Identification and Mitigation of Reactive Metabolites of 2

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Identification and Mitigation of Reactive Metabolites of 2Aminoimidazole-Containing Microsomal Prostaglandin E Synthase-1 Inhibitors Terminated due to Clinical Drug-Induced Liver Injury Bryan H. Norman, Matthew J Fisher, Matthew A. Schiffler, Steven L. Kuklish, Norman E. Hughes, Boris A Czeskis, Kenneth C Cassidy, Trent L Abraham, Jeffrey J Alberts, and Debra Luffer-Atlas J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01806 • Publication Date (Web): 09 Feb 2018 Downloaded from http://pubs.acs.org on February 11, 2018

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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Medicinal Chemistry

Identification and Mitigation of Reactive Metabolites of 2-AminoimidazoleContaining Microsomal Prostaglandin E Synthase-1 Inhibitors Terminated due to Clinical Drug-Induced Liver Injury. Bryan H. Norman,*† Matthew J. Fisher,† Matthew A. Schiffler,*† Steven L. Kuklish,† Norman E. Hughes,† Boris A. Czeskis,ǁ Kenneth C. Cassidy,ǁ Trent L. Abraham,ǁ Jeffrey J. Alberts,ǁ Debra LufferAtlasǁ †

Discovery Chemistry Research and Technologies and ǁDrug Disposition, Lilly Research Laboratories, A

Division of Eli Lilly and Company, Indianapolis, Indiana 46285, USA Abstract Two 2-aminoimidazole-based inhibitors, LY3031207 (1) and LY3023703 (2), of the microsomal prostaglandin E synthase-1 (mPGES-1) enzyme were found to cause drug-induced liver injury (DILI) in humans. We studied imidazole ring substitutions to successfully mitigate reactive metabolite (RM) formation. These studies support the conclusion that RM formation may play a role in the observations of DILI and the consideration of 2-aminoimidazoles as structure alerts, due to the high likelihood of bioactivation to generate RMs. Introduction In recent years, there has been significant interest in the factors and mechanisms associated with druginduced liver injury (DILI) since preclinical assessments of drug candidates often do not predict human DILI.1 Several assessment and mitigation strategies have been reported that focus on approaches such as structure alert avoidance, reactive metabolite (RM) mitigation and decreasing the potential of drug candidates to inhibit liver transporters, such as the bile salt export pump (BSEP).2 Additionally, many groups focus on delivering drug candidates with a very low projected human dose, as the empirical

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association of high daily dose with DILI is strong. Structure alerts can be described as specific structural moieties that, when present in drugs (or drug candidates), have been associated with adverse drug reactions (ADRs).2a Frequently, structure alerts align with known reactive metabolite risks due to the bioactivation of specific functional groups present in the drug or drug candidate. Currently, there is no consensus on how to definitively categorize structure alerts on the basis of DILI risk potential. Furthermore, there are many safe and successful drugs that contain structure alerts, form RMs, inhibit liver transporters and/or have high recommended daily doses. However, since these attributes confer no advantage to a drug candidate, many try to avoid these risks whenever possible. When not possible, the decision to develop a drug candidate with known risks becomes part of a complex risk-benefit assessment based on other important factors, such as the seriousness of the disease (e.g. life-threatening vs. non-lifethreatening), duration of treatment (acute vs. chronic) and unmet medical need. In 2011 and 2012, two 2-aminoimidazole-based inhibitors (LY3031207, 1 and LY3023703, 2)3 of the microsomal prostaglandin E synthase-1 (mPGES-1) enzyme entered clinical development for the treatment of osteoarthritis pain (Figure 1). While these agents had robust safety margins in preclinical safety assessment studies, both were ultimately terminated from clinical development due to the emergence of liver safety signals, such as increased alanine aminotransferase (ALT), during phase 1 multiple ascending dose studies.3c, 3d In an effort to understand the origins of these signals, we have studied these agents (and several related analogs) in various preclinical models often used to assess DILI risk, especially with regard to their propensity to form RMs.

Figure 1. mPGES-1 inhibitors terminated because of human DILI.


