabolic effects of two closely related thiophene compounds, tienilic acid (TA) and tienilic acid isomer (TAI). The metabonomic data were anchored with traditional clinical chemistry and histopathologic analyses. TA was removed from the market as a result of suspected immune-mediated hepatotoxicity, whereas TAI is an intrinsic hepatotoxin. Equimolar doses of TA and TAI were administered to Sprague-Dawley rats, and sampling was conducted at 2, 6, and 24 h post-treatment. Histopathologic analyses revealed development of a significant hepatic lesion 24 h post-TAI treatment with a parallel increase in plasma alanine aminotransferase (ALT) activity. In contrast, TA was not associated with the development of a hepatic lesion or an increase in plasma ALT activity. High-resolution NMR spectral metabolic profiles were generated for liver extracts, plasma, and urine at multiple time points. Multivariate statistical tools were applied to model the metabolic profiles and identify discriminatory metabolites that reflected both the adaptation to TA administration and the onset and progression of TAI-induced hepatotoxicity. TAI was shown to induce marked metabolic effects on the metabolome at all time points, with dramatic metabolic perturbations at 24 h post-treatment correlating with the histopathologic and clinical chemistry evidence of a hepatic lesion. The TAI-induced metabolic perturbations provided evidence for the generation of electrophilic reactive metabolites and a significant impairment of bioenergetic metabolic pathways. TA induced early metabolic perturbations that were largely resolved by 24 h post-treatment, suggesting the reestablishment of metabolic homeostasis and the ability to adapt to the intervention, with hepatic hypotaurine potentially representing a means of assessment of hepatic adaptation. This comparative metabonomic approach enabled the discrimination of metabolic perturbations that were common to both treatments and were interpreted as nontoxic thiophene-induced perturbations. Importantly, this approach enabled the identification of temporal metabolic perturbations that were unique to TAI or TA treatment and hence were of relevance to the development of toxicity or the ability to adapt. This approach is applicable to the future study of pharmacologically and structurally similar compounds and represents a refined means of identification of biomarkers of toxicity.

Identification of protein targets of reactive metabolites of tienilic acid in human hepatocytes.

Tienilic acid (TA) is a uricosuric diuretic that was withdrawn from the market only months after its introduction because of reports of serious incidents of drug-induced liver injury including some fatalities. Its hepatotoxicity is considered to be primarily immunoallergic in nature. Like other thiophene compounds, TA undergoes biotransformation to a S-oxide metabolite which then reacts covalently with cellular proteins. To identify protein targets of TA metabolites, we incubated [(14)C]-TA with human hepatocytes, separated cellular proteins by 2D gel electrophoresis, and analyzed proteins in 36 radioactive spots by tryptic digestion followed by LC-MS/MS. Thirty-one spots contained at least one identifiable protein. Sixteen spots contained only one of 14 nonredundant proteins which were thus considered to be targets of TA metabolites. Six of the 14 were also found in other radioactive spots that contained from 1 to 3 additional proteins. Eight of the 14 had not been reported to be targets for any reactive metabolite other than TA. The other 15 spots each contained from 2 to 4 identifiable proteins, many of which are known targets of other chemically reactive metabolites, but since adducted peptides were not observed, the identity of the adducted protein(s) in these spots is ambiguous. Interestingly, all the radioactive spots corresponded to proteins of low abundance, while many highly abundant proteins in the mixture showed no radioactivity. Furthermore, of approximately 16 previously reported protein targets of TA in rat liver ( Methogo, R., Dansette, P., and Klarskov, K. ( 2007 ) Int. J. Mass Spectrom. , 268 , 284 -295 ), only one (fumarylacetoacetase) is among the 14 targets identified in this work. One reason for this difference may be statistical, given that each study identified a small number of targets from among thousands present in hepatocytes. Another may be the species difference (i.e., rat vs human), and still another may be the method of detection of adducted proteins (i.e., Western blot vs C-14). Knowledge of human target proteins is very limited. Of more than 350 known protein targets of reactive metabolites, only 42 are known from humans, and only 21 of these are known to be targets for more than one chemical. Nevertheless, the demonstration that human target proteins can be identified using isolated hepatocytes in vitro should enable the question of species differences to be addressed more fully in the future.

