JPWO2020059841A1 - Prion disease remedy - Google Patents

Abstract

To provide a compound that has an effect of suppressing the production of abnormal prions and is useful as a therapeutic agent for prion diseases. Formula (I): [In the formula, each symbol is as described in the specification. ], And a salt thereof, and a prion disease therapeutic agent containing the compound as an active ingredient.

Description

The present invention relates to a compound having an effect of suppressing the production of abnormal prion and a therapeutic agent for prion disease containing the compound as an active ingredient.

Prion disease is a fatal neurodegenerative disease that is thought to be caused by the accumulation of abnormal prion proteins in the brain. Currently, there is no effective treatment method for prion disease, so the development of a drug for treating prion disease is considered to greatly contribute to future medical treatment. In addition, since prion disease occurs in a wide range of animals such as livestock such as cattle, sheep and deer, pets and wild animals, it is considered that it can also be used as an animal drug.
Various substances have been listed as candidates for the development of therapeutic agents for prion diseases, but (1) the anti-prion activity is insufficient, and (2) the molecular structure is not easy to optimize. The main organ affected by prion disease is the brain, but its effect is weak in vivo due to its low blood-brain barrier permeability, and (4) it has side effects such as liver dysfunction. However, it has not been put into practical use as a therapeutic drug.
The present inventors have found that the compound described in Patent Document 1 exerts an excellent effect in preventing the onset and progression of prion disease (Compound 1, GN8) (Non-Patent Document 1). Further, an improved compound thereof has been found (Patent Document 2 and Patent Document 3). However, from the viewpoint of pharmaceutical products, these compounds have problems such as being difficult to water due to their symmetric structure. In order to improve the problems of these compounds, the present inventors are also developing compounds obtained by prion in silico drug discovery as a new drug discovery development, and normal prions are being developed using the method of logical drug discovery. A compound with a molecular weight of 500 or less, which is predicted to bind to a protein, was searched for by docking simulation, and the effect of the prion disease model in an experimental system using the low molecular weight compounds (NPRs) was reported (Patent Document 4, Non-Patent Document 2). ). However, none of the compounds have solved all of the problems. The present inventors improved the accuracy of the number of compound libraries and calculation parameters in order to search for further effective compounds, evaluated the efficacy of the compounds obtained after recalculation in the experimental system of the prion disease model, and excellent effects. The structure was developed using the compound having the above as a lead compound, and a novel compound as a candidate for a therapeutic agent for prion disease listed in the present invention was found.

Japanese Unexamined Patent Publication No. 2009-13126 WO2010 / 131717 WO2015 / 119111 Japanese Unexamined Patent Publication No. 2018-35099

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1502-1507 EBioMedicine 9 (2016) 238-249

An object of the present invention is to provide a compound which has an effect of suppressing the production of abnormal prions and is useful as a therapeutic agent for prion diseases.

As a result of diligent studies to solve the above problems, the present inventors have found that the compound represented by the following formula (I) has an effect of suppressing the production of abnormal prions, and have completed the present invention.
That is, the present invention is as follows.
[1] Equation (I):

[During the ceremony,
Ring A represents a C _5-10 hydrocarbon ring or a 5- to 10-membered heterocycle;
R ¹ represents a hydrogen atom, a C _1-6 alkyl group or an optionally substituted 5- to 6-membered monocyclic aromatic heterocyclic group;
R ² indicates a hydrogen atom or an oxo group;
Alternatively, R ¹ and R ² combine to form a C _4-8 hydrocarbon ring;
R ³ indicates a hydrogen atom, a C _1-6 alkyl group or an optionally substituted amino group;
Y is -NR ⁴ R ⁵ or

Show;
R ⁴ and R ⁵ each independently represent a hydrogen atom or an optionally substituted C _1-6 alkyl group;
X indicates O or NR ⁶ ;
Ring B represents a 4- to 8-membered nitrogen-containing non-aromatic heterocycle;
n indicates 0 or 1;
R ⁶ represents a C _1-6 alkyl group, a C _3-10 cycloalkyl group or an optionally substituted C _1-6 alkyl-carbonyl group. ]
Compounds represented by (However, the following compounds:

except for. ) Or a salt thereof (may be abbreviated as "Compound (I)" in the present specification).
[2] Ring A is a C _6-10 allene ring or a 5- to 10-membered non-aromatic heterocycle;
R ¹ is substituted with 1 to 3 substituents selected from (1) hydrogen atom, (2) C _1-6 alkyl group or (3) (a) 5 to 6 member monocyclic aromatic heterocyclic group. A 5- to 6-membered monocyclic aromatic heterocycle optionally substituted with 1 to 3 substituents selected from the C _1-6 alkyl group and (b) C _{3-10 cycloalkyl group.} Is the basis;
R ² is hydrogen atom or an oxo group; or,
R ¹ and R ² combine to form a C _4-8 cycloalkene ring;
R ³ is 1 to 3 substituents selected from (1) hydrogen atom, (2) C _1-6 alkyl group or (3) (a) 5 to 6 member monocyclic non-aromatic heterocyclic group. optionally substituted with 1-3 substituents selected from optionally substituted C _1-6 alkyl group and (b) 5 to 6-membered monocyclic non-aromatic heterocyclic group C _1-6 An amino group optionally substituted with one or two substituents selected from alkyl-carbonyl groups;
Y is -NR ⁴ R ⁵ or

And;
R ⁴ and R ⁵ are independently composed of (1) hydrogen atom or (2) (a) mono- or di-C _1-6 alkylamino group and (b) 5- to 6-membered monocyclic non-aromatic group. _{A C 1-6} alkyl group optionally substituted with 1-3 substituents selected from the heterocyclic group;
Ring B is a 4- to 8-membered nitrogen-containing non-aromatic heterocycle;
X is O or NR ⁶ ;
n is 0 or 1;
R ⁶ is 1 to 3 substitutions selected from (1) C _1-6 alkyl group, (2) C _3-10 cycloalkyl group or (3) 5 to 6 member monocyclic non-aromatic heterocyclic group. _{A C 1-6} alkyl-carbonyl group that may be substituted with a group;
[1] The compound or salt thereof.
[3] Equation (I):

[During the ceremony,
Ring A represents a C _5-10 hydrocarbon ring or a 5- to 10-membered heterocycle;
R ¹ represents a hydrogen atom, a C _1-6 alkyl group or an optionally substituted 5- to 6-membered monocyclic aromatic heterocyclic group;
R ² indicates a hydrogen atom or an oxo group;
Alternatively, R ¹ and R ² combine to form a C _4-8 hydrocarbon ring;
R ³ indicates a hydrogen atom, a C _1-6 alkyl group or an optionally substituted amino group;
Y is -NR ⁴ R ⁵ or

Show;
R ⁴ and R ⁵ each independently represent a hydrogen atom or an optionally substituted C _1-6 alkyl group;
X indicates O or NR ⁶ ;
Ring B represents a 4- to 8-membered nitrogen-containing non-aromatic heterocycle;
n indicates 0 or 1;
R ⁶ represents a C _1-6 alkyl group, a C _3-10 cycloalkyl group or an optionally substituted C _1-6 alkyl-carbonyl group. ]
A drug for treating prion disease, which contains the compound represented by (1) or a salt thereof as an active ingredient.

The compound of the present invention has an effect of suppressing the production of abnormal prions and is useful as a therapeutic agent for prion diseases.

FIG. 1 shows the direct binding of a compound to recombinant PrP using SPR analysis. FIG. 2 shows the effect of NPR-130 and NPR-162 on cytotoxicity in non-prion infected cells (N2a-58). FIG. 3 shows the dose response of anti-prion compounds (NPR-130 and NPR-162) to ^{aberrant prion protein (PrP Sc} ) in prion-infected cells (N2a-FK). FIG. 4 shows images of the effects of anti-prion compounds (NPR-130 and NPR-162) on prion-infected cells (N2a-FK) observed under a microscope. FIG. 5 shows the evaluation of NPR-130 and -162 using a prion bioassay system. FIG. 6 shows the expression ^{of PrP Sc} at 100 dpi in prion-infected mice treated with NPR-130 or -162 and in the brain and spleen at the end of infection. FIG. 7 shows pathological changes in the brain and spleen of 100 d.p.i. of prion-infected mice treated with NPR-130 or -162. FIG. 8 shows the effect of NPRS compounds (26 types) on ^{PrP Sc} in cells continuously infected with prions (N2a-FK). FIG. 9 shows anti-prion compounds (NPRS-3, -6, -7, -9, -11, -17, -19, -20) against ^{aberrant prion protein (PrP Sc} ) in prion-infected cells (N2a-FK). , -21, -23, -24, -25) dose response. FIG. 10 shows the binding affinity screening for PrP of the compound NPRS group using SPR analysis. FIG. 11 shows the direct binding of the compound to recombinant PrP using SPR analysis. FIG. 12 shows the direct binding of the compound to recombinant PrP using SPR analysis.

Hereinafter, the present invention will be described in detail.
Hereinafter, the definition of each substituent used in the present specification will be described in detail. Unless otherwise specified, each substituent has the following definitions.

In the present specification, the "C _1-6 alkyl group" includes, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 1-ethylpropyl and hexyl. , Isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 2-ethylbutyl.
In the present specification, the "C _3-10 cycloalkyl group" includes, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, bicyclo [2.2.1] heptyl, bicyclo [ 2.2.2] Octyl, Bicyclo [3.2.1] Octyl, Adamantyl can be mentioned.
In the present specification, the "C _1-6 alkyl-carbonyl group" includes, for example, acetyl, propanoyl, butanoyl, 2-methylpropanol, pentanoyl, 3-methylbutanoyl, 2-methylbutanoyl, 2,2-. Examples thereof include dimethylpropanoyl, hexanoyl and heptanoyle.

In the present specification, the "5- to 6-membered monocyclic aromatic heterocyclic group" refers to, for example, 1 to 4 heteroatoms selected from a nitrogen atom, a sulfur atom and an oxygen atom in addition to a carbon atom as a ring-constituting atom. Examples thereof include a 5-membered 6-membered monocyclic aromatic heterocyclic group containing.
Preferable examples of the "5- to 6-membered monocyclic aromatic heterocyclic group" include thienyl, frill, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isooxazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridadinyl, 1, Examples thereof include 2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, triazolyl, tetrazolyl and triazinyl.

In the present specification, as the "5- to 6-membered monocyclic non-aromatic heterocyclic group", for example, 1 to 4 hetero selected from a nitrogen atom, a sulfur atom and an oxygen atom in addition to a carbon atom as a ring-constituting atom. Examples thereof include a 5-membered 6-membered monocyclic non-aromatic heterocyclic group containing an atom.
Preferable examples of the "5- to 6-membered monocyclic non-aromatic heterocyclic group" are tetrahydrothienyl, tetrahydrofuranyl, pyrrolinyl, pyrrolidinyl, imidazolinyl, imidazolidinyl, oxazolinyl, oxazolidinyl, pyrazolinyl, pyrazolydinyl, thiazolinyl, thiazolidinyl, tetrahydro. Isothiazolyl, tetrahydrooxazolyl, tetrahydroisooxazolyl, piperidinyl, piperazinyl, tetrahydropyranyl, dihydropyridinyl, dihydrothiopyranyl, tetrahydropyrimidinyl, tetrahydropyridadinyl, dihydropyranyl, tetrahydropyranyl , Tetrahydropyranyl, morpholinyl, thiomorpholinyl.

In the present specification _{, examples of the "C 5-10} hydrocarbon ring" include a C _5-10 cycloalkane ring, a C _5-10 cycloalkene ring, and a C _6-10 arene ring.
Preferable examples of the "C _5-10 cycloalkane ring" include a cyclopentane ring, a cyclohexane ring, a cycloheptane ring, a cyclooctane ring, a cyclononane ring, and a cyclodecane ring.
Preferable examples of the "C _5-10 cycloalkene ring" include a cyclopentene ring, a cyclohexene ring, a cycloheptene ring, a cyclooctene ring, a cyclononene ring, and a cyclodecene ring.
Preferable examples of the "C _6-10 arene ring" include a benzene ring and a naphthalene ring.

In the present specification _{, examples of the "C 4-8} hydrocarbon ring" include a C _4-8 cycloalkane ring, a C _4-8 cycloalkene ring, and a C ₆ arene ring.
Preferable examples of the "C _4-8 cycloalkane ring" include a cyclobutane ring, a cyclopentane ring, a cyclohexane ring, a cycloheptane ring, and a cyclooctane ring.
Preferable examples of the "C _4-8 cycloalkene ring" include a cyclobutene ring, a cyclopentene ring, a cyclohexene ring, a cycloheptene ring, and a cyclooctene ring.
Preferable examples of the "C ₆ arene ring" include a benzene ring.

