Electrophilicity of Pyridazine-3-carbonitrile, Pyrimidine-2-carbonitrile

Article pubs.acs.org/crt

Electrophilicity of Pyridazine-3-carbonitrile, Pyrimidine-2-carbonitrile, and Pyridine-carbonitrile Derivatives: A Chemical Model To Describe the Formation of Thiazoline Derivatives in Human Liver Microsomes Sarmistha Sinha,† Deepak Ahire,† Santosh Wagh,† Dibakar Mullick,‡ Ramesh Sistla,§ Kumaravel Selvakumar,‡ Janet Caceres Cortes,∥ Siva Prasad Putlur,† Sandhya Mandlekar,⊥ and Benjamin M. Johnson*,# †

Pharmaceutical Candidate Optimization, ‡Medicinal Chemistry Department, and §Advanced Biotechnology Department, Biocon Bristol-Myers Squibb R&D Center (BBRC), Syngene International Ltd, Plot No. 2 & 3, Bommasandra IV Phase, Jigani Link Road, Bangalore 560100, India ∥ Department of Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Co., Route 206 & Province Line Road, Princeton, New Jersey 08543, United States ⊥ Pharmaceutical Candidate Optimization, Bristol-Myers Squibb India Ltd. BBRC, Bangalore 560100, India # Department of Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Co., 5 Research Parkway, Wallingford, Connecticut 06492, United States S Supporting Information *

ABSTRACT: Certain aromatic nitriles are well-known inhibitors of cysteine proteases. The mode of action of these compounds involves the formation of a reversible or irreversible covalent bond between the nitrile and a thiol group in the active site of the enzyme. However, the reactivity of these aromatic nitrile-substituted heterocycles may lead inadvertently to nonspecific interactions with DNA, protein, glutathione, and other endogenous components, resulting in toxicity and complicating the use of these compounds as therapeutic agents. In the present study, the intrinsic reactivity and associated structure−property relationships of cathepsin K inhibitors featuring substituted pyridazines [6-phenylpyridazine-3-carbonitrile, 6-(4-fluorophenyl)pyridazine-3-carbonitrile, 6-(4-methoxyphenyl)pyridazine-3-carbonitrile, 6-p-tolylpyridazine-3-carbonitrile], pyrimidines [5-ptolylpyrimidine-2-carbonitrile, 5-(4-fluorophenyl)pyrimidine-2-carbonitrile], and pyridines [5-p-tolylpicolinonitrile and 5-(4fluorophenyl)picolinonitrile] were evaluated using a combination of computational and analytical approaches to establish correlations between electrophilicity and levels of metabolites that were formed in glutathione- and N-acetylcysteine-supplemented human liver microsomes. Metabolites that were characterized in this study featured substituted thiazolines that were formed following rearrangements of transient glutathione and N-acetylcysteine conjugates. Peptidases including γ-glutamyltranspeptidase were shown to catalyze the formation of these products, which were formed to lesser extents in the presence of the selective γ-glutamyltranspeptidase inhibitor acivicin and the nonspecific peptidase inhibitors phenylmethylsulfonyl fluoride and aprotinin. Of the chemical series mentioned above, the pyrimidine series was the most susceptible to metabolism to thiazoline-containing products, followed, in order, by the pyridazine and pyridine series. This trend was in keeping with the diminishing electrophilicity across these series, as demonstrated by in silico modeling. Hence, mechanistic insights gained from this study could be used to assist a medicinal chemistry campaign to design cysteine protease inhibitors that were less prone to the formation of covalent adducts.

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INTRODUCTION

been revealed by studies of the bioactivation of xenobiotics and endogenous compounds.1−7,9 Such compounds may represent a

Compounds that contain electrophilic functional groups, like alkyl halides and Michael acceptors, are prone to react in vivo and are usually avoided in drug design for safety reasons. Much of what is known about the effects of such reactive compounds has © 2014 American Chemical Society

Received: June 30, 2014 Published: November 5, 2014 2052

dx.doi.org/10.1021/tx500256j | Chem. Res. Toxicol. 2014, 27, 2052−2061

Chemical Research in Toxicology

Article

Figure 1. Nitrile-substituted heterocyclic compounds evaluated in this study.

for nitriles that might have the potential for reactivity as a result of their connectivity to neighboring chemical groups. To this end, Macfaul et al.12 described a simple method to assess potential drug-related liabilities by measuring the chemical reactivity of nitriles in phosphate buffer, using glutathione and cysteine as trapping agents. This method did not account for the contributions of drug metabolizing enzymes, which might influence the rate of conjugate formation and improve (or detract from) the utility of the model as a risk-assessment tool. The present study represents an extension of Macfaul et al.’s work, where nitrile-substituted pyridazines [6-phenylpyridazine-3carbonitrile (1), 6-(4-fluorophenyl)pyridazine-3-carbonitrile (2), 6-(4-methoxyphenyl)pyridazine-3-carbonitrile (3), 6-ptolylpyridazine-3-carbonitrile (4)], pyrimidines [5-p-tolylpyrimidine-2-carbonitrile (5), 5-(4-fluorophenyl)pyrimidine-2-carbonitrile (6)], and pyridines [5-p-tolylpicolinonitrile (7) and 5-(4-fluorophenyl)picolinonitrile (8)] (Figure 1) were incubated in GSH- and N-acetylecysteine (NAC)-supplemented human liver microsomes, and the roles of hepatic enzymes in the formation of metabolites were characterized. Furthermore, the reactivity toward GSH and NAC and associated structure−property relationships of these substituted heterocycles were explored in the context of a physiologically relevant system, and the utility of the model as a risk-assessment tool was evaluated and interpreted in the context of computational assessments of substrate electrophilicity. The substrates that were selected for this study included 2-cyanopyrimidines, a series that has been explored in the design of cathepsin K inhibitors,10−17 and two similar series based on cyano-substituted pyridazine and pyridine motifs.11,16 It was hypothesized that the formation of GSH- and NAC-derived conjugates would depend on the electrophilicity of the corresponding substrates and might also be influenced by hepatic enzymes. This hypothesis was evaluated by estimating the electrophilicity of the cyano carbon atom of different ligands and by calculating the free energy of formation of GSH-related conjugates using quantum chemical methods and then

