Systematic Evaluation of the Metabolism and Toxicity of

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Systematic Evaluation of the Metabolism and Toxicity of Thiazolidinone and Imidazolidinone Heterocycles Shi Qing Tang, Yong Yang Irvin Lee, David Sheela Packiaraj, Han Kiat Ho,* and Christina Li Lin Chai* Department of Pharmacy, Faculty of Science, National University of Singapore, 18 Science Drive 4, Singapore 117543 S Supporting Information *

ABSTRACT: The thiazolidine and imidazolidine heterocyclic scaffolds, i.e., the rhodanines, 2,4-thiazolidinediones, 2-thiohydantoins, and hydantoins have been the subject of debate on their suitability as starting points in drug discovery. This attention arose from the wide variety of biological activities exhibited by these scaffolds and their frequent occurrence as hits in screening campaigns. Studies have been conducted to evaluate their value in drug discovery in terms of their biological activity, chemical reactivity, aggregation-based promiscuity, and electronic properties. However, the metabolic profiles and toxicities have not been systematically assessed. In this study, a series of five-membered multiheterocyclic (FMMH) compounds were selected for a systematic evaluation of their metabolic profiles and toxicities on TAMH cells, a metabolically competent rodent liver cell line and HepG2 cells, a model of human hepatocytes. Our studies showed that generally the rhodanines are the most toxic, followed by the thiazolidinediones, thiohydantoins, and hydantoins. However, not all compounds within the family of heterocycles were toxic. In terms of metabolic stability, 5-substituted rhodanines and 5benzylidene thiohydantoins were found to have short half-lives in the presence of human liver microsomes (t1/2 < 30 min) suggesting that the presence of the endocyclic sulfur and thiocarbonyl group or a combination of C5 benzylidene substituent and thiocarbonyl group in these heterocycles could be recognition motifs for P450 metabolism. However, the stability of these compounds could be improved by installing hydrophilic functional groups. Therefore, the toxicities and metabolic profiles of FMMH derivatives will ultimately depend on the overall chemical entity, and a blanket statement on the effect of the FMMH scaffold on toxicity or metabolic stability cannot and should not be made.

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INTRODUCTION Rhodanines, 2,4-thiazolidinediones, 2-thiohydantoins, and hydantoins are five-membered multiheterocyclic (FMMH) scaffolds that are commonly used in drug discovery.1−4 Compounds containing these scaffolds are identified to exhibit a wide variety of biological activities. Some examples of their biological applications include their use as anticonvulsant, muscle relaxant, antiplatelet, anti-inflammatory, antihyperglycaemic, aldose reductase inhibitory, antimicrobial, and anticancer agents.1−4 A number of FMMH derivatives, such as phenytoin, enzalutamide, and pioglitazone have also been approved as therapeutic drugs by the FDA,5 and epalrestat has been approved to be used in Japan6 (Figure 1). The diversity in the reported biological activities of FMMH derivatives suggests that the scaffolds confer the potential to interact with a wide variety of molecular targets, and this is evident in the increasing prevalence of FMMH compounds as screening hits against a plethora of molecular targets.7 As such, some medicinal chemists consider FMMHs to be “privileged structures” that are capable of providing ligand points for more than one type of receptors or enzyme targets.8−11 However, this has raised concerns on the suitability of FMMHs as starting points for drug development.7,10,12,13 It was reported that rhodanines could interact nonspecifically with off-target © 2015 American Chemical Society

Figure 1. FDA approved therapeutic drugs containing FMMH scaffolds.

proteins, making compounds containing this scaffold nonoptimizable for drug development.2,10,14 Separately, the prevalence of thiazolidinediones in screening hits also makes this heterocyclic system questionable for hit-to-lead optimization.7 As a result, some have classified these scaffolds as pan Received: June 10, 2015 Published: September 24, 2015 2019

DOI: 10.1021/acs.chemrestox.5b00247 Chem. Res. Toxicol. 2015, 28, 2019−2033

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Chemical Research in Toxicology

Figure 2. Proposed metabolic activation of troglitazone, rosiglitazone, and pioglitazone through oxidation of the 2,4-thiazolidinedione ring.

3) were synthesized, and their metabolic stabilities against P450 metabolism and toxicities against two metabolically competent

assay interference compounds (PAINS) and hold the opinion that FMMHs have little value in drug development and should be removed from screening libraries.7,10,12−14 Thus, the value of FMMHs as scaffolds in drug discovery has been heavily debated among the medicinal chemistry community. In 2012, Mendgen et al. conducted a systematic structure− activity relationship study of FMMH against different enzymatic targets in order to evaluate their activity toward nucleophiles, aggregation-based promiscuity, and electronic properties.10 They suggested that general condemnation of FMMH scaffolds in drug discovery was inappropriate and would deprive medicinal chemists from accessing these attractive building blocks.10 However, Mendgen et al. did not evaluate the toxicity and metabolism of FMMHs, and information on metabolism and toxicity of FMMHs is only available for specific drugs and their derivatives. Those studies suggest that knowledge on metabolism and toxicities of FMMHs are important as FMMHs could confer toxicity through metabolism.15−24 The antiepileptic drug, phenytoin (Figure 1), that contains a hydantoin scaffold, was studied by several research groups to determine the underlining mechanism for the development of skin rashes observed in 5−10% of patients.18 These studies showed that phenytoin undergoes hydroxylation to form 5-(4-hydroxyphenyl-)-5phenylhydantoin (HPPH) that could be further oxidized to the reactive catechol, resulting in the formation of a drug− protein adduct.18 Evidence of FMMH ring opening during metabolism to form reactive metabolites was also reported for the antidiabetic drug pioglitazone and its derivatives (Figure 2).15,17,20,21,25 The formation of the reactive isocyanate was associated with the reported fatal cases of hepatotoxicity.15,17,20,21,25 Although insights regarding the metabolism and toxicities of FMMHs could be gained from the abovereported studies, this information is inadequate for a systematic evaluation of the structure−metabolism and structure−toxicity relationships of FMMHs and their derivatives. Knowledge on the metabolism and toxicity of FMMHs is necessary in order to evaluate the suitability of these scaffolds in drug discovery. Thus, this study aims to compare and contrast the metabolic profiles and toxicities of FMMHs to help bridge the gap in knowledge regarding these scaffolds. Such information would allow a thorough consideration of the relevance of FMMH as scaffolds in drug discovery. Five series of FMMHs with different N3 and C5 substituents based on thiazolidinone and imidazolidinone scaffolds (Figure

