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Discovery of a Hydroxypyridinone APJ Receptor Agonist as a Clinical Candidate James A. Johnson,* Soong-Hoon Kim, Ji Jiang, Monique Phillips, William A. Schumacher, Jeffrey S. Bostwick, Peter S. Gargalovic, Joelle M. Onorato, Chiuwa E. Luk, Claudia Generaux, Yan He, Xue-Qing Chen, Carrie Xu, Michael A. Galella, Tao Wang, David A. Gordon, Ruth R. Wexler, and Heather J. Finlay

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ABSTRACT: Apelin-13 is an endogenous peptidic agonist of the apelin receptor (APJ) receptor with the potential for improving cardiac function in heart failure patients. However, the low plasma stability of apelin-13 necessitates continuous intravenous infusion for therapeutic use. There are several approaches to increase the stability of apelin-13 including attachment of pharmacokinetic enhancing groups, stabilized peptides, and Fc-fusion approaches. We sought a small-molecule APJ receptor agonist approach to target a compound with a pharmacokinetic profile amenable for chronic oral administration. This manuscript describes sequential optimization of the pyrimidinone series, leading to pyridinone 14, with in vitro potency equivalent to the endogenous ligand apelin-13 and with an excellent oral bioavailability and PK profile in multiple preclinical species. Compound 14 exhibited robust pharmacodynamic effects similar to apelin-13 in an acute rat pressure−volume loop model and was advanced as a clinical candidate.

■ INTRODUCTION Heart failure (HF) is a complex chronic syndrome characterized by insufficient cardiac output (CO) to maintain normal organ perfusion and function. Worldwide, an estimated 24 million people are diagnosed with HF, a number that is projected to increase.1,2 Within the HF disease spectrum, approximately one half of HF patients have left ventricular ejection fraction (EF) below 50% as a direct result of significant impairment in cardiac contractility, a hallmark of HF with reduced EF (HFrEF). Standard of care for HFrEF patients includes blockers of the renin−angiotensin−aldosterone system (RAAS) such as angiotensin-converting enzyme inhibitors (ACEi) or angioten- sin II type 1 receptor (AT1R) blockers (ARBs), which lower blood pressure and attenuate cardiac remodeling.3 Co- medications are commonly prescribed such as diuretics to reduce edema, vasodilators to reduce blood pressure and lower cardiac load, and β-blockers to reduce stress on the heart by lowering heart rate (HR) and other adrenergic responses. Currently available inotropic agents have shown short-term symptomatic relief for HF patients, but long-term use leads to an increase in mortality.4,5 Despite the current standard-of-care regime, only half of patients diagnosed with HFrEF will survive beyond 5 years.6,7 Further approaches are needed to more effectively address HF symptoms and decrease mortality in this area of clear unmet medical need. The apelin−apelin receptor (APJ) system plays a critical role

in cardiovascular physiology and regulation of homeostasis.8

APJ is a class A G-protein-coupled receptor (GPCR) with the peptides apelin and elabela identified as endogenous ligands, of which apelin is the most studied.9−14 Pre-pro-apelin is produced as a 77 amino acid peptide that is cleaved by a family of endopeptidases to smaller peptides of varying activity.15 Of these fragments, the pyroglutamated form of apelin-13, [Pyr1]apelin-13, has the highest affinity for APJ and is the primary peptide circulating in human plasma and heart.16,17 APJ is a Gi-coupled receptor, and its binding with [Pyr1]apelin-13 causes inhibition of intracellular cyclic adenosine mono- phosphate (cAMP) production, β-arrestin recruitment, and receptor internalization.18 Numerous studies utilizing [Pyr1]- apelin-13 in preclinical animal models of cardiovascular diseases have shown benefit. For example, treatment using [Pyr1]apelin- 13 in a high-fat diet mouse ischemia/reperfusion model resulted in significantly reduced infarct size and myocardial apoptosis.19

Hypertensive rats with HF induced by a two-kidney, one-clip method had significantly improved systolic and diastolic function when infused with [Pyr1]apelin-13.20 Most impor-

Received: October 29, 2020 Published: March 9, 2021

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a small-molecule APJ receptor agonist a

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APJ is a class A G-protein-coupled receptor (GPCR) apelin and elabela identified as endogenous ligands,

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primary peptide circulating in human plasma and heart. Gi-coupled receptor, s inhibition of intracellular cyclic adenosine mono-

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tantly, acute studies in HFrEF patients using intravenous (i.v.) administration of [Pyr1]apelin-13 demonstrated increased CO along with peripheral and coronary vasodilation, which was sustained for the duration of the 6 h infusion without significant effects on blood pressure or HR.21,22

The therapeutic potential of APJ agonism by [Pyr1]apelin-13 is encouraging; however, low plasma stability makes it unsuitable for oral administration and chronic treatment.23

Synthetic modifications to the peptide have been made in an effort to increase plasma half-life including macrocyclization and backbone modifications, conjugation with polyethylene glycol or fatty acid scaffolds, as well as Fc-fusion constructs.24−32 An agonistic antibody for APJ has also recently been reported.33

During the course of our studies, additional small-molecule APJ receptor agonists have been reported in the literature, and subsequent to our work, the structure of the clinical lead AMG 986 (Figure 1) was disclosed.34 Studies of AMG 986 using acute i.v. dosing in a rat model of diastolic dysfunction demonstrated improvement in stroke volume (SV) and EF while lowering systemic vascular resistance.35 Chronic oral dosing of the closely related tool molecule AM-8123 in a rat myocardial infarction model resulted in improved heart function and reduced collagen deposition without evidence of hypertrophy. In addition to triazoles,36−47 various cores such as benzimidazoles,48−51

pyrazoles, and pyrimidinones52−57 have recently been reported from different entities. We recently disclosed a series of pyrimidinone-based APJ

agonists which demonstrated good oral exposure in rat PK studies.58 We sought to increase the chemical stability of this series by exploring the pyridinone core (Figure 2).59 Extensive optimization at the C3 and C6 positions for potency and improved PK profile led to compound 14, which was advanced as a clinical candidate.

■ RESULTS AND DISCUSSION Structure−Activity Relationships. The potency for

compounds was measured by the ability to inhibit forskolin- induced cAMP production in HEK293 cells expressing human APJ (hAPJ) receptor. Ester 1 was used as an intermediate to explore substitution at the C3 position. Oxadiazoles 3 and 4 had significantly better potency than the triazole 2 (Table 1). The metabolic stability of the series remained low in rat liver microsomes. The 1,3,4-oxadiazole 4 was the most potent of the heterocycles prepared and was selected as the preferred linker to evaluate the pendant benzyl group substitution. Surveying chlorine substitution on the benzyl ring revealed

ortho and meta substitution (compounds 5 and 6) to afford similar to moderate improvement in potency (Table 2). Para-Cl substitution (compound 7) provided a significant increase in potency, albeit with no improvement in metabolic stability. Removing the bridging methylene by directly attaching a para- Cl phenyl to the oxadiazole (compound 8) resulted in a significant loss in potency. Concurrent with optimization at the C3 position, modifica-

tion of the C6 position of pyridinone was explored (Table 3). Earlier SAR work in the pyrimidinone series indicated that small lipophilic groups were potent at this position and were, therefore, targeted in the pyridinone series.58 While a cyclo- propyl ring attached directly to the pyridinone (compound 9) resulted in a loss in potency, a cyclopentyl ring at this position was tolerated (compound 10). The methoxyethyl (compound 11) did not improve potency but did increase solubility and metabolic stability relative to the butyl side chain (compound 7). We were pleased to find that the ethoxymethyl (compound 12) maintained these favorable properties while improving potency, and this side chain was therefore utilized in further optimization efforts. With the ethoxymethyl at C6 in place, additional modification

of the pendant benzyl ring was explored. Replacing the 4- chlorophenyl group of compound 12 with 5-(2-chloropyridine) (compound 13) resulted in an improvement in solubility while maintaining potency (Table 4). The 5-(3-chloropyridine) (compound 14) not only further improved solubility but also resulted in a significant potency improvement. Compound 14 was determined to be a fully competitive

orthosteric ligand, displacing radiolabeled [Pyr1]apelin-13 from the hAPJ receptor, with a Ki similar to the endogenous ligand

Figure 1. Representative APJ agonists from the literature with reported activity.

Figure 2. Pyridinone core optimization.

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(Table 5). Compound 14 was also equipotent to [Pyr1]apelin- 13 in its ability to inhibit forskolin-mediated cAMP production across species. No evidence of biased signaling was observed, as exemplified by the similarities of compound 14 and [Pyr1]- apelin-13 to stimulate β-arrestin recruitment.60 Based on the profile of potency and metabolic stability, compound 14 was selected for investigative in vivo studies. Chemistry. The syntheses of the pyridinone core are

described in Scheme 1. Directed ortho-metallation of

dimethylresorcinol followed by alkylation with ethyl bromoa- cetate afforded 15. Condensation of the anion of the ethyl phenylacetate with the requisite acid chloride afforded a mixture of β-keto ester tautomers 16a−e. Amination using ammonia converted 16a−e to the enamines 17a−e. The pyridinones 1 and 18b−e were synthesized by acylation of 17a−e with ethyl malonyl chloride, followed by intramolecular cyclization in the presence of NaOEt.

