Hit-to-Lead Optimization and Discovery of 5 - ACS Publications

Article Cite This: J. Med. Chem. 2017, 60, 9040-9052

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Hit-to-Lead Optimization and Discovery of 5‑((5-([1,1′-Biphenyl]-4yl)-6-chloro‑1H‑benzo[d]imidazol-2-yl)oxy)-2-methylbenzoic Acid (MK-3903): A Novel Class of Benzimidazole-Based Activators of AMPActivated Protein Kinase Ping Lan,*,† F. Anthony Romero,† Dariusz Wodka,† Andrew J. Kassick,† Qun Dang,⊥ Tony Gibson,⊥ Daniel Cashion,⊥ Gaochao Zhou,‡ Yuli Chen,‡ Xiaoping Zhang,‡ Aihua Zhang,‡ Ying Li,‡ Maria E. Trujillo,§ Qing Shao,§ Margaret Wu,§ Shiyao Xu,∥ Huaibing He,∥ Deidre MacKenna,⊥ Jocelyn Staunton,⊥ Kevin T. Chapman,† Ann Weber,† Iyassu K. Sebhat,*,† and Gergely M. Makara† †

Department of Medicinal Chemistry, ‡Department of Biology, §Department of Pharmacology, and ∥Department of Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, MRL, Merck & Co., Inc., 2000 Galloping Hill Road, Kenilworth, New Jersey 07033, United States ⊥ Metabasis Therapeutics, Inc., 11119 North Torrey Pines Road, La Jolla California 92037, United States S Supporting Information *

ABSTRACT: AMP-activated protein kinase (AMPK) plays an essential role as a cellular energy sensor and master regulator of metabolism in eukaryotes. Dysregulated lipid and carbohydrate metabolism resulting from insulin resistance leads to hyperglycemia, the hallmark of type 2 diabetes mellitus (T2DM). While pharmacological activation of AMPK is anticipated to improve these parameters, the discovery of selective, direct activators has proven challenging. We now describe a hit-to-lead effort resulting in the discovery of a potent and selective class of benzimidazole-based direct AMPK activators, exemplified by 5-((5([1,1′-biphenyl]-4-yl)-6-chloro-1H-benzo[d]imidazol-2-yl)oxy)-2-methylbenzoic acid, 42 (MK-3903). Compound 42 exhibited robust target engagement in mouse liver following oral dosing, leading to improved lipid metabolism and insulin sensitization in mice.

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INTRODUCTION Since its discovery in 1973,1,2 extensive research has contributed to our understanding of the biology of 5′adenosine monophosphate-activated protein kinase (AMPK) including its fundamental role as a major sensor of cellular energetics and regulator of metabolism in eukaryotes.3−5 AMPK is a conserved serine/threonine protein kinase. Activation of AMPK in liver inhibits fatty acid synthesis (FAS) and stimulates fatty acid oxidation (FAO) by phosphorylating and inactivating both isoforms of acetyl CoA carboxylase ACC1 and ACC2, the first enzymes shown to be downstream targets for AMPK.6,7 ACCs are rate-limiting enzymes for the synthesis of malonyl-CoA, which is a potent inhibitor of fatty acid oxidation and a precursor in fatty acid biosynthesis.8 Activation of AMPK in primary culture of hepatocytes was shown to reduce gene expression of the key gluconeogenic enzymes, phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6 Pase).9,10 In skeletal muscle, AMPK activation stimulates glucose uptake acutely © 2017 American Chemical Society

because of increased translocation of the glucose transporter (GLUT4) to the plasma memberane.11 Gene expression of GLUT 4 is up-regulated via AMPK activation as well.12 Dysregulation of metabolism in man leads to metabolic syndrome, a condition characterized by insulin resistance. Elevated ectopic intracellular lipids are considered a major cause of insulin resistance. If left untreated, insulin resistance leads to hyperglycemia, which defines type 2 diabetes mellitus (T2DM), due in part to the inability of insulin to suppress hepatic glucose production.13 The combined metabolic changes in lipid and glucose homeostasis induced by AMPK activation are anticipated to contribute to increased metabolic flexibility and insulin sensitivity. Indeed, insulin sensitivity of muscle, liver, and whole body was increased after a single dose of 5aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), Received: September 12, 2017 Published: October 16, 2017 9040

DOI: 10.1021/acs.jmedchem.7b01344 J. Med. Chem. 2017, 60, 9040−9052

Journal of Medicinal Chemistry

Article

Figure 1. Representative AMPK direct activators.

a widely used phosphorylated AMP anologue for AMPK activation, in insulin-resistant rat model.14 Conversely, there are potential concerns associated with systemic AMPK activation, in particular, with AMPK activation in the heart.15,16 Mutations in the AMPK γ2-subunit lead to PRKAG2 cardiomyopathy. These mutations appear to prevent the binding of inhibitory ATP to the allosteric nucleotidebinding regulatory site, resulting in nonphysiological activation of AMPK.17 Given the potential association of adverse effects with systemic AMPK activation, AMPK isoform-selective or tissue-selective activation appeared an attractive approach for developing activators. A number of agents, including two antihyperglycemic drugs, have been reported to activate AMPK in cells, tissues, and/or in vivo.10,18 While the detailed modes of action for many of these agents may not be fully delineated, most of them activate AMPK in an indirect manner.19,20 The discovery of direct activators of AMPK has proven to be challenging, in part due to its structural complexity. The enzyme is a heterotrimer comprised of a catalytic (α1 or α2), a “scaffold” (β1 or β2), and a regulatory (γ1 or γ2 or γ3) subunit, all of which are encoded by separate genes. Excluding splice variants, there are a total of 12 AMPK complexes in mammals.21 It is not clear which isoform(s) are required to realize the maximum benefits of AMPK activation in vivo. Furthermore, distinct complex/ subunit distributions exist in different tissues of human and other preclinical animal species. For example, human skeletal muscle contains predominately α2β2-containing complexes.22,23 The β1-containing complexes, including α1β1γ1 and α2β1γ1, account for 95% of total AMPK activity in rodent liver extract.24 However, recently published reports suggest α1β2γ1 to be the major isoform in human liver.25,26 Despite major efforts to identify direct AMPK activators, few molecules have been reported.27−29 Some of the more widely used compounds include thienopyridone 1 (A-769662),30 the first small-molecule direct AMPK activator with β1 selectivity, the phosphonic acid 2 (identified from a library of AMP mimetics)31 and PT1-related AMPK activator 3.32 Repeated attempts to identify direct AMPK activators through traditional high-throughput-screens (HTS) of the MSD compound collection failed to generate progressable hits. The team therefore opted to take a less traditional approach involving the bioassay of a proprietary fragment collection (informer library) at high concentration. Using this approach, we identified several hits, established the tractability of the SAR of a hit bearing a benzimidazole core (Figure 1, compound 4), efficiently optimized that series, and ultimately discovered suitable tool compounds for proof-of-concept studies, all in less than one year. These efforts, as well as the results of initial

biological evaluation of an optimized tool compound 42 (MK3903),33 are now detailed here.

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RESULTS AND DISCUSSION Following multiple unsuccessful HTS campaigns to identify direct AMPK activators, we conducted an informer library screen to find nontraditional chemotypes as a starting point for optimization efforts. This informer library comprised a subset of our internal compound collection, with molecular weights of >200 Da and containing up to 22 heavy atoms. The heavy atom count was used as the molecular weight upper filter to avoid biasing against heavier elements such as chlorine. The in silico construction of the library utilized typical physicochemical property filters such as solubility and log P. The collection was intentionally designed to straddle the boundary between fragment-like and lead-like libraries.34 By biasing the deck toward larger and less hydrophilic fragments than those typically used in NMR- or X-ray-based screens, we hoped to increase the likelihood of identifying hits with 30% activation vs maximal activation attained by AMP) were identified from the informer screen. One of these was the benzimidazole derivative 5,6-dichlorobenzimidazole-2-propionic acid with an EC50 of ∼30 μM (compound 4, Table 1). The low molecular weight and micromolar potency of 4 translated to a ligand efficiency index (LE)35 of 0.38, which we considered a good starting point for optimization. An investigation of the SAR of hit 4 was therefore initiated, beginning with an examination of the effect of aromatic ring substitution. Compounds 7−11 were synthesized following known procedures (Scheme 1).36 Commercially available phenylenediamines 5 with various substituents were condensed with succinic anhydride at high temperature. The amide intermediates 6, when heated with acetic acid, cyclized to give the benzimidazole 2-propionic acids. For compounds 12 and 13, the 4-chloro-5-bromobenzimidazole (intermediate 11) was further derivatized via Suzuki cross-coupling37 with the corresponding boronic acids. All analogues were assayed against 9041