In the present specification, the "5- to 10-membered heterocycle" includes, for example, 1 to 4 heteroatoms selected from a nitrogen atom, a sulfur atom and an oxygen atom in addition to a carbon atom as constituent atoms. Examples include 10-membered aromatic heterocycles and 5- to 10-membered non-aromatic heterocycles.
Suitable examples of the "5- to 10-membered aromatic heterocycle" are thiophene, furan, pyrrole, imidazole, pyrazole, thiazole, isothiazole, oxazole, isooxazole, pyridine, pyrazine, pyrimidine, pyridazine, 1, 2, 5- to 6-membered monocyclic aromatic heterocycles such as 4-oxazole, 1,3,4-oxadiazole, 1,2,4-thiazole, 1,3,4-thiazole, triazole, tetrazole, triazine, etc. ;
Benzothiophene, benzofuran, benzoimidazole, benzoxazole, benzoisoxazole, benzothiazole, benzoisothiazole, benzotriazole, imidazolepyridine, thienopyridine, flopyridine, pyrrolopyridine, pyrazolopyridine, oxazolopyridine, thiazolopyridine, imidazolepyrimidine, Imidazopyrimidine, thienopyrimidine, flopyrimidine, pyrolopyrimidine, pyrazolopyrimidine, oxazolopyrimidine, thiazolopyrimidine, pyrazolopyrimidine, pyrazorotridin, indole, isoindole, 1H-indazole, purine, isoquinoline, quinoline, phthalazine, naphthylidine. , 8 to 10-membered fused aromatic heterocycles such as quinoxalin, quinazoline, cinnoline and the like.
Preferable examples of the "5- to 10-membered non-aromatic heterocycle" are tetrahydrothiophene, tetrahydrofuran, pyrrolin, pyrrolidine, imidazoline, imidazolidine, oxazoline, oxazolidine, pyrazoline, pyrazolidine, thiazolin, thiazolidine, tetrahydroisothiazole, tetrahydro. Oxazole, tetrahydroisoxazole, piperidine, piperazin, tetrahydropyridine, dihydropyridine, dihydrothiopyran, tetrahydropyranmidin, tetrahydropyrandazine, dihydropyran, tetrahydropyran, tetrahydropyran, morpholine, thiomorpholin, azepan, diazepan, azepine, azocan, diazocan, 5- to 8-membered monocyclic non-aromatic heterocycles such as oxepan;
Dihydrobenzofuran, dihydrobenzimidazole, dihydrobenzoxazole, dihydrobenzothiazole, dihydrobenzoisothiazole, tetrahydroisoquinoline, tetrahydroquinoline, 4H-quinoline, indolin, isoinazoline, tetrahydrothieno [2,3-c] pyridine, tetrahydroquinoxaline, tetrahydro Examples thereof include 9 to 10-membered fused non-aromatic heterocycles such as phthalazine, tetrahydronaphthylidine, tetrahydroquinazoline, tetrahydrocinnoline, octahydroisoquinoline, benzpyridine and dihydroquinoline.

In the present specification, the "4- to 8-membered nitrogen-containing non-aromatic heterocycle" contains, for example, two nitrogen atoms or one nitrogen atom and one oxygen atom in addition to the carbon atom as constituent atoms. Examples include 4- to 8-membered nitrogen-containing non-aromatic heterocycles.
Preferable examples of the "4- to 8-membered nitrogen-containing aromatic heterocycle" include diazetidine (eg, 1,3-diazetidine), oxazetidine (eg, 1,3-oxazetidine), tetrahydropyrazole, tetrahydroimidazole, oxazolidine, and the like. Isoxazolidine, piperazin, morpholine, oxazepan (eg 1,4-oxazepan), diazepan (eg 1,4-diazepan), diazacyclooctane (eg 1,5-diazacyclooctane), oxazocan (eg, 1,5-diazacyclooctane). 1,4-oxazocan).

In the present specification, the "optionally substituted amino group" includes, for example, a C _1-6 alkyl group, a C _3-10 cycloalkyl group and a C _1-6 alkyl-carbonyl group, respectively, which may be substituted. Examples thereof include amino groups which may be substituted with one or two substituents selected from the above.
Suitable examples of optionally substituted amino groups are amino groups, optionally _{substituted mono- or di-C 1-6} alkylamino groups, optionally substituted mono- or di-C _{3 -10} cycloalkylamino groups, optionally substituted mono- or di-C _1-6 alkyl-carbonylamino groups can be mentioned.

In the present specification, the "mono- or di-C _1-6 alkylamino group" includes, for example, methylamino, ethylamino, propylamino, isopropylamino, butylamino, dimethylamino, diethylamino, dipropylamino and dibutylamino. , N-ethyl-N-methylamino.
In the present specification, examples of the "mono- or di-C _3-10 cycloalkylamino group" include cyclopropylamino and cyclohexylamino.
In the present specification, examples of the "mono- or di-C _1-6 alkyl-carbonylamino group" include acetylamino, propanoylamino and butanoylamino.

In the present specification, examples of the substituent of "may be substituted" include a halogen atom, a hydroxy group, a cyano group, a nitro group, a carboxy group, a C _1-6 alkyl group, and a C _3-10 cycloalkyl. Examples include groups, C _1-6 alkyl-carbonyl groups, 5- to 6-membered monocyclic aromatic heterocyclic groups, 5- to 6-membered monocyclic non-aromatic heterocyclic groups, and optionally substituted amino groups. The number of substituents is, for example, 1 to 5, preferably 1 to 3. When a plurality of substituents are present, each substituent may be the same or different.

The definition of each symbol in the formula (I) will be described in detail below.
Ring A represents a C _5-10 hydrocarbon ring or a 5- to 10-membered heterocycle.
As the "C _5-10 hydrocarbon ring" represented _{by ring A, a C 6-10} arene ring (eg, a benzene ring) is preferable.
As the "5- to 10-membered heterocycle" represented by ring A, a 5- to 10-membered non-aromatic heterocycle (eg, benzopyran ring, dihydroquinoline ring) is preferable.
Ring A is preferably a C _6-10 allene ring (eg, benzene ring) or a 5- to 10-membered non-aromatic heterocycle (eg, benzopyran ring, dihydroquinoline ring), more preferably C _6-10. It is an arene ring (eg, a benzene ring).

R ¹ represents a hydrogen atom, a C _1-6 alkyl group or an optionally substituted 5- to 6-membered monocyclic aromatic heterocyclic group, and R ² represents a hydrogen atom or an oxo group, or R. ¹ and R ² combine to form a C _4-8 hydrocarbon ring.

Represented by R ¹ of the "5 to optionally substituted 6-membered monocyclic aromatic heterocyclic group" as "substituent" substituted C _1-6 alkyl group (e.g., methyl, tert-butyl, 2-methylbutane-2-yl) and C _3-10 cycloalkyl groups (eg, cyclohexyl) are preferred.
R ¹ is
Preferably, (1) a hydrogen atom, (2) a C _1-6 alkyl group (eg, methyl) or (3) (a) an optionally substituted C _1-6 alkyl group (eg, methyl, tert-butyl). , 2-Methylbutane-2-yl) and (b) 5- to 6-membered monocyclic fragrances optionally substituted with 1-3 substituents selected from _{C 3-10 cycloalkyl groups (eg, cyclohexyl).} Group heterocyclic group (eg, tetrazolyl),
More preferably, it is selected from (1) a hydrogen atom, (2) a C _1-6 alkyl group (eg, methyl) or (3) (a) a 5- to 6-membered monocyclic aromatic heterocyclic group (eg, frills). _{C 1-6} alkyl groups (eg, methyl, tert-butyl, 2-methylbutane-2-yl) and (b) C _3-10 cycloalkyl groups, which may be substituted with 1 to 3 substituents. A 5- to 6-membered monocyclic aromatic heterocyclic group (eg, tetrazolyl) which may be substituted with 1 to 3 substituents selected from (eg, cyclohexyl).
More preferably, it may be substituted with 1 to 3 substituents selected from (1) hydrogen atom or (2) (a) 5- to 6-membered monocyclic aromatic heterocyclic groups (eg, frills). With _{1-3 substituents selected from C 1-6} alkyl groups (eg, methyl, tert-butyl, 2-methylbutane-2-yl) and (b) C _3-10 cycloalkyl groups (eg, cyclohexyl). It is a 5- to 6-membered monocyclic aromatic heterocyclic group (eg, tetrazolyl) which may be substituted.

As the "C _4-8 hydrocarbon ring" ^{formed by bonding R 1} and R ² _{, a C 4-8} cycloalkene ring (eg, a cyclohexene ring) is preferable.

R ³ represents a hydrogen atom, a C _1-6 alkyl group or an optionally substituted amino group.
The "substituent" of the "optionally substituted amino group" represented by ^{R 3} _{includes a optionally substituted C 1-6} alkyl group (eg, ethyl) and a optionally substituted C _{1-. 6} Alkyl-carbonyl groups (eg, methylcarbonyl, ethylcarbonyl) are preferred.
R ³ is
Preferably, (1) a hydrogen atom, (2) a C _1-6 alkyl group (eg, methyl, ethyl) or (3) (a) optionally substituted C _1-6 alkyl group (eg, ethyl) and (B) An amino group optionally substituted with one or two substituents selected from the optionally substituted _{C 1-6 alkyl-carbonyl group (eg, methylcarbonyl, ethylcarbonyl).}
More preferably, (1) a hydrogen atom, (2) a C _1-6 alkyl group (eg, methyl, ethyl) or (3) (a) a 5- to 6-membered monocyclic non-aromatic heterocyclic group (eg, pyrrolidinyl). _{C 1-6} alkyl group (eg, ethyl) and (b) 5- to 6-membered monocyclic non-aromatic heterocyclic group (eg, pyrrolidinyl) which may be substituted with 1 to 3 substituents selected from). , Piperidinyl) and optionally substituted with _{1 or 2 substituents selected from C 1-6} alkyl-carbonyl groups (eg, methylcarbonyl, ethylcarbonyl). It is an amino group that may be present
_{More preferably, a C 1-6} alkyl-carbonyl group which may be substituted with 1 to 3 substituents selected from 5- to 6-membered monocyclic non-aromatic heterocyclic groups (eg, pyrrolidinyl, piperidinyl) (eg, pyrrolidinyl, piperidinyl). An amino group which may be substituted with one or two substituents selected from (eg, methylcarbonyl, ethylcarbonyl).

Y is -NR ⁴ R ⁵ or

, R ⁴ and R ⁵ each independently represent a hydrogen atom or an optionally substituted C _1-6 alkyl group, where X represents O or NR ⁶ and ring B is 4-8. It represents a member nitrogen-containing non-aromatic heterocycle, where n represents 0 or 1, and R ⁶ is a C _1-6 alkyl group, a C _3-10 cycloalkyl group or an optionally substituted C _1-6 alkyl. -Indicates a carbonyl group.

The "substituent" of the "optionally substituted C _1-6 alkyl group" represented by R ⁴ or R ⁵ _{includes mono- or di-C 1-6} alkylamino groups (eg, diethylamino) and 5 to 5 to. A 6-membered monocyclic non-aromatic heterocyclic group (eg, morpholinyl) is preferred.
R ⁴ and R ⁵ are preferably independent of each other, preferably (1) a hydrogen atom or (2) (a) a mono- or di-C _1-6 alkylamino group (eg, diethylamino) and (b) 5 to. _{A C 1-6} alkyl group (eg, ethyl, n-propyl) optionally substituted with 1-3 substituents selected from 6-membered monocyclic non-aromatic heterocyclic groups (eg, morpholinyl). ..

As the "4- to 8-membered non-aromatic heterocycle" represented by ring B, a morpholine ring is preferable when X represents O, and a piperazine ring is preferable when ^{X represents NR 6.}

"Optionally substituted C _1-6 alkyl - carbonyl group" represented by R ⁶ as "substituent" of 5 to 6-membered monocyclic non-aromatic Hajime Tamaki (e.g., pyrrolidinyl) are preferred.
R ⁶ is,
Preferably, (1) a C _1-6 alkyl group (eg, methyl, isopropyl), (2) a C _3-10 cycloalkyl group (eg, cyclopentyl, cyclohexyl) or (3) a 5- to 6-membered monocyclic non-aromatic group. _{A C 1-6} alkyl-carbonyl group (eg, methylcarbonyl) which may be substituted with 1 to 3 substituents selected from the group heterocyclic groups (eg, pyrrolidinyl).
More preferably, it is (1) a C _1-6 alkyl group (eg, isopropyl) or (2) a C _3-10 cycloalkyl group (eg, cyclopentyl, cyclohexyl).
More preferably, it is a C _3-10 cycloalkyl group (eg, cyclopentyl, cyclohexyl).

Preferable examples of compound (I) include the following compounds.
[Compound I-1]
Ring A is a C _6-10 allene ring (eg, benzene ring) or a 5- to 10-membered non-aromatic heterocycle (eg, benzopyran ring, dihydroquinoline ring);
R ¹ is (1) a hydrogen atom, (2) a C _1-6 alkyl group (eg, methyl) or (3) (a) an optionally substituted C _1-6 alkyl group (eg, methyl, tert-). 5- to 6-membered monocyclic group optionally substituted with 1-3 substituents selected from butyl, 2-methylbutane-2-yl) and (b) C _{3-10 cycloalkyl groups (eg, cyclohexyl).} It is an aromatic heterocyclic group (eg, tetrazolyl);
R ² is hydrogen atom or an oxo group; or,
R ¹ and R ² combine to form a C _4-8 cycloalkene ring (eg, cyclohexene ring);
R ³ is (1) a hydrogen atom, (2) a C _1-6 alkyl group (eg, methyl, ethyl) or (3) (a) an optionally substituted C _1-6 alkyl group (eg, ethyl). And (b) an amino group optionally substituted with one or two substituents selected from the optionally substituted _{C 1-6 alkyl-carbonyl group (eg, methylcarbonyl, ethylcarbonyl);}
Y is -NR ⁴ R ⁵ or

And;
R ⁴ and R ⁵ are independent of (1) hydrogen atom or (2) (a) mono- or di-C _1-6 alkylamino group (eg, diethylamino) and (b) 5-6 members. _{A C 1-6} alkyl group (eg, ethyl, n-propyl) optionally substituted with 1-3 substituents selected from cyclic non-aromatic heterocyclic groups (eg, morpholinyl);
Ring B is a 4- to 8-membered nitrogen-containing non-aromatic heterocycle (eg, morpholine ring, piperazine ring);
X is O or NR ⁶ ;
n is 0 or 1;
R ⁶ is (1) a C _1-6 alkyl group (eg, methyl, isopropyl), (2) a C _3-10 cycloalkyl group (eg, cyclopentyl, cyclohexyl) or (3) a 5- to 6-membered monocyclic non-cyclic group. _{A C 1-6} alkyl-carbonyl group (eg, methylcarbonyl) which may be substituted with 1-3 substituents selected from aromatic heterocyclic groups (eg, pyrrolidinyl);
Compound (I).