Figure 3. Series of FMMHs designed for comparison of their metabolic stability and toxicity on TAMH cells.

liver cell lines, transforming growth factor (TGF) α overexpressing mouse hepatocyte (TAMH) cells and HepG2 cells, were tested. These substituents include N3-carboxymethyl, C5benzylidene, C5-propylidene, and C5-benzyl substituents. The C5-benzylidene and N3-carboxymethyl substituents were chosen due to the presence of similar substituents in existing FMMH drugs and the inclusion of the C5-benzyl and C5propylidene substituents could allow an extension of the study of substituent effects on the metabolic stabilities and toxicities of the compounds. For the toxicity studies, the TAMH cell line was chosen because it is metabolically competent and expresses drug-metabolizing enzymes, such as murine cytochrome P450 isozymes, which is homologous to human CYP2E1 and CYP3A4. The TAMH cell line is also stable and maintains a differentiated phenotype over multiple passages. The HepG2 cell line is a model of human hepatocytes and is frequently used as models for toxicity testing.26−28 P450-mediated metabolism was evaluated in this study as most of the toxicity related metabolism of FMMH derivatives were found to be due to phase 1 metabolism as illustrated in the examples above.15,17,20,21,25 Our studies suggest that sulfur-containing scaffolds, which are the rhodanines, thiohydantoins, and thiazolidinediones, are more likely to be metabolically unstable 2020


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Chemical Research in Toxicology or toxic to liver cells. In contrast to the findings by Mendgen et al., we discerned that selected arylidene and alkylidene rhodanines are potential Michael acceptors that may contribute to nonspecific activities toward biological nucleophiles, leading to liver cell toxicities.10 However, the equivalent thiohydantoins, thiazolidinediones, and hydantoins have less propensity to be Michael acceptors, and this finding is in accordance with the observations reported by Mendgen et al.10 Although our studies suggest that sulfur-containing arylidene and alkylidene FMMHs are more likely to be metabolically unstable and toxic as well as potential Michael acceptors, a generalization on these cannot and should not be made. The reactivities, metabolic stabilities, and toxicities can be reduced or removed through a judicious choice of substituents on the FMMHs in question. Thus, FMMHs could still be attractive hit compounds owing to their ease of derivatization and synthetic tractability.

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(2,4-dioxothiazolidin-3-yl) acetate, was isolated by column chromatography (0% to 80% ethyl acetate/petroleum ether, bp 60−80 °C). To a solution of methyl 2-(2,4-dioxothiazolidin-3-yl) acetate (0.5 g, 2.643 mmol) in glacial acetic acid (10.6 mL) was added aqueous HCl (12 N, 2.64 mL), and the mixture was refluxed for 2 h. After evaporation to dryness in vacuo, the crude solid was washed with diethyl ether and dried to yield the pure yellow solid in 17% yield. General Procedure for the Knoevenagel Condensation of Aldehydes with Thiazolidinedione. To a mixture of 2,4-thiazolidinedione (0.5 g, 4.27 mmol) and anhydrous sodium acetate (4.0 equiv) in glacial acetic acid (0.2 M) was added aldehyde (0.5 equiv). The reaction mixture was refluxed overnight, then cooled to room temperature, and poured into cold water. The solid was filtered and recrystallized from hot EtOH. (Z)-2-(5-Benzylidene-2,4-dioxothiazolidin-3-yl) Acetic Acid (8). To a solution of 2,4-thiazolidinedione 6 (0.8 g, 3.90 mmol) in acetone (46.8 mL) was added anhydrous K2CO3 (2 equiv). Methyl bromoacetate (2 equiv) was then added to the mixture dropwise, and the mixture was refluxed overnight. After cooling to room temperature, the mixture was filtered and the solvent removed in vacuo. The product, (Z)-methyl 2-(5-benzylidene-2,4-dioxothiazolidin3-yl)acetate, was recrystallized from hot EtOH. To a solution of (Z)methyl 2-(5-benzylidene-2,4-dioxothiazolidin-3-yl)acetate (0.1 g, 0.361 mmol) in glacial acetic acid (1.5 mL) was added aqueous HCl (12 N, 0.36 mL), and the mixture was refluxed for 2 h. After cooling to room temperature, the mixture was added to 50 mL of water at room temperature, and the precipitate that formed was filtered and dried to yield a white solid in 73% yield. Ethyl 2-(5-oxo-2-thioxoimidazolidin-1-yl) Acetate (30). To a solution of HCO2H·H2N-Gly-Gly-OEt in CH2Cl2 (0.35 M), was added triethylamine (2.0 equiv), followed by thio-carbonyl diimidazole (1.1 equiv). The reaction was stirred at room temperature for 15 h. Then, the mixture was washed with 5% citric acid solution, saturated NaHCO3 solution and brine, and dried over anhydrous Na2SO4. The mixture was filtered, and the solvent was evaporated. The crude product was purified with column chromatography (50% EtOAc/Pet ether) to obtain a yellow solid in 48% yield. 2-(5-Oxo-2-thioxoimidazolidin-1-yl) Acetic Acid (9). Ester 30 was dissolved acetone/3 N HCl (2:1) (0.2 M) and refluxed at 75 °C. The reaction was monitored with TLC. When the reaction was complete, the mixture was cooled to room temperature and diluted with water. The mixture was filtered, and the crude product was obtained as a solid. The solid was recrystallized in hot EtOH to obtain a yellow solid in 94% yield. General Procedure for the Knoevenagel Condensation of Aldehydes with Thiohydantoin. To a mixture of thiohydantoin (1.0 g, 8.61 mmol) and anhydrous sodium acetate (4.0 equiv) in glacial acetic acid (0.2 M) was added aldehyde (0.5 equiv). The reaction mixture was refluxed overnight, then cooled to room temperature and poured into cold water. The solid was recrystallized in hot EtOH to obtain the pure product. (Z)-2-(4-Benzylidene-5-oxo-2-thioxoimidazolidin-1-yl) acetic acid (12). To a solution of thiohydantoin 30 in EtOH (0.2 M), was added benzaldehyde (1.0 equiv) and piperidine (2.5 equiv). The reaction mixture was stirred for 18 h. Then, the mixture was cooled to room temperature, and EtOH was removed in vacuo. The crude product was purified with column chromatography (25 to 50% EtOAc/Pet ether). The esters obtained were then dissolved acetone/3 N HCl (2:1) (0.2 M) and refluxed at 75 °C. The reaction was monitored with TLC. When the reaction was complete, the mixture was cooled to room temperature and diluted with water. The mixture was extracted with EtOAc. The combined organic layer was washed with 1 N HCl and brine, and dried with anhydrous Na2SO4. The solvent was removed in vacuo. The crude product was purified with column chromatography (10% MeOH/CH2Cl2) to obtain a yellow solid in 37% yield. Methyl 2-(2,5-Dioxoimidazolidin-1-yl) Acetate (31). To a solution of hydantoin (2.0 g, 2.00 mmol) in dry N,N-dimethylformamide (50 mL) in a sealed tube was added anhydrous K2CO3 (1.5 equiv). Methyl bromoacetate (1.5 equiv) was then added dropwise to the mixture, which was then stirred overnight at 60 °C. Upon cooling to room

