Discovery of the First Potent and Orally Available Agonist of the

Jun 2, 2014 - Pharmaceutical Research Division, Takeda Pharmaceutical Company Ltd., 26-1, ... G-protein-coupled receptor 52 (GPR52) is an orphan Gs-...
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Discovery of the First Potent and Orally Available Agonist of the Orphan G‑Protein-Coupled Receptor 52 Masaki Setoh,*,† Naoki Ishii,† Mitsunori Kono,† Yuhei Miyanohana,† Eri Shiraishi,† Toshiya Harasawa,†,§ Hiroyuki Ota,† Tomoyuki Odani,† Naoyuki Kanzaki,† Kazunobu Aoyama,† Teruki Hamada,† and Masakuni Kori‡ †

Pharmaceutical Research Division, Takeda Pharmaceutical Company Ltd., 26-1, Muraoka-higashi-2-chome, Fujisawa, Kanagawa 251-8555, Japan ‡ CMC Center, Takeda Pharmaceutical Company Ltd., 17-85, Jusohonmachi-2-chome, Yodogawa-ku, Osaka 532-8686, Japan S Supporting Information *

ABSTRACT: G-protein-coupled receptor 52 (GPR52) is an orphan Gs-coupled G-protein-coupled receptor. GPR52 inhibits dopamine D2 receptor signaling and activates dopamine D1/N-methyl-D-aspartate receptors via intracellular cAMP accumulation, and therefore, GPR52 agonists may have potential as a novel class of antipsychotics. A series of GPR52 agonists with a bicyclic core was designed to fix the conformation of the phenethyl ether moiety of compounds 2a and 2b. 3-[2-(3-Chloro-5-fluorobenzyl)-1-benzothiophen7-yl]-N-(2-methoxyethyl)benzamide 7m showed potent activity (pEC50 = 7.53 ± 0.08) and good pharmacokinetic properties. Compound 7m significantly suppressed methamphetamine-induced hyperactivity in mice after oral administration of 3 mg/kg without disturbance of motor function.

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INTRODUCTION Schizophrenia is a chronic and devastating mental disorder, which affects approximately 1% of the global population irrespective of ethnic, economical, or cultural boundaries.1,2 The core symptoms of schizophrenia are broadly classified as (1) positive symptoms such as delusions, hallucinations, and paranoia; (2) negative symptoms such as apathy and lack of social interaction; and (3) cognitive impairment such as memory and attention deficits.3 Current treatment for schizophrenia mainly includes atypical antipsychotic agents including serotonin−dopamine antagonists (SDA, 5-HT2A and D2 receptors antagonists, e.g., risperidone), multiacting receptor-targeted antipsychotics (MARTA, e.g., olanzapine), and dopamine system stabilizers (DSS, e.g., aripiprazole). These agents are effective against positive symptoms but show limited efficacy in the treatment of negative symptoms and cognitive impairment. Moreover, these antipsychotics are associated with side effects such as weight gain, diabetes, QT prolongation, hyperprolactinemia, and extrapyramidal syndromes.4−7 Therefore, there are still the unmet medical needs in the current therapies, and development of a new class of antipsychotics with efficacy for negative symptoms and/or cognitive impairment as well as without side effects is required. G-protein-coupled receptor 52 (GPR52) is an orphan Gscoupled G-protein coupled receptor.8 Although its natural ligand has not been identified, we have recently discovered a surrogate ligand9 and analyzed GPR52 expression pattern in human, mouse, and rat tissues and showed that GPR52 is © 2014 American Chemical Society

highly expressed in the brain. Examination of localization of GPR52 mRNA in the rat brain using in situ hybridization showed that GPR52 is highly expressed in the mesolimbic system which is responsible for positive symptoms and is colocalized with dopamine D2 receptor. In addition, GPR52 is partially colocalized with dopamine D1 receptors in the prefrontal cortex, which is involved in cognitive function. On the basis of these findings, activation of Gs-coupled GPR52 could inhibit D2 receptor signaling as well as activate D1/ NMDA function via intracellular cAMP accumulation. Therefore, we expect that GPR52 agonists could be a novel class of antipsychotics to serve the unmet medical needs. We conducted high-throughput screening (HTS) for GPR52 agonists and discovered a hit compound 1 with a moderate potency (pEC50 < 5, 33% at 10 μM). Our initial modifications of the hit compound 1 afforded N-(2-hydroxyethyl)benzamide derivatives 2a and 2b with more potent agonistic activity (pEC50 of 6.19 ± 0.01 and 6.46 ± 0.09, respectively) than compound 1.10 In an effort to improve the agonistic activity, new core scaffolds were designed using compounds 2a and 2b as lead compounds. In this paper, we examined if fixing the conformation of the phenethyl ether moiety of compounds 2a and 2b through construction of a bicyclic central core could lead to enhancement of the agonistic activity. We designed 6−5 ring benzofurans 7a and 7b as the first tool compounds by Received: February 23, 2014 Published: June 2, 2014 5226

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Figure 1. Design of fused bicyclic compounds.

cyclization between the 1-position of the pyridine ring and the α-position of the phenethyl ether moiety (Figure 1). The global minimum conformations of the designed benzofurans 7a and 7b and compound 2a were calculated using the MNDO-PM3 (MOPAC, version 7.01) method in MOE11 (Figure 2a).11,12 When the central cores of the minimum conformations of 2a, 7a, and 7b were superimposed, a large gap was observed between the location of the benzene ring in the benzyl group of 7a and 7b and that of the benzene ring in the phenethyl group of 2a. On the basis of conformation analysis, we expected that the conformational restriction of the benzene ring in the benzyl group by fixing the phenethyl ether moiety would have an effect on the GPR52 agonistic activity and designed various 6−5 ring system bicyclic compounds 7a−f. Among them, benzofuran (7a), benzothiophene (7c), and indazole (7e) derivatives, which have a heteroatom at the corresponding position of the oxygen atom of the ether moiety of compounds 2a and 2b, were designed to examine the effect of the heteroatom at the position. Benzofuran (7b), benzothiophene (7d), and indazole (7f) derivatives, in which the corresponding heteroatom was replaced by carbon and moved to the upper right of the core template (dotted circles in Figure 1), were synthesized to investigate the effect on the activity. We also designed dihydrobenzofurans 7g and 7h. Conformational analyses of 2a and dihydrobenfuran 7g using MOE indicated that the location of the benzene ring in the benzyl group of 7g was located in the vicinity of the benzene ring in the phenethyl group of compound 2a (Figure 2b). Therefore, consideration of GPR52 agonistic activity of compounds 2a, 7a, and 7g would demonstrate the appropriate location of the benzene ring in the benzyl group for the agonistic activity. Furthermore, benzofurans 7i and 7j were designed by introducing the methyl group at the 3-position (Y1 or Y2) of the benzofurans 7a and 7b to clarify the tolerability for the GPR52 agonistic activity. Especially, introduction of the methyl group at the Y1 position (3-position of the benzofuran 7i) would restrict the rotation of the benzamide group at the 4-position

Figure 2. (a) Overlay of compounds 2a (red), 7a (blue), and 7b (brown). The picture was generated using Moe 2010.10. (b) Compounds 2a (red), 7a (blue), and 7g (yellow). The picture was generated using Moe 2010.10.

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Scheme 1a

Reagents and conditions: (a) 3-(R′OCO)phenylboronic acid, Pd(PPh3)4, DME, 2 M Na2CO3 aq, 85 °C; (b) XPhB(OH)2, Pd(PPh3)4, DME, 2 M Na2CO3 aq, 85 °C; (c) 2 N NaOH, EtOH or THF−MeOH, room temperature; (d) RNH2, HOBt, WSC, DMF, room temperature. a

(RNH2) to afford the desired amide derivatives 7a−f and 7i−n. The benzofuran derivatives 6a and 6b were reduced with triethylsilane in TFA to yield the 2,3-dihydrobenzofuran derivatives 6g and 6h.13 Compounds 6g and 6h were condensed with 2-aminoethanol to afford the desired amide derivatives 7g and 7h (Scheme 2). The synthesized target compounds are listed in Table 1. Synthesis of the benzofurans 3a, 3b, 3i, and 3j described above is depicted in Scheme 3. By use of Rap−Stoermer condensation14 from (3-trifluoromethyl)phenacyl bromide 9, prepared by bromination15 of 3-trifluoromethylacetophenone 8 and commercially available 2-hydroxybenzaldehyde or acetophenone derivatives 10a, 10b, and 10i, and 10j, prepared by bromination of 1-(2-hydroxyphenyl)ethanone, 2-aroylbenzofurans 11a, 11b, 11i, and 11j were synthesized. Subsequent Wolff−Kishner reduction of the compounds 11a, 11b, 11i, and 11j afforded the 2-(3-trifluoromethyl)benzylbenzofurans 3a, 3b, 3i, and 3j. Preparation of benzothiophene derivatives 4c, 4d, and 4n is illustrated in Scheme 4. Carboxylic acids 12c, 12d, and 12n16 were reduced with borane−THF complex to give alcohols 13c,

and vary the dihedral angle between the benzofuran and the benzamide. On the other hand, introduction of the methyl group at the Y2 position (3-position of the benzofuran 7j) may suggest the presence of a hydrophobic pocket around the Y2 position. In this paper, we describe the discovery of the first GPR52 agonists. Synthesis, structure−activity relationship, and pharmacological effects of representative compounds are also discussed.