[Compound I-2]
Ring A is a C _6-10 allene ring (eg, benzene ring) or a 5- to 10-membered non-aromatic heterocycle (eg, benzopyran ring, dihydroquinoline ring);
R ¹ is selected from (1) hydrogen atom, (2) C _1-6 alkyl group (eg, methyl) or (3) (a) 5 to 6 member monocyclic aromatic heterocyclic group (eg, frill). _{C 1-6} alkyl groups (eg, methyl, tert-butyl, 2-methylbutane-2-yl) and (b) C _3-10 cycloalkyl groups, which may be substituted with 1 to 3 substituents. A 5- to 6-membered monocyclic aromatic heterocyclic group (eg, tetrazolyl) which may be substituted with 1-3 substituents selected from (eg, cyclohexyl);
R ² is hydrogen atom or an oxo group; or,
R ¹ and R ² combine to form a C _4-8 cycloalkene ring (eg, cyclohexene ring);
R ³ is (1) a hydrogen atom, (2) a C _1-6 alkyl group (eg, methyl, ethyl) or (3) (a) a 5- to 6-membered monocyclic non-aromatic heterocyclic group (eg, pyrrolidinyl). _{C 1-6} alkyl group (eg, ethyl) and (b) 5- to 6-membered monocyclic non-aromatic heterocyclic group (eg, pyrrolidinyl) which may be substituted with 1 to 3 substituents selected from). , Piperidinyl) and optionally substituted with _{1 or 2 substituents selected from C 1-6} alkyl-carbonyl groups (eg, methylcarbonyl, ethylcarbonyl). It is an amino group that may be;
Y is -NR ⁴ R ⁵ or

And;
R ⁴ and R ⁵ are independent of (1) hydrogen atom or (2) (a) mono- or di-C _1-6 alkylamino group (eg, diethylamino) and (b) 5-6 members. _{A C 1-6} alkyl group (eg, ethyl, n-propyl) optionally substituted with 1-3 substituents selected from cyclic non-aromatic heterocyclic groups (eg, morpholinyl);
Ring B is a 4- to 8-membered nitrogen-containing non-aromatic heterocycle (eg, morpholine ring, piperazine ring);
X is O or NR ⁶ ;
n is 0 or 1;
R ⁶ is (1) a C _1-6 alkyl group (eg, methyl, isopropyl), (2) a C _3-10 cycloalkyl group (eg, cyclopentyl, cyclohexyl) or (3) a 5- to 6-membered monocyclic non-cyclic group. _{A C 1-6} alkyl-carbonyl group (eg, methylcarbonyl) which may be substituted with 1-3 substituents selected from aromatic heterocyclic groups (eg, pyrrolidinyl);
Compound (I).

[Compound I-3]
Ring A is a C _6-10 arene ring (eg, a benzene ring);
R ¹ is selected from (1) hydrogen atom, (2) C _1-6 alkyl group (eg, methyl) or (3) (a) 5 to 6 member monocyclic aromatic heterocyclic group (eg, frill). _{C 1-6} alkyl groups (eg, methyl, tert-butyl, 2-methylbutane-2-yl) and (b) C _3-10 cycloalkyl groups, which may be substituted with 1 to 3 substituents. A 5- to 6-membered monocyclic aromatic heterocyclic group (eg, tetrazolyl) which may be substituted with 1-3 substituents selected from (eg, cyclohexyl);
R ² is a hydrogen atom; or
R ¹ and R ² combine to form a C _4-8 cycloalkene ring (eg, cyclohexene ring);
R ³ may be substituted with 1-3 substituents selected from 5- to 6-membered monocyclic non-aromatic heterocyclic groups (eg, pyrrolidinyl, piperidinyl) C _1-6 alkyl-carbonyl groups (eg, pyrrolidinyl, piperidinyl). An amino group which may be substituted with one or two substituents selected from (eg, methylcarbonyl, ethylcarbonyl);
Y is -NR ⁴ R ⁵ or

And;
R ⁴ and R ⁵ are independent of (1) hydrogen atom or (2) (a) mono- or di-C _1-6 alkylamino group (eg, diethylamino) and (b) 5-6 members. _{A C 1-6} alkyl group (eg, ethyl, n-propyl) optionally substituted with 1-3 substituents selected from cyclic non-aromatic heterocyclic groups (eg, morpholinyl);
Ring B is a 4- to 8-membered nitrogen-containing non-aromatic heterocycle (eg, piperazine ring);
X is NR ⁶ ;
n is 0;
R ⁶ is (1) a C _1-6 alkyl group (eg, isopropyl) or (2) a C _3-10 cycloalkyl group (eg, cyclopentyl, cyclohexyl);
Compound (I).

[Compound I-4]
Ring A is a C _6-10 arene ring (eg, a benzene ring);
R ¹ may be substituted with (1) a hydrogen atom or (2) 1 to 3 substituents selected from (a) 5- to 6-membered monocyclic aromatic heterocyclic groups (eg, frills). With _{1-3 substituents selected from C 1-6} alkyl groups (eg, methyl, tert-butyl, 2-methylbutane-2-yl) and (b) C _3-10 cycloalkyl groups (eg, cyclohexyl). It is a 5- to 6-membered monocyclic aromatic heterocyclic group (eg, tetrazolyl) that may be substituted;
R ² is a hydrogen atom; or
R ¹ and R ² combine to form a C _4-8 cycloalkene ring (eg, cyclohexene ring);
R ³ may be substituted with 1-3 substituents selected from 5- to 6-membered monocyclic non-aromatic heterocyclic groups (eg, pyrrolidinyl, piperidinyl) C _1-6 alkyl-carbonyl groups (eg, pyrrolidinyl, piperidinyl). An amino group which may be substituted with one or two substituents selected from (eg, methylcarbonyl, ethylcarbonyl);
Y is

And;
Ring B is a 4- to 8-membered nitrogen-containing non-aromatic heterocycle (eg, piperazine ring);
X is NR ⁶ ;
n is 0;
R ⁶ is a C _3-10 cycloalkyl group (eg, cyclopentyl, cyclohexyl);
Compound (I).

[Compound I-5]
Ring A is a C _6-10 arene ring (eg, a benzene ring);
R ¹ may be substituted with (1) a hydrogen atom or (2) 1 to 3 substituents selected from (a) 5- to 6-membered monocyclic aromatic heterocyclic groups (eg, frills). With _{1-3 substituents selected from C 1-6} alkyl groups (eg, methyl, tert-butyl, 2-methylbutane-2-yl) and (b) C _3-10 cycloalkyl groups (eg, cyclohexyl). It is a 5- to 6-membered monocyclic aromatic heterocyclic group (eg, tetrazolyl) that may be substituted;
R ² is a hydrogen atom; or
R ¹ and R ² combine to form a C _4-8 cycloalkene ring (eg, cyclohexene ring);
R ³ may be substituted with 1 to 3 substituents selected from (a) 5- to 6-membered monocyclic non-aromatic heterocyclic groups (eg, pyrrolidinyl) (eg, C _1-6 alkyl groups). _{, Ethyl) and (b) C 1-6} alkyl-may be substituted with 1-3 substituents selected from 5- to 6-membered monocyclic non-aromatic heterocyclic groups (eg, pyrrolidinyl, piperidinyl). An amino group optionally substituted with one or two substituents selected from carbonyl groups (eg, methylcarbonyl, ethylcarbonyl);
Y is -NR ⁴ R ⁵ or

And;
R ⁴ and R ⁵ are each independently composed of 1 to 3 substituents selected from (1) a hydrogen atom or (2) a 5- to 6-membered monocyclic non-aromatic heterocyclic group (eg, morpholinyl). It is a C _1-6 alkyl group (eg, n-propyl) that may be substituted;
Ring B is a 4- to 8-membered nitrogen-containing non-aromatic heterocycle (eg, piperazine ring);
X is NR ⁶ ;
n is 0 or 1;
R ⁶ is a C _3-10 cycloalkyl group (eg, cyclopentyl, cyclohexyl);
Compound (I).

Specific examples of the compound (I) include the compounds of Examples 1 to 28 described later.

When the compound (I) is a salt, such salts include, for example, a salt with an inorganic base, a salt with an organic base, a salt with an inorganic acid, a salt with an organic acid, a basic or acidic amino acid. Examples include salt.

Preferable examples of salts with inorganic bases include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt and magnesium salt; aluminum salt; ammonium salt.

Suitable examples of salts with organic bases include trimethylamine, triethylamine, pyridine, picolin, ethanolamine, diethanolamine, triethanolamine, tromethamine [tris (hydroxymethyl) methylamine], tert-butylamine, cyclohexylamine, benzylamine, Examples thereof include salts with dicyclohexylamine and N, N-dibenzylethylenediamine.

Preferable examples of salts with inorganic acids include salts with hydrogen chloride, hydrogen bromide, nitric acid, sulfuric acid and phosphoric acid.

Suitable examples of salts with organic acids are formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid. , Salt with p-toluene sulfonic acid.

Preferable examples of salts with basic amino acids include salts with arginine, lysine and ornithine.

Preferable examples of the salt with an acidic amino acid include a salt with aspartic acid and glutamic acid.

Among these salts, pharmaceutically acceptable salts are preferable. Pharmaceutically acceptable salts include salts with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, etc., if the compound has a basic functional group; or acetic acid, phthalic acid. , Fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid, p-toluenesulfonic acid and other organic acids. If the compound has an acidic functional group, it is an inorganic salt such as an alkali metal salt (eg, sodium salt, potassium salt, etc.), an alkaline earth metal salt (eg, calcium salt, magnesium salt, barium salt, etc.). ; Ammonium salt and the like can be mentioned.

Further, Compound (I), an isotope ^{(eg, 3 H, 13 C, 14} C, 18 F, 35 S, 125 I) may be labeled with the like.
Furthermore, compound (I) may be a hydrate, a non-hydrate, a non-solvate, or a solvate.
Further, ^{a deuterium converter obtained by converting 1} H to ² H (D) is also included in the compound (I).

If compound (I) has an asymmetric center, isomers such as enantiomers or diastereomers may be present. All such isomers and mixtures thereof are included within the scope of the invention. In addition, isomers may be produced by conformation or tautomerism, and such isomers or mixtures thereof are also included in the compound (I) of the present invention.

Next, a method for producing the compound (I) of the present invention will be described.
For example, ^{compound (I) in which R 1} may be substituted tetrazolyl and n is 0 can be prepared according to the following scheme.

(In the formula, R indicates a substituent of "tetrazolyl which may be substituted", and other symbols are as defined above.)
The reaction conditions can be appropriately determined with reference to, for example, Examples.

Further, for example, ^{the compound (I) in which R 1} and R ² are bonded to form a C _4-8 hydrocarbon ring and n is 0 can be produced according to the following scheme.

(In the formula, each symbol is as defined above.)
The reaction conditions can be appropriately determined with reference to, for example, Examples.

Alternatively, for example, compound (I) can be prepared according to the following scheme.

(In the formula, m indicates 0 or 1, Z indicates a leaving group such as a halogen atom (eg, chlorine atom), a methanesulfonate group, and other symbols are as defined above.)
The reaction conditions can be appropriately determined with reference to, for example, Examples.

By converting the substituent of the compound (I) thus obtained by applying a means known per se (that is, introduction of a substituent or conversion of a functional group), another substance contained in the compound (I) can be obtained. Compounds or salts thereof can also be produced.
As a method for introducing a substituent or converting a functional group, a known general method is used. For example, a halogen atom (eg, fluorine, chlorine, bromine, iodine) or a halogenated C _1-6 alkyl may be used. Methyl group, cyclopropyl group, vinyl group, cyano group, formyl group, carbonyl group, carboxyl group of sulfonyl-oxy group [eg, methanesulfonyloxy, ethanesulfonyloxy, trichloromethanesulfonyloxy, trifluoromethanesulfonyloxy (triflate)] , Conversion to hydroxyl group, amino group, boryl group, etc., conversion of formyl group to ethynyl group by Saifers-Gilbert carbon increase reaction, conversion to carboxy group by hydrolysis of ester, conversion to carbamoyl group by amidation of carboxy group Conversion, conversion of carboxy group to hydroxymethyl group, reduction of carbonyl group to methylene group, reduction of carbonyl group or conversion to alcohol by alkylation, reductive amination of carbonyl group, carbonyl group Oxymation of, amino group acylation, amino group ureaization, amino group sulfonylation, amino group alkylation, active halogen substitution or amination with amine, hydroxy group alkylation, hydroxy group substitution or amino Is mentioned.
When introducing a substituent or converting a functional group, if there is a reactive site where a reaction other than the intended one occurs, a protecting group is introduced into the reactive site in advance by a means known per se as necessary. After carrying out the desired reaction, the protecting group can also be removed by means known per se to produce a compound included in the scope of the present invention.
For example, when the raw material compound or the intermediate has an amino group, a carboxyl group or a hydroxyl group as a substituent, these groups may be protected by a protecting group generally used in peptide chemistry and the like. In this case, the target compound can be obtained by removing the protecting group as necessary after the reaction.

The compound (I) obtained by the above-mentioned production method can be isolated and purified by known means such as solvent extraction, pH conversion of a solution, dissolution, crystallization, recrystallization and chromatography.

