RU2840772C1 - Substituted piperidine compounds and use thereof - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to substituted piperidine compounds having orexin receptor type 2 activating activity. Disclosed is a compound selected from the group consisting of (2R)-2-cyclopropyl-2-{(1R,3S,5S)-3-[(3S,4R)-1-(5-fluoropyrimidin-2-yl)-3-methoxypiperidin-4-yl]-8-azabicyclo[3.2.1]octan-8-yl}acetamide of formula (I), (R)-2-((1R,3S,5S)-3-((3S,4R)-1-(5-fluoropyrimidin-2-yl)-3-methoxypiperidin-4-yl)-8-azabicyclo[3.2.1]octan-8-yl)-3-methylbutanamide of formula (II), (R)-2-((1R, 3S, 5S)-3-((3S, 4R)-1-(5-chloropyrimidin-2-yl)-3-ethoxypiperidin-4-yl)-8-azabicyclo[3.2.1]octan-8-yl)-2-cyclopropylacetamide of formula (III) and (R)-2-cyclopropyl-2-((1R,3S,5S)-3-((2S,4S) -1-(5-fluoropyrimidin-2-yl)-2-methylpiperidin-4-yl)-8-azabicyclo[3.2.1]octan-8-yl)acetamide of formula (IV), or a pharmaceutically acceptable salt thereof. Pharmaceutical compositions, therapeutic agents, methods of treating narcolepsy and cataplexy and use of compounds of formula (I) for preparing pharmaceutical compositions are also provided.

EFFECT: disclosed compound has type 2 orexin receptor activating activity and can be used as a therapeutic agent for treating narcolepsy and cataplexy.

16 cl, 3 dwg, 3 tbl, 13 ex

Description

AREA OF TECHNOLOGY

[0001] The present invention relates to substituted piperidine compounds having orexin receptor type 2 activating activity and pharmaceutically acceptable salts thereof, as well as medical use. Furthermore, the present invention relates to medicaments containing the compounds as active ingredients.

LEVEL OF TECHNOLOGY

[0002] Orexin-A (OX-A) and orexin-B (OX-B), two types of intracerebral neuropeptides specifically produced by specific neurons located in the external hypothalamic area of the brain, have been identified as endogenous ligands of orexin receptors (PTL 1-4), which are G protein-coupled receptors found primarily in the brain (see PTL 5 and NPL 1). Two subtypes of orexin receptors are known: OX ₁ receptors of the 1 subtype (OX1R) and OX ₂ receptors of the 2 subtype (OX2R). Orexins have been shown to promote feeding behavior in rats (NPL 1).

[0003] Mutation of the OX2R gene has been reported as a cause of narcolepsy in dogs (NPL 2), and orexin knockout mice exhibit narcolepsy-like symptoms very similar to human or canine narcolepsy (NPL 3). Studies using transgenic mice with modified orexin neurons and double-transgenic mice generated by crossing these mice with transgenic mice overexpressing orexin have shown that the narcolepsy-like symptoms exhibited by modification of orexin neurons are rescued by constant expression of orexin (NPL 4). Similarly, intracerebroventricular administration of OX-A to transgenic mice with modified orexin neurons has been found to suppress the cataplexy-like (affective cataplexy) inhibitory effect and to improve narcolepsy symptoms such as increased alertness (NPL 4). Studies in OX2R knockout mice also suggest that OX2R is important for maintaining alertness (NPL 5). Damage to orexin nerves has also been suggested to be a cause of daytime sleepiness in patients with Parkinson's disease (NPL 6). Furthermore, it has been suggested that plasma OX-A levels are low in patients with sleep apnea syndrome (NPL 7). Based on this, it has been suggested that OX2R agonists may serve as treatments for narcolepsy or therapeutic agents for other sleep disorders in which hypersomnia is observed (NPL 8).

[0004] Therefore, it is believed that compounds with OX2R activating activity can be used as therapeutic agents for the treatment of narcolepsy, idiopathic hypersomnia, hypersomnia, sleep apnea syndrome, states of impaired consciousness such as coma, narcolepsy syndrome accompanied by narcolepsy-like symptoms, and hypersomnia syndrome accompanied by daytime hypersomnia (e.g., Parkinson's disease, Guillain-Barré syndrome, and Kleine-Levin syndrome).

[0005] TAK-925, a compound with OX2R activating activity, has entered phase I trials (intravenous infusion) in healthy humans and patients with narcolepsy.

List of references

Patent Literature Sources

[0006]

[PTL 1] International Patent Application Publication No. WO 1996/34877.

[PTL 2] Unexamined Publication of Japanese Patent Application HEI No. 10-327888.

[PTL 3] Unexamined Publication of Japanese Patent Application HEI No. 10-327889.

[PTL 4] Unexamined Publication of Japanese Patent Application HEI No. 11-178588.

[PTL 5] Unexamined Publication of Japanese Patent Application HEI No. 10-229887.

Non-patent literature sources

[0007]

[NPL 1] Sakurai T. et al, Cell, 1998, 92, 573-585.

[NPL 2] Lin L. et al, Cell, 1999, 98, 365-376.

[NPL 3] Chemelli R.M. et al, Cell, 1999, 98, 437-451.

[NPL 4] Mieda M. et al, Proc. Natl. Acad. Sci. USA, 2004, 101, 4649-4654.

[NPL 5] Willie J.T. et al, Neuron, 2003, 38, 715-730.

[NPL 6] Thannickal T.C. et al, Brain. 2007, 130, 1586-1595.

[NPL 7] Busquets X. et al, Respiration, 2004, 71, 575-579.

[NPL 8] Mieda M. et al, CNS Drugs, 2013, 27, 83-90.

BRIEF DESCRIPTION

[0008] The aim of the present invention is to provide piperidine compounds having orexin receptor type 2 activating activity and pharmaceutically acceptable salts thereof, as well as pharmaceutical compositions containing them.

[0009] In particular, the present invention relates to the following items 1-21.

1. A compound selected from the group consisting of (2R)-2-cyclopropyl-2-{(1R,3S,5S)-3-[(3S,4R)-1-(5-fluoropyrimidin-2-yl)-3-methoxypiperidin-4-yl]-8-azabicyclo[3.2.1]octan-8-yl}acetamide represented by the following formula (I):

(R)-2-((1R,3S,5S)-3-((3S,4R)-1-(5-fluoropyrimidin-2-yl)-3-methoxypiperidin-4-yl)-8-azabicyclo[3.2.1]octan-8-yl)-3-methylbutanamide represented by the following formula (II):

(R)-2-((1R,3S,5S)-3-((3S,4R)-1-(5-chloropyrimidin-2-yl)-3-ethoxypiperidin-4-yl)-8-azabicyclo[3.2.1]octan-8-yl)-2-cyclopropylacetamide, represented by the following formula (III):

and (R)-2-cyclopropyl-2-((1R,3S,5S)-3-((2S,4S)-1-(5-fluoropyrimidin-2-yl)-2-methylpiperidin-4-yl)-8-azabicyclo[3.2.1]octan-8-yl)acetamide represented by the following formula (IV):

or a pharmaceutically acceptable salt thereof.

2. (2R)-2-Cyclopropyl-2-{(1R,3S,5S)-3-[(3S,4R)-1-(5-fluoropyrimidin-2-yl)-3-methoxypiperidin-4-yl]-8-azabicyclo[3.2.1]octan-8-yl}acetamide, represented by the following formula (I):

or a pharmaceutically acceptable salt thereof.

3. (R)-2-((1R,3S,5S)-3-((3S,4R)-1-(5-Fluoropyrimidin-2-yl)-3-methoxypiperidin-4-yl)-8-azabicyclo[3.2.1]octan-8-yl)-3-methylbutanamide, represented by the following formula (II):

or a pharmaceutically acceptable salt thereof.

4. (R)-2-((1R,3S,5S)-3-((3S,4R)-1-(5-Chloropyrimidin-2-yl)-3-ethoxypiperidin-4-yl)-8-azabicyclo[3.2.1]octan-8-yl)-2-cyclopropylacetamide represented by the following formula (III):

or a pharmaceutically acceptable salt thereof.

5. (R)-2-Cyclopropyl-2-((1R,3S,5S)-3-((2S,4S)-1-(5-fluoropyrimidin-2-yl)-2-methylpiperidin-4-yl)-8-azabicyclo[3.2.1]octan-8-yl)acetamide, represented by the following formula (IV):

or a pharmaceutically acceptable salt thereof.

6. A pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof according to any one of paragraphs 1-5 above.

7. An orexin receptor type 2 agonist comprising a compound or a pharmaceutically acceptable salt thereof according to any one of paragraphs 1-5 above.

8. A therapeutic agent for the treatment of narcolepsy, comprising a compound or a pharmaceutically acceptable salt thereof according to any one of paragraphs 1 to 5 above.

9. A method for treating narcolepsy comprising administering to a subject a pharmacologically effective dose of a compound or a pharmaceutically acceptable salt thereof according to any one of paragraphs 1-5 above.

10. A method for activating an orexin receptor type 2 comprising administering to a subject a pharmacologically effective dose of a compound or a pharmaceutically acceptable salt thereof according to any one of paragraphs 1-5 above.

11. A method of treating narcolepsy comprising administering to a subject a compound or a pharmaceutically acceptable salt thereof according to any one of paragraphs 1-5 above.

12. Use of a compound or a pharmaceutically acceptable salt thereof according to any one of paragraphs 1-5 above for the manufacture of a pharmaceutical composition intended for the treatment of narcolepsy.

13. A compound or pharmaceutically acceptable salt thereof according to any one of paragraphs 1-5 above for use in the treatment of narcolepsy.

14. A therapeutic agent for the treatment of cataplexy, comprising a compound or a pharmaceutically acceptable salt thereof according to any one of paragraphs 1 to 5 above.

15. A method for treating cataplexy comprising administering to a subject a pharmacologically effective dose of a compound or a pharmaceutically acceptable salt thereof according to any one of paragraphs 1-5 above.

16. A compound or pharmaceutically acceptable salt thereof according to any one of paragraphs 1-5 above for use in the treatment of cataplexy.

17. Use of a compound or a pharmaceutically acceptable salt thereof according to any one of paragraphs 1-5 above for the manufacture of a pharmaceutical composition intended for the treatment of cataplexy.

18. A therapeutic agent for the treatment of hypersomnia syndrome, comprising a compound or a pharmaceutically acceptable salt thereof according to any one of paragraphs 1-5 above.

19. A method for treating hypersomnia syndrome, comprising administering to a subject a pharmacologically effective dose of a compound or a pharmaceutically acceptable salt thereof according to any one of paragraphs 1-5 above.

20. A compound or a pharmaceutically acceptable salt thereof according to any one of paragraphs 1-5 above for use in the treatment of hypersomnia syndrome.

21. Use of a compound or a pharmaceutically acceptable salt thereof according to any one of paragraphs 1-5 above for the manufacture of a pharmaceutical composition intended for the treatment of hypersomnia syndrome.