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CHEMISTRY Synthesis of the designed compounds is outlined in Schemes 1 and 2. Preparation of starting materials 3a, 3b, 3e, 3f, 3i, 3j, 4c, 4d, and 4n is described in the sections below. Suzuki coupling reaction of bromides or triflates 3a, 3b, 3e, 3f, 3i, and 3j with 3(ethoxycarbonyl)phenylboronic acid gave benzoates 5a, 5b, 5e, 5f, 5i, and 5j (method A). Suzuki coupling reaction of bromides 4c, 4d, and 4n with phenylboronic acids gave benzoates 5c, 5d, 5m, and 5n (method B). Basic hydrolysis of the benzoates 5a− f, 5i, 5j, 5m, and 5n afforded corresponding carboxylic acids 6a−f, 6i, 6j, 6m, and 6n, which were condensed with amines 5228

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Scheme 2a

a

Reagents and conditions: (a) triethylsilane, TFA, room temperature; (b) 2-aminoethanol, HOBt, WSC, DMF, room temperature.

because of the steric hindrance, while that between the 3nonsubstituted benzofuran core of 7a (blue) and the 4-phenyl ring was 61°. The difference in the dihedral angle between the 3-methylbenzofuran 7i (green) and 3-nonsubstituted benzofuran 7a (blue) significantly affected the potential to exhibit agonistic activity. Orthogonal configuration of the phenyl group at 4-position was unfavorable for binding to the GPR52 receptor. We then modified the amide moiety of the benzothiophene derivative 7d as a representative compound. GPR52 agonistic activities of the benzothiophene derivatives (7k−n) are summarized in Table 3. Conversion of the N-2-hydroxyethyl group of the lead compound (7d) into an N-2-carbamoylmethyl (7k) or an N-2-methoxyethyl group (7l) maintained potent GPR52 agonistic activity (pEC50 of 7.26 ± 0.19 and 7.37 ± 0.16, respectively). Modification of the amide chain did not show a significant effect on the activity. GPR52 might not have the interaction sites with the amide chain. 3-Chloro-5fluorobenzyl derivative (7m, pEC50 = 7.53 ± 0.08) was as potent as 3-trifluoromethyl derivative (7l, pEC50 = 7.37 ± 0.16). Introduction of fluorine at the 4-position (7n, pEC50 = 7.08 ± 0.16) also resulted in comparable potent GPR52 activity. Thus, we identified a series of the benzothiophenes (7d, 7k− n) as the first potent GPR52 agonists and the representative compound 7m with good oral pharmacokinetic profile (Cmax =108.1 ng/mL; AUC0−8h = 613.7 ng·h/mL; F (bioavailability) = 73%, mouse cassette dosing at 0.1 mg/kg, iv and 1 mg/kg, po) and good brain penetration (brain/plasma AUC ratio B/P = 0.94, in mice after oral administration of 30 mg/kg) which could be used for in vivo pharmacological evaluation. Compound 7m showed potent activity with pEC50 of 7.53 ± 0.08 and showed no cAMP production using CHO cells expressing human dopamine D 1 receptor (Figure 4). Compound 7m affects cAMP accumulation through direct interaction with GPR52. Furthermore, compound 7m did not interact with other receptor (such as D1, D2, 5-HT2, AMPA, and NMDA). Antipsychotic activity of 7m was assessed in methamphetamine (MAP) (2 mg/kg, sc) induced hyperlocomotion17 test in mice. The compound 7m (3, 10, and 30 mg/kg po) dose-dependently attenuated MAP-induced hyper-

13d, and 13n. Suzuki coupling reaction of the alcohols 13c, 13d, and 13n with 3-(ethoxycarbonyl)phenylboronic acid gave benzoates 14c, 14d, and 14n, which in turn provided the bromides 4c, 4d, and 4n upon reaction with PBr3. The 2-benzyl-2H-indazole derivatives 3e and 3f were synthesized by regioselective benzylation at the 2-position of commercially available bromo-2H-indazoles 15e and 15f under heating condition (Scheme 5).

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BIOLOGICAL RESULTS AND DISCUSSION GPR52 agonistic activities of the synthesized compounds were determined based on the cAMP production using CHO cells expressing human GPR52 (Tables 2 and 3). All of the synthesized compounds exhibited GPR52 agonistic activity. Of particular note, the benzofuran derivatives (7a, pEC50 = 7.55 ± 0.25; 7b, pEC50 = 7.46 ± 0.16), the benzothiophene derivative (7c, pEC50 = 7.42 ± 0.20; 7d, pEC50 = 7.43 ± 0.21), and the indazole derivative (7e, pEC50 = 7.41 ± 0.12) showed potent GPR52 agonistic activity. The results suggest that the benzene ring in the benzyl group of compounds 7c−e exists in the same territory as that of compound 7a (Figure 2). Compounds 7b, 7d, and 7f showed comparable activity to 7a, 7c, and 7e, respectively, indicating that the position of the heteroatom on the templates did not have a significant impact on the activity and GPR52 might not have the sites that interact with the heteroatom of these compounds. On the other hand, the dihydrobenzofuran derivatives (compounds 7g [pEC50 = 6.19 ± 0.05] and 7h [pEC50 = 6.68 ± 0.07]) showed much weaker activity than the benzofuran derivatives 7a and 7b. These results suggested that the location of the benzene ring in the benzyl group of compound 7a had a greater impact on the GPR52 agonistic activity than those of compounds 2a and 7g (Figure 2b). Substituent effect at the 3-position on the benzofurans 7a and 7b was examined by introducing a methyl group. Compound 7j (Y2 = Me) (pEC50 = 7.09 ± 0.14) was fully tolerated, but compound 7i (Y1 = Me) resulted in a decrease of activity (pEC50 < 5). Conformational analyses of simple fragments of 7a and 7i were performed in MOE (Figure 3). The dihedral angle between 3-methylbenzofuran core of 7i (green) and the 4-phenyl ring was nearly orthogonal (85°) 5229

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Journal of Medicinal Chemistry Table 1. Synthesized Compounds 7a−n

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CONCLUSIONS

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EXPERIMENTAL SECTION

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The first GPR52 agonists were successfully discovered and evaluated. Initial modification of the hit compound 1 obtained using HTS afforded the key lead compounds 2a and 2b. On the basis of a superimposition study of 2a and the designed benzofuran derivative 7a, several conformationally constrained bicyclic derivatives, 7a−j, were synthesized as a novel series of GPR52 agonists. Further modification of the benzothiophene derivatives provided the representative GPR52 agonist 7m (pEC50 = 7.53 ± 0.08) which showed good brain penetration and pharmacokinetic properties after oral administration in mice. MAP-induced hyperactivity was suppressed in mice after oral administration of 3 mg/kg compound 7m. Furthermore, compound 7m showed low risk of impaired motor function even at a high dose. The discovery of GPR52 agonists has potential to lead to a novel class of antipsychotics which could address the current unmet medical needs.

a

General. Reactions were run using the commercially available starting materials and solvents without further purification. In the following experimental, melting points were determined on a Büchi B545 and were uncorrected. Proton nuclear magnetic resonance (1H NMR) spectra were recorded on a Varian Mercury-300 (300 MHz) or a Bruker AV-300M spectrometer. Chemical shifts are given in parts per million (ppm) with tetramethylsilane as an internal standard. Abbreviations are used as follows: s = singlet, d = doublet, t = triplet, q = quartet, quin = quintet, m = multiplet, dd = doublets of doublet, dt = doublets of triplet, dq = doublets of quartet, brs = broad singlet. Coupling constants (J) are given in hertz (Hz). Liquid chromatography−mass spectrometry (LC/MS) analysis was performed on a Shimadzu LC-20AD, equipped with a L-column 2 ODS (3.0 mm × 50 mm i.d., 3 μm particle size, CERI, Japan), eluting with 5 mM AcONH4 in ultrapure water/acetonitrile = 90/10 (mobile phase A) and 5 mM AcONH4 in ultrapure water/acetonitrile = 10/90 (mobile phase B), using the following elution gradient of 5% B to 90% B over 0.9 min followed by 90% B isocratic over 1.1 min at a flow rate of 1.5 mL/min (detection at 220 or 254 nm). Mass spectra were recorded using a Shimadzu LCMS-2020 with electrospray ionization. Thin-layer chromatography (TLC) was carried out using Merck Kieselgel 60, 63−200 mesh F254 plates or Fuji Silysia Chemical Ltd. 100−200 mesh NH plates. Chromatographic purification was carried out on PurifPack (SI 60 μm or NH 60 μm, Fuji Silysia Chemical Ltd.). All of the final products undergoing biological testing were >95% pure as demonstrated by analysis carried out using analytical high-performance liquid chromatography (HPLC). The HPLC analyses were performed using a Shimadzu UFLC instrument equipped with a L-column 2 ODS (3.0 mm × 50 mm, 2 μm). Elution was with a gradient of 5−90% solvent B in solvent A (solvent A was 0.1% TFA in water, and solvent B was 0.1% TFA in acetonitrile) at a flow rate of 1.2 mL/min with UV detection at 220 nm. All commercially available solvents and reagents were used without further purification unless otherwise stated. Yields were not optimized. Method A. 3-{2-[3-(Trifluoromethyl)benzyl]-1-benzofuran-4yl}benzoic Acid (6a). A mixture of 3a (3.40 g, 9.58 mmol), [3(ethoxycarbonyl)phenyl]boronic acid (2.23 g, 11.5 mmol), and Pd(PPh3)4 (553 mg, 0.48 mmol) in 2 N Na2CO3 (30 mL)−DME (30 mL) was refluxed for 16 h under N2 atmosphere. Then the reaction mixture was concentrated under reduced pressure, diluted with water, and extracted with EtOAc. The extract was washed with brine, then dried over MgSO4 and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (EtOAc−hexane = 2:3) to give 3.30 g of 5a. To a solution of this compound was added 2 N NaOH (8 mL, 16 mmol) in EtOH (50 mL) at room temperature. The mixture was stirred for 2 h at 60 °C. After concentration, the mixture was diluted with water, acidified with 1 N HCl, and extracted with EtOAc. The extract was