EXPERIMENTAL PROCEDURES

Synthesis of FMMHs Compounds. All reagents and solvents were purchased from Sigma-Aldrich or Alfa Aesar and were used without further purification unless otherwise specified. Dry DMF and CH2Cl2 were dried using a GlassContour Solvent Purification System. All other solvents of analytical (AR) and HPLC grades were used without further purification. Thin layer chromatography (TLC) was performed on Merck precoated silica gel plates. Visualization was accomplished with UV light or by staining with KMnO4 or cerium ammonium molybdate solution. Compounds were purified by flash chromatography on column using Merck silica gel 60 (230−400 mesh) unless otherwise specified. Low-resolution mass spectra were recorded by electrospray ionization on an Applied Biosystems MDS SCIEX API 2000 mass spectrometer. High-resolution mass spectra were recorded by electrospray ionization-time-of-flight mode on an Agilent 6210 mass spectrometer. Analytical HPLC was performed with a Shimadzu system and a Phenomenex Kinetex 2.6 μm C18 100 AA (150 × 4.60 mm, 2.6 μm) column to check the purity of the compounds. The system conditions were 35−90% ACN (0.1% formic acid)/H2O (0.1% formic acid) at a flow rate of 0.7 mL/min for 15 min (Method A); 20% ACN (0.1% formic acid)/15−75% MeOH/H2O at a flow rate of 0.7 mL/min for 15 min (Method B). All compounds were found to have >95% purity. NMR spectra were recorded at 400 MHz for 1H and at 100 MHz for 13C on a Bruker spectrometer with CDCl3, DMSO-d6, or CD3OD as solvent. Analytical data can be found in Supporting Information. 2-(4-Oxo-2-thioxothiazolidin-3-yl) Acetic Acid (1). Compound 1 was synthesized following a literature procedure.29 General Procedure for the Knoevenagel Condensation of Aldehydes with Rhodanines. The compounds were synthesized following a literature procedure.29,30 To a solution of rhodanine (1.0 equiv) in glacial acetic acid (0.2 M) was added NaOAc (4.0 equiv) and aldehyde (0.5 equiv). The resulting mixture was refluxed for 18 h and cooled to room temperature. Acetic acid was removed in vacuo. The residue was partitioned in 1 N HCl and EtOAc (1:1). The aqueous layer was then extracted with EtOAc and dried with Na2SO4. The mixture was filtered, and the solvent was removed in vacuo. The crude product was purified with column chromatography (50% EtOAc/Pet ether or 10% MeOH/CH2Cl2) or by recrystallization with hot EtOH/1 N HCl. 2-(2,4-Dioxothiazolidin-3-yl) Acetic Acid (5). To a solution of 2,4thiazolidinedione (1.0 g, 8.54 mmol) in dry N,N-dimethylformamide (0.1 M) was added anhydrous K2CO3 (1.5 equiv), and the mixture was stirred at 60 °C. Methyl bromoacetate (1.2 equiv) was then added dropwise to the mixture, which was stirred overnight at 60 °C. Upon cooling to room temperature, distilled water (21 mL) was added, and the crude product was extracted with ethyl acetate (3 × 50 mL). The combined organic layer was washed with 5% aqueous citric acid and brine, then dried over anhydrous MgSO4. The mixture was filtered, and the solvent was evaporated in vacuo. The pure product, methyl 22021


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Chemical Research in Toxicology temperature, the solvent was removed in vacuo, then distilled water (50 mL) was added. The aqueous phase was then saturated with NaCl, and the crude product was extracted with ethyl acetate (4 × 50 mL). The combined organic layer was washed with 5% aqueous citric acid and brine, and dried over anhydrous MgSO4. The mixture was filtered, and the solvent was evaporated in vacuo. A white solid was isolated by column chromatography (0% to 80% ethyl acetate/petroleum ether) in 22% yield. 2-(2,5-Dioxoimidazolidin-1-yl) Acetic Acid (13). To a solution of hydantoin 31 (0.1 g, 0.581 mmol) in glacial acetic acid (2.32 mL) was added aqueous HCl (12 N, 0.58 mL), and the mixture was refluxed for 2 h. After cooling to room temperature, the mixture was evaporated to dryness in vacuo to obtain a yellow solid in 83% yield. General Procedure for the Knoevenagel Condensation of Aldehydes with Hydantoin. A solution of hydantoin (0.5 g, 4.99 mmol) in water (10 mL) was heated to 70 °C and its pH adjusted to 7 with saturated NaHCO3. A solution of ethanolamine (1.5 equiv) in EtOH (10 mL) was added, and the mixture was then heated to 90 °C. Aldehyde (1.1 equiv) was added dropwise, and the reaction mixture was refluxed overnight, then cooled to room temperature and poured into cold water. The solid was filtered and recrystallized from hot ethanol. (Z)-2-(4-Benzylidene-2,5-dioxoimidazolidin-1-yl) Acetic Acid (16). To a solution of hydantoin 31 in EtOH (0.2 M), was added the benzaldehyde (1.0 equiv) and piperidine (2.5 equiv). The reaction mixture was stirred for 3 days. Then, the mixture was cooled to room temperature, and EtOH was removed in vacuo. The crude product was purified with column chromatography (25 to 50% EtOAc/Pet ether). The ester obtained was then dissolved acetone/3 N HCl (2:1) (0.2 M) and refluxed at 75 °C. The reaction was monitored with TLC. When the reaction was complete, the mixture was cooled to room temperature and diluted with water. The mixture was extracted with EtOAc. The combined organic layer was washed with 1 N HCl and brine, and dried with anhydrous Na2SO4. The solvent was removed in vacuo. The crude product was purified with column chromatography (10% MeOH/CH2Cl2) to obtain a white solid in 37% yield. General Procedure for the Reduction of Benzylidene FMMH. To a solution of benzylidene FMMH (100 mg) in acetic acid (0.2 M) was added Zn (1.5 equiv), and the reaction mixture was refluxed for 18 h. The reaction was then cooled to 50 °C, added with MeOH (5× of acetic acid), and refluxed for 5 min. The reaction mixture was filtered, and the solvent was removed in vacuo. The crude product was purified with column chromatography (50% EtOAc/Pet ether). 5-Benzylimidazolidine-2,4-dione. To a solution of D- or Lphenylalanine (500 mg, 3.0 mmol) in 20 mL of water was added potassium cyanate (491 mg, 6.0 mmol). The reaction was stirred at 50 °C for 30 min. Then, the reaction was concentrated to 5 mL and added with 1 N HCl to form precipitate. Subsequently, 10 mL of 2 N HCl was added, and the reaction was refluxed for 2 h. The reaction was then cooled to room temperature, and the solid was filtered and washed with water. Determination of Toxicity on TAMH and HepG2 Cells. HEPES-buffered Dulbecco’s modified Eagle’s Media/Ham’s F12 (DMEM/F12) and soybean trypsin inhibitor were purchased from Gibco (Grand Island, NY). Insulin, transferrin, and selenium (ITS) premix was from BD Biosciences (San Jose, CA). Phosphate buffered saline (PBS, as 10× solution) was from Vivantis (Selangor, Malaysia). Dimethyl sulfoxide (DMSO) was from Merck (Darmstadt, Germany). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was from Duchefa Biochemie (Haarlem, The Netherlands). All other reagents and chemicals were from Sigma-Aldrich (St. Louis, MO) unless otherwise stated. The toxicities of the compounds were assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay with TAMH and Hep G2 cells.31 TAMH cells were cultured in serum-free, HEPES-buffered DMEM/F12 medium supplemented with ITS (5 μg/mL insulin, 5 μg/mL transferrin, and 5 ng/mL selenium), 100 nM dexamethasone, 10 mM nicotinamide, and 2 mg/L gentamicin. HepG2 cells were cultured in MEM with 10% FBS. Trypsin-EDTA was used to passage cells at 70−90% confluence, and it was inhibited by 0.5 mg/mL soybean