[0010] The substituted piperidine compounds represented by the formulas (I), (II), (III) and (IV) (hereinafter referred to as compounds (I), (II), (III) and (IV)), or pharmaceutically acceptable salts thereof, of the present invention have orexin receptor type 2 activating activity, as shown by the activity data in the pharmacological test examples described below. The compounds (I), (II), (III) and (IV) of the present invention have orexin receptor type 2 activating activity and can be used as therapeutic agents for narcolepsy.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[0011] Fig. 1 is an ORTEP diagram showing the results of X-ray crystallographic analysis of the compound obtained in Example 1.

Fig. 2 is an ORTEP diagram showing the results of X-ray crystallographic analysis of the compound obtained in Example 3 (a dimer containing two added acetonitrile molecules).

Fig. 3 is an ORTEP diagram showing the results of X-ray crystallographic analysis of the compound obtained in Example 4.

DETAILED DESCRIPTION

[0012] The present invention will now be explained in detail.

[0013] Polymorphic crystals of the compounds of the present invention may also exist, and any crystalline form or mixture thereof, as well as amorphous forms, may be used without any limitation, and the compounds of the present invention also include both anhydrous and solvated (especially hydrated) forms.

[0014] Isotopically labeled forms of compounds (I), (II), (III) and (IV) are also encompassed by the present invention. An isotopically labeled compound is the same as any of compounds (I), (II), (III) and (IV) except that one or more atoms are replaced by an atom having a different atomic mass or mass number than the atomic mass or mass number that is normally found in nature. Examples of isotopes that may be included in the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, phosphorus, sulfur, iodine and chlorine, and in ^{particular 2} H, ³ H, ¹¹ C, ¹⁴ C, ¹⁵ N, 18 O, ¹⁸ F, ³² P, ³⁵ S, ¹²³ I and ¹²⁵ I.

[0015] Isotope-labeled compounds, such as those incorporating radioactive isotopes such as ³ H and/or ¹⁴ C, are useful for tissue distribution assays of drugs and/or substrates. ^{The 3} H and ¹⁴ C isotopes are considered useful due to their ease of preparation and detection. ^{The 11} C and ¹⁸ F isotopes are considered useful for PET (positron emission tomography), while the ¹²⁵ I isotope is considered useful for SPECT (single photon emission computed tomography); and all are useful for brain imaging. Substitution of ² H and similar isotopes with heavier isotopes offers advantages for certain types of treatment, such as longer in vivo half-life and lower dosage requirements due to higher metabolic stability, and are therefore considered useful in some circumstances. Such isotopically labeled compounds can be prepared in a uniform manner by the procedures disclosed in the examples below, using readily usable isotopically labeled reagents in place of non-isotopically labeled reagents.

[0016] The "pharmaceutically acceptable salt" mentioned herein is not particularly limited as long as it is formed with the compound of the present invention, and specific examples include acid addition salts such as inorganic acid salts, organic acid salts, or acidic amino acid salts.

[0017] Unless otherwise specified, the term "pharmaceutically acceptable salt" as used herein refers to a salt in which the number of acid molecules relative to one molecule of compound is, without limitation, preferably from about 0.5 to about 2 acid molecules relative to one molecule of compound, or more preferably about 0.5, about 1, or about 2 acid molecules relative to one molecule of compound for a salt formed in the appropriate proportion.

[0018] Preferred examples of inorganic acid salts include hydrochlorides, hydrobromides, sulfates, nitrates and phosphates, and preferred examples of organic acid salts include acetates, succinates, fumarates, maleates, tartrates, citrates, lactates, stearates, benzoates, methanesulfonates, p -toluenesulfonates and benzenesulfonates.

[0019] Preferred examples of acidic amino acid salts include aspartic acid salts and glutamic acid salts.

[0020] When any of the compounds (I), (II), (III) and (IV) of the present invention is obtained in the form of a free form, it can be converted into an acceptable salt or hydrate of the compound (I), (II), (III) or (IV) by a conventional method.

[0021] When any of the compounds (I), (II), (III) and (IV) of the present invention is obtained in the form of a salt of the compound (I), (II), (III) or (IV) or a hydrate of the compound (I), (II), (III) or (IV), it can be converted into the free form of the compound (I), (II), (III) or (IV) in a conventional manner.

[0022] The various isomers (e.g. optical isomers, rotational isomers and stereoisomers) obtained for the compounds (I), (II), (III) and (IV) of the present invention can be purified and isolated using conventional separation means such as, for example, recrystallization, a diastereomer salt method, enzymatic resolution or chromatographic methods (e.g. thin layer chromatography, column chromatography or gas chromatography).

[0023] [Composition]

The pharmaceutical composition of the present invention can be prepared by mixing a pharmaceutically acceptable additive with a compound selected from the group of compounds (I), (II), (III), and (IV), or a pharmaceutically acceptable salt thereof. The pharmaceutical composition of the present invention can be prepared by a known method such as the method described in the General Preparation Guidelines in the Japanese Pharmacopoeia, 17th Edition.

[0024] The pharmaceutical composition of the present invention can be suitably administered to a patient according to the dosage form.

[0025] The dosage of any of the compounds (I), (II), (III) and (IV) or pharmaceutically acceptable salts thereof of the present invention may vary depending on the severity of the symptoms, the age, sex and body weight of the patient, the dosage form or kind of salt and the specific type of disease, but it is generally from about 30 μg to 10 g, preferably from 100 μg to 5 g and even more preferably from 100 μg to 1 g for oral administration, and from about 30 μg to 1 g, preferably 100 μg to 500 mg and even more preferably from 100 μg to 300 mg for administration by injection per day for an adult in a single or divided doses.

[0026] As used herein, "orexin receptor type 2 agonist" refers to an agent that binds to orexin receptor type 2 and has an orexin receptor type 2 activating effect, as well as a ligand in vivo.

[0027] The compounds of the present invention can also be used as chemical probes for capturing target proteins of physiologically active small molecules. In particular, the compounds of the present invention can be converted into affinity chromatography probes or photoaffinity probes by introducing labeling groups or linkers into those parts of the compounds that are different from their structural parts necessary for exhibiting their activity, using the method described in J. Mass Spectrum. Soc. Jpn. Vol. 51, No. 5 2003, p 492-498, or, for example, in WO 2007/139149.

[0028] The label group or linker used in the chemical probe may be a group, for example, from any of the following (1) to (5):

(1) protein labeling groups, including photoaffinity labeling groups (e.g., benzoyl, benzophenone, azide, carbonyl azide, diaziridine, enone, diazo and nitro groups) and chemical affinity groups (e.g., ketone groups with an alpha carbon atom substituted by a halogen atom, carbamoyl, ester and alkylthio groups, α,β-unsaturated ketones, Michael acceptors such as esters and oxirane);

(2) cleavable linkers such as -S-S-, -O-Si-O-, monosaccharides (such as glucose or galactose) and disaccharides (such as lactose) and oligopeptide linkers cleavable by enzymatic reactions;

(3) marker groups for fluorescence in situ hybridization, such as biotin, 3-(4,4-difluoro-5,7-dimethyl-4H-3a and 4a-diaza-4-bora-s-indacen-3-yl)propionyl;

(4) detectable markers, including radioactive label groups such as ¹²⁵ I, ³² P, ³ H, and ¹⁴ C; fluorescent label groups such as fluorescein, rhodamine, dansyl, umbelliferone, 7-nitrofurazanyl, and 3-(4,4-difluoro-5,7-dimethyl-4H-3a and 4a-diaza-4-bora-s-indacen-3-yl)propionyl groups; chemiluminescent groups such as lumiferin and luminol; and heavy metal ions such as lanthanide metal ions and radium ion; or

(5) groups associated with solid phase carriers such as glass beads, glass plates, microtiter plates, agarose beads, agarose sheets, polystyrene beads, polystyrene sheets, nylon beads and nylon sheets.

[0029] A probe obtained by introducing a label group selected from items (1) to (5) above into the compound of the present invention by the method described in the above-mentioned literature can be used as a chemical probe for identifying labeled proteins that are useful for discovering new innovative targets in drug development.

EXAMPLES

[0030] Compounds (I), (II), (III) and (IV) of the present invention can be produced by the methods described in the following examples, and the effects of the compounds can be confirmed by the methods described in the following test examples. However, these specific examples are merely illustrative and are not intended to limit the present invention in any way, and various modifications can also be made without departing from the scope of the present invention.

[0031] The compounds mentioned with reference to published documents were prepared by the method described in these documents.

[0032] The abbreviations used throughout this document are those commonly known to those skilled in the art. In particular, the following are used.

n -: normal

tert -: tertiary

¹ H-NMR: proton nuclear magnetic resonance spectrometry

MS: mass spectrometry

HPLC: High Performance Liquid Chromatography

[0033] The term "room temperature" as used in all examples and preparation examples generally refers to a range from about 10°C to 35°C. Percentages are weight percent unless otherwise noted.

[0034] Chemical shifts in proton nuclear magnetic resonance spectra are reported in units of δ (ppm) relative to tetramethylsilane, and coupling constants are reported in hertz (Hz). Patterns are represented as s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br: broad, and brs: broad singlet.

[0035] Mass spectrometry was performed using Acquity UPLC™ or Acquity UPC ² ™ from Waters Co.

[0036] For the chromatography, Silica Gel60 (70-230 mesh or 230-400 mesh AS™) from Merck or PSQ60B from Fuji Silysia Chemical, Ltd. was used as the silica gel, or a prepacked column {Column: Hi-Flash™ Column (silica gel) from Yamazen, size: S (16 × 60 mm), M (20 × 75 mm), L (26 × 100 mm), 2L (26 × 150 mm) or 3L (46 × 130 mm), or Biotage™ SNAP Ultra Silica Cartridge from Biotage Co., size: 10 g, 25 g or 50 g} was used. Supercritical fluid chromatography (SFC) fractionation was performed using Prep100q from Waters Co.

[0037] As the NH silica gel, CHROMATOREX NH-DM2035 manufactured by Fuji Silysia Chemical Ltd. was used, or a prepacked column {column: Hi-Flash™ Column (Amino) manufactured by Yamazen, size: S (16 × 60 mm), M (20 × 75 mm), L (26 × 100 mm), 2L (26 × 150 mm), or 3L (46 × 130 mm), or Presep™ (Luer Lock) NH ₂ (HC) manufactured by Wako Pure Chemical Industries, Ltd., size: type M (14 g/25 mL), type L (34 g/70 mL), type 2L (50 g/100 mL), or type 3L (110 g/200 mL)} was used.

[0038] The nomenclature of the following compounds corresponds to the nomenclature given in the E-Notebook version 12 or 13 (Perkin-Elmer), except for reagents commonly used.

