Design, Synthesis, and Evaluation of Piperazinyl Pyrrolidin-2-ones as

Article Cite This: J. Med. Chem. 2018, 61, 9205−9217

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Design, Synthesis, and Evaluation of Piperazinyl Pyrrolidin-2-ones as a Novel Series of Reversible Monoacylglycerol Lipase Inhibitors Jumpei Aida,* Makoto Fushimi, Tomokazu Kusumoto, Hideyuki Sugiyama, Naoto Arimura, Shuhei Ikeda, Masako Sasaki, Satoshi Sogabe,† Kazunobu Aoyama,† and Tatsuki Koike Research, Takeda Pharmaceutical Co., Ltd., 26-1, Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa 251-8555, Japan

J. Med. Chem. 2018.61:9205-9217. Downloaded from pubs.acs.org by UNIV OF TEXAS AT EL PASO on 10/27/18. For personal use only.

S Supporting Information *

ABSTRACT: Monoacylglycerol lipase (MAGL) is a major serine hydrolase that hydrolyzes 2-arachidonoylglycerol (2-AG) to arachidonic acid (AA) and glycerol in the brain. Because 2-AG and AA are endogenous biologically active ligands in the brain, inhibition of MAGL is an attractive therapeutic target for CNS disorders, particularly neurodegenerative diseases. In this study, we report the structure-based drug design of novel piperazinyl pyrrolidin-2-ones starting from our hit compounds 2a and 2b. By enhancing the interaction of the piperazinyl pyrrolidin-2-one core and its substituents with the MAGL enzyme via design modifications, we identified a potent and reversible MAGL inhibitor, compound (R)-3t. Oral administration of compound (R)3t to mice decreased AA levels and elevated 2-AG levels in the brain.

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INTRODUCTION Monoacylglycerol lipase (MAGL) is a major serine hydrolase that hydrolyzes 2-arachidonoylglycerol (2-AG, 1a) to arachidonic acid (AA, 1b) and glycerol in the brain.1 AA and its metabolite eicosanoid have been reported to cause neural inflammation, which can lead to neurodegenerative diseases such as Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease.2−5 On the other hand, 2-AG-mediated activation of cannabinoid receptors is known to be neuroprotective and prevent neuroinflammation following tissue damage and in neuronal disease models. Therefore, inhibition of MAGL is expected to be an attractive therapeutic approach for the treatment of neurodegenerative diseases.5−7 To date, diverse MAGL inhibitors including maleimide 1c, urea 1d, ketone 1e, carbamates 1f and 1g, and oxadiazolone 1h have been reported (Figure 1).8−13 Most of these MAGL inhibitors are irreversible inhibitors that form covalent bonds with the catalytic Ser122 residue of the MAGL enzyme.12,14,15 These compounds helped clarify the biological activities of MAGL such as its antinociceptive, anti-inflammatory, and neuroprotective properties.11,16,17 However, most of these irreversible inhibitors failed to enter clinical trials because of issues related to enzyme selectivity, toxicity, among others.18,19 Alternatively, several groups have recently explored reversible MAGL inhibitors.20−24 Among them is the amide derivative 1i, whose reversibility was revealed by its cocrystal structure with the MAGL enzyme.20 This crystal structure encouraged us to develop novel reversible inhibitors that have better selectivity and toxicity profiles than irreversible inhibitors. Herein, we report our efforts to develop a novel series of reversible MAGL © 2018 American Chemical Society

inhibitors using a structure-based drug design (SBDD) approach.

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RESULTS AND DISCUSSION

Study Conception. A high-throughput screening campaign of our compound library led to the identification of pyrrolidinone derivatives 2a (IC50 = 3800 nM) and 2b (10% inhibition at 10 μM) with moderate MAGL inhibitory activity (Figure 2a). The MAGL inhibitory activities were evaluated by measuring the amount of AA produced from 2-AG using recombinant His-hMAGL enzyme. In this assay, test compounds are normally preincubated for 60 min with the enzyme; however, to characterize the time-dependent inhibition of these compounds, their inhibitory activities were also evaluated without preincubation (0 min). The results showed that compound 2a inhibited MAGL in a time-independent manner (IC50 = 2200 nM at 0 min, 3800 nM at 60 min), while the known MAGL inhibitor 1h13 inhibited MAGL in a timedependent manner (IC50 = 1700 nM at 0 min, 31 nM at 60 min). These results suggested that our hit compounds 2a and 2b bind MAGL reversibly without forming covalent bonds, which encouraged us to use these as our starting point. To enhance the inhibitory activity, we adopted an SBDD approach utilizing the reported cocrystal structure of 1i and the MAGL enzyme (Figure 2b).19 First, we assumed that the amide carbonyl group of 2b interacts with Ser122 in the Received: May 24, 2018 Published: September 25, 2018 9205

DOI: 10.1021/acs.jmedchem.8b00824 J. Med. Chem. 2018, 61, 9205−9217

Journal of Medicinal Chemistry

Article

Figure 1. Arachidonoylglycerol (2-AG, 1a), arachidonic acid (AA, 1b), and reported MAGL inhibitors 1c−i.

catalytic site and that the lipophilic phenyoxyphenyl group positions itself in the large hydrophobic pocket of MAGL (Figure 2c). This binding mode mimics the transition state of the covalent MAGL inhibitors that form covalent binding with Ser122.12,14 Next, to enhance the inhibitory activity, we proposed introducing a substituent to the amphiphilic pocket of MAGL, where the pyrimidinyl piperazine unit of 1i interacts with Arg75 and Tyr194 (Figure 2b). The overlay study of this cocrystal structure with the presumed binding mode of compound 2b led us to believe that the pyrimidinyl piperazine unit of 1i was located at position 4 of the pyrrolidinone ring of 2b. Therefore, we designed compound 3a, which has a piperazine-pyrimidine unit at position 4 of pyrrolidinone ring (Figure 2d). Chemistry. The designed compounds were synthesized following Scheme 1 or Scheme 2. N-Aryl-2,4-pyrrolidinediones 6a and 6b were synthesized by the condensation of methyl 4chloro-3-methoxy-2-(E)-butenoate 4 and the corresponding arylamines by hydrolysis under acidic condition (Scheme 1). Reductive amination with piperazine units afforded compounds 3b, 7a, and 7b. Compound 3a was synthesized by the deprotection of intermediated 7a, followed by Buchwald− Hartwig reaction with 2-chloropyrimidine. Amidation of 7c with corresponding carboxylic acids yielded compounds 3c−h. Biaryl compounds 3l−q were synthesized from 7b by Suzuki− Miyaura palladium coupling reaction. Michael reaction of 2-furanone 8 with piperazines provided key intermediates 3-piperazinyl butyrolactones 9a and 9b (Scheme 2). Target compounds 3i−k and intermediates 11a− c were synthesized via direct amidation with aluminum reagents followed by the Mitsunobu reaction. Finally, Suzuki−Miyaura reactions yielded 3r−t from the corresponding halogenated intermediates 11a−c. Optical resolution of 3t afforded (R)-3t and (S)-3t. Biological Evaluation. The MAGL inhibitory activities of the synthesized compounds were measured using RapidFire high-throughput mass spectrometry assay with recombinant His-hMAGL protein and 2-AG (preincubation time with the test compounds and the enzyme: 60 min). Compound 3a (IC50 = 140 nM) exhibited significantly improved MAGL inhibitory activity compared with 2b (Table 1). This result supports our design concept, which aimed to include interaction with the amphiphilic pocket of MAGL

Figure 2. Design of novel MAGL inhibitors: (a) structures of hit compounds 2a and 2b; (b) 2D image of the cocrystal structure of 1i (PDB code 3PE6); (c) hypothetical docking model of hit compound 2b with MAGL; (d) design of compound 3a.

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Scheme 1. Synthetic Route of Pyrrolidinone Derivatives via N-Aryl-2,4-pyrrolidinedionesa

(a) (1) Amines, triethylamine, acetonitrile, reflux, (2) acetic acid, toluene, 50 °C; (b) hydrochloric acid, toluene, 50 °C; (c) piperazines, sodium cyanoborohydride, acetic acid, rt to 50 °C; (d) 4 M HCl in EtOAc, EtOAc, 60 °C; (e) 2-chloropyrimidine, Pd2(dba)3, Xantophos, Cs2CO3, dioxane, 100 °C; (f) carboxylic chlorides or carboxylic acids, condensation reagents, bases, rt; (g) boronic acid reactants, palladium reagents, phosphine ligand, bases, solvents, heat; (h) (1) bis(pinacolate)diborane, PdCl2(dppf), KOAc, DMF, 80 °C; (2) 2-bromo-3-methylpyridine, (A-taPhos)2PdCl2, Cs2CO3, DMF, water, 80 °C. a

Next, we explored the substituent targeted to the hydrophobic pocket of MAGL as shown in Table 2. Conversion of the 4-phenoxyphenyl group of 3e to a 3-phenoxyphenyl group (3i, IC50 = 11 nM) enhanced the inhibitory activity 3-fold. This result led us to explore substituents at position 3 of the central benzene ring. Replacement of the phenoxy group of compound 3i with benzyloxy (3j, IC50 = 44 nM) lowered the activity, whereas replacement with phenyl (3k, IC50 = 8.9 nM) enhanced the activity. The biaryl derivative 3k was a potent MAGL inhibitor; therefore, we further explored substituents on the distal phenyl ring of 3k by introducing a chloro group to each position. The chloro derivatives all showed potent MAGL inhibitory activity (o-chloro 3l, IC50 = 0.64 nM; mchloro 3m, IC50 = 2.2 nM; p-chloro 3n, IC50 = 9.8 nM). Among these compounds, 3l had the most potent inhibitory activity, down to a subnanomolar level, suggesting that the dihedral angle of the biphenyl moiety is important for potent activity. The cocrystal structure of 3l and the MAGL enzyme was obtained and confirmed our design hypothesis that 3l binds MAGL at the active site noncovalently (PDB code 5ZUN, Figure 3). The pyrrolidinone oxygen makes hydrogen bonds with the side chain of the catalytic Ser122 residue and the main