Yield from esters (5c, 5d, 5m, and 5n). bRacemate.

locomotion (Figure 5). Moreover, compound 7m did not show significant cataleptogenic effects even at a dose of 100 mg/kg (Figure 6) and had a low risk of extrapyramidal side effects (EPS). These data suggest that the downstream functions by activation of GPR52 could be different from those of D2 receptor blockade. 5230

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Scheme 3a

a Reagents and conditions: (a) CuBr2, EtOAc, 80 °C; (b) 9, K2CO3, acetonitrile, 80 °C; (c) (11a, 11b, and 11j) hydrazine hydrate, ethylene glycol,130 °C, then potassium hydroxide, 160 °C; (11i) (1) hydrazine hydrate, ethylene glycol,130 °C, then potassium hydroxide, 160 °C; (2) boron tribromide, dichloromethane, room temperature, (3) Tf2O, pyridine, room temperature.

Scheme 4a

Reagents and conditions: (a) 1.2 M borane−THF complex, 60 °C; (b) 3-(ethoxycarbonyl)phenylboronic acid, Pd(PPh3)4, 2 M Na2CO3 aq, DME, 85 °C; (c) PBr3, Et2O, room temperature. a

Scheme 5a

described for 6a in 72% yield starting from compound 3b as colorless crystals. Mp: 169−170 °C (EtOAc−hexane). 1H NMR (300 MHz, CDCl3) δ: 4.19 (s, 2H), 6.47 (s, 1H), 7.29 (t, J = 7.5 Hz, 1H), 7.38− 7.62 (m, 7H), 8.07 (d, J = 7.8 Hz, 1H), 8.14 (d, J = 7.8 Hz, 1H), 8.60 (t, J = 1.5 Hz, 1H). 3-{2-[3-(Trifluoromethyl)benzyl]-2H-indazol-4-yl}benzoic Acid (6e). Compound 6e was prepared in a manner similar to that described for 6a in 73% yield starting from compound 3e. 1H NMR (300 MHz, DMSO-d6) δ: 5.80 (s, 2H), 7.23 (d, J = 6.4 Hz, 1H), 7.37 (dd, J = 8.7, 6.4 Hz, 1H), 7.54−7.72 (m, 5H), 7.79 (s, 1H), 7.95−8.04 (m, 2H), 8.24 (t, J = 1.6 Hz, 1H), 8.78 (d, J = 0.8 Hz, 1H). 3-{2-[3-(Trifluoromethyl)benzyl]-2H-indazol-7-yl}benzoic Acid (6f). Compound 6f was prepared in a manner similar to that described for 6a in 65% yield starting from compound 3f. 1H NMR (300 MHz, DMSO-d6) δ: 5.83 (s, 2H), 7.13−7.24 (m, 1H), 7.48−7.82 (m, 7H), 7.91−7.97 (m, 1H), 8.25−8.31 (m, 1H), 8.61−8.68 (m, 2H). 3-{3-Methyl-2-[3-(trifluoromethyl)benzyl]-1-benzofuran-4yl}benzoic Acid (6i). Compound 6i was prepared in a manner similar to that described for 6a in 49% yield starting from compound 3i as colorless crystals. Mp: 169−170 °C (EtOAc−hexane). 1H NMR (300 MHz, CDCl3) δ: 1.82 (s, 3H), 4.12 (s, 2H), 7.10 (dd, J = 7.2, 1.2 Hz,

a Reagents and conditions: (a) 3-(trifluoromethyl)benzyl bromide, DMF, 50 °C.

washed with water and brine and then dried over MgSO4 and concentrated under reduced pressure. The residue was crystallized from EtOAc−hexane to give 6a (2.3 g, 61%) as colorless crystals. Mp: 138−139 °C (EtOAc−hexane). 1H NMR (300 MHz, CDCl3) δ: 4.19 (s, 2H), 6.63 (s, 1H), 7.20−7.78 (m, 8H), 7.85 (dd, J = 7.8, 1.2 Hz, 1H), 8.13 (dd, J = 7.8, 1.2 Hz, 1H), 8.37 (s, 1H). 3-{2-[3-(Trifluoromethyl)benzyl]-1-benzofuran-7-yl}benzoic Acid (6b). Compound 6b was prepared in a manner similar to that 5231

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Table 2. GPR52 Agonistic Activities of Bicyclic Compoundsa

a

Data are expressed as the mean ± SEM from two independent experiments performed in duplicate.

Table 3. GPR52 Agonistic Activities of Benzothiophene Derivativesa

a

Data are expressed as the mean ± SEM from two independent experiments performed in duplicate.

Figure 4. cAMP production assay of compound 7m using CHO cells expressing human GPR52 or dopamine D1 receptor (DRD1). The data are the mean ± SD (n = 4). mmol) in Et2O (13 mL) was added dropwise PBr3 (126 μL, 1.34 mmol). The mixture was stirred at room temperature for 1.5 h, diluted with EtOAc, washed with saturated aqueous NaHCO3 and brine, dried over MgSO4, and concentrated to give crude product of 4c (448 mg). A mixture of this compound, [3-(trifluoromethyl)phenyl]boronic acid (272 mg, 1.43 mmol), and Pd(PPh3)4 (55 mg, 0.048 mmol) in 2 M Na2CO3 solution (2.4 mL)-DME (15 mL) was refluxed for 12 h under N2 atmosphere. Then the reaction mixture was concentrated under reduced pressure, diluted with water, and extracted with EtOAc. The extract was dried over MgSO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane−EtOAc = 95:5 to 80:20) to give 5c (392 mg, 70%, two steps). 1H NMR (300 MHz, CDCl3) δ: 1.40 (t, J = 7.1 Hz, 3H), 4.26 (s, 2H), 4.40 (q, J = 7.2 Hz, 2H), 7.14 (d, J = 0.8 Hz, 1H), 7.29−7.53 (m, 6H), 7.53−7.58 (m, 1H), 7.70−7.78 (m, 2H), 8.05−8.10 (m, 1H), 8.21−8.24 (m, 1H).

Figure 3. Minimized structures: fragments of benzofuran 7a (blue) and 3-methylbenzofuran 7i (green). The picture was generated using Moe 2010.10. 1H), 7.23−7.30 (m, 1H), 7.37−7.57 (m, 6H), 7.67 (dt, J = 7.5, 1.5 Hz, 1H), 8.13 (dd, J = 7.8, 1.5 Hz, 1H), 8.17 (t, J = 1.5 Hz, 1H). 3-{3-Methyl-2-[3-(trifluoromethyl)benzyl]-1-benzofuran-7yl}benzoic Acid (6j). Compound 6j was prepared in a manner similar to that described for 6a in 70% yield starting from compound 3j as colorless crystals. Mp: 200−201 °C (EtOAc−hexane). 1H NMR (300 MHz, CDCl3) δ: 2.28 (s, 3H), 4.17 (s, 2H), 7.32 (t, J = 7.8 Hz, 1H), 7.40−7.60 (m, 7H), 8.05 (dt, J = 7.5, 1.5 Hz, 1H), 8.11 (dt, J = 7.8, 1.5 Hz, 1H), 8.58 (t, J = 1.5 Hz, 1H). Method B. Ethyl 3-{2-[3-(Trifluoromethyl)benzyl]-1-benzothiophen-4-yl}benzoate (5c). To a solution of 14c (399 mg, 1.28 5232

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Journal of Medicinal Chemistry