[0039] Example of obtaining 1

Synthesis of 2-bromo-2-cyclopropylacetamide

Oxalyl chloride (CAS No. 79-37-8) (3.11 mL, 36.3 mmol) and N,N-dimethylformamide (60.0 μL, 0.775 mmol) were added to a solution of cyclopropylacetic acid (CAS No. 5239-82-7) (3.30 g, 33.0 mmol) in 1,2-dichloroethane (60 mL), and the mixture was stirred for 40 min at room temperature. Hydrobromic acid (56.0 mg, 0.330 mmol) and N-bromosuccinimide (CAS No. 128-08-5) (7.04 g, 39.6 mmol) were added to the reaction mixture, which was then heated under reflux for 18 h. The reaction mixture was added to ammonia (28% aqueous solution, 60 ml, 2.77 mol) at 0°C, and then ethyl acetate and sodium hydroxide (2N aqueous solution) were added for separation. The organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The resulting solid was triturated with ethyl acetate/ n -heptane, and the precipitate was filtered off. The resulting solid was dried under reduced pressure to give the title compound (1.20 g).

MS (ESI)mass/charge: 178[M+H] ⁺

[0040] Receiving example 2

Synthesis of 3-methoxyisonicotinonitrile

Sodium ethoxide (CAS#124-41-4) (3.90 g, 72.2 mmol) was added to a solution of 3-chloro-4-cyanopyridine (CAS#68325-15-5) (5.00 g, 36.1 mmol) in tetrahydrofuran (36.0 mL) at room temperature, and the mixture was heated under reflux for 1 hour. Water and ethyl acetate were added to the reaction mixture, and the organic layer was separated. The organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The resulting solid was triturated with ethyl acetate/ n -heptane, and the precipitate was filtered off. The resulting solid was dried under reduced pressure to give the title compound (1.91 g).

¹ H-NMR (400 MHz, CDCl ₃ ) δ (ppm): 4.06 (s, 3H), 7.44 (d, J=5.0 Hz, 1H), 8.37 (d, J=4.5 Hz, 1H), 8.49 (s, 1H).

MS (ESI)mass/charge: 135[M+H] ⁺

[0041] Example of obtaining 3

Synthesis of (R)-2-bromo-3-methylbutanamide

Oxalyl chloride (9.28 mL, 108 mmol) and N,N-dimethylformamide (30.0 μL, 0.387 mmol) were added to a solution of (R)-2-bromo-3-methylbutyric acid (CAS No. 76792-22-8) (9.80 g, 54.1 mmol) in methylene chloride (100 mL) at 0 °C, and the mixture was stirred for 6 h at room temperature. The reaction mixture was added to ammonia (28% aqueous solution, 50.0 mL, 2.31 mol) at 0 °C, and then extracted with methylene chloride. The organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The resulting solid was triturated with ethyl acetate/ n -heptane, and the precipitate was filtered off. The resulting solid was dried under reduced pressure to give the title compound (8.20 g).

¹ H-NMR (400 MHz, CDCl ₃ ) δ (ppm): 1.01 (d, J=6.3 Hz, 3H), 1.07 (d, J=6.8 Hz, 3H), 2.34-2.39 (m, 1H), 4.28 (d, J=4.5 Hz, 1H), 5.58 (brs, 1H), 6.41 (brs, 1H).

MS (ESI) mass/charge: 180[M+H] ⁺

[0042] Example of obtaining 4

Synthesis of (1R,3s,5S)-3-((3S,4R)-1-(5-fluoropyrimidin-2-yl)-3-methoxypiperidin-4-yl)-8-azabicyclo[3.2.1]octane

[0043] (1) Synthesis of tert -butyl-(1R,3r,5S)-3-hydroxy-3-(3-methoxypyridin-4-yl)-8-azabicyclo[3.2.1]octane-8-carboxylate

After adding 3-methoxyisonicotinonitrile (1.19 g, 8.88 mmol) and bis(pinacolato)diboron (CAS No. 73183-34-3) (2.25 g, 8.88 mmol) to a solution of N-( tert -butoxycarbonyl)nortropinone (CAS No. 185099-67-6) (1.00 g, 4.44 mmol) in methyl tert -butyl ether (18.0 mL), the mixture was heated under reflux for 16 h. Sodium carbonate (2 mol/L aqueous solution, 20.0 mL) was added to the reaction mixture at 0 °C, and the mixture was stirred for 20 min at 0 °C. Ethyl acetate was added to the reaction mixture, and the organic layer was separated. The organic layer was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, 0-20% methanol/ethyl acetate) to give the title compound (1.12 g).

¹ H-NMR (400 MHz, CDCl ₃ ) δ (ppm): 1.48 (s, 9H), 1.77 (m, 2H), 1.94-1.99 (m, 2H), 2.21-2.32 (m, 2H), 2.37-2.48 (m, 1H), 2.63-2.74 (m, 1H), 2.78 (s, 1H), 3.96 (s, 3H), 4.22-4.29 (m, 1H), 4.31-4.42 (m, 1H), 7.28 (d, J=5.4 Hz, 1H), 8.22-8.25 (m, 2H).

MS (ESI)mass/charge: 335[M+H] ⁺

[0044] (2) Synthesis of tert -butyl-(1R,5S)-3-(3-methoxypyridin-4-yl)-8-azabicyclo[3.2.1]oct-2-ene-8-carboxylate

Concentrated sulfuric acid (1.60 mL, 30.0 mmol) was added to tert -butyl (1R,3r,5S)-3-hydroxy-3-(3-methoxypyridin-4-yl)-8-azabicyclo[3.2.1]octane-8-carboxylate (610 mg, 1.82 mmol), and the mixture was stirred for 40 min at room temperature. The reaction mixture was added to a solution of potassium hydroxide (5.00 g, 89.1 mmol) in water (10.0 mL) at 0 °C. Tetrahydrofuran (10.0 mL) and di- tert- butyl dicarbonate (CAS No. 24424-99-5) (478 mg, 2.19 mmol) were added to the reaction mixture, which was then stirred for 10 min at room temperature. Ethyl acetate was added to the reaction mixture and the organic layer was separated. The organic layer was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, 5-100% ethyl acetate/ n -heptane) to give the title compound (420 mg).

¹ H-NMR (400 MHz, CDCl ₃ ) δ (ppm): 1.46 (s, 9H), 1.72-1.81 (m, 1H), 1.97-2.03 (m, 2H), 2.05-2.36 (m, 2H), 2.94-3.26 (m, 1H), 3.87 (s, 3H), 4.21-4.61 (m, 2H), 6.30 (brs, 1H), 6.99 (d, J=4.5 Hz, 1H), 8.17 (d, J=5.0 Hz, 1H), 8.21 (s, 1H).

MS (ESI)mass/charge: 317[M+H] ⁺

[0045] (3) Synthesis of tert -butyl-(1R,5S)-3-(3-methoxypyridin-4-yl)-8-azabicyclo[3.2.1]octane-8-carboxylate

After adding 10% palladium carbon (AD, 52.7% aqueous, 284 mg, 0.126 mmol, product of Kawaken Fine Chemicals Co., Ltd.) to a solution of tert -butyl (1R,5S)-3-(3-methoxypyridin-4-yl)-8-azabicyclo[3.2.1]oct-2-ene-8-carboxylate (400 mg, 1.26 mmol) in methanol (3.00 mL), the mixture was stirred for 1 hour at room temperature under a hydrogen atmosphere. The reaction mixture was filtered through Celite™, and the residue was washed with ethyl acetate. The resulting filtrate was concentrated under reduced pressure to give a mixture of the endo form and the exo form (endo:exo=1:2, 398 mg).

¹ H-NMR (400 MHz, CDCl ₃ ) δ (ppm): 1.49 (s, 6H), 1.50 (s, 3H), 1.61-2.13 (m, 22/3H), 2.40-2.45 (m, 2/3H), 2.96-3.01 (m, 1/3H), 3.47-3.59 (m, 2/3H), 3.88 (s, 1H), 3.90 (s, 2H), 4.16-4.28 (m, 1H), 4.34 (brs, 1H), 7.04 (d, J=4.5 Hz, 2/3H), 7.06 (d, J=5.0 Hz, 1/3H), 8.13-8.23 (m, 2H).

MS (ESI)mass/charge: 319[M+H] ⁺

[0046] (4) Synthesis of tert -butyl-(1R,3s,5S)-3-(3-methoxypyridin-4-yl)-8-azabicyclo[3.2.1]octane-8-carboxylate

Potassium tert -butoxide (281 mg, 2.50 mmol) was added to a solution of tert -butyl (1R,5S)-3-(3-methoxypyridin-4-yl)-8-azabicyclo[3.2.1]octane-8-carboxylate (398 mg, 1.25 mmol) in tert -butanol (4.00 mL), and the mixture was heated under reflux for 17 h. Ethyl acetate and brine were added to the reaction mixture, and the organic layer was separated. Then, the organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to give the title compound (385 mg).

¹ H-NMR (400 MHz, CDCl ₃ ) δ (ppm): 1.49 (s, 9H), 1.58-2.06 (m, 8H), 3.49-3.56 (m, 1H), 3.90 (s, 3H), 4.24 (brs, 1H), 4.34 (brs, 1H), 7.04 (d, J=5.0 Hz, 1H), 8.17-8.19 (m, 2H).

MS (ESI)mass/charge: 319[M+H] ⁺

[0047] (5) Synthesis of tert -butyl (1R,3s,5S)-3-(1-(5-fluoropyrimidin-2-yl)-3-methoxypiperidin-4-yl)-8-azabicyclo[3.2.1]octane-8-carboxylate

After adding 10% palladium on carbon (AD, 52.7% aqueous, 7.91 g, 3.52 mmol, product of Kawaken Fine Chemicals Co., Ltd.) to a solution of tert -butyl (1R,3s,5S)-3-(3-methoxypyridin-4-yl)-8-azabicyclo[3.2.1]octane-8-carboxylate (11.2 g, 35.2 mmol) in acetic acid (100 mL), the mixture was stirred for 18 h at 70 °C under hydrogen atmosphere. The reaction mixture was filtered through Celite™, and the residue was washed with ethyl acetate. The filtrate was concentrated under reduced pressure. Ethyl acetate and sodium hydroxide (2 N) were added to the residue, and the organic layer was separated. The organic layer was dried with ISOLUTE™ HM-N, filtered and concentrated under reduced pressure. After N,N-dimethylformamide (50.0 mL), 2-chloro-5-fluoropyrimidine (CAS#62802-42-0) (5.21 mL, 42.2 mmol) and potassium carbonate (7.29 g, 52.8 mmol) were added to the residue, the mixture was stirred for 40 min at 80 °C. Ethyl acetate and brine were added to the reaction mixture and the organic layer was separated. The organic layer was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, 10-60% ethyl acetate/ n -heptane) to give the title compound (9.84 g).

¹ H-NMR (400 MHz, CDCl ₃ ) δ (ppm): 1.19-1.30 (m, 3H), 1.46 (s, 9H), 1.46-1.65 (m, 6H), 1.81-1.97 (m, 3H), 2.68 (d, J=14.5 Hz, 1H), 2.71-2.81 (m, 1H), 3.30 (s, 3H), 3.35 (brs, 1H), 4.08-4.31 (m, 2H), 4.64-4.77 (m, 1H), 5.04-5.18 (m, 1H), 8.13 (s, 2H).