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amine 14 and the corresponding arylaldehydes (for 16, 17), or via nucleophilic substitution of bromo-substituted alkylcarboxylic acids by the thiol moiety in 5,6-dichloro-1H-benzimidazole2-thiol 18 (for 19, 21) (Table 2). Compounds where benzoic acids were directly attached to the benzimidazole 2-position (16, 17) lost activity. Interestingly, introduction of a sulfur atom in the 2-position linker (compound 19) resulted in significant improvement in both maximum activation percentage and potency relative to 4. Oxidation of the thio group to a sulfoxide (20) led to a loss of activity. We expanded the SAR by looking at additional acid linkers containing the newly discovered thio substituent. The route used for previous analogues (Scheme 2) was unsuitable for this purpose, so we looked to develop a more efficient synthesis that would allow late-stage incorporation of the benzimidazole 2substituent. As illustrated in Scheme 3, benzimidazole-2-thiol 18 was converted to 2-methylsulfonylbenzimidazole 22 via methylation with methyl iodide followed by oxidation with mCPBA. The desired 2-arylthio benzimidazoles were obtained following reaction of 22 with various substituted arylthiophenols.38 Generally, arylthio substitution was well tolerated. Acid substitution in the meta- or ortho-positions (26, 27) resulted in significantly improved potency compared to the initial hit, and homologation of the acid appeared to be well tolerated (25 vs 27 (Table 3)). We next examined whether the potency gains realized from our investigation of SAR in the benzimidazole 2- and 5positions would be additive. The key intermediates 32 and 33 provided ideal starting points to quickly access the fully assembled analogues (35−38 (Table 4)) via standard crosscoupling conditions (Scheme 4). The resultant hybrid compounds had significantly improved potency, with EC50 values in the nanomolar range. The most potent analogue, containing a 2-hydroxylbiphenyl group (38), exhibited an EC50 of 29 nM. While less potent, compound 35 activated the complex 320% versus the % maximal activation observed with the physiological activator AMP. An important metabolic consequence of AMPK activation is the site-specific phosphorylation/inhibition of ACC. This leads to the inhibition of fatty acid synthesis, also known as de novo lipogenesis (DNL).39 Measures of compound effects on DNL thus served as a useful, proximal pharmacodynamic marker of AMPK target engagement and activation. Administration of 30 mg/kg compound 36 to mice intraperitoneally (ip) led to ∼40% inhibition of DNL. A much higher oral dose of 100 mg/ kg oral was required for more modest reductions in DNL (∼20%). Pharmacokinetic (PK) studies established that compound 36 had high clearance (60 mL/min/kg) and poor bioavailability (7%) (Table 5). Examination of P-glycoprotein 1 (P-gp) susceptibility and permeability of the compound demonstrated very high transport in Lilly Laboratories porcine

Table 1. Effect of 3-(1H-Benzo[d]imidazol-2-yl)propanoic Acids on pAMPK7 Activity

Percent maximum activation referred to activation level at 50 μM. Values are the average of at least two experiments, each using a 10point titration. cNot determined.

a b

pAMPK 7, and the maximum activation at a concentration of 50 μM is shown in Table 1. Compounds without substitution (7) or with substituents such as methoxy (8) or small alkyl groups (9) showed very little activation. The presence of a single chlorine atom on the aromatic ring (10) was sufficient to allow some degree of activation, which underscored the importance of this substituent. When this chlorine was retained and the other position was substituted with a phenyl ring (12), a modest (∼3-fold) activity gain was realized vs 4. An additional small potency enhancement was observed upon replacement of the phenyl ring with a biphenyl group (13). In parallel, some of our efforts were directed toward modifications in the benzimidazole 1- and 2-positions (Scheme 2). A complete loss of activity was observed when the benzimidazole −NH was methylated (data not shown). A number of benzimidazoles containing modified acid linkages were quickly prepared via the condensation of o-phenylenedi-

Scheme 1. Synthesis of 3-(1H-Benzo[d]imidazol-2-yl)propanoic Acidsa

Reagents and conditions: (a) succinic anhydride, NMP, 80 °C; (b) acetic acid, reflux; (c) corresponding boronic acid, Pd(PPh3)4, K2CO3, dioxane, 80 °C. a

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Scheme 2. Syntheses of 3-(1H-Benzo[d]imidazol-2-yl) Acids with Various Linkersa

a

Reagents and conditions: (a) Na2S2O5, EtOH/H2O, 80 °C; (b) LiOH, MeOH/TH/H2O, 40 °C; (c) KOH, EtOH, rt; (d) m-CPBA, DCM, rt.

Table 2. Effect of (1H-Benzo[d]imidazol-2-yl) Acids with Various Linkers on pAMPK7 Activity

Table 3. Effect of 1H-Benzo[d]imidazol-2-yl)thioaryl Acids on pAMPK7 Activity

Percent maximum activation referred to activation level at 50 μM. Values are the average of at least 2 experiments, each using a 10-point titration. cNot Determined.

a b

Percent maximum activation referred to activation level at 50 μM. Values are the average of at least two experiements, each using a 10point titration. cNot determined.

a b

Table 4. Effect of (6-Chloro-5-aryl-1H-benzo[d]imidazol-2yl)thioacetic Acids and Thiobenzoic Acids on pAMPK7 Activity

Scheme 3. Synthesis of 1H-Benzo[d]imidazol-2-yl)thioaryl Acidsa

a

Reagents and conditions: (a) Mel, K2CO3, acetone, rt; (b) m-CPBA, DCM, rt; (c) arylthiols, EtOH, 70 °C, followed by hydrolysis, LiOH, MeOH/THF/H2O, 40 °C, when needed.

kidney (LLC-PK1) control cells. These data implicated 36 as a substrate of another transporter, which was subsequently identified as the breast cancer resistance protein (BCRP/ ABCG2). BCRP is expressed in the luminal plasma membrane of enterocytes, where it is responsible for the efflux of substrates into the intestine. The transporter is also expressed in hepatocytes, where it pumps substrates into bile.40 When compound 36 was dosed in BCRP knockout (KO) mice, a ∼30-fold reduction in clearance and commensurate ∼10-fold increase in bioavailability was observed, suggesting that BCRP

a

Values are the average of at least two experiments, each using a 10point titration. bPercent maximum activation refers to the highest activation level observed in the titration concentration range (∼2 nM to 50 μM).

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Scheme 4. Synthesis of (6-Chloro-5-aryl-1H-benzo[d]imidazol-2-yl)thioacetic Acids and Thiobenzoic Acidsa

Reagents and conditions: (a) NIS, acetic acid, 50 °C; (b) Fe/NH4Cl, EtOH/water, 50 °C; (c) CS2, aq KOH, EtOH, reflux; (d) tert-butyl bromoacetate, THF, Cs2CO3, rt; (e) corresponding alkyne, Cul, PdCl2(PPh3)2, DMF, Et3N, 120 °C or corresponding arylboronic acid, Pd(PPh3)4, toluene, aq K2CO3, 80 °C, followed by TFA/water, rt; (f) Mel, m-CPBA, DCM, rt; (g) corresponding arylboronic acid, Pd(PPh3)4, aq K2CO3, dioxane, 120 °C; (h) 3-mercaptobenzoic acid, EtOH, 70 °C. a

benzimidazole 5-position. This effort delivered potent AMPK activators with single- and double-digit nanomolar EC50 values. Table 6 details the data for the resulting compounds and highlights the impact of single atom modifications. The replacement of the sulfur for an oxygen atom resulted in at least 1 order of magnitude gain in activity (e.g., ∼13-fold gain for compound 41 vs 37). Further enhancement of activity was accomplished when small substituents were added to the benzoic acid moiety. For example, the introduction of a para-Cl or para-Me led to ∼2−7-fold improvement in potency (42 and 43 vs 41). In line with previous SAR, the introduction of the hydroxy group at the ortho position of the biphenyl group furnished compounds with further improved potency (47). Smaller benzimidazole 5-substituents were also tolerated, with the fused heterocyclic methylindole group affording one of the most potent activators (48). A crystal structure of compound 48, in complex with human pAMPK, was recently published (Figure 2).41 This study helped to define a novel allosteric activation site on pAMPK and provided detailed structural information that helps to explain the potency of the series. As part of our optimization efforts, we monitored ligand efficiency metrics. While the high LE of the hit compound was well conserved, the progression of lipophilic ligand efficiencies (LLEs) remained relatively unchanged or slightly decreased for later compounds. LLE is an index combining both in vitro potency and lipophilicity.35 The concurrent increase in potency

Table 5. PK Parameters for Compound 36 in Wild-Type and BCRP Knock-Out Micea CLp (mL/min/kg) t1/2 (h) F (%) Vdss (L/kg) AUCN (PO) (μM·h·kg/mg)

WT

KO

60 2.8 7 6.7 0.05

2.9 10.8 77 2.6 11.4

a

CLp, plasma clearance; t1/2, terminal half-life; F, bioavailability; Vdss, volume of distribution at steady state; AUCN (PO), normalized area under plasma concentration vs time curve following PO dosing.