The compound (I) of the present invention has an effect of suppressing the production of abnormal prions and is useful as a therapeutic agent for prion diseases and the like.
Although "prion disease" is classified into sporadic, familial (hereditary) and acquired (infectious), the compounds of the present invention are preferably administered to familial prion disease and acquired prion disease, and are family members. It is preferably administered due to sex prion disease.
Prion diseases include sheep scrapy, bovine spongy encephalopathy, Creutzfeldt-Jakob disease, chronic wasting disease, transmissible mink encephalopathy, cat spongiform encephalopathy, Gerstmann-Sträsler-Scheinker syndrome, and fatal familial insomnia. The compound of the present invention is suitable for Creutzfeldt-Jakob disease and the like.
As used herein, "treatment" includes improvement of symptoms, prevention of aggravation, maintenance of remission, and prevention of recurrence.
The compound (I) of the present invention exhibits an effect of suppressing the production of abnormal prion, and the compound (I) of the present invention has efficacy expression, pharmacokinetics (eg, absorbability, distribution, metabolism, excretion), solubility ( (Eg, water-soluble), interaction with other drugs (eg, drug-metabolizing enzyme inhibitory action), safety (eg, acute toxicity, chronic toxicity, genetic toxicity, reproductive toxicity, cardiotoxicity, carcinogenicity, central toxicity) It is also useful as a medicine because it is excellent in stability (eg, chemical stability, stability to enzymes).
Therefore, compound (I) of the present invention is used to suppress the production of aberrant prions in mammals (eg, mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys, humans). Can be used.

The compound (I) of the present invention can be administered orally or parenterally to a mammal (preferably human) as a pharmaceutical, as it is or by blending a pharmacologically acceptable carrier.
Hereinafter, a medicine containing the compound (I) of the present invention (may be abbreviated as “the medicine of the present invention”) will be described in detail. Dosage forms of the pharmaceuticals of the present invention include, for example, tablets (eg, sugar-coated tablets, film-coated tablets, sublingual tablets, buccal tablets, intraoral rapid-disintegrating tablets), pills, granules, powders, capsules (eg, soft capsules). Agents, microcapsules), syrups, emulsions, suspending agents, film agents (eg, orally disintegrating film, oral mucosal patch film) and other oral agents. The dosage forms of the pharmaceuticals of the present invention include, for example, injections, infusions, transdermal agents (eg, iontophoresis percutaneous agents), suppositories, ointments, nasal agents, transpulmonary agents, and eye drops. Parenteral preparations such as, etc. are also mentioned. Further, the pharmaceutical product of the present invention may be a release-controlled preparation such as a rapid-release preparation or a sustained-release preparation (eg, sustained-release microcapsule).

The pharmaceutical product of the present invention can be produced by a known production method generally used in the field of pharmaceutical technology (eg, the method described in the Japanese Pharmacopoeia). Further, in the pharmaceutical of the present invention, if necessary, excipients, binders, disintegrants, lubricants, sweeteners, surfactants, suspending agents, emulsifiers, colorants commonly used in the pharmaceutical field are used. , Preservatives, fragrances, flavoring agents, stabilizers, thickeners and other additives can be appropriately contained.
Examples of the above-mentioned pharmacologically acceptable carrier include these additives.

For example, tablets can be made with excipients, binders, disintegrants, lubricants and the like, and pills and granules can be made with excipients, binders, disintegrants and the like. Further, powders and capsules can be produced using excipients and the like, syrups can be produced using sweeteners and the like, and emulsions or suspensions can be produced using suspending agents, surfactants, emulsifiers and the like.

Examples of excipients include lactose, sucrose, glucose, starch, sucrose, microcrystalline cellulose, citrus powder, mannitol, sodium hydrogencarbonate, calcium phosphate, calcium sulfate.
Examples of the binder include 5 to 10% by weight starch paste solution, 10 to 20% by weight Arabica rubber solution or gelatin solution, 1 to 5% by weight tragant solution, carboxymethyl cellulose solution, sodium alginate solution, and glycerin.
Examples of disintegrants include starch and calcium carbonate.
Examples of lubricants include magnesium stearate, stearic acid, calcium stearate, and purified talc.
Examples of sweeteners include glucose, fructose, invert sugar, sorbitol, xylitol, glycerin, and simple syrup.
Examples of surfactants include sodium lauryl sulfate, polysorbate 80, sorbitan monofatty acid ester, and polyoxyl 40 stearate.
Examples of suspending agents include gum arabic, sodium alginate, sodium carboxymethyl cellulose, methyl cellulose and bentonite.
Examples of emulsifiers include gum arabic, tragant, gelatin and polysorbate 80.

For example, when the pharmaceutical agent of the present invention is a tablet, the tablet can be added to the compound (I) of the present invention, for example, an excipient (eg, lactose, sucrose, starch), a disintegrant (eg,) according to a method known per se. , Starch, calcium carbonate), binder (eg starch, gum arabic, carboxymethyl cellulose, polyvinylpyrrolidone, hydroxypropyl cellulose) or lubricant (eg talc, magnesium stearate, polyethylene glycol 6000) and compression molding Then, if necessary, it can be produced by coating by a method known per se for taste masking, enteric or persistent purposes. Examples of the coating agent used for coating include hydroxypropylmethyl cellulose, ethyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, polyoxyethylene glycol, Tween 80, Pluronic F68, cellulose acetate phthalate, hydroxypropylmethyl cellulose phthalate, hydroxymethyl cellulose acetate succinate, and the like. Eudragit (Roam, Germany, methacrylic acid / acrylic acid copolymer) and dyes (eg, Bengala, titanium dioxide) can be used.

In addition to intravenous injections, the injections include subcutaneous injections, intradermal injections, intramuscular injections, intraperitoneal injections, drip injections and the like.

Such injections are prepared by a method known per se, that is, by dissolving, suspending or emulsifying compound (I) of the present invention in a sterile aqueous or oily solution. Examples of the aqueous solution include physiological saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride) and the like. The aqueous solution contains suitable solubilizing agents such as alcohols (eg ethanol), polyalcohols (eg propylene glycol, polyethylene glycol), nonionic surfactants (eg polysorbate 80, HCO-50). You may. Examples of the oily liquid include sesame oil and soybean oil. The oily liquid may contain a suitable dissolution aid. Examples of the solubilizing agent include benzyl benzoate and benzyl alcohol. In addition, the injection includes a buffer (eg, phosphate buffer, sodium acetate buffer), a soothing agent (eg, benzalkonium chloride, prokine hydrochloride), a stabilizer (eg, human serum albumin, polyethylene glycol). , Preservatives (eg, benzyl alcohol, phenol) and the like may be blended. The prepared injection solution can usually be filled in an ampoule.

The content of the compound (I) of the present invention in the pharmaceutical product of the present invention varies depending on the form of the pharmaceutical product, but is usually about 0.01 to about 100% by weight, preferably about 2 to 2 to the total weight of the pharmaceutical product. It is about 85% by weight, more preferably about 5 to about 70% by weight.

The content of the additive in the pharmaceutical product of the present invention varies depending on the form of the preparation, but is usually about 1 to about 99.9% by weight, preferably about 10 to about 90% by weight, based on the whole preparation. be.

The compound (I) of the present invention is stable, has low toxicity, and can be used safely. The daily dose of compound (I) of the present invention varies depending on the patient's condition and body weight, the type of compound, the route of administration, etc., but for example, when orally administered to a patient for the purpose of treating cancer, an adult (body weight). Approximately 60 kg) The daily dose of compound (I) of the present invention is from about 1 to about 1000 mg, preferably from about 3 to about 300 mg, more preferably from about 10 to about 200 mg, once or twice or more. It can be administered in 3 divided doses.

When the compound (I) of the present invention is administered parenterally, it is usually administered in the form of a liquid preparation (eg, an injection). The single dose of compound (I) of the present invention varies depending on the subject to be administered, the target organ, symptoms, the administration method, etc., but for example, it is usually about 0.01 to about 100 mg per 1 kg of body weight, preferably about 0.01. It is preferred to administer to about 50 mg, more preferably about 0.01 to about 20 mg of compound (I) of the invention by intravenous injection.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited by the following examples, and is appropriately modified to the extent that it can meet the above-mentioned and the following purposes. Of course, it is possible to carry out, and all of them are included in the technical scope of the present invention.

(Reference Example 1) Synthesis of 4-(((tert-butyldimethylsilyl) oxy) methyl) aniline

(4-Aminophenol) Methanol (1.2 g) is dissolved in N, N-dimethylformamide (20 mL), tert-butyldimethylsilyl chloride (1.5 g) and imidazole (1.3 g) are added, and the mixture is added at room temperature. The mixture was stirred for 20 hours. The reaction mixture was diluted with water (50 mL) and then extracted with ethyl acetate. The extract was washed with saturated brine and dried over magnesium sulfate. After distilling off the solvent under reduced pressure, the residue was purified by column chromatography (developing solvent: ethyl acetate ester: hexane = 1:10) to obtain a yellow liquid product (1.9 g; yield 83%). ..
^{1 1} HNMR (500MHz, CDCl ₃ ) δ0.09 (s, 6H), 0.93 (s, 9H), 3.62 (br.s, NH ₂ ), 4.63 (s, 2H), 6.66 (D, J = 7.8Hz, 2H), 7.11 (d, J = 8.6Hz, 2H).

(Reference Example 2) Synthesis of 2-bromo-N- (4-(((tert-butyldimethylsilyl) oxy) methyl) phenyl) acetamide

4-(((tert-butyldimethylsilyl) oxy) methyl) aniline (1.9 g) is dissolved in dichloromethane (20 mL), cooled to 0 ° C., and then 2-bromoacetylbromid (1.1 mL), triethylamine ( 2.2 mL) was added, and the mixture was stirred at room temperature for 12 hours. The reaction mixture was diluted with water (50 mL) and then extracted with dichloromethane. The extract was washed with saturated brine and dried over magnesium sulfate. After distilling off the solvent under reduced pressure, the residue was purified by column chromatography (developing solvent: ethyl acetate ester: hexane = 1: 4) to obtain a yellow solid product (1.5 g; yield 53%). ..
^{1 1} HNMR (500MHz, CDCl ₃ ) δ0.10 (s, 6H), 0.95 (s, 9H), 4.04 (s, 2H), 4.72 (s, 2H), 7.32 (d, J = 8.3Hz, 2H), 7.50 (d, J = 8.3Hz, 2H), 8.11 (br.s, NH).

(Reference Example 3) Synthesis of N- (4-(((tert-butyldimethylsilyl) oxy) methyl) phenyl) -2- (pyrrolidin-1-yl) acetamide

2-bromo-N- (4-(((tert-butyldimethylsilyl) oxy) methyl) phenyl) acetylamide (1.5 g) was dissolved in tetrahydrofuran (30 mL), pyrrolidine (415 μL), potassium carbonate (1.2 g). ) Was added, and the mixture was stirred at room temperature for 20 hours. The reaction mixture was diluted with water (50 mL) and then extracted with ethyl acetate. The extract was washed with saturated brine and dried over magnesium sulfate. After distilling off the solvent under reduced pressure, the residue was purified by column chromatography (developing solvent: ethyl acetate ester: methanol = 10: 1) to obtain a yellow liquid product (910 mg; yield 62%).
^{1 1} HNMR (500MHz, CDCl ₃ ) δ0.10 (s, 6H), 0.95 (s, 9H), 1.85-1.88 (m, 4H), 2.70-2.71 (m, 4H) ), 3.28 (s, 2H), 4.71 (s, 2H), 7.29 (d, J = 8.1Hz, 2H), 7.54 (d, J = 8.3Hz, 2H), 9.09 (br.s, NH).

(Reference Example 4) Synthesis of N- (4- (hydroxymethyl) phenyl) -2- (pyrrolidin-1-yl) acetamide

N-(4-(((tert-butyldimethylsilyl) oxy) methyl) phenyl) -2- (pyrrolidin-1-yl) acetylamide (910 mg) was dissolved in tetrahydrofuran (20 mL) and TBAF was dissolved in tetrahydrofuran (1M, 1M,). 3.1 mL) was added, and the mixture was stirred at room temperature for 4 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (developing solvent: ethyl acetate ester: methanol = 10: 1) to obtain a yellow liquid product (596 mg; yield 98%).
^{1 1} HNMR (500MHz, CDCl ₃ ) δ1.82-1.91 (m, 4H), 2.64-2.74 (m, 4H), 3.29 (s, 2H), 4.67 (s, 2H) ), 7.34 (d, J = 8.4Hz, 2H), 7.59 (d, J = 8.3Hz, 2H), 9.13 (br.s, NH).

(Reference Example 5) Synthesis of N- (4-formylphenyl) -2- (pyrrolidin-1-yl) acetamide

N- (4- (Hydroxymethyl) phenyl) -2- (pyrrolidin-1-yl) acetamide (184 mg) was dissolved in dichloromethane (4 mL), Dess-Martin peryodinane (399 mg) was added, and the mixture was added at room temperature for 12 hours. Stirred. The reaction mixture was diluted with saturated aqueous sodium hydrogen carbonate solution (20 mL) and then extracted with ethyl acetate. The extract was washed successively with water and saturated brine, and dried over magnesium sulfate. After distilling off the solvent under reduced pressure, the residue was purified by column chromatography (developing solvent: ethyl acetate ester: methanol = 10: 1) to obtain a yellow liquid product (120 mg; yield 66%).
^{1 1} HNMR (500 MHz, CDCl ₃ ) δ1.891-1.92 (m, 4H), 2.72-2.78 (m, 4H), 3.36 (s, 2H), 7.78 (d, J) = 8.6Hz, 2H), 7.87 (d, J = 8.6Hz, 2H), 9.93 (s, 1H).

(Reference Example 6) Synthesis of N- (4-(((tert-butyldimethylsilyl) oxy) methyl) phenyl) -2- (piperidine-1-yl) acetamide

2-bromo-N- (4-(((tert-butyldimethylsilyl) oxy) methyl) phenyl) acetylamide (600 mg) is dissolved in tetrahydrofuran (10 mL), and pyrrolidine (165 μL) and potassium carbonate (460 mg) are added. , Stirred at room temperature for 20 hours. The reaction mixture was diluted with water (20 mL) and then extracted with ethyl acetate. The extract was washed with saturated brine and dried over magnesium sulfate. After distilling off the solvent under reduced pressure, the residue was purified by column chromatography (developing solvent: ethyl acetate ester: methanol = 10: 1) to obtain a yellow liquid product (343 mg; yield 57%).
^{1 1} HNMR (500MHz, CDCl ₃ ) δ0.10 (s, 6H), 0.94 (s, 9H), 1.46-1.53 (m, 1H), 1.63-1.68 (m, 4H) ), 2.52-2.58 (m, 4H), 3.07 (s, 2H), 4.71 (s, 2H), 7.29 (d, J = 8.3Hz, 2H), 7. 54 (d, J = 8.6Hz, 2H), 9.26 (br.s, NH).