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89−90 °C. 1H NMR (300 MHz, CDCl3) δ: 1.41 (t, J = 7.1 Hz, 3H), 1.95 (t, J = 6.0 Hz, 1H), 4.41 (q, J = 7.1 Hz, 2H), 4.94 (dd, J = 6.2, 1.0 Hz, 2H), 7.12 (dd, J = 9.6, 8.2 Hz, 1H), 7.31 (dd, J = 8.1, 4.8 Hz, 1H), 7.40−7.42 (m, 1H), 7.56 (t, J = 7.7 Hz, 1H), 7.86 (dq, J = 7.7, 1.0 Hz, 1H), 8.09 (dt, J = 7.9, 1.4 Hz, 1H), 8.31 (t, J = 1.8 Hz, 1H). 3-{2-[3-(Trifluoromethyl)benzyl]-2,3-dihydro-1-benzofuran7-yl}benzoic Acid (6h). To a mixture of 6b (1.00 g, 2.52 mmol) in TFA (10 mL) was added triethylsilane (0.8 mL, 5.0 mmol) at room temperature, and the mixture was refluxed for 3 h. The reaction mixture was concentrated at reduced pressure, diluted with water, and extracted with EtOAc. The extract was washed with saturated aqueous NaHCO3 and brine, then dried over MgSO4 and concentrated under reduced pressure. The residue was crystallized from hexane to give 6h (0.8 g, 80%). Mp: 156−157 °C (hexane). 1H NMR (300 MHz, CDCl3) δ: 2.99−3.11 (m, 2H), 3.20−3.40 (m, 2H), 4.90 (br s, 1H), 5.00−5.12 (m, 1H), 6.95 (t, J = 7.5 Hz, 1H), 7.16 (d, J = 7.2 Hz, 1H), 7.34 (d, J = 8.1 Hz, 1H), 7.38−7.54 (m, 5H), 7.95 (d, J = 7.8 Hz, 1H), 8.05 (d, J = 7.8 Hz, 1H), 8.46 (s, 1H). 3-{2-[3-(Trifluoromethyl)benzyl]-2,3-dihydro-1-benzofuran4-yl}benzoic Acid (6g). Compound 6g was prepared in a manner similar to that described for 6h in 79% yield starting from compound 6a. 1H NMR (300 MHz, CDCl3) δ: 2.98−3.16 (m, 2H), 3.19 (dd, J = 15.2, 7.5 Hz, 1H), 3.39 (dd, J = 15.3, 8.7 Hz, 1H), 5.00−5.40 (m, 2H), 6.81 (d, J = 7.8 Hz, 1H), 6.93 (d, J = 7.8 Hz, 1H), 7.21 (t, J = 8.1 Hz, 1H), 7.38−7.57 (m, 5H), 7.66 (d, J = 7.8 Hz, 1H), 8.08 (d, J = 7.5 Hz, 1H), 8.19 (s, 1H). N-(2-Hydroxyethyl)-3-{2-[3-(trifluoromethyl)benzyl]-1-benzofuran-4-yl}benzamide (7a). A mixture of 6a (200 mg, 0.50 mmol), HOBt (75 mg, 0.56 mmol), WSC (106 mg, 0.56 mmol) in DMF (3 mL) was stirred at room temperature overnight. The mixture was poured into water and extracted with EtOAc. The extract was washed with saturated aqueous NaHCO3 and water, dried over MgSO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (EtOAc−hexane = 2:8 to 10:0) to give 7a (154 mg, 70%) as colorless crystals. Mp: 135−136 °C (EtOAc−hexane). 1H NMR (300 MHz, CDCl3) δ: 2.43 (t, J = 4.8 Hz, 1H), 3.66 (q, J = 4.8 Hz, 2H), 3.86 (q, J = 4.8 Hz, 2H), 4.18 (s, 2H), 6.60 (s, 1H), 6.64 (br s, 1H), 7.26−7.32 (m, 2H), 7.38−7.60 (m, 6H), 7.68−7.81 (m, 2H), 8.01 (s, 1H). LC/MS m/z 440.3 (M + H). N-(2-Hydroxyethyl)-3-{2-[3-(trifluoromethyl)benzyl]-1-benzofuran-7-yl}benzamide (7b). Compound 7b was prepared in a manner similar to that described for 7a in 52% yield starting from compound 6b as colorless crystals. Mp: 140−141 °C (EtOAc− hexane). 1H NMR (300 MHz, CDCl3) δ: 2.54 (t, J = 5.1 Hz, 1H), 3.65 (q, J = 5.1 Hz, 2H), 3.85 (q, J = 5.1 Hz, 2H), 4.19 (s, 2H), 6.45 (s, 1H), 6.63 (br s, 1H), 7.29 (d, J = 7.8 Hz, 1H), 7.38−7.61 (m, 7H), 7.77 (dt, J = 7.8, 1.5 Hz, 1H), 7.95 (dt, J = 7.8, 1.5 Hz, 1H), 8.20 (t, J = 1.5 Hz, 1H). LC/MS m/z 440.3 (M + H). N-(2-Hydroxyethyl)-3-{2-[3-(trifluoromethyl)benzyl]-2H-indazol-4-yl}benzamide (7e). Compound 7e was prepared in a manner similar to that described for 7a in 74% yield starting from compound 6e as colorless crystals. Mp: 136−137 °C (EtOAc− hexane). 1H NMR (300 MHz, DMSO-d6) δ: 3.31−3.41 (m, 2H), 3.50−3.58 (m, 2H), 4.74 (t, J = 5.7 Hz, 1H), 5.79 (s, 2H), 7.26 (d, J = 6.2 Hz, 1H), 7.33−7.42 (m, 1H), 7.53−7.72 (m, 5H), 7.79 (s, 1H), 7.85−7.94 (m, 2H), 8.18 (t, J = 1.6 Hz, 1H), 8.57 (t, J = 5.7 Hz, 1H), 8.80 (d, J = 0.8 Hz, 1H). LC/MS m/z 440.3 (M + H). N-(2-Hydroxyethyl)-3-{2-[3-(trifluoromethyl)benzyl]-2H-indazol-7-yl}benzamide (7f). Compound 7f was prepared in a manner similar to that described for 7a in 78% yield starting from compound 6f as colorless crystals. Mp: 136−137 °C (EtOAc− hexane). 1H NMR (300 MHz, DMSO-d6) δ: 3.33−3.41 (m, 2H), 3.54 (q, J = 6.0 Hz, 2H), 4.75 (t, J = 6.0 Hz, 1H), 5.83 (s, 2H), 7.19 (dd, J = 8.3, 7.2 Hz, 1H), 7.49−7.65 (m, 4H), 7.64−7.73 (m, 1H,), 7.74−7.87 (m, 3H), 8.27 (d, J = 8.3 Hz, 1H), 8.40 (s, 1H), 8.52 (t, J = 6.0 Hz, 1H), 8.65 (s, 1H). LC/MS m/z 440.3 (M + H). N-(2-Hydroxyethyl)-3-{2-[3-(trifluoromethyl)benzyl]-2,3-dihydro-1-benzofuran-4-yl}benzamide (7g). Compound 7g was prepared in a manner similar to that described for 7a in 59% yield starting from compound 6g as colorless crystals. Mp: 120−121 °C

Figure 5. Dose-dependent inhibition of MAP-induced hyperlocomotion by compound 7m in mice. Mice were administered compound 7m (1−30 mg/kg, po) and placed in activity chambers for 60 min. After this period, MAP (2 mg/kg, sc) was injected and measurement was started. Data are expressed as the mean ± SEM (n = 6−7 per group). Data were analyzed by Williams test: (∗) P < 0.025 vs the control group treated with vehicle and MAP.

Figure 6. Low cataleptogenic potential of compound 7m in mice. Compound 7m (10−100 mg/kg, po) and haloperidol (0.1 mg/kg, ip) were evaluated for cataleptic potential in mice. All animals were tested 1 and 2 h after dosing. Data are expressed as the mean ± SEM (n = 6 per group). Data were analyzed by Williams (compound 7m) or Student’s t-test (haloperidol): (∗) P < 0.05 vs the corresponding vehicle control. Ethyl 3-{2-[3-(Trifluoromethyl)benzyl]-1-benzothiophen-7yl}benzoate (5d). A mixture of 4d (1.61 g, 4.29 mmol), [3(trifluoromethyl)phenyl]boronic acid (0.98 g, 5.15 mmol), Pd(PPh3)4 (0.15 g, 0.13 mmol), Na2CO3 (0.91 g, 8.58 mmol), water (10 mL), and DME (30 mL) was refluxed for 14 h under N2 atmosphere. Then the reaction mixture was concentrated under reduced pressure, diluted with water, and extracted with EtOAc. The extract was washed with water, dried over MgSO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane−EtOAc = 10:1) to give 5d (1.36 g, 72%) as an oil. 1H NMR (300 MHz, CDCl3) δ: 1.39 (t, J = 6.9 Hz, 3H), 4.27 (s, 2H), 4.93 (d, J = 6.9 Hz, 2H), 7.10 (s, 1H), 7.32 (d, J = 8.1 Hz, 1H), 7.40−7.60 (m, 6H), 7.68 (d, J = 7.5 Hz, 1H), 7.86 (d, J = 8.1 Hz, 1H), 8.06 (d, J = 6.3 Hz, 1H), 8.33 (s, 1H). Ethyl 3-{2-(3-Chloro-5-fluorobenzyl)-1-benzothiophen-7-yl}benzoate (5m). Compound 5m was prepared in a manner similar to that described for 5d in 79% yield starting from compound 4d. 1H NMR (300 MHz, CDCl3) δ: 1.39 (t, J = 7.1 Hz, 3H), 4.18 (s, 2H), 4.40 (q, J = 7.1 Hz, 2H), 6.85−6.91 (m, 1H), 6.93−6.99 (m, 1H), 7.06 (s, 1H), 7.13 (s, 1H), 7.31−7.35 (m, 1H), 7.44 (t, J = 7.7 Hz, 1H), 7.54 (t, J = 7.7 Hz, 1H), 7.70 (dd, J = 8.0, 1.1 Hz, 1H), 7.85−7.90 (m, 1H), 8.05−8.09 (m, 1H), 8.32−8.35 (m, 1H). Ethyl 3-{4-Fluoro-2-(hydroxymethyl)-1-benzothiophen-7yl}benzoate (5n). Compound 5n was prepared in a manner similar to that described for 5d in 80% yield starting from compound 4n. Mp: 5233