plays an important role in the elimination and/or absorption of 36. Given the identified transporter liabilities, we continued with efforts to optimize structures. To this end, we examined the utility of methylsulfone intermediate 34 to access 2-aryloxy analogues. Initial attempts to directly apply this approach were unsuccessful, but modified solvent-free conditions utilizing elevated reaction temperatures afforded the desired products in modest yields (Scheme 5, step b).38 An alternate route was developed later using SEM-protected benzimidazole 39, which afforded higher yields under milder conditions. Using both approaches, we quickly synthesized a group of O-linked analogues with various aryl or alkyne groups in the

Scheme 5. Synthesis of 5,6-Substituted ((1H-Benzo[d]imidazol-2-yl)oxy)benzoic Acidsa

Reagents and conditions: (a) corresponding arylboronic acid or ester, Pd(PPh3)4, K3PO4, dioxane, 100 °C, or corresponding alkynes, CuI, PdCl2(PPh3)2, Et3N, DMF, 120 °C; (b) corresponding methyl 3-hydroxybenzoate, neat, 130 °C, followed by hydrolysis; (c) Et3N, SEM-Cl, THF, rt; (d) methyl 5-hydroxy-2-methylbenzoate, K2CO3, DMF, rt; (e) corresponding arylboronic ester, Pd(PPh3)4, K2CO3, DMF, 120 °C; (f) TBAF, THF, 80 °C, followed by 2.5 N aq NaOH, 45 °C a

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Table 6. Effect of 5,6-Substituted ((1H-Benzo[d]imidazol-2-yl)oxy)benzoic Acids on pAMPK7 Activity

a

Values are the average of at least two experiments, each using a 10-point titration. bPercent maximum activation referred to the highest activation level observed in the titration concentration range (∼0.8 nM to 16 μM).

Table 7. Effect of Compound 42 at the 12 Recombinant Human pAMPK Complexes human pAMPK complex

subunit composition

1 2 3 4 5 6 7 8 9 10 11 12

Figure 2. Ligand efficiencies during hit-to-lead progression.

α1 α1 α1 α1 α1 α1 α2 α2 α2 α2 α2 α2

β1 β1 β1 β2 β2 β2 β1 β1 β1 β2 β2 β2

EC50 (nM) (%max)a

γ1 γ2 γ3 γ1 γ2 γ3 γ1 γ2 γ3 γ1 γ2 γ3

8 (217%) 21 (242%) 19 (61%) 40 (50%) 118 (36%) 2860 (8%) 9 (320%) 22 (175%) 26 (209%) 39 (94%) 23 (145%) 35 (135%)

a EC50 values are the average of at least two experiments, each using a 10-point titration. %max refers to the highest activation level observed in the titration concentration range (∼2 nM to 50 μM).

and lipophilicity suggested that the binding affinity improvement realized with later compounds came mainly from enhanced lipophilic interactions between the compounds and the putative binding sites. Compound 42 was selected, together with other candidate compounds, for further in vitro and in vivo profiling. Activation was assessed for all 12 recombinant human pAMPK complexes. Compound 42 activates 10 of the 12 pAMPK complexes with EC50 values in the range of 8−40 nM and maximal activation >50% (Table 7.). The compound partially activates pAMPK5 (36% max), which is only a minor component of human and mouse liver, and it does not activate pAMPK6, which is not detected in liver.24 The pharmacokinetics of 42 in C57BL/6 mice, Sprague− Dawley rats, and beagle dogs were characterized by moderate systemic plasma clearance (5.0−13 mL/min/kg), a volume of distribution at steady state of 0.6−1.1 L/kg, and a terminal halflife of ∼2 h (Table 8). Compound 42 had low oral bioavailability (8.4%) in C57BL/6 mice, but the oral exposure was later improved using other vehicles (data not shown). Oral bioavailability in rats and dogs was improved (27−78%).

Table 8. Pharmacokinetic Parameters of Compound 42 in Preclinical Speciesa species pharmacokinetic parameter

mouse

rat

dog

dose IV/PO (mg/kg) CLp (mL/min/kg) Vdss (L/kg) t1/2 (h) Cmax (μM) tmax (h) F (%) oral AUCN (μM·h·kg/mg)

2/10 6.8 0.7 2.1 2.0 1.0 8.4 0.5

1/4 5.0 0.6 1.7 4.8 0.5 25 2.6

0.5/1 13 1.1 2.4 1.1 0.5 78 2.3

a

CLp, plasma clearance; Vdss, volume distribution at steady state; t1/2,: terminal half-life; Cmax, maximum concentration; tmax, time of maximum concentration; F, bioavailability; AUCN, normalized area under plasma concentration vs time curve.

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Compound 42 demonstrated low permeability (Papp = 6 × 10−6 cm/s) in LLC-PK1 cells42 and is a substrate of human liver uptake transporters OATP1B1 and OATP1B3 (organic anion transporter proteins).43 42 is a weak reversible inhibitor of CYP3A4 and 2D6 in human liver microsomes (apparent IC50 > 50 μM) and did not exhibit time-dependent inhibition of CYP3A4 activity. In the in vitro hPXR assay 42 had an EC50 of >30 μM, suggesting that it is not a potent PXR agonist.44 The Panlab screen and follow up evaluation showed that 42 binds moderately to the prostanoid DP2 (CRTH2) receptor (binding IC50 = 1.8 μM) but not in the presence of 10% human serum (binding IC50 > 86 μM).45 42 was also screened at Upstate against a panel of kinase targets, identifying p38-regulated/ activated protein kinase (PRAK) as the only hit with IC50 < 10 μM. As discussed above, the level of phosphorylated ACC (pACC), a downstream AMPK substrate, is an indicator of intracellular AMPK activation. The pACC to total ACC ratio, measured with a novel Meso Scale Discovery (MSD) assay, was used as a proximal target engagement readout for AMPK activation in vivo. Diet induced obese (DIO) mice (n = 8) were treated with 42 (30 mg/kg BID) for 15 days. Liver and muscle were harvested 2 h following the final dose. pACC/ACC ratios were significantly increased in both liver (3.1-fold) and skeletal muscle (1.6-fold) (Figure 3).

Figure 4. Acute effects of 42 on hepatic fatty acid synthesis in db/+ mice. *p < 0.05 compared to vehicle; **p < 0.01 compared to vehicle; ***p < 0.001 compared to vehicle.

mg/kg QD po) for 12 days were subjected to an oral glucose tolerance test (oGTT) following a 4 h fast on day 12. The effects on blood glucose and insulin 20 min after bolus glucose administration were combined to provide an insulin resistance index (IRI) (Figure 5).47 A dose dependent reduction in IRI

Figure 5. Chronic effect of compound 42 on a measure of insulin sensitivity in DIO mice following 12 days of dosing. oGTT IRI = measured glucose (mM) × measured insulin (ng/mL). *p < 0.05; **p < 0.01; ***p < 0.001.

was observed, mainly driven by suppression of insulin excursion during the oGTT. There were no significant effects on blood glucose. A modest effect on body weight at 30 mg/kg BID (3.1% loss of body weight) complicated interpretation of the data for that dose group, but significant effects on IRI were observed without body weight change for the 10 mg/kg BID and 30 mg/kg QD groups. Taken together, these data suggest that compound 42 holds promise as an insulin sensitizing agent.

Figure 3. Effect of compound 42 (30 mg/kg BID, 15 days) administration on pACC/ACC in (a) liver and (b) muscle of DIO mice 2 h after the final dose.

In addition, we measured inhibition of de novo fatty acid synthesis (FAS) in liver, one functional consequence of increased ACC phosphorylation. Acute oral administration of compound 42 (3, 10, and 30 mg/kg) to high-fructose fed db/+ mice resulted in significant inhibition of hepatic FAS for all three doses. Specifically, db/+ mice were fed for 1 week on a diet enriched with 64% fructose to elevate rates of FAS. FAS was measured both 2 and 8 h after compound dosing. In this dose titration study, 42 inhibited hepatic FAS by 60%, 61%, and 66% 2 h after doses of 3, 10, and 30 mg/kg. At 8 h after dosing, inhibition of FAS by 42 was 29%, 37%, and 49% at these respective doses (Figure 4.). Compound 42 was also assessed for its chronic effects on insulin sensitization in DIO mice. DIO Mice are normoglycemic but hyperinsulinemic, serving as a good model of insulin resistance or prediabetes.46 DIO mice treated with compound 42 (3−30 mg/kg BID and 30

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CONCLUSION A biochemical informer (fragment) library screen was used to identify a novel class of AMPK activators where prior HTS approaches had failed. Starting from a low affinity and low molecular weight hit, we were able to optimize the benzimidazole pharmacophore by successfully utilizing both replacement and extension strategies. The first breakthrough came from the replacement of the 2-benzimidazole carbon linkage with thiobenzoic acid, which effectively propelled the potency of the resulting analogues into the low micromolar range. The second significant improvement was realized when the benzimidazole core was extended at the 5-position, 9046