(Reference Example 7) Synthesis of N- (4- (hydroxymethyl) phenyl) -2- (piperidine-1-yl) acetamide

N-(4-(((tert-butyldimethylsilyl) oxy) methyl) phenyl) -2- (piperidine-1-yl) acetylamide (343 mg) was dissolved in tetrahydrofuran (10 mL) and TBAF was dissolved in tetrahydrofuran (1M, 1M,). 1.1 mL) was added, and the mixture was stirred at room temperature for 12 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (developing solvent: ethyl acetate ester: methanol = 10: 1) to obtain a yellow liquid product (227 mg; yield 96%).
^{1 1} HNMR (500MHz, CDCl ₃ ) δ1.60-1.70 (m, 1H), 1.86-1.88 (m, 2H), 2.69-2.72 (m, 2H), 3.29 (S, 2H), 4.67 (s, 2H), 7.36 (d, J = 8.3Hz, 2H), 7.59 (d, J = 8.6Hz, 2H), 9.12 (br) .S, NH).

(Reference Example 8) Synthesis of N- (4-formylphenyl) -2- (piperidine-1-yl) acetamide

N- (4- (Hydroxymethyl) phenyl) -2- (piperidine-1-yl) acetamide (227 mg) was dissolved in dichloromethane (4 mL), Dess-Martin peryodinane (465 mg) was added, and the mixture was added at room temperature for 12 hours. Stirred. The reaction mixture was diluted with saturated aqueous sodium hydrogen carbonate solution (20 mL) and then extracted with ethyl acetate. The extract was washed successively with water and saturated brine, and dried over magnesium sulfate. After distilling off the solvent under reduced pressure, the residue was purified by column chromatography (developing solvent: ethyl acetate ester) to obtain a yellow solid product (120 mg; yield 53%).
^{1 1} HNMR (500MHz, CDCl ₃ ) δ1.52-1.56 (m, 2H), 1.65-1.70 (m, 4H), 2.54-2.62 (m, 4H), 3.12 (S, 2H), 7.76 (d, J = 8.8Hz, 2H), 7.88 (d, J = 8.6Hz, 2H), 9.61 (br.s, NH), 9.94 (S, 1H).

(Reference Example 9) Synthesis of 2-bromo-N- (4-(((tert-butyldimethylsilyl) oxy) methyl) phenyl) propanamide

4-(((tert-butyldimethylsilyl) oxy) methyl) aniline (950 mg) is dissolved in dichloromethane (10 mL), cooled to 0 ° C., and then 2-bromopropionyl bromide (632 μL), triethylamine (1.1 mL). Was added, and the mixture was stirred at room temperature for 12 hours. The reaction mixture was diluted with water (50 mL) and then extracted with dichloromethane. The extract was washed with saturated brine and dried over magnesium sulfate. After distilling off the solvent under reduced pressure, the residue was purified by column chromatography (developing solvent: ethyl acetate ester: hexane = 1:10) to obtain a yellow solid product (895 mg; yield 60%).
^{1 1} HNMR (500MHz, CDCl ₃ ) δ0.10 (s, 6H), 0.94 (s, 9H), 1.98 (d, J = 7.1Hz, 3H), 4.56 (q, J = 7) .1Hz, 2H), 4.72 (s, 2H), 7.31 (d, J = 8.3Hz, 2H), 7.50 (d, J = 8.3Hz, 2H), 8.04 (br) .S, NH).

(Reference Example 10) Synthesis of N-(4-(((tert-butyldimethylsilyl) oxy) methyl) phenyl) -2- (pyrrolidin-1-yl) propanamide

2-Bromo-N- (4-(((tert-butyldimethylsilyl) oxy) methyl) phenyl) propanamide (950 mg) is dissolved in tetrahydrofuran (15 mL), and pyrrolidine (238 μL) and potassium carbonate (668 mg) are added. The mixture was stirred at room temperature for 20 hours. The reaction mixture was diluted with water (50 mL) and then extracted with ethyl acetate. The extract was washed with saturated brine and dried over magnesium sulfate. After distilling off the solvent under reduced pressure, the residue was purified by column chromatography (developing solvent: ethyl acetate ester: methanol = 10: 1) to obtain a colorless liquid product (726 mg; yield 83%).
^{1 1} HNMR (500MHz, CDCl ₃ ) δ0.10 (s, 6H), 0.94 (s, 9H), 1.38 (d, J = 7.1Hz, 3H), 1.82-1.85 (m) , 4H), 2.61-2.68 (m, 4H), 3.05 (q, J = 6.8Hz, 1H), 4.71 (s, 2H), 7.29 (d, J = 8) .6Hz, 2H), 7.54 (d, J = 8.6Hz, 2H), 8.96 (br.s, NH).

(Reference Example 11) Synthesis of N- (4- (hydroxymethyl) phenyl) -2- (pyrrolidin-1-yl) propanamide

N-(4-(((tert-butyldimethylsilyl) oxy) methyl) phenyl) -2- (pyrrolidin-1-yl) propanamide (720 mg) was dissolved in tetrahydrofuran (20 mL) and TBAF was dissolved in tetrahydrofuran (1 M). , 2.5 mL) was added, and the mixture was stirred at room temperature for 12 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (developing solvent: ethyl acetate ester: methanol = 10: 1) to obtain a yellow liquid product (489 mg; yield 99%).
^{1 1} HNMR (500MHz, CDCl ₃ ) δ1.39 (d, J = 6.8Hz, 3H), 1.83-1.85 (m, 4H), 2.61-2.68 (m, 4H), 3 .06 (q, J = 7.1Hz, 1H), 4.66 (s, 2H), 7.34 (d, J = 8.4Hz, 2H), 7.59 (d, J = 8.3Hz, 2H), 9.01 (br.s, NH).

(Reference Example 12) Synthesis of N- (4-formylphenyl) -2- (pyrrolidin-1-yl) propanamide

Dissolve N- (4- (hydroxymethyl) phenyl) -2- (pyrrolidin-1-yl) propanamide (489 mg) in dichloromethane (10 mL), add Dess-Martin peryodinane (1.0 g), and bring to room temperature. And stirred for 12 hours. The reaction mixture was diluted with saturated aqueous sodium hydrogen carbonate solution (20 mL) and then extracted with ethyl acetate. The extract was washed successively with water and saturated brine, and dried over magnesium sulfate. After distilling off the solvent under reduced pressure, the residue was purified by column chromatography (developing solvent: ethyl acetate ester: methanol = 10: 1) to obtain a yellow liquid product (345 mg; yield 71%).
^{1 1} HNMR (500MHz, CDCl ₃ ) δ1.40 (d, J = 6.9Hz, 3H), 1.85-1.87 (m, 4H), 2.64-2.71 (m, 4H), 3 .14 (q, J = 6.8Hz, 1H), 7.77 (d, J = 8.3Hz, 2H), 7.87 (d, J = 8.6Hz, 2H), 9.40 (s, 1H), 9.93 (s, 1H).

(Reference Example 13) Synthesis of N- (4-acetylphenyl) 2-bromoacetamide

Dissolve 1- (4-amino) acetophenone (1.1 g) in dichloromethane (20 mL), cool to 0 ° C., add 2-bromoacetamide (1.1 mL) and triethylamine (2.2 mL), and bring to room temperature. And stirred for 12 hours. The reaction mixture was diluted with water (50 mL) and then extracted with dichloromethane. The extract was washed with saturated brine and dried over magnesium sulfate. After distilling off the solvent under reduced pressure, the residue was purified by column chromatography (developing solvent: ethyl acetate ester: hexane = 1: 1) to obtain a white solid product (783 mg; yield 38%).
^{1 1} HNMR (500MHz, CDCl ₃ ) δ2.61 (s, 3H), 4.06 (s, 2H), 7.66 (d, J = 8.8Hz, 2H), 7.98 (d, J = 8) .8Hz, 2H), 8.27 (br.s, NH).

(Reference Example 14) Synthesis of N- (4-acetylphenyl) -2- (pyrrolidin-1-yl) acetamide

N- (4-Acetylphenyl) 2-bromoacetamide (750 mg) was dissolved in tetrahydrofuran (20 mL), pyrrolidine (288 μL) and potassium carbonate (809 mg) were added, and the mixture was stirred at room temperature for 20 hours. The reaction mixture was diluted with water (50 mL) and then extracted with ethyl acetate. The extract was washed with saturated brine and dried over magnesium sulfate. After distilling off the solvent under reduced pressure, the residue was purified by column chromatography (developing solvent: ethyl acetate ester: methanol = 4: 1) to obtain a white solid product (577 mg; yield 80%).
^{1 1} HNMR (500MHz, CDCl ₃ ) δ1.88-1.89 (m, 4H), 2.59 (s, 3H), 2.70-2.75 (m, 4H), 3.32 (s, 2H) ), 7.69 (d, J = 8.8Hz, 2H), 7.96 (d, J = 9.1Hz, 2H), 9.36 (br.s, NH).

(Reference Example 15) Synthesis of 2-bromo-N- (5-oxo-5,6,7,8-tetrahydronaphthalene-2-yl) acetamide

6-Amino-3,4-dihydronaphthalene-1 (2H) -one (1.2 g) was dissolved in dichloromethane (20 mL), cooled to 0 ° C., then 2-bromoacetamide (1.1 mL), triethylamine (2). .2 mL) was added, and the mixture was stirred at room temperature for 12 hours. The reaction mixture was diluted with water (50 mL) and then extracted with dichloromethane. The extract was washed with saturated brine and dried over magnesium sulfate. After distilling off the solvent under reduced pressure, the residue was purified by column chromatography (developing solvent: ethyl acetate ester: hexane = 1: 1) to obtain a white solid product (753 mg; yield 34%).
^{1 1} HNMR (500MHz, CDCl ₃ ) δ2.122.17 (m, 2H), 2.65-2.68 (m, 2H), 2.97-2.99 (m, 2H), 4.05 (S, 2H), 7.33 (dd, J = 2.2,8.6Hz, 1H), 7.68 (s, 1H), 8.05 (d, J = 8.6Hz, 1H), 8 .24 (br.s, NH).

(Reference Example 16) Synthesis of N- (5-oxo-5,6,7,8-tetrahydronaphthalene-2-yl) -2- (pyrrolidin-1-yl) acetamide

2-Bromo-N- (5-oxo-5,6,7,8-tetrahydronaphthalene-2-yl) acetamide (730 mg) was dissolved in tetrahydrofuran (15 mL), and pyrrolidine (256 μL) and potassium carbonate (718 mg) were added. In addition, the mixture was stirred at room temperature for 20 hours. The reaction mixture was diluted with water (50 mL) and then extracted with ethyl acetate. The extract was washed with saturated brine and dried over magnesium sulfate. After distilling off the solvent under reduced pressure, the residue was purified by column chromatography (developing solvent: ethyl acetate ester: methanol = 4: 1) to obtain a yellow liquid product (514 mg; yield 73%).
^{1 1} HNMR (500MHz, CDCl ₃ ) δ1.87-1.90 (m, 4H), 2.11-2.16 (m, 2H), 2.62-2.65 (m, 2H), 2.69 -2.75 (m, 4H), 2.95-2.98 (m, 2H), 3.31 (s, 2H), 7.32 (dd, J = 2.2,8.6Hz, 1H) , 7.75 (s, 1H), 8.02 (d, J = 8.6Hz, 1H), 9.33 (br.s, NH).

Example 1 of 3-((1- (tert-butyl) -1H-tetrazole-5-yl) (4-cyclohexylpiperazin-1-yl) methyl) -6-methyl-2H-chromen-2-one Synthetic

6-Methyl-2-oxo-2H-chromen-3-carbaldehyde (46 mg) was dissolved in methanol (1.5 mL), 1-cyclohexylpiperazin (42 mg), tert-butyl isocyanide (29 μL), trimethylsilyl azide (33 μL). Was added, and the mixture was stirred at 50 ° C. for 10 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (developing solvent: ethyl acetate ester: hexane = 1: 1) to obtain a yellow liquid product (52 mg; yield 62%).
^{1 1} HNMR (500MHz, CDCl ₃ ) δ1.08-1.21 (m, 5H), 1.59-1.82 (m, 4H), 1.82 (s, 9H), 2.16-2.20 (M, 1H), 2.41 (s, 3H), 2.56-2.76 (m, 8H), 5.93 (s, 1H), 7.21 (d, J = 8.1Hz, 1H) ), 7.34-7.37 (m, 2H), 8.49 (s, 1H).

(Example 2) 3-((1- (tert-butyl) -1H-tetrazole-5-yl) (4- (2- (pyrrolidin-1-yl) acetyl) piperazine-1-yl) methyl) -6 -Synthesis of ethyl-2-oxo-1,2-dihydroquinoline

6-Ethyl-2-oxo-1,2-dihydroquinoline-3-carbaldehyde (50 mg), 1- (piperazine-1-yl) ethane-1-one (49 m), tert-butyl isocyanide (46 μL), trimethylsilyl azide Using (36 μL), the reaction treatment was carried out according to the method described in Example 1 to obtain the title object (13 mg, yield 10%).
^{1 1} HNMR (500MHz, CDCl ₃ ) δ0.89 (t, J = 7.1Hz, 3H), 1.68-1.73 (m, 4H), 1.81 (s, 9H), 2.472 .54 (m, 4H), 2.60-2.68 (m, 2H), 2.72 (q, J = 7.6Hz, 2H), 2,78-2.87 (m, 2H), 3 .24 (s, 2H), 3.55-3.63 (m, 4) H), 6.19 (s, 1H), 7.23 (d, J = 8.3Hz, 2H), 7.40 (D, J = 8.1 Hz, 2H), 8.39 (s, 1H), 11.5 (br. S, NH).