Table 1. C3 SAR

aCompound-stimulated inhibition of forskolin-mediated cAMP production utilizing HEK293 cells expressing human or rat APJ were used to calculate the EC50 and measured in at least triplicate.

bMetabolic stability was reported as percent of parent compound remaining after 10 min incubation with NADPH-supplemented human (H) and rat (R) liver microsomes.

Table 2. Oxadiazole Substitution

aCompound-stimulated inhibition of forskolin-mediated cAMP production utilizing HEK293 cells expressing human or rat APJ were used to calculate the EC50 and measured in at least triplicate.

bMetabolic stability was reported as percent of parent compound remaining after 10 min incubation with NADPH-supplemented human (H) and rat (R) liver microsomes.

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(Table

The synthesis of the heterocyclic linkers is described in Scheme 2. Condensation of ester 1 with ethyl 2-phenyl- acetimidate afforded triazole 2, while condensation with N′- hydroxy-2-phenylacetimidamide afforded the 1,2,4-oxadiazole 3. The 1,3,4-oxadiazole 4 was prepared by a two-step procedure of hydrazide formation from ester 1, followed by T3P-mediated coupling and cyclization using phenylacetic acid. The two-step method of hydrazide formation, then coupling,

and dehydration was used for 1,3,4-oxadiazoles shown in Scheme 3.

Crystal Structure. A single-crystal structure of compound 14 was obtained, which showed orthogonality of the pyridinone ring and lower 2,6-dimethoxy phenyl (Figure 3). The solid-state structure was the pyridinone tautomer, with the oxadiazole

Table 3. C6 SAR

aCompound-stimulated inhibition of forskolin-mediated cAMP production utilizing HEK293 cells expressing human or rat APJ were used to calculate the EC50 and measured in at least triplicate.

bMetabolic stability was reported as percent of parent compound remaining after 10 min incubation with NADPH-supplemented human (H) and rat (R) liver microsomes.

Table 4. Oxadiazole Substitution

aCompound-stimulated inhibition of forskolin-mediated cAMP production utilizing HEK293 cells expressing human or rat APJ were used to calculate the EC50 and measured in at least triplicate.

bMetabolic stability was reported as percent of parent compound remaining after 10 min incubation with NADPH-supplemented human (H) and rat (R) liver microsomes.

Table 5. Comparison of Compound 14 to [Pyr1]apelin-13

assaya 14 [Pyr1]apelin-13

hAPJ Ki (nM)b 0.074 ± 0.055 0.17 ± 0.039 APJ cAMP EC50 (nM) human 0.023 ± 0.018 0.05 ± 0.07 rat 0.024 ± 0.0074 0.06 ± 0.06 dog 0.03 ± 0.01 0.06 ± 0.03 monkey 0.08 ± 0.04 0.14 ± 0.27 hAPJ β-arrestin EC50 (nM) 7.1 ± 3.2 18 ± 12.4

an ≥ 4 for human and rat assays and n = 3 for monkey and dog assays. b[Pyr1]-apelin 13 was used as a positive control.

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forming an intramolecular hydrogen bond to the phenolic proton (pKa 4.3). Pharmacokinetics. The pharmacokinetics of compound 14

were evaluated in rat and cynomolgus monkey following a 3 mg/ kg oral dose administered as micro-suspensions (Table 6). The Cmax and AUCINF in rat were 5.6 μM and 34 μM

.h, respectively, with an oral bioavailability (F) of 77%. For monkey, decreased Cmax and AUCINF were observed (1.5 μM and 15 μM

.h, respectively), but the overall bioavailability was still high (56%). The renal clearance of 14 was low with 4% of 14 eliminated as parent in urine for rat and 12% in monkey (biliary excretion was not determined). Metabolism. Compound 14 was found to be stable when

incubated with rat, monkey, and human liver microsomes for 45 min at 30 μM substrate concentration, with >95% remaining across species. Demethylation of the resorcinol ring to form non- interconverting atropisomers was the major metabolite in rat (3.3%), monkey (2.3%), and human (0.2%) liver microsomes. No glutathione adducts or glucuronides were detected during biotransformation studies. Pharmacodynamics. Compound 14 was advanced to the

acute rat pressure−volume loop (PV-loop) model to assess cardiovascular pharmacodynamics in anesthetized normal rats.61 Compound 14 was administered by i.v. infusion for 15 min (10 μg/kg/min), followed by a 15 min observation period

(Figure 4) while hemodynamic parameters including CO, left ventricular contractility (LV dP/dt), HR, and mean arterial blood pressure (MAP) were recorded. Compound 14 produced an increase in CO of 12%, with free fraction adjusted C[plasma] = 18 nM at the 5 min time point.62 A similar effect was also observed on cardiac contractility with a maximal response on LV +dP/dt of 9% using this dosing paradigm. The magnitude of these hemodynamic effects are consistent to those observed with [Pyr1]apelin-13 in preclinical models as well as in human subjects.21−23,63−65 There was a minor increase in HR over vehicle (3%) and a mild but transient decrease in MAP. These data suggest that the effects of compound 14 on increasing CO and dP/dt were primarily due to a combination of increased cardiac contractility and peripheral vasodilatation. This represents a potential improvement in contrast to currently used inotropes such as dobutamine, which increases CO by mechanisms that significantly increase HR.66 An account of compound 14 describing a sustained increase in SV and CO in a preclinical HF rat model with chronic dosing has recently been published.62

■ CONCLUSIONS In summary, we have developed SAR in the pyridinone series and optimized for APJ receptor agonist potency and metabolic stability culminating in compound 14 with potency and a

Scheme 1. Synthesis of Pyridinone Coresa

aReagents: (a) (1) nBuLi, CuI, THF; (2) ethyl bromoacetate, −78 °C to RT, 86%; (b) (1) LiHMDS, THF,−78 °C to RT; (2) XCO2Cl, −78 °C to RT, 88% for 16a, 71% for 16b, 95% for 16c, 28% for 16d, 54% for 16e; (c) HCO2NH4, EtOH, 68% for 17a, 34% for 17b, 49% for 17c, 63% for 17d, 70% for 17e; (d) (1) ethyl malonyl chloride, 1 N NaHCO3, DCM; (2) NaOEt, EtOH; 35% for 1, 28% for 18b, 23% for 18c, 25% for 18d, 35% for 18e.

Scheme 2. Synthesis of Triazole, 1,2,4-Oxadiazole, and 1,3,4-Oxadiazole Compoundsa

aReagents: (a) (1) NH2NH2, EtOH, 98%; (2) ethyl 2-phenylacetimidate, DIEA, isopropyl alcohol, 120 °C (microwave), 56% for 2; (b) N′- hydroxy-2-phenylacetimidamide, 120 °C, 26% for 3; (c) (1) NH2NH2, EtOH, 98%; (2) phenylacetic acid, T3P, dioxane, 160 °C (microwave), 72% for 4.

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cardiovascular effect similar to the endogenous ligand [Pyr1]- apelin-13. Compound 14 has increased chemical and metabolic stability and is greater than 100-fold more potent compared to our previously reported pyrimidinone series while improving pharmacokinetic properties.58 In addition, compound 14 has an improved pharmacokinetic profile across species and pharma-

codynamics effects that enabled an acceptable projected human dose for clinical studies.67

■ EXPERIMENTAL SECTION Chemistry. All solvents and reagents were used as obtained.