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procedure by utilizing benzene-1,2-diamine as the starting material. 1H NMR (500 MHz, acetone-d6) δ 7.49 (dd, J = 6.0, 3.2 Hz, 2H), 7.14 (dd, J = 6.0, 3.2 Hz, 2H), 3.17 (t, J = 7.0 Hz, 2H), 2.90 (t, J = 7.0 Hz, 2H). 3-(5-Methoxy-1H-benzo[d]imidazol-2-yl)propanoic Acid (8). The title compound was prepared in the manner similar to the general procedure by utilizing 4-methoxybenzene-1,2-diamine as the starting material. 1H NMR (500 MHz, acetone-d6) δ 7.38 (d, J = 8.7 Hz, 1H), 7.03 (d, J = 2.4 Hz, 1H), 6.78 (dd, J = 8.7, 2.4 Hz, 1H), 3.79 (s, 3H), 3.14 (t, J = 7.0 Hz, 2H), 2.87 (t, J = 7.0 Hz, 2H). 3-(5,6-Dimethyl-1H-benzo[d]imidazol-2-yl)propanoic Acid (9). The title compound was prepared in the manner similar to the general procedure by utilizing 4,5-dimethylbenzene-1,2-diamine as the starting material. 1H NMR (500 MHz, CD3OD) δ 7.31 (s, 2H), 3.16 (d, J = 7.3 Hz, 2H), 2.81 (d, J = 7.8 Hz, 2H), 2.35 (s, 6H). 3-(5-Chloro-1H-benzo[d]imidazol-2-yl)propanoic Acid (10). The title compound was prepared in the manner similar to the general procedure by utilizing 4-chlorobenzene-1,2-diamine as the starting material. 1H NMR (500 MHz, DMSO-d6) δ 7.86 (dd, J = 2.0, 0.6 Hz, 1H), 7.77 (dd, J = 8.7, 0.6 Hz, 1H), 7.53 (dd, J = 8.7, 2.0 Hz, 1H), 3.30 (t, J = 7.1 Hz, 2H), 3.01 (t, J = 7.1 Hz, 2H). 3-(6-Bromo-5-chloro-1H-benzo[d]imidazol-2-yl)propanoic Acid (11). The title compound was prepared in the manner similar to the general procedure by utilizing 4-bromo-5-chlorobenzene-1,2diamine as the starting material (10 mg, 8% yield). 1H NMR (500 MHz, DMSO-d6) δ 7.85 (s, 1H), 7.73 (s, 1H), 3.00 (t, J = 7.1 Hz, 2H), 2.68 (t, J = 7.1 Hz, 2H). 3-(5-Chloro-6-phenyl-1H-benzo[d]imidazol-2-yl)propanoic Acid (12). 3-(6-Bromo-5-chloro-1H-benzo[d]imidazol-2-yl)propanoic acid (0.39 mmol, 1 equiv) (11) and phenylboronic acid (1 equiv) were dissolved in anhydrous dioxane (3 mL), then catalyst Pd(PPh3)4 (5 mol %) was added followed by 1 M K2CO3 (2 equiv). The resulting mixture was stirred at 80 °C overnight. The reaction was concentrated to dryness, and the desired product was isolated by preparative HPLC (38 mg, 32% yield). 1H NMR (500 MHz, DMSO-d6) δ 7.87 (s, 1H), 7.62 (s, 1H), 7.48−7.39 (m, 5H), 3.20 (t, J = 7.1 Hz, 2H), 2.88 (t, J = 7.1 Hz, 2H). 3-(6-([1,1′-Biphenyl]-4-yl)-5-chloro-1H-benzo[d]imidazol-2yl)propanoic Acid (13). The title compound was prepared in the manner similar to compound 12 by utilizing [1,1′-biphenyl]-4ylboronic acid as the starting material (69 mg, 47% yield). 1H NMR (500 MHz, DMSO-d6) δ 7.91 (s, 1H), 7.79−7.77 (m, 2H), 7.75−7.73 (m, 2H), 7.70 (s, 1H), 7.56−7.54 (m, 2H), 7.51−7.48 (m, 2H), 7.40− 7.37 (m, 1H), 3.22 (t, J = 7.1 Hz, 2H), 2.90 (t, J = 7.1 Hz, 2H). Preparation of 2-(5,6-Dichloro-1H-benzo[d]imidazol-2-yl)benzoic Acid (16). To a solution of 4,5-dichlorobenzene-1,2-diamine (0.2 mmol, 1 equiv) in ethanol (2 mL) and water (1 mL) was added methyl 2-formylbenzoate (1 equiv) followed by sodium metabissulfite (2 equiv). The mixture was stirred at 80 °C for overnight and then was concentrated to dryness. LiOH (10 equiv) was added to the afforded residue, followed by addition of solvents MeOH, THF, and water (1 mL each). The mixture was stirred at 40 °C. After overnight, the reaction was concentrated to dryness and the desired product was isolated by preparative HPLC (10 mg, 16% yield). 1H NMR (500 MHz, DMSO-d6) δ 8.76 (t, J = 1.7 Hz, 1H), 8.41−8.39 (m, 1H), 8.07 (dt, J = 7.8, 1.4 Hz, 1H), 7.87 (s, 2H), 7.70 (t, J = 7.8 Hz, 1H). 3-(5,6-Dichloro-1H-benzo[d]imidazol-2-yl)benzoic Acid (17). The title compound was prepared in the manner similar to the procedure as 16 by utilizing methyl 3-formylbenzoate as the starting material (7 mg, 12% yield). 1H NMR (500 MHz, DMSO-d6) δ 8.76 (t, J = 1.7 Hz, 1H), 8.40 (dt, J = 7.8, 1.5 Hz, 1H), 8.07 (dt, J = 7.8, 1.4 Hz, 1H), 7.87 (s, 2H), 7.69 (t, J = 7.8 Hz, 1H). Preparation of 3-((5,6-Dichloro-1H-benzo[d]imidazol-2-yl)thio)propanoic Acid (19). To a solution of 5,6-dichloro-1Hbenzo[d]imidazole-2-thiol (18) (0.2 mmol, 1 equiv) in ethanol (1 mL) was added 3-bromopropanoic acid (1 equiv) and KOH (2 equiv). The mixture was stirred overnight at rt. The reaction was concentrated to dryness, and the desired product was isolated by preparative HPLC (14 mg, 20% yield). 1H NMR (500 MHz, DMSO-d6) δ 7.70 (s, 2H), 3.42 (t, J = 6.9 Hz, 2H), 2.76 (t, J = 6.9 Hz, 2H).

appending a hydrophobic moiety that nearly doubled the size of the structures. The gain in size was crucial for manifesting potency, facilitating interaction with a highly hydrophobic pocket generated at the interface between the carbohydratebinding module of the β-subunit and the kinase domain of the α-subunit.41 The last potency boost came from a single atom replacement of sulfur with oxygen at the benzimidazole 2position, furnishing compounds with single-digit nanomolar potencies. Overall, these optimization efforts led to 4 orders of magnitude improvement in potency versus the original hit and ∼4-fold increase in the %maximal activation achieved by the physiological activator AMP. These efforts identified molecules suitable for in vivo biological studies. Chronic oral administration of compound 42 robustly increased ACC phosphorylation in liver with more modest effects in skeletal muscle. Treatment of various mouse models with compound 42 resulted in expected alterations in lipid metabolism and improvements in a measure of insulin sensitization. Based in part on these promising preclinical results, compound 42 was designated as a development candidate to support further research on this novel class of AMPK activators. The results of the extensive followup studies employing 42 and other compounds derived from this class of AMPK activators will be reported separately.48

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EXPERIMENTAL SECTION

General Experimental Methods. Commercially available starting materials, reagents, and solvents were used as received. Anhydrous reactions were performed under an inert atmosphere such as nitrogen. Microwave reactions were performed using a Biotage Initiator microwave reactor. Reaction progress was generally monitored by LCMS (Waters Acquity UPLC). Flash column chromatography purification of intermediates was performed on an ISCO CombiFlashRf system using prepacked ISCO silica gel columns. Elution was performed using a gradient of hexane/ethyl acetate. Preparative reverse-phase HPLC purification of final products was performed on a Waters AutoPurification System with a Waters CSH (C-18, 100 mm × 19 mm, 5 μm) reverse-phase column. Elution was performed using a gradient of acetonitrile/water (0.16% TFA) over an 8 min period at a flow rate of 25 mL/min. The fractions were detected with PDA and MS detectors. Fractions containing the desired material were concentrated using GeneVac HT-24 to obtain the final products. The purity analyses of all final compounds were performed on Waters Acquity UPLC equipped with BEH-C18 column (50 mm × 1.0 mm, 1.7 μm), using gradients 10:90 to 95:5 v/v CH3CN/H2O + v 0.05% TFA over 1.6 min period at a flow rate of 0.3 mL/min, with UV wavelength 254 nm. All tested compounds exhibited >95% purity under the LC conditions. Proton NMRs were recorded on a Bruker 500 MHz spectrometer and were analyzed using MestReNova program. Chemical shifts are expressed in ppm and referenced to deuterated dimethyl sulfoxide (DMSO-d6) or acetone (acetone-d6) or methanol (CD3OD). All reported yields are not optimized. General Procedure for Preparation of 3-(1H-benzo[d]imidazol-2-yl)propanoic Acids. To a solution of benzene-1,2diamine (0.2 mmol, 1 equiv) in NMP (1 mL) was added succinic anhydride (0.2 mmol, 1 equiv). The mixture was stirred for overnight at 80 °C. Then acetic acid (1 mL) was added, and the afforded solution was stirred for overnight under reflux. The reaction was concentrated to dryness, and the desired product was isolated by preparative HPLC. 3-(5,6-Dichloro-1H-benzo[d]imidazol-2-yl)propanoic Acid (4). The title compound was prepared in the manner similar to the general procedure by utilizing 4,5-dichlorobenzene-1,2-diamine as the starting material. 1H NMR (500 MHz, acetone-d6) δ 7.69 (s, 2H), 3.17 (t, J = 7.0 Hz, 2H), 2.90 (t, J = 7.0 Hz, 2H) . 3-(1H-Benzo[d]imidazol-2-yl)propanoic Acid (7). The title compound was prepared in the manner similar to the general 9047