burden for living organisms because of the potential for covalent modification of essential cellular molecules like DNA and proteins, and, in certain cases, the formation of these complexes can lead to adverse events. Glutathione (GSH), a thiolcontaining tripeptide that is present in cells at concentrations of 1−10 mM, plays an important role in protecting cells by trapping reactive electrophiles in the form of stable metabolites that can be detected readily using LC-MS.4−6 For this reason, screening for GSH conjugates of new chemical entities in vitro is done regularly to help assess the suitability of compounds as drug candidates.7−9 Despite this practice, the pharmaceutical industry maintains a considerable interest in the discovery and development of covalent inhibitors: drugs that contain an electrophilic group that can react either reversibly or irreversibly with a nucleophile in the active site of a target protein. This approach has a long history that extends back to the discovery of aspirin and includes several of the world’s top-selling medicines such as omeprazole and clopidogrel.8 Additionally, chemotypes that feature electrophilic moieties have been explored as therapeutic agents in the areas of osteoporosis and osteoarthritis, where they act as inhibitors of cysteine proteases. For example, several electrophilic nitrilecontaining compounds have been identified as inhibitors of cathepsin K, and, among these, three chemotypes have been explored extensively: cyanamides, nitrile-appended aromatic heterocycles, and aminoacetonitriles.11,16 The mode of action of these compounds involves the formation of a reversible or irreversible covalent bond between the nitrile and a cysteine residue within the catalytic site.10−17 A computational approach was devised by Oballa et al. to calculate the theoretical reactivity of such compounds with a cysteine residue, demonstrating that reactivity was governed by the intrinsic electrophilicity of the aromatic nitrile-substituted heterocycles.11 However, such compounds that exhibit biological activity against cysteine proteases may also have the potential to react with endogenous thiols and other nucleophiles like proteins, peptides, and DNA.18,19 Therefore, in vitro risk assessment tools are essential 2053



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tube was sealed with a Teflon screw cap and stirred at 80 °C for 6 h. The reaction mixture was cooled to room temperature, water (10 mL) was added, and the mixture was extracted with ethyl acetate (2 × 10 mL). The combined organic layer was washed with brine (10 mL), dried over Na2SO4, filtered, and concentrated. The crude product was purified by flash chromatography to afford the desired compound as a white solid with 77% yield. The MS and 1H NMR (400 MHz, CDCl3) values of 2 were as follows: [M + H] + = 202; 1H NMR (400 MHz, CDCl3): δ 8.16 (m, 2H), 7.96 (d, J = 8.8 Hz, 1H), 7.87 (d, J = 8.8 Hz, 1H), 7.27 (m, 2H). The rest of the compounds, 1 and 3−8, were prepared in a similar fashion (Scheme 2). Analytical data of these compounds are provided as Supporting Information. General in Vitro Incubations. Each compound, 1−8 and 4-methoxybenzonitrile (negative control, 30 μM), was incubated with human liver microsomes (1 mg/mL) and GSH (3 mM) in sodium phosphate buffer (100 mM; pH 7.4). The total reaction volume was 500 μL. The mixture was incubated for 60 min at 37 °C at 60 rpm in an orbital incubator shaker (Innova 40, New Brunswick Scientific, CT, USA). Similarly, each compound, 1−8 and 4-methoxybenzonitrile (negative control), was incubated with GSH (3 mM) in sodium phosphate buffer (100 mM; pH 7.4) without human liver microsomes for 60 min at 37 °C at 60 rpm. Control experiments were performed in a similar fashion without the compounds. At the 0 and 60 min time points, the reaction was terminated by adding a 200 μL aliquot from the incubation to an equal volume of acetonitrile. The reaction mixture was vortex-mixed and centrifuged at 20 827g for 10 min. The resulting supernatants were analyzed by LC-MS/MS. Additionally, each compound, 1−8 (30 μM), was incubated with NAC (3 mM) with and without human liver microsomes (1 mg/mL) in a similar fashion as described above for subsequent LC-MS/MS analysis. Compound 6 (30 μM) was incubated with stable-labeled GSH (glycine-13C2,15N; 3 mM) in sodium phosphate buffer (100 mM; pH 7.4) with human liver microsomes (1 mg/mL) in a total reaction volume of 500 μL. The incubations and sample processing were carried out in a similar fashion as described above, and samples were analyzed by LC-MS/MS. As an additional control for enzyme activity, human liver microsomes were heated at 80 °C for 5 min and then incubated with 6 (30 μM) and GSH (3 mM). The incubation conditions, time, and sample processing methods were identical to those used in the microsomal incubations described above. Samples were analyzed using LC-MS/MS. In Vitro Incubations with Inhibitors. To a microsomal suspension (1 mg/mL) in sodium phosphate buffer (100 mM; pH 7.4), either acivicin24−26 or a mixture of PMSF and aprotinin27 was added to give a final concentration of 5 mM of each, and the suspensions were preincubated for 20 min. Incubations were initiated with the addition of 6 and GSH to a final concentration of 30 μM. Control experiments were performed in a similar fashion without the inhibitors. Incubations and sample processing were conducted as described under General in Vitro Incubations, and samples were analyzed using LC-MS/MS. Kinetics Studies. Compounds 2, 6, and 8 were selected as representatives from each series for an assessment of kinetics. Each compound (1 μM) was incubated with cysteine (3 mM) in 100 mM

comparing the computational and experimental results to each other. Additional experiments to characterize the role of enzymes in these reactions, to measure disappearance kinetics, and to define the origin of the GSH- and NAC-derived moieties were also carried out. As a complicating factor in such studies, aliphatic and aromatic nitriles are known to react chemically with cysteine and then rearrange to form thiazole derivatives (Scheme 1).11 Scheme 1. Reaction between a Nitrile-Containing Compound and Cysteine To Form a Thiazoline Derivative

For example, the formation of thiazole derivatives in vivo from aliphatic nitriles such as odanacatib, a cathepsin K inhibitor,20 has been reported. To help characterize the reactivity of these nitrilesubstituted heterocycles further, the mechanism of formation of GSH-derived thiazoline derivatives was investigated using 6 as a model substrate.