trypsin inhibitor in PBS before the cells were plated. Cultures were maintained in a humidified incubator with 5% carbon dioxide/95% air at 37 °C. The compounds were first screened at 200 μM, and following that, the compounds that showed cell viability less than 50% of the cells were tested for their LC50 values. Determination of P450 Metabolic Stability. Fifty-donor pooled mixed gender human liver microsomes (P450) (20 mg/mL) were purchased from XenoTech (Lenexa, KS). Nicotinamide adenine dinucleotide phosphate (NADP+) was from ENZO (Farmingdale, NY). UDPGA was from Nacalai Tesque (Kyoto, Japan). HPLC/ spectro grade acetonitrile was from Tedia Company (Fairfield, OH). All other reagents and chemicals were from Sigma-Aldrich (St. Louis, MO) unless otherwise stated. The metabolic stabilities of the compounds were assessed by incubating 3 μM of the compound in acetonitrile with pooled P450 in a system consisting of 100 mM potassium phosphate buffer (pH 7.4), 0.5 mg/mL P450, 1.3 mM NADP+, 3.3 mM glucose-6-phosphate, 0.4 U/mL glucose-6-phosphate dehydrogenase, 3.3 mM magnesium chloride, and 0.05 mM sodium citrate at 37 °C. The reaction was quenched in ice cold acetonitrile containing 2 μM of internal standard (pheyntoin) at time points of 0, 5, 10, 15, 20, 30, and 60 min. The samples were then vortexed for 30 s and centrifuged at 13,000 rpm for 10 min at 4 °C. The supernatant was collected and the concentration of the compounds tested was determined using high performance liquid chromatography−tandem mass spectrometry (LC-MS/MS). A plot of the natural logarithm of the area ratio against time was obtained using GraphPad Prism 5 (San Diego, CA, USA), and the slope of the linear regression (−k, where k is the elimination rate constant) was used to determine the in vitro half-life (t1/2). For compounds with t1/2 < 60 min, the in vitro intrinsic clearance (CLint) was determined. All compounds tested were stable over the incubation period in their respective negative controls, which did not contain the NADPHregenerating system. Metabolite Identification and GSH Trapping Assay of Metabolically Unstable Compounds. The metabolite identification of the metabolically unstable compounds were carried out by incubating 50 μM of the compound with P450 in a system consisting of 100 mM potassium phosphate buffer (pH 7.4), 0.5 mg/mL P450, 1.3 mM NADP+, 3.3 mM glucose-6-phosphate, 0.4 U/mL glucose-6phosphate dehydrogenase, 3.3 mM magnesium chloride, and 0.05 mM sodium citrate with or without the presence of 5 mM GSH at 37 °C for 1 h. The samples were quenched in 4× ice-cold acetonitrile, vortexed for 30 s, and centrifuged at 13,000 rpm for 10 min. The supernatant was collected, and the solvents were evaporated under N2 at 30 °C to dryness. Then, the samples were reconstituted in 200 μL of water/acetonitrile (95:5) and filtered. The filtered samples were subjected to LC-MS/MS for analysis. LC-MS/MS Analysis for Metabolic Stability Assays. Quantitative LC-MS/MS of the samples from the metabolic stability assay was performed on an Agilent 1200 Series LC system (Santa Clara, CA, USA) connected to a AB Sciex Qtrap 3200 (AB Sciex, Framingham, MA). The column used was a Phemonenex Luna C18 100 Å, 3.0 μm, 50 mm × 3.00 mm column (Torrance, CA, USA). The mobile phase system was first run at 2% acetonitrile (0.1% formic acid)/water (0.1% formic acid) with a flow rate of 450 μL/min at 27 °C. The organic phase was then increased to 98% in 1 min and maintained for 2 min. Then, the organic phase was decreased back to 2%. The total run time was 9.5 min. Negative ion ESI LC-MS/MS in multiple reaction monitoring (MRM) mode was carried out to quantify the amount of compound in each sample. The declustering potential (DP), entrance potential (EP), collision energy (CE), and collision cell exit potential (CXP) were optimized for each individual compound. LC-MS/MS Analysis for Metabolite Identification Assays. LCMS/MS of the samples from the metabolite identification assay was performed on an Agilent 1290 Infinity LC system connected to a AB Sciex Qtrap 5500 (AB Sciex, Framingham, MA). The column used was a Phemomenex Kinetex C18 100 Å, 1.7 μm, 100 mm × 2.10 mm column (Torrance, CA, USA). The mobile phase system was first run at 2% acetonitrile (0.1% formic acid)/water (0.1% ammonium formate) for 2 min, with a flow rate of 450 μL/min at 30 °C. Then, 2022