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3-((5,6-Dichloro-1H-benzo[d]imidazol-2-yl)sulfinyl)propanoic Acid (20). Compound 3-((5,6-dichloro-1H-benzo[d]imidazol-2-yl)thio)propanoic acid (19) (0.2 mmol, 1 equiv) was dissolved in DCM (4 mL), then m-CPBA (1 equiv) was added. The mixture was stirred at rt overnight. The reaction was concentrated to dryness, and the desired product was isolated by preparative HPLC (20 mg, 30% yield). 1H NMR (500 MHz, acetone-d6) δ 7.90 (s, 2H), 3.70−3.64 (m, 1H), 3.43 (ddd, J = 13.7, 7.9, 6.4 Hz, 1H), 2.93−2.87 (m, 1H), 2.66 (ddd, J = 17.5, 7.9, 6.4 Hz, 1H). 2-((5,6-Dichloro-1H-benzo[d]imidazol-2-yl)thio)acetic Acid (21). The title compound was prepared in the manner similar to the procedure as 19 by utilizing 2-bromoacetic acid as the starting material (32 mg, 50% yield). 1H NMR (500 MHz, DMSO-d6) δ 7.69 (s, 2H), 4.15 (s, 2H). Preparation of Intermediate 5,6-Dichloro-2-(methylsulfonyl)-1H-benzo[d]imidazole (22). Step1, 5,6-Dichloro-2-(methylthio)-1H-benzo[d]imidazole: K2CO3 (14 mmol, 2 equiv), followed by iodomethane (8.4 mmol, 1.2 equiv), was added to a solution of 18 (7 mmol, 1 equiv) in acetone (20 mL) at 0 °C. The reaction was stirred at rt overnight. Volatiles were removed, and the residue was partitioned between EtOAc and water. Concentration afforded the desired product as a white foam, which was subjected to chromatography on a column of SiO2 eluting with a gradient from 30% to 100% EtOAc in hexanes to provide the title compound. Step 2, 5,6-Dichloro-2-(methylsulfonyl)-1H-benzo[d]imidazole: mChloroperbenzoic acid (10 mmol, 2 equiv) was added to a suspension of 5,6-dichloro-2-(methylthio)-1H-benzo[d]imidazole (5 mmol, 1 equiv) in DCM (50 mL). The reaction was stirred at rt overnight then washed with 1N aqueous NaOH. The organic phase was concentrated. The residue was subjected to chromatography on a column of SiO2 eluting with a gradient from 10% to 100% EtOAc in hexanes to provide the title compound (500 mg, 41% yield). 1H NMR (500 MHz, DMSO-d6) δ 8.00 (s, 2H), 3.51 (s, 3H). General Procedure for Preparation of 1H-Benzo[d]imidazol2-yl)thioaryl Acids. To a solution of intermediate 22 (0.2 mmol, 1 equiv) in ethanol (1.5 mL) was added the corresponding mercaptobenzoic acid (1 equiv). The mixture was stirred at 70 °C overnight. The reaction was concentrated to dryness, and the desired product was isolated by preparative HPLC. 4-((5,6-Dichloro-1H-benzo[d]imidazol-2-yl)thio)benzoic Acid (23). The title compound was prepared in the manner similar to the general procedure by utilizing 4-mercaptobenzoic acid as the starting material (26 mg, 40% yield). 1H NMR (500 MHz, DMSO-d6) δ 7.95−7.93 (m, 2H), 7.79 (s, 2H), 7.59−7.57 (m, 2H). 2-((5,6-Dichloro-1H-benzo[d]imidazol-2-yl)thio)thiazole-4carboxylic Acid (24). The title compound was prepared in the manner similar to the general procedure by utilizing ethyl 2mercaptothiazole-4-carboxylate as the starting material, followed by hydrolysis with addition of a solution of LiOH (5 equiv) in mixed solvents MeOH/THF/water for 2 h (14 mg, 20% yield). 1H NMR (500 MHz, CD3OD) δ 8.45 (s, 1H), 7.75 (s, 2H). 2-(3-((5,6-Dichloro-1H-benzo[d]imidazol-2-yl)thio)phenyl)acetic Acid (25). The title compound was prepared in the manner similar to the general procedure by utilizing 2-(3-mercaptophenyl)acetic acid as the starting material (34 mg, 48% yield). 1H NMR (500 MHz, acetone-d6) δ 7.63−7.61 (m, 3H), 7.54−7.51 (m, 1H), 7.43− 7.40 (m, 2H), 3.68 (s, 2H). 2-((5,6-Dichloro-1H-benzo[d]imidazol-2-yl)thio)benzoic Acid (26). The title compound was prepared in the manner similar to the general procedure by utilizing 2-mercaptobenzoic acid as the starting material (32 mg, 47% yield). 1H NMR (500 MHz, DMSO-d6) δ 7.96 (dd, J = 7.8, 1.6 Hz, 1H), 7.86 (br s, 1H), 7.43 (ddd, J = 8.1, 7.3, 1.6 Hz, 1H), 7.33 (td, J = 7.5, 1.2 Hz, 1H), 6.92 (dd, J = 8.1, 1.1 Hz, 1H). 3-((5,6-Dichloro-1H-benzo[d]imidazol-2-yl)thio)benzoic Acid (27). The title compound was prepared in the manner similar to the general procedure by utilizing 3-mercaptobenzoic acid as the starting material (27 mg, 41% yield). 1H NMR (500 MHz, DMSO-d6) δ 8.04 (t, J = 1.8 Hz, 1H), 7.98−7.96 (m, 1H), 7.81 (ddd, J = 7.8, 2.0, 1.1 Hz, 1H), 7.75 (s, 2H), 7.59−7.56 (m, 1H).