MS (ESI)mass/charge: 421[M+H] ⁺

[0048] (6) Synthesis of tert -butyl (1R,3s,5S)-3-((3S,4R)-1-(5-fluoropyrimidin-2-yl)-3-methoxypiperidin-4-yl)-8-azabicyclo[3.2.1]octane-8-carboxylate

The compound tert -butyl (1R,3s,5S)-3-(1-(5-fluoropyrimidin-2-yl)-3-methoxypiperidin-4-yl)-8-azabicyclo[3.2.1]octane-8-carboxylate (7.00 g, 16.6 mmol) was fractionated 100 mg at a time by supercritical fluid chromatography using CHIRALPAK ^R IC/SFC (3 cm × 25 cm) with Daicel (mobile phase: CO ₂ : methanol (90:10), 120 bar, 40°C, flow rate: 100 ml/min) to give the title compound (3.02 g) upon subsequent elution with a retention time of 8.45 minutes.

¹ H-NMR (400 MHz, CDCl ₃ ) δ (ppm): 1.17-1.29 (m, 3H), 1.46 (s, 9H), 1.46-1.66 (m, 6H), 1.85-1.97 (m, 3H), 2.68 (d, J=15.0 Hz, 1H), 2.75 (td, J=12.9, 3.2 Hz, 1H), 3.30 (s, 3H), 3.35 (brs, 1H), 4.11-4.31 (m, 2H), 4.66-4.76 (m, 1H), 5.10 (dt, J=14.4, 2.6 Hz, 1H), 8.13 (s, 2H).

MS (ESI) mass/charge: 443[M+Na] ⁺

(Analytical conditions) Supercritical fluid chromatography using CHIRALPAK™ IC-3 (3.0 mm × 50 mm) with Daicel (mobile phase: _CO2 :methanol (85:15), 40°C, flow rate: 1.2 ml/min, detection: UV (254 nm)).

(Analysis results) The retention time of the title compound was 1.34 minutes and the optical purity was >99% ee.

[0049] (7) Synthesis of (1R,3s,5S)-3-((3S,4R)-1-(5-fluoropyrimidin-2-yl)-3-methoxypiperidin-4-yl)-8-azabicyclo[3.2.1]octane

Trifluoroacetic acid (5.00 mL, 64.9 mmol) was added to tert -butyl (1R,3s,5S)-3-((3S,4R)-1-(5-fluoropyrimidin-2-yl)-3-methoxypiperidin-4-yl)-8-azabicyclo[3.2.1]octane-8-carboxylate (1.00 g, 2.38 mmol), and the mixture was stirred for 20 min at room temperature. The reaction mixture was concentrated under reduced pressure. Aqueous saturated 2N sodium hydroxide was added to the residue, and extraction was performed with ethyl acetate (3 times). The combined organic layers were dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to give the title compound (620 mg).

MS (ESI)mass/charge: 321[M+H] ⁺

[0050] Example of obtaining 5

Synthesis of (S)-2-bromo-2-cyclopropylacetamide

[0051] (1) Synthesis of (S)-3-(2-cyclopropylacetyl)-4-phenyloxazolidin-2-one

Pivaloyl chloride (302 mL, 2470 mmol) was added to tetrahydrofuran (7000 mL) at room temperature. Cyclopropylacetic acid (238 mL, 2450 mmol) was added at room temperature, and the mixture was cooled to 0 °C. After triethylamine (350 mL) was added dropwise, triethylamine (400 mL, total amount: 5340 mmol) was added over 10 min, and the mixture was stirred for 76 min at 0 °C. Then, after (S)-4-phenyloxazolidin-2-one (350 g, 2140 mmol) was added to the reaction mixture in one go, lithium chloride (109 g, 2570 mmol) was further added in one go. The reaction mixture was stirred for 18 hours at room temperature, and then ethyl acetate (7000 ml) and water (3500 ml) were added, and the mixture was stirred for 40 minutes at room temperature. The organic layer was separated and washed with aqueous 5% sodium hydrogen carbonate solution (3500 ml) and water (1750 ml) in that order. The resulting organic layer was concentrated to 1750 ml. The azeotropic procedure of adding ethyl acetate (2100 ml) and concentrating to 1750 ml was repeated 3 times, after which ethyl acetate (1050 ml) was added and the mixture was concentrated to 1750 ml. The resulting concentrate was stirred, and n -heptane (1000 ml) was added dropwise. The resulting suspension was stirred for 10 minutes, and n -heptane (2500 ml) was further added dropwise. The liquid mixture was stirred overnight at room temperature and then stirred for an additional 3.5 h at 0°C. The resulting solid was filtered using a glass filter and washed with ethyl acetate/ n -heptane (550 mL of a 1:3 mixture). The resulting solid was dried under reduced pressure to give the title compound (407 g).

¹ H-NMR (400 MHz, CDCl ₃ ) δ (ppm): 0.09-0.24 (m, 2H) 0.46-0.58 (m, 2H) 1.00-1.13 (m, 1H), 2.74-2.83 (m, 1H), 2.88-2.98 (m, 1H), 4.25-4.32 (m, 1H), 4.70 (t, J=8.83 Hz, 1H), 5.45 (dd, J=8.83, 3.85 Hz, 1H), 7.28-7.43 (m, 5H).

[0052] (2) Synthesis of (S)-2-bromo-2-cyclopropylacetamide

A 1 M dichloromethane solution of dibutyl boron triflate (500 mL, 500 mmol) was added dropwise to a solution of (S)-3-(2-cyclopropylacetyl)-4-phenyloxazolidin-2-one (100 g, 408 mmol) in dichloromethane (1000 mL) over 40 min under ice-cooling, and the mixture was stirred for 10 min under ice-cooling. After N,N-diisopropylethylamine (92 mL, 530 mmol) was added dropwise over 25 min under ice-cooling, the mixture was stirred for 1 h under ice-cooling. Then, the reaction mixture was cooled to an internal temperature of -72 °C in a dry ice-ethanol bath. Then N-bromosuccinimide (80 g, 448 mmol) was added at one time and the mixture was stirred for 1 hour and 20 minutes in a dry ice-ethanol bath. After adding 28-30% aqueous ammonia (800 mL, 6390 mmol) and tetrahydrofuran (1000 mL), the mixture was stirred for 2 hours in a water bath. The organic layer and the aqueous layer were separated, and the aqueous layer was extracted 3 times with ethyl acetate (500 mL). The resulting organic layer was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (4 kg silica gel, 30-40% ethyl acetate/ n -heptane) to give 132 g of crude product. After adding tert -butyl methyl ether (1440 ml) to the obtained crude product and stirring for 1 hour at 50°C to dissolve, the mixture was further stirred for 1 day at room temperature. The resulting solid was filtered and washed with tert -butyl methyl ether (200 ml). The filtrate and the washing solution were combined, ethyl acetate (700 ml) and activated carbon (SEISEI SHIRASAGI, 26 g) were added, and the mixture was stirred for 30 minutes at room temperature. The activated carbon was removed by filtration through Celite ^R , and the activated carbon was washed with ethyl acetate (700 ml). After combining the filtrate and the washing solution, the mixture was concentrated under reduced pressure to give the title compound (97.1 g, content 65.9%).

¹ H-NMR (400 MHz, CDCl ₃ ) δ (ppm): 0.40-0.52 (m, 1H), 0.63-0.73 (m, 1H), 0.77-0.86 (m, 1H), 0.86-0.97 (m, 1H), 1.41-1.50 (m, 1H), 3.59-3.83 (m, 1H), 5.34-5.71 (m, 1H), 5.94-6.30 (m, 1H).

MS (ESI) mass/charge: 180[M+H] ⁺

(Analytical conditions) Chromatography using CHIRALPAK™ IA (0.46 cm × 25 cm × 2) with Daicel (mobile phase: ethanol: n -hexane (10:90), 40°C, flow rate: 0.8 ml/min, detection: UV (210 nm)).

(Analysis results) The retention time of the title compound was 24.5 minutes.

[0053] Example 1

Synthesis of (R)-2-cyclopropyl-2-((1R,3S,5S)-3-((3S,4R)-1-(5-fluoropyrimidin-2-yl)-3-methoxypiperidin-4-yl)-8-azabicyclo[3.2.1]octan-8-yl)acetamide

After adding 2-bromo-2-cyclopropylacetamide (433 mg, 2.43 mmol), cesium carbonate (793 mg, 2.43 mmol) and silver oxide (564 mg, 2.43 mmol) to a solution of (1R,3S,5S)-3-((3S,4R)-1-(5-fluoropyrimidin-2-yl)-3-methoxypiperidin-4-yl)-8-azabicyclo[3.2.1]octane (260 mg, 0.811 mmol) in acetonitrile (4.00 mL), the mixture was stirred for 40 h at room temperature. The reaction mixture was purified by direct column chromatography (silica gel, 20% methanol/ethyl acetate). The resulting product was dissolved in methanol (10 mL) and fed into a Waters Porapak Rxn™ CX (two 2 g cartridges). Each solid phase was washed with methanol (20 mL), and then each product was eluted with ammonia (2 mol/L methanol solution, 20 mL), and the eluates were combined and concentrated under reduced pressure. The residue was dissolved in methanol and fractionated by supercritical fluid chromatography using CHIRALPAK™ (OD-H/SFC (2 cm × 25 cm) with Daicel (mobile phase: _CO2 :methanol (85:15), 120 bar, 40°C, flow rate: 70 mL/min.), at 10 mg/500 μL (methanol) per cycle to afford the compound with a retention time of 6.41 minutes (120 mg) as an enantiomeric mixture. The obtained compound was dissolved in methanol and fractionated by supercritical fluid chromatography using CHIRALPAK™ (IG/SFC (2 cm × 25 cm) with Daicel (mobile phase: _CO2 :methanol (75:25), 120 bar, 40°C, flow rate: 70 ml/min) at 13 mg/500 µl (methanol) per cycle to give a compound consisting mainly of the subsequently eluting component with a retention time of 9.11 minutes (75 mg). The resulting compound was dissolved in methanol and fractionated by supercritical fluid chromatography using CHIRALPAK™ (IG/SFC (2 cm × 25 cm) with Daicel (mobile phase: _CO2 :methanol (75:25), 120 bar, 40°C, flow rate: 70 ml/min) at 4 mg/100 µl (methanol) per cycle to give a compound consisting mainly of the subsequently eluting component with a retention time of 7.52 minutes (52 mg). The resulting compound was dissolved in methanol and fractionated by supercritical fluid chromatography using CHIRALPAK™ (IG/SFC (2 cm × 25 cm) with Daicel (mobile phase: CO ₂ :methanol (75:25), 120 bar, 40°C, flow rate: 70 ml/min) at 7 mg/500 µl (methanol) per cycle to give the title compound, consisting mainly of the subsequently eluting component, with a retention time of 7.18 minutes (31.3 mg).