dx.doi.org/10.1021/jm5002919 | J. Med. Chem. 2014, 57, 5226−5237

Journal of Medicinal Chemistry

Article

(EtOAc−hexane). 1H NMR (300 MHz, CDCl3) δ: 2.42 (br s, 1H), 2.94−3.06 (m, 2H), 3.17 (dd, J = 14.7, 7.5 Hz, 1H), 3.34 (dd, J = 15.3, 8.4 Hz, 1H), 3.65 (q, J = 5.1 Hz, 2H), 3.85 (br s, 2H), 4.98−5.05 (m, 1H), 6.62 (br s, 1H), 6.80 (d, J = 8.1 Hz, 1H), 6.90 (d, J = 7.5 Hz, 1H), 7.20 (t, J = 8.1 Hz, 1H), 7.38−7.57 (m, 6H), 7.72 (d, J = 7.2 Hz, 1H), 7.85 (s, 1H). LC/MS m/z 442.3 (M + H). N-(2-Hydroxyethyl)-3-{2-[3-(trifluoromethyl)benzyl]-2,3-dihydro-1-benzofuran-7-yl}benzamide (7h). Compound 7h was prepared in a manner similar to that described for 7a in 55% yield starting from compound 6h as colorless crystals. Mp: 161−162 °C (EtOAc−hexane). 1H NMR (300 MHz, CDCl3) δ: 2.60 (br s, 1H), 2.97−3.08 (m, 2H), 3.18−3.38 (m, 2H), 3.63 (q, J = 5.1 Hz, 2H), 3.83 (q, J = 5.1 Hz, 2H), 5.00−5.13 (m, 1H), 6.60 (br s, 1H), 6.93 (t, J = 7.5 Hz, 1H), 7.14 (d, J = 7.5 Hz, 1H), 7.32 (d, J = 7.8 Hz,,1H), 7.28− 7.52 (m, 5H), 7.70 (d, J = 7.5 Hz, 1H), 7.85 (t, J = 6.9 Hz, 1H), 8.08 (s, 1H). LC/MS m/z 442.4 (M + H). N-(2-Hydroxyethyl)-3-{3-methyl-2-[3-(trifluoromethyl)benzyl]-1-benzofuran-4-yl}benzamide (7i). Compound 7i was prepared in a manner similar to that described for 7a in 54% yield starting from compound 6i as colorless crystals. Mp: 139−141 °C (EtOAc−hexane). 1H NMR (300 MHz, CDCl3) δ: 1.79 (s, 3H), 2.40 (br s, 1H), 3.65 (q, J = 5.4 Hz, 2H), 3.85 (t, J = 5.4 Hz, 2H), 4.11 (s, 2H), 6.63 (br s, 1H), 7.08 (d, J = 7.5 Hz, 1H), 7.21−7.28 (m, 1H), 7.37−7.60 (m, 7H), 7.79−7.83 (m, 2H). LC/MS m/z 454.4 (M + H). N-(2-Hydroxyethyl)-3-{3-methyl-2-[3-(trifluoromethyl)benzyl]-1-benzofuran-7-yl}benzamide (7j). Compound 7j was prepared in a manner similar to that described for 7a in 62% yield starting from compound 6j as colorless crystals. Mp: 158−159 °C (EtOAc−hexane). 1H NMR (300 MHz, CDCl3) δ: 2.26 (s, 3H), 2.57 (br s, 1H), 3.63 (q, J = 5.7 Hz, 2H), 3.83 (q, J = 5.7 Hz, 2H), 4.17 (s, 2H), 6.61 (br s, 1H), 7.30 (t, J = 7.8 Hz, 1H), 7.40−7.56 (m, 7H), 7.75 (d, J = 7.8 Hz, 1H), 7.93 (d, J = 7.8 Hz, 1H), 8.18 (t, J = 1.5 Hz, 1H). LC/MS m/z 454.3 (M + H). N-(2-Hydroxyethyl)-3-{2-[3-(trifluoromethyl)benzyl]-1-benzothiophen-7-yl}benzamide (7d). To a solution of ethyl 3-{2-[3(trifluoromethyl)benzyl]-1-benzothiophen-7-yl}benzoate (5d) (0.25 g, 0.57 mmol) in MeOH (5 mL)−THF (2 mL) was added 1 N NaOH (1.70 mL, 1.70 mmol) in EtOH (50 mL). The mixture was stirred for 30 min at 60 °C. After concentration, the mixture was diluted with water, acidified with 1 N HCl, and extracted with EtOAc. The extract was washed with water and brine and then dried over MgSO4 and concentrated under reduced pressure. The residue was dissolved in DMF (5 mL). To the solution were added 2-aminoethanol (0.041 mL, 0.68 mmol), WSC (0.15 g, 0.85 mmol), and HOBt (0.12 g, 0.85 mmol). The mixture was stirred for 15 h at room temperature. The reaction solution was diluted with water and extracted with EtOAc. The extract was washed with water, dried over MgSO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane−EtOAc = 1:1) to give 7d (0.25 g, 97%) as an oil. 1H NMR (300 MHz, CDCl3) δ: 2.24 (t, J = 4.8 Hz, 1H), 3.65 (q, J = 5.1 Hz, 1H), 3.85 (q, J = 5.1 Hz, 1H), 4.27 (s, 2H), 6.62 (br s, 1H), 7.11 (s, 1H), 7.31 (d, J = 7.5 Hz, 1H), 7.40−7.60 (m, 6H), 7.69 (d, J = 7.5 Hz, 1H), 7.78−7.85 (m, 2H), 8.05 (s, 1H). LC/MS m/z 456.3 (M + H). N-(2-Hydroxyethyl)-3-{2-[3-(trifluoromethyl)benzyl]-1-benzothiophen-4-yl}benzamide (7c). Compound 7c was prepared in a manner similar to that described for 7d in 85% yield starting from compound 5c as an oil. 1H NMR (300 MHz, CDCl3) δ: 2.41 (t, J = 4.9 Hz, 1H), 3.61−3.72 (m, 2H), 3.81−3.91 (m, 2H), 4.27 (s, 2H), 6.62 (br s, 1H), 7.13 (d, J = 0.8 Hz, 1H), 7.28−7.59 (m, 7H), 7.66− 7.71 (m, 1H), 7.73−7.78 (m, 1H), 7.79−7.84 (m, 1H), 7.93−7.96 (m, 1H). LC/MS m/z 456.3 (M + H). N-(2-Amino-2-oxoethyl)-3-{2-[3-(trifluoromethyl)benzyl]-1benzothiophen-7-yl}benzamide (7k). Compound 7k was prepared in a manner similar to that described for 7d in 94% yield starting from compound 5d as a solid. Mp: 147−148 °C (EtOAc−hexane). 1H NMR (300 MHz, CDCl3) δ: 4.18 (d, J = 4.8 Hz, 2H), 4.27 (s, 1H), 5.45 (br s, 1H), 6.00 (br s, 1H), 6.90−7.00 (m, 1H), 7.10 (s, 1H), 7.31 (d, J = 7.2 Hz, 1H), 7.40−7.60 (m, 6H), 7.68 (d, J = 8.1 Hz, 1H), 7.80−7.90 (m, 2H), 8.10 (m, 1H). LC/MS m/z 469.3 (M + H).