Preparation of Intermediate tert-Butyl 2-((6-Chloro-5-iodo1H-benzo[d]imidazol-2-yl)thio)acetate (32). Step 1, 5-Chloro-4iodo-2-nitroaniline (29): To a solution of 5-chloro-2-nitroaniline 28 (145 mmol, 1 equiv) in AcOH (250 mL) was added Niodosuccinimide (145 mmol, 1 equiv). The mixture was stirred overnight at 50 °C, cooled down to rt, and filtered. The solid residue was washed with AcOH, water, saturated aqueous NaHCO3, and water and then dried to afford the desired product as a brown solid, which was used in the next step without further purification. Step 2, 4-Chloro-5-iodobenzene-1,2-diamine (30): To a suspension of 29 (122 mmol) in EtOH (800 mL) and water (150 mL) was added iron powder (673 mmol) and NH4Cl (306 mmol). The mixture was heated under nitrogen at 50 °C overnight. Additional iron powder (673 mmol) and NH4Cl (306 mmol) were added, and heating was continued for 45 h. The reaction mixture was cooled, filtered, and concentrated. The residue was dissolved in ethyl acetate and washed with sodium bicarbonate solution. The organic phase was concentrated to afford the desired product as a gray solid, which was used in the next step without further purification. Step 3, 5-Chloro-6-iodo-1,3-dihydro-2H-benzimidazole-2-thione (31): KOH (238 mmol) in water (50 mL), followed by carbon disulfide (238 mmol), was added to a solution of 30 (198 mmol) in EtOH (300 mL). The mixture was heated at reflux for 3 h, cooled, and filtered. To the filtrate was added water (300 mL) and then AcOH (25 mL) in water (50 mL). The precipitate was collected, washed with water and a small amount of EtOH, and dried to afford the desired product as a brown powder, which was used in the next step without further purification. Step 4, tert-Butyl 2-(6-chloro-5-iodo-1H-benzo[d]imidazol-2ylthio)acetate (32): Cs2CO3 (7.08 mmol), followed by tert-butyl bromoacetate (3.54 mmol), was added to a solution of 31 (3.54 mmol) in THF (20 mL) at 0 °C. The reaction was stirred at rt for 0.5 h. Volatiles were removed, and the residue was partitioned between EtOAc and water. Concentration afforded the desired product as a white power. LC-MS: calculated for C13H14ClIN2O2S 423.95, observed m/e 424.8 (M + H)+. Preparation of Intermediate 6-Chloro-5-iodo-2-(methylsulfonyl)-1H-benzo[d]imidazole (33). Step 1, 6-Chloro-5-iodo-2(methylthio)-1H-benzimidazole: K2CO3 (1.61 mmol), followed by iodomethane (1.61 mmol), was added to a solution of 31 (3.22 mmol) in acetone (20 mL) at 0 °C. The reaction was stirred at rt for 1 h. Additional K2CO3 (1.61 mmol) and iodomethane (1.61 mmol) were added, and stirring continued at rt overnight. Volatiles were removed, and the residue was partitioned between EtOAc and water. Concentration afforded the desired product as a white foam, which was used in the next step without further purification. Step 2, 6-Chloro-5-iodo-2-(methylsulfonyl)-1H-benzimidazole: mChloroperbenzoic acid (6.16 mmol) was added to a suspension of 6chloro-5-iodo-2-(methylthio)-1H-benzimidazole (3.08 mmol) in DCM (50 mL). The reaction stirred at rt for 10 min then washed with 10% aqueous NaHCO3. The organic phase was concentrated. The residue was triturated with MeOH (3 mL) and filtered to afford the title compound as white powder (0.8 g, 70% yield). 1H NMR (500 MHz, DMSO-d6) δ 8.31 (s, 1H), 8.00 (s, 1H), 3.53 (s, 3H). Preparation of 2-((6-Chloro-5-(phenylethynyl)-1H-benzo[d]imidazol-2-yl)thio)acetic Acid (35). A 2 mL Biotage microwave vial was charged with copper iodide (10 mol %) and Pd(PPh3)2Cl2 (5 mol %). A solution of 32 (0.059 mmol, 1 equiv) in DMF (0.5 mL) was added to the vial, followed by a solution of ethynylbenzene (0.118 mmol, 2 equiv) in DMF (250 μL) and TEA (0.41 mmol). The resulting suspension was heated in a microwave synthesizer at 120 °C for 5 min. The solvent was removed in vacuo to give dark-brown oil. The afforded residue was then dissolved in 9:1 TFA/water (2 mL) and stirred at rt for 2 h. The solution was evaporated to dryness in vacuo to give oily residue, which was purified by preparative HPLC to give the title compound (2.4 mg, 12% yield). 1H NMR (500 MHz, DMSO-d6) δ 7.68 (d, J = 0.5 Hz, 1H), 7.60 (d, J = 0.5 Hz, 1H), 7.57−7.55 (m, 2H), 7.45−7.41 (m, 3H), 3.65 (s, 2H). Preparation of 2-((5-([1,1′-Biphenyl]-4-yl)-6-chloro-1Hbenzo[d]imidazol-2-yl)thio)acetic Acid (36). A solution of aq 9048



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potassium carbonate (0.15 mmol, 1M), Pd(PPh3)4 (0.0002 mmol), [1,1′-biphenyl]-4-ylboronic acid (0.05 mmol), and 32 (0.05 mmol) in toluene (0.25 mL) was heated at 80 °C for 15 h. The aqueous phase was removed, and the organic phase was concentrated to afford a solid residue. A solution of the residue in TFA (0.9 mL) and water (0.1 mL) was then stirred at room temperature for 2 h and concentrated. The resulted solid was purified with HPLC to give the desired product (6.5 mg, 33% yield). 1H NMR (500 MHz, DMSO-d6) δ 7.74−7.71 (m, 4H), 7.66 (s, 1H), 7.53 (s, 1H), 7.52−7.46 (m, 4H), 7.39−7.36 (m, 1H), 4.18 (s, 2H). Preparation of 3-((5-([1,1′-Biphenyl]-4-yl)-6-chloro-1Hbenzo[d]imidazol-2-yl)thio)benzoic Acid (37). A solution of aq potassium carbonate (0.15 mL, 1M), Pd(PPh3)4 (5% mmol), 4biphenylboronic acid (0.075 mmol, 1.5 equiv), and 33 (0.05 mmol) in dioxane (0.25 mL) was heated in a Biotage microwave at 120 °C for 10 min. The mixture was concentrated to dryness. The afforded residue was dissolved in ethanol (1 mL) followed by addition of 3mercaptobenzoic acid (2 equiv). The mixture was heated at 70 °C for overnight then cooled and concentrated to remove solvent. The desired product was isolated by preparative HPLC (9 mg, 40% yield). 1 H NMR (500 MHz, DMSO-d6) δ 8.05 (s, 1H), 7.97 (d, J = 5 Hz, 1H), 7.80 (d, J = 10 Hz, 1H), 7.77−7.74 (m, 5H), 7.59 (t, J = 10 Hz, 1H), 7.56−7.49 (m, 5H), 7.40 (t, J = 10 Hz, 1H). Preparation of 3-((6-Chloro-5-(2′-hydroxy-[1,1′-biphenyl]-4yl)-1H-benzo[d]imidazol-2-yl)thio)benzoic Acid (38). Step 1, 4′Bromobiphenyl-2-ol: Potassium phosphate (2 M in water) (10.9 mmol) and Pd(PPh3)4 (0.18 mmol) were added to a solution of 1bromo-4-iodobenzene (7.25 mmol) and 2-hydroxybenzeneboronic acid (7.25 mmol) in dioxane (50 mL). The reaction was heated at 100 °C for 1 h. Volatiles were removed, and the residue was purified by chromatography over silica eluting with 0−50% EtOAc/hexane to afford the desired product as a light-yellow oil. Step 2, 4′-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)biphenyl-2ol: Potassium acetate (3.73 mmol) and dichloro[1,1′-bis(diphenylphosphino) ferrocene]palladium(II) DCM adduct (31 μmol) were added to a solution of product from step 1 (1.24 mmol) and bis(pinacolato)diboron (1.37 mmol) in DME (3 mL). The reaction was heated at 150 °C under microwave irradiation for 10 min. Reaction mixture was filtered through a pad of Celite and purified by chromatography over silica eluting with 0−50% EtOAc/hexane to furnish the title compound as a white solid. Step 3, 3-((6-Chloro-5-(2′-hydroxy-[1,1′-biphenyl]-4-yl)-1Hbenzo[d]imidazol-2-yl)thio)benzoic acid (38): A solution of aq potassium carbonate (0.15 mL, 1M), Pd(PPh3)4 (5% mmol), product from step 2 (0.075 mmol, 1.5 equiv), and 33 (0.05 mmol) in dioxane (0.25 mL) was heated in a Biotage microwave at 120 °C for 10 min. The mixture was concentrated to dryness, and the residue was dissolved in ethanol (1 mL) followed by addition of 3mercaptobenzoic acid (2 equiv). The mixture was heated at 70 °C for overnight, then cooled and concentrated to remove solvent. The desired product was isolated by preparative HPLC (8 mg, 33% yield). 1 H NMR (500 MHz, DMSO-d6) δ 8.01 (t, J = 1.7 Hz, 1H), 7.92 (dt, J = 7.7, 1.4 Hz, 1H), 7.64−7.59 (m, 4H), 7.48−7.44 (m, 4H), 7.31 (dd, J = 7.6, 1.7 Hz, 1H), 7.17−7.14 (m, 1H), 6.97 (dd, J = 8.1, 1.2 Hz, 1H), 6.88 (td, J = 7.4, 1.2 Hz, 1H). Preparation of 5-((5-([1,1′-Biphenyl]-4-yl)-6-chloro-1Hbenzo[d]imidazol-2-yl)oxy)-2-methylbenzoic Acid (42). A solution of potassium phosphate, tribasic (2 M solution in water) (126 mmol), Pd(PPh3)4 (1.7 mmol), 4-biphenylboronic acid (63.1 mmol), and 33 (42.1 mmol) in dioxane (300 mL) was heated at 100 °C for 5 h. The aqueous phase was removed, and the organic phase was concentrated, diluted with EtOAc and DCM, filtered, and concentrated to afford a white solid. The white solid was then mixed with methyl 5-hydroxy-2-methylbenzoate (18.28 mmol) in DCM (100 mL), and the mixture was concentrated. The resulting oily residue was placed under nitrogen in a sealed vessel and heated at 130 °C for 3 h, then cooled and mixed with EtOAc and water. The mixture was filtered, and the organic phase was concentrated. Chromatography over silica eluting with 40% EtOAc/hexane afforded a white foam product. A solution of this product in MeOH/water (1:1) (250 mL)