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Chemical Research in Toxicology Table 1. % TAMH Cell Viability at 200 μM and LC50 Values of FMMHs Derivatives

toxicity

a

1

2

a

compd

R

R

X

Y

milogP

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

H benzylidene propylidene benzylidene H benzylidene propylidene benzylidene H benzylidene propylidene benzylidene H benzylidene propylidene benzylidene benzyl benzyl benzyl S-benzyl R-benzyl

CH2CO2H H H CH2CO2H CH2CO2H H H CH2CO2H CH2CO2H H H CH2CO2H CH2CO2H H H CH2CO2H H H H H

S S S S O O O O S S S S O O O O S O S O

S S S S S S S S NH NH NH NH NH NH NH NH S S NH NH

−1.21 1.82 1.11 1.14 −1.75 1.48 0.77 −0.80 −1.95 1.08 0.36 −1.96 −2.49 0.74 0.02 0.05 2.00 1.46 1.26 0.72

% viability at 200 μM

LC50 (μM)

96.2 7.40 9.29 90.0 102 44.1 36.0 110 81.2 52.4 46.9 67.4 102 106 72.4 74.3 33.9 44.6 74.1 75.9 82.3

>200 28.8 ± 11 22.5 ± 3.1 >200 >200 144 ± 28 81.8 ± 0.8 >200 >200 >200 >200 >200 >200 >200 >200 >200 107 ± 3.1 99.7 ± 0.5 >200 >200

milogP calculated using the online Molinspiration property calculator (Slovak Republic).

the organic phase was increased to 95% in 13 min and maintained for 2 min. Subsequently, the organic phase was decreased to 2% in 1 min. The total run time was 22.0 min. An enhanced mass scan followed by enhanced product ion (EMS-EPI) analysis was carried out to identify metabolites in each sample while neutral loss followed by enhanced product ion (NL-EPI) analysis was carried out to identify the structures of GSH trapped metabolites. The EMS scan was run in positive mode at a scan range of m/z 50 to m/z 600 and a scan rate of 10000 Da/s with DP of 70 V, EP of 10 V, and CE of 10 V. The NL scan of m/z 129 was run in negative mode at a scan range of m/z 200 to m/z 1000 and a scan rate of 2000 Da/s with DP of 70 V, EP of 10 V, CE of 10 V, and CXP of 12 V. Information-dependent acquisition (IDA) was used to trigger the acquisition of EPI spectra for ions greater than 5000 cps with exclusion of ions for 5 s after three occurrences for the two most intense peaks for both NL and EMS scans. The EPI scan was run in positive mode for daughter ions from m/z 50 to m/z 600 at a scan rate of 10 000 Da/s and a LIT fill time of 1.0 ms with DP of 70 V, EP of 10 V and CE of 23 V. Negative ion ESI LC-MS/MS in multiple reaction monitoring (MRM) mode was carried out to determine the retention times of 4hydroxy-5-benzylidene, 2-hydroxy benzylidene, 4-hydroxy benzyl and 2-hydroxy benzyl rhodanine, and 5-benzyl thiohydantoin. The declustering potential (DP), entrance potential (EP), collision energy (CE), and collision cell exit potential (CXP) were optimized for each individual compound. Dansyl-GSH (dGSH) Depletion Assay. Dansyl-GSH (dGSH) was reported by Gan et al. to be a trapping agent for the quantitative estimation and identification of reactive metabolites in 2005.32 Thus, dGSH was used in this assay to quantify the amount of reactive metabolites formed from the metabolism of FMMH. Dansyl-GSH (dGSH) was synthesized following the procedure reported by Gan et al.32 The dGSH depletion assay was carried out by incubating 50 μM of the compound with P450 in a system consisting of 100 mM potassium phosphate buffer (pH 7.4), 0.5 mg/mL P450, 1.3 mM NADP+, 3.3

mM glucose-6-phosphate, 0.4 U/mL glucose-6-phosphate dehydrogenase, 3.3 mM magnesium chloride, and 0.05 mM sodium citrate with or without the presence of 100 μM GSH at 37 °C for 1 h. The samples were quenched in 4× ice-cold methanol containing 50 μM DTT, vortexed for 30 s and centrifuged at 13,000 rpm for 10 min. The samples were subjected to HPLC (Shimadzu, Japan) for analysis. A fluorescence detector (Shidmazu, Prominence RF-20A) was used to detect and quantify the amount of dGSH. A Phenomenax Kinetex 2.6 μm C18 100 Å (150 × 4.60 mm, 2.6 μm) column was used to separate the dGSH adducts using a gradient mobile phase system with a flow rate of 1.0 mL/min. The mobile phase system was first run at 20% acetonitrile (0.1% formic acid)/water (0.1% formic acid) for 2 min, Then, the organic phase was increased to 50% in 10 min. Subsequently, the organic phase was increased to 90% in 10 min and maintained at the concentration for 5 min. Then, the organic phase was decreased to 20% in 1 min. The total run time was 30.0 min. The amount of dGSH depleted was quantified by comparing the peak area in the fluorescence chromatograph with a standard curve using dGSH as an external standard. N-Ethylmaleimide (NEM) was used as the positive control for this experiment. Statistical Analysis. The lipophilicities of compounds were evaluated using the milogP values. The higher the milogP values, the higher the lipophilicity. The milogP of the compounds were calculated using the online Molinspiration software (Slovak Republic). The correlation between LC50 and milogP was analyzed using Spearman’s rank order correlation (rs). The correlation was calculated using GraphPad Prism 5 (San Diego, CA, USA). Statistical significance refers to P < 0.05 (two-tailed).