(Arg75 and Tyr194) in addition to the binding with Ser122 in the catalytic domain. Replacement of the pyrimidine ring of 3a with a phenyl (3b, 37% inhibition at 10 μM) reduced the inhibitory activity, indicating that the pyrimidine ring of 3a hydrogen-bonds with Arg75 as we expected and that this interaction is important for potent activity. On the basis of these results, we explored the amide derivatives 3c−f, which possess a carbonyl group in the same position as the nitrogen atom of 3a. A carbonyl group in this position is expected to function as a hydrogen bond acceptor (HBA) with Arg75. We introduced different sized rings such as phenyl (3c), thiophene (3d), thiazole (3e), and oxazole (3f). Compounds 3c−f exhibited MAGL inhibitory activities (IC50 = 38 nM to 2000 nM), and the thiazole derivative 3e (IC50 = 38 nM) exhibited a more potent activity than the pyrimidine derivative 3a (IC50 = 140 nM). These results suggest that the carbonyl group of this amide series acts as an HBA for Arg75 and that the thiazole ring best fits the amphiphilic pocket of MAGL. Introduction of a methyl group at position 4 (3g, IC50 = 2200 nM) and position 5 (3h, 38% inhibition at 10 μM) of the thiazole ring was not beneficial. On the basis of these results, the thiazole amide was the substituent selected to occupy the amphiphilic pocket and was used in further investigation of this series. 9207



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Scheme 2. Synthetic Route of Pyrrolidinone Derivatives via 3-Piperazinyl Butyrolactonesa

a

(a) Piperazines, MeOH, rt; (b) arylamines, trialkylaluminum reagents, solvents, heating; (c) Mitsunobu reagents, THF, rt; (d) (1) arylamines, trialkylaluminum reagents, solvents, heating; (2) Mitsunobu reagents, THF, rt; (e) boronic acid reactants, (A-taPhos)2PdCl2, Cs2CO3, solvents, heating; (f) optical resolution by SFC.

the 4-methyl-2-pyridinyl derivative 3p (IC50 = 53 nM) had significantly decreased MAGL inhibitory activity, whereas the 2-methyl-3-pyridinyl derivative 3q (IC50 = 9.2 nM) retained potent inhibitory activity. Next, we modified the central phenyl group keeping the 2-methylpyridine-3-yl group in the distal position. To enhance the in vitro inhibitory activity, a 4fluorophenyl analog 3r and a 5-fluorophenyl analog 3s, which were expected to interact lipophilically with Leu241, were designed. As expected, the 5-fluorophenyl analog 3s (IC50 = 6.5 nM) had enhanced MAGL inhibitory activity, while the 4fluorophenyl analog 3r (IC50 = 13 nM) had decreased in vitro potency. Having identified an optimal biaryl moiety, we revisited the thiazole carbonyl moiety. Replacement of the thiazole carbonyl group with a pyrimidine ring, which sits in an alternative active region of the amphiphilic pocket of MAGL, yielded 3t that had a good balance between potent inhibitory activity (IC50 = 5.0 nM) and good metabolic stability (65 μL min−1 mg−1). Optical resolution of 3t produced both enantiomers, and (R)-3t exhibited potent MAGL inhibitory activity (IC50 = 3.6 nM) with good metabolic stability in human microsomes (29 μL min−1 mg−1), whereas (S)-3t shows slight MAGL activity (IC50 > 10 000 nM). This result appears to be consistent with those obtained with the cocrystal structure analysis during which only the R isomer of 3l was observed. To further investigate the binding profile of compound (R)3t with MAGL, a dissociation experiment was conducted using the dilution method. The results showed a dissociation rate constant (koff) of 3.1 × 10−3 s−1 for (R)-3t. The dissociation half-life (t1/2) calculated from koff was 3.7 min. These results

chains of Ala51 and Met123. In addition, the biphenyl unit on the left-hand side settles in the lipophilic pocket and the ochlorophenyl group is directed toward the lid domain. The ochloro substitution enhanced MAGL inhibitory activity likely owing to the lipophilic interaction with Ile179. The thiazolyl carbonyl piperazine 3l occupies the amphiphilic pocket, where the carbonyl group forms a direct hydrogen bond with the side chain of Arg57 and water-mediated hydrogen bonds with the side chains of Glu53 and His272. There is a possibility that the MAGL enzyme recognizes the chirality at position 4 of the pyrrolidinone ring because a cocrystal structure was observed only for (R)-3l despite having performed the cocrystal investigation using racemic 3l. Compound 3l exhibited potent MAGL inhibitory activity; however, its metabolic stability in human liver microsomes (181 μL min−1 mg−1) required improvement in order to develop an orally available MAGL inhibitor. To improve metabolic stability, we replaced the distal phenyl ring of 3l with a pyridine ring to reduce the lipophilicity of the molecule. First, the chloro group of compound 3l (log D = 3.18) was displaced with a methyl group to avoid having a halopyridine moiety, which is well-known for its reactivity. The resulting o-methyl compound 3o (IC50 = 1.2 nM) showed potent inhibitory activity (Table 3). To lower the polar surface area value to improve blood−brain barrier (BBB) permeability, a nitrogen atom was introduced at the sterically hindered position, yielding the pyridine derivatives 3p and 3q (log D = 1.85 for both). As expected, both compounds showed improved metabolic stability (52 μL min−1 mg−1 for 3p and 30 μL min−1 mg−1 for 3q). Regarding the MAGL inhibitory activity, 9208



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Table 1. Inhibitory Activities of 3a−h against Human MAGL

Table 2. Inhibitory Activities of 3e and 3i−n against human MAGL

a

IC50 values and 95% confidence intervals were calculated from duplicate measurements using a four-parameter logistic curve using XLfit software (IDBS, London, U.K.). b% Inhibition at 10 μM.

confirm that compound (R)-3t is a reversible MAGL inhibitor. Although MAGL is responsible for ∼85% of 2-AG hydrolysis in mouse brain, it is reported that there are some minor enzymes that hydrolyze 2-AG, such as α,β-hydrolase domaincontaining protein 12 (ABHD12), ABHD6, and fatty acid amide hydrolase (FAAH).1 For the assessment of selectivity for these enzyme, we evaluated in vitro inhibitory activity of (R)-3t against FAAH. Compound (R)-3t did not show significant inhibititory activity against FAAH (IC50 > 10 μM) under the assay condition reported previously.25 Finally, a pharmacokinetic (PK) and pharmacodynamic (PD) study of compound (R)-3t was performed in mice. High exposure levels of (R)-3t were observed not only in the plasma but also in the brain 1 h after oral administration to mice (Table 4; dose, 10 mg/kg; plasma concentrations, 1.01 μg/mL; brain concentration, 0.656 μg/g). To confirm the PD of (R)3t, its effects on the tissue content of 2-AG and AA were evaluated in mice brains 1 h after oral administration.

a

IC50 values and 95% confidence intervals were calculated from duplicate measurements using a four-parameter logistic curve using XLfit software (IDBS, London, U.K.).

Compound (R)-3t significantly decreased AA levels (25% reduction) and elevated 2-AG levels (340% increase) compared with the control (Figure 4). Difference of the dynamic changes between AA and 2-AG levels by the treatment of (R)-3t might be due to the different degree of MAGL contribution to modulate these components. Covalent MAGL inhibitors such as 1f have been reported to show antinociceptive responses in addition to elevation of 2-AG 9209



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Figure 3. Cocrystal structure of 3l and MAGL enzyme (PDB code 5ZUN): (a) X-ray cocrystal structure; (b) 2D image of the cocrystal structure.

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levels;26 hence, (R)-3t is expected to also produce some pharmacological effects through the modulation of AA and 2AG levels.

EXPERIMENTAL SECTION

Chemistry: General. Melting points were determined with a Büchi melting point apparatus B-545 and are uncorrected. 1H NMR spectra were obtained at a Bruker DPX-300 spectrometer or Bruker Avance NMRS-400. Chemical shifts are given in δ values (ppm) using tetramethylsilane as the internal standard. Peak multiplicities are expressed as follows: s, singlet; d, doublet; t, triplet; q, quartet; dd, doublet of doublet; br, broad; brs, broad singlet; m, multiplet. Elemental analyses were carried out by Takeda Pharmaceutical Company Ltd., Medicinal Chemistry Research Laboratories. MS spectra were recorded using a Shimadzu LCMS-2020 with electrospray ionization. Reactions were followed by TLC on silica gel 60 F 254 precoated TLC plates (E. Merck) or NH TLC plates (Fuji Silysia Chemical Ltd.). Chromatographic separations were carried out on

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CONCLUSION We have developed a potent and reversible MAGL inhibitor (R)-3t starting from pyrrolidinone hit compounds 2a and 2b using an SBDD approach. Piperazinyl pyrrolidinone (R)-3t significantly influenced AA (25%) and 2-AG levels (340%) in mice brains. The design of these molecules is a promising development toward potential treatments for neurodegenerative diseases. Further evaluation of (R)-3t as a reversible MAGL inhibitor will be reported in due course. 9210



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Table 3. MAGL Inhibitory Activities and Human Metabolic Stabilities of 3l and 3o−t

Table 4. Pharmacokinetic and Pharmacodynamic Study of (R)-3t

hMSb

Cplasmac

Cbrainc

IC50 (nM)

(μL min−1 mg−1)

(μg/mL)

(μg/g)

3.6 (0.57−0.71)

29

1.01

0.656

hMAGL a

a

IC50 values and 95% confidence intervals were calculated from duplicate measurements using a four-parameter logistic curve using XLfit software (IDBS, London, U.K.). bClearance in human liver microsomes. cCompound (R)-3t was suspended in 0.5% methylcellulose aqueous solution for oral administration (10 mg/kg, C57Bl/ 6 mice, n = 3).