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EXPERIMENTAL PROCEDURES

Materials. Arylboronic acids, 4-methoxybenzonitrile, and Cs2CO3 were purchased from Aldrich Chemical Co. (Bangalore, India). 5-Bromopicolinonitrile and PdCl2(dppf)−CH2Cl2 complex were purchased from Alfa Aesar (Hyderabad, India). 5-Bromopyrimidine-2carbonitrile was purchased from OChem Incorporation (Chicago, IL, USA), 8-oxobenzyl guanine was purchased from Toronto Research Chemicals (Toronto, Canada), and 6-chloropyridazine-3-carbonitrile was prepared using a reported procedure.21−23 Human liver microsomes were purchased from Invitrogen Bioservices India Pvt. Ltd. Dimethylformamide (DMF), arylboronic acid, cesium carbonate (Cs2CO3), glutathione (GSH), stable-isotope-labeled glutathione (glycine-13C2,15N), cysteine, acivicin, phenylmethanesulfonyl fluoride (PMSF), and aprotinin were purchased from Sigma-Aldrich (Bommasandra Industrial Area, Bangalore, India). HPLC grade acetonitrile and formic acid were purchased from Merck Specialties Private Ltd. (Worli, Mumbai, India). Preparation of 6-(4-Fluorophenyl)pyridazine-3-carbonitrile. Compound 2 was synthesized by a coupling reaction between 6-chloropyridazine-3-carbonitrile and (4-fluorophenyl)boronic acid. An oven-dried Schlenk tube under argon was charged with 6-chloropyridazine-3-carbonitrile (80.0 mg, 0.573 mmol), cesium carbonate (374 mg, 1.15 mmol), (4-fluorophenyl)boronic acid (88.0 mg, 0.631 mmol), PdCl2(dppf)CH2Cl2 (41.9 mg, 0.0570 mmol), DMF (1 mL), and water (0.1 mL). The solution was purged with argon, and the

Scheme 2. Synthetic Scheme Illustrating the Preparation of Carbonitriles Featuring Pyridazine, Pyrimidine, and Pyridine Moieties

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phosphate buffer (pH 7.4) in duplicate. The total reaction volume was 1 mL. The mixture was incubated for 60 min at 37 °C at 60 rpm. At each time point (0, 2.5, 5, 15, 30, 45, and 60 min), a 50 μL aliquot was withdrawn from the reaction mixture and treated with 150 μL acetonitrile containing 8-oxobenzyl guanine (1 μM) as an internal standard. The reaction mixture was vortex mixed and centrifuged at 20 827g for 10 min. The resulting supernatant was analyzed by LC-MS. LC-MS/MS Analysis. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was carried out using an Agilent 1100 HPLC (Agilent Technologies Inc., Wilmington, DE, USA) connected in-line to an Agilent photodiode array detector and an LTQ Velos Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) fitted with an electrospray ionization source and operated in positive-ion mode. Chromatographic separations were performed on a Thermo Hypersil Gold C18 (150 × 3 mm; 5 μm) column maintained at 40 °C. The mobile phase consisted of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile) delivered at a flow rate of 0.4 mL/min with a linear gradient as follows: 2 to 60% B over 10 min, 60% B isocratic for 20 min, 98% B isocratic for 2 min, and 2% isocratic for 8 min. For electrospray mass spectrometry, nitrogen was used as both the sheath gas and auxiliary gas, and helium was used for collisioninduced dissociation. The capillary temperature and source voltage were set to 300 °C and 5 kV, respectively. The Orbitrap was operated in Fourier transform mass spectrometry mode to enable the acquisition of both full-scan mass spectra and product-ion MS/MS spectra with mass accuracies within 5 ppm. A typical data-dependent MS method included three scan events: (1) full-scan MS over the range of m/z 100−800, (2) MS2 of the most intense ions exceeding 1000 cps in the previous fullscan mass spectrum, and (3) MS3 of the most intense product ions from the MS2 scan. Dynamic exclusion criteria included mass widths of 5 ppm, a repeat count of 2, and a repeat duration of 6 s. The MS2 and MS3 normalized collision energies were ramped over the range of 35−45%. Data acquisition and processing were carried out using Xcalibur software (version 1.3, Thermo Fisher Scientific). Isolation of NAC Conjugate of 6 for NMR. Compound 6 (∼150 μg) was incubated in rat liver microsomes (1 mg/mL) supplemented with NAC (3 mM) for 60 min at 37 °C at 60 rpm in an orbital shaker. The total reaction volume was 15 mL. The reaction was terminated by adding an equal volume of cold acetonitrile. Then, the reaction mixture was vortexed and centrifuged at 20 827g for 10 min. The resulting supernatants were concentrated to 1−1.5 mL under nitrogen using a TurboVap (Caliper Life Sciences, Hopkinton, MA, USA) maintained at 25 °C. During evaporation, acetonitrile was added three to four times (1:2/1:3 v/v) to remove excess protein. Finally, the concentrated sample was subjected to HPLC as described in the LC-MS section, and the NAC conjugate was isolated using a fraction collector (Gilson Inc., Middleton, WI, USA). All fractions containing the desired NAC adduct were pooled, and the solvent was evaporated to dryness under nitrogen in a TurboVap. The purified sample containing the NAC adduct was analyzed by NMR. Nuclear Magnetic Resonance (NMR). The isolated metabolite and parent samples were dissolved in DMSO-d6 (50 μL) and placed in a 1.7 mm NMR tube. The samples were analyzed using a Bruker Avance 600 MHz NMR spectrometer equipped with a 1.7 mm TCI triple resonance cryoprobe (Bruker, Billerica, MA, USA) at 27 °C. Proton spectra were recorded using the single pulse (zg30) sequence and multiple solvent suppression (lc1pncwps). 13C chemical shift data were obtained from a one-dimensional carbon spectrum of the parent and heteronuclear single quantum coherence (HSQC) and multiple-bond correlation spectroscopy (HMBC) of the metabolite. Peak assignments were made based on 1H, 1H−1H gradient correlation spectroscopy (COSY), 1H−13C-edited DEPT HSQC, and HMBC spectroscopic analysis. ACD/NMR prediction and processing software from Advanced Chemistry Development Inc. (Toronto, ON, Canada) was used to help interpret the NMR data. Free Energy Calculations. Density functional theory calculations were carried out to estimate the reaction energies between the reactants (thiol and the nitrile moieties) and the conjugates. GSH was represented by a cysteine residue to simplify the calculations. Cysteine was selected as an alternative to ethanethiol because the marginal computational cost

was low and it represented a closer approximation to GSH. The free energies of both the reactants and the products were calculated in the gas phase with B3LYP/6-31G(**) basis sets. The Jaguar 8.0 module from the Schrödinger suite was used for these calculations with the accuracy level of calculations set to fully analytic. The approximate free energy of formation (ΔG) was calculated from the gas-phase energies (G) as follows: ΔG = Gproduct − G(cys+ligand). The electrophilicity of the carbon atom in the cyano group on different ligands was also estimated by quantum chemical optimization of the ligands using density functional theory with B3LYP/6-31G(**) basis sets. The Jaguar 8.0 module from the Schrödinger suite was used for these calculations. Subsequent to the optimization, the Fukui functions were calculated. Fukui functions, f+ and f−, indicate the electrophilicity and nucleophility potential of atoms, respectively.28