Reactions involving air- or moisture-sensitive reagents were carried out under a nitrogen atmosphere. Microwave reactions were performed using Biotage Initiator reactors. Reaction progress was monitored by TLC analysis (2.5 × 7.5 cm pre-coated plates, Merck KgaA, TLC silica gel 60 F254) and reverse-phase analytical HPLC equipped with a mass detector (Shimadzu HPLC system running Discovery VP software, coupled with a Waters Micromass ZQ mass spectrometer running Mass Lynx version 3.5 software). NMR spectra were recorded in a deuterated solvent with a Bruker or JEOL Fourier transform spectrometer operating at frequencies as follows: 1H NMR: 400 MHz (Bruker or JEOL) or 500 MHz (JEOL). 13C NMR: 100 or 125 MHz (Bruker or JEOL). Spectral data are reported in the following format: chemical shift (multiplicity, coupling constants, and number of hydrogens). Chemical shifts are specified in ppm downfield of a tetramethylsilane internal standard (δ units, tetramethylsilane = 0 ppm) and/or referenced to solvent peaks, which in 1H NMR spectra appear at 2.49 ppm for CD2HSOCD3, 3.30 ppm for CD2HOD, and 7.24 ppm for CHCl3, and which in

13C NMR spectra appear at 39.7 ppm for CD3SOCD3 and 49.0 ppm for CD3OD. All

13C NMR spectra were proton decoupled. All coupling constants (J) are reported in hertz. The purity of compounds 1−14 were ≥95% as determined by one of the following analytical HPLC methods: method A: column: Waters Sunfire C18; 3.5 μm, 30 × 150 mm; solvent A: 95:5:0.05 [water/ acetonitrile/trifluoroacetyl (TFA)]; solvent B: 95:5:0.05 (acetonitrile/ water/TFA); flow rate: 1 mL/min; gradient: 10−100% B over 12 min with 3 min hold time; wavelength: 220 nm. Method B: column: Waters XBridge Phenyl; 3.5 μm, 30 × 150 mm; solvent A: 95:5:0.05 (water/ acetonitrile/TFA); solvent B: 95:5:0.05 (acetonitrile/water/TFA); flow rate: 1 mL/min; gradient: 10 to 100% B over 12 min with 3 min hold time; wavelength: 220 nm. Method C: Column: Waters XBridge C18, 2.1 mm × 50 mm, 1.7 μm particles; mobile phase A: 5:95 acetonitrile/water with 0.1% trifluoroacetic acid; mobile phase B: 95:5 acetonitrile/water with 0.1% trifluoroacetic acid; temperature: 50 °C; gradient: 0% B to 100% B over 3 min, then a 0.50 min hold at 100% B; flow: 1 mL/min; detection: MS and UV (220 nm). Method D: Waters Acquity UPLC BEH C18, 2.1 × 50 mm, 1.7 μm particles; mobile phase A: 5:95 acetonitrile/water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile/water with 10 mM ammonium acetate; temperature: 50 °C; gradient: 0−100% B over 3 min, then a 0.75 min hold at 100% B; flow: 1.11 mL/min; detection: UV at 220 nm. Method E: Waters XBridge C18, 2.1 mm × 50 mm, 1.7 μm particles; mobile phase A: 5:95 acetonitrile/water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile/water with 10 mM ammonium acetate; temperature: 50 °C; gradient: 0% B to 100% B over 3 min, then a 0.50 min hold at 100% B; flow: 1 mL/min; detection: MS and UV (220 nm). Method F: Waters Acquity UPLC BEH C18, 2.1 × 50 mm, 1.7 μm particles; mobile phase A: 5:95 acetonitrile/water with 0.1% trifluoroacetic acid; mobile phase B: 95:5 acetonitrile/water with 0.1%

Scheme 3. Synthesis of 1,3,4-Oxadiazole Compoundsa

aReagents: (a) (1) NH2NH2, EtOH; (2) ZCO2H, T3P, dioxane, 160 °C.

Figure 3. Crystal structure of compound 14a. aThe crystal structure of compound 14 has been deposited with the CCDC (Cambridge Crystallographic Data Center) with deposition code CCDC 2015092, resolution of 0.83 Å.

Table 6. PK Parameters for Compound 14

species rata monkeyb

dose (mg/kg) 3 (p.o.)c 3 (p.o.)c

Cmax (μM) 5.6 ± 2.8 1.5 AUCINF (μM·h) 34 ± 6.9 15.3 T1/2 (h)

d 2.5 ± 0.2 3.6 F (%) 77 56

aMale Sprague-Dawley, n = 3. bMale cynomolgus, n = 2. cVehicle: (micro-suspension) 0.5/0.1/99.4 v/v/v Methocel A4M/Tween 80/ water. dDetermined by i.v. dosing at 1 mg/kg using 5/5/90 w/w/w DMAC/Cremophor EL/sodium carbonate buffer pH 9 vehicle for rat and 10/40/50 w/w/w ethanol/PEG400/0.1 M sodium carbonate buffer pH 9 for monkey.

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trifluoroacetic acid; temperature: 50 °C; gradient: 0−100% B over 3 min, then a 0.75 min hold at 100% B; flow: 1.11 mL/min; detection: UV at 220 nm. Method G: instrument: Agilent HPLC, Column: Phenonemax Kinetex XB-C18, 100 × 4.6 mm, 2.6 μm, flow conditions: 1.0 mL/min, 25 °C, mobile phase: A: 0.05% TFA in water, B: 0.05% TFA in acetonitrile, gradient profile: time (min): 0, 10, 14, (A = 100-B) % B: 25 95, 95%, post run time: 6 min (under the initial mobile-phase conditions), injection details: 2 μL of ∼0.5 mg/mL in MeOH. Method H: instrument: Agilent HPLC, Column: Water Xbridge Phenyl, 150 (L) × 4.6 mm (ID), 3.5 μm, flow conditions: 1.0 mL/min, 25 °C, mobile phase: A: 0.05%TFA in water, B: 0.05%TFA in acetonitrile, gradient profile: time (min): 0, 10, 12 (A = 100-B) % B: 25, 95, 95%, post run time: 6 min (under the initial mobile-phase conditions), injection details: 1 μL of ∼0.5 mg/mL in MeOH−ACN(50:50). Scheme 1 Compounds (1, 15, 16a−e, 17a−e, and 18b−e).

Ethyl 2-(2,6-Dimethoxyphenyl)acetate (15). To a solution of 1,3- dimethoxybenzene (3.3 mL, 25 mmol) in tetrahydrofuran (THF) (40 mL) was added dropwise 2.5 M nBuLi in hexanes (10 mL, 25 mmol) over a 10 min period, and then the mixture was stirred for 2 h. Crushed copper(I) iodide (2.4 g, 13 mmol) was added slowly, and then the mixture was stirred for 1 h, turning homogeneous. The mixture was cooled to −78 °C, and then ethyl bromoacetate (2.8 mL, 25 mmol) was added dropwise over 20 min. The cold bath was removed, and the mixture was allowed to warm to room temperature. The mixture was quenched by addition of water, diluted with Et2O, and filtered through Celite. The filtrate was diluted with 1.5 N K2HPO4 and extracted with Et2O (2×). The extracts were washed with brine, dried (MgSO4), filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography eluting with 0−15% EtOAc/ hexanes to afford compound 15 (4.8 g, 86% yield) as a light brown oil, which solidified upon standing. MS m/z: 225.1 [M + H]+. 1H NMR (500 MHz, CDCl3): δ 7.23 (t, J = 8.4 Hz, 1H), 6.58 (d, J = 8.3 Hz, 2H), 4.17 (q, J = 7.2 Hz, 2H), 3.83 (s, 6H), 3.71 (s, 2H), 1.27 (t, J = 7.2 Hz, 3H). Ethyl 2-(2,6-Dimethoxyphenyl)-4-ethoxy-3-hydroxybut-2-enoate

(16e). To a solution of compound 15 (5.0 g, 22 mmol) in THF (50 mL) at −78 °C was added dropwise 1.0 M LiHMDS in THF (56 mL, 56 mmol), and the mixture was stirred for 10 min at room temperature for

1 h. The mixture was cooled to −78 °C, and then 2-ethoxyl acetyl chloride (3.7 mL, 33 mmol) was added dropwise and the mixture was allowed to warm to 0 °C and stirred for 15 min. The mixture was quenched with satd NH4Cl and extracted with EtOAc (3×). The combined extracts were washed with brine, dried (Na2SO4), filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography eluting with 0−30% EtOAc/hexanes to afford an isomeric mixture of compound 16e (3.7 g, 54% yield) as a clear colorless oil. MS m/z: 311.1 [M + H]+. 1H NMR of major isomer (500 MHz, CDCl3): δ 7.28−7.23 (m, 1H), 6.61−6.53 (m, 2H), 4.34− 4.05 (m, 4H), 3.88−3.70 (m, 6H), 3.59−3.32 (m, 2H), 1.30−1.08 (m, 6H).

Ethyl 3-Amino-2-(2,6-dimethoxyphenyl)-4-ethoxybut-2-enoate (17e). The isomeric mixture of compound 16e (6.2 g, 20 mmol) and ammonium formate (7.6 g, 120 mmol) in absolute ethanol (50 mL) was stirred at room temperature. After 24 h, the reaction mixture was filtered and concentrated under reduced pressure. The residue was dissolved in water and extracted with EtOAc (3×). The combined extracts were dried (Na2SO4), filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography eluting with 0−35% EtOAc/hexanes to afford compound 17e (4.3 g, 70% yield) as a clear colorless oil. MS m/z: 310.1 [M + H]+. 1H NMR (500 MHz, CDCl3): δ 7.25−7.20 (m, 1H), 6.55 (d, J = 8.5 Hz, 2H), 4.26− 4.02 (m, 2H), 3.85−3.71 (m, 8H), 3.41 (d, J = 6.9 Hz, 2H), 1.27−1.04 (m, 6H).