(Example 3) N-(4-((1- (tert-butyl) -1H-tetrazole-5-yl) (4-cyclohexylpiperazin-1-yl) methyl) phenyl) -2- (pyrrolidin-1-yl) Il) Synthesis of acetamide

Performed using N- (4-formylphenyl) -2- (pyrrolidin-1-yl) acetamide (35 mg), 1-cyclohexylpiperazine (25 m), tert-butyl isocyanide (20 μL), trimethylsilyl azide (30 μL) The reaction treatment was carried out according to the method described in Example 1 to obtain the title object (24 mg, yield 31%).
^{1 1} HNMR (500MHz, CDCl ₃ ) δ1.03-1.30 (m, 5H), 1.54-1.66 (m, 2H), 1.70 (s, 9H), 1.73-1.90 (M, 7H), 2.16-2.22 (m, 1H), 2.38-2.72 (m, 12H), 3.27 (s, 2H), 5.05 (s, 1H), 7.46 (d, J = 8.6Hz, 2H), 7.56 (d, J = 8.8Hz, 2H), 9.13 (br.s, NH).

(Example 4) N- (4-((1- (cyclohexyl-1H-tetrazole-5-yl) (4-cyclohexylpiperazin-1-yl) methyl) phenyl) -2- (pyrrolidin-1-yl) acetamide Synthesis of

Example 1 using N- (4-formylphenyl) -2- (pyrrolidin-1-yl) acetamide (46 mg), 1-cyclohexylpiperazine (34 mg), cyclohexyl isocyanide (25 μL), trimethylsilyl azide (40 μL). The reaction treatment was carried out according to the method described in 1) to obtain the title object (70 mg, yield 65%).
^{1 1} HNMR (500MHz, CDCl ₃ ) δ1.06-1.28 (m, 10H), 1.42-2.05 (m, 14H), 2.22-2.46 (m, 3H), 2.62 -2.73 (m, 11H), 3.28 (s, 2H), 4.51-4.65 (m, 1H), 4.87 (s, 1H), 7.40 (d, J = 8) .6Hz, 2H), 7.58 (d, J = 8.6Hz, 2H), 9.15 (br.s, NH).

(Example 5) N-(4-((1- (tert-butyl) -1H-tetrazole-5-yl) ((3-morpholinopropyl) amino) methyl) phenyl) -2- (pyrrolidin-1-yl) ) Synthesis of acetamide

N- (4-formylphenyl) -2- (pyrrolidin-1-yl) acetamide (75 mg), 3-morpholinopropane-1-amine (46 mg), tert-butyl isocyanide (43 μL), trimethylsilyl azide (64 μL) are used. Then, the reaction treatment was carried out according to the method described in Example 1 to obtain the title object (82 mg, yield 53%).
^{1 1} HNMR (500MHz, CDCl ₃ ) δ1.50-1.68 (m, 11H), 1.85-1.87 (m, 4H), 2.36-2.42 (m, 6H), 2.58 (T, J = 6.9Hz, 2H), 2.67-2.70 (m, 4H), 3.27 (s, 2H), 3.68-3.71 (m, 4H), 5.22 (S, 1H), 7.28 (d, J = 8.6Hz, 2H), 7.57 (d, J = 8.8Hz, 2H), 9.14 (br.s, NH).

(Example 6) N- (4-((1- (tert-butyl) -1H-tetrazole-5-yl) (4-cyclohexylpiperazin-1-yl) methyl) phenyl) -2- (piperidine-1-yl) Il) Synthesis of acetamide

Performed using N- (4-formylphenyl) -2- (piperidine-1-yl) acetamide (49 mg), 1-cyclohexylpiperazine (34 m), tert-butyl isocyanide (27 μL), trimethylsilyl azide (40 μL) The reaction treatment was carried out according to the method described in Example 1 to obtain the title object (70 mg, yield 70%).
^{1 1} HNMR (500MHz, CDCl ₃ ) δ1.09-1.26 (m, 6H), 1.46-1.49 (m, 2H), 1.62-1.67 (m, 4H), 1.70 (S, 9H), 1.75-1.84 (m, 4H), 2.18-2.20 (m, 1H), 2.43-2.62 (m, 12H), 3.06 (s) , 2H), 5.05 (s, 1H), 7.46 (d, J = 8.6Hz, 2H), 7.55 (d, J = 8.8Hz, 2H), 9.30 (br.s) , NH).

Example 7 of N-(4-((1-cyclohexyl-1H-tetrazole-5-yl) (4-cyclohexylpiperazin-1-yl) methyl) phenyl) -2- (piperidine-1-yl) acetamide Synthetic

Example 1 using N- (4-formylphenyl) -2- (piperidine-1-yl) acetamide (49 mg), 1-cyclohexylpiperazine (34 mg), cyclohexyl isocyanide (25 μL), trimethylsilyl azide (40 μL). The reaction treatment was carried out according to the method described in 1) to obtain the title object (32 mg, yield 29%).
^{1 1} HNMR (500MHz, CDCl ₃ ) δ1.01-1.58 (m, 12H), 1.61-1.98 (m, 14H), 2.18-2, 25 (m, 1H), 2.33 -2.41 (m, 2H), 2.50-2.67 (m, 10H), 3.07 (s, 2H), 4.59-4.68 (m, 1H), 4.89 (s) , 1H), 7.41 (d, J = 8.6Hz, 2H), 7.57 (d, J = 8.6Hz, 2H), 9.31 (br.s, NH).

(Example 8) N-(4-((1- (tert-butyl) -1H-tetrazole-5-yl) ((2- (diethylamino) ethyl) amino) phenyl) -2- (piperidine-1-yl) ) Synthesis of acetamide

Example 1 using N- (4-formylphenyl) -2- (piperidine-1-yl) acetamide (49 mg), diethylaminoethylamine (23 mg), tert-butyl isocyanide (27 μL), trimethylsilyl azide (40 μL). The reaction treatment was carried out according to the method described in 1 to obtain the title object (20 mg, yield 21%).
^{1 1} HNMR (500MHz, CDCl ₃ ) δ0.96 (t, J = 7.1H, 6H), 1.45-1.52 (m, 3H), 1.62-1.65 (m, 3H), 1 .65 (s, 9H), 2.45 (q, J = 7.1Hz, 4H), 2.49-2.60 (m, 8H), 3.06 (s, 2H), 5.30 (s) , 1H), 7.30 (d, J = 8.6Hz, 2H), 7.55 (d, J = 8.6Hz, 2H), 9.29 (br.s, NH).

(Example 9) N- (4-((1- (tert-butyl) -1H-tetrazole-5-yl) (4-cyclohexylpiperazin-1-yl) methyl) phenyl) -2- (pyrrolidin-1-yl) Il) Synthesis of propanamide

Using N- (4-formylphenyl) -2- (pyrrolidin-1-yl) propanamide (49 mg), 1-cyclohexylpiperazine (34 mg), tert-butyl isocyanide (27 μL), trimethylsilyl azide (40 μL), The reaction treatment was carried out according to the method described in Example 1 to obtain the title object (38 mg, yield 36%).
^{1 1} HNMR (500MHz, CDCl ₃ ) δ1.07-1.24 (m, 5H), 1.34-1.36 (m, 3H), 1.58-1.60 (m, 1H), 1.70 (S, 9H), 1.73-1.90 (m, 8H), 2.16-2.25 (m, 1H), 2.38-2.47 (m, 2H), 2.58-2 .63 (11H), 5.04 (s, 1H), 7.45 (d, J = 8.3Hz, 2H), 7.56 (d, J = 8.6Hz, 2H), 9.01 (br .S, NH).

(Example 10) N-(4-((1- (tert-butyl) -1H-tetrazole-5-yl) ((3-morpholinopropyl) amino) methyl) phenyl) -2- (pyrrolidin-1-yl) ) Synthesis of propanamide

N- (4-formylphenyl) -2- (pyrrolidin-1-yl) propanamide (49 mg), 3-morpholinopropane-1-amine (29 mg), tert-butyl isocyanide (27 μL), trimethylsilyl azide (40 μL) Using, the reaction treatment was carried out according to the method described in Example 1 to obtain the title object (42 mg, yield 42%).
^{1 1} HNMR (500MHz, CDCl ₃ ) δ1.35 (dd, J = 2.5, 6.9Hz, 3H), 1.64 (s, 9H), 1.64-1.68 (m, 2H), 1 .80-1.82 (m, 4H), 2.35-2.40 (m, 6H), 2.55-2.63 (m, 6H), 3.03-3.05 (m, 1H) , 3.66-3.69 (m, 4H), 5.21 (s, 1H), 7.26 (dd, J = 2.0, 8.6Hz, 2H), 7.57 (d, J = 8.8Hz, 2H), 9.01 (br.s, NH).

Example 11 of N-(4-((1-cyclohexyl-1H-tetrazole-5-yl) ((3-morpholinopropyl) amino) methyl) phenyl) -2- (pyrrolidin-1-yl) propanamide Synthetic

Using N- (4-formylphenyl) -2- (pyrrolidin-1-yl) propanamide (49 mg), 3-morpholinopropane-1-amine (29 mg), cyclohexyl isocyanide (25 μL), trimethylsilyl azide (40 μL) Then, the reaction treatment was carried out according to the method described in Example 1 to obtain the title object (40 mg, yield 38%).
^{1 1} HNMR (500MHz, CDCl ₃ ) δ1.19-1.28 (m, 5H), 1.33 (d, J = 7.1Hz, 3H), 1.49-1.56 (m, 2H), 1 .67-1.87 (m, 9H), 2.33-2.53 (m, 12H), 3.03 (q, J = 6.9Hz, 1H), 3.65-3.66 (m, 4H), 4.25-4.34 (m, 1H), 5.14 (d, J = 3.2Hz, 1H), 7.26 (d, J = 8.4Hz, 2H), 7.55 ( d, J = 8.6Hz, 2H), 9.04 (br.s, NH).

(Example 12) Synthesis of 1-(((1- (tert-butyl) -1H-tetrazol-5-yl) (phenyl) methyl) -4-cyclohexylpiperazine)

Reaction treatment was performed with benzaldehyde (32 mg), 1-cyclohexylpiperazine (50 mg), tert-butyl isocyanide (40 μL) and trimethylsilyl azide (60 μL) according to the method described in Example 1 to obtain the title product (42 mg, yield). The rate was 37%).
^{1 1} HNMR (500MHz, CDCl ₃ ) δ1.04-1.24 (m, 5H), 1.58-1.60 (m, 1H), 1.67 (s, 9H), 1.73-1.86 (M, 4H), 2.29-2.33 (m, 1H), 2.42-2.54 (m, 2H), 2.59-2.72 (m, 6H), 5.08 (s) , 1H), 7.27-7.33 (m, 3H), 7.45-7.47 (m2H).

Example 13 Synthesis of N-(4-((1- (tert-butyl) -1H-tetrazole-5-yl) (4-cyclohexylpiperazin-1-yl) methyl) phenyl) acetamide

Reaction treatment was performed with N- (4-formylphenyl) acetamide (48 mg), 1-cyclohexylpiperazine (50 mg), tert-butyl isocyanide (40 μL), and trimethylsilyl azide (60 μL) according to the method described in Example 1. , 100 mg of the title object, yield 76%) was obtained.
^{1 1} HNMR (500MHz, CDCl ₃ ) δ1.06-1.23 (m, 5H), 1.57-1.65 (m, 1H), 1.69 (s, 9H), 1.75-1.89 (M, 4H), 2.19 (s, 3H), 2.24-2.29 (m, 1H), 2.43-2.63 (m, 8H), 5.06 (s, 1H), 7.44 (d, J = 8.3Hz, 2H), 7.55 (d, J = 8.6Hz, 2H).

(Example 14) N-(4-((1- (tert-butyl) -1H-tetrazole-5-yl) (4-methylpiperazin-1-yl) methyl) phenyl) -2- (pyrrolidin-1-yl) Il) Synthesis of acetamide

Performed using N- (4-formylphenyl) -2- (pyrrolidin-1-yl) acetamide (46 mg), 1-methylpiperazine (25 mg), tert-butyl isocyanide (25 μL), trimethylsilyl azide (40 μL) The reaction treatment was carried out according to the method described in Example 1 to obtain the title object (33 mg, yield 37%).
^{1 1} HNMR (500MHz, CDCl ₃ ) δ1.68 (s, 9H), 1.83-1.85 (m, 4H), 2.22 (s, 3H), 2.36-2.48 (m, 6H) ), 2.60-2.68 (m, 6H), 3.25 (s, 2H), 5.09 (s, 1H), 7.42 (d, J = 8.8Hz, 2H), 7. 55 (d, J = 8.8Hz, 2H), 9.12 (br.s, NH).

(Example 15) N-(4-((1- (tert-butyl) -1H-tetrazole-5-yl) (4-isopropylpiperazine-1-yl) methyl) phenyl) -2- (pyrrolidin-1-yl) Il) Synthesis of acetamide

Performed using N- (4-formylphenyl) -2- (pyrrolidin-1-yl) acetamide (46 mg), 1-isopropylpiperazine (26 mg), tert-butyl isocyanide (25 μL), trimethylsilyl azide (40 μL) The reaction treatment was carried out according to the method described in Example 1 to obtain the title object (41 mg, yield 44%).
^{1 1} HNMR (500MHz, CDCl ₃ ) δ0.99 (d, J = 6.4Hz, 6H), 1.68 (s, 9H), 1.83-1.85 (m, 4H), 2.37-2 .68 (m, 13H), 3.25 (s, 2H), 5.05 (s, 1H), 7.44 (d, J = 8.6Hz, 2H), 7.54 (d, J = 8) .6Hz, 2H), 9.12 (br.s, NH).