was mixed with NaOH (5 M in water) (23.46 mL, 117 mmol). The mixture was heated at 70 °C for 1 h. The mixture was then cooled, diluted with water, and acidified with 2 M aqueous HCl. The title compound was isolated by preparative HPLC as a white powder (3.8 g, 22% yield). 1H NMR (500 MHz, DMSO-d6): δ 13.09 (br s, 1H), 7.78 (d, 1H, J = 2.7 Hz), 7.75−7.70 (m, 4 H), 7.56 (s, 1H), 7.54−7.46 (m, 5 H), 7.43−7.36 (m, 3 H), 2.54 (s, 3 H). 13C NMR (125 MHz, DMSO-d6): δ 168.2, 158.2, 151.5, 140.2, 139.5, 139.3, 137.0, 133.5, 133.3, 132.1, 130.7, 129.5, 128.0, 127.1, 126.8, 124.7, 124.3, 122.3, 21.1. HRMS m/z observed 455.1176 (M + H+, C27H19ClN2O3, theoretical 455.1162). 3-((5-([1,1′-Biphenyl]-4-yl)-6-chloro-1H-benzo[d]imidazol-2yl)oxy)benzoic Acid (41). The title compound was prepared in the manner similar to the procedures described in compound 42 by utilizing methyl 3-hydroxybenzoate as the starting material (20 mg, 24% yield). 1H NMR (500 MHz, DMSO-d6) δ 7.92 (s, 1H), 7.88 (d, J = 7.7 Hz, 1H), 7.72−7.59 (m, 6H), 7.52−7.46 (m, 5H), 7.38 (t, J = 7.5 Hz, 2H). 5-((5-([1,1′-Biphenyl]-4-yl)-6-chloro-1H-benzo[d]imidazol-2yl)oxy)-2-chlorobenzoic Acid (43). The title compound was prepared in the manner similar to the procedures described in compound 42 by utilizing methyl 2-chloro-5-hydroxybenzoate as the starting material (50 mg, 35% yield). 1H NMR (500 MHz, acetone-d6) δ 8.03 (d, J = 2.9 Hz, 1H), 7.74 (t, J = 7.6 Hz, 5H), 7.67 (d, J = 8.8 Hz, 1H), 7.61 (s, 1H), 7.55 (d, J = 7.9 Hz, 2H), 7.49 (t, J = 7.5 Hz, 3H), 7.39−7.36 (m, 1H). 5-((6-Chloro-5-(phenylethynyl)-1H-benzo[d]imidazol-2-yl)oxy)-2-methylbenzoic Acid (44). A Biotage microwave vial was charged with copper iodide (10 mol %) and Pd(PPh3)2Cl2 (5 mol %). A solution of intermediate 33 (0.4 mmol, 1 equiv) in DMF (2 mL) was added to the vial, followed by a solution of ethynylbenzene (0.8 mmol, 2 equiv) in DMF (250 μL) and TEA (200 μL). The resulting mixture was heated in a microwave synthesizer at 120 °C for 5 min. The solvent was removed in vacuo to give dark-brown oil which was used in the synthesis of the title compound without further purification. The title compound was prepared in the manner similar to the procedures described in compound 42 by utilizing methyl 5hydroxy-2-methylbenzoate as the starting material (36 mg, 18% yield). 1 H NMR (500 MHz, DMSO-d6) δ 7.54 (s, 1H), 7.49−7.46 (m, 4H), 7.36−7.35 (m, 3H), 7.21−7.16 (m, 2H), 2.42 (s, 3H). 2-Chloro-5-((6-chloro-5-(phenylethynyl)-1H-benzo[d]imidazol-2-yl)oxy)benzoic Acid (45). The title compound was prepared in the manner similar to the procedures described in compound 44 by utilizing methyl 2-chloro-5-hydroxybenzoate as the starting material. 1H NMR (500 MHz, DMSO-d6) δ 7.85 (d, J = 2.7 Hz, 1H), 7.67−7.56 (m, 6H), 7.43 (d, J = 5.7 Hz, 3H). 5-((5-(4-(1H-Pyrazol-5-yl)phenyl)-6-chloro-1H-benzo[d]imidazol-2-yl)oxy)-2-methylbenzoic Acid (46). The title compound was prepared in the manner similar to the procedures described in compound 42 by utilizing (4-(1H-pyrazol-5-yl)phenyl)boronic acid as the starting material (30 mg, 25% yield). 1H NMR (500 MHz, DMSO-d6) δ 7.87−7.85 (m, 2H), 7.78 (d, J = 2.7 Hz, 1H), 7.73 (d, J = 2.2 Hz, 1H), 7.55 (s, 1H), 7.51 (dd, J = 8.3, 2.8 Hz, 1H), 7.47−7.45 (m, 2H), 7.40 (d, J = 8.5 Hz, 1H), 6.75 (d, J = 2.2 Hz, 1H), 7.37 (s, 1H), 2.54 (s, 3H). 5-((6-Chloro-5-(2′-hydroxy-[1,1′-biphenyl]-4-yl)-1H-benzo[d]imidazol-2-yl)oxy)-2-methylbenzoic Acid (47). The title compound was prepared in the manner similar to the procedures described in compound 42 by utilizing intermediate 4′-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)-[1,1′-biphenyl]-2-ol as the starting material (44 mg, 24%). 1H NMR (500 MHz, acetone-d6) δ 7.99 (d, J = 2.7 Hz, 1H), 7.69−7.67 (m, 2H), 7.58−7.55 (m, 2H), 7.49 (s, 1H), 7.48 (t, J = 1.9 Hz, 1H), 7.44 (d, J = 0.5 Hz, 1H), 7.42 (d, J = 8.4 Hz, 1H), 7.37 (dd, J = 7.6, 1.7 Hz, 1H), 7.21−7.17 (m, 1H), 7.01 (dd, J = 8.1, 1.2 Hz, 1H), 6.95 (td, J = 7.5, 1.2 Hz, 1H), 2.60 (s, 3H). 5-((6-Chloro-5-(1-methyl-1H-indol-5-yl)-1H-benzo[d]imidazol-2-yl)oxy)-2-methylbenzoic Acid (48). Step 1, 6-Chloro5-iodo-2-(methylsulfonyl)-1-{[2-(trimethylsilyl)ethoxy]methyl}-1Hbenzimidazole (39): Et 3 N (20.95 mL, 150 mmol) and 2(trimethylsilyl)ethoxy methyl chloride (17.29 mL, 98 mmol) were 9049



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added to a solution of intermediate 33 (75 mmol) in THF (200 mL). The reaction was stirred at rt for 1 h. Volatiles were removed and the residue partitioned between EtOAc and water. The organic phase was washed with 2 N aqueous HCl and brine, dried (MgSO4), and concentrated to afford the title compound as a white solid. Step 2, Methyl 5-[(6-chloro-5-iodo-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-benzimidazol-2-yl)oxy]-2-methylbenzoate (40): K2CO3 (16.64 mmol) was added to a solution of methyl 5-hydroxy-2methylbenzoate (6.57 mmol) and 39 (4.31 mmol) in DMF (30 mL). The reaction was stirred at rt for 24 h. Volatiles were removed, and the residue was acidified with 2 N aqueous HCl and extracted with EtOAc. The organics were washed with water and brine, dried (MgSO4), and concentrated to afford the title compound as a brown solid, which was used without further purification. Step 3, Methyl 5-[(6-chloro-5-(1-methyl-1H-indol-5-yl)-1-{[2(trimethylsilyl)ethoxy]methyl}-1H-benzimidazol-2-yl)oxy]-2-methylbenzoate: A 500 mL flask was charged with 40 (4.31 mmol), 1methylindole-5-boronic acid pinacol ester (5.7 mmol), Pd(PPh3)4 (0.65 mmol), DMF (50 mL), and 1 M aqueous K2CO3 (13 mL). The reaction was degassed with N2 and then heated at 120 °C for 45 min. The reaction was concentrated then partitioned between H2O (100 mL) and EtOAc (200 mL). The aqueous phase was extracted with EtOAc (2 × 100 mL). The combined organic layers were washed with brine, dried (MgSO4), filtered, and concentrated. Chromatography over silica eluting with 10−30% EtOAc/hexanes afforded the desired product as a yellow-orange foam. Step 4, 5-((6-Chloro-5-(1-methyl-1H-indol-5-yl)-1H-benzo[d]imidazol-2-yl)oxy)-2-methylbenzoic acid (48): To a solution of product from step 3 (2.9 mmol) in THF (30 mL) was added TBAF (1.0 M in THF) (13 mmol) dropwise via syringe. The reaction was heated at 80 °C for 45 min. More TBAF (3 mL) was added, and heating continued for 4 h. The reaction was cooled and diluted with EtOAc (100 mL) and saturated aqueous KHSO4 (∼pH 3). The aqueous phase was extracted with EtOAc (100 mL). The combined organic layers were washed with H2O and brine, dried (Na2SO4), filtered, and concentrated. The residue was dissolved in MeOH (50 mL) and treated with 2.5 N aqueous NaOH. The reaction was heated at 45 °C for 1 h. Volatiles were removed, and the residue was dissolved in H2O (150 mL). The aqueous phase was washed with EtOAc (2 × 100 mL), acidified to pH ∼ 1 with 2 N aqueous HCl, and extracted with EtOAc (2 × 100 mL). The combined organics were washed with H2O and brine, dried (Na2SO4), filtered, and concentrated. The title compound was isolated by preparative HPLC (660 mg, 53% yield). 1H NMR (500 MHz, DMSO-d6) δ 7.78 (d, J = 2.7 Hz, 1H), 7.54−7.46 (m, 4H), 7.40 (d, J = 8.5 Hz, 1H), 7.36 (d, J = 3.1 Hz, 1H), 7.32 (s, 1H), 7.18 (dd, J = 8.4, 1.7 Hz, 1H), 6.45 (dd, J = 3.1, 0.8 Hz, 1H), 3.82 (s, 3H), 2.54 (s, 3H).