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RESULTS

Detection of Conjugates in Human Liver Microsomes and Structure Elucidation of 6A and 6B Based on HRMS. In human liver microsomes, two GSH-derived conjugates of 1−8 (denoted 1A, 1B, 2A, etc., where the number corresponds to the substrates) were detected by UV and MS (Table 1 and Figures 2 and 3A for 6; Supporting Information for the remaining compounds). The masses of all GSH-derived conjugates A and B were 161.0146 and 103.9932 u higher than their respective parent compounds 1−8, respectively. Moreover, the retention time, exact mass, and product-ion spectrum of each NAC-derived conjugate was identical to that of its corresponding GSH-derived metabolite B, suggesting that one of the metabolites produced during reactions with both GSH and NAC was the same. In phosphate buffer that was supplemented with either GSH or NAC, but not human liver microsomes, all compounds were still converted to their respective metabolites A and B, respectively; however, the abundance of these products was lower in this system. No conjugates were found in controls prepared using 4-methoxybenzonitrile as a substrate or in incubations where no substrates were present. Trace quantities of these conjugates were detected in some of the samples that were withdrawn at the 0 min time point. In liver microsomes, the formation of these conjugates was independent of the P450 cofactor NADP(H). The MS/MS product-ion spectra of 6 and its metabolites 6A and 6B are shown in Figure 3A (extracted-ion chromatograms and MS/MS spectra of the remaining compounds and their metabolites are shown in the Supporting Information). The product ions of m/z 343 and 315 were produced by water loss and decarboxylation from the glycine moiety, respectively. The fragment ion of m/z 258 resulted from the cleavage of carboxamide bond and corresponded to fluorophenylpyrimidinethiazoline moiety. The product ion of m/z 200 resulted from cleavage of the thiazoline ring and loss of acetamidoacetic acid moiety, a fragmentation pathway that was also observed during an MS/MS product-ion scan of the parent compound, 6. The product ion spectra of metabolites A and B of other compounds (Supporting Information) exhibited similar fragmentation patterns to those of 6A and 6B. To characterize its structure further, 6B was prepared on a larger scale, isolated, and analyzed by 1H, 13C, and COSY NMR (Figure 3B and Table 2). Characterization of 6B by NMR. The 1H and 13C chemical shifts of both 6 and 6B are listed in Table 2. Comparison of these spectra revealed small chemical shift differences for the protons of the fluorophenyl pyrimidine ring, with the largest change of 0.09 ppm being observed for protons 2 and 4. New proton resonances, labeled 17′, 17″, and 18, were observed between 3 and 5 ppm. These protons exhibit COSY correlations (Figure 3B) 2055



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In Vitro Incubation with Stable-Labeled GSH (Glycine-13C2,15N). The involvement of endogenous cysteinylglycine in the formation of 6A was assessed by incubating 6 with stablelabeled GSH in human liver microsomes. The incubations resulted in the formation of [13C2,15N]-6A, a metabolite whose mass-to-charge ratio was greater, by m/z 3, than that of the conjugate generated in the incubations with unlabeled GSH in human liver microsomes (Figure 3A). Since all three labels were present on the glycine moiety of GSH, the measured mass of 6B was observed to be the same after each incubation. Incubations in Heat-Inactivated Human Liver Microsomes. In order to investigate the contribution of microsomal enzymes in the formation of thiazoline derivatives, 6 was incubated with heat-inactivated human liver microsomes supplemented with GSH (Table 3). Both 6A and 6B were formed in this incubation. However, the amounts that were observed were more than 80% lower than those observed in active human liver microsomes. Incubations with Inhibitors and Human Liver Microsomes. Compound 6 was incubated with acivicin (5 mM), a selective inhibitor of γ-glutamyl transpeptidase,24−26 together with GSH in human liver microsomes in order to investigate the role of γ-glutamyl transpeptidase in the formation of 6A and 6B (Table 4). The amount of 6A that was formed was more than 50% lower in the presence of acivicin. However, no effect of acivicin was noted in the formation of 6B. Similarly 6 was incubated with a mixture of aprotinine (5 mM) and PMSF (5 mM), nonspecific peptidase inhibitors,27 to assess the role of peptidases in the formation of 6A and 6B (Table 4). It was found that formation of both cyclized adducts, 6A and 6B, were almost completely inhibited in the presence of these inhibitors. Kinetics Studies. Compounds 2, 6, and 8 were selected as representatives of the pyridazine, pyrimidine, and pyridine series, respectively, for use in a kinetics assessment. The half-lives of these compounds were measured in the presence of aqueous cysteine (pH 7.4). In this experiment, the half-life of 2 was 11 ± 0.09 min, the half-life of 6 was 8 ± 0.38 min, and the half-life of 8 was >1 h. Modeling Studies. Nucleophilic attack by GSH on the carbon of the nitrile group is a key step in the formation of the thiazoline derivatives shown in Scheme 3. Hence, predicting the energy of formation of the nitrile with GSH was the focus of the effort. Since quantum chemical calculations are computationally intensive, cysteine was used as a surrogate for GSH in order to reduce the number of atoms in these calculations. The order of adduct formation was predicted according to the free energy of the reaction: the lower the free energy, the more favored the adduct formation. Figure 4 shows that the predicted free energies of adduct formation of 1−8 correlate well with the experimental measurements with an R2 of 0.79. Gas-phase energies were calculated in each case and, since the effort was aimed at rank-ordering compounds, it was assumed that the hydration effects would not alter the correlation dramatically. In order to estimate the electrophilicity of the cyano carbon atoms of the different compounds, the Fukui functions were calculated after the quantum chemical optimization. Fukui functions, f+ and f−, for each atom provide useful reactivity indices for nucleophilic and electrophilic attack, respectively.28 Using this approach, the nitrile groups of pyrimidine compounds 5 and 6 were found to be the most electrophilic followed by those of the pyridines (7 and 8) and pyridazines (1−4).