Figure 4. Brain AA and 2-AG levels of mice treated with (R)-3t (10 mg/kg, po). Data are shown as the mean with error bars showing the SEM, n = 3 ((∗) p < 0.05 versus vehicle; (∗∗∗) p < 0.001 versus vehicle (Student’s t-test).). and solvent B was 0.1% TFA in acetonitrile) through an L-column 2 ODS (3.0 mm × 50 mm, 2 mm) column at 1.2 mL min. Area % purity was measured at 254 nm. The purities of all tested compounds in biological systems were confirmed to be more than 95% pure as determined by analytical HPLC except as noted. Yields refer to isolated and purified products derived from nonoptimized procedures. Reagents and solvents were obtained from commercial sources and used without further purification. General Procedure A: Combinatorial Suzuki−Miyaura Coupling Reaction. A mixture of compound 7b (0.039 g, 0.08 mmol), corresponding boronic reagent (0.096 mmol), (A-taPhos)2PdCl2 (5.66 mg, 8.00 μmol), K2CO3 (0.022 g, 0.160 mmol) in DME (1 mL)/water (10 μL) was heated by microwave irradiation at 130 °C for 30 min. After filtration, the filtrate was partitioned between EtOAc and water. The organic phase was concentrated by air-blowing. The residue was purified by Gilson (A = 10 mM NH4HCO3, B = ACN, B% = (5−100)/5 min) to give a product. General Procedure B: Combinatorial Direct Amidation. A mixture of compound 9a (33.8 mg, 120 μmol), corresponding arylamine (0.12 mmol), and DABAL-Me3 (24.6 mg, 0.10 mmol) in THF (1 mL) was heated at 100 °C for 20 min under microwave irradiation (Synthos 3000, 4x48MC). The reaction mixture was quenched with Na2SO4·10 H2O (200 mg) and stirred for 1 h. The reaction mixture was diluted with THF (2 mL), filtered through Celite, and the filtrate was concentrated by blowing away with the air at 60 °C. The residue was purified by preparative HPLC (YMCTriartC18, eluted with MeCN/10 mM NH4HCO3 aqueous solution). The desired fraction was concentrated by blowing away with the air at 60 °C to give acyclic amide intermediate. Di-tert-butyl

a

IC50 values and 95% confidence intervals were calculated from duplicate measurements using a four-parameter logistic curve using XLfit software (IDBS, London, U.K.). bClearance in human liver microsomes. c% inhibition at 10 μM. silica gel 60 (0.063−0.200 or 0.040−0.063 mm, E. Merck) or basic silica gel (Chromatorex NH, 100−200 mesh, Fuji Silysia Chemical Ltd.) using the indicated eluents. The HPLC analyses were performed using a Shimadzu UFLC instrument. Elution was done with a gradient of 5−90% solvent B in solvent A (solvent A was 0.1% TFA in water, 9211



Article

azodicarboxylate (32.8 mg, 0.142 mmol) was added to a solution of the intermediate and triphenylphosphine polymer-bound (PS-TPP) (44.5 mg, 3.2 mmol/g) in THF (1 mL) at rt. The mixture was stirred at rt for 16 h. The reaction mixture was filtered. The filtrate was concentrated by blowing away with the air at 60 °C. The residue was purified by preparative HPLC (YMCTriartC18, eluted with MeCN/ 10 mM NH4HCO3 aqueous solution). The desired fraction was concentrated by blowing away with the air at 60 °C to give corresponding product. 1-(4-Phenoxyphenyl)-4-[4-(pyrimidin-2-yl)piperazin-1-yl]pyrrolidin-2-one 3a. To the stirred solution of compound 7c (230 mg, 0.68 mmol) in dioxane (10 mL) and 2-chloropyrimidine (94 mg, 0.82 mmol) was added Cs2CO3 (554.7 mg, 1.71 mmol), and the reaction mixture was degassed for 30 min. After that xantphos (39 mg, 0.06 mmol) and Pd2(dba)3 (32 mg, 0.03 mmol) were added to the reaction mixture and heated at 100 °C for 2 h. The reaction mixture was then cooled to rt and concentrated under reduced pressure. Crude mixture was diluted with EtOAc (100 mL) and washed with water (2 × 50 mL). Organic part was separated, dried with Na2SO4, and concentrated under reduced pressure to get crude which was purified by preparative HPLC (Gemini C18, eluted with MeCN/10 mM NH4OAc aqueous solution) to afford compound 3a (49 mg, 17%) as off-white solid. 1H NMR (400 MHz, DMSO-d6) δ 2.47−2.56 (5H, m), 2.66−2.72 (1H, m), 3.22 (1H, t, J = 7.4 Hz), 3.74−3.78 (5H, m), 3.96−3.98 (1H, m), 6.62 (1H, t, J = 4.7 Hz), 6.97 (2H, d, J = 8.2 Hz), 7.03 (2H, d, J = 8.9 Hz), 7.12 (1H, t, J = 7.3 Hz), 7.37 (2H, t, J = 7.9 Hz), 7.68 (2H, d, J = 8.9 Hz), 7.68 (2H, d, J = 4.7 Hz), 8.36 (2H, d, J = 4.6 Hz). MS: [M + H]+ 416.2. 1-(4-Phenoxyphenyl)-4-(4-phenylpiperazin-1-yl)pyrrolidin2-one 3b. To a mixture of compound 6a (50 mg, 0.19 mmol) in THF (1.9 mL) was added 1-phenylpiperazine (0.034 mL, 0.22 mmol) at rt. The mixture was stirred at rt for 2 h. The mixture was heated to reflux for 1 h. To the mixture were added acetic acid (0.032 mL, 0.56 mmol) and sodium cyanoborohydoride (0.035 g, 0.56 mmol) at rt. The mixture was stirred at rt for 5 days. To the mixture was added 2 M aqueous NaOH solution (0.27 mL) at 0 °C. The mixture was diluted with water and saturated brine at rt, extracted with EtOAc, dried over Na2SO4, filtered, concentrated in vacuo, and purified by column chromatography (NH-silica gel, eluted with 66−96% EtOAc in hexane) to yield compound 3b (35 mg, 45%) as a white solid after trituration with EtOAc/hexane. 1H NMR (300 MHz, CDCl3) δ 2.61−2.73 (5H, m), 2.74−2.86 (1H, m), 3.19−3.36 (5H, m), 3.83 (1H, dd, J = 9.5, 6.8 Hz), 3.91−4.01 (1H, m), 6.84−7.14 (8H, m), 7.23−7.37 (4H, m), 7.49−7.58 (2H, m). MS: [M + H]+ 414.1. 4-(4-Benzoylpiperazin-1-yl)-1-(4-phenoxyphenyl)pyrrolidin-2-one 3c. To a solution of compound 7c (50 mg, 0.13 mmol) and TEA (0.093 mL, 0.67 mmol) in DMA (1 mL) was added benzoyl chloride (0.023 mL, 0.20 mmol) at ambient temperature. The mixture was stirred at ambient temperature overnight. The mixture was quenched with water and extracted with EtOAc. The organic layer was separated, washed with water and brine successively, dried over Na2SO4, and concentrated in vacuo. The residue was purified by column chromatography (NH silica gel, eluted with 10−100% EtOAc in hexane). The residue was crystallized with IPE-IPA-hexane to give compound 3c (31.0 mg, 52%) as an off-white solid. 1H NMR (300 MHz, CDCl3) δ 2.33−2.68 (5H, m), 2.68−2.83 (1H, m), 3.14−3.33 (1H, m), 3.37−4.05 (6H, m), 6.93−7.05 (4H, m), 7.06−7.14 (1H, m), 7.28−7.37 (2H, m), 7.38−7.46 (5H, m), 7.48−7.55 (2H, m). MS: [M + H]+ 442.2. 1-(4-Phenoxyphenyl)-4-[4-(2-thienylcarbonyl)piperazin-1yl]pyrrolidin-2-one 3d. Compound 3d was synthesized by a similar procedure to compound 3c in 53% yield as an off-white solid. 1H NMR (300 MHz, CDCl3) δ 2.48−2.68 (5H, m), 2.72−2.83 (1H, m), 3.28 (1H, quin, J = 7.5 Hz), 3.70−3.88 (5H, m), 3.88−3.98 (1H, m), 6.95−7.14 (6H, m), 7.28−7.37 (3H, m), 7.44−7.56 (3H, m). MS: [M + H]+ 448.1. 1-(4-Phenoxyphenyl)-4-[4-(1,3-thiazol-2-ylcarbonyl)piperazin-1-yl]pyrrolidin-2-one 3e. To a solution of compound 7c (200 mg, 0.53 mmol) and thiazole-2-carboxylic acid (83 mg, 0.64 mmol) in DMF (3 mL) were added HATU (305 mg, 0.80 mmol) and