N-(2-Methoxyethyl)-3-{2-[3-(trifluoromethyl)benzyl]-1-benzothiophen-7-yl}benzamide (7l). Compound 7l was prepared in a manner similar to that described for 7d in 90% yield starting from compound 5d as colorless crystals. Mp: 113−114 °C (EtOAc− hexane). 1H NMR (300 MHz, CDCl3) δ: 3.37 (s, 3H), 3.56 (d, J = 5.0 Hz, 2H), 3.67 (q, J = 5.0 Hz, 2H), 4.27 (s, 2H), 6.53 (br s, 1H), 7.10 (s, 1H), 7.32 (d, J = 7.5 Hz, 1H), 7.40−7.60 (m, 6H), 7.69 (d, J = 7.8 Hz, 1H), 7.75−7.85 (m, 2H), 8.04 (s, 1H). LC/MS m/z 470.3 (M + H). 3-[2-(3-Chloro-5-fluorobenzyl)-1-benzothiophen-7-yl]-N-(2methoxyethyl)benzamide (7m). Compound 7m was prepared in a manner similar to that described for 7d in 63% yield starting from compound 5m as colorless crystals. Mp: 94−95 °C (hexane−Et2O). 1 H NMR (300 MHz, CDCl3) δ: 3.37 (s, 3H), 3.53−3.60 (m, 2H), 3.63−3.71 (m, 2H), 4.17 (s, 2H), 6.53 (br s, 1H), 6.85−6.91 (m, 1H), 6.93−6.99 (m, 1H), 7.04−7.07 (m, 1H), 7.12−7.14 (m, 1H), 7.31− 7.35 (m, 1H), 7.39−7.47 (m, 1H), 7.54 (t, J = 7.4 Hz, 1H), 7.70 (dd, J = 7.7, 1.1 Hz, 1H), 7.78−7.85 (m, 2H), 8.03−8.07 (m, 1H). LC/MS m/z 454.2 (M + H). N-(2-Amino-2-oxoethyl)-3-{4-fluoro-2-[3-(trifluoromethyl)benzyl]-1-benzothiophen-7-yl}benzamide (7n). Compound 7n was prepared in a manner similar to that described for 7d in 37% yield starting from compound 5n as a solid. Mp: 157−158 °C (EtOAc− hexane). 1H NMR (300 MHz, CDCl3) δ: 4.20 (d, J = 4.9 Hz, 2H), 4.27 (s, 2H), 5.53 (br s, 1H), 6.20 (br s, 1H), 7.03−7.14 (m, 2H), 7.22 (s, 1H), 7.23−7.29 (m, 1H), 7.39−7.58 (m, 5H), 7.75−7.86 (m, 2H), 8.04−8.09 (m, 1H). LC/MS m/z 487.3 (M + H). 2-Bromo-1-[3-(trifluoromethyl)phenyl]ethanone (9). A mixture of 1-[3-(trifluoromethyl)phenyl]ethanone (8) and CuBr2 in AcOEt (50 mL) was refluxed for 16 h. The solid was filtered off. The filtrate was washed with saturated aqueous NaHCO3, dried, and concentrated. The residue was used without further purification. 1-(3-Bromo-2-hydroxyphenyl)ethanone (10j). To a mixture of 1-(2-hydroxyphenyl)ethanone (4.00 g, 29.5 mmol) and diisopropylamine (0.42 mL, 2.95 mmol) in CS2 (50 mL) was added NBS (5.25 g, 29.5 mmol) at 0 °C. The mixture was stirred for 1 h at room temperature, taken up in water, and extracted with EtOAc. The extract was washed with saturated aqueous NaHCO3 and water, then dried over MgSO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (EtOAc−hexane = 0:10 to 2:8) to give 10j (1.60g, 25%). 1H NMR (300 MHz, CDCl3) δ: 2.66 (s, 3H), 6.82 (t, J = 7.8 Hz, 1H), 7.69−7.75 (m, 2H), 12.9 (s, 1H). (4-Bromo-1-benzofuran-2-yl)[3-(trifluoromethyl)phenyl]methanone (11a). To a solution of 2-bromo-6-hydroxybenzaldehyde (10a) (4.0 g, 19.9 mmol) in acetonitrile (50 mL) were added 9 (6.38 g, 23.9 mmol) and K2CO3 (3.30 g, 23.9 mmol) at room temperature. The mixture was refluxed for 16 h and taken up in water and extracted with EtOAc. The combined organic extracts were washed with water and brine, dried over MgSO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane−EtOAc = 90:10 to 40:60) to give 11a (4.4 g, 60%). Mp: 91−92 °C (MeOH). 1H NMR (300 MHz, CDCl3) δ: 7.39 (t, J = 8.1 Hz, 1H), 7.49−7.62 (m, 3H), 7.71 (t, J = 7.5 Hz, 1H), 7.91 (d, J = 7.8, 1.2 Hz, 1H), 8.25 (d, J = 7.8 Hz, 1H), 8.32 (s, 1H). (7-Bromo-1-benzofuran-2-yl)[3-(trifluoromethyl)phenyl]methanone (11b). Compound 11b was prepared in a manner similar to that described for 11a in 69% yield starting from compound 10b as colorless crystals. Mp: 75−78 °C (MeOH). 1H NMR (300 MHz, CDCl3) δ: 7.24 (t, J = 7.5 Hz, 1H), 7.66−7.74 (m, 4H), 7.90 (d, J = 7.8 Hz, 1H), 8.36 (d, J = 7.8 Hz, 1H), 8.50 (s, 1H). (4-Methoxy-3-methyl-1-benzofuran-2-yl)[3(trifluoromethyl)phenyl]methanone (11i). Compound 11i was prepared in a manner similar to that described for 11a in 69% yield starting from compound 10i as colorless crystals. Mp: 91−92 °C (MeOH). 1H NMR (300 MHz, CDCl3) δ: 2.82 (s, 3H), 3.96 (s, 3H), 6.66 (d, J = 8.1 Hz, 1H), 7.09 (d, J = 8.4 Hz, 1H), 7.39 (t, J = 8.1 Hz, 1H), 7.64 (t, J = 8.1 Hz, 1H), 7.82 (d, J = 8.1 Hz, 1H), 8.24 (dd, J = 7.8 Hz, 1H), 8.31 (s, 1H). 5234

dx.doi.org/10.1021/jm5002919 | J. Med. Chem. 2014, 57, 5226−5237

Journal of Medicinal Chemistry

Article

(7-Bromo-3-methyl-1-benzofuran-2-yl)[3-(trifluoromethyl)phenyl]methanone (11j). Compound 11j was prepared in a manner similar to that described for 11a in 88% yield starting from compound 10j as colorless crystals. Mp: 97−98 °C (MeOH). 1H NMR (300 MHz, CDCl3) δ: 2.70 (s, 3H), 7.20−7.27 (m, 1H), 7.63−7.71 (m, 3H), 7.87 (d, J = 7.8 Hz, 1H), 8.39 (d, J = 7.8 Hz, 1H), 8.57 (s, 1H). 4-Bromo-2-[3-(trifluoromethyl)benzyl]-1-benzofuran (3a). A mixture of 11a (4.4 g, 11.9 mmol) and hydrazine monohydrate (2.38 g, 47.6 mmol) in ethylene glycol (50 mL) was heated to 130 °C for 2 h. After the mixture was cooled to room temperature, potassium hydroxide (2.00 g, 35.7 mmol) was added to the mixture. The mixture was heated to 160 °C for 2 h. The reaction solution was taken up in water and extracted with EtOAc. The extract was washed with water and brine, then dried over MgSO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane−EtOAc = 95:5 to 30:70) to give 3a (3.4 g, 80%) as an oil. 1H NMR (300 MHz, CDCl3) δ: 4.16 (s, 2H), 6.46 (s, 1H), 7.09 (t, J = 8.1 Hz, 1H), 7.31−7.36 (m, 2H), 7.40−7.61 (m, 4H). 7-Bromo-2-[3-(trifluoromethyl)benzyl]-1-benzofuran (3b). Compound 3b was prepared in a manner similar to that described for 3a in 74% yield starting from compound 11b as an oil. 1H NMR (300 MHz, CDCl3) δ: 4.20 (s, 2H), 6.40 (s, 1H), 7.06 (t, J = 7.5 Hz, 1H), 7.35−7.59 (m, 6H). 7-Bromo-3-methyl-2-[3-(trifluoromethyl)benzyl]-1-benzofuran (3j). Compound 3j was prepared in a manner similar to that described for 3a in 75% yield starting from compound 10j as an oil. 1H NMR (300 MHz, CDCl3) δ: 2.20 (s, 3H), 4.18 (s, 2H), 7.08 (t, J = 7.5 Hz, 1H), 7.34−7.53 (m, 6H). 4-Methoxy-3-methyl-2-[3-(trifluoromethyl)benzyl]-1-benzofuran. This compound was prepared in a manner similar to that described for 3a in 74% yield starting from compound 11i as an oil. 1H NMR (300 MHz, CDCl3) δ: 2.37 (s, 3H), 3.89 (s, 3H), 4.08 (s, 2H), 6.59 (d, J = 8.1 Hz, 1H), 6.97 (d, J = 7.8 Hz, 1H), 7.08 (t, J = 7.5 Hz, 1H), 7.35−7.50 (m, 4H). 3-Methyl-2-[3-(trifluoromethyl)benzyl]-1-benzofuran-4-ol. To a solution of 4-methoxy-3-methyl-2-[3-(trifluoromethyl)benzyl]-1benzofuran (1.51 g, 4.68 mmol) in dichloromethane (30 mL) was added dropwise boron tribromide (1.0 M dichloromethane solution, 5.0 mL, 5.0 mmol) at 0 °C in Ar atmosphere. The mixture was stirred at ambient temperature for 3 h. The reaction mixture was quenched with saturated aqueous NaHCO3. The organic layer was separated, and the aqueous layer was extracted with dichloromethane. The combined organic layer was washed with brine, dried over MgSO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane−EtOAc = 90:10 to 20:80) to give the titled compound (1.38 g, 96%). Mp: 91−92 °C (EtOAc− hexane). 1H NMR (300 MHz, CDCl3) δ: 2.41 (s, 3H), 4.09 (s, 2H), 5.05 (s, 1H), 6.50 (d, J = 8.4 Hz, 1H), 6.92−7.05 (m, 2H), 7.30−7.51 (m, 4H). 3-Methyl-2-[3-(trifluoromethyl)benzyl]-1-benzofuran-4-yl Trifluoromethanesulfonate (3i). To a solution of 3-methyl-2-[3(trifluoromethyl)benzyl]-1-benzofuran-4-ol (1.38 g, 4.50 mmol) in pyridine (15 mL) was added dropwise trifluoromethanesulfonic anhydride (0.83 mL, 4.95 mmol) at 0 °C, and the mixture was stirred for 4 h at room temperature. The reaction mixture was quenched with water, and crude product was extracted with EtOAc. The organic layer was washed with 1 N HCl and saturated aqueous NaHCO3, then dried over MgSO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane−EtOAc = 90:10 to 50:50) to give 3i (1.40 g, 71%) as an oil. 1H NMR (300 MHz, CDCl3) δ: 2.40 (s, 3H), 4.13 (s, 2H), 7.13 (d, J = 7.2 Hz, 1H), 7.23 (t, J = 8.1 Hz, 1H), 7.30−7.51 (m, 5H). (7-Bromo-1-benzothiophen-2-yl)methanol (13d). To a 1.2 M borane−THF solution (6.86 mL, 8.10 mmol) was added 7-bromo-1benzothiophene-2-carboxylic acid (12d) (0.52 g, 2.02 mmol) at 0 °C, and the mixture was then stirred for 3 h at 60 °C. The reaction mixture was quenched by the slow addition of 1 N HCl and extracted with EtOAc. The extract was washed with water, dried over MgSO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane−EtOAc = 4:1) and