Table 1. Positive-Ion MS and MS/MS Data and Relative UV Abundance of Metabolites of 1−8 Formed Following Reaction with GSH and NAC in Human Liver Microsomes metabolite

observed precursor ion (m/z)

theoretical precursor ion (m/z)

1A

343.0852

343.0859

1B 2A 2B 3A 3B 4A 4B 5A 5B 6A 6B 7A 7B 8A 8B metabolite 1A 1B 2A 2B 3A 3B 4A 4B 5A 5B 6A 6B 7A 7B 8A 8B

MS/MS fragments (m/z)

325, 297, 251, 240, 182 286.0636 286.0645 242, 196, 182 361.0757 361.0765 343, 315, 269, 258, 200 304.0545 304.0551 260, 214, 200 373.0963 373.0965 355, 327, 281, 276, 212 316.0745 316.0750 272, 226, 212 357.1001 357.1010 339, 311, 265, 254, 196 300.0789 300.0801 256, 210, 196 357.1006 357.1010 339, 311, 265, 254, 196 300.0800 300.0801 254, 196 361.0760 361.0765 343, 315, 269, 258, 200 304.0547 304.0551 258, 200 356.1055 356.1063 338, 310, 264, 253, 195 299.0840 299.0849 255, 195 360.0804 360.0813 342, 314, 268, 257, 199 303.0590 303.0598 259, 199 relative abundance in the relative abundance in the presence of GSH (%) presence of NAC (%) 31 not observed by UV 47 not observed by UV 19 5 18 5 67 11 78 10 3 not observed by UV 9 not observed by UV

36 65 45 27 69 72 3 6

among one another, consistent with the presence of the thiazoline carboxylic acid moiety. Additional key data supporting the structure of 6B includes changes in the chemical shifts associated with the pyrimidine moiety (Table 2 and Figure 3B). N1 and N5 of the conjugate exhibited an upfield chemical shift of 5.5 ppm compared to the parent. Similarly, C6 and C7 of the conjugate exhibited downfield chemical shifts of 15.2 and 48.3 ppm compared to parent, respectively. Additionally, new peaks were observed in the 1H−13C DEPT-HSQC and HMBC spectra of 6B, consistent with the presence of the thiazoline carboxylic acid moiety. The NMR data confirmed that the structure of 6B was 2-(5-(4-fluorophenyl)pyrimidin-2-yl)-4,5-dihydrothiazole-4carboxylic acid. Additionally, this data also suggested that the structure of 6A was 2-(5-(4-fluorophenyl)pyrimidin-2-yl)-4,5dihydrothiazole-4-carboxamido acetic acid (Figure 3A,B), as the mass difference between 6A and 6B was equal to the mass of glycine. 2056



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Figure 2. LC-UV Analysis (284 nm) of supernatants that were collected following incubations of 6 in human liver microsomes supplemented with either GSH (A) or N-acetylcysteine (B), showing the formation of conjugates (A) 6A and 6B and (B) 6B.

Figure 3. (A) MS/MS product-ion spectra of 6A and 6B and structural interpretation of fragment ions. The measured mass-to-charge ratios of all ions are within 5 ppm of the corresponding theoretical values. (B) COSY spectrum of 6B showing the presence of new resonances in the 3−6 ppm region. Correlations are observed between protons 17′, 17″, and 18, consistent with the presence of the thiazoline carboxylic acid moiety.

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DISCUSSION

functional groups that are prone to bioactivation are usually avoided in new chemical entities for safety reasons. However, some functional groups can participate in reactions with GSH without bioactivation by cytochromes P450.35 The extent of such reactions may depend on the electronic architecture of the substrate. Such a trend was observed in the present study, where the formation of GSH- and NAC-derived conjugates was demonstrated and correlated with the structures of multiple N-heterocycles in a risk-assessment exercise. To this end, HRMS and NMR data was used to elucidate the structures

Much of what is known about the biological effects of electrophiles comes from the bioactivation literature. The formation of reactive intermediates during the metabolism of xenobiotics is usually considered to be an undesirable event in drug discovery, since such intermediates can form covalent adducts with DNA or proteins. Alternatively, electrophiles can undergo covalent adduction with GSH, leading to depletion of GSH levels and leaving the liver vulnerable to hepatotoxicity.29−34 Therefore, 2057



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suggested that this metabolite formed via an oxime and/or a nitrile oxide intermediate.36 Formation of this intermediate was proposed to require P450, although it should be noted that additional steps were necessary in this sequence, since the starting material was a primary amine instead of a nitrile. In the present study, formation of thiazoline metabolites was NADP(H)-independent, suggesting that no such intermediate was involved. The mechanism of formation of the thiazoline metabolites was postulated to entail intramolecular cyclization of the S-linked GSH conjugate and concomitant elimination of pyroglutamic acid and ammonia, resulting in a dihydrothiazole carboxamido intermediate that subsequently underwent loss of glycine to yield a thiazoline carboxyl derivative.37 However, the role of enzymes in the formation of the thiazoline metabolites was not demonstrated. In the present study, the mechanism of formation of these thiazoline metabolites was investigated using 6 as a model substrate. The metabolism study of 6 using labeled GSH, which demonstrated the formation of a product that was greater in mass by 3 u than the product of a study using unlabeled GSH, established that the formation of 6A in human liver microsomes was GSH-mediated and was not dependent on some other endogenous cysteine-related substrate such as cysteinylglycine. Moreover, the thiazoline metabolites were also detected in lower quantities following reactions in phosphate buffer; however, their abundance was increased by the addition of human liver microsomes and decreased by the addition of heatinactivated human liver microsomes. Therefore, these results demonstrated the role of microsomal enzymes like γ-glutamyltranspeptidase in catalyzing the formation of these thiazoline metabolites. Indeed, the formation of 6A was inhibited in the presence of acivicin, suggesting a role for γ-glutamyltranspeptidase in catalyzing the removal of the γ-glutamyl moiety from the GSH conjugate. It is unlikely that this enzyme was further involved in the cyclization of the final product, since the amine intermediate would have intrinsically improved nucleophilicity vs the amide, and intramolecular reaction to form 6A would be favored entropically. Conversely, the formation of 6B in human liver microsomes was not inhibited by acivicin and therefore occurred independently of γ-glutamyltranspeptidase. Other peptidases also appeared to assist in the formation of both 6A and 6B, since a cocktail of PMSF and aprotinin inhibited these reactions. Therefore, the formation of the thiazoline metabolites was dependent on both chemical and enzymatic factors as depicted in Scheme 3. In this interpretation, the reaction was initiated by nucleophilic attack of GSH on the nitrile carbon to produce an intact GSH conjugate as an intermediate step, likely in reversible fashion. Subsequently, γ-glutamyltranspeptidase and another unspecified peptidase catalyzed the removal of the glutamic acid moiety from the GSH conjugate,36,37,39 resulting in the formation of a stable product. Again, the formation of the thiazoline ring is proposed to occur spontaneously for the reasons cited above. In the final step, 6B was produced from 6A by loss of glycine, a reaction that was subject to inhibition by PMSF/aprotenine and therefore was likely peptidase-dependent. A similar mechanism could be drawn to explain the formation of this metabolite from NAC, where removal of the N-acetyl moiety could be catalyzed by an amidase, esterase, or N-deacetylase. The formation of these thiazoline metabolites differed across the series, decreasing in the order pyrimidine > pyridazine ≫ pyridine (Table 1), consistent with decreasing electrophilicity. The general trend is consistent with that reported by Oballa et al.11 It is also consistent with our own data on the kinetics of disappearance of representative compounds in the presence of