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At the time of these clinical investigations, the 2-aminoimidazole functionality was not considered a structure alert, nor had it been reported to form RMs. Nonetheless, the substructure is isoelectronic with 2-aminothiazole, which is a well recognized structure alert and has been associated with RM formation and DILI.4 Obach and co-workers reported on the differential liver safety profiles of the non-steroidal anti-inflammatory drugs (NSAIDs) sudoxicam 3 and meloxicam 4, both of which contain the 2aminothiazole substructure and have maximum daily recommended doses under 50 mg (Scheme 1).5 The clinical development of 3 was terminated due to severe hepatotoxicity while 4 is considered to be a safe marketed drug. Obach and co-workers reported that the preclinical and clinical metabolic pathways of these agents and their propensity to form RMs may align with the observed clinical safety outcomes. Preclinically, both undergo oxidative metabolism on the thiazole ring to form the intermediate 4,5thiazole epoxides 5 and 6, which can react to form covalent bonds to proteins. However, meloxicam differs from sudoxicam by the addition of a single methyl group on the thiazole ring, providing an additional, non-RM-forming metabolic pathway, as summarized in Scheme 1. Specifically, methyl hydroxylation to form 10, followed by further oxidation to acid 11, is a pathway only possible for meloxicam. Support for the formation of thiazole epoxide metabolites was established by the observation of diols (7 and 8) and the common acylthiourea 9, which is formed after ring fragmentation and loss of the dicarbonyl species. These pathways were consistent across human liver microsomes (HLMs), nonclinical in vivo studies (rodents), and in humans. It was shown that the oxidative metabolism on the methyl group of 4 offered another metabolic pathway that minimized RM formation and may have contributed to its improved liver safety profile. More recently, there has been a report of RM formation (human and rodent microsomes) with some unsubstituted 2-aminoimidazoles.6 Due to the structural similarity and electronics between 2-aminoimidazoles and 2-aminothiazoles, we explored the potential that a similar metabolic pathway may be associated with the observed clinical hepatotoxicity of 2aminoimidazoles 1 and 2.



Scheme 1. Sudoxicam (3) and Meloxicam (4) have divergent metabolic pathways that align with observed human DILI.

Results Preclinical and clinical metabolites of 1. Drug candidate 1 is an inhibitor of mPGES-1 (inhibition of PGE2 formation in human whole blood IC50 = 0.24 µM) with a good preclinical safety profile.3a, 3b However, during the multiple ascending dose clinical trial (25, 75 and 225 mg per day for one month), several human subjects experienced significantly increased ALT levels (5-45x ULN) and DILI-related symptoms, such as hepatocellular injury and hypersensitivity. These clinical observations, along with the full clinical and non-clinical safety profiles, have been described in a recent manuscript by Jin and coworkers.3d They also described the identification of the metablolites, which are summarized, along with the preclinical in vitro and in vivo metabolites, in Figure 2. We have characterized the metabolites that were identified after incubation in rat, dog and human hepatocytes, as well as in human liver microsomes (HLMs) fortified with glutathione. Finally, we characterized metabolites that were observed in vivo from the plasma of rats and dogs. Table 1 shows the metabolites that were identified in each of these systems. All metabolites were identified on the basis of LCMS, tandem mass spectral (MS/MS) fragmentation


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patterns, HRMS and, in the case of 1-M4, independent total synthesis. The data supporting all metabolite assignments can be found in the Supporting Information (Figures S1-S5; Tables S1-S3). For metabolites 1-M1-1-M5, there was good alignment between the in vitro, in vivo and clinical metabolites. A conjugated metabolite (1-M7) was identified in human urine and several conjugated metabolites were identified in vitro. Based on precedent from the established metabolism of 3 and 4, it seems most likely that the conjugated metabolites of 1, along with 1-M4, 1-M5 and 1-M6, were derived from an intermediate epoxide 1-M10, which was not specifically detected.

Figure 2. Chemical structures of all in vitro and in vivo metabolites of 1. *Structure confirmed by independent total synthesis.



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Table 1. Metabolites of 1. In vitroa Metabolite

Hepatocytes

In vivob

HLM+GSH

Rat

Dog

Human

1-M1

R, D, H

P

P

P, U

1-M2

R, D

P

P

U

1-M3

R, D, H

P

P

U

1-M4

R, D, H

P

P

P, U

1-M5

D

P

P

P, U

1-M6

U

1-M7

H

1-M8

H

1-M9 1-M13

U

R H

1-M14

P

P

1-M15

P

P

a

In vitro data indicates the detection of metabolites in rat (R), dog (D) and human (H) hepatocytes, as well

has human (H) liver microsomes. bIn vivo data indicates the detection of metabolites in plasma (P) and urine (U) for the indicated species. Only human urine was assessed.