Can in vitro metabolism-dependent covalent binding data in liver microsomes distinguish hepatotoxic from nonhepatotoxic drugs? An analysis of 18 drugs with consideration of intrinsic clearance and daily dose.

In vitro covalent binding assessments of drugs have been useful in providing retrospective insights into the association between drug metabolism and a resulting toxicological response. On the basis of these studies, it has been advocated that in vitro covalent binding to liver microsomal proteins in the presence and the absence of NADPH be used routinely to screen drug candidates. However, the utility of this approach in predicting toxicities of drug candidates accurately remains an unanswered question. Importantly, the years of research that have been invested in understanding metabolic bioactivation and covalent binding and its potential role in toxicity have focused only on those compounds that demonstrate toxicity. Investigations have not frequently queried whether in vitro covalent binding could be observed with drugs with good safety records. Eighteen drugs (nine hepatotoxins and nine nonhepatotoxins in humans) were assessed for in vitro covalent binding in NADPH-supplemented human liver microsomes. Of the two sets of nine drugs, seven in each set were shown to undergo some degree of covalent binding. Among hepatotoxic drugs, acetaminophen, carbamazepine, diclofenac, indomethacin, nefazodone, sudoxicam, and tienilic acid demonstrated covalent binding, while benoxaprofen and felbamate did not. Of the nonhepatotoxic drugs evaluated, buspirone, diphenhydramine, meloxicam, paroxetine, propranolol, raloxifene, and simvastatin demonstrated covalent binding, while ibuprofen and theophylline did not. A quantitative comparison of covalent binding in vitro intrinsic clearance did not separate the two groups of compounds, and in fact, paroxetine, a nonhepatotoxin, showed the greatest amount of covalent binding in microsomes. Including factors such as the fraction of total metabolism comprised by covalent binding and the total daily dose of each drug improved the discrimination between hepatotoxic and nontoxic drugs based on in vitro covalent binding data; however, the approach still would falsely identify some agents as potentially hepatotoxic.

Involvement of cytochrome P450-mediated metabolism in tienilic acid hepatotoxicity in rats.

Tienilic acid is reported to be converted into electrophilic metabolites by cytochrome P450 (CYP) in vitro. In vivo, however, the metabolites have not been detected and their effect on liver function is unknown. We previously demonstrated that tienilic acid decreased the GSH level and upregulated genes responsive to oxidative/electrophilic stresses, such as heme oxygenase-1 (Ho-1), glutamate-cysteine ligase modifier subunit (Gclm) and NAD(P)H dehydrogenase quinone 1 (Nqo1), in rat liver, as well as inducing hepatotoxicity by co-treatment with the glutathione biosynthesis inhibitor l-buthionine-(S,R)-sulfoximine (BSO). In this study, for the first time, we identified a glutathione-tienilic acid adduct, a stable conjugate of putative electrophilic metabolites with glutathione (GSH), in the bile of rats given a single oral dose of tienilic acid (300mg/kg). Furthermore, a tienilic acid-induced decrease in the GSH level and upregulation of Ho-1, Gclm and Nqo1 were completely blocked by pretreatment with the CYP inhibitor 1-aminobenzotriazole (ABT, 66mg/kg, i.p.). The increase in the serum ALT level and hepatocyte necrosis resulting from the combined dosing of BSO and tienilic acid was prevented by ABT, despite a low hepatic GSH level. These findings suggest that the electrophilic metabolites of tienilic acid produced by CYP induce electrophilic/oxidative stresses in the rat liver and this contributes to the hepatotoxicity of tienilic acid under impaired GSH biosynthesis.

Synthesis, and structure-activity relationship for C(4) and/or C(5) thienyl substituted pyrimidines, as a new family of antimycobacterial compounds.