Ethyl 5-(2,6-Dimethoxyphenyl)-6-(ethoxymethyl)-4-hydroxy-2- oxo-1,2-dihydropyridine-3-carboxylate (18e). To a solution of compound 17e (5.2 g, 17 mmol) in a mixture of dichloromethane (DCM) (100 mL) and 1 N NaHCO3 (97 mL, 97 mmol) was added dropwise ethyl malonyl chloride (8.5 mL, 17 mmol) and the mixture was vigorously stirred for 2 h. The layers were separated and the aqueous layer was extracted with DCM (2×). The combined extracts were washed with satd NH4Cl and brine, dried (Na2SO4), filtered, and concentrated under reduced pressure. The residue was dissolved in absolute EtOH (100 mL), then sodium ethoxide in ethanol (21 weight % in ethanol, 25 mL, 67 mmol) was added, and the reaction mixture was stirred at room temperature. After 16 h, the reaction mixture was concentrated and then taken up in water (250 mL) and ethyl acetate

Figure 4. Hemodynamic response of compound 14 in anesthetized rats (acute PV loop model)a. aEffects of compound 14 (10 μg/kg/min) and 0.1 M Na2CO3, pH 10 vehicle (100 μL/kg/min) on CO, dP/dt, HR, and mean aortic pressure in isoflurane-anesthetized male Sprague-Dawley rats (n = 8 in each group) with 15 min infusion, followed by a 15 min washout period.

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(250 mL). The layers were separated and the aqueous phase was acidified with conc HCl until pH 3−4, and then the solid compound 18e was collected by filtration (1.4 g). The aqueous layer was extracted with DCM (3 × 100 mL), dried over Na2SO4, concentrated, and purified using silica gel chromatography (50% ethyl acetate/hexanes to ethyl acetate and 10% methanol/ethyl acetate) to isolate additional compound 18e (0.73 g). The product isolated from filtration and chromatography was combined to afford compound 18e (2.1 g, 35% yield) as a white solid. MS m/z: 378.08 [M + H]+. 1H NMR (500 MHz, CDCl3): δ 13.72 (s, 1H), 7.37 (t, J = 8.4 Hz, 1H), 6.66 (d, J = 8.3 Hz, 2H), 4.49 (q, J = 7.2 Hz, 2H), 4.11 (s, 2H), 3.78 (s, 6H), 3.51 (q, J = 7.2 Hz, 2H), 1.46 (t, J = 7.2 Hz, 3H), 1.24 (t, J = 7.0 Hz, 3H). Compounds 1 and 18b−d were prepared as described in the general

procedure given for compound 18e. Ethyl 6-Butyl-5-(2,6-dimethoxyphenyl)-4-hydroxy-2-oxo-1,2-di-

hydropyridine-3-carboxylate (1). 1H NMR (500 MHz, DMSO-d6): δ 13.15 (br s, 1H), 11.45 (br s, 1H), 7.34 (t, J = 8.4 Hz, 1H), 6.71 (d, J = 8.5 Hz, 2H), 4.32 (q, J = 7.2 Hz, 2H), 3.69 (s, 6H), 2.16−2.07 (m, 2H), 1.36−1.28 (m, 2H) 1.30 (t, J = 7.2 Hz, 3H), 1.08 (sxt, J = 7.4 Hz, 2H), 0.66 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz, DMSO-d6): δ 173.5, 172.0, 160.0, 158.2, 154.5, 129.9, 109.5, 103.9, 102.8, 95.9, 60.8, 55.5, 30.3, 29.5, 21.5, 14.2, 13.3. HRMS (ESI) calcd for C20H25NO6 [M + H]+calcd, 376.17546; found, 376.17596. Purity = 96% (method A). Ethyl 6-Cyclopropyl-5-(2,6-dimethoxyphenyl)-4-hydroxy-2-oxo-

1,2-dihydropyridine-3-carboxylate (18b). MS m/z = 360.1 [M + H]+. 1H NMR (500 MHz, CDCl3): δ 7.39−7.32 (m, 1H), 6.64 (d, J = 8.3 Hz, 2H), 4.56−4.29 (m, 2H), 3.77 (s, 6H), 3.75−3.70 (m, 1H), 1.47−1.39 (m, 3H), 0.87−0.78 (m, 4H). Ethyl 6-Cyclopentyl-5-(2,6-dimethoxyphenyl)-4-hydroxy-2-oxo-

1,2-dihydropyridine-3-carboxylate (18c). MS m/z: 388.3 [M + H]+. 1H NMR (500 MHz, CDCl3): δ 7.37 (t, J = 8.4 Hz, 1H), 6.66 (d, J = 8.5 Hz, 2H), 4.48 (q, J = 7.0 Hz, 2H), 3.77 (s, 6H), 2.79−2.69 (m, 1H), 1.90−1.81 (m, 2H), 1.80−1.72 (m, 2H), 1.57−1.50 (m, 4H), 1.45 (t, J = 7.2 Hz, 3H). Ethyl 5-(2,6-Dimethoxyphenyl)-4-hydroxy-6-(2-methoxyethyl)-2-

oxo-1,2-dihydropyridine-3-carboxylate (18d). MS m/z: 378.08 [M + H]+.·1H NMR (500 MHz, CDCl3): δ 13.63 (s, 1H), 9.72 (br s, 1H), 7.36 (t, J = 8.4 Hz, 1H), 6.65 (d, J = 8.3 Hz, 2H), 4.46 (q, J = 7.2 Hz, 2H), 3.78 (s, 6H), 3.55 (t, J = 5.5 Hz, 2H), 3.38 (s, 3H), 2.51 (t, J = 5.5 Hz, 2H), 1.45 (t, J = 7.2 Hz, 3H). Schemes 2 and 3 Compounds (2−13). 3-(5-Benzyl-4H-1,2,4-

triazol-3-yl)-6-butyl-5-(2,6-dimethoxyphenyl)-4-hydroxypyridin- 2(1H)-one (2). To a suspension of compound 1 (50 mg, 0.13 mmol) in ethanol (0.75 mL) was added hydrazine (0.084 mL, 2.6 mmol), and the mixture was stirred for 30 min at room temperature. The mixture was concentrated under reduced pressure to afford 6-butyl-5-(2,6- dimethoxyphenyl)-4-hydroxy-2-oxo-1,2-dihydropyridine-3-carbohy- drazide (47 mg, 98% yield) as a white solid. MS m/z: 362.1 [M + H]+. 1H NMR (400 MHz, DMSO-d6): δ 15.52 (s, 1H), 11.78 (br s, 1H), 10.89 (t, J = 4.4 Hz, 1H), 7.34 (t, J = 8.4 Hz, 1H), 6.71 (d, J = 8.4 Hz, 2H), 4.72 (d, J = 4.8 Hz, 2H), 3.68 (s, 6H), 2.18−2.09 (m, 2H), 1.32 (quin, J = 7.5 Hz, 2H), 1.14−1.02 (m, 2H), 0.66 (t, J = 7.4 Hz, 3H). To a solution of 6-butyl-5-(2,6-dimethoxyphenyl)-4-hydroxy-2-oxo-1,2- dihydropyridine-3-carbohydrazide (6.0 mg, 0.017 mmol) and ethyl 2- phenylacetimidate (2.7 mg, 0.017 mmol) in 2-propanol (0.3 mL) was added diisopropylethyl amine (DIEA) (0.10 mL, 0.57 mmol), and the mixture was heated at 120 °C using microwave irradiation for 20 min. The mixture was concentrated under reduced pressure and then purified by reverse-phase HPLC to afford compound 2 (5.3 mg, 56% yield). 1H NMR (500 MHz, CD3OD): δ 7.38 (t, J = 8.4 Hz, 1H), 7.33− 7.26 (m, 4H), 7.24−7.17 (m, 1H), 6.73 (d, J = 8.5 Hz, 2H), 4.10 (s, 2H), 3.75 (s, 6H), 2.34−2.25 (m, 2H), 1.51−1.39 (m, 2H), 1.20 (sxt, J = 7.4 Hz, 2H), 0.76 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz, CD3OD): δ 168.3, 162.4, 159.1, 158.7, 152.6, 150.3, 137.6, 129.8, 128.4, 128.2, 126.2, 109.9, 106.1, 103.6, 94.1, 54.8, 33.4, 30.7, 30.1, 21.8, 12.5. HRMS (ESI) calcd for C26H28N4O4 [M + H]

+, 461.21833; found, 461.21831. Purity = 97% (method C). 3-(3-Benzyl-1,2,4-oxadiazol-5-yl)-6-butyl-5-(2,6-dimethoxyphen-

yl)-4-hydroxypyridin-2(1H)-one (3). A vial containing compound 1 (25 mg, 0.067 mmol) and N′-hydroxy-2-phenylacetimidamide (50 mg,