Preclinical and clinical metabolites of 2. Drug candidate 2 is a highly potent mPGES-1 inhibitor (inhibition of PGE2 formation in human whole blood IC50 = 0.012 µM) with a good preclinical safety profile.3a, 3b The improved potency of 2, relative to 1, translated to a significantly lower projected human efficacious dose of 3 mg per day. Nonetheless, during the multiple ascending dose clinical trial (2.5, 7.5,


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15, and 30 mg per day for one month), one subject manifested significantly increased ALT levels (10x ULN) at a dose of 30 mg per day. Due to the clinical safety experience with the previous related mPGES1 inhibitor, 1, as well as detection of circulating metabolites that were consistent with RM formation, 2 was terminated from clinical development. Similar to 1, we have characterized the metabolites of 2 that were identified in humans, after incubation in rat, dog and human hepatocytes, human liver microsomes (HLMs) fortified with glutathione and from the plasma of rats and dogs (Figure 3a). Figure 3b shows the in vitro metabolites from the related analog 12. Table 2 shows the metabolites of both 2 and 12 that were identified in each system, and were identified on the basis of LCMS, tandem mass spectral (MS/MS) fragmentation patterns, HRMS and, in the case of 2-M1-2-M3, independent total synthesis. The data supporting all metabolite assignments can be found in the Supporting Information (Figures S6-S10; Tables S-S3). While no glutathione conjugates were identified in humans, nor in preclinical species, several were found in vitro. Based on our previous experience with 1, we believe that acylguanidine 2M3, detected in vitro and in humans, was most likely derived from an intermediate imidazole epoxide. In an effort to rule out the possibility that conjugates of 2 could be formed from conjugate addition to the acrylamide metabolite 2-M4, t-butyl analog 12 was profiled and found to form glutathione conjugates identical to those found for 2.



Figure 3. (a) Chemical structures of metabolites of 2. *Structures confirmed by independent total synthesis. (b) Chemical structures of metabolites of 12.


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Table 2. Metabolites of 2 and 12. In vitroa Metabolite

Hepatocytes

In vivob

HLM+GSH

Rat

Dog

Human

2-M1

R, H

P

P

P, U

2-M2

R, D, H

P

P

P, U

2-M3

R, D, H

U

2-M4

R

P

2-M5

P

2-M6

R, H

2-M7

R, H

U

P

P

2-M10

P

P

1-M11

P

P

H

2-M8

H

2-M9

H

12-M1

R, D, H

12-M2

R, D, H

12-M3

R, D, H

12-M4

D

H

12-M5

H

12-M6

H

a

In vitro data indicates the detection of metabolites in rat (R), dog (D) and human (H) hepatocytes, as well

has human (H) liver microsomes. bIn vivo data indicates the detection of metabolites in plasma (P) and urine (U) for the indicated species. Only human urine was assessed.



Reactive Metabolite Studies with related 2-Aminoimidazoles. We are not aware of any drugs containing the 2-aminoimidazole moiety. Thus, this substructure has not been extensively profiled for RM risk and there currently exist no clear RM mitigation strategies. Since there was good alignment between the preclinical and clinical metabolism of two distinct 2-aminoimidazoles (1 and 2), we profiled additional analogs to assess the impact of structure on RM formation. For these studies, we used the hepatocyte metabolite ID and glutathione trapping assays as sentry models to determine relative RM risk in substituted 2-aminoimidazole analogs of 1 (Table 3). Based on the hydroxylated metabolites of 13 and 14, methyl substitution provided a metabolic “soft spot” that could divert metabolism away from ring epoxidation. However, based on the formation of acylguanidines, imidazole ring epoxidation remained a significant metabolic pathway for methyl substituted 2-aminoimidazoles. There was no evidence of GSH conjugation in hepatocytes, however, both 13 and 14 showed multiple GSH (and related) conjugates in the trapping assay. The fragmentation patterns are consistent with incorporation onto the imidazole ring. Compound 15 demonstrated that a methyl hydroxylated analog is further oxidized to the carboxylic acid and did not form epoxide intermediates nor GSH conjugates in the trapping assay. Furthermore, formation of the carboxylic acid results in a more electron deficient imidazole, making it theoretically less prone to ring epoxidation. This hypothesis is supported by additional 2-aminoimidazoles 16, 17 and 18, which did not form epoxides, nor GSH conjugates, perhaps due to the incorporation of electron withdrawing groups. This may be a good mitigation strategy for 2-aminoimidazole bioactivation and is consistent with reports by others7 in a number of other heterocyclic systems.


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Table 3. Substituted 2-aminoimidazoles and metabolites detected after incubation in human and rat hepatocytes and human liver microsomes containing glutathione.