crystallized from hexane−EtOAc to give 13d (0.41 g, 83%). Mp: 82−83 °C. 1H NMR (300 MHz, CDCl3) δ: 1.94 (t, J = 6.0 Hz, 1H), 4.94 (d, J = 6.0 Hz, 2H), 7.21 (d, J = 7.8 Hz, 1H), 7.31 (s, 1H), 7.47 (d, J = 7.8 Hz, 1H), 7.67 (d, J = 7.8 Hz, 1H). (4-Bromo-1-benzothiophen-2-yl)methanol (13c). Compound 13c was prepared in a manner similar to that described for 13d in 68% yield starting from compound 12c. 1H NMR (300 MHz, CDCl3) δ: 1.91−2.00 (m, 1H), 4.95 (dd, J = 6.0, 0.8 Hz, 2H), 7.13−7.19 (m, 1H), 7.36−7.38 (m, 1H), 7.50 (dd, J = 7.4, 0.8 Hz, 1H), 7.74 (d, J = 8.0 Hz, 1H). (7-Bromo-4-fluoro-1-benzothiophen-2-yl)methanol (13n). Compound 13n was prepared in a manner similar to that described for 13d in 60% yield starting from compound 12n. Mp: 80−81 °C. 1H NMR (300 MHz, DMSO-d6) δ: 2.01 (t, J = 6.0 Hz, 1H), 4.95 (dd, J = 6.0, 0.9 Hz, 2H), 6.92 (dd, J = 9.6, 8.1 Hz, 1H), 7.34−7.42 (m, 2H). Ethyl 3-[2-(Hydroxymethyl)-1-benzothiophen-7-yl]benzoate (14d). A mixture of 13d (1.8 g, 7.40 mmol), [3-(ethoxycarbonyl)phenyl]boronic acid (1.72 g, 8.88 mmol), Pd(PPh3)4 (0.26 g, 0.22 mmol), Na2CO3 (1.57 g, 14.8 mmol), water (10 mL), and DME (30 mL) was refluxed for 16 h under N2 atmosphere. Then the reaction mixture was concentrated under reduced pressure, diluted with water, and extracted with EtOAc. The extract was washed with water, dried over MgSO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane−EtOAc = 3:1) to give 14d (2.20 g, 95%) as an oil. 1H NMR (300 MHz, CDCl3) δ: 1.41 (t, J = 7.2 Hz, 3H), 1.93 (t, J = 6.0 Hz, 1H), 4.41 (q, J = 7.2 Hz, 2H), 4.93 (d, J = 6.0 Hz, 2H), 7.30 (s, 1H), 7.36 (d, J = 6.9 Hz, 1H), 7.45 (t, J = 7.5 Hz, 1H), 7.56 (t, J = 7.8 Hz, 1H), 7.74 (d, J = 7.8 Hz, 1H), 7.91 (d, J = 7.8 Hz, 1H), 8.08 (d, J = 7.8 Hz, 1H), 8.36 (s, 1H). Ethyl 3-[2-(Hydroxymethyl)-1-benzothiophen-4-yl]benzoate (14c). Compound 14c was prepared in a manner similar to that described for 14d in 91% yield starting from compound 13c. 1H NMR (300 MHz, CDCl3) δ: 1.39 (t, J = 7.1 Hz, 3H), 2.13 (br s, 1H), 4.39 (q, J = 7.1 Hz, 2H), 4.89 (br s, 2H), 7.25 (d, J = 3.3 Hz, 1H), 7.30− 7.43 (m, 2H), 7.53 (t, J = 7.7 Hz, 1H), 7.68−7.75 (m, 1H), 7.82 (d, J = 7.7 Hz, 1H), 8.04−8.09 (m, 1H), 8.21 (t, J = 1.6 Hz, 1H). Ethyl 3-[4-Fluoro-2-(hydroxymethyl)-1-benzothiophen-7yl]benzoate (14n). Compound 14n was prepared in a manner similar to that described for 14d in 80% yield starting from compound 13n. Mp: 89−90 °C. 1H NMR (300 MHz, CDCl3) δ: 1.41 (t, J = 7.1 Hz, 3H), 1.95 (t, J = 6.0 Hz, 1H), 4.41 (q, J = 7.1 Hz, 2H), 4.94 (dd, J = 6.2, 1.0 Hz, 2H), 7.12 (dd, J = 9.6, 8.2 Hz, 1H), 7.31 (dd, J = 8.1, 4.8 Hz, 1H), 7.40−7.42 (m, 1H), 7.56 (t, J = 7.7 Hz, 1H), 7.86 (dq, J = 7.7, 1.0 Hz, 1H), 8.09 (dt, J = 7.9, 1.4 Hz, 1H), 8.31 (t, J = 1.8 Hz, 1H). Ethyl 3-[2-(Bromomethyl)-1-benzothiophen-7-yl]benzoate (4d). To a solution of 14d (2.2 g, 7.04 mmol) in Et2O (20 mL) was added dropwise PBr3 (0.7 mL, 7.39 mmol). The mixture was stirred at room temperature for 2 h, diluted with EtOAc, washed with saturated aqueous NaHCO3 and brine, dried over MgSO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane−EtOAc = 10:1) to give 4d (1.91 g, 72%) as colorless crystals. Mp: 96−97 °C. 1H NMR (300 MHz, CDCl3) δ: 1.41 (t, J = 7.2 Hz, 3H), 4.41 (q, J = 7.2 Hz, 2H), 4.77 (s, 2H), 7.38−7.45 (m, 2H), 7.46 (t, J = 7.5 Hz, 1H), 7.54 (t, J = 7.8 Hz, 1H), 7.73 (d, J = 7.5 Hz, 1H), 7.90 (d, J = 7.8 Hz, 1H), 8.10 (d, J = 7.5 Hz, 1H), 8.35 (s, 1H). Ethyl 3-[2-(Bromomethyl)-4-fluoro-1-benzothiophen-7-yl]benzoate (4n). Compound 4n was prepared in a manner similar to that described for 4d in 42% yield starting from compound 14n. Mp: 139−140 °C. 1H NMR (300 MHz, CDCl3) δ: 1.41 (t, J = 7.1 Hz, 3H), 4.41 (q, J = 7.1 Hz, 2H), 4.75 (s, 2H), 7.12 (dd, J = 9.3, 8.1 Hz, 1H), 7.33 (dd, J = 8.1, 4.8 Hz, 1H), 7.50 (s, 1H), 7.56 (t, J = 7.8 Hz, 1H), 7.84 (d, J = 7.5 Hz, 1H), 8.09 (d, J = 7.8 Hz, 1H), 8.30 (d, J = 1.8 Hz, 1H). 7-Bromo-2-[3-(trifluoromethyl)benzyl]-2H-indazole (3e). A mixture of 7-bromo-1H-indazole (15e) (1.00 g, 5.08 mmol) and 1(bromomethyl)-3-(trifluoromethyl)benzene (1.16 mL, 7.61 mmol) in DMF (2.0 mL) was heated at 50 °C for 20 h. The reaction solution was poured into water and extracted with EtOAc. The extract was 5235