0.33 mmol) was sealed and then stirred at 120 °C for 3 h. The mixture was purified by reverse-phase HPLC to afford compound 3 (8.0 mg, 26% yield). 1H NMR (500 MHz, DMSO-d6): δ 12.47 (br s, 1H), 11.87 (br s, 1H), 7.41−7.30 (m, 5H), 7.29−7.22 (m, 1H), 6.72 (d, J = 8.5 Hz, 2H), 4.14 (s, 2H), 3.67 (s, 6H), 2.20−2.10 (m, 2H), 1.34 (quin, J = 7.5 Hz, 2H), 1.09 (sxt, J = 7.3 Hz, 2H), 0.66 (t, J = 7.3 Hz, 3H). 13C NMR (126 MHz, DMSO-d6): δ 174.0, 169.1, 165.7, 159.4, 158.2, 154.5, 135.5, 130.1, 129.0, 128.6, 127.0, 109.2, 103.9, 103.2, 93.0, 55.6, 30.7, 30.5, 29.6, 21.5, 13.3. HRMS (ESI) calcd for C26H27N3O5 [M + H]

+

calcd, 462.20235; found, 462.20322. Purity ≥99% (method D). 3-(5-Benzyl-1,3,4-oxadiazol-2-yl)-6-butyl-5-(2,6-dimethoxyphen-

yl)-4-hydroxypyridin-2(1H)-one (4). To a solution of 6-butyl-5-(2,6- dimethoxyphenyl)-4-hydroxy-2-oxo-1,2-dihydropyridine-3-carbohy- drazide (15 mg, 0.042 mmol, prepared as described for compound 2) in dioxane (0.4 mL) was added phenylacetic acid (6.2 mg, 0.046 mmol), followed by a 50% solution of T3P in ethyl acetate (0.075 mL, 0.13 mmol), and the mixture was heated by microwave irradiation at 160 °C for 1 h. The mixture was concentrated under reduced pressure and then purified using reverse-phase HPLC to afford compound 4 (14 mg, 72% yield). 1H NMR (500 MHz, DMSO-d6): δ 11.78 (br s, 1H), 11.69 (br s, 1H), 7.41−7.27 (m, 6H), 6.73 (d, J = 8.3 Hz, 2H), 4.38 (s, 2H), 3.69 (s, 6H), 2.20−2.10 (m, 2H), 1.33 (quin, J = 7.6 Hz, 2H), 1.09 (sxt, J = 7.4 Hz, 2H), 0.66 (t, J = 7.3 Hz, 3H). 13C NMR (126 MHz, DMSO-d6, 60 °C): δ 168.0, 164.0, 163.7, 159.5, 158.5, 152.3, 134.5, 129.9, 128.8, 128.6, 127.1, 110.1, 104.3, 103.4, 92.6, 55.7, 30.7, 30.4, 29.6, 21.5, 13.2. HRMS (ESI) calcd for C26H27N3O5 [M + H]

+, 462.20235; found, 462.20343. Purity = 99% (method D).

Compounds 5−13 were prepared from compounds 1 and 18b−e as described in the general procedure given for compound 4.

6-Butyl-3-(5-(2-chlorobenzyl)-1,3,4-oxadiazol-2-yl)-5-(2,6-dime- thoxyphenyl)-4-hydroxypyridin-2(1H)-one (5). 1H NMR (500 MHz, DMSO-d6): δ 7.58−7.44 (m, 2H), 7.42−7.33 (m, 3H), 6.73 (d, J = 8.5 Hz, 2H), 4.48 (s, 2H), 3.70 (s, 6H), 2.21−2.10 (m, 2H), 1.34 (quin, J = 7.6 Hz, 2H), 1.09 (sxt, J = 7.3 Hz, 2H), 0.66 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz, DMSO-d6): δ 167.8, 163.8, 162.9, 159.5, 158.3, 152.4, 133.4, 132.2, 131.6, 130.0, 129.5, 129.4, 127.6, 109.4, 104.0, 103.3, 92.5, 55.6, 30.3, 29.7, 29.1, 21.5, 13.3. HRMS (ESI) calcd for C26H26ClN3O5 [M + H]

+, 496.16338; found, 496.16299. Purity = 99% (method E).

6-Butyl-3-(5-(3-chlorobenzyl)-1,3,4-oxadiazol-2-yl)-5-(2,6-dime- thoxyphenyl)-4-hydroxypyridin-2(1H)-one (6). 1H NMR (500 MHz, DMSO-d6): δ 7.54−7.46 (m, 1H), 7.43−7.33 (m, 4H), 6.73 (d, J = 8.5 Hz, 2H), 4.42 (s, 2H), 3.69 (s, 6H), 2.21−2.09 (m, 2H), 1.34 (quin, J = 7.6 Hz, 2H), 1.09 (sxt, J = 7.3 Hz, 2H), 0.66 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz, DMSO-d6): δ 167.8, 163.8, 163.4, 159.5, 158.3, 152.4, 136.9, 133.2, 130.5, 130.0, 128.9, 127.7, 127.2, 109.5, 104.0, 103.3, 92.6, 55.6, 30.3, 30.1, 29.7, 21.5, 13.3. HRMS (ESI) calcd for C26H26ClN3O5 [M + H]

+, 496.16338; found, 496.16311. Purity ≥ 99% (method E).

6-Butyl-3-(5-(4-chlorobenzyl)-1,3,4-oxadiazol-2-yl)-5-(2,6-dime- thoxyphenyl)-4-hydroxypyridin-2(1H)-one (7). 1H NMR (500 MHz, DMSO-d6): δ 7.58−7.44 (m, 2H), 7.42−7.33 (m, 3H), 6.73 (d, J = 8.5 Hz, 2H), 4.48 (s, 2H), 3.70 (s, 6H), 2.21−2.10 (m, 2H), 1.34 (quin, J = 7.6 Hz, 2H), 1.09 (sxt, J = 7.3 Hz, 2H), 0.66 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz, DMSO-d6): δ 167.8, 163.8, 163.6, 159.5, 158.3, 152.4, 133.5, 131.9, 130.8, 130.0, 128.6, 109.5, 104.0, 103.3, 92.6, 55.6, 30.3, 29.9, 29.7, 21.5, 13.3. HRMS (ESI) calcd for C26H26ClN3O5 [M + H]+, 496.16338; found, 496.16446. Purity = 95% (method E).

6-Butyl-3-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)-5-(2,6-dime- thoxyphenyl)-4-hydroxypyridin-2(1H)-one (8). 1H NMR (500 MHz, DMSO-d6): δ 8.06 (d, J = 8.5 Hz, 2H), 7.74 (d, J = 8.5 Hz, 2H), 7.37 (t, J = 8.4 Hz, 1H), 6.75 (d, J = 8.3 Hz, 2H), 3.72 (s, 6H), 2.24−2.13 (m, 2H), 1.36 (quin, J = 7.6 Hz, 2H), 1.11 (sxt, J = 7.4 Hz, 2H), 0.68 (t, J = 7.3 Hz, 3H). 13C NMR (126 MHz, DMSO-d6): δ 167.9, 163.6, 161.6, 159.7, 158.3, 152.7, 136.6, 130.0, 129.7, 128.3, 122.2, 109.4, 104.0, 103.4, 92.7, 55.6, 30.4, 29.7, 21.5, 13.4. HRMS (ESI) calcd for C25H24ClN3O5 [M + H]

+, 482.14773; found, 482.14756. Purity 98.6% (method C).

3-(5-(4-Chlorobenzyl)-1,3,4-oxadiazol-2-yl)-6-cyclopropyl-5-(2,6- dimethoxyphenyl)-4-hydroxypyridin-2(1H)-one (9). 1H NMR (500

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MHz, CDCl3): δ 9.13 (br s, 1H), 7.51−7.29 (m, 5H), 6.74 (d, J = 8.5 Hz, 2H), 4.39 (s, 2H), 3.71 (s, 6H), 1.47 (br s, 1H), 0.97 (d, J = 4.3 Hz, 2H), 0.76 (d, J = 6.4 Hz, 2H). 13C NMR (126 MHz, CDCl3): δ 169.1, 164.8, 163.2, 160.1, 158.8, 147.1, 132.3, 130.3, 130.2, 129.1, 109.8, 106.2, 104.1, 56.0, 31.1, 12.7, 7.5. HRMS (ESI) calcd for C25H23ClN3O5 [M + H]

+, 480.1326; found, 480.1328. Purity = 99% (method F). 3-(5-(4-Chlorobenzyl)-1,3,4-oxadiazol-2-yl)-6-cyclopentyl-5-(2,6-

dimethoxyphenyl)-4-hydroxypyridin-2(1H)-one (10). 1H NMR (500 MHz, CDCl3): δ 9.73 (br s, 1H), 7.39 (br t, J = 8.2 Hz, 1H), 7.33 (s, 4H), 6.68 (d, J = 8.3 Hz, 2H), 4.30 (s, 2H), 3.77 (s, 6H), 2.79 (br t, J = 8.8 Hz, 1H), 1.96−1.78 (m, 5H), 1.78−1.65 (m, 2H), 1.63−1.47 (m, 2H). 13C NMR (126 MHz, CDCl3): δ 169.2, 164.8, 163.3, 160.6, 158.8, 154.2, 133.6, 132.2, 130.3, 130.1, 129.1, 109.9, 104.0, 93.2, 55.9, 41.4, 32.0, 31.1, 26.0. HRMS (ESI) calcd for C27H27ClN3O5 [M + H]