Parent

Metabolites detected in

m/z

GSH Conjugates

hepatocytes

m/z

detected in HLMs

311 13-M1 (P + CysGly - 2H)

525

13-M2 (P + GSH – 2H)

654

13-M3 (P + GSH – 2H)

654

13-M4 (P + O + GSH)

672

365 13 (m/z 349)

365

311 14-M1 (P + CysGly – 538 2H)

14

379

(m/z 363) 668 14-M2 (P + GSH – 2H)



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379

379

None

-

None

376

None

15 (m/z 365)

None 16 (m/z 378)

17 (m/z 360)

470

None 18 m/z 470


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379

270

Discussion and Conclusions Based on our in vitro, in vivo, and human metabolic data for 1, we hypothesize the metabolic pathway shown in Scheme 2. In summary, we propose that 1 undergoes oxidation on both the t-butyl group to form 1-M1 and the imidazole to form epoxide 1-M10. The undetected epoxide 1-M10 can undergo several potential subsequent transformations, such as hydrolysis to the diastereomeric diols 1-M2 and 1M3, which can fragment to form the stable acylguanidine 1-M5. This pathway is directly analogous to the formation of acylthioureas of 2-aminothiazoles, such as 3.5 Since we did not detect the intermediate epoxide, 1-M5 became an important signature metabolite consistent with epoxide formation. Alternatively, the epoxide can react directly with hepatocyte proteins to form covalent adducts, which would not be detected in plasma. It can also be trapped by a soft nucleophile such as glutathione or related circulating thiols to form 1-M11-1-M13. These compounds, after loss of water, could form 1-M71-M9. Finally, the epoxide can isomerize to form the hydroxyimidazole 1-M4, drawn as the enol tautomer. The structure of 1-M4 was definitively assigned on the basis of an independent synthesis, whereby the synthetic material shared identical properties with the metabolite. The metabolic pathway described here is consistent with all of the data from our metabolite identification studies. Metabolite 1M6 was not detected preclinically but was detected in clinical samples and could be formed from metabolite 1-M1 and/or 1-M5.



Scheme 2. Proposed metabolic pathway for 1. *Structures confirmed by independent total synthesis. Based on the data and our studies with 1, we hypothesize the metabolic pathway shown in Scheme 3 for 2. The parent 2 undergoes amide bond hydrolysis to form 2-M6 and, presumably, benzoic acid derivative 2-M12 (not detected). We noted two different hydroxylation products, 2-M1 and 2-M2, both of which were confirmed by independent syntheses. Acrylamide 2-M4 may have been formed by dehydration of one or both of these precursors. It seems likely that the formation of acylguanidine 2-M3 is the result of imidazole epoxidation (to form 2-M13), followed by hydrolysis to diol 2-M14 and fragmentation to 2-M3 (confirmed by independent synthesis). We were unable to definitively assign the location of conjugate 2M7. Due to 4,5-disubstitution on the imidazole, glutathione addition to the imidazole epoxide 2-M13


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could not dehydrate to form a glutathione conjugated imidazole. Thus, we originally hypothesized that the glutathione may add to the acrylamide 2-M4 to form an aliphatic conjugate 2-M7a. However, we could not rule out the potential for a multistep mechanism whereby glutathione may be attached to the imidazole methyl substitutent, or onto the imidazole itself (2-M7b). Both possibilities are outlined in Scheme 3 (red box). Our studies with the t-butyl analog 12 are consistent with glutathione conjugation onto the imidazole portion of the molecule, since the formation of an acrylamide is not possible for 12. Furthermore, our studies with substituted aminoimidazoles provide some rationale for incorporation of the glutathione onto the imidazole (vida infra).

Scheme 3. Proposed metabobolic pathway for 2. *Structures confirmed by independent total synthesis.



Unlike some 5-ring heterocycles, such as thiazoles, thiophenes, thiazoladinediones, pyrroles and furans,2a, 4, 8

the 2-aminoimidazole has not been described as a structure alert. To our knowledge, the first reported