triethylamine (0.373 mL, 2.67 mmol) at ambient temperature. The mixture was stirred at ambient temperature overnight. The mixture was quenched with water and extracted with EtOAc. The organic layer was separated, washed with water and saturated brine successively, dried over sodium sulfate, and concentrated in vacuo. The residue was purified by column chromatography (NH silica gel, eluted with 20−100% EtOAc in hexane). The residue was crystallized with IPA-IPE-hexane to give compound 3e (170 mg, 71%) as an offwhite solid. 1H NMR (300 MHz, CDCl3) δ 2.54−2.71 (5H, m), 2.72−2.85 (1H, m), 3.22−3.36 (1H, m), 3.74−3.87 (2H, m), 3.87− 3.99 (2H, m), 4.35−4.49 (1H, m), 4.49−4.63 (1H, m), 6.96−7.06 (4H, m), 7.06−7.13 (1H, m), 7.30−7.37 (2H, m), 7.49−7.56 (3H, m), 7.88 (1H, d, J = 3.4 Hz). MS: [M + H]+ 449.0. 4-[4-(Oxazole-2-carbonyl)piperazin-1-yl]-1-(4phenoxyphenyl)pyrrolidin-2-one 3f. To a mixture of compound 7c (50 mg, 0.13 mmol) in THF (1.34 mL) were added oxazole-2carboxylic acid (23 mg, 0.20 mmol), triethylamine (0.130 mL, 0.94 mmol), 1-hydroxybenzotriazole hydrate (31 mg, 0.20 mmol), and 1ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (38 mg, 0.20 mmol) at rt. The mixture was stirred at rt for 17 h. The mixture was diluted with water, saturated brine, and saturated aqueous sodium hydrogen carbonate solution at rt, extracted with EtOAc, dried over sodium sulfate, filtered, concentrated in vacuo, and purified by column chromatography (NH-silica gel, eluted with 50−100% EtOAc in hexane) to yield compound 3f (38 mg, 66%) as a yellow amorphous solid. 1H NMR (300 MHz, CDCl3) δ 2.50−2.70 (5H, m), 2.72−2.86 (1H, m), 3.29 (1H, quin, J = 7.4 Hz), 3.72−3.86 (2H, m), 3.85−3.99 (2H, m), 4.14−4.26 (1H, m), 4.26−4.41 (1H, m), 6.95− 7.05 (4H, m), 7.06−7.14 (1H, m), 7.25 (1H, s), 7.29−7.38 (2H, m), 7.49−7.57 (2H, m), 7.78 (1H, s). MS: [M + H]+ 433.0. 4-(4-[(4-Methyl-1,3-thiazol-2-yl)carbonyl]piperazin-1-yl)-1(4-phenoxyphenyl)pyrrolidin-2-one 3g. Compound 3g was synthesized by a similar procedure to compound 3f in 38% yield as a white powder. 1H NMR (300 MHz, CDCl3) δ 2.48 (3H, s), 2.53− 2.72 (5H, m), 2.71−2.85 (1H, m), 3.20−3.37 (1H, m), 3.71−3.85 (2H, m), 3.86−3.99 (2H, m), 4.31−4.50 (1H, m), 4.50−4.66 (1H, m), 6.96−7.14 (6H, m), 7.29−7.37 (2H, m), 7.49−7.57 (2H, m). MS: [M + H]+ 463.0. 4-(4-[(5-Methyl-1,3-thiazol-2-yl)carbonyl]piperazin-1-yl)-1(4-phenoxyphenyl)pyrrolidin-2-one 3h. Compound 3h was synthesized by a similar procedure to compound 3f in 37% yield as a white powder. 1H NMR (300 MHz, CDCl3) δ 2.46−2.70 (8H, m), 2.71−2.84 (1H, m), 3.27 (1H, dt, J = 14.8, 7.8 Hz), 3.72−3.99 (4H, m), 4.32−4.60 (2H, m), 6.95−7.06 (4H, m), 7.06−7.14 (1H, m), 7.28−7.38 (2H, m), 7.48−7.56 (3H, m). MS: [M + H]+ 463.0. 1-(3-Phenoxyphenyl)-4-[4-(1,3-thiazol-2-ylcarbonyl)piperazin-1-yl]pyrrolidin-2-one 3i. To a solution of ADDP (86 mg, 0.37 mmol) in THF (2 mL) was added tri-n-butylphosphine (0.092 mL, 0.37 mmol) at 0 °C. The mixture was stirred at ambient temperature for 30 min. After that, a solution of compound 10a (58 mg, 0.12 mmol) in THF (2 mL) was added. The mixture was stirred at ambient temperature overnight. The volatile material was removed by evaporation, and the residue was purified by column chromatography (NH silica gel, eluted with 10−75% EtOAc in hexane). The residue was crystallized by IPE-IPA-hexane to give compound 3i (23.0 mg, 41%) as an off-white solid. 1H NMR (300 MHz, CDCl3) δ 2.47−2.85 (6H, m), 3.14−3.34 (1H, m), 3.69−4.00 (4H, m), 4.30− 4.64 (2H, m), 6.79 (1H, d, J = 8.0 Hz), 7.02 (2H, d, J = 8.3 Hz), 7.11 (1H, t, J = 7.4 Hz), 7.27−7.41 (5H, m), 7.54 (1H, d, J = 3.4 Hz), 7.88 (1H, d, J = 3.0 Hz). MS: [M + H]+ 448.9. 1-[3-(Benzyloxy)phenyl]-4-[4-(1,3-thiazol-2-ylcarbonyl)piperazin-1-yl]pyrrolidin-2-one 3j. Compound 3j was synthesized by general procedure B combinatorially in 6% yield. MS: [M + H]+ 463.0. 4.1.31.2 1-(Biphenyl-3-yl)-4-[4-(1,3-thiazol-2-ylcarbonyl)piperazin-1-yl]pyrrolidin-2-one 3k. Compound 3k was synthesized by general procedure B combinatorially in 9% yield. Purity 91%. MS: [M + H]+ 433.0. 1-(2′-Chlorobiphenyl-3-yl)-4-[4-(1,3-thiazol-2-ylcarbonyl)piperazin-1-yl]pyrrolidin-2-one 3l. A mixture of compound 7b 9212



Article

1-[4-Fluoro-3-(2-methylpyridin-3-yl)phenyl]-4-[4-(1,3-thiazol-2-ylcarbonyl)piperazin-1-yl]pyrrolidin-2-one 3r. Compound 3r was synthesized by a similar procedure to compound 3l in 69% yield as an off-white solid. 1H NMR (300 MHz, CDCl3) δ 2.46 (3H, s), 2.54−2.72 (5H, m), 2.73−2.86 (1H, m), 3.23−3.35 (1H, m), 3.74−3.86 (2H, m), 3.86−4.00 (2H, m), 4.34−4.49 (1H, m), 4.51−4.64 (1H, m), 7.14−7.24 (2H, m), 7.48 (1H, dd, J = 6.4, 2.6 Hz), 7.51−7.56 (2H, m), 7.62 (1H, ddd, J = 8.9, 4.2, 3.0 Hz), 7.88 (1H, d, J = 3.0 Hz), 8.55 (1H, dd, J = 4.9, 1.5 Hz). MS: [M + H]+ 466.1. 1-[3-Fluoro-5-(2-methylpyridin-3-yl)phenyl]-4-[4-(1,3-thiazol-2-ylcarbonyl)piperazin-1-yl]pyrrolidin-2-one 3s. Compound 3s was synthesized by a similar procedure to compound 3l in 81% yield as a white powder. 1H NMR (300 MHz, CDCl3) δ 2.51 (3H, s), 2.58−2.73 (5H, m), 2.75−2.86 (1H, m), 3.29 (1H, quin, J = 7.6 Hz), 3.75−4.03 (4H, m), 4.38−4.62 (2H, m), 6.80−6.89 (1H, m), 7.19 (1H, dd, J = 7.7, 4.9 Hz), 7.31 (1H, d, J = 1.7 Hz), 7.48−7.56 (3H, m), 7.88 (1H, d, J = 3.0 Hz), 8.53 (1H, dd, J = 4.9, 1.7 Hz). MS: [M + H]+ 466.0. 1-[3-Fluoro-5-(2-methylpyridin-3-yl)phenyl]-4-[4-(pyrimidin-2-yl)piperazin-1-yl]pyrrolidin-2-one 3t. Compound 3t was synthesized by a similar procedure to compound 3l in 78% yield as a white powder. 1H NMR (300 MHz, CDCl3) δ 2.52 (3H, s), 2.54− 2.62 (4H, m), 2.65−2.76 (1H, m), 2.76−2.89 (1H, m), 3.20−3.34 (1H, m), 3.75−4.02 (6H, m), 6.47−6.55 (1H, m), 6.78−6.88 (1H, m), 7.15−7.24 (1H, m), 7.31 (1H, s), 7.48−7.56 (2H, m), 8.32 (2H, d, J = 4.7 Hz), 8.53 (1H, dd, J = 4.8, 1.8 Hz). MS: [M + H]+ 433.2. (R)-1-[3-Fluoro-5-(2-methylpyridin-3-yl)phenyl]-4-[4-(pyrimidin-2-yl)piperazin-1-yl]pyrrolidin-2-one (R)-3t. A racemate of compound 3t (4.40 g, 10.2 mmol) was separated by preparative HPLC (CHIRALCEL OD, 50 mm i.d. × 500 mm L, Daicel Corporation, mobile phase, EtOH/hexane = 500/500). The material that showed shorter retention time was recrystallized with EtOAc/ heptane to give compound (R)-3t (1.66 g, 34%) as an off-white solid. 1 H NMR (300 MHz, CDCl3) δ 2.52 (3H, s), 2.54−2.62 (4H, m), 2.64−2.75 (1H, m), 2.77−2.88 (1H, m), 3.16−3.34 (1H, m), 3.81 (1H, dd, J = 9.3, 6.9 Hz), 3.85−3.92 (4H, m), 3.93−4.02 (1H, m), 6.46−6.55 (1H, m), 6.80−6.88 (1H, m), 7.19 (1H, dd, J = 7.6, 5.0 Hz), 7.29−7.33 (1H, m), 7.47−7.57 (2H, m), 8.31 (2H, d, J = 4.7 Hz), 8.53 (1H, dd, J = 4.9, 1.7 Hz). MS: [M + H]+ 433.2. Elemental Anal. Calcd for C24H25N6OF: C 66.65; H 5.83; N 19.43. Found: C 66.41; H 5.72; N 19.30. (S)-1-[3-Fluoro-5-(2-methylpyridin-3-yl)phenyl]-4-[4-(pyrimidin-2-yl)piperazin-1-yl]pyrrolidin-2-one (S)-3t. Compound (S)-3t was optically separated by a similar procedure to compound (R)-3t in 39% yield as a beige solid. The absolute configuration of (S)-3t was determined to be S configuration by X-ray crystallographic analysis. 1H NMR (300 MHz, CDCl3) δ 2.52 (3H, s), 2.54−2.62 (4H, m), 2.65−2.75 (1H, m), 2.76−2.87 (1H, m), 3.19−3.33 (1H, m), 3.78−3.91 (5H, m), 3.93−4.01 (1H, m), 6.49−6.56 (1H, m), 6.80−6.88 (1H, m), 7.19 (1H, dd, J = 7.6, 5.0 Hz), 7.29−7.33 (1H, m), 7.47−7.57 (2H, m), 8.31 (2H, d, J = 4.7 Hz), 8.53 (1H, dd, J = 4.9, 1.7 Hz). MS: [M + H]+ 433.2. 4-Methoxy-1-(4-phenoxyphenyl)-1H-pyrrol-2(5H)-one 5a. To a mixture of 4-phenoxyaniline (10.0 g, 54.0 mmol) in MeCN (40.0 mL) was added a mixture of methyl 4-chloro-3-methoxy-2-(E)butenoate 4 (9.18 mL, 67.5 mmol) in MeCN (40.0 mL) at 0 °C. To the mixture was added a mixture of triethylamine (8.28 mL, 59.4 mmol) in MeCN (20.0 mL) at 0 °C. The mixture was heated to reflux for 4 h. The mixture was cooled to rt. The precipitates were removed by filtration. To the filtrate was added water (80 mL). The mixture was acidified to pH 3 using conc hydrogen chloride. The mixture was extracted with EtOAc, dried over Na2SO4, filtered, and concentrated in vacuo. To the mixture were added toluene (80.0 mL) and acetic acid (2.0 mL) at rt. The mixture was heated to 50 °C for 1.5 h. The mixture was diluted with water, brine, and saturated aqueous NaHCO3 solution at rt, extracted with EtOAc, dried over Na2SO4, filtered, concentrated in vacuo, and purified by column chromatography (silica gel, eluted with 49−70% EtOAc in hexane) to yield compound 5a (7.43 g, 49%) as a yellow solid after trituration with