¹ H-NMR (400 MHz, CDCl ₃ ) δ (ppm): 0.28-0.37 (m, 1H), 0.45-0.52 (m, 1H), 0.53-0.58 (m, 1H), 0.60-0.68 (m, 1H), 0.78 (dd, J=8.8, 4.3 Hz, 1H), 1.20-1.27 (m, 2H), 1.32-1.45 (m, 2H), 1.46-1.52 (m, 2H), 1.59-1.65 (m, 3H), 1.70-1.80 (m, 2H), 1.81-1.88 (m, 1H), 2.08 (d, J=9.1 Hz, 1H), 2.68 (dd, J=14.3, 1.1 Hz, 1H), 2.75 (td, J=12.8, 2.9 Hz, 1H), 3.21-3.24 (m, 1H), 3.30 (s, 3H), 3.36 (brs, 1H), 3.86-3.91 (m, 1H), 4.65-4.76 (m, 1H), 5.10 (dt, J=14.2, 2.4 Hz, 1H), 5.14-5.24 (m, 1H), 6.92-7.02 (m, 1H), 8.14 (s, 2H).

MS (ESI)mass/charge: 418[M+H] ⁺

(Analytical conditions) Chromatography using CHIRALPAK™ IA (0.46 cm × 15 cm) with Daicel (mobile phase: ethanol:hexane (20:80), 40°C, flow rate: 1 ml/min, detection: UV (254 nm)).

(Analysis results) The retention time of the title compound was 4.91 minutes and the optical purity was >99% ee.

[0054] Preparation of a single crystal of (R)-2-cyclopropyl-2-((1R,3S,5S)-3-((3S,4R)-1-(5-fluoropyrimidin-2-yl)-3-methoxypiperidin-4-yl)-8-azabicyclo[3.2.1]octan-8-yl)acetamide and crystallographic analysis using X-rays

The title compound obtained in Example 1 (2.97 mg) was dissolved in methanol (1 ml). After placing 500 μl of the solution in a vial, the cap was carefully closed (solvent evaporation method). After 1 day, a single crystal of the title compound was obtained in the vial. The obtained single crystal was subjected to crystallographic analysis using X-rays under the following conditions. The X-ray crystal structure of the title compound is shown in Fig. 1.

Analytical instrument: XtaLAB PRO P200 MM007HF (Rigaku, Japan)

Software: CrysAlisPro (Rigaku Oxford Diffraction).

X-ray: Monochromatic Cu-Kα with multilayer mirror (40 V/30 mA).

Measurement: the mode of oscillation of the ω axis.

Camera length: 35 mm.

Measurement temperature: -170°C.

[0055] According to IUPAC, (R)-2-cyclopropyl-2-((1R,3S,5S)-3-((3S,4R)-1-(5-fluoropyrimidin-2-yl)-3-methoxypiperidin-4-yl)-8-azabicyclo[3.2.1]octan-8-yl)acetamide synthesized in Example 1 is called (2R)-2-cyclopropyl-2-{(1R,3S,5S)-3-[(3S,4R)-1-(5-fluoropyrimidin-2-yl)-3-methoxypiperidin-4-yl]-8-azabicyclo[3.2.1]octan-8-yl}acetamide.

[0056] Example 2

Synthesis of (R)-2-((1R,3S,5S)-3-((3S,4R)-1-(5-fluoropyrimidin-2-yl)-3-methoxypiperidin-4-yl)-8-azabicyclo[3.2.1]octan-8-yl)-3-methylbutanamide

After adding (R)-2-bromo-3-methylbutanamide (326 mg, 1.81 mmol), cesium carbonate (590 mg, 1.81 mmol) and silver oxide (419 mg, 1.81 mmol) to a solution of (1R,3s,5S)-3-((3S,4R)-1-(5-fluoropyrimidin-2-yl)-3-methoxypiperidin-4-yl)-8-azabicyclo[3.2.1]octane (290 mg, 0.905 mmol) in acetonitrile (8.00 mL), the mixture was stirred for 40 h at room temperature. The reaction mixture was filtered through silica gel and the residue was washed with 20% methanol/ethyl acetate. The filtrate was concentrated under reduced pressure. The residue was dissolved in methanol (5 mL) and added to a Waters Porapak Rxn™ CX (4 g batch). The solid phase was washed with methanol (40 mL) and then the product was eluted with ammonia (2 N methanol solution, 40 mL) and the eluate was concentrated under reduced pressure. The residue was fractionated by supercritical fluid chromatography using a CHIRALPAK™ (IG/SFC (2 cm × 25 cm) with Daicel (mobile phase: _CO2 :methanol (80:20), 120 bar, 40°C, flow rate: 70 mL/min.) at 5 mg per cycle to afford the title compound with a retention time of 4.41 minutes (154 mg).

¹ H-NMR (400 MHz, CDCl ₃ ) δ (ppm): 0.95 (d, J=6.8 Hz, 3H), 1.02 (d, J=6.8 Hz, 3H), 1.15-1.56 (m, 8H), 1.75 (td, J=10.8, 6.1 Hz, 3H), 1.86-2.10 (m, 2H), 2.67 (d, J=13.1 Hz, 1H), 2.75 (td, J=12.9, 3.2 Hz, 1H), 2.95 (d, J=4.1 Hz, 1H), 3.19-3.27 (m, 1H), 3.30 (s, 3H), 3.33-3.46 (m, 2H), 4.70 (dt, J=13.3, 2.2 Hz, 1H), 5.06-5.16 (m, 1H), 5.30-5.36 (m, 1H), 6.79 (brd, J=5.0 Hz, 1H), 8.14 (s, 2H).

MS (ESI)mass/charge: 421[M+H] ⁺

[0057] Example 3

Synthesis of (R)-2-((1R,3S,5S)-3-((3S,4R)-1-(5-chloropyrimidin-2-yl)-3-ethoxypiperidin-4-yl)-8-azabicyclo[3.2.1]octan-8-yl)-2-cyclopropylacetamide

[0058] (1) Synthesis of tert -butyl-(1R,3s,5S)-3-((3S,4R)-3-methoxypiperidin-4-yl)-8-azabicyclo[3.2.1]octane-8-carboxylate hemi(2S,3S)-2,3-bis((4-methylbenzoyl)oxy)succinate

A mixture of acetic acid (160 ml) with tert -butyl (1R,3s,5S)-3-(3-methoxypyridin-4-yl)-8-azabicyclo[3.2.1]octane-8-carboxylate (20 g, 62.8 mmol) and 10% palladium-carbon (type AD, approximately 50% aqueous, 6 g, product of Kawaken Fine Chemicals Co., Ltd.) was stirred overnight at 70°C under a hydrogen atmosphere. The palladium-carbon was removed by filtration and washed with methanol (10 times the volume), and the filtrate was concentrated by approximately 2 times. After isopropyl acetate (15 times the volume) and n -heptane (3 times the volume) were added to the resulting concentrate, the mixture was washed with 48% aqueous sodium hydroxide solution (54.7 ml). Then, after adding n -heptane (100 ml) and washing with water (5 times volume), it was concentrated by about 5 times. The azeotropic procedure of adding isopropyl acetate (5 times volume) to the resulting concentrate and concentrating by about 5 times was repeated twice to obtain tert -butyl (1R,3s,5S)-3-(3-methoxypiperidin-4-yl)-8-azabicyclo[3.2.1]octane-8-carboxylate (15.1 g) as an isopropyl acetate solution.

After adding (-)-di-p-toluoyl-L-tartaric acid (2.70 g, 6.98 mmol) to a solution of the obtained tert -butyl (1R,3s,5S)-3-(3-methoxypiperidin-4-yl)-8-azabicyclo[3.2.1]octane-8-carboxylate (7.55 g, 23.3 mmol) and (+)-dipivaloyl-D-tartaric acid (1.85 g, 5.82 mmol) in isopropyl acetate (151 ml), acetonitrile (30.2 ml) and methanol (38 ml), the mixture was stirred for 3 days at room temperature to obtain a solid.

The resulting solid was filtered and then washed with a mixed solution of isopropyl acetate and acetonitrile (9/1). The resulting stock solution was concentrated 10 times, and isopropyl acetate (10 times the volume), aqueous 5 N sodium hydroxide solution (4.65 mL, 23.269 mmol), and water (4 times the volume) were added. After removing the aqueous layer, it was washed with water (4 times the volume). Then, after concentrating by about 5 times, isopropyl acetate (10 times volume) was added and azeotropic distillation was performed by about 5 times, (+)-di-p-toluoyl-D-tartaric acid (3.15 g, 8.144 mmol) was added to a solution of (+)-dipivaloyl-D-tartaric acid (1.852 g, 5.817 mmol) in methanol (38 mL), acetonitrile (30.2 mL) and isopropyl acetate (151 mL), and the mixture was stirred overnight at room temperature. Then, (+)-di-p-toluoyl-D-tartaric acid compounds (0.05 equivalent) and isopropyl acetate (10 times volume) were added. The total amount was recovered by concentration, neutralization with sodium hydroxide and washing with purified water, then added (+)-Di-p-toluoyl-D-tartaric acid (3.15 g, 8.144 mmol) was added to a solution of (+)-dipivaloyl-D-tartaric acid (0.2 equivalent) in isopropyl acetate (151 mL), acetonitrile (30.2 mL) and methanol (38 mL), and the mixture was stirred overnight at room temperature. The compound (+)-di-p-toluoyl-D-tartaric acid (0.05 equivalent) was added to the reaction mixture, and the mixture was stirred for 4 hours at room temperature. The compounds (+)-di-p-toluoyl-D-tartaric acid (0.025 equivalent) and isopropyl acetate (10 times the volume) were added to the reaction mixture, and the mixture was stirred overnight at room temperature. The total amount was recovered by concentration, neutralization with sodium hydroxide and washing with purified water, and (+)-Di-p-toluoyl-D-tartaric acid (3.15 g, 8.14 mmol) was added to a solution of (+)-dipivaloyl-D-tartaric acid (0.2 equivalent) in isopropyl acetate (151 mL), acetonitrile (30.2 mL) and methanol (38 mL), and the mixture was stirred overnight at room temperature. Then, isopropyl acetate (10 times its volume) was added. After 7 h, the resulting solid was filtered and washed with isopropyl acetate to give the title compound (3.45 g).

(Analytical conditions) Chromatography using CHIRALPAK™ IA (0.46 cm × 25 cm) with Daicel (mobile phase: ethanol: n -hexane (20:80), 40°C, flow rate: 1.0 ml/min, detection: UV (254 nm)).

(Analysis results) The retention time of the title compound was 8.52 minutes, and the optical purity was 99.2% ee.