evidence of RM liability in 2-aminoimidazoles came in 2014, when Srivastava and co-workers outlined a pathway where various 2-aminoimidazoles were trapped by glutathione (GSH) and N-acetyl cysteine (NAC) in human liver microsomes.6 One of the pathways described was via trapping of a 4,5-epoxide, similar to our observations. In this case, the epoxide forming metabolite was avoided by the addition of methyl groups onto the ring. However, while this mitigated epoxidation on the ring, they hypothesized a mechanism of oxidation of a methyl group, followed by loss of water to generate new imine methide RMs, which trapped GSH and NAC. In contrast, we show that while methyl hydroxylation of 13 and 14 was observed in hepatocyte incubations, the formation of acylguanidine metabolite 1-M5 suggests the formation of imidazole epoxide intermediates (Scheme 4). GSH trapping experiments provide data in support of the imine methide pathway as well, as proposed in Scheme 4. In the case of 13, two different GSH conjugates were formed and the structures proposed as 13-M2 and 13-M3. This is consistent with isomeric conjugates and two competing pathways. We hypothesize that one conjugate is formed via the epoxide pathway and the other via the imine methide pathway. The detection of 13-M4 provides evidence for epoxide pathway for the metabolism of 13. While we couldn’t definitively assign the conjugates of 13 and 14, fragment m/z 252 is consistent with conjugation on the imidazole. In the case of 14, only single conjugates were formed. Thus, we hypothesize that 14 functions only by the imine methide pathway. Since we showed that 2 was likely conjugated on the imidazole ring, the imine methide pathway may play a role in its oxidation and conjugation as well.


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Scheme 4. Potential competing pathways for substituted 2-aminoimidazoles.

It is noteworthy that the hydroxylated analog 15 did not generate any conjugates in the hepatocyte incubations nor in the GSH trapping assay (Table 3). This seems inconsistent with the mechanism proposed by Srivastava and outlined in Scheme 4. It is possible that methyl oxidation of 13 and 14 produces the imine methide metabolite directly by a hydrogen atom abstraction, followed by single electron oxidation. This mechanism has been previously hypothesized by Rettie and co-workers.9 For compounds 16-18, the lack of observed metabolites indicative of imidazole epoxidation suggests that electron withdrawing groups may mitigate RM formation in 2-aminoimidazoles. The causes of human DILI are multifactorial and associated with many variables, such as dose, metabolic profile, off-target activities that impact liver function, and patient susceptibility. Historically, most have relied on standard non-clinical safety assessment studies to discharge DILI risk and this has resulted in the termination of many drug candidates prior to human studies. Nonetheless, due to the difficulty associated



with preclinical predictive methods and the fact that humans are frequently more sensitive to liver injury compared to lower mammals, DILI remains a recurring problem in drug development. Amongst the variables associated with DILI, understanding the metabolic profile and likelihood of forming RMs has become an important factor in assessing DILI risk. Over the past several years, due to technological advances associated with high throughput assays and detection capabilities, the ability to assess and identify the likely human metabolites using in vitro and in vivo methodologies have become more routine. Drug discovery teams now have the benefit of mitigating RM potential very early in their optimization efforts. Furthermore, these advances have resulted in improved understanding of the functional groups that may result in RM formation. Over the years, many groups in pharma have described preclinical DILI mitigation strategies. In addition to relying on liver signals in non-clinical safety assessment studies, some groups have focused on a variety of in vitro and in vivo methods to assess liver safety.2 Most of these approaches have recommended the avoidance or reduction of RM potential as a key part of their preclinical DILI mitigation strategies. It is tempting to try to use preclinical RM data in a quantitative sense to assess relative human risk. Some have attempted to define safe and unsafe threshold levels of radiolabelled drug binding to hepatocytes and/or microsomes. However, in a detailed analysis of those data, there was no correlation between amount of covalently bound drug and preclinical liver signals.10 Subsequently, it has been shown that covalent binding better correlates with clinical DILI when human doses and exposures are taken into account and the total covalent binding burden is assessed.2b Recently, Brink and coworkers showed that GSH trapping experiments adequately predicted covalent binding of radiolabeled drug in many instances.2d Thus, since GSH trapping experiments can be applied to early preclinical SARs in iterative fashion, this seems to be an excellent risk reduction approach. Finally, patient susceptibility, sensitivity and ability to acclimate to RMs almost certainly plays a role, confounding any definition of safe RM levels. Thus, the most cautious approach of avoiding structure alerts and removing RM potential during lead optimization may be most appropriate. Furthermore, it has been demonstrated that it is


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frequently possible to tune RM potential by changing the steric and/or electronic properties of the susceptible functional groups.