(50 mg, 0.10 mmol), (2-chlorophenyl)boronic acid (24.3 mg, 0.16 mmol), (A-taPhos)2PdCl2 (7.3 mg, 10 μmol), Cs2CO3 (67.6 mg, 0.21 mmol), and toluene (3.0 mL)/water (1.0 mL) was heated at 100 °C for 10 min under microwave irradiation. The organic layer was purified by column chromatography (NH silica gel, eluted with 0− 15% MeOH in EtOAc) and recrystallized from EtOAc−hexane to give compound 3l (40.0 mg, 83%) as a white powder. 1H NMR (300 MHz, CDCl3) δ 2.50−2.72 (5H, m), 2.74−2.85 (1H, m), 3.28 (1H, quin, J = 7.6 Hz), 3.84 (3H, dd, J = 9.5, 6.7 Hz), 3.95−4.06 (1H, m), 4.38−4.60 (2H, m), 7.23 (1H, d, J = 1.1 Hz), 7.28−7.37 (3H, m), 7.41−7.50 (2H, m), 7.54 (1H, d, J = 3.0 Hz), 7.58 (1H, t, J = 1.9 Hz), 7.70 (1H, ddd, J = 8.2, 2.2, 0.9 Hz), 7.88 (1H, d, J = 3.2 Hz). MS: [M + H]+ 467.0. 1-(3′-Chlorobiphenyl-3-yl)-4-[4-(1,3-thiazol-2-ylcarbonyl)piperazin-1-yl]pyrrolidin-2-one 3m. A mixture of compound 7b (50.0 mg, 0.10 mmol), 3-chlorophenylboronic acid (48.6 mg, 0.31 mmol), Pd2(dba)3 (9.5 mg, 10.4 μmol), SPhos (12.8 mg, 0.03 mmol), and 2 M Na2CO3 aqueous solution (0.155 mL) in toluene (1.0 mL) was stirred at 100 °C under N2 for 3 h. The mixture was diluted with EtOAc and water and extracted with EtOAc. The organic layer was separated, washed with water and brine successively, dried over Na2SO4, and concentrated in vacuo. The residue was purified by column chromatography (NH silica gel, eluted with 50−100% EtOAc in hexane). The residue was purified by column chromatography (silica gel, eluted with 0−20% MeOH in EtOAc) to give compound 3m (20.0 mg, 41%) as an off-white solid after triturating with iPrOAc-IPE. 1H NMR (300 MHz, CDCl3) δ 2.57−2.74 (5H, m), 2.75−2.87 (1H, m), 3.24−3.37 (1H, m), 3.76−3.97 (3H, m), 3.97− 4.07 (1H, m), 4.38−4.63 (2H, m), 7.32−7.49 (5H, m), 7.54 (1H, d, J = 3.0 Hz), 7.55−7.64 (2H, m), 7.78 (1H, t, J = 1.8 Hz), 7.88 (1H, d, J = 3.2 Hz). MS: [M + H]+ 467.1. 1-(4′-Chlorobiphenyl-3-yl)-4-[4-(1,3-thiazol-2-ylcarbonyl)piperazin-1-yl]pyrrolidin-2-one 3n. Compound 3n was synthesized by general procedure A combinatorially in 54% yield. MS: [M + H]+ 467.1. 1-(2′-Methylbiphenyl-3-yl)-4-[4-(1,3-thiazol-2-ylcarbonyl)piperazin-1-yl]pyrrolidin-2-one 3o. Compound 3o was synthesized by general procedure A combinatorially in 50% yield. MS: [M + H]+ 447.1. 1-[3-(3-Methylpyridin-2-yl)phenyl]-4-[4-(1,3-thiazol-2ylcarbonyl)piperazin-1-yl]pyrrolidin-2-one 3p. To a solution of compound 6b (50 mg, 0.10 mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′bi(1,3,2-dioxaborolane) (39.5 mg, 0.16 mmol), KOAc (30.5 mg, 0.31 mmol) in DMF (1 mL) was added PdCl2(dppf) (7.58 mg, 10.4 μmol) at ambient temperature. The mixture was stirred at 80 °C for 1 h and then allowed to rt. To the mixture were added 2-bromo-3methylpyridine (0.035 mL, 0.31 mmol), Cs2CO3 (67.6 mg, 0.21 mmol), (A-taPhos)2PdCl2 (7.0 mg, 10.4 μmol), and water (0.1 mL) successively. The mixture was stirred at 80 °C under N2 for 3 h. The mixture was diluted with EtOAc and water and extracted with EtOAc. The organic layer was separated, washed with water and brine successively, dried over Na2SO4, and concentrated in vacuo. The residue was purified by column chromatography (NH silica gel, eluted with 50−100% EtOAc in hexane) to give compound 3p (5.5 mg, 12%) as an off-white solid after triturating with IPE/i-PrOAc. 1H NMR (300 MHz, CDCl3) δ 2.38 (3H, s), 2.54−2.71 (5H, m), 2.72− 2.85 (1H, m), 3.20−3.35 (1H, m), 3.74−3.95 (3H, m), 3.97−4.06 (1H, m), 4.36−4.47 (1H, m), 4.48−4.60 (1H, m), 7.19 (1H, dd, J = 7.6, 4.8 Hz), 7.32 (1H, d, J = 7.7 Hz), 7.41−7.50 (1H, m), 7.54 (1H, d, J = 3.2 Hz), 7.59 (1H, d, J = 8.3 Hz), 7.66−7.74 (2H, m), 7.88 (1H, d, J = 3.2 Hz), 8.52 (1H, d, J = 3.8 Hz). MS: [M + H]+ 448.2. 1-[3-(2-Methylpyridin-3-yl)phenyl]-4-[4-(1,3-thiazol-2ylcarbonyl)piperazin-1-yl]pyrrolidin-2-one 3q. Compound 3q was synthesized by a similar procedure to compound 3l in 71% yield as a white powder. 1H NMR (300 MHz, CDCl3) δ 2.52 (3H, s), 2.57−2.72 (5H, m), 2.74−2.85 (1H, m), 3.29 (1H, t, J = 7.2 Hz), 3.77−4.04 (4H, m), 4.36−4.61 (2H, m), 7.08−7.15 (1H, m), 7.19 (1H, dd, J = 7.7, 4.9 Hz), 7.45 (1H, t, J = 7.9 Hz), 7.50−7.55 (2H, m), 7.57−7.65 (2H, m), 7.88 (1H, d, J = 3.2 Hz), 8.51 (1H, dd, J = 4.8, 1.8 Hz). MS: [M + H]+ 448.1 9213