Combination of the Suzuki cross-coupling and nucleophilic aromatic substitution of hydrogen (SN(H)) reactions proved to be a convenient method for the synthesis of C(4) and/or C(5) mono(thienyl) and di(thienyl) substituted pyrimidines from commercially available 5-bromopyrimidine. All new pyrimidines were found to be active in micromolar concentrations in vitro against H37Rv, avium, terrae, rifampicin and isoniazid-resistance strains of Mycobacterium tuberculosis. The data for acute in vivo toxicity in mice have been obtained for these compounds which appear to be promising antitubercular agents.

Studies on uricosuric diuretics. 4. Three-dimensional structure-activity relationships and receptor mapping of (aryloxy)acetic acid diuretics.

Attempts to develop new (aryloxy)acetic acids with a better profile of diuretic and uricosuric activities as well as with fewer side effects have produced a series of compounds in which the ring system has been varied. Diuretic screening of these analogues in rats indicated that the great difference in the activity between these compounds might be ascribed to a difference in the ring system rather than that in the substituent effect and that the annulation hypothesis described before is not necessarily applicable to all of these compounds. This prompted us to study the relationship between the structure and the diuretic activity of the (aryloxy)acetic acids. An active model (receptor model) was created with the indanone moiety of R-(-)-3 and the dihydrobenzofuran-2-carboxylic acid moiety of S-(+)-4. The three-dimensional structure-activity study of known compounds 2-4, and 5a using the active model showed that the degree of fitting to the model is related to the diuretic activity of these compounds. This was also confirmed for compounds 6a, 6b, 9a, 10a, 11a, 12a, 13a, 14a, 15a, and 16a, and the relation between the structure and the diuretic activity was rationalized qualitatively. With these insights in mind, a modified receptor model was constructed. We believe that this model is useful for a prediction of the activity of compounds not yet synthesized as well as for designing new (aryloxy)acetic acid diuretics.

A novel approach to cyclin-dependent kinase 5/p25 inhibitors: A potential treatment for Alzheimer's disease.

Based on the earlier results of our in-house database and compound library, a series of novel clubbed thienyl triazoles was designed which may emerge as potential cdk5/p25 inhibitors, for the treatment of Alzheimer's disease. A benign synthesis was planned so as to take an advantage of MAOS (Microwave Assisted Organic Synthesis) method. Evaluation of the SAR of this series has allowed the identification of compounds 4, 5, 7 and 8 from series I while 13, 14, 16 and 17 from series II as significant cdk5/p25 inhibitors and thus have potential as possible treatments for Alzheimer's disease.

The substrate binding site of human liver cytochrome P450 2C9: an approach using designed tienilic acid derivatives and molecular modeling.

Biochemical experiments, using the well-defined human liver CYP2C9 expressed in yeast, and molecular modeling techniques were used to derive a predictive model for substrates of CYP2C9. The ability of 10 2-aroylthiophenes related to tienilic acid to act as substrates for CYP2C9 was studied. Four of them were original compounds that were synthesized and completely characterized by several spectroscopic techniques. In these 10 compounds various chemical functions, such as ester, amide, alcohol, phenol, ether or tetrazole functions, replaced the OCH2COOH function of tienilic acid. Among them, only the derivatives containing an acidic function (carboxylic acids, phenol, and tetrazole whose pKaS are 4.8, 6.3, and 3.8, respectively) underwent a 5-hydroxylation of their thiophene ring like tienilic acid. Despite their close structural analogy with tienilic acid, all of the other compounds not only did not undergo any 5-hydroxylation of their thiophene ring but also failed to act as inhibitors of CYP2C9. These results strongly suggested that the presence, at pH 7.4, of a negative charge on the substrate is a very important feature in its recognition by CYP2C9. In fact, the four new substrates of CYP2C9 described in this study, a carboxylic acid, phenol, and tetrazole derivative, each of which is related to tienilic acid, and the antiinflammatory drug, suprofen (with Km between 12 and 130 microM and kcat between 0.2 and 1.3 min-1), as well as almost all CYP2C9 substrates reported in the literature, exhibit a pKa below 7 (except phenytoin whose pKa is 8.1). They mainly exist as anions at physiological pH. By using molecular modeling techniques, 12 CYP2C9 substrates were superimposed with respect to their hydroxylation site and fitted onto templates, which were rigid molecules such as (S)-warfarin and phenytoin. It was thus possible to arrange them in order that all their anionic sites were at a distance around 4 A from a common point (a putative cationic site of the protein) in space. These results provide a model of the substrate binding site of CYP2C9, in which substrates interact through their anionic site A- with a cationic residue of the CYP2C9 protein C+. In that model, the distance between the hydroxylation site (Hy) and the anionic site (A-) is 7.8 +/- 1.6 A, and the

Electrospray ionization mass spectrometric analysis of intact cytochrome P450: identification of tienilic acid adducts to P450 2C9.