Table 2. Proton, Carbon, and Nitrogen Chemical Shift Assignments for 6B

metabolite position

δ (1H)

group

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

N CH C CH N C C N C CH CH C CH CH F S CH2

18 19

CH CO

δ (13C)

δ (15N) 292.7

9.26 (s) 9.26 (s)

155.1 132.0 155.1 292.7 157.6 164.6

7.96 (m) 7.41 (t, J = 8.6 Hz) 7.41 (t, J = 8.6 Hz) 7.96 (m)

3.48 (overlap), 3.64 (t, J = 8.2 Hz) 5.06 (t, J = 8.2 Hz)

129.6 129.0 116.0 162.8 116.0 129.0

35.3 83.6 172.0

Table 3. Comparison of Formation of 6A and 6B in Human Liver Microsomes, Heated Human Liver Microsomes, and Buffer 6A 6B

buffer

denatured human liver microsomes

human liver microsomes

28% 8%

11% 0.9%

70% 12%

Table 4. Formation of 6A and 6B in the Presence of Acivicin, an Inhibitor of γ-Glutamyltranspepetidase, and a Mixture Containing the Peptidase Inhibitors PMSF and Aprotinin

6 6A 6B

human liver microsomes only

human liver microsomes + acivicin

human liver microsomes + aprotinin + PMSF

28% 55% 8%

47% 23% 9%

98% 2% 0%

of thiazoline-containing metabolites that were formed by reactions between these nitrile-substituted heterocycles and GSH (Figure 3 and Scheme-3) in human liver microsomes. These metabolites exhibited 2-(4,5-dihydrothiazole-4-carboxamido)acetic acid (A) and 4,5-dihydrothiazole-4-carboxylic acid (B) moieties. Additional information was obtained by carrying out incubations of these heterocycles with NAC, which provided a similar but simplified route to the B group of metabolties that represented the final products of these reactions. Similar metabolites have been reported on multiple occasions. For example, DPC423 (1-[3-(aminomethyl)phenyl]-N-[3-fluoro-2′(methylsulfonyl)[1,1′-biphenyl]-4-yl]-3-(trifluoromethyl)-1Hpyrazole-5-carboxamide), a potent inhibitor of blood coagulation factor Xa, produced a thiazoline metabolite in the rat, and it was 2058



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Scheme 3. Proposed Mechanism of Formation of 6A and 6B on Reaction of 6 with GSH in Human Liver Microsomes

Figure 4. Predicted free energies of reactions between cysteine and compounds 1−8 and correlation with yield of metabolites A, as measured experimentally. 2059



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would correlate better with free energy of formation.38 This is in keeping with the approach of Oballa et al. that recognized the role of thermodynamic factors in governing the equilibrium of these reversible reactions. However, the results of the present study are also consistent with those of Berteotti et al., which showed that the activation energies en route to thio adducts were lower for cyano pyrimidines than for cyano pyridines. In conclusion, the mechanism of formation of thiazoline metabolites from nitrile-substituted N-heterocycles and GSH was proposed following computational studies and experiments using human liver microsomes. Specifically, the structure−property relationships of these compounds were investigated by modeling the electrophilicity of the nitrile groups using a computational approach, and then output from that model was correlated with metabolite formation, as estimated experimentally using LC-MS. This approach demonstrated that electrophilicity of the N-heterocycles played a key role in determining the extent of formation of the thiazoline metabolites. The lowering of electrophilicity of such N-heterocycles, either by replacement of the pyrimidine ring with pyridine or by para-phenyl substitution with electron-donating groups, could be used as a strategy to minimize the conversion of these compounds to thiazoline metabolites. Hence, the mechanistic insights gained from this study could be used to assist a medicinal chemistry effort to design a refined chemotype with improved stability toward glutathione and other biological thiols.

cysteine, where half-lives increased in the order 6 (pyrimidine) < 2 (pyridazine) < 8 (pyridine). On the basis of these results, 2-cyanopyrimidine was interpreted to be the most electrophilic functionality due to the presence of its two electron-withdrawing nitrogen atoms adjacent to the 2-carbon, suggesting that the electrophilicity of the nitrile was sensitive to inductive effects. By comparison, the pyridazine series was less electrophilic than that of the pyrimidine series due to the altered position of the second nitrogen atom distal from the 2-carbon, diminishing this effect. By extension, the pyridine series was less succeptible to nucleophilic attack owing to fewer electron-withdrawing nitrogen atoms as ring constituents compared to pyrimidine and an accordingly diminished inductive effect on the nitrile group. The functional groups at the para position of the phenyl ring also appeared to regulate the electrophilicity of the nitrile and influence the rates of reaction with GSH. As a general trend, p-fluoro substitution of the phenyl moiety increased electrophilicity of the cyano group via an electron-withdrawing resonance effect, resulting in higher amounts of adduct, whereas p-methyl and p-methoxyl substitution lowered the electrophilicity of the cyano group by hyperconjugation, resulting in lower amounts of adduct. The calculated gas-phase reaction energies, which accounted for both the makeup of the heterocycles and the pendant substituents on the phenyl group, were consistent with the trend in nitrile electrophilicity, as evidenced by relative extents of reaction observed among the pyrimidine, pyridazine, and pyridine compounds. Solvation effects were not accounted for these calculations, since the aim was to rank-order compounds (not necessarily to calculate energies with a high degree of accuracy), and it was inferred that solvation effects would not influence this outcome. In each series, electron-donating substituents on the phenyl ring were found to reduce conjugate formation, whereas electron-withdrawing substituents facilitated it. The reaction energies predicted this trend with reasonable accuracy. The computed energies are shown in Figure 4. As mentioned in Oballa et al.,11 the reaction energies would not be expected to correspond to empirical reaction rates in a quantitative sense but may be used to rank-order compounds within a series, an expectation that is consistent with the results of this study. It is also noteworthy that the present computational study aimed to predict the extent of product formation, which is a thermodynamic quantity, as opposed estimating reactivity via electrophilicity, a kinetic parameter. Time-dependent depletion of compounds from the pyrimidine, pyridine, and pyridazine series in buffer and human liver microsomes was performed. Higher turnover of 6 in human liver microsomes indicated that liver enzymes indeed catalyzed the formation of the thiazoline metabolites. Compound 6 exhibited the highest turnover and formed the highest amounts of thiazoline metabolites in microsomes, demonstrating that the nitrile on the pyrimidine core was the most electrophilic and that reaction with GSH was catalyzed by microsomal enzymes. The Fukui indices for these three compounds agreed with the observed high turnover of the cyano pyrimidine. The cyano carbon that forms the adduct had a value of 0.000435 in the context of the pyrimidine ring and scored lower as a substituent of the pyridine (0.000387) and pyridazine (0.000355) rings. Recently, Berteotti et al.28 reported a computational study on the reactivity of nitriles. In this study, activation energies were estimated via modeling of the transition state of the nitrile− cysteine reaction and correlated with a kinetic measure of nitrile reactivity. In contrast, the adduct yields that were measured in the present study were interpreted as thermodynamic quantities that