(Example 16) N-(4-((1- (tert-butyl) -1H-tetrazole-5-yl) (4-cyclopentylpiperazin-1-yl) methyl) phenyl) -2- (pyrrolidin-1-yl) Il) Synthesis of acetamide

Performed using N- (4-formylphenyl) -2- (pyrrolidin-1-yl) acetamide (46 mg), 1-cyclopentylpiperazine (31 mg), tert-butyl isocyanide (25 μL), trimethylsilyl azide (40 μL) The reaction treatment was carried out according to the method described in Example 1 to obtain the title object (31 mg, yield 36%).
^{1 1} HNMR (500MHz, CDCl ₃ ) δ1.28-1.35 (m, 2H), 1.45-1.54 (m, 2H), 1.60-1.66 (m, 2H), 1.68 (S, 9H), 1.76-1.84 (m, 6H), 2.40-2.67 (m, 13H), 3.24 (s, 2H), 5.05 (s, 1H), 7.43 (d, J = 8.6Hz, 2H), 7.54 (d, J = 8.6Hz, 2H), 9/11 (br.s, NH).

(Example 17) Synthesis of N-(4-((1- (tert-butyl) -1H-tetrazole-5-yl) (morpholino) methyl) phenyl) -2- (pyrrolidin-1-yl) acetamide

Example 1 using N- (4-formylphenyl) -2- (pyrrolidin-1-yl) acetamide (46 mg), morpholine (17 mg), tert-butyl isocyanide (25 μL), trimethylsilyl azide (40 μL). The reaction treatment was carried out according to the method described to obtain the title object (48 mg, yield 56%).
^{1 1} HNMR (500MHz, CDCl ₃ ) δ1.70 (s, 9H), 1.87-2.00 (m, 4H), 2.39-2.42 (m, 2H), 2.63-2.66 (M, 2H), 2.77-2.79 (m, 4H), 3.37 (s, 2H), 3.65-3.67 (m, 4H), 5.14 (s, 1H), 7.39 (d, J = 8.8Hz, 2H), 7.57 (d, J = 8.6Hz, 2H), 9.55 (br.s, NH).

(Example 18) N- (4-((4-Cyclohexylpiperazin-1-yl) (1- (fran-2-ylmethyl) -1H-tetrazole-5-yl) methyl) phenyl) -2- (pyrrolidine-) 1-Il) Synthesis of acetamide

N- (4-formylphenyl) -2- (pyrrolidin-1-yl) acetamide (82 mg), 1-cyclohexylpiperazine (67 mg), 2- (isocyanaminomethyl) furan (52 mg), trimethylsilyl azide (80 μL) is used. Then, the reaction treatment was carried out according to the method described in Example 1 to obtain the title object (70 mg, yield 33%).
^{1 1} HNMR (500MHz, CDCl ₃ ) δ1.18-1.28 (m, 5H), 1.61-1.63 (m, 1H), 1.71-1.87 (m, 8H), 2.22 -2.27 (m, 1H), 2.40-2.73 (m, 12H), 3.28 (s, 2H), 3.57-3.60 (m, 1H), 4.93 (s) , 1H), 5.45 (d, J = 15.6Hz, 1H), 5.75 (d, J = 15.6Hz, 1H), 6.35-6.37 (m, 2H), 7.37 -7.40 (m, 3H), 7.57 (d, J = 8.8Hz, 2H), 9.15 (br.s, NH).

(Example 19) N- (4-((4-Cyclohexylpiperazin-1-yl) (1-tert-pentyl-1H-tetrazole-5-yl) methyl) phenyl) -2- (pyrrolidin-1-yl) Synthesis of acetamide

Performed using N- (4-formylphenyl) -2- (pyrrolidin-1-yl) acetamide (46 mg), 1-cyclohexylpiperazine (34 mg), tert-pentylisocyanide (28 μL), trimethylsilyl azide (40 μL) The reaction treatment was carried out according to the method described in Example 1 to obtain the title object (43 mg, yield 41%).
^{1 1} HNMR (500MHz, CDCl ₃ ) δ0.58 (t, J = 7.6Hz, 3H), 1.07-1.26 (m, 5H), 1.57-1.60 (m, 1H), 1 .64 (s, 3H), 1.71 (s, 3H), 1.76-1.86 (m, 8H), 1.91-2.04 (m, 2H), 2.15-2.24 (M, 1H), 2.43-2.67 (m, 12H), 3.25 (s, 2H), 4.98 (s, 1H), 7.44 (d, J = 8.6Hz, 2H) ), 7.57 (d, J = 8.8Hz, 2H), 9.12 (br.s, NH).

(Reference Example 17) Synthesis of tert-butyl (4- (chloromethyl) phenyl) carbamate

tert-butyl (4- (hydroxymethyl) phenyl) carbamate (1.4 g) was dissolved in dichloromethane (20 mL), thionyl chloride (1.0 mL) and 5 drops of pyridine were added, and the mixture was stirred at room temperature for 12 hours. After distilling off the reaction solution under reduced pressure, the residue was purified by column chromatography (developing solvent: ethyl acetate ester: hexane = 1: 1) to obtain a yellow liquid product (125 mg; yield 8%).
^{1 1} HNMR (500 MHz, CDCl ₃ ) δ1.53 (s, 9H), 4.57 (s, 2H), 6.49 (br.s, NH), 7.31-7.37 (m, 4H).

(Reference Example 18) Synthesis of tert-butyl (4-((4-cyclohexylpiperazin-1-yl) methyl) phenyl) carbamate

tert-butyl (4- (chloromethyl) phenyl) carbamate (125 mg) was dissolved in dichloromethane (5 mL), 1-cyclohexylpiperazine (87 mg) and triethylamine (86 μL) were added, and the mixture was stirred at room temperature for 12 hours. The reaction mixture was diluted with water (20 mL) and then extracted with dichloromethane. The extract was washed with saturated brine and dried over magnesium sulfate. After distilling off the solvent under reduced pressure, the residue was purified by column chromatography (developing solvent: ethyl acetate ester) to obtain a white solid product (85 mg; yield 44%).
^{1 1} HNMR (500MHz, CDCl ₃ ) δ1.11-1.28 (m, 5H), 1.52 (s, 9H), 1.60-1.63 (m, 1H), 1.77-1.89 (M, 4H), 2.19-2.25 (m, 1H), 2.41-2.63 (m, 8H), 3.44 (s, 2H), 6.43 (br.s, NH) ), 7.23-7.31 (m, 4H).

(Reference Example 19) Synthesis of 4-((4-cyclohexylpiperazin-1-yl) methyl) phenyl) aniline

Dissolve tert-butyl (4-((4-cyclohexylpiperazin-1-yl) methyl) phenyl) carbamate (85 mg) in dichloromethane (5 mL), add trifluoroacetic acid (1 mL), and stir at room temperature for 5 hours. bottom. The solvent was distilled off under reduced pressure, diluted with saturated aqueous sodium hydrogen carbonate solution (20 mL), and extracted with dichloromethane. The extract was washed successively with water and saturated brine, and dried over magnesium sulfate. After distilling off the solvent under reduced pressure, a yellow liquid product (62 mg; 99% yield) was obtained.
^{1 1} HNMR (500MHz, CDCl ₃ ) δ1.120-2.18 (m, 10H), 2.51-2.86 (m, 6H), 3.46-3.70 (m, 5H), 6.66 (D, J = 8.6Hz, 2H), 7.04-7.10 (m, 4H).

(Reference Example 20) Synthesis of 2-bromo-N-4-((4-cyclohexylpiperazin-1-yl) methyl) phenyl) acetamide

4-((4-Cyclohexylpiperazin-1-yl) methyl) phenyl) aniline (62 mg) is dissolved in dichloromethane (5 mL), cooled to 0 ° C., then 2-bromoacetylbromid (30 μL), triethylamine (64 μL). Was added, and the mixture was stirred at room temperature for 12 hours. The reaction mixture was diluted with 10 mL of water and then extracted with dichloromethane. The extract was washed with saturated brine and dried over magnesium sulfate. After distilling off the solvent under reduced pressure, the residue was purified by column chromatography (developing solvent: ethyl acetate ester: methanol = 4: 1) to obtain a white solid product (64 mg; yield 71%).
^{1 1} HNMR (500 MHz, CD ₃ OD) δ1.10-1.34 (m, 5H), 1.64-1.67 (m, 1H), 1.83-1.88 (m, 2H), 1. 93-2.01 (m, 2H), 2.45-2.80 (m, 9H), 3.54 (s, 2H), 3.97 (s, 2H), 7.30 (d, J = 8.6Hz, 2H), 7.54 (d, J = 8.6Hz, 2H).

(Example 20) Synthesis of N-4-((4-cyclohexylpiperazin-1-yl) methyl) phenyl) -2- (pyrrolidin-1-yl) acetamide

2-Bromo-N-4-((4-cyclohexylpiperazin-1-yl) methyl) phenyl) acetylamide (64 mg) is dissolved in tetrahydrofuran (3 mL), pyrrolidine (16 μL) and potassium carbonate (45 mg) are added. The mixture was stirred at room temperature for 20 hours. The reaction mixture was diluted with water (10 mL) and then extracted with ethyl acetate. The extract was washed with saturated brine and dried over magnesium sulfate. After distilling off the solvent under reduced pressure, the residue was purified by column chromatography (developing solvent: ethyl acetate ester: methanol = 2: 1) to obtain a white solid product (51 mg; yield 83%).
^{1 1} HNMR (500MHz, CDCl ₃ ) δ1.08-1.28 (m, 5H), 1.61-1.63 (m, 1H), 1.77-1.89 (m, 8H), 2.19 -2.27 (m, 1H), 2.41-2.73 (m, 12H), 3.27 (s, 2H), 3.47 (s, 2H), 7.28 (d, J = 7) .8Hz, 2H), 7.52 (d, J = 7.8Hz, 2H), 9.07 (br.s, NH).

(Example 21) Synthesis of N- (5- (4-cyclohexylpiperazin-1-yl) -5,6,7,8-tetrahydronaphthalene-2-yl) acetamide

N- (5-oxo-5,6,7,8-tetrahydronaphthalene-2-yl) acetamide (800 mg) was dissolved in tetrahydrofuran (8 mL), 1-cyclohexylpiperazine (1.5 g) was added, and -80 was added. Cooled to ° C. Titanium tetrachloride (219 μL) was added, and the mixture was stirred for 2 hours and then at room temperature for 20 hours. The reaction mixture was diluted with aqueous ammonia and ethyl acetate (1: 1; 8 mL) and then extracted with ethyl acetate. The extract was washed with saturated brine and dried over magnesium sulfate. After distilling off the solvent under reduced pressure, the residue is dissolved in methanol (10 mL), a hydrochloric acid methanol solution (1 M, 8 mL) and a NaCl _{solution of NaCNBH 3} (1.0 M, 8 mL) are added, and the mixture is stirred at room temperature for 12 hours. bottom. The reaction mixture was distilled off under reduced pressure, the reaction mixture was diluted with water (20 mL), and then extracted with ethyl acetate. The extract was washed successively with water and saturated brine, and dried over magnesium sulfate. After distilling off the solvent under reduced pressure, the residue was purified by column chromatography (developing solvent: dichloromethane: methanol = 4: 1) to obtain a yellow solid product (359 mg; yield 25%).
^{1 1} HNMR (500MHz, CDCl ₃ ) δ1.10-1.28 (m, 5H), 1.64-1.72 (m, 3H), 1.77-2.05 (m, 8H), 2.17 (S, 3H), 2.28-2.37 (m, 1H), 2.50-2.77 (m, 10H), 3.75-3.78 (m, 1H), 7.06 (br) .S, NH), 7.21-7.24 (m, 2H), 7.61 (d, J = 8.3Hz, 1H).

(Example 22) Synthesis of N- (4- (1- (4-cyclohexylpiperazin-1-yl) ethyl) phenyl) -2- (pyrrolidin-1-yl) acetamide

N-(4-acetylphenyl) -2- (pyrrolidin-1-yl) acetamide (500 mg), 1-cyclohexyl-piperazine (1.0 g), titanium tetrachloride (100 [mu] L), NaCNBH ₃ solution in tetrahydrofuran (1.0 M, 4 mL ) Was used for reaction treatment according to the method described in Example 20 to obtain the title object (157 mg, yield 20%).
^{1 1} HNMR (500MHz, CDCl ₃ ) δ1.14-1.30 (m, 5H), 1.65-2.05 (m, 8H), 2.67-2.84 (m, 13H), 3.26 (S, 2H), 3.76-3.82 (m, 1H), 7.36 (d, J = 8.3Hz, 2H), 7.59 (d, J = 8.1Hz, 2H), 9 .00 (br.s, NH).

(Example 23) Synthesis of N- (5- (4-cyclohexylpiperazin-1-yl) -5,6,7,8-tetrahydronaphthalene-2-yl) -2- (pyrrolidin-1-yl) acetamide

N- (5-oxo-5,6,7,8-tetrahydronaphthalene-2-yl) -2- (pyrrolidin-1-yl) acetamide (500 mg), 1-cyclohexylpiperazin (929 mg), titanium tetrachloride (100 μL) ), using NaCNBH ₃ solution in tetrahydrofuran (1.0 M, 4 mL), the reaction was carried out according to the method described in example 22 to give the title target compound (210 mg, 27% yield).
^{1 1} HNMR (500MHz, CDCl ₃ ) δ1.13-1.38 (m, 5H), 1.65-1.70 (m, 3H), 1.84-1.89 (m, 8H), 1.95 -1.98 (m, 2H), 2.04-2.11 (m, 2H), 2.67-2.90 (m, 13H), 3.27 (s, 2H), 3.80-3 .82 (m, 1H), 7.24 (s, 1H), 7.40 (d, J = 8.3Hz, 1H), 7.55 (d, J = 8.6Hz, 1H), 9.01 (Br.s, NH).