A general scheme for the purification of baculovirus-expressed cytochrome P450s (P450s) from the crude insect cell pastes has been designed which renders the P450s suitable for analysis by high-performance liquid chromatography (HPLC) electrospray ionization mass spectrometry (ESI-MS). An HPLC/ESI-MS procedure has been developed to analyze small amounts of intact purified P450 (P450s cam-HT, 1A1, 1A2, 2A6, 2B1, 2C9, 2C9 C175R, 3A4, 3A4-HT) and rat NADPH cytochrome P450 reductase (P450 reductase). The experimentally determined and predicted (based on the amino acid sequences) molecular masses (MMs) of the various proteins had identical rank orders. For each individual protein, the difference between the experimentally determined (+/-SD, based on experiments performed on at least 3 different days) and predicted MMs ranged from 0.002 to 0.035%. Each experimentally determined MM had a standard deviation of less than 0.09% (based on the charge state distribution). Application of this HPLC/ESI-MS technique made the detection of the covalent modification to P450 2C9 following mechanism-based inactivation by tienilic acid possible. In the absence of glutathione, three P450 2C9 species were detected that produced ESI mass spectra corresponding to native P450 2C9 and both a monoadduct and a diadduct of tienilic acid to P450 2C9. In the presence of glutathione, only native P450 2C9 and the monoadduct were detected. Based on the observed mass shifts for the P450 2C9/tienilic acid adducts, a mechanism for the inactivation of P450 2C9 by tienilic acid is proposed.

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.

Tienilic acid (TA) is responsible for an immune-mediated drug-induced hepatitis in humans, while its isomer (TAI) triggers a direct hepatitis in rats. In this study, we describe an immunological approach developed for studying the specificity of the covalent binding of these two compounds. For this purpose, two different coupling strategies were used to obtain TA-carrier protein conjugates. In the first strategy, the drug was linked through its carboxylic acid function to amine residues of carrier proteins (BSA-N-TA and casein-N-TA), while in the second strategy, the thiophene ring of TA was attached to proteins through a short 3-thiopropanoyl linker, the corresponding conjugates (BSA-S-5-TA and betaLG-S-5-TA) thus preferentially presenting the 2, 3-dichlorophenoxyacetic moiety of the drug for antibody recognition. The BSA-S-5-TA conjugate proved to be 30 times more immunogenic than BSA-N-TA. Anti-TA-protein adduct antibodies were obtained after immunization of rabbits with BSA-S-5-TA (1/35000 titer against betaLG-S-5-TA in ELISA). These antibodies strongly recognized the 2, 3-dichlorophenoxyacetic moiety of TA but poorly the part of the drug engaged in the covalent binding with the proteins. This powerful tool was used in immunoblots to compare TA or TAI adduct formation in human liver microsomes as well as on microsomes from yeast expressing human liver cytochrome P450 2C9. TA displayed a highly specific covalent binding focused on P450 2C9 which is the main cytochrome P450 responsible for its hepatic activation in humans. On the contrary, TAI showed a nonspecific alkylation pattern, targeting many proteins upon metabolic activation. Nevertheless, this nonspecific covalent binding could be completely shifted to a thiol trapping agent like GSH. The difference in alkylation patterns for these two compounds is discussed with regard to their distinct toxicities. A relationship between the specific covalent binding of P450 2C9 by TA and the appearance of the highly specific anti-LKM2 autoantibodies (known to specifically recognize P450 2C9) in patients affected with TA-induced hepatitis is strongly suggested.