[0059] (2) Synthesis of tert -butyl (1R,3s,5S)-3-((3S,4R)-1-(5-chloropyrimidin-2-yl)-3-methoxypiperidin-4-yl)-8-azabicyclo[3.2.1]octane-8-carboxylate

N,N-Dimethylformamide (10 mL) containing 2,5-dichloropyrimidine (288 mg, 1.93 mmol), tert -butyl (1R,3s,5S)-3-((3S,4R)-3-methoxypiperidin-4-yl)-8-azabicyclo[3.2.1]octane-8-carboxylate hemi(2S,3S)-2,3-bis((4-methylbenzoyl)oxy)succinate (500 mg, 0.966 mmol) and potassium carbonate (267 mg, 1.93 mmol) were stirred for 24 h at 80 °C. The reaction mixture was cooled to room temperature, and then water was added and extraction was performed 3 times with ethyl acetate. The organic layer was collected and concentrated. The resulting residue was purified by column chromatography (silica gel, 10-100% ethyl acetate/ n -heptane) to give the title compound (368 mg).

¹ H-NMR (400 MHz, CDCl ₃ ) δ (ppm): 1.24-1.27 (m, 2H), 1.41-1.49 (m, 2H), 1.46 (s, 9H), 1.61 (brs, 4H), 1.82-1.98 (m, 4H), 2.68 (d, J=14.04 Hz, 1H), 2.75 (td, J=12.91, 3.17 Hz, 1H), 3.30 (s, 3H), 3.36 (brs, 1H), 4.11-4.32 (m, 2H), 4.68-4.80 (m, 1H), 5.12 (dt, J=14.27, 2.61 Hz, 1H), 8.16 (s, 2H).

[0060] (3) Synthesis of tert -butyl (1R,3s,5S)-3-((3S,4R)-1-(5-chloropyrimidin-2-yl)-3-hydroxypiperidin-4-yl)-8-azabicyclo[3.2.1]octane-8-carboxylate

A mixture of tert -butyl (1R,3s,5S)-3-((3S,4R)-1-(5-chloropyrimidin-2-yl)-3-methoxypiperidin-4-yl)-8-azabicyclo[3.2.1]octane-8-carboxylate (300 mg, 0.687 mmol) and 48% hydrobromic acid (5 mL) was stirred for 20 min at room temperature and then heated for 2.5 h at 100°C. Then, it was stirred overnight at room temperature, and the resulting reaction mixture was concentrated under reduced pressure. To the resulting residue were added tetrahydrofuran (10 mL) and an aqueous saturated sodium hydrogen carbonate solution, and then di- tert-butyl dicarbonate (165 mg, 0.755 mmol) was added at room temperature. After confirming the completion of the reaction by UPLC, ethyl acetate and water were added, and the organic layer was separated. The aqueous layer was extracted again with ethyl acetate, and combined with the previously obtained organic layer, and dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. The resulting residue was washed with a mixed solvent of 10% ethyl acetate/ n -heptane to give the title compound (226 mg).

¹ H-NMR (400 MHz, CDCl ₃ ) δ (ppm): 1.28-1.34 (m, 2H), 1.46 (s, 9H), 1.59-1.72 (m, 6H), 1.75-1.97 (m, 4H), 2.70-2.83 (m, 1H), 2.91 (dd, J=14.04, 0.91 Hz, 1H), 3.97-4.03 (m, 1H), 4.09-4.34 (m, 2H), 4.68-4.79 (m, 1H), 4.81-4.91 (m, 1H), 8.18 (s, 2H).MS (ESI) mass/charge: 423[M+H] ⁺

[0061] (4) Synthesis of tert -butyl (1R,3s,5S)-3-((3S,4R)-1-(5-chloropyrimidin-2-yl)-3-ethoxypiperidin-4-yl)-8-azabicyclo[3.2.1]octane-8-carboxylate

Sodium hydride (60%, dispersed in liquid paraffin, 7.09 mg, 0.177 mmol) was added to a solution of tert -butyl (1R,3s,5S)-3-((3S,4R)-1-(5-chloropyrimidin-2-yl)-3-hydroxypiperidin-4-yl)-8-azabicyclo[3.2.1]octane-8-carboxylate (50 mg, 0.118 mmol) in N,N-dimethylformamide (1 ml) under ice-cooling. After stirring for 30 min, ethyl iodide (0.019 ml, 0.236 mmol) was added, and the mixture was stirred for 16 h at room temperature. After adding water and ethyl acetate to the reaction mixture, the organic layer was separated and concentrated. The resulting residue was purified by column chromatography (silica gel, 1-20% ethyl acetate/ n -heptane) to give the title compound (42.5 mg).

MS (ESI)mass/charge: 451[M+H] ⁺

[0062] (5) Synthesis of (R)-2-((1R,3S,5S)-3-((3S,4R)-1-(5-chloropyrimidin-2-yl)-3-ethoxypiperidin-4-yl)-8-azabicyclo[3.2.1]octan-8-yl)-2-cyclopropylacetamide

After treating tert -butyl (1R,3s,5S)-3-((3S,4R)-1-(5-chloropyrimidin-2-yl)-3-ethoxypiperidin-4-yl)-8-azabicyclo[3.2.1]octane-8-carboxylate (42.5 mg, 0.094 mmol) with trifluoroacetic acid (1 mL) for 30 min, the mixture was concentrated. The resulting residue was dissolved in methanol and added to Waters Porapak Rxn™ CX (0.4 g). The solid phase was washed with methanol (6 mL), and then the product was eluted with ammonia (2 mol/L methanol solution, 6 mL), and the eluate was concentrated under reduced pressure. A mixture of the obtained residue (S)-2-bromo-2-cyclopropylacetamide (51.6 mg, 0.188 mmol), obtained by the same procedure as in Preparation Example 5, potassium carbonate (26.0 mg, 0.188 mmol) and acetonitrile (2 mL) was stirred for 17 days at room temperature. The reaction mixture was filtered using acetonitrile (3 mL) and the resulting filtrate was fractionated by supercritical fluid chromatography using CHIRALPAK ^R (IA/SFC (3 cm × 25 cm) with Daicel (mobile phase: CO ₂ : methanol (75:25), 120 bar, 40 °C, flow rate: 100 mL/min.) at 500 μL per cycle to give the title compound with a retention time of 7.55 minutes (22.2 mg).

¹ H-NMR (400 MHz, CDCl ₃ ) δ (ppm): 0.26-0.38 (m, 1H), 0.40-0.58 (m, 2H), 0.59-0.68 (m, 1H), 0.71-0.83 (m, 1H), 0.98-1.92 (m, 12H), 1.04 (t, J=7.02 Hz, 3H), 2.07 (brd, J=9.06 Hz, 1H), 2.61-2.81 (m, 2H), 3.13-3.30 (m, 2H), 3.45 (brs, 1H), 3.61-3.75 (m, 1H), 3.84-3.92 (m, 1H), 4.66-4.76 (m, 1H), 4.93-5.11 (m, 1H), 5.17-5.32 (m, 1H), 6.90-7.03 (m, 1H), 8.16 (s, 2H).

MS (ESI)mass/charge: 448[M+H] ⁺

(Analytical conditions) Supercritical fluid chromatography using CHIRALPAK™ IA-3 (0.46 cm × 25 cm) with Daicel (mobile phase: _CO2 :methanol (80:20), 40°C, flow rate: 1.2 ml/min, detection: UV (257 nm)).

(Analysis results) The retention time of the title compound was 2.19 minutes and the optical purity was >99.9% ee.

[0063] Preparation of a single crystal of (R)-2-((1R,3S,5S)-3-((3S,4R)-1-(5-chloropyrimidin-2-yl)-3-ethoxypiperidin-4-yl)-8-azabicyclo[3.2.1]octan-8-yl)-2-cyclopropylacetamide and crystallographic analysis using X-rays

The title compound obtained in Example 3 (1.37 mg) was dissolved in acetonitrile (600 μl). After placing 200 μl of the solution into the vial, the cap was carefully closed (solvent evaporation method). After 1 day, a single crystal of the title compound (a dimer crystal with two acetonitrile groups added) was obtained in the vial. The obtained single crystal was subjected to crystallographic analysis using X-rays under the following conditions. The X-ray crystal structure of the title compound is shown in Fig. 2.

Analytical instrument: XtaLAB PRO P200 MM007HF (Rigaku, Japan)

Software: CrysAlisPro (Rigaku Oxford Diffraction).

X-ray: Monochromatic Cu-Kα with multilayer mirror (40 V/30 mA).

Measurement: the mode of oscillation of the ω axis.

Camera length: 35 mm.

Measurement temperature: -170°C.

[0064] Example 4

Synthesis of (R)-2-cyclopropyl-2-((1R,3S,5S)-3-((2S,4S)-1-(5-fluoropyrimidin-2-yl)-2-methylpiperidin-4-yl)-8-azabicyclo[3.2.1]octan-8-yl)acetamide

[0065] (1) Synthesis of ethyl (1R,3s,5S)-3-((S)-2-methyl-1,2,3,6-tetrahydropyridin-4-yl)-8-azabicyclo[3.2.1]octane-8-carboxylate

Ethyl (1R,3s,5S)-3-(tosyloxy)-8-azabicyclo[3.2.1]octane-8-carboxylate (CAS No. 2236076-85-8) (200 mg, 0.566 mmol), nickel(II) bromide complex of ethylene glycol dimethyl ether (17.5 mg, 0.057 mmol), 4,4-di- tert -butyl-2,2-dipyridyl (15.2 mg, 0.057 mmol), potassium iodide (94.0 mg, 0.566 mmol), and manganese (62.2 mg, 1.13 mmol) were added to a 13 mm screw-cap test tube. Then, a solution of tert -butyl (S)-2-methyl-4-(((trifluoromethyl)sulfonyl)oxy)-3,6-dihydropyridine-1(2H)-carboxylate (CAS#876922-74-6) (195 mg, 0.566 mmol) in N,N-dimethylacetamide (4.0 mL) and 4-ethylpyridine (0.064 mL, 0.566 mmol) was added under a stream of nitrogen. The resulting mixture was stirred for 12.5 h at 80 °C under a nitrogen atmosphere. The reaction mixture was cooled to room temperature and then diluted with ethyl acetate. Insoluble materials were removed by filtration through a cotton plug, and the eluate was partitioned between water and ethyl acetate. The aqueous layer was separated and extracted with ethyl acetate. The organic layers were combined, washed 3 times with water and concentrated under reduced pressure to afford the coupling product as a crude product.

Trifluoroacetic acid (1.0 mL) was added to a solution of the obtained crude product in dichloromethane (4.0 mL), and the mixture was stirred for 1 h at room temperature. The reaction mixture was purged with nitrogen to distill off the solvent, and the residue was diluted with methanol. The resulting methanol solution was transferred to a Waters PoraPak Rxn™ CX (20 cc (2 g) cartridge). After washing the solid with methanol (20.0 mL), it was eluted with ammonia (2 N methanol solution, 20 mL). The eluate was concentrated under reduced pressure to give the crude product of the title compound (120 mg).