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F. Anthony Romero: 0000-0001-7917-1459 Notes

The authors declare the following competing financial interest(s): The authors are, or were, employees of Merck & Co. Inc., Kenilworth, NJ USA during this work, and may own shares of Merck & Co. stock.

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ACKNOWLEDGMENTS We thank the staff of the purification group at Merck & Co., Inc., Kenilworth, NJ, USA, for their help with final product purification. We also thank Dr. John Debenham and Dr. Robert Myers for their support and review of the manuscript. This work was fully funded by Merck & Co. Inc., Kenilworth, NJ, USA.

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ABBREVIATIONS USED ACC1, ACC2, acetyl-CoA carboxylase 1 and 2; AMPK, 5′adenosine monophosphate-activated protein kinase; AICAR, 5aminoimidazole-4-carboxamide-1-β-D-ribofuranoside; BCRP/ ABCG2, breast cancer resistance protein/ATP-binding cassette subfamily G member 2; C57BL/6, C57 black 6 mouse strain; DNL, de novo lipogenesis; DP2 (or CRTH2), prostaglandin D2 receptor 2; FAO, fatty acid oxidation; FAS, fatty acid synthesis; GLUT4, glucose transporter type 4; G6 Pase, glucose 6-phosphatase; IRI, insulin resistance index; KO, knockout; LLC-PK1, Lilly Laboratories cell- porcine kidney 1; Mpk, milligrams per kilograms; ND, not determined; NIS, Niodosuccinimide; OATP1B1, organic anion transporter protein B1; OGTT, oral glucose tolerance test; PEPCK, phosphoenolpyruvate carboxykinase; PRAK, p38-regulated/activated protein kinase; PRKAG2, protein kinase, amp-activated, noncatalytic, γ2; pACC, phophorylated acetyl-CoA carboxylase; pAMPK, phosphorylated AMP kinase; PXR, pregnane X receptor; SEM, 2-(trimethylsilyl)ethoxymethyl.

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(1) Beg, Z. H.; Allmann, D. W.; Gibson, D. M. Modulation of 3hydroxy-3-methylglutaryl coenzyme A reductase activity with cAMP and wth protein fractions of rat liver cytosol. Biochem. Biophys. Res. Commun. 1973, 54, 1362−1369. (2) Carlson, C. A.; Kim, K. H. Regulation of hepatic acetyl coenzyme A carboxylase by phosphorylation and dephosphorylation. J. Biol. Chem. 1973, 248, 378−380. (3) Ruderman, N.; Prentki, M. AMP kinase and malonyl-CoA: targets for therapy of the metabolic syndrome. Nat. Rev. Drug Discovery 2004, 3, 340−351. (4) Hardie, D. G. AMPK: a key regulator of energy balance in the single cell and the whole organism. Int. J. Obes. 2008, 32, S7−S12. (5) Lage, R.; Dieguez, C.; Vidal-Puig, A.; Lopez, M. AMPK: a metabolic gauge regulating whole-body energy homeostasis. Trends Mol. Med. 2008, 14, 539−549. (6) Hardie, D. G. The AMP-activated protein kinase pathway − new players upstream and downstream. J. Cell Sci. 2004, 117, 5479−5487. (7) Ha, J.; Daniel, S.; Broyles, S. S.; Kim, K. H. Critical phosphorylation sites for acetyl-CoA carboxylase activity. J. Biol. Chem. 1994, 269, 22162−22168. (8) Abu-Elheiga, L.; Matzuk, M. M.; Abo-Hashema, K. A. H.; Wakil, S. J. Continuous fatty acid oxidation and reduced fat storage in mice lacking acetyl-CoA carboxylase 2. Science 2001, 291, 2613−2616. (9) Lochhead, P. A.; Salt, I. P.; Walker, K. S.; Hardie, D. G.; Sutherland, C. 5-Aminoimidazole-4-carboxamide riboside mimics the effects of insulin on the expression of the 2 key gluconeogenic genes PEPCK and glucose-6-phosphatase. Diabetes 2000, 49, 896−903. (10) Zhou, G.; Myers, R.; Li, Y.; Chen, Y.; Shen, X.; Fenyk-Melody, J.; Wu, M.; Ventre, J.; Doebber, T.; Fujii, N.; Musi, N.; Hirshman, M.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b01344. Details of in vitro and in vivo assays, 1H NMR of final compounds and full characterization of compound 42 (PDF) Molecular formula strings (CSV)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*For P. L.: phone, (908)740-0042; E-mail ping_lan@merck. com. *For I. K. S.: phone, (201)245-0118; Email, iyassu@kallyope. com. ORCID

Ping Lan: 0000-0001-8728-180X 9050



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(42) Compound permeability was determined in bidirectional LLCPK1 cell model using the protocol described in the reference: Balimane, P. V.; Han, Y.-H.; Chong, S. Current industrial practices of assessing permeability and p-glycoprotein interaction. AAPS J. 2006, 8 (1), E1−13 Briefly, a monolayer of LLC-PK1 cells was formed when the cells were seeded onto polycarbonate filter membrane and cultured. Solution of test compound in HBSS buffer was added to either the apical (A) or the basolateral (B) compartment of the culture plate. Aliquots were taken from wells on both sides at the end of incubation, and the concentrations of test compound were analyzed by HPLC. The apparent permeation (Papp) represents the average of the Papps from A to B and from B to A.. (43) The transporter uptake assay was performed according to published procedures: Brouwer, K. L.; Keppler, D.; Hoffmaster, K. A.; Bow, D. A. J.; Cheng, Y.; Lai, Y.; Palm, J. E.; Stieger, B.; Evers, R. In vitro methods to support transporter evaluation in drug discovery and development. Clin. Pharmacol. Ther. 2013, 94, 95−112 Briefly, the labeled compound [3H]-42 was incubated with wild-type Madin− Darby canine kidney (MDCK II) cells or transfected MDCK II cells stably expressing human organic anion transporting polypeptide (OATP1B1 or OATP1B3). The accumulation of the test compound in cells over the incubation period (< 10 minutes) were assessed by scintillation counting.. (44) Cytochrome P450 (CYP) inhibition and induction assays were available at Evotec as a contract service. Potential CYP inhibition was assessed using panel of CYP reversible assay and CYP3A4 timedependent inhibition assay in human liver microsomes. The potential of a compound to cause CYP induction via activation of human pregnane X receptor (hPXR) was assessed utilizing stably transfected human hepatoma cell line and a luciferase reporter gene assay. A summary of each assay protocol and the references for each assay are listed in http://www.cryprotex.com/admepk (45) For Panlab selectivity counterscreen, all radioligand binding or enzymatic assays utilized were available at MDS Pharma Services (current name: Eurofins Panlab Taiwan, Ltd.) as a contract service. A summary of each assay protocol and the reference for each assay are listed in the Eurofins Panlab Taiwan catalogue. (46) Yang, C.-Y.; Liao, J. K. A mouse model of diet-induced obesity and insulin resistance. Methods Mol. Biol. 2012, 821, 421−433. (47) Matsuda, M.; Defronzo, R. A. Insulin sensitivity indices obtained from oral glucose tolerance testing. Diabetes Care 1999, 22, 1462− 1470. (48) Myers, R. W.; Guan, H.-P.; Ehrhart, J.; Petrov, A.; Prahalada, S.; Tozzo, E.; Yang, X.; Kurtz, M. M.; Trujillo, M.; Gonzalez Trotter, D.; Feng, D.; Xu, S.; Eiermann, G.; Holahan, M. A.; Rubins, D.; Conarello, S.; Niu, X.; Souza, S. C.; Miller, C.; Liu, J.; Lu, K.; Feng, W.; Li, Y.; Painter, R. E.; Milligan, J. A.; He, H.; Liu, F.; Ogawa, A.; Wisniewski, D.; Rohm, R.; Wang, L.; Bunzel, M.; Qian, Y.; Zhu, W.; Wang, H.; Bennet, B.; LaFranco Scheuch, L. L.; Fernandez, G. E.; Li, C.; Klimas, M.; Zhou, G.; van Heek, M.; Biftu, T.; Weber, A.; Kelley, D. E.; Thornberry, N.; Erion, M. D.; Kemp, D. M.; Sebhat, I. K. Systemic pan-AMPK activator MK-8722 improves glucose homeostasis but induces cardiac hypertrophy. Science 2017, 357, 507−511.

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