Experimental Section General Methods. All reagents and anhydrous solvents were obtained from commercial sources and used without further purification unless noted otherwise. 1H NMR spectra were recorded on a Bruker 300 MHz spectrometer, a Bruker 400 MHz spectrometer, a Bruker 500 MHz spectrometer, or a Varian 400 MHz spectrometer. 1H NMR chemical shifts are reported in ppm with the solvent as the internal standard (DMSO-d5 2.49 ppm, CHCl3 7.26 ppm). 13C NMR chemical shifts are reported in ppm with the solvent as the internal standard (DMSO-d6 39.52 ppm). 19F NMR chemical shifts are reported in ppm relative to an external standard of CFCl3. 31P NMR chemical shifts are reported in ppm relative to an external standard of 85% H3PO4. Compounds were analyzed for purity by HPLC and HPLC-MS, and unless otherwise stated, purities of synthesized compounds were all found to be >95% by the following HPLC method: Agilent 1100 HPLC with VWD detectors, Waters Exterra MS 4.6 mm × 150 mm × 3.5 µm C18 column; eluent 0.1% formic acid in a gradient of 5% to 100% CH3CN in water over 3.75 min; flow rate 1.0 mL/min; column temperature 50 °C; λ 300 and 214 nm. All SMILES strings generated using OPSIN.11 Chemicals. The preparation of compounds 1 and 2 have been previously described.3a, 12 The preparation of intermediates and metabolites 1-M14,4a 1-M15,3a 2-M53a have been previously described. 2-(Difluoromethyl)-N-(4-methyl-5-(4-(trifluoromethyl)phenyl)-1H-imidazol-2-yl)-5(pivalamidomethyl)nicotinamide (12). Compound 12 was prepared in 27% yield, according to the procedure described for the preparation of 2.12b 1H NMR (400 MHz, DMSO-d6): δ 8.60 (s, 1H), 8.18 (t, J = 5.6 Hz, 1H), 8.01 (s, 1H), 7.79 (d, J = 8.1 Hz, 2H), 7.71 (d, J = 8.1 Hz, 2H), 7.35 (br s, 1H), 4.35 (d, J = 5.6 Hz, 2H), 2.46 (s, 3H), 1.10 (s, 9H). LC/MS (ESI+): 510 (M+H)+.



2-Chloro-N-(4-methyl-1H-imidazol-2-yl)-5-(pivalamidomethyl)benzamide (13). 1-M14 (1.00 g, 3.71 mmol) was treated with 2-amino-4-methylimidazole hydrochloride (1.49 g, 11.1 mmol), BOP (2.46 g, 5.56 mmol), N-methylmorpholine, 1.02 mL, 9.23 mmol), and DMF (37 mL). The mixture was heated to 60 °C with stirring overnight. Then, EtOAc (30 mL) was added, and the mixture was washed with 5% aqueous LiCl (3 × 20 mL). The combined aqueous layers were extracted with EtOAc (2 × 50 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude material was subjected to flash chromatography on silica gel, eluting with a gradient of 0–5% MeOH in dichloromethane, to furnish an off-white solid, which was further purified by reverse-phase chromatography on C18 silica gel, eluting with 0.1% formic acid in a gradient of 10–100% CH3CN/water to furnish 13 as a white solid (41.9 mg, 3.2% yield). 1H NMR (400 MHz, CD3OD): δ 8.25–8.07 (m, 2H), 7.52–7.40 (m, 2H), 7.40–7.32 (m, 1H), 6.62 (s, 1H), 4.37 (d, J = 5.0 Hz, 2H), 2.21 (s, 3H), 1.20 (s, 9H). LC/MS (ESI+): 349/351 (M+H)+, 371/373 (M+Na)+. 2-Chloro-N-(4,5-dimethyl-1H-imidazol-2-yl)-5-(pivalamidomethyl)benzamide (14). 1-M14 (706 mg, 2.62 mmol) and 2-amino-4,5-dimethylimidazole (240 mg, 2.10 mmol) were treated with DMF (6.3 mL), followed by TBTU (971 mg, 2.93 mmol) and DIEA (1.28 mL, 7.33 mmol). The resulting suspension was heated to 80 °C with stirring for 7 hours, then was cooled to room temperature with stirring overnight. The mixture was partitioned between water and EtOAc, then the organic phase was washed with saturated aqueous NaHCO3, water, and saturated aqueous NaCl. The organic phase was concentrated under reduced pressure, then the crude material was subjected to flash chromatography on silica gel, eluting with a gradient of 0–2% (7M NH3 in CH3OH) in EtOAc. The fractions containing 14 were subjected to preparatory centrifugal thin-layer chromatography on a silica gel plate (2 mm thickness), eluting with a gradient of 0–5% (7M NH3 in CH3OH) in CH2Cl2. The fractions containing 14 were concentrated under reduced pressure, and the resulting crystals were washed with 2:1 hexanes:CH2Cl2 and isolated by filtration to furnish 14 as a light tan solid (215 mg, 28% yield). 1H NMR (400 MHz, DMSO-d6): δ 11.40 (s, 2H), 8.09 (t, J = 5.6 Hz, 1H), 7.38 (d, J = 8.1 Hz, 1H), 7.35 (d, J = 2.1 Hz, 1H), 7.21 (dd, J = 8.2, 2.1