+, 508.1639; found, 508.1640. Purity ≥ 99% (method B). 3-(5-(4-Chlorobenzyl)-1,3,4-oxadiazol-2-yl)-5-(2,6-dimethoxy-

phenyl)-4-hydroxy-6-(2-methoxyethyl)pyridin-2(1H)-one (11). 1H NMR (400 MHz, CDCl3): δ 7.44−7.38 (m, 1H), 7.37−7.29 (m, 5H), 7.30−7.28 (m, 1H), 6.69−6.67 (m, 1H), 6.66 (s, 1H), 4.30 (s, 2H), 3.77 (s, 6H), 3.56 (t, J = 5.9 Hz, 2H), 3.35 (s, 3H), 2.68 (t, J = 5.8 Hz, 2H). 13C NMR (126 MHz, CDCl3): δ 169.4, 164.2, 163.7, 160.2, 158.5, 149.7, 133.7, 132.0, 130.6, 130.4, 129.1, 109.0, 106.9, 104.0, 93.55, 70.0, 59.0, 55.9, 34.1, 31.1. HRMS (ESI) calcd for C25H25ClN3O6 [M + H]

+, 498.1432; found, 498.1435. Purity ≥ 99% (method B). 3-(5-(4-Chlorobenzyl)-1,3,4-oxadiazol-2-yl)-5-(2,6-dimethoxy-

phenyl)-6-(ethoxymethyl)-4-hydroxypyridin-2(1H)-one (12). MS m/ z: 498.1 [M + H]+. 1H NMR (500 MHz, DMSO-d6): δ 7.53−7.29 (m, 5H), 6.74 (d, J = 8.5 Hz, 2H), 4.40 (s, 2H), 3.96 (s, 2H), 3.69 (s, 6H), 3.27 (d, J = 7.0 Hz, 2H), 1.00 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, DMSO-d6): δ 167.6, 163.9, 163.4, 159.1, 158.2, 146.8, 133.4, 131.9, 130.8, 130.2, 128.6, 108.5, 104.2, 94.0, 65.7, 65.3, 55.7, 29.9, 14.7. Anal. Calcd for C25H24ClN3O6: C, 60.03; H, 4.88; N, 8.52. Found C, 60.27; H, 4.92; N, 8.52. Purity ≥ 99% (method F). 3-(5-((6-Chloropyridin-3-yl)methyl)-1,3,4-oxadiazol-2-yl)-5-(2,6-

dimethoxyphenyl)-6-(ethoxymethyl)-4-hydroxypyridin-2(1H)-one (13). 1H NMR (500 MHz, DMSO-d6): δ 8.49 (s, 1H), 7.92 (d, J = 7.6 Hz, 1H), 7.56 (d, J = 8.2 Hz, 1H), 7.37 (t, J = 8.2 Hz, 1H), 6.74 (d, J = 8.5 Hz, 2H), 4.48 (s, 2H), 3.97 (s, 2H), 3.70 (s, 6H), 3.41−3.20 (m, 2H), 1.00 (t, J = 6.9 Hz, 3H). 13C NMR (126 MHz, DMSO-d6): δ 167.6, 163.5, 163.4, 159.2, 158.2, 150.3, 149.2, 146.8, 140.6, 130.2, 129.9, 124.2, 108.5, 104.1, 86.6, 65.7, 65.3, 55.7, 27.3, 14.7. HRMS (ESI) calcd for C24H24ClN6O6 [M + H]

+, 499.1384; found, 499.1387. Purity ≥ 99% (method A). 3-(5-((5-Chloropyridin-2-yl)methyl)-1,3,4-oxadiazol-2-yl)-5-(2,6-

dimethoxyphenyl)-6-(ethoxymethyl)-4-hydroxypyridin-2(1H)-one (14). To a solution of compound 18e (1.0 g, 2.7 mmol) in methanol (20 mL) and DMF (10 mL) was added hydrazine (0.83 mL, 27 mmol), and the mixture was stirred for 16 h and then evaporated under reduced pressure. The residue was suspended in methanol and then filtered to afford 5-(2,6-dimethoxyphenyl)-6-(ethoxymethyl)-4-hydroxy-2-oxo- 1,2-dihydropyridine-3-carbohydrazide (0.81 g, 82% yield) as a white solid. MS m/z: 364.2 [M + H]+. 1H NMR (500 MHz, DMSO-d6): δ 7.34 (t, J = 8.4 Hz, 1H), 6.71 (d, J = 8.5 Hz, 2H), 4.75 (d, J = 5.0 Hz, 2H), 3.94 (s, 2H), 3.68 (s, 6H), 3.25 (q, J = 7.1 Hz, 2H), 0.98 (t, J = 6.9 Hz, 3H). To a suspension of 5-(2,6-dimethoxyphenyl)-6-(ethoxy- methyl)-4-hydroxy-2-oxo-1,2-dihydropyridine-3-carbohydrazide (1.6 g, 4.4 mmol) and 2-(5-chloropyridin-2-yl)acetic acid (0.99 g, 5.5 mmol) in dioxane (40 mL) was added 50% T3P in EtOAc (16 mL, 26 mmol), and the mixture was heated at 100 °C for 24 h. The mixture was allowed to cool to room temperature and then evaporated under reduced pressure. The residue was purified using silica gel chromatography eluting with 2−5% methanol/DCM. The pooled fractions containing the product were evaporated under reduced pressure and then recrystallized from DCM/methanol to isolate compound 14 (0.95 g, 43% yield). MS m/z: 497.0 [M + H]+. 1H NMR (500 MHz, DMSO-d6): δ 11.97−11.84 (m, 1H), 11.46 (br s, 1H), 8.57 (d, J = 2.5 Hz, 1H), 7.98 (dd, J = 8.4, 2.6 Hz, 1H), 7.56 (d, J = 8.3 Hz, 1H), 7.37 (t, J = 8.4 Hz, 1H), 6.74 (d, J = 8.3 Hz, 3H), 4.59 (s, 2H), 3.95

(s, 2H), 3.70 (s, 6H), 3.27 (q, J = 6.9 Hz, 2H), 0.99 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, DMSO-d6): δ 167.6, 163.6, 162.9, 159.0, 158.2, 153.3, 147.8, 146.9, 136.9, 130.2, 130.0, 124.9, 108.5, 104.0, 94.0, 65.7, 65.3, 55.7, 32.8, 14.7. Anal. Calcd for C24H23ClN4O6: C, 57.70; H, 4.66; N, 11.20. Found C, 57.44; H, 4.51; N, 11.13. Purity = 99% (method H).

Metabolic Stability in Liver Microsomes. Compounds were tested using a high-throughput metabolic stability (Met Stab) assay measuring % remaining of the intact compound after a 10 min incubation with β-nicotinamide adenine dinucleotide phosphate (NADPH)-fortified liver microsomes (1 mg/mL) in 100 mM sodium phosphate buffer, pH 7.4, 5 mM MgCl2, and 1 mM NADPH.

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Solubility. Solubility measurements were carried out by suspending excess solids in 50 mM phosphate buffer (pH 7.4) and agitating at ambient temperature for 24 h. Excess solids were isolated by centrifugation, and the solubility of the compound in the supernatant was measured. Reversed-phase liquid chromatography was performed on a 5 cm × 4.6 mm Phenomenex Luna C18 column with a gradient using acetonitrile−water [0.1% formic acid (v/v)] 5−95% over a 14 min run time at a flow rate of 1.0 mL/min. UV detection was at 254 nm, and ESI-POS was performed using a single-point calibration of a standard prepared in methanol.

APJ Binding Assays. Cell Membrane Extract Preparation. Stable human embryonic kidney 293 (HEK293 ZF) cell lines were cultured to express human, monkey, dog, or rat APJ cells. APJ-expressing cells were grown in minimum essential media (Thermo Fisher Scientific, catalog #11095-072) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Sigma, catalog #12106C), 2 mM L-glutamine (Thermo Fisher Scientific, catalog #25030-081), and 0.3 mg/mL of hygromycin B (Thermo Fisher Scientific, catalog #10687-010) and incubated at 37 °C with 5% CO2. Cell monolayers were harvested, washed twice with phosphate-buffered saline (PBS; Thermo Fisher Scientific, catalog #14190-250), resuspended in a homogenization buffer [50 mM N-(2- hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES), pH 7.2; 1 mM ethylenediaminetetraacetic acid (EDTA); 2 mM MgCl2; 250 mM sucrose supplemented with protease inhibitors; Roche, catalog #11836170001], and homogenized using Dounce homogenizers. The homogenate was centrifuged repeatedly (500g, 4 °C for 5 min) and the pooled supernatant was recentrifuged (50,000g, 4 °C for 1 h). The membrane pellet was washed and resuspended in homogenization buffer.