MS (ESI)mass/charge: 279[M+H] ⁺

[0066] (2) Synthesis of ethyl (1R,3s,5S)-3-((2S,4S)-1-(5-fluoropyrimidin-2-yl)-2-methylpiperidin-4-yl)-8-azabicyclo[3.2.1]octane-8-carboxylate

A mixture of ethyl (1R,3s,5S)-3-((S)-2-methyl-1,2,3,6-tetrahydropyridin-4-yl)-8-azabicyclo[3.2.1]octane-8-carboxylate (120 mg, 0.431 mmol), 10% palladium on carbon (48.47% aqueous solution of the product, 183 mg, 0.083 mmol, product of NE Chemcat Corp.) and methanol (4.0 mL) was stirred for 6.5 h under hydrogen atmosphere. Insoluble materials were removed by filtration through Celite™ and concentrated under reduced pressure to give the reduced form (113 mg) as a cis / trans mixture.

MS (ESI)mass/charge: 281[M+H] ⁺

A mixture of the obtained reduced form (113 mg), 2-chloro-5-fluoropyrimidine (0.048 mL, 0.517 mmol), cesium carbonate (281 mg, 0.862 mmol) and N,N-dimethylacetamide (2.0 mL) was stirred for 8 h at 100 °C. The mixture was purified by column chromatography (silica gel, 0% - 30% ethyl acetate/ n -heptane) to give the title compound (82.0 mg) as a cis / trans mixture.

MS (ESI)mass/charge: 377[M+H] ⁺

(Analytical conditions) Supercritical fluid chromatography using CHIRALPAK™ IF-3 (3.0 mm × 50 mm) with Daicel (mobile phase: _CO2 :methanol (70:30), 40°C, flow rate: 1.2 ml/min, detection: UV (210-400 nm)).

(Analysis results) The retention time of the title compound was 1.02 minutes, and the retention time of the trans form was 1.23 minutes. The cis : trans ratio was 4:5 (peak area ratio), and the optical purity was >99% ee.

The resulting cis / trans mixture was fractionated by supercritical fluid chromatography using CHIRALPAK ^R (IF/SFC (3 cm × 25 cm) with Daicel (mobile phase: _CO2 :methanol (70:30), 120 bar, 40°C, flow rate: 100 ml/min.) at 10 mg per cycle to give the first eluting title compound with a retention time of 5.55 minutes (34.0 mg).

¹ H-NMR (400 MHz, CDCl ₃ ) δ (ppm): 1.17 (d, J=6.34 Hz, 3H), 1.23 (t, J=7.25 Hz, 3H), 1.19-2.02 (m, 14H), 3.09 (ddd, J=13.70, 10.99, 5.66 Hz, 1H), 4.10 (q, J=7.10 Hz, 2H), 4.17-4.30 (m, 3H), 4.34 (dd, J=13.59, 7.25 Hz, 1H), 8.13 (s, 2H).

MS (ESI)mass/charge: 377[M+H] ⁺

(Analytical conditions) Supercritical fluid chromatography using CHIRALPAK™ IF-3 (3.0 mm × 50 mm) with Daicel (mobile phase: _CO2 :methanol (70:30), 40°C, flow rate: 1.2 ml/min, detection: UV (245 nm)).

(Analysis results) The retention time of the title compound was 1.02 min, the cis : trans ratio was > 99:1, and the optical purity was > 99% ee.

[0067] (3) Synthesis of (1R,3s,5S)-3-((2S,4S)-1-(5-fluoropyrimidin-2-yl)-2-methylpiperidin-4-yl)-8-azabicyclo[3.2.1]octane

A mixture of ethyl (1R,3s,5S)-3-((2S,4S)-1-(5-fluoropyrimidin-2-yl)-2-methylpiperidin-4-yl)-8-azabicyclo[3.2.1]octane-8-carboxylate (34 mg, 0.09 mmol) and 48% hydrobromic acid (2.0 ml) was stirred for 1 hour at 100°C. Aqueous 5 N sodium hydroxide solution was added to the reaction mixture at 0°C for neutralization. The mixture was extracted 3 times with dichloromethane, and the organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure to give the title compound (21.7 mg).

MS (ESI)mass/charge: 305[M+H] ⁺

[0068] (4) Synthesis of (R)-2-cyclopropyl-2-((1R,3S,5S)-3-((2S,4S)-1-(5-fluoropyrimidin-2-yl)-2-methylpiperidin-4-yl)-8-azabicyclo[3.2.1]octan-8-yl)acetamide

A mixture of (1R,3s,5S)-3-((2S,4S)-1-(5-fluoropyrimidin-2-yl)-2-methylpiperidin-4-yl)-8-azabicyclo[3.2.1]octane (5.00 mg, 0.016 mmol), potassium carbonate (4.54 mg, 0.033 mmol), (S)-2-bromo-2-cyclopropylacetamide (8.24 mg, 0.033 mmol) and acetonitrile (1.0 mL) was stirred at room temperature for 7 days. Potassium carbonate (4.54 mg, 0.033 mmol) and (S)-2-bromo-2-cyclopropylacetamide (8.24 mg, 0.033 mmol) were added to the reaction mixture, which was then stirred at room temperature for 3 days. An aqueous ammonium chloride solution was added to the reaction mixture to suspend the reaction mixture. The mixture was extracted with ethyl acetate, and the organic layer was washed with water and concentrated under reduced pressure to obtain a crude product.

The resulting crude product was diluted with methanol and added to a Waters PoraPak Rxn™ CX (6 cc cartridge (400 mg)). After washing the solid with methanol (6.0 mL), it was eluted with ammonia (2 N methanol solution, 6 mL). The eluate was concentrated under reduced pressure and the resulting residue was purified by thin layer chromatography (NH, dichloromethane) to give the title compound (3.74 mg).

¹ H-NMR (400 MHz, CDCl ₃ ) δ (ppm): 0.25-0.36 (m, 1H), 0.39-0.51 (m, 1H), 0.51-0.59 (m, 1H), 0.59-0.70 (m, 1H), 0.70-0.84 (m, 1H), 1.18 (d, J=6.34 Hz, 3H), 1.21-1.55 (m, 9H), 1.61-1.95 (m, 5H), 2.04 (d, J=9.06 Hz, 1H), 3.10 (ddd, J=13.70,10.99, 5.21 Hz, 1H), 3.15-3.28 (m, 1H), 3.80-3.94 (m, 1H), 4.17-4.29 (m, 1H), 4.35 (dd, J=13.59, 7.25 Hz, 1H), 5.18 (brs, 1H), 6.94 (brs, 1H), 8.14 (s, 2H).

MS (ESI)mass/charge: 402[M+H] ⁺

[0069] Preparation of a single crystal of (R)-2-cyclopropyl-2-((1R,3S,5S)-3-((2S,4S)-1-(5-fluoropyrimidin-2-yl)-2-methylpiperidin-4-yl)-8-azabicyclo[3.2.1]octan-8-yl)acetamide and crystallographic analysis using X-rays

The title compound obtained in Example 4 (0.81 mg) was dissolved in methanol (600 µl). A vial containing a 200 µl portion thereof was carefully placed in a slightly larger vial containing 2 ml of tert -butyl methyl ether and capped (vapor diffusion method). After 12 days, a single crystal of the title compound was obtained in the vial. The single crystal obtained was subjected to X-ray crystallographic analysis under the following conditions. The X-ray crystal structure of the title compound is shown in Fig. 3.

Analytical instrument: XtaLAB PRO P200 MM007HF (Rigaku, Japan)

Software: CrysAlisPro (Rigaku Oxford Diffraction).

X-ray: Monochromatic Cu-Kα with multilayer mirror (40 V/30 mA).

Measurement: the mode of oscillation of the ω axis.

Camera length: 35 mm.

Measurement temperature: -170°C.

[0070] Examples of pharmacological tests

The following pharmacological testing was carried out using the compounds from Examples 1-4.

[0071] Test Example 1-1. Evaluation of Activity Against OX1R and OX2R

HEK293 (human embryonic kidney 293) cells overexpressing human OX1R (hOX1R) or human OX2R (hOX2R) were seeded in 384-well microplates (Greiner) at 10,000 cells per well and cultured for 1 day in high-glucose DMEM (FujiFilm Corp.-Wako Pure Chemical Industries Ltd.) supplemented with 10% FBS (Thermo Scientific) and 1% penicillin-streptomycin (FujiFilm Corp.-Wako Pure Chemical Industries, Ltd.). After removing the medium, 40 μl of assay buffer (20 mM HEPES (Sigma-Aldrich Japan, KK), Hanks' balanced salt solution (Gibco), 0.1% BSA (Sigma-Aldrich Japan, KK), 0.1% Pluronic F-127 (Invitrogen)) containing Calcium 4 dye (Molecular Device Corporation) and 2.5 mM probenecid (Sigma-Aldrich Japan, KK) were added, and the plate was incubated for 60 minutes. After an additional 20 μl of assay buffer was added, 20 μl of assay buffer containing the test compound was added, and the reaction was initiated. The change in intracellular calcium ion concentration during the reaction was measured based on the ratio of fluorescence intensity according to the fluorescence value with excitation at 480 nm and detection at 540 nm using FDSS7000 (Hamamatsu Photonics, KK). The test compound was dissolved in DMSO to a concentration of 10 mM and diluted with assay buffer to a final concentration of 3 × 10 ^-10 M to 1 × 10 ^-7 M (final DMSO concentration 0.1%). The half-maximal effective concentration (EC ₅₀ value) was determined from the fluorescence value upon addition of the test compound at different concentrations, with the fluorescence value in the well with the added buffer containing no compound as 0% and the fluorescence value in the well with 10 nM OX-A (Peptide Research Lab) as 100%. The EC ₅₀ value of each compound is shown in Table 1.

[0072] Table 1

Example No. hOX1R EC ₅₀ (nM) hOX2R EC ₅₀ (nM) 1 >100 0.99 2 >100 3.08

[0073] Test Example 1-2 Evaluation of Activating Activity for OX1R and OX2R

Human embryonic kidney 293 cells (HEK293) with forced expression of hOX1R or hOX2R were seeded in 384-well microplates (Greiner) at 10,000 cells per well and cultured for 1 day in high-glucose DMEM (FujiFilm Corp.-Wako Pure Chemical Industries Ltd.) supplemented with 10% FBS (Thermo Scientific) and 1% penicillin-streptomycin (FujiFilm Corp.-Wako Pure Chemical Industries, Ltd.). After removing the medium, 30 μl of assay buffer (20 mM HEPES (Sigma-Aldrich Japan, KK), Hanks' balanced salt solution (Gibco), 0.1% BSA (Sigma-Aldrich Japan, KK), 0.1% Pluronic F-127 (Invitrogen)) containing Calcium 4 dye (Molecular Device Corporation) and 2.5 mM probenecid (Sigma-Aldrich Japan, KK) were added and the mixture was incubated for 60 minutes. A 30-μl portion of the assay buffer containing the test compound was added and the reaction was initiated. The change in intracellular calcium ion concentration due to the reaction was measured based on the ratio of fluorescence intensity according to the fluorescence value with excitation at two wavelengths of 480 nm and 540 nm using FDSS7000 (Hamamatsu Photonics, KK). The test compound was dissolved in DMSO to a concentration of 10 mM and diluted with assay buffer to a final concentration of 3 × 10 ^-11 M to 1 × 10 ^-5 M (final DMSO concentration 0.1%). The EC ₅₀ value was determined from the fluorescence value when the test compound was added at different concentrations, with the fluorescence value in the well with the added buffer containing no compound as 0% and the fluorescence value in the well with OX-A at a concentration of 30 nM (Peptide Research Lab) as 100%. The EC ₅₀ value of each compound is shown in Table 2.