Article

EtOAc/hexane. 1H NMR (300 MHz, DMSO-d6) δ 3.85 (3H, s), 4.48 (2H, s), 5.32 (1H, s), 6.92−6.99 (2H, m), 6.99−7.05 (2H, m), 7.06− 7.13 (1H, m), 7.32−7.41 (2H, m), 7.66−7.74 (2H, m). MS: [M + H]+ 281.8. 1-(3-Iodophenyl)-4-methoxy-1H-pyrrol-2(5H)-one 5b. Compound 5b was synthesized by a similar procedure to compound 5a in 45% yields as a white powder. 1H NMR (300 MHz, CDCl3) δ 3.87 (3H, s), 4.24 (2H, s), 5.18 (1H, s), 7.05 (1H, t, J = 8.1 Hz), 7.40 (1H, dt, J = 7.8, 1.2 Hz), 7.61−7.75 (1H, m), 7.96 (1H, t, J = 1.9 Hz). MS: [M + H]+ 316.0. 4-Hydroxy-1-(4-phenoxyphenyl)-1H-pyrrol-2(5H)-one 6a. A mixture of compound 5a (7.43 g, 26.4 mmol) in hydrochloric acid (37%, 46.4 mL, 26.4 mmol)/toluene (91.2 mL) was heated to 50 °C for 21 h. The mixture was concentrated in vacuo, diluted with saturated aqueous NaHCO3 solution, water, and brine, extracted with EtOAc, dried over Na2SO4, filtered, and concentrated in vacuo to yield compound 6a (6.89 g, 98%) as a light brown solid after trituration with EtOAc/hexane. 1H NMR (300 MHz, DMSO-d6) δ 4.37 (2H, s), 4.96 (1H, brs), 6.88−7.16 (5H, m), 7.29−7.45 (2H, m), 7.69 (2H, d, J = 8.7 Hz), 11.87 (1H, brs). MS: [M + H]+ 268.2. 4-Hydroxy-1-(3-iodophenyl)-1H-pyrrol-2(5H)-one 6b. Compound 6b was synthesized by a similar procedure to compound 6a in 95% yields as a white powder. 1H NMR (300 MHz, CDCl3) δ 3.33 (2H, s), 4.31 (2H, t, J = 1.1 Hz), 7.06−7.16 (1H, m), 7.57 (1H, dd, J = 7.9, 1.5 Hz), 7.62−7.76 (1H, m), 8.01 (1H, t, J = 1.9 Hz). MS: [M − H]− 300.1. tert-Butyl 4-[5-Oxo-1-(4-phenoxyphenyl)pyrrolidin-3-yl]piperazine-1-carboxylate 7a. Compound 7a was synthesized by a similar procedure to compound 3b in 66% yield as a white solid. 1H NMR (300 MHz, CDCl3) δ 1.43−1.50 (9H, m), 2.46 (4H, brs), 2.56−2.68 (1H, m), 2.70−2.81 (1H, m), 3.14−3.32 (1H, m), 3.47 (4H, brs), 3.77 (1H, dd, J = 9.5, 6.4 Hz), 3.84−3.98 (1H, m), 6.95− 7.06 (4H, m), 7.06−7.14 (1H, m), 7.29−7.37 (2H, m), 7.48−7.57 (2H, m). MS: [M + H]+ 438.1. 1-(3-Iodophenyl)-4-[4-(1,3-thiazol-2-ylcarbonyl)piperazin1-yl]pyrrolidin-2-one 7b. Compound 7b was synthesized by a similar procedure to compound 3b in 67% yield as a white powder. 1 H NMR (300 MHz, CDCl3) δ 2.52−2.70 (5H, m), 2.72−2.87 (1H, m), 3.26 (1H, quin, J = 7.5 Hz), 3.70−4.01 (4H, m), 4.35−4.63 (2H, m), 7.09 (1H, t, J = 8.1 Hz), 7.50 (1H, d, J = 7.9 Hz), 7.55 (1H, d, J = 3.4 Hz), 7.64 (1H, dd, J = 8.3, 1.9 Hz), 7.85−7.94 (2H, m). MS: [M + H]+ 483.1. 1-(4-Phenoxyphenyl)-4-(piperazin-1-yl)pyrrolidin-2-one Hydrochloride 7c. To a mixture of compound 7a (2.90 g, 6.63 mmol) in EtOAc (88 mL) was added 4 M hydrogen chloride in EtOAc (16.6 mL, 66.3 mmol) at rt. The mixture was stirred at rt for 30 min and at 60 °C for 30 min. To the mixture was added 4 M hydrogen chloride in EtOAc (16.56 mL, 66.26 mmol) at 60 °C. The mixture was heated to 60 °C for 14 h. To the mixture was added 4 M hydrogen chloride in EtOAc (33.1 mL, 132.51 mmol) at 60 °C. The mixture was heated to reflux for 6 h. To the mixture was added 2 M hydrogen chloride in MeOH (66.3 mL, 133 mmol) at rt. The mixture was heated to reflux for 2 h. The mixture was concentrated in vacuo to yield compound 7c (2.46 g, 99%) as a white solid after trituration with EtOAc/hexane. 1H NMR (300 MHz, DMSO-d6) δ 2.75−2.97 (3H, m), 3.02−3.26 (4H, m), 3.33 (2H, brs), 3.86−4.24 (3H, m), 6.94−7.20 (5H, m), 7.33−7.43 (2H, m), 7.52 (1H, brs), 7.63−7.70 (2H, m), 9.48 (2H, brs). MS: [M + H]+ 338.2. 4-[4-(1,3-Thiazol-2-ylcarbonyl)piperazin-1-yl]dihydrofuran2(3H)-one 9a. To a solution of piperazin-1-yl(1,3-thiazol-2-yl)methanone (2.00 g, 10.1 mmol) in MeOH (4 mL) was added 2(5H)furanone 8 (0.852 mL, 12.2 mmol) in at 0 °C. The mixture was stirred at ambient temperature overnight. To the mixture was added IPA (4 mL) and IPE (2 mL). The mixture was stirred at rt for 1 h. The precipitate was collected by filtration and washed with IPA/IPE (v/v = 50/50) to give compound 9a (2.11 g, 74%) as an off-white solid. 1H NMR (300 MHz, CDCl3) δ 2.43−2.76 (6H, m), 3.38 (1H, quin, J = 7.2 Hz), 3.84 (2H, brs), 4.22 (1H, dd, J = 9.4, 6.4 Hz), 4.44 (3H, dd, J = 9.2, 7.0 Hz), 7.55 (1H, d, J = 3.0 Hz), 7.88 (1H, d, J = 3.0 Hz). MS: [M + H]+ 282.1.

4-[4-(Pyrimidin-2-yl)piperazin-1-yl]dihydrofuran-2(3H)-one 9b. Compound 9b was synthesized by a similar procedure to compound 9a in 85% yield as an off-white solid. 1H NMR (300 MHz, CDCl3) δ 2.44−2.62 (5H, m), 2.62−2.72 (1H, m), 3.28−3.43 (1H, m), 3.77−3.92 (4H, m), 4.24 (1H, dd, J = 9.2, 6.6 Hz), 4.45 (1H, dd, J = 9.3, 7.0 Hz), 6.51 (1H, t, J = 4.7 Hz), 8.24−8.37 (2H, m). MS: [M + H]+ 249.1. 4-Hydroxy-N-(3-phenoxyphenyl)-3-[4-(thiazole-2carbonyl)piperazin-1-yl]butanamide 10a. To a solution of 3phenoxyaniline (42.7 mg, 0.23 mmol) in toluene (2 mL) was added 1.8 M AlMe3 toluene solution (0.233 mL, 0.42 mmol) at ambient temperature under Ar. The mixture was stirred at ambient temperature for 1 h. To the resulting mixture was added a solution of compound 9a (59 mg, 0.21 mmol) in toluene (1 mL). The mixture was stirred at 70 °C for 3 h. The residue was purified by column chromatography (NH silica gel, eluted with 50−100% EtOAc in hexane and then 0−30% MeOH in EtOAc) to give compound 10a (58.0 mg, 59%) as a pale yellow oil. 1H NMR (300 MHz, CDCl3) δ 2.31 (1H, dd, J = 15.9, 5.3 Hz), 2.64 (1H, dd, J = 15.9, 8.3 Hz), 2.74− 2.85 (2H, m), 2.85−3.02 (2H, m), 3.17−3.30 (1H, m), 3.64−3.75 (2H, m), 3.75−3.90 (2H, m), 4.44 (2H, brs), 6.68−6.79 (1H, m), 6.98−7.05 (2H, m), 7.05−7.12 (1H, m), 7.15 (1H, s), 7.26−7.37 (4H, m), 7.53−7.58 (1H, m), 7.86−7.91 (1H, m), 9.25 (1H, s). MS: [M + H]+ 467.2. N-(3-Bromo-4-fluorophenyl)-4-hydroxy-3-[4-(thiazole-2carbonyl)piperazin-1-yl]butanamide 10b. To a mixture of compound 9a (100 mg, 0.36 mmol) and 3-bromo-4-fluoroaniline (108 mg, 0.57 mmol) in toluene (2 mL) was added DABAL-Me3 (72.9 mg, 0.28 mmol) at rt, and the mixture was stirred at 80 °C for 1 h. To the mixture were added EtOAc, THF, and Na2SO4·10H2O (550 mg, 1.71 mmol), and the mixture was stirred at rt for 1 h. The insoluble material was removed by filtration, and the filtrate was concentrated in vacuo. The residue was purified by column chromatography (silica gel, eluted with 0−20% MeOH in EtOAc) to give compound 10b (168 mg, 100%) as a pale yellow oil. 1H NMR (300 MHz, CDCl3) δ 2.35 (1H, dd, J = 15.8, 5.4 Hz), 2.66 (1H, dd, J = 15.8, 8.2 Hz), 2.76−2.98 (4H, m), 3.19−3.32 (1H, m), 3.66−3.79 (3H, m), 3.79−3.99 (2H, m), 4.35−4.64 (2H, m), 7.06 (1H, t, J = 8.5 Hz), 7.32−7.40 (1H, m), 7.53−7.58 (1H, m), 7.85 (1H, dd, J = 6.1, 2.5 Hz), 7.89 (1H, d, J = 3.2 Hz), 9.28 (1H, s). MS: [M + H]+ 472.1. N-(3-Fluoro-5-iodophenyl)-4-hydroxy-3-[4-(thiazole-2carbonyl)piperazin-1-yl)]butanamide 10c. Compound 10c was synthesized by a similar procedure to compound 10b in 96% yield as a pale yellow oil. 1H NMR (300 MHz, CDCl3) δ 1.61 (1H, brs), 2.35 (1H, dd, J = 15.9, 5.3 Hz), 2.66 (1H, dd, J = 16.0, 8.2 Hz), 2.80−3.00 (4H, m), 3.23 (1H, dd, J = 8.3, 6.2 Hz), 3.62−4.00 (4H, m), 4.34− 4.64 (2H, m), 7.07−7.21 (1H, m), 7.44 (1H, dt, J = 10.5, 2.0 Hz), 7.56 (2H, d, J = 3.2 Hz), 7.89 (1H, d, J = 3.2 Hz), 9.50 (1H, brs). MS: [M + H]+ 519.1. N-(3-Bromo-5-fluorophenyl)-4-hydroxy-3-[4-(pyrimidin-2yl)piperazin-1-yl]butanamide 10d. Compound 10d was synthesized by a similar procedure to compound 10b in 73% yield as a pale yellow oil. 1H NMR (300 MHz, CDCl3) δ 2.39 (1H, dd, J = 16.2, 4.4 Hz), 2.62−2.71 (1H, m), 2.72−2.83 (2H, m), 2.89−3.02 (2H, m), 3.14−3.26 (1H, m), 3.69−3.80 (2H, m), 3.82−4.01 (4H, m), 6.50− 6.59 (1H, m), 6.92−7.01 (1H, m), 7.34−7.45 (2H, m), 8.28−8.39 (2H, m), 10.07 (1H, brs). MS: [M + H]+ 438.1. 1-(3-Bromo-4-fluorophenyl)-4-[4-(1,3-thiazol-2-ylcarbonyl)piperazin-1-yl]pyrrolidin-2-one 11a. Compound 11a was synthesized by a similar procedure to compound 3i in 59% yield as an offwhite solid. 1H NMR (300 MHz, CDCl3) δ 2.54−2.71 (5H, m), 2.73−2.83 (1H, m), 3.21−3.35 (1H, m), 3.76 (1H, dd, J = 9.4, 6.6 Hz), 3.84 (1H, brs), 3.87−3.96 (2H, m), 4.34−4.48 (1H, m), 4.49− 4.65 (1H, m), 7.12 (1H, dd, J = 9.0, 8.0 Hz), 7.52−7.60 (2H, m), 7.80 (1H, dd, J = 6.0, 2.6 Hz), 7.88 (1H, d, J = 3.2 Hz). MS: [M + H]+ 453.0. 1-(3-Fluoro-5-iodophenyl)-4-[4-(thiazole-2-carbonyl)piperazin-1-yl]pyrrolidin-2-one 11b. Compound 11b was synthesized by a similar procedure to compound 3i in 43% yield as a white powder. 1H NMR (300 MHz, CDCl3) δ 2.55−2.71 (5H, m), 2.74− 9214