Radioligand Binding Assays. Assays were carried out at room temperature in a binding buffer [25 mM HEPES, pH 7.2; 10 mM MgCl2; 2 mM EGTA; and 0.1% bovine serum albumin (BSA; Sigma, catalog #A8412)] using 96-well Corning plates (Sigma, catalog #3605). The test compound was prepared in dimethyl sulfoxide (DMSO; final DMSO concentration ≤0.5%).

An APJ-expressing cell membrane was added, and the reaction was subsequently stopped by separating the free and bound compounds using a 0.3% polyethyleneimine (Sigma, catalog #P3143)-soaked UniFilter-96 GF/B plate (PerkinElmer, catalog #6005177) mounted on a UniFilter-96 cell harvester (PerkinElmer). The samples were washed repeatedly, and radioactivity in the filter plate was measured using a TopCount with 40 μL/well of Microscint 20 (PerkinElmer, catalog #6013621). Exact [3H]apelin-13 concentrations used were determined using a liquid scintillation analyzer (Tri-Carb 3110TR; PerkinElmer).

For the kinetic binding assays, the observed rate constant (Kobs) for each [Pyr1]apelin-13 concentration was determined using nonlinear regression curve fit for one-phase association method. The Kobs values from three independent association binding experiments were plotted as a function of their corresponding [3H]apelin-13 concentrations using a linear regression equation. The association rate constant (Kon) of (Pyr1) apelin-13 was determined by the resulting slope of the linear fit, and the dissociation rate constant (Koff) was estimated from the Y-axis intercept. The half-life (T1/2) was determined as 0.693/Koff. [

3H]apelin- 13 Koff and T1/2 were also measured directly by dissociation experiments. The membrane was incubated with 0.5 nM [3H]apelin- 13 for 45 min at room temperature with or without excess unlabeled [Pyr1]apelin-13 (80 nM); [Pyr1]apelin-13 (80 nM) was then added at different time points from 2 to 120 min. Membrane-bound counts per

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minute were plotted against their corresponding time points and analyzed using nonlinear regression for exponential two-phase decay. A range of compound 14 concentrations was added to cell

membrane extract containing [3H] apelin-13 [∼2-fold equilibrium dissociation constant (Kd)]. Excess unlabeled [Pyr

1]apelin-13 (80 nM) was used to determine specific binding from 0 to 120 min. Values were fit globally using nonlinear regression for competitive binding kinetics, which applied the Kon and Koff constraints for [

3H]apelin-13 determined in the association binding experiments. β-Arrestin Recruitment Assay. Chinese hamster ovary (CHO)-K1

PathHunter cells stably co-expressing the enzyme acceptor-tagged β- arrestin2 and hAPJ containing a ProLink tag at the C-terminus (DiscoverX, catalog #93−0250C2) were used. Cells were grown for ≥2 days before assay in the F-12 medium (Invitrogen, catalog #11765) supplemented with 10% heat-inactivated FBS (Sigma, catalog #F4135), 1× penicillin−streptomycin−glutamine (Invitrogen, catalog #10378), 0.8 mg/mL of geneticin (Invitrogen, catalog #10131), and 0.3 mg/mL of hygromycin B (Invitrogen, catalog #10687) at 37 °C with 5% CO2. Cells were then plated (10,000 cells/well) in 50 μL complete growth medium in amine 384-well (BD Purecoat, catalog #356719) or poly-D- lysine (Greiner, catalog #781946) plates. After overnight incubation at 37 °C, 5% CO2, the medium was

replaced with 15 μL of Hank’s balanced salt solution (HBSS)/HEPES/ 0.1% BSA assay buffer. The cells were incubated with test compounds serially diluted in DMSO (30 nL/well) at 37 °C and 5% CO2 for 90 min, and the reaction stopped with a PathHunter Detection Reagent. After a 1 h incubation in the dark at room temperature, chemiluminescent signals (EnVision, PerkinElmer) were used to calculate EC50. Compound potency was determined as described for the cAMP assay. cAMP Inhibition Assays. Cells were grown as described (see “APJ

Binding Assays”, above) for at least 2−4 days prior to assays. Cells were collected, centrifuged (1000 rpm, 5 min), and resuspended in an assay buffer of HBSS with Ca2+ and Mg2+ (Gibco, catalog #14025), 20 mM HEPES (Gibco, catalog #15630), and 0.1% fatty acid-free BSA (Sigma, catalog #A9205). The test compounds serially diluted in DMSO were dispensed into 384-well ProxiPlates (PerkinElmer, catalog #6008289) at 25 or 50 nL/well. To this, 2.5 μL/well of the assay buffer with 0.1 mM IBMX (Sigma, catalog #I5879) and 4 μM forskolin (Sigma, catalog #F6886) was added to the ProxiPlates via a Multi-drop Combi-reagent dispenser (Thermo Scientific, catalog #5840300). The HEK293 ZF cells (2.5 μL/well) were then added, and the plates were incubated at room temperature for 30 min. Intracellular cAMP levels were determined using the Dynamic cAMP kit (Cisbio, catalog #62AM4PEJ). Per well, 2.5 μL of cAMP d2 conjugate followed by 2.5 μL/well of cryptate-conjugated anti-cAMP antibody (both diluted in lysis buffer included in the Cisbio cAMP kit) was added to the cells. Following a 1 h incubation at room temperature, time-resolved fluorescence intensity was measured (337 nm excitation and dual emission at 615 and 665 nm; EnVision, PerkinElmer). A calibration curve was constructed with an external cAMP standard (1 μM to 0.1 pM) by plotting the fluorescent intensity ratio (665:615 nm emission) against cAMP concentrations. Data from individual concentration− response reactions were fitted to a four-parameter sigmoidal dose− response equation using Bristol Myers Squibb analysis software. The arithmetic mean and standard deviation of the half maximal effective concentration (EC50) from the individual fits were calculated using multiple test data. Compounds were tested in at least triplicate using independent experiments. In Vivo Characterization Studies. All animal studies were

performed under the approval of the Bristol Myers Squibb (BMS) Animal Care and Use Committee and in accordance with the American Association for Accreditation of Laboratory Animal Care (AAALAC). Male Sprague-Dawley rats (≥250 g; Harlan, Charles River, or Envigo) were used in all in vivo characterization studies to avoid the estrous cycle, possibly complicating the pharmacology. Rat and Monkey PK Protocol. Male Sprague-Dawley rats (290 to

345 g; N = 3) received the compound by oral gavage (3 mg/kg micronized suspension; mean particle size: 2.5 μm) following an overnight fast. Drug was formulated in methylcellulose, Tween 80, and

water (0.5:0.1:99.4, v/v/v). Serial blood samples (∼0.2 mL) were collected from the jugular vein into K2EDTA-containing tubes at predose and at 0.25, 0.5, 1, 2, 4, 7, 24, and 48 h post dose. Plasma samples, obtained by centrifugation at 4 °C (1500−2000g), were stored at −80 °C until analysis by LC/MS/MS. Male cynomolgus monkeys (4.6 and 4.7 kg; N = 2) received the compound by oral gavage, following an overnight fast (3 mg/kg micronized suspension; mean particle size = 4.2 μm). Drug was formulated in methylcellulose, Tween 80, and water (0.5:0.1:99.4, v/v/v). Serial blood samples (∼0.3 mL) were collected from the jugular vein into K2EDTA-containing tubes at predose and at 0.25, 0.5, 1, 2, 3, 5, 7, 24, 30, 48, and 72 h post dose. Plasma samples, obtained by centrifugation at 4 °C (1500−2000g), were stored at −80 °C until analysis by LC/MS/MS.

Acute Rat PV Loop Protocol. Surgical and catheterization methods: Sprague-Dawley rats were anesthetized with 4% isoflurane, and surgical anesthesia was maintained with 2% isoflurane. The left femoral artery was exposed, isolated, and catheterized with a Millar SPR-671(1.4F) pressure catheter, which was advanced into the abdominal aorta for measurement of aortic blood pressure. The right jugular vein and the right common carotid artery were exposed via a midline incision in the ventral neck. Each vessel was isolated and cleaned of surrounding fascia, and the artery was separated from the vagus nerve. The right jugular vein was cannulated with a length of PE50 for administration of test substances. The right carotid artery was catheterized with a 2F Millar pressure−volume transducer (model SPR- 838), which was advanced into the left ventricle for measurement of cardiac left ventricular pressure and conductance. General experimental protocol: following at least a 15 min equilibration period, an infusion compound 14 at 10 μg/kg/min × 15 min or vehicle (0.1 M Na2CO3, pH 10, 100 μL/kg/min × 15 min) was begun. After the infusion was stopped, the pressure−volume recording was monitored for an additional 15 min. Following the observation period, saline calibration (for calculation of parallel conductance, Vp) was performed according to manufacturer’s instructions. From one rat, at the end of the experiment, 3 mL of blood was withdrawn from the abdominal vena cava onto 0.1 mL heparin (1000 u/mL) and used to construct a standard curve of volume versus conductance. The equation for the line constructed through these points was used to calculate volume from the conductance data collected during the experiment. This equation was used to calculate volume data from the conductance data collected from the subsequent rats run that day. Results: sixteen (16) Harlen Sprague- Dawley rats were randomly selected to receive either vehicle (0.1 M Na2CO3, pH 10), 100 μL/kg/min × 15 min (n = 8), or compound 14 at 10 μg/kg/min × 15 min (n = 8). Following surgical instrumentation, isoflurane anesthesia was delivered at 2% and maintained at that concentration for the duration of the experiment. CO, SV, maximum and minimum dP/dt, HR, mean aortic pressure (MAP), and left ventricular end diastolic pressure were derived from the pressure− volume data. Significant differences from baseline values were observed for drug-treated rats compared with vehicle-treated rats for CO, SV, dP/dt max, dP/dt min, HR, and MAP. The average % change in area under the curve (AUC) measured over the 15 min of drug administration was 8.4% (SV), 11.7% (CO), 8.7% (dP/dt max), 3.2% (dP/dt min), 3.0% (HR), and −2.8% (MAP) for the compound 14 treated rats. One-way analysis of variance (ANOVA) was used with Tukey’s or Dunnett’s tests for multiple comparisons (GraphPad Prism version 5.0 and above). D’Agostino-Pearson normality test was used to assess normality of data. A P-value of <0.05 was considered significant.