[0074] Table 2

Example No. hOX1R EC ₅₀ (nM) hOX2R EC ₅₀ (nM) 1 4700 2,3 3 1400 4.3 4 3900 4.4

[0075] Test Case 2: Increase in Spontaneous Movement

Similar to an increase in body temperature or an increase in cardiovascular parameters such as blood pressure, an increase in movement in mice is one of the indicators of alertness. In this test example, the alertness effect was assessed by measuring the spontaneous movement of mice. Male C57BL/6NCrl mice (18-19 weeks old, Charles River Laboratories, Japan Inc., 4 mice in each group) were used for the experiment. Spontaneous movement was measured using a movement measuring device (VersaMax Oven Field, AccuScan Instruments, Inc.) by irradiating infrared rays on the side sections of the measuring cage and quantifying the number of times the mice passed through the irradiation. After placing the mice in the measuring cage and conditioning for 3 hours, the compound (10 mg/kg) was orally administered. Spontaneous movement was measured 2 hours after administration. The test compound-administered group was administered a solution of the test compound dissolved in 0.1 mol/L hydrochloric acid containing 5% (v/v) DMSO and 5% (v/v) Kolliphor™ EL. In the control group, mice were administered only the vehicle without the test compound.

[0076] The results are shown in Table 3. The spontaneous movement was presented as a percentage for the group administered with the test compound, and the spontaneous movement in the control group was taken as 100%.

[0077] Table 3

Test compound Spontaneous movement
(% control) solvent 100 Example 1 865 Example 2 253

[0078] As shown in Table 3, the compounds of the present invention increased the spontaneous movement in mice. In other words, the compounds of the present invention were shown to have an alerting effect.

[0079] Test Example 3. Awakening-promoting effect of orally administering the compound of the present invention to wild-type mice

Wild-type (WT) male C57BL/6 mice were used as experimental animals. Electrodes for measuring electroencephalogram (EEG) and electromyogram (EMG) were surgically implanted into 13-week-old mice under isoflurane anesthesia. The solvent (0 mg/kg) or a solution of the compound from Example 1 dissolved in solvent (1, 3, or 10 mg/kg) was administered orally 30-15 minutes before turning off the lights. The solvent was 0.1 mol/L hydrochloric acid containing 5% (v/v) DMSO and 5% (v/v) Kolliphor™ EL. EEG and EMG were recorded for approximately 24 hours, starting from 1 hour before turning off the lights. Mice were used repeatedly with a washout period of 2 days or more. The electroencephalogram and electromyogram data obtained for each mouse were used to evaluate the sleep stage of each interval (10 seconds) using a sleep analysis software (SleepSign: Kissei Comtec Co., Ltd.). The time (sleep onset latency) until the onset of onset sleep after lights-off (sleep for 8 or more intervals starting after non-REM sleep) was measured for each mouse. Using 16 mice in each administration group, the sleep onset latencies of the vehicle administration group (control group) and the compound administration group of Example 1 were compared using Dunnett's multiple comparison test after survival time analysis taking into account the number of experiments and the use of the same individuals, with a significance level of 5% on both sides.

[0080] The sleep latency values in mice administered with the vehicle and the compound of Example 1 at a dose of 1, 3 and 10 mg/kg were 0.23 hours, 0.28 hours, 0.44 hours and 2.07 hours, respectively. In the groups administered with the compound of Example 1 at a dose of 3 or 10 mg/kg, the sleep onset latency was significantly increased compared to the group administered with the vehicle. In particular, it was found that when the compound of Example 1 was administered orally, the sleep onset latency was prolonged in a dose-dependent manner.

[0081] Test Example 4. Awakening effect and prolongation of state without manifestation of cataplexy upon oral administration of the compound of the present invention to orexin-deficient mice ( orexin/ataxin -3 Tg/+ mice)

Orexin/ataxin-3 Tg/+ mice (hereinafter referred to as "Tg mice"; Hara et al., Neuron, 30, 345-54, 2001) with a C57BL/6 genetic background were used as experimental animals. Electrodes for measuring electroencephalogram and electromyogram were surgically implanted into 12-week-old mice (± 2 weeks) under isoflurane anesthesia. The solvent (0 mg/kg) or sample solution (the compound from Example 1 dissolved in solvent: 0.3, 1, or 3 mg/kg) was administered orally 30-15 min before turning off the lights. The solvent used was 0.1 mol/L hydrochloric acid solution containing 5% (v/v) DMSO and 5% (v/v) Kolliphor™ EL. Electroencephalogram and electromyogram were recorded for approximately 24 hours, starting from 1 hour before lights out. Mice were used repeatedly with a washout period of 2 days or more. EEG and EMG data obtained for each mouse were used to assess the sleep stage of each bin (10 seconds) using sleep analysis software (SleepSign: Kissei Comtec Co., Ltd.), up to a maximum of 4 hours (threshold). For the purpose of this experiment, cataplexy-like symptoms were defined as REM sleep occurring immediately after wakefulness (direct transition from wakefulness to REM sleep (DREM)) for a continuous period of 4 bins or longer. DREM is an analog of cataplexy in mice (Exp Neurol. 2009; 217:46-54). For each mouse, the time to onset of sleep after lights-out (sleep for 8 consecutive intervals or longer, excluding DREM) (sleep onset latency) and the time to onset of DREM (DREM latency) were measured. The number of mice was 14 in the vehicle-treated group and in the groups administered with the compound of Example 1. Sleep onset latency and DREM latency in the vehicle-treated control group and the compound-treated group were compared using Dunnett's multiple comparison test after survival time analysis taking into account the number of experiments and the use of the same individuals, with a significance level of 5% on both sides for each.

[0082] The sleep onset latency values in Tg mice administered with vehicle and the compound of Example 1 at a dose of 0.3, 1 and 3 mg/kg were 0.21 hours, 0.31 hours, 0.64 hours and 2.42 hours, respectively, indicating a significant increase in sleep onset latency in the groups administered with the compound of Example 1 at a dose of 1 and 3 mg/kg. In particular, it was found that when the compound of Example 1 was administered orally, the sleep onset latency was prolonged in a dose-dependent manner from 1 mg/kg in orexin-deficient mice.

[0083] The DREM latency values for the vehicle administration and the compound of Example 1 at 0.3, 1 and 3 mg/kg in Tg mice were 1.16 hours, 1.50 hours, 2.26 hours and 4.00 hours, respectively, confirming that the DREM latency was significantly and dose-dependently increased in the groups receiving the compound of Example 1 at 0.3, 1 and 3 mg/kg compared to the vehicle administration group. In other words, the states without exhibiting cataplexy-like behavior in mice were maintained by administering the compounds of the present invention in a dose-dependent manner.

Claims (32)

1. A compound selected from the group consisting of (2R)-2-cyclopropyl-2-{(1R,3S,5S)-3-[(3S,4R)-1-(5-fluoropyrimidin-2-yl)-3-methoxypiperidin-4-yl]-8-azabicyclo[3.2.1]octan-8-yl}acetamide represented by the following formula (I): (R)-2-((1R,3S,5S)-3-((3S,4R)-1-(5-fluoropyrimidin-2-yl)-3-methoxypiperidin-4-yl)-8-azabicyclo[3.2.1]octan-8-yl)-3-methylbutanamide represented by the following formula (II): (R)-2-((1R,3S,5S)-3-((3S,4R)-1-(5-chloropyrimidin-2-yl)-3-ethoxypiperidin-4-yl)-8-azabicyclo[3.2.1]octan-8-yl)-2-cyclopropylacetamide, represented by the following formula (III): and (R)-2-cyclopropyl-2-((1R,3S,5S)-3-((2S,4S)-1-(5-fluoropyrimidin-2-yl)-2-methylpiperidin-4-yl)-8-azabicyclo[3.2.1]octan-8-yl)acetamide represented by the following formula (IV): or a pharmaceutically acceptable salt thereof. 2. (2R)-2-Cyclopropyl-2-{(1R,3S,5S)-3-[(3S,4R)-1-(5-fluoropyrimidin-2-yl)-3-methoxypiperidin-4-yl]-8-azabicyclo[3.2.1]octan-8-yl}acetamide, represented by the following formula (I): or a pharmaceutically acceptable salt thereof. 3. (R)-2-((1R,3S,5S)-3-((3S,4R)-1-(5-Fluoropyrimidin-2-yl)-3-methoxypiperidin-4-yl)-8-azabicyclo[3.2.1]octan-8-yl)-3-methylbutanamide, represented by the following formula (II): or a pharmaceutically acceptable salt thereof. 4. (R)-2-((1R,3S,5S)-3-((3S,4R)-1-(5-Chloropyrimidin-2-yl)-3-ethoxypiperidin-4-yl)-8-azabicyclo[3.2.1]octan-8-yl)-2-cyclopropylacetamide represented by the following formula (III): or a pharmaceutically acceptable salt thereof. 5. (R)-2-Cyclopropyl-2-((1R,3S,5S)-3-((2S,4S)-1-(5-fluoropyrimidin-2-yl)-2-methylpiperidin-4-yl)-8-azabicyclo[3.2.1]octan-8-yl)acetamide, represented by the following formula (IV): or a pharmaceutically acceptable salt thereof. 6. An orexin receptor type 2 agonist comprising a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-5. 7. A pharmaceutical composition for the treatment of narcolepsy, comprising a pharmacologically effective dose of a compound or a pharmaceutically acceptable salt thereof according to claim 2 and one or more pharmaceutically acceptable additives. 8. A therapeutic agent for the treatment of narcolepsy, containing a pharmacologically effective dose of a compound or a pharmaceutically acceptable salt thereof according to claim 2. 9. A method for treating narcolepsy comprising administering to a subject a pharmacologically effective dose of the compound or a pharmaceutically acceptable salt thereof according to claim 2. 10. A compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5 for use in the treatment of narcolepsy. 11. Use of a compound or a pharmaceutically acceptable salt thereof according to claim 2 for the manufacture of a pharmaceutical composition intended for the treatment of narcolepsy. 12. A pharmaceutical composition for the treatment of cataplexy, comprising a pharmacologically effective dose of a compound or a pharmaceutically acceptable salt thereof according to claim 2 and one or more pharmaceutically acceptable additives. 13. A therapeutic agent for the treatment of cataplexy, comprising a pharmacologically effective dose of a compound or a pharmaceutically acceptable salt thereof according to claim 2. 14. A method for treating cataplexy, comprising administering to a subject a pharmacologically effective dose of a compound or a pharmaceutically acceptable salt thereof according to claim 2. 15. A compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5 for use in the treatment of cataplexy. 16. Use of a compound or a pharmaceutically acceptable salt thereof according to claim 2 for the manufacture of a pharmaceutical composition intended for the treatment of cataplexy.