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Journal of Medicinal Chemistry washed with water, dried over MgSO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (EtOAc−hexane = 1:9) to give 3e (983 mg, 58%) as an oil. 1H NMR (300 MHz, CDCl3) δ: 5.72 (s, 2H), 6.91−7.00 (m, 1H), 7.42−7.56 (m, 3H), 7.56−7.65 (m, 3H), 7.96 (s, 1H). 4-Bromo-2-[3-(trifluoromethyl)benzyl]-2H-indazole (3f). Compound 3f was prepared in a manner similar to that described for 3e in 55% yield starting from compound 15f. 1H NMR (300 MHz, CDCl3) δ: 5.64 (s, 2H), 7.12−7.19 (m, 1H), 7.22−7.28 (m, 1H), 7.42−7.54 (m, 2H), 7.57−7.70 (m, 3H), 7.97 (s, 1H). Evaluation of GPR52 Agonistic Activities. CHO cells expressing human GPR52 (10 000 cells) were incubated with test compounds for 30 min at 37 °C in 30 μL/well assay buffer (Hanks’ balanced salt solution (HBSS) containing 5 mM HEPES (pH7.6), 0.5% BSA, 100 μM IBMX, and 100 μM Ro20-1724). Then 10 μL/well of 0.1 U/μL anti cAMP acceptor beads (PerkinElmer) and 10 μL/well of 0.1 U/μL biotinylated cAMP donor beads (PerkinElmer) were added and incubated overnight at room temperature. cAMP concentrations were determined by measuring AlphaScreen signal with Envision (PerkinElmer). pEC50 values were calculated by using nonlinear regression analysis with XLfit (IDBS). pEC50 is the concentration of the compounds that produces a response that is 50% of its maximal effect, expressed as negative logarithm. Emax (%) is the efficacy maximum of the compounds in the cAMP assay relative to 1 μM 7a as 100% and DMSO as 0%. MAP-Induced Hyperlocomotion Test. All animal protocols were approved by the Experimental Animal Care and Use Committee of Takeda Pharmaceutical Company Ltd. Male ICR mice (7−8 weeks) were used for all experiments. The locomotor activity in mice was measured using a locomotor activity monitor, MDC-LT (Brain Science Idea Co., Ltd.). Mice were individually placed in transparent polycarbonate cages (30 cm × 40 cm × 20 cm). After habituation for 60 min, compound 7m (1−30 mg/kg, po) was administered. MAP (2 mg/kg, sc) was injected 60 min after the drug treatment. Activity counts were monitored for 150 min after administration of 7m. Catalepsy Test. Male ICR mice (6−7 weeks old) were used. All animals were tested 1 and 2 h after administration of compound 7m (10−100 mg/kg, po) or haloperidol (0.1 mg/kg, ip). The mice were lifted by the tail and placed with their front paws on a steel bar and the hind legs on the plane surface. The time spent for the mice to show a cataleptic posture, which was defined as an immobile posture while keeping both forelimbs on the bar, was measured with a maximum limit of 30 s. The procedure was repeated twice more, and the cataleptic response time was averaged for each mouse.

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ABBREVIATIONS USED

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REFERENCES

cAMP, 3′,5′-cyclic adenosine monophosphate; EC50, half maximal effective concentration; 5-HT, 5-hydroxytryptamine (serotonin); mRNA, messenger ribonucleic acid; NMDA, Nmethyl-D-aspartate; MNDO, modified neglect of diatomic overlap; PM3, parametrized model number 3; TFA, trifluoroacetic acid; Pd(PPh3)4, tetrakis(triphenylphosphine)palladium(0); DME, 1,2-dimethoxyethane; Na2CO3, sodium carbonate; NaOH, sodium hydroxide; EtOH, ethyl alcohol; THF, tetrahydrofuran; MeOH, methyl alcohol; WSC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; HOBt, 1hydroxybenzotriazole; DMF, N,N-dimethylformamide; CuBr2, copper(II) bromide; EtOAc, ethyl acetate; K2CO3, potassium carbonate; Tf2O, trifluoromethanesulfonic anhydride; PBr3, phosphorus tribromide; CHO, Chinese hamster ovary; SEM, standard error of the mean; AMPA, 2-amino-3-(3-hydroxy-5methyl-4-isoxazolyl)propionic acid; AUC, area under the curve; AcONH4, ammonium acetate; mp, melting point; NaHCO3, sodium hydrogen carbonate; MgSO4, magnesium sulfate; HCl, hydrochloric acid; HEPES, 2-[4-(2-hydroxyethyl)-1piperazinyl]ethanesulfonic acid; BSA, bovine serum albumin; IBMX, 3-isobutyl-1-methylxanthine

(1) First, M. B.; Tasman, A. DSM-IV-TR Mental Disorders: Diagnosis, Etology and Treatment; Wiley: Chichester, U.K., 2004 pp 639−701. (2) Saha, S.; Chant, D.; Welham, J.; McGrath, J. A systematic review of the prevalence of schizophrenia. PLoS Med. 2005, 2, e141. (3) Thaker, G. K.; Carpenter, W. T., Jr. Advances in schizophrenia. Nat. Med. 2001, 7, 667−671. (4) Leucht, S.; Wahlbeck, K.; Hamann, J.; Kissling, W. New generation antipsychotics versus low-potency conventional antipsychotics: a systematic review and meta-analysis. Lancet 2003, 361, 1581−1589. (5) Newcomer, J. W. Second-generation (atypical) antipsychotics and metabolic effects: a comprehensive literature review. CNS Drugs 2005, 19 (Suppl. 1), 1−93. (6) Casey, D. E. Metabolic issues and cardiovascular disease in patients with psychiatric disorders. Am. J. Med. 2005, 118 (Suppl. 2), 15S−22S. (7) Taylor, D. M. Antipsychotics and QT prolongation. Acta Psychiatr. Scand. 2003, 107, 85−95. (8) Sawzdargo, M.; Nguyen, T.; Lee, D. K.; Lynch, K. R.; Cheng, R.; Heng, H. H. Q.; George, S. R.; O’Dowd, B. F. Identification and cloning of three novel human G protein-coupled receptor genes GPR52, ΨGPR53 and GPR55: GPR55 is extensively expressed in human brain. Mol. Brain Res. 1999, 64, 193−198. (9) Komatsu, H.; Maruyama, M.; Yao, S.; Shinohara, T.; Sakuma, K.; Imaichi, S.; Chikatsu, T.; Kuniyeda, K.; Foo, K. S.; Lam, S. P.; Zhuo, K.; Lay, S. M.; Tan, M.; Matsumoto, Y.; Hashimoto, T.; Miyajima, N.; Ito, Y.; Ogi, K.; Habata, Y.; Mori, M. Anatomical transcriptome of G protein-coupled receptors leads to the identification of a novel therapeutic candidate GPR52 for psychiatric disorders. PLoS One 2014, 9, e90134. (10) Setoh, M.; Kobayashi, T.; Tanaka, T.; Baba, A. Compound having 6-membered aromatic ring. WO 2009/107391, 2009. (11) Molecular Operating Environment, version 2010.10; Chemical Computing Group, Inc.: Montreal, Quebec, Canada, 2010. (12) Stewart, J. J. P. Optimization of parameters for semiempirical methods II. Applications. J. Comput. Chem. 1989, 10, 209−220. (13) Lau, C. K.; Belanger, P. C.; Dufresne, C.; Scheigetz, J.; Thtrien, M.; Fitzsimmons, B.; Young, R. N.; Ford-Hutchinson, A. W.; Riendeau, D.; Denis, D.; Guay, J.; Charleson, S.; Piechuta, H.; McFarlane, C. S.; Chiu, S-H. L.; Eline, D.; Alvaro, R. F.; Miwa, G.; Walsh, J. L. Development of 2,3-dihydro-6-(3-phenoxypropyl)-2-(2-

ASSOCIATED CONTENT

S Supporting Information *

Elemental analysis results of the synthesis of compounds 7a, 7b, and 7e−n. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +81-466-32-1122. Fax: +81-466-29-4454. E-mail: [email protected]. Present Address §

T.H.: Shiga University of Medical Science, Seta Tsukinowacho, Otsu, Shiga 520-2192, Japan. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Takanobu Kuroita and Dr. Kazuyoshi Aso for helpful discussions during the preparation of the manuscript, and Yuji Shimizu for generation of data in the GPR52 agonistic activities. 5236

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phenylethyl)-5-benzofuranol (L-670,630) as a potent and orally active inhibitor of 5-lipoxygenase. J. Med. Chem. 1992, 35, 1299−1318. (14) Stille, J. R.; Ward, J. A.; Leffelman, C.; Sullivan, K. A. 5Formylsalicylaldehyde as a linker for the synthesis of benzofuran containing insulin sensitivity enhancer compounds. Tetrahedron Lett. 1996, 37, 9267−9270. (15) King, L. C.; Ostrum, K. O. Selective bromination with copper(II) bromide. J. Org. Chem. 1964, 29, 3459−3461. (16) Agejas-Chicharro, J.; Bueno Melendo, A. B.; Camp, N. P.; Gilmore, J.; Jimenez-Aguado, A. M.; Lamas-Peteira, C.; MarcosLlorente, A.; Mazanetz, M. P.; Montero Salgado, C.; Timms, G. H.; Williams, A. C. Pharmaceutical compounds. US 2004/122001, 2004. (17) Suzuki, Y.; Funakoshi, T.; Chaki, S.; Kawashima, N.; Ogawa, S.; Kumagai, T.; Nakazato, A.; Komurasaki, T.; Okuyama, S. In vitro and in vivo pharmacological profile of 4-(4-fluorobenzylidene)-1-[2-[5-(4fluorophenyl)-1H-pyrazol-4-yl] ethyl] piperidine (NRA0161). Life Sci. 2002, 71, 2603−2615.

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