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RESULTS Toxicity of FMMHs on TAMH and HepG2 Cells. In the initial screen, compounds were tested at a single concentration (200 μM) to evaluate the threshold for toxicity. In this study, compounds that reduced cell viability by 50% at 200 μM and 2023


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Chemical Research in Toxicology Table 2. % HepG2 Cell Viability at 200 μM and LC50 Values of FMMHs Derivatives

toxicity

a

1

2

compd

R

R

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21



a

X

Y

milogP



−1.21 1.82 1.11 1.14 −1.75 1.48 0.77 −0.8 −1.95 1.08 0.36 −1.96 −2.49 0.74 0.02 0.05 2 1.46 1.26 0.72

% viability at 200 μM

LC50 (μM)

84.5 64.1 70.6 83.0 96.9 99.3 79.5 87.1 90.8 95.5 85.1 90.5 96.2 82.9 89.7 92.3 59.8 79.6 79.3 81.3 111.1

>200 >200 >200 >200 >200 >200 >200 >200 >200 >200 >200 >200 >200 >200 >200 >200 >200 >200 >200 >200


Table 3. Metabolic Half-Life (t1/2) and Intrinsic Clearance (CLint) of FMMHs Derivatives

metabolic stability

a

1

2

a

compd

R

R

X

Y

milogP

t1/2 (min)

CLint (mL/min/mg protein)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21





−1.21 1.82 1.11 1.14 −1.75 1.48 0.77 −0.80 −1.95 1.08 0.36 −1.96 −2.49 0.74 0.02 0.05 2.00 1.46 1.26 0.72

>60 24.6 26.9 >60 >60 >60 >60 >60 >60 9.8 >60 >60 N.D. >60 N.D. >60 24.7 >60 >60 >60

N.D. 0.056 0.052 N.D. N.D. N.D. N.D. N.D. N.D. 0.141 N.D. N.D. N.D. N.D. N.D. N.D. 0.056 N.D. N.D. N.D.


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Figure 4. XIC trace and EMS spectra of metabolites of compound 2.

between 80 and 145 μM and are thus only moderately toxic compared to rhodanines 2 and 3. However, no distinct LC50 curve was observed for 5-benzylidene thiohydantoin 10, and it was deemed to be not toxic below 200 μM (LC50 > 200 μM). In the initial screen for the HepG2 cell line, none of the compounds showed less than 50% cell viability (Table 2). Only 3 compounds showed less than 75% cell viability when incubated with compounds at 200 μM. These are the C5substituted rhodanine compounds, 2, 3, and 17 (64.1%, 70.6%, and 59.8%, respectively). Metabolic Stability of FMMHs. The metabolic stability of FMMHs were evaluated by determining the half-life (t1/2) of the compounds upon P450 incubation at 37 °C for 60 min. Apart from hydantoin 13, where the mass of the compound was

exhibited LC50 values of less than 50 μM are categorized as toxic; compounds that reduced cell viability by 25% and exhibited LC50 values between 50 and 200 μM are categorized as moderately toxic, and compounds that did not show cell viability at the highest dose of 200 μM are categorized as nontoxic. From this screen, only seven compounds reduced the viability of the TAMH cells by more than 50%, i.e., the C5substituted rhodanines (compounds 2, 3, and 17), thiazolidinediones (compounds 6, 7, and 18), and thiohydantoin 10 (Table 1). These potentially toxic compounds were subjected to LC50 determination. Only 5-benzylidene and 5-propylidene rhodanines (2 and 3) exhibited significant toxicity, i.e., at 28.8 μM and 22.5 μM, respectively. The LC50 of 5-benzyl rhodanine 17 and the C5-substituted thiazolidinediones (6, 7, and 18) are 2025


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Metabolite Identification of Metabolically Unstable Compounds. Attempts were then made to identify the metabolites for compounds 2, 3, 10, and 17. The compounds were incubated with P450 at 37 °C for 60 min, and positive EMS-EPI scan mode was used to identify the metabolites. For 5-benzylidene and 5-benzyl rhodanines (2 and 17), oxidation products with masses of 238.0 and 240.0, respectively, were observed at 6.2 and 5.5 min (Figures 4 and 5). Plausible structures for these metabolites are the hydroxylated products of the benzene ring (Figure 7a and b). In contrast, a metabolite

too low to obtain reliable readings, the metabolic stabilities of all compounds were successfully measured (Table 3). Similar to the trend for toxicity on TAMH cells, most of the compounds were found to be metabolically stable compared to their respective negative controls, with k close to 0 min−1, and do not have a measurable t1/2. Only the 5-benzylidene, 5-propylidene, 5-benzyl rhodanines (compounds 2, 3, and 17, respectively) and 5-benzylidene thiohydantoin (compound 10) have t1/2 of 24.6, 26.9, 24.7, and 9.8 min, respectively. 2026


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subjected to MRM scan mode to determine their retention times. The MRM scan methods of compounds 22−27 were also performed on the P450 samples. In the EMS scan of rhodanine 2, an oxidation product with a retention time of 6.18 min was detected. However, the retention times of the proposed metabolites rhodanines 22, 23, and 24 were at 8.66, 7.93, and 8.14 min, respectively (Table 4). Thus, the oxidation product detected via the EMS scan in the P450 sample was not the predicted hydroxylation products. When the MRM scan files of rhodanines 22, 23, and 24 were run on the P450 sample, a product with the retention time of 7.96 min was detected. Therefore, rhodanine 23 with the retention time of 7.93 min is one of the metabolites of rhodanine 2. Similarly for rhodanine 17, the oxidation product

resulting from the incubation of 5-benzylidene thiohydantoin 10 with P450 displayed a mass of 207.0 at 6.0 min consistent with a reduced product (Figure 6). This could be the saturated product 19 (Figure 7c). No identifiable metabolites were detected for 5-propylidene rhodanine 3. Validation of Metabolites Identified for Compounds 2, 10, and 17. On the basis of the fragmentation pattern of the mass spectra for the metabolites identified for 2, 10, and 17 (Figure 7), we proposed that the metabolites for rhodanines 2 and 17 were hydroxylated at the benzene ring to form rhodanines 22, 23, or 24 and 25, 26, or 27 while the metabolite of thiohydantoin 10 was compound 19. To confirm the proposed oxidation and reduction products, authentic 22−27 (Figure 8) were synthesized. Compounds 22−27 were then 2027


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Figure 7. EPI spectra of proposed metabolite of (a) compound 2, (b) compound 17, and (c) compound 10. Proposed structures of metabolites and fragmentation pathways are shown.

detected using the EMS scan was not the hydroxylation product 25, 26, or 27 as they have different retention times. However, rhodanine 26 with a retention time of 7.10 min is one of the oxidation products of rhodanine 17 as a product with the retention time of 7.11 min was detected when the MRM scan files of rhodanines 25, 26, and 27 were run on the P450 samples. Therefore, based on the observations above, one of the metabolites from the P450 metabolism of rhodanines 2 and 17 arose from the parahydroxylation of the benzene ring of

rhodanines 2 and 17. The identity of the metabolites detected with the EMS scan has not been ascertained. As for 5-benzylidene thiohydantoin 10, the reduction product was indeed the saturated compound 19 as the retention time of the metabolite in the EMS scan is the same as the retention time of compound 19, which is at 6 min. GSH Trapping of Reactive Metabolites. GSH-trapping experiments on compounds 2, 3, 10, and 17 were carried out to identify the reactive metabolites generated by P450. One 2028


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reactivity with dGSH or a rapid reversibility of any products formed.