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Hz, 1H), 4.22 (d, J = 6.0 Hz, 2H), 2.00 (s, 6H), 1.09 (s, 9H). LC/MS (ESI+): 363/365 (M+H)+, 385/387 (M+Na)+. 2-Chloro-N-(4-(hydroxymethyl)-1H-imidazol-2-yl)-5-(pivalamidomethyl)benzamide (15). 2-ChloroN-(4-(dimethoxymethyl)-1H-imidazol-2-yl)-5-(pivalamidomethyl)benzamide was prepared from 1-tertbutoxycarbonyl-2-amino-4-(dimethoxymethyl)-1H-imidazole13 (565 mg, 2.1 mmol) and 1-M14 (287 mg, 2.1 mmol) essentially according to the procedure described for 13. The resulting product was dissolved in CH3OH (5 mL) and treated with sodium borohydride (152 mg, 4.0 mmol) and stirred at room temperature for 10 min. Then, saturated aqueous NH4Cl was added, and the mixture was concentrated under reduced pressure to remove the methanol, then the remaining aqueous phase was extracted with EtOAc (2 × 20 mL), separated, dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting material was subjected to flash chromatography on silica gel, eluting with 20:1 CH2Cl2:CH3OH. The relevant fractions were concentrated under reduced pressure to furnish 15 as a white solid (160 mg, 53% yield). 1H NMR (400 MHz, DMSO-d6): δ 11.71 (br s, 2H), 8.14 (t, J = 6.0 Hz, 1H), 7.47 (d, J = 8.3 Hz, 1H), 7.40 (d, J = 1.0 Hz, 1H), 7.31 (dd, J = 8.0, 1.0 Hz, 1H), 6.67 (s, 1H), 4.87 (br s, 1H), 4.34 (s, 2H), 4.27 (d, J = 6.0 Hz, 2H), 1.13 (s, 9H). LC/MS (ESI+): 365/367 (M+H)+. 2-(2-Chloro-5-(pivalamidomethyl)benzamido)-1H-imidazole-4-carboxamide (16). Ethyl 2-(2-Chloro5-(pivalamidomethyl)benzamido)-1H-imidazole-4-carboxylate (3.18 g) was prepared from ethyl 2aminoimidazole-4-carboxylate and 1-M14 essentially according to the procedure described for 13. Then, ethyl 2-(2-Chloro-5-(pivalamidomethyl)benzamido)-1H-imidazole-4-carboxylate (1.50 g, 3.69 mmol) was dissolved in methanol (12 mL) and THF (24 mL) and treated with 2N aqueous NaOH (12 mL, 24 mmol) and stirred at room temperature overnight. Then, 1N aqueous HCl was added with stirring until the pH reached 7.0 The mixture was extracted with EtOAc (3 × 100 mL), and the organic layers were combined, dried over Na2SO4, filtered, and concentrated under reduced pressure to furnish 2-(2-Chloro-5(pivalamidomethyl)benzamido)-1H-imidazole-4-carboxylic acid as a white solid (682 mg, 37% yield). Then, 2-(2-Chloro-5-(pivalamidomethyl)benzamido)-1H-imidazole-4-carboxylic acid (200 mg, 0.45



mmol) was dissolved in THF (2 mL) and treated with DMF (5 µL), cooled to 0 °C, and treated with oxalyl chloride (38.9 µL, 0.45 mmol) and allowed to warm to room temperature with stirring for one hour. Water (5 mL) was added, and the organic phase was extracted with 3:1 CHCl3:iPrOH (3 × 20 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude material was purified by reverse-phase chromatography on C18 silica gel, eluting with 0.1% formic acid in a gradient of 25–100% CH3CN/water, to furnish 16 as an off-white solid (7.0 mg, 4.1% yield). 1H NMR (400 MHz, CD3OD): δ 7.51 (s, 1H), 7.49 (s, 1H), 7.47 (d, J = 8.4 Hz, 1H), 7.40 (dd, J = 8.4, 1.6 Hz, 1H), 4.38 (s, 2H), 1.20 (s, 9H). LC/MS (ESI+): 378/380 (M+H)+, 400/402 (M+Na)+, 755/757 (2M+H)+, 777/779 (2M+Na)+. 2-Chloro-N-(4-cyano-1H-imidazol-2-yl)-5-(pivalamidomethyl)benzamide (17). Prepared from 2amino-4-cyanoimidazole (60 mg, 0.56 mmol) and 1-M14 essentially according to the procedure described for 13. Yield = 5.0 mg (2.5%).