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ASSOCIATED CONTENT

S Supporting Information *

LC-MS chromatograms and MS/MS product-ion spectra of all metabolites as well as NMR spectra of 6 and 6B. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors thank Ramaswamy A. Iyer, Yue-Zhong Shu, Jonathan L. Josephs, and William Griffith Humphreys for helpful suggestions and discussion. The authors also acknowledge Silvi Ann Chacko and Senthil Kumar Murugesan for helping to conduct experiments and LC-MS method development.

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ABBREVIATION NAC, N-acetylcysteine; PMSF, phenylmethanesulfonyl fluoride; DMF, dimethylformamide; Cs2CO3, cesium carbonate; HSQC, heteronuclear single quantum coherence; HMBC, heteronuclear multiple bond corelation spectroscopy; COSY, correlated spectroscopy; DEPT, distortionless enhancement by polarization transfer; HRMS, high-resolution mass spectroscopy

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REFERENCES

(1) Guengerich, F. P. (2005) Principles of covalent binding of reactive metabolites and examples of activation of bis-electrophiles by conjugation. Arch. Biochem. Biophys. 433, 369−378. (2) Liebler, D. C. (2007) Protein damage by reactive electrophiles: targets and consequences. Chem. Res. Toxicol. 21, 117−128. (3) Anders, M. (2004) Glutathione-dependent bioactivation of haloalkanes and haloalkenes. Drug Metab. Rev. 36, 583−594.

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(4) Anders, M. (2007) Chemical toxicology of reactive intermediates formed by the glutathione-dependent bioactivation of halogencontaining compounds. Chem. Res. Toxicol. 21, 145−159. (5) Marnett, L. (1998) Chemistry and biology of DNA damage by malondialdehyde. IARC Sci. Publ., 17−27. (6) Guengerich, F. P. (2005) Activation of alkyl halides by glutathione transferases. Methods Enzymol. 401, 342−353. (7) Kalgutkar, A. S., Gardner, I., Obach, R. S., Shaffer, C. L., Callegari, E., Henne, K. R., Mutlib, A. E., Dalvie, D. K., Lee, J. S., Nakai, Y., O’Donnell, J. P., Boer, J., and Harriman, S. P. (2005) A comprehensive listing of bioactivation pathways of organic functional groups. Curr. Drug Metabol. 6, 161−225. (8) Singh, J., Petter, R. C., Baillie, T. A., and Whitty, A. (2011) The resurgence of covalent drugs. Nat. Rev. Drug Discovery 10, 307−317. (9) Park, B. K., Boobis, A., Clarke, S., Goldring, C. E., Jones, D., Kenna, J. G., Lambert, C., Laverty, H. G., Naisbitt, D. J., Nelson, S., NicollGriffith, D. A., Obach, R. S., Routledge, P., Smith, D. A., Tweedie, D. J., Vermeulen, N., Williams, D. P., and Baillie, T. A. (2011) Managing the challenge of chemically reactive metabolites in drug development. Nat. Rev. Drug Discovery 10, 292−306. (10) Morley, A. D., Kenny, P. W., Burton, B., Heald, R. A., Macfaul, P. A., Mullett, J., Page, K., Porres, S. S., Ribeiro, L. R., and Smith, P. (2009) 5-Aminopyrimidin-2-ylnitriles as cathepsin K inhibitors. Bioorg. Med. Chem. Lett. 19, 1658−1661. (11) Oballa, R. M., Truchon, J.-F., Bayly, C. I., Chauret, N., Day, S., Crane, S., and Berthelette, C. (2007) A generally applicable method for assessing the electrophilicity and reactivity of diverse nitrile-containing compounds. Bioorg. Med. Chem. Lett. 17, 998−1002. (12) Macfaul, P. A., Morley, A. D., and Crawford, J. J. (2009) A simple in vitro assay for assessing the reactivity of nitrile containing compounds. Bioorg. Med. Chem. Lett. 19, 1136−1138. (13) Deaton, D. N., and Tavares, F. X. (2005) Design of cathepsin K inhibitors for osteoporosis. Curr. Top. Med. Chem. 5, 1639−1675. (14) Grabowskal, U., Chambers, T. J., and Shiroo, M. (2005) Recent developments in cathepsin K inhibitor design. Curr. Opin. Drug Discovery Dev. 8, 619−630. (15) Altmann, E., Cowan-Jacob, S. W., and Missbach, M. (2004) Novel purine nitrile derived inhibitors of the cysteine protease cathepsin K. J. Med. Chem. 47, 5833−5836. (16) Rankovic, Z., Cai, J., Kerr, J., Fradera, X., Robinson, J., Mistry, A., Hamilton, E., Mcgarry, G., Andrews, F., and Caulfield, W. (2010) Design and optimization of a series of novel 2-cyano-pyrimidines as cathepsin K inhibitors. Bioorg. Med. Chem. Lett. 20, 1524−1527. (17) Ehmke, V., Quinsaat, J. E., Rivera-Fuentes, P., Heindl, C., Freymond, C., Rottmann, M., Brun, R., Schirmeister, T., and Diederich, F. (2012) Tuning and predicting biological affinity: aryl nitriles as cysteine protease inhibitors. Org. Biomol. Chem. 10, 5764−5768. (18) Park, B. K., Kitteringham, N. R., Maggs, J. L., Pirmohamed, M., and Williams, D. P. (2005) The role of metabolic activation in druginduced hepatotoxicity. Annu. Rev. Pharmacol. Toxicol. 45, 177−202. (19) Shin, N. Y., Liu, Q., Stamer, S. L., and Liebler, D. C. (2007) Protein targets of reactive electrophiles in human liver microsomes. Chem. Res. Toxicol. 20, 859−867. (20) Kassahun, K., Black, W. C., Nicoll-Griffith, D., Mcintosh, I., Chauret, N., Day, S., Rosenberg, E., and Koeplinger, K. (2011) Pharmacokinetics and metabolism in rats, dogs, and monkeys of the cathepsin K inhibitor odanacatib: demethylation of a methylsulfonyl moiety as a major metabolic pathway. Drug Metab. Dispos. 39, 1079− 1087. (21) Leclerc, R., and Uguen, D. (1994) A convenient preparation of optically pure 3-hydroxyglutaric acid derivatives. Tetrahedron Lett. 35, 1999−2002. (22) White, E. H., and Worther, H. (1966) Analogs of firefly luciferin. III. J. Org. Chem. 31, 1484−1488. (23) Goodman, A. J., Stanforth, S. P., and Tarbit, B. (1999) Desymmetrization of dichloroazaheterocycles. Tetrahedron 55, 15067−15070. (24) Mutlib, A., Shockcor, J., Chen, S.-Y., Espina, R., Lin, J., Graciani, N., Prakash, S., and Gan, L.-S. (2001) Formation of unusual glutamate