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DISCUSSION A total of 21 compounds across 4 FMMH scaffolds based on the thiazolidines and imidazolidines were synthesized to evaluate the hepatotoxicities and metabolic stabilities of the FMMHs and assess their suitability as scaffolds for drug discovery and development. The results from the toxicity studies showed that only the C5-substituted FMMHs are toxic to the TAMH cells (Compounds 2, 3, 6, 7, 17, and 18) and that only C5substituted rhodanines showed toxicities (albeit low) to HepG2 cells (compounds 2, 3, and 17). From the TAMH cells studies, it was observed that among the four FMMHs scaffolds, similarly substituted rhodanines are the most toxic (LC50 ca. 20−110 μM), followed by the thiazolidinediones that are moderately toxic (LC50 ca. 80−145 μM), and the thiohydantoins and hydantoins that are nontoxic (LC50 > 200 μM). The results from HepG2 cells also supported the observation from the TAMH cells that the rhodanine derivatives are the most toxic among the four scaffolds, but the magnitude of these toxicities is lower in comparison. As lipophilicity is known to be a key parameter in conferring toxicity to liver cells, we compared the lipophilicity of the compounds in order to rationalize the observed trend.33,34 The milogP values were calculated using the best available model Molinspiration program (Table 1). miLogP is a property for the measurement of the lipophilicity of a compound through fitting the calculated logP with experimental logP for a set of 12,000 compounds. Spearman’s rank order correlation between the LC50 values and milogP was calculated using GraphPad Prism 5 to evaluate their relationship.35 The calculation revealed that the toxicities of FMMHs are partially correlated to their lipophilicities with the rs value of −0.66. This indicates that the higher the lipophilicity of the FMMHs, the greater the toxicity. Such correlation between lipophilicity and toxicity could also be found in the 2phenylaminophenylacetic acid derived NSAIDS, local anesthetic agents, flavonols, and compounds from company databases.33,34,36 Therefore, for FMMHs with the same substituents, rhodanines are the most toxic followed by the thiazolidinediones, thiohydantoins, and hydantoins, and their lipophilicities increase with the same trend. Analysis of the toxicity results within the FMMHs showed that toxicity is strongly affected by the substituents. Among the rhodanines and thiazolidinediones, the C5-substituted rhodanines and thiazolidinediones (compounds 2, 3, 6, 7, 17, and 18) exhibit TAMH cell toxicity, but the rhodanines and thiazolidinediones (compounds 1, 4, 5, and 8) with the hydrophilic N3-carboxymethyl substituent are nontoxic. In this case, the installed N3-carboxymethyl substituent decreases the lipophilicities of the compounds, rendering the compounds nontoxic. Within the C5-substituted rhodanines and thiazolidinediones, the 5-benzylidene and 5-propylidene substituted compounds (2 and 6, and 3 and 7) were also more toxic than the 5-benzyl substituted compounds (17 and 18). As these compounds possess similar lipophilicities, such differences in toxicities maybe a result of the chemical reactivity or metabolism of the compounds. In order to determine the P450 metabolic stability across the FMMHs derivatives and to assess if there was a correlation between P450 metabolic stabilities and the toxicities observed, the metabolic profiles of the compounds were evaluated. The

Figure 8. Proposed oxidation products for rhodanines 2 and 17.

Table 4. Retention Times of the Metabolites of Compounds 2, 10, and 17 and the Retention Times of Compounds 19, 22, 23, 24, 25, 26, and 27 compd

mass detected in EMS+

RT (min)

MRMa

2

238

6.18

17

240

5

10 19 22 23 24 25 26 27

207

6

22 23 24 25 26 27 19

RT (min) 6.03, 6.03, 6.03, 5.23, 5.23, 5.23, 6 6 8.66 7.93 8.14 8.05 7.11 7.46

7.96 7.96 7.96 7.10, 8.07 7.10, 8.07 7.10, 8.07

MRM scan mode method file of the compounds indicated in this row was used to run the P450 samples.

a

glutathionylated product was captured for 5-benzylidene rhodanine 2 and 5-propylidene rhodanine, both with and without the presence of activated P450. The masses of the GSconjugates were 529.1 and 481.0, respectively (Figures S1 and S2 in Supporting Information, which correspond to compounds 28 and 29 (Figure 9). These GS-conjugates identified for rhodanines 2 and 3 suggest that the compounds 2 and 3 are likey Michael acceptors and that their reactivities may contribute to their toxicities observed in TAMH cells. No GS-conjugates were identified for 5-benzylidene thiohydantoin 10 and 5-benzyl rhodanine 17 indicating that the metabolites generated were not reactive. Dansyl-GSH (dGSH) Depletion Studies. The dGSH depletion studies were carried out in order to quantify the amount of reactive metabolites formed for rhodanines 2 and 3. Such quantification would allow us to correlate the formation of reactive metabolites with the toxicities observed. N-Ethylmaleimide (NEM) was used as a positive control to validate the assay employed. As shown in Figure 10b and c, a new adduct peak was formed when dGSH was incubated with NEM in both inactivated and activated P450. A total of 37% of dGSH depletion was observed for the negative control where P450 was inactivated, and 29% of dGSH depletion was observed for the sample where P450 was activated. Dimerization of dGSH was also observed in the P450 activated samples (Figure 10a and c). For rhodanines 2 and 3, no depletion of dGSH was detected when the compounds were incubated with dGSH in both inactivated and activated P450, indicating a lack of 2029


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Figure 9. EPI spectra of proposed GS-conjugate of (a) compound 2 and (b) compound 3. Proposed GS-conjugate structures 28 and 29 and the fragmentation pathway are shown.