1

H NMR (300 MHz, DMSO-d6): δ 7.71 (s, 1H), 7.52–7.50 (m, 2H),

7.45–7.43 (m, 1H), 4.41 (s, 2H), 1.24 (s, 9H). LC/MS (ESI+): 360/362 (M+H)+. 2-(2-Chloro-5-(pivalamidomethyl)benzamido)-N-phenyl-1H-imidazole-4-carboxamide (18). Prepared from 1-M14 (200 mg, 0.449 mmol) and aniline (206 µL, 2.24 mmol) essentially according to the procedure described for 13. Yield = 20 mg (11%). 1H NMR (400 MHz, CD3OD): δ 7.68 (d, J = 7.9 Hz, 2H), 7.60 (s, 1H), 7.52–7.47 (m, 2H), 7.41 (dd, J = 8.4, 1.8 Hz, 1H), 7.34 (t, J = 7.9 Hz, 2H), 7.12 (t, J = 7.4 Hz, 1H), 4.39 (s, 2H), 1.20 (s, 9H). LC/MS (ESI+): 360/362 (M+H)+.

Supporting Information Supplementary information contains additional figures and tables showing MS spectra, HPLC chromatograms and compound characterization data. This material is available free of charge via the Internet at http://pubs.acs.org.


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Corresponding Author E-mail: [email protected] Phone: (317) 433-4994. Abbreviations Used mPGES-1, microsomal prostaglandin E synthase-1; DILI, drug-induced liver injury; RM, reactive metabolite; BSEP, bile salt export pump; ALT, alanine aminotransferase; HLM, human liver microsomes; ULN, upper limit of normal; GSH, glutathione; NAC, N-acetyl cysteine; EtOAc, ethyl acetate; DMF, N, N-dimethylformamide; THF, tetrahydrofuran; HOBt, 1-hydroxybenzotriazole; EDC, N-(3dimethylaminopropyl)-N’-ethylcarbodiimide; BOP-Cl, bis(2-oxo-3-oxazolidinyl)phosphinic chloride. References (1) (a) Lewis, J. H. The art and science of diagnosing and managing drug-induced liver injury in 2015 and beyond. Clin Gastroenterol Hepatol 2015, 13 (12), 2173-2189 e2178; (b) Tujios, S.; Fontana, R. J. Mechanisms of drug-induced liver injury: from bedside to bench. Nat Rev Gastroenterol Hepatol 2011, 8 (4), 202-211; (c) Sarges, P.; Steinberg, J. M.; Lewis, J. H. Drug-Induced Liver Injury: Highlights from a review of the 2015 literature. Drug Safety 2016, 39 (9), 801-821; (d) Chen, M.; Suzuki, A.; Borlak, J.; Andrade, R. J.; Lucena, M. I. Drug-induced liver injury: Interactions between drug properties and host factors. J Hepatol 2015, 63 (2), 503-514. (2) (a) Stepan, A. F.; Walker, D. P.; Bauman, J.; Price, D. A.; Baillie, T. A.; Kalgutkar, A. S.; Aleo, M. D. Structural alert/reactive metabolite concept as applied in medicinal chemistry to mitigate the risk of idiosyncratic drug toxicity: a perspective based on the critical examination of trends in the top 200 drugs marketed in the United States. Chem Res Toxicol 2011, 24 (9), 1345-1410; (b) Thompson, R. A.; Isin, E. M.; Li, Y.; Weidolf, L.; Page, K.; Wilson, I.; Swallow, S.; Middleton, B.; Stahl, S.; Foster, A. J.; Dolgos, H.; Weaver, R.; Kenna, J. G. In vitro approach to assess the potential for risk of idiosyncratic adverse reactions caused by candidate drugs. Chem Res Toxicol 2012, 25 (8), 1616-1632; (c) Sakatis, M. Z.; Reese, M. J.; Harrell, A. W.; Taylor, M. A.; Baines, I. A.; Chen, L.; Bloomer, J. C.; Yang, E. Y.; Ellens,



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Identification and Mitigation of Reactive Metabolites of 2

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