Compound 14 Crystallographic Data Collection and Refine- ment Statistics. Compound 14 crystals were obtained from an acetone/methanol (v/v = 1/1) solution after 1 day of slow evaporation of the mother liquor at room temperature. A yellowish prism-shaped single crystal of approximate dimensions 0.22 × 0.21 × 0.04 mm was used to collect single-crystal X-ray diffraction (XRD) data. The XRD data collection was carried out using a Bruker-AXS Microstar rotating anode equipped with Cu Kα radiation (λ = 1.54178 Å), a Kappa2000 diffractometer, and an APEX II detector. The crystal was not cooled during data collection, that is, data collection occurred at ambient conditions or ∼23 °C. The XRD data were acquired using sets of Ω and Φ scans, each at a frame width of 2°. Indexing and processing of the

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measured intensity data were performed using the BrukerSAINT- APEX2 software package/program suite (Bruker (2012). Bruker- SAINT-APEX2. Bruker AXS Inc., Madison, Wisconsin, USA). A direct method solution was calculated using SHELXS-97 [Sheldrick, G.M. (1997) SHELXS97 and SHELXL97. Program for Crystal Structure Solution and Refinement. University of Göttingen, Göttingen], which provided the atomic positions from the E-map. Full-matrix least-square refinement and different Fourier cycles were performed using SHELXL-2014/6 (Sheldrick, G.M. (2008) SHELX Version 2014/6. Acta Crystallographica, A64, 112−122). The non-hydrogen atoms for compound 14 were refined with anisotropic displacement parameters. Hydrogen atoms were placed in ideal positions and refined as riding atoms with relative isotropic displacement parameters. Single-crystal X- ray data for compound 14: crystal data (CCDC deposition no. 2015092): C24H23ClN4O6, FW = 498.91, triclinic, space group = P1bar, Z = 2, a = 8.71600(10) Å, b = 10.5824(2) Å, c = 13.8013(2) Å, a = 71.2620(10)°, b = 81.8360(10)°, g = 77.8870(10)°, V = 1174.84(3) Å3, T = ∼296 K, Cu Kα, 10136 reflections, 3601 unique (Rint = 0.0223), R1 = 0.0396, wR2 = 0.440 for 3175 observed reflections with I > 2σ(I), 321 refined parameters, GoF = 1.071. Thermal ellipsoid crystal structure of compound 14 in the Supporting Information S17.

■ ASSOCIATED CONTENT *sı Supporting Information The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01878.

1H, 13C NMR, and HPLC spectra for compounds 1−14 and thermal ellipsoid crystal structure of compound 14 (PDF) Molecular formula strings (CSV)

■ AUTHOR INFORMATION Corresponding Author James A. Johnson − Bristol Myers Squibb Company, Research and Development, Princeton, New Jersey 08543-5400, United States; orcid.org/0000-0003-4969-5280; Phone: 609- 252-6043; Email: [email protected]; Fax: 609- 818-3550

Authors Soong-Hoon Kim − Bristol Myers Squibb Company, Research and Development, Princeton, New Jersey 08543-5400, United States; orcid.org/0000-0002-6809-6577

Ji Jiang − Bristol Myers Squibb Company, Research and Development, Princeton, New Jersey 08543-5400, United States

Monique Phillips − Bristol Myers Squibb Company, Research and Development, Princeton, New Jersey 08543-5400, United States

†William A. Schumacher − Bristol Myers Squibb Company, Research and Development, Princeton, New Jersey 08543- 5400, United States

Jeffrey S. Bostwick − Bristol Myers Squibb Company, Research and Development, Princeton, New Jersey 08543-5400, United States

Peter S. Gargalovic − Bristol Myers Squibb Company, Research and Development, Princeton, New Jersey 08543-5400, United States

Joelle M. Onorato − Bristol Myers Squibb Company, Research and Development, Princeton, New Jersey 08543-5400, United States

Chiuwa E. Luk − Bristol Myers Squibb Company, Research and Development, Princeton, New Jersey 08543-5400, United States

Claudia Generaux − Bristol Myers Squibb Company, Research and Development, Princeton, New Jersey 08543-5400, United States

Yan He − Bristol Myers Squibb Company, Research and Development, Princeton, New Jersey 08543-5400, United States

Xue-Qing Chen − Bristol Myers Squibb Company, Research and Development, Princeton, New Jersey 08543-5400, United States

Carrie Xu − Bristol Myers Squibb Company, Research and Development, Princeton, New Jersey 08543-5400, United States

Michael A. Galella − Bristol Myers Squibb Company, Research and Development, Princeton, New Jersey 08543-5400, United States

Tao Wang − Bristol Myers Squibb Company, Research and Development, Princeton, New Jersey 08543-5400, United States

David A. Gordon − Bristol Myers Squibb Company, Research and Development, Princeton, New Jersey 08543-5400, United States

Ruth R. Wexler − Bristol Myers Squibb Company, Research and Development, Princeton, New Jersey 08543-5400, United States

Heather J. Finlay − Bristol Myers Squibb Company, Research and Development, Princeton, New Jersey 08543-5400, United States; orcid.org/0000-0003-2309-0136

Complete contact information is available at: https://pubs.acs.org/10.1021/acs.jmedchem.0c01878

Author Contributions J.A.J. and S.-H.K. contributed equally. All authors have given approval to the final version of the manuscript. Notes The authors declare the following competing financial interest(s): JAJ, S-HK, JJ, MP, PSG, JMO, CG, YH, X-QC, MAG, TW, DAG, RRW, HJF are current and WAS, JSB, CEL, CX are former employees of Bristol Myers Squibb. †Deceased 19 January 2019. The crystal structure of compound 14 with atomic coordinates has been deposited with the CCDC (Cambridge Crystallo- graphic Data Center) with deposition code CCDC 2015092. Authors will release the atomic coordinates upon article publication.

■ ACKNOWLEDGMENTS The authors thank Anne Rose, Chris Freeden, and Pamela Abraham for pharmacokinetic studies, Pancras Wong for in vivo support, Sarah Traeger for NMR assistance, Robert Langish and Linping Wang for support with HRMS, Gerry G. Everlof for solubility determination, and the Department of Discovery Synthesis (DDS) and the Biocon Bristol Myers Squibb Research and Development Center (BBRC) for the preparation of intermediates.

■ ABBREVIATIONS ACEi, angiotensin-converting enzyme inhibitor; APJ, apelin receptor; ARB, angiotensin II receptor blocker; AT1R, angiotensin II type 1 receptor; AUCINF, area under the plasma drug concentration−time curve from time zero to infinity; cAMP, cyclic adenosine monophosphate; Cl, clearance; Cmax, maximum serum concentration; CO, cardiac output; DCM,

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dichloromethane; DIEA, diisopropylethyl amine; DMAC, N,N- dimethylacetamide; ERK, extracellular signal-regulated kinase; EtOH, ethanol; F, bioavailability; GPCR, G-protein-coupled receptor; HEK, human embryonic kidney; HF, heart failure; HR, heart rate; IPA, isopropyl alcohol; i.v., intravenous; LiHMDS, lithium hexamethyldisilazide; LV, left ventricle; MAP, mean arterial pressure; NADPH, nicotinamide adenine dinucleotide phosphate; NaOEt, sodium ethoxide; PD, pharmacodynamic; PK, pharmacokinetic; p.o., by mouth; PV loop, pressure−volume loop; [Pyr1], pyroglutamyl1; RAAS, renin−angiotensin−aldosterone system; T1/2, half-life; THF, tetrahydrofuran; T3P, propylphosphonic anhydride; Vss, volume of distribution

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