Article

2.85 (1H, m), 3.27 (1H, quin, J = 7.6 Hz), 3.68−3.97 (4H, m), 4.36− 4.64 (2H, m), 7.21−7.25 (1H, m), 7.52−7.61 (2H, m), 7.63 (1H, s), 7.88 (1H, d, J = 3.2 Hz). MS: [M + H]+ 501.1. 1-(3-Bromo-5-fluorophenyl)-4-[4-(pyrimidin-2-yl)piperazin1-yl]pyrrolidin-2-one 11c. Compound 11c was synthesized by a similar procedure to compound 3i in 65% yield as a white powder. 1H NMR (300 MHz, CDCl3) δ 2.47−2.63 (4H, m), 2.63−2.73 (1H, m), 2.74−2.86 (1H, m), 3.18−3.31 (1H, m), 3.71−3.80 (1H, m), 3.81− 3.97 (5H, m), 6.49−6.54 (1H, m), 7.00−7.08 (1H, m), 7.47−7.57 (2H, m), 8.32 (2H, d, J = 4.8 Hz). Found 420.1. Measurements of in Vitro MAGL Inhibitory Activity. Preparation of Enzyme Fraction. Human MGLL cDNA was obtained by PCR using human ORF xlone (DNA form; clone ID, 100004585) as a template. For PCR, two kinds of primers, 5′CCACCATCATCACGGATCCATGCCAGAGGAAAGTTCCCCCA-3′ [SEQ id no. 1] and 5′-TGGTGCTCGAGTGCGGCCGCTCAGGGTGGGGACGCAGTTC-3′ [SEQ id no. 2], and PrimeSTAR MAX DNA polymerase (Takara Bio Inc.) were used, and (1) reaction at 98 °C for 1 min, (2) 25 cycles of reaction at 98 °C for 10 s and 68 °C for 10 s as one cycle, and (3) reaction at 72 °C for 1 min were performed. The obtained PCR product was digested with Bam HI and Not I (Takara Bio Inc.), inserted into the Bam HI/Not I site of pET21HH(V) (pET21a (Novagen) inserted with His χ6 and TEV protease recognition sequence) by using Ligation High (Toyobo Co., Ltd.), and introduced into ECOS JM109 (Nippon Gene Co., Ltd.), whereby expression plasmid pET21HH(V)/His-hMGLLv2 for Escherichia coli was constructed. Recombinant His-hMAGL protein was prepared by transforming ECOS Competent E. coli BL21(DE3) (Nippon Gene Co., Ltd.) with the pET21HH(V)/His-hMGLLv2 plasmid prepared above. E. coli obtained by transformation was inoculated to 10 mL of LB medium (1% peptone, 0.5% yeast extract, 0.5% sodium chloride, 0.01% ampicillin) and cultured at 30 °C for 16 h. The obtained culture medium (5 mL) was transplanted into a 2 L Sakaguchi flask containing 1 L of main fermentation medium (1.05% M9 medium broth (AMRESCO LLC), 0.5% yeast extract, 1.5% sorbitol, 1.5% casamino acid, 0.024% magnesium sulfate, 0.01% antifoaming agent PE-L (Wako Pure Chemical Industries, Ltd.), 0.01% ampicillin) and shaking culture at 37 °C, and 150 rpm was started. When the turbidity of the culture medium reached about 500 Klett unit, the culture temperature was lowered to 16 °C, isopropyl-pD-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, and the mixture was further cultured for 19 h. After the completion of culture, the culture medium was centrifuged (4 °C, 6000 rpm, 10 min) to give His-hMAGL expression E. coli. Then, HishMAGL expression E. coli was suspended in 50 mM Tris-HCl (pH 8.0, 100 mL) containing 1% Triton X-100, 20 mM imidazole, 3 mM DTT, 5 U/mL Benzonase (Merck), and 150 mM NaCl, sufficiently cooled, and subjected to sonication at AMPLITUDE = 60%, 15 s/ ON, 30 s/OFF for 3 min using 3/4 in. solid type crushed horn of BRANSON digital sonifier 450 (Central Scientific Commerce, Inc.). Furthermore, the homogenate was centrifuged (4 °C, 15 000 rpm, 15 min) and the supernatant was obtained. As the purification apparatus, AKTA explorer 10s (GE Healthcare Japan Corporation) was used at 4 °C. To the obtained supernatant was added 5 M NaCl to the final salt concentration of 0.3 M, and the mixture was flown through and adsorbed to 5 mL of Ni-NTA Superflow cartridges (QIAGEN) equilibrated in advance with buffer A (50 mM Tris-HCl (pH 8.0) containing 0.05% TritonX-100, 1 mM DTT, 300 mM NaCl). The column was sufficiently washed with buffer A containing 20 mM imidazole, and His-hMAGL was eluted with buffer A containing imidazole at a final concentration of 250 mM. The eluate was further subjected to gel filtration using HiLoad 26/600 Superdex 200 pg (GE Healthcare Japan Corporation) equilibrated with 50 mM Tris-HCl, pH 8.0, containing 10% glycerol, 0.05% Triton X-100, 1 mM DTT, 150 mM NaCl. The eluted fraction was concentrated by Amicon Ultra-15 10K (Merck Millipore) to give purified His-hMAGL protein. The protein concentration was measured by BCA protein assay kit (Thermo Fisher Scientific) using BSA as the standard. Measurement of MAGL Inhibitory Activity by Mass Spectrometry. His-hMAGL was diluted with enzyme reaction

buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.025% (w/v) Triton X-100, 0.01% bovine serum albumin) to a concentration of 7.5 ng/ mL. To each well of a 384-well assay plate (Greiner 781280) was added a solution (5 μL) of a test compound dissolved in DMSO, which was diluted with the above-mentioned enzyme reaction buffer, then His-hMAGL solution (5 μL) diluted to a concentration of 7.5 ng/mL was added and the mixture was incubated at rt for 60 min. Thereafter, to each well was added 150 μL of 2-AG (Tocris Bioscience) by 5 μL, and the mixture was incubated at rt for 10 min. Then, 2% formic acid (Wako Pure Chemical Industries, Ltd.) was added by 10 μL to discontinue the reaction. Furthermore, MeCN (50 μL) containing 3 μM AA-d8 (Cayman Chemical Company) was added, and the mixture was stirred. The amount of AA in the obtained enzyme reaction mixture was calculated by measuring by RapidFire mass spectrometry and correcting by the amount of AA-d8. High throughput online solid phase extraction was performed using RapidFire 300 system (Agilent Technologies, Inc.). Samples were loaded on SPE C4 cartridge (Agilent Technologies, Inc.) and desalted with 0.2% (ν/ν) acetic acid (Wako Pure Chemical Industries, Ltd.) in ultrapure water/acetonitrile (70/30, ν/ν) at a flow rate of 1.5 mL/ min, eluted at a flow rate of 0.5 mL/min with 0.2% (ν/ν) acetic acid dissolved in acetonitrile/ultrapure water (90/10, ν/ν), and injected into the mass spectrometry part. The injection needle was washed with ultrapure water (500 ms) and MeCN (500 ms) to minimize carryover. The suction time (injection loop 5 μL), load/cleansing time, elution time, and re-equilibration time were adjusted to 300, 3000, 4250, and 1000 ms, respectively, and the total cycle time was adjusted to about 10.0 s. The RapidFire300 system was controlled by RapidFire UI software version 3.6 (Agilent Technologies, Inc.). The mass spectrometry of the resultant product was performed using API4000 triple quadrupole mass spectrometer (AB SCIEX) equipped with an electrospray ion sauce (Turboion Spray) in a negative selected reaction monitoring (SRM) mode. The conditions of SRM are shown below. The parameters of the instrument were optimized as follows: capillary temperature 600 °C, ion spray voltage −4.5 kV, collision gas 8 psi, curtain gas 15 psi, ion source gas 1 60 psi, ion source gas 2 60 psi. The mass spectrometer was controlled by Analyst software version 1.5.1 (AB SCIEX). The peak area integration was analyzed using RapidFire integrator software version 3.6 (Agilent Technologies, Inc.). MAGL inhibitory rate (%) was calculated according to the following calculation formula. (1 − (AA production amount of test compound addition group − AA production amount of enzyme addition-free group) ÷ (AA production amount of test compound addition-free group − AA production amount of enzyme addition-free group)) × 100. The 50% inhibitory concentration (IC50) was calculated by fitting using XLfit (IDBS Ltd.) and dose response one-site four-parameter logistic model. Metabolic Stability Assay. In vitro oxidative metabolic studies of the test compounds were carried out using hepatic microsomes obtained from humans. The reaction mixture with a final volume of 0.05 mL consisted of 0.2 mg/mL hepatic microsomes in 50 mM KH2PO4−K2HPO4 phosphate buffer (pH 7.4) and 1 μM test compound. The reaction was initiated by the addition of an NADPH-generating system containing 25 mM MgCl2, 25 mM glucose 6-phosphate, 2.5 mM β-NADP+, and 7.5 units/mL glucose 6phosphate dehydrogenase at 20 vol % of the reaction mixture. After addition of the NADPH-generating system, the mixture was incubated at 37 °C for 0, 15, and 30 min. The reaction was terminated by addition of an equivalent volume of acetonitrile. After the samples were mixed and centrifuged, the supernatant fractions were analyzed using LC−MS/MS. For metabolic clearance determinations, chromatograms were analyzed to determine the disappearance of the parent compound from the reaction mixtures. All incubations were carried out in duplicate. Measurement of in Vitro Kinetic Parameters with MAGL. To determine the kinetics parameters with a dilution assay, compounds were dissolved in DMSO and then diluted in the assay buffer. Ten microliters of compound solution was added to a 96-well plate, and then 10 μL of 750 ng/mL human MAGL solution was added to the plate and incubated at rt for 1 h. The concentration of the compound 9215



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was equal to 10-fold the IC50 value upon incubation for 60 min. After this incubation, 4 μL of compound−enzyme mixture was transferred to a 96-well plate and 396 μL of substrate solution 450 μM 2-AG was added to the well. By its rapid dilution, the concentration of the inhibitor dropped to 10-fold below the IC50. The reaction was stopped by the addition of acetonitrile containing AA-d8. The amount of AA in the obtained enzyme reaction mixture was calculated by measuring by RapidFire mass spectrometry and correcting by the amount of AA-d8. The progress curves were fitted to the following equations to determine the values for koff, the apparent rate constant from initial rate v0 to steady-state rate vs, and dissociation half-life t1/2. P = vst + (v0 − vs)(1 − e−koff t )/koff + P0

t1/2 =

ln 2 koff

Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b00824. Crystallography preparation and data (a cocrystal of MALG with 3l and a single crystal of (S)-3t) (PDF) Molecular formula strings and some data (CSV) Accession Codes

The coordinates of the crystal structures of MAGL in complex with compounds 3l (5ZUN) have been deposited in the Protein Data Bank. Authors will release the atomic coordinates and experimental data upon article publication.

(1)

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(2)

AUTHOR INFORMATION

Corresponding Author

where t is time, P is product, and P0 is product at time t = 0. The kon value was calculated from Ki and koff values. Measurements of in Vivo MAGL Inhibitory Activity. Animal. Male C57BL/6J mice were supplied by CLEA Japan Inc. (Tokyo, Japan). The mice were housed in groups of 3/cage in a lightcontrolled room (12 h light/dark cycle with lights on at 07:00). Food and water were provided ad libitum. Mice used in the experiments were 8 weeks old and had completed an acclimation period of at least 1 week. The care and use of the animals and the experimental protocols used in this research were approved by the Experimental Animal Care and Use Committee of Takeda Pharmaceutical Company Limited. Pharmacokinetic Analysis in Mouse. Compound (R)-3t was suspended in distilled water with 0.5% methylcellulose. Compound (R)-3t (10 mg/kg) was orally administered to mice at a volume of 10 mL/kg body weight. Plasma and brain samples were collected at 1 h after administration. The cerebrum hemisphere was homogenized with 4-fold (v/w) of saline. The concentrations of (R)-3t in the collected samples were measured by high performance liquid chromatography/tandem mass spectrometry (LC/MS/MS). Measurements of 2-AG and AA in Mouse Brain. Compound (R)-3t was suspended in distilled water with 0.5% methylcellulose. Compound (R)-3t (10 mg/kg) was orally administered to mice at a volume of 10 mL/kg body weight. Brain samples were collected at 1 h after administration. The cerebrum hemisphere was homogenized with 9-fold (v/w) of isopropanol and then centrifuged. The supernatant was mixed with internal standard solution (AA-d8 and 2-AG-d8 in isopropanol). The sample solution was applied to a highperformance liquid chromatography/tandem mass spectrometry (LC/ MS/MS) analysis. Chromatographic separation was performed by a gradient elution on a reverse phase column, Xbridge C18 (2.5 μm, 2.1 mm × 50 mm, Waters, Milford, MA), under the column temperature of 60 °C. The solvent system consisted of 0.01% acetic acid−1 mM NH3−2 μM EDTA−2Na in distilled water (solvent A) and 0.001% acetic acid−0.2 mM NH3 in ethanol−isopropanol (3:2, ν/ν) (solvent B) with a flow rate of 0.5 mL/min. The following gradient program was applied to the chromatographic separation: 0−1 min, 1% solvent B; 1−1.2 min, 1−55% solvent B; 1.2−2.7 min, 55−75% solvent B; 2.7−3.5 min, 75−99% solvent B; 3.5−6 min, 99% solvent B; and 6−8 min, 1% solvent B. The eluate from the liquid chromatography system was directly introduced to an electrospray ionization on TSQ Vantage mass spectrometer (Thermo Fisher Scientific, San Jose, CA) with a simultaneous polarity switching, where AA and its internal standard (AA-d8) were ionized by negative ionization mode, and 2-AG and its internal standard (2-AG-d8) were ionized by positive ionization mode. The calibration curve was drawn with a weighting of 1/x, and confirmed by assessing the accuracy within a range of ±15% and the linearity of R2 > 0.995. The amount of change was calculated based on the value obtained for the control group (0.5% methylcellulose solution administration group).

*Phone: +81 466 32 1068. Fax: +81 466 29 4544. E-mail: [email protected]. ORCID

Jumpei Aida: 0000-0002-7546-1915 Satoshi Sogabe: 0000-0003-2393-9582 Present Address †

S. S. and K. A.: Axcelead Drug Discovery Partners, Inc., 26-1, Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa 251-0012, Japan. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. J. A., M. F., T. K., H. S., S.I., and T.K. contributed to the design and synthesis of compounds. N.A., M.S., S.S., and K.A. contributed to in vitro and in vivo studies. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge Drs. Mitsuhiro Nishitani and Motoo Iida for the excellent technical assistance with determining the absolute configuration; Drs Shinobu Sasaki and Yoji Hayase for the excellent technical assistance with conducting combinatorial chemistry; Drs. Kotaro yokoyama, Yoshinori Satomi, Megumi Hirayama, and Hiroyuki Kobayashi for the excellent technical assistance with analyzing 2-AG and AA; Drs. Tomohiro Kaku, Takanobu Kuroita, Takashi Miki, and Hidekazu Tokuhara for valuable suggestions to drug design.

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ABBREVIATIONS USED ADDP, 1,1′-(azodicarbonyl)dipiperidine; dppf, 1,1′-bis(diphenylphosphino)ferrocene; HATU, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; 2-AG, 2-arachidonoylglycerol; A- ta Phos, 4-(dimethylaminophenyl)di-tertbutylphosphine; MeCN, acetonitrile; DABAL-Me3, bis(trimethylaluminum)-1,4-diazabicyclo[2.2.2]octane; dba, dibenzylideneacetone; IPE, diisopropyl ether; koff, dissociation rate constant; IPA, isopropyl alcohol; MAGL, monoacylglycerol lipase; ORF, open reading frame

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Design, Synthesis, and Evaluation of Piperazinyl Pyrrolidin-2-ones as

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