Figure 10. Fluorescence chromatogram of (a) dGSH incubated with activated P450; (b) dGSH and NEM incubated with inactivated P450; and (c) dGSH and NEM incubated with activated P450.

metabolically unstable. This suggests that the observed high toxicities on TAMH cells and low toxicities on HepG2 cells exhibited by the rhodanine derivatives may be related to the susceptibility of these rhodanines toward P450 metabolism to generate reactive metabolites. In our subsequent experiments to identify possible reactive metabolite-mediated toxicity, we were

half-lives of the compounds in the presence of P450 were determined as an indicator of overall metabolic stability. We reason this to yield a representative profile because the known pharmacokinetics of related compounds are primarily metabolized by Phase I pathways. We identified that the C5 substituted rhodanines (compounds 2, 3, and 17) are 2030


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toxic than others. In the former, phase 1 metabolic profiling of the FMMHs identified 5-substituted rhodanines to be unstable and prone to oxidation and 5-benzylidene thiohydantoin to be the most unstable and prone to reduction. These results suggest that the presence of both endocylic sulfur and the thiocarbonyl group or a combination of C5-benzylidene and the thiocarbonyl group in FMMH rings may be recognition motifs for P450. In contrast, the thiazolidinediones and hydantoins are all metabolically stable. In terms of toxicities, among the four FMMH scaffolds evaluated, the rhodanines were found to be the most toxic compounds in both TAMH and HepG2 cells. However, it is not clear if the toxicities can be attributed to the inherent chemical reactivities of the compounds in question, or due to the formation of reactive metabolites. Regardless of the mechanisms of toxicities, our studies have highlighted that it is possible to improve the metabolic and toxicity profiles of rhodanines by installing hydrophilic substituents as demonstrated by rhodanine 4. This ability to “tune” the properties of compounds through its substituents suggests that in the context of toxicities and metabolic stabilities at least, rhodanines (and related FMMHs) should not be excluded as scaffolds for drug discovery and development. FMMHs could still be attractive hit compounds in drug discovery owing to their ease of derivatization and synthetic tractability.

able to discern that the benzene hydroxylation at the para position was one of the metabolic pathways for both rhodanines 2 and 17. This metabolic pathway is in accordance with the metabolic pathway of epalrestat where mono- or dihydroxylated metabolites were identified.16 However, GSH trapping studies of reactive metabolites show that glutathionylated adducts were formed with rhodanines 2 and 3, in the presence of both activated and nonactivated P450. This suggests that the two rhodanines are Michael acceptors under these conditions. An implication of this is that the observed toxicities of 5-benzylidene rhodanine 2 and 5propylidene rhodanine 3 on TAMH cells (LC50 = 28.8 and 22.5 μM, respectively) may be due to Michael acceptor reactivities of rhodanines 2 and 3 which in turn may result in nonspecific reactivity toward biological nucleophiles in TAMH cells. Attempts to quantify the Michael adducts formed using the dansyl GSH (dGSH) depletion assay showed that when using a 2-fold excess of dGSH, no depletion of dGSH was observed for both rhodanines 2 and 3. These results suggest that under these conditions, the amount of Michael adducts formed between the rhodanine derivatives and dGSH is either not detectable or is reversible. The observation from the dGSH depletion studies is in agreement with the claims by Mendegen et al., which states that the intrinsic Michael acceptor reactivities of FMMHs are insignificant.10 Moreover, our observations from the GSH trapping assays are also in agreement with the findings of Arsovska et al. where 5benzylidene rhodanine was found not to be totally unreactive toward cysteamine, and thus, there is a probability for the GSadduct to be captured in the LC-MS/MS analysis.37 However, in view of the possibility of retro-Michael reactions, the reaction conditions (concentrations, reagents, e.g., GSH versus dGSH, etc.) may influence the formation and thus the detection of the GS-adducts. In view of this, it is premature to conclude that the observed toxicities of rhodanines 2 and 3 are due to the inherent chemical reactivities of the vinylidene moiety. As the toxicities of rhodanines 2 and 3 are significantly lower on the less metabolically active HepG2 cells (only 64.1% and 70.6% cell viability at 200 μM cell viability, respectively) as compared to TAMH cells, the contribution of reactive metabolite formation to toxicity still cannot be excluded. Nonetheless, the depletion studies suggest that the observed half-lives of the compounds might not be due to the reaction between the FMMH derivatives with P450 but rather due to the metabolism of the compounds by P450. Interestingly, thiohydantoin 10, which is nontoxic to cells, is the least stable compound with a half-life of 9.8 min, suggesting that thiohydantoin 10 was likely transformed into chemically inert metabolites. This proposition was proven when we successfully identified 5-benzyl thiohydantoin 19 as the metabolite for thiohydantoin 10. The identification of 5-benzyl thiohydantoin 19 is unexpected as vinyl groups are generally metabolized into epoxides following P450 incubation. It is speculated that the reduction of the CC into C−C is mediated by reductases found in P450 as is evident from the BYZX ((E)-2-(4-((diethylamino)methyl)benzylidene)-5,6-dimethoxy-2,3-dihydroinden-one) and shogaol metabolism studies by Yu et al. and Chen et al., respectively.38,39 From our toxicity and metabolic stability studies, we infer that thiohydantoin 19 is nontoxic and metabolically stable against P450 metabolism. Our studies above with the FMMHs show that some scaffolds are more prone to P450 metabolism and/or are more

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.5b00247. Spectroscopic data for compounds; TIC and XIC trace and neutral loss spectra of compounds 2 and 3; and precursor and product ions utilized for MRM analysis in the determination of Phase I metabolic stability (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*H.H.K. Tel: 65-6516 7963. E-mail: [email protected]. *C.L.L.C. Tel: 65-6601 1061. E-mail: [email protected]. Funding

This work was supported by a start-up grant [R-148-000-146133] to C.L.L.C. and a grant to H.H.K. [R-148-000-187-112]. Notes

The authors declare no competing financial interest.

■

ABBREVIATIONS FMMH, five-membered multiheterocycle; TAMH, transforming growth factor mouse α hepatocyte; FDA, food and drug administration; PAINS, pan assay interference compounds; DMF, dimethylformamide; AR, analytical reagents; ACN, acetonitrile; HRMS, high resolution mass spectrometry; calcd, calculated; ESI, electron-spray ionization; MRM, multiple reaction monitoring; EMS, enhanced mass scan; EPI, enhanced product ion; NL, neutral loss; BYXZ, ((E)-2-(4((diethylamino)methyl)benzylidene)-5,6-dimethoxy-2,3-dihydroinden-one; DTT, DL-dithiothreitol; NEM, N-ethylmaleimide

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