conjugates of 1-[3-(aminomethyl) phenyl]-N-[3-fluoro-2′-(methylsulfonyl)-[1,1′-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (DPC 423) and its analogs: the role of γ-glutamyltranspeptidase in the biotransformation of benzylamines. Drug Metab. Dispos. 29, 1296−1306. (25) Antoine, B., Rahimi-Pour, A., Siest, G., Magdalou, J., and Galteau, M. M. (1987) Differential time-course of induction of rat liver gammaglutamyltransferase and drug-metabolizing enzymes in the endoplasmic reticulum, golgi and plasma membranes after a single phenobarbital injection. Evaluation of protein variations by two-dimensional electrophoresis. Cell Biochem. Funct. 5, 217−231. (26) Raulf, M., Stuning, M., and Konig, W. (1985) Metabolism of leukotrienes by L-gamma-glutamyl-transpeptidase and dipeptidase from human polymorphonuclear granulocytes. Immunology 55, 135−147. (27) Miles, K. K., Kessler, F. K., Smith, P. C., and Ritter, J. K. (2006) Characterization of rat intestinal microsomal UDP-glucuronosyltransferase activity toward mycophenolic acid. Drug Metab. Dispos. 34, 1632− 1639. (28) Berteotti, A., Vacondio, F., Lodola, A., Bassi, M., Silva, C., Mor, M., and Cavalli, A. (2014) Predicting the reactivity of nitrile-carrying compounds with cysteine: a combined computational and experimental study. ACS Med. Chem. Lett. 5, 501−505. (29) Ju, C., and Uetrecht, J. (2002) Mechanism of idiosyncratic drug reactions: reactive metabolite formation, protein binding and the regulation of the immune system. Curr. Drug Metab. 3, 367−377. (30) Liebler, D. C., and Guengerich, F. P. (2005) Elucidating mechanisms of drug-induced toxicity. Nat. Rev. Drug Discovery 4, 410− 420. (31) Uetrecht, J. P. (1999) New concepts in immunology relevant to idiosyncratic drug reactions: the “danger hypothesis” and innate immune system. Chem. Res. Toxicol. 12, 387−395. (32) Cohen, S. D., Pumford, N. R., Khairallah, E. A., Boekelheide, K., Pohl, L. R., Amouzadeh, H., and Hinson, J. A. (1997) Selective protein covalent binding and target organ toxicity. Toxicol. Appl. Pharmacol. 143, 1−12. (33) Mitchell, J., Jollow, D., Potter, W., Davis, D., Gillette, J., and Brodie, B. (1973) Acetaminophen-induced hepatic necrosis. I. Role of drug metabolism. J. Pharmacol. Exp. Ther. 187, 185−194. (34) Mitchell, J., Jollow, D., Potter, W., Gillette, J., and Brodie, B. (1973) Acetaminophen-induced hepatic necrosis. IV. Protective role of glutathione. J. Pharmacol. Exp. Ther. 187, 211−217. (35) Kalgutkar, A. S., Mascitti, V., Sharma, R., Walker, G. W., Ryder, T., McDonald, T. S., Chen, Y., Preville, C., Basak, A., McClure, K. F., Kohrt, J. T., Robinson, R. P., Munchhof, M. J., and Cornelius, P. (2011) Intrinsic electrophilicity of a 4-substituted-5-cyano-6-(2-methylpyridin3-yloxy)pyrimidine derivative: structural characterization of glutathione conjugates in vitro. Chem. Res. Toxicol. 24, 269−278. (36) Mutlib, A., Chen, S., Espina, R., Shockcor, J., Prakash, S., and Gan, L. (2002) P450-mediated metabolism of 1-[3-(aminomethyl)phenyl]N-[3-fluoro-2′-(methylsulfonyl)-[1,1′-biphenyl]-4-yl]-3 (trifluoromethyl)-1H-pyrazole-5-carboxamide (DPC 423) and its analogues to aldoximes. Characterization of glutathione conjugates of postulated intermediates derived from aldoximes. Chem. Res. Toxicol. 15, 63−75. (37) Kera, Y., Kiriyama, T., and Komura, S. (1985) Conjugation of acetaldehyde with cysteinylglycine, the first metabolite in glutathione breakdown by gamma-glutamyltranspeptidase. Agents Actions 17, 48− 52. (38) Baidya, M., and Mayr, H. (2008) Nucleophilicities and carbon basicities of DBU and DBN. Chem. Commun. 15, 1792−1794. (39) Chandra, A. K., and Nguyen, M. T. (2002) Use of local softness for the interpretation of reaction mechanisms. Int. J. Mol. Sci. 3, 310− 323.

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Electrophilicity of Pyridazine-3-carbonitrile, Pyrimidine-2-carbonitrile

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