(Example 24) 4-((1- (tert-butyl) -1H-tetrazole-5-yl) (4-cyclohexylpiperazin-1-yl) methyl) -N-2- (pyrrolidin-1-yl) ethyl ) Synthesis of aniline

N-(4-((1- (tert-butyl) -1H-tetrazole-5-yl) (4-cyclohexylpiperazin-1-yl) methyl) phenyl) -2- (pyrrolidin-1-yl) acetamide (62 mg) ) Was dissolved in tetrahydrofuran (2 mL), lithium aluminum hydride (5 mg) was added, and the mixture was stirred by heating and refluxing for 1 hour. The reaction solution was returned to room temperature, and an aqueous sodium hydroxide solution (3 drops) was added to stop the reaction. The precipitate was removed by filtration, and the residue was purified by column chromatography (developing solvent: methanol) to obtain 15 mg (yield 25%) of a colorless liquid product.
^{1 1} HNMR (300MHz, CDCl ₃ ) δ0.99-1.23 (m, 5H), 1.58-1.62 (m, 1H), 1.67 (s, 9H), 1.74-1.86 (M, 8H), 2.16-2.24 (m, 1H), 2.36-2.74 (m, 14H), 3.13-3.17 (m, 2H), 4.37 (br) .S, NH), 4.92 (s, 1H), 6.55 (d, J = 8.8Hz, 2H), 7.23 (d, J = 8.8Hz, 2H).

(Reference Example 21) Synthesis of 2-bromo-N- (4- (2-((tert-butyldimethylsilyl) oxy) ethyl) phenyl) acetamide

4-(2-((tert-butyldimethylsilyl) oxy) ethyl) aniline (1.55 g) was dissolved in dichloromethane (20 mL), cooled to 0 ° C., and then 2-bromoacetylbromid (770 μL), triethylamine ( 1.6 mL) was added, and the mixture was stirred at room temperature for 12 hours. The reaction mixture was diluted with water (50 mL) and then extracted with dichloromethane. The extract was washed with saturated brine and dried over magnesium sulfate. After distilling off the solvent under reduced pressure, the residue was purified by column chromatography (developing solvent: ethyl acetate ester: hexane = 1:10) to obtain 1.5 g (yield 53%) of a yellow solid product.
^{1 1} HNMR (300MHz, CDCl ₃ ) δ-0.01 (s, 6H), 0.87 (s, 9H), 2.80 (t, J = 7.1Hz, 2H), 3.79 (t, J) = 7.1Hz, 2H), 4.03 (s, 2H), 7.20 (d, J = 8.2Hz, 2H), 7.50 (d, J = 8.8Hz, 2H), 8.09 (Br.s, NH).

(Reference Example 22) Synthesis of N- (4- (2-hydroxyethyl) phenyl) -2- (pyrrolidin-1-yl) acetamide

2-bromo-N- (4- (2-((tert-butyldimethylsilyl) oxy) ethyl) phenyl) acetylamide (1.5 g) was dissolved in tetrahydrofuran (30 mL), pyrrolidine (393 μL), potassium carbonate (825 mg). ) Was added, and the mixture was stirred at room temperature for 20 hours. The reaction mixture was diluted with water (50 mL) and then extracted with ethyl acetate. The extract was washed with saturated brine and dried over magnesium sulfate. After distilling off the solvent under reduced pressure, the residue was purified by column chromatography (developing solvent: ethyl acetate ester: methanol = 4: 1) to obtain 204 mg (yield 21%) of a yellow liquid product.
^{1 1} HNMR (300MHz, CDCl ₃ ) δ1.84-1.89 (m, 4H), 2.68-2.72 (m, 4H), 2.85 (t, J = 6.5Hz, 2H), 3 .29 (s, 2H), 3.85 (t, J = 7.1Hz, 2H), 7.20 (d, J = 8.2Hz, 2H), 7.53 (d, J = 8.8Hz, 2H), 9.08 (br.s, NH).

(Reference Example 23) Synthesis of 4- (2- (pyrrolidin-1-yl) acetamide) phenethyl methanesulfonate

N- (4- (2-Hydroxyethyl) phenyl) -2- (pyrrolidin-1-yl) acetamide (170 mg) was dissolved in dichloromethane (5 mL), cooled to 0 ° C., and then methanesulfonic acid chloride (94 mg). , Triethylamine (141 μL) was added, and the mixture was stirred at room temperature for 12 hours. The reaction mixture was diluted with water (20 mL) and then extracted with dichloromethane. The extract was washed with saturated brine and dried over magnesium sulfate. After distilling off the solvent under reduced pressure, the residue was used for the next reaction without purification.

(Example 25) Synthesis of N- (4- (2- (4-cyclohexylpiperazin-1-yl) ethyl) phenyl) -2- (pyrrolidin-1-yl) acetamide

The residue (156 mg) obtained in Reference Example 23 was dissolved in acetonitrile (10 mL), 1-cyclohexylpiperazine (81 mg) and potassium carbonate (66 mg) were added, and the mixture was stirred under heating under reflux for 12 hours. The reaction mixture was diluted with water (20 mL) and then extracted with ethyl acetate ester. The extract was washed with saturated brine and dried over magnesium sulfate. After distilling off the solvent under reduced pressure, the residue was purified with (developing solvent: ethyl acetate ester: methanol = 4: 1) to obtain 9 mg (yield 5%) of a yellow solid product.
^{1 1} HNMR (300MHz, CDCl ₃ ) δ1.13-1.34 (m, 5H), 1.63-1.67 (m, 1H), 1.75-2.09 (m, 8H), 2.36 -2.49 (m, 1H), 2.59-2.81 (m, 16H), 3.28 (s, 2H), 7.16 (d, J = 8.8Hz, 2H), 7.49 (D, J = 8.2Hz, 2H), 9.06 (br.s, NH).

(Example 26) N- (4-((1- (tert-butyl) -1H-tetrazole-5-yl) (4-cyclohexylpiperazin-1-yl) methyl) -N- (2- (pyrrolidin-1) -Il-1-yl) Ethyl) Synthesis of aniline

N-(4-((1- (tert-butyl) -1H-tetrazole-5-yl) (4-cyclohexylpiperazin-1-yl) methyl) phenyl) -2- (pyrrolidin-1-yl) acetamide (62 mg) ) Was dissolved in tetrahydrofuran (2 mL), LAH (10 mg) was added, and the mixture was stirred at 60 ° C. for 30 minutes. The reaction solution was returned to room temperature, a 1N NaOH aqueous solution was added, and then the mixture was filtered through cerite. After distilling off the filtrate under reduced pressure, the residue was purified by column chromatography (developing solvent: ethyl acetate ester: hexane = 1: 1) to obtain 15 mg (yield 25%) of a yellow liquid product.
^{1 1} HNMR (300MHz, CDCl ₃ ) δ1.08-1.23 (m, 5H), 1.58-1.62 (m, 2H), 1.67 (s, 9H), 1.74-1.88 (M, 7H), 2.16-2.19 (m, 1H), 2.42-2.74 (m, 14H), 3.13-3.17 (m, 2H), 4.37 (br) .t, NH), 4.92 (s, 1H), 6.54 (d, J = 8.8Hz, 2H), 7.22 (d, J = 8.2Hz, 2H).

(Reference Example 24) Synthesis of 2-bromo-N- (4- (2-((tert-butyldimethylsilyl) oxy) ethyl) phenyl) acetamide

4-(((tert-butyldimethylsilyl) oxy) ethyl) aniline (1.9 g) is dissolved in dichloromethane (40 mL), cooled to 0 ° C., and then 2-bromoacetylbromid (770 μL), triethylamine (1. 6 mL) was added, and the mixture was stirred at room temperature for 12 hours. The reaction mixture was diluted with water (50 mL) and then extracted with dichloromethane. The extract was washed with saturated brine and dried over magnesium sulfate. After distilling off the solvent under reduced pressure, the residue was purified by column chromatography (developing solvent: ethyl acetate ester: hexane = 1:10) to obtain 1.5 g (yield 53%) of a yellow solid product.
^{1 1} HNMR (300MHz, CDCl ₃ ) δ-0.01 (s, 6H) 0.87 (s, 9H), 2.80 (t, J = 7.1Hz, 2H), 3.78 (t, J = 7.1Hz, 2H), 4.03 (s, 1H), 7.20 (d, J = 8.2Hz, 2H), 7.46 (d, J = 8.8Hz, 2H), 8.09 (d, J = 8.8Hz, 2H) br.s, NH).

(Reference Example 25) Synthesis of N- (4- (2-hydroxyethyl) phenyl) -2- (pyrrolidin-1-yl) acetamide

2-bromo-N- (4- (2-((tert-butyldimethylsilyl) oxy) ethyl) phenyl) acetylamide (1.5 g) was dissolved in tetrahydrofuran (20 mL), pyrrolidine (393 μL), potassium carbonate (828 mg). ) Was added, and the mixture was stirred at room temperature for 20 hours. The reaction mixture was diluted with water (50 mL) and then extracted with ethyl acetate. The extract was washed with saturated brine and dried over magnesium sulfate. After distilling off the solvent under reduced pressure, the residue was purified by column chromatography (developing solvent: ethyl acetate ester: methanol = 10: 1) to obtain 204 mg (yield 21%) of a yellow liquid product.
^{1 1} HNMR (300MHz, CDCl ₃ ) δ1.84-1.89 (m, 4H), 2.68-2.72 (m, 4H), 2.85 (t, J = 6.5Hz, 2H), 3 .29 (s, 2H), 3.85 (t, J = 6.4Hz, 2H), 7.20 (d, J = 8.2Hz, 2H), 7.53 (d, J = 8.8Hz, 2H), 9.01 (br.s, NH).

(Reference Example 26) Synthesis of 4- (2- (pyrrolidin-1-yl) acetamide) phenethyl methanesulfonate

N- (4- (2-Hydroxyethyl) phenyl) -2- (pyrrolidin-1-yl) acetamide (170 mg) is dissolved in dichloromethane (5 mL), and triethylamine (141 μL) and methanesulfonyl chloride (64 μL) are added. The mixture was stirred at room temperature for 12 hours. After distilling off the reaction solution under reduced pressure, the residue was subjected to column chromatography (developing solvent: ethyl acetate ester: methanol = 4: 1), and 156 mg of the obtained crude product was used for the next reaction.

Example 27 Synthesis of N- (4- (2- (4-cyclohexylpiperazin-1-yl) ethyl) phenyl) -2- (pyrrolidin-1-yl) acetamide

The crude 4- (2- (pyrrolidin-1-yl) acetamide) phenethyl methanesulfonate (156 mg) obtained in Reference Example 26 was dissolved in acetonitrile (10 mL), and potassium carbonate (66 mg) and 1-cyclohexylpiperazine (1-cyclohexylpiperazine) were dissolved in acetonitrile (10 mL). 81 mg) was added, and the mixture was stirred at 70 ° C. for 12 hours. The reaction mixture was diluted with water (50 mL) and then extracted with ethyl acetate. The extract was washed with saturated brine and dried over magnesium sulfate. After distilling off the solvent under reduced pressure, the residue was purified by column chromatography (developing solvent: ethyl acetate ester: methanol = 4: 1) to obtain 9 mg (yield 5%) of a yellow liquid product.
^{1 1} HNMR (300MHz, CDCl ₃ ) δ1.19-1.35 (m, 5H), 1.63-1.67 (m, 1H), 1.76-1.98 (m, 8H), 1.94 -2.03 (m, 1H), 2.43-2.81 (m, 16H), 3.27 (s, 2H), 7.16 (d, J = 8.8Hz, 2H), 7.49 (D, J = 8.2Hz, 2H), 9.06 (br.s, NH).

(Example 28) Synthesis of (5- (4-cyclohexylpiperazin-1-yl) -N- (2- (pyrrolidin-1-yl) ethyl) -5,6,7,8-tetrahydronaphthalene-2-aniline

N- (5- (4-Cyclohexylpiperazin-1-yl) -5,6,7,8-tetrahydronaphthalene-2-yl) -2- (pyrrolidin-1-yl) acetamide (85 mg), LAH (15 mg) Was used for reaction treatment according to the method described in Example 22 to obtain the title object (35 mg, yield 43%).
^{1 1} HNMR (500MHz, CDCl ₃ ) δ1.08-1.28 (m, 5H), 1.61-1.66 (m, 3H), 1.77-1.81 (m, 6H), 1.90 -1.94 (m, 4H), 2.20-2.24 (m, 2H), 2.52-2.82 (m, 15H), 3.18 (t, J = 6.2Hz, 2H) , 3.70-3.72 (m, 1H), 6.33 (d, J = 2.5Hz, 1H), 6.49 (dd, J = 2.7, 8.6Hz, 1H), 7. 44 (d, J = 8.3Hz, 1H).

(Test example)
<Materials and methods>
1. 1. Reagents and Antibodies Candidate compounds with anti-prion effects (NPR-130 and -162) were purchased from ASINEX. Using NPR-130 and -162 as lead compounds, the compounds of the present application (NPRS-1 (Example 1), -2 (Example 2), -3 (Example 3), -4 (Example 4), -5 ( Examples 5), -6 (Example 6), -7 (Example 7), -8 (Example 8), -9 (Example 9), -10 (Example 10), -11 (Example 10). 11), -12 (Example 12), -13 (Example 13), -14 (Example 21), -15 (Example 14), -16 (Example 15), -17 (Example 16). , -18 (Example 17), -19 (Example 18), -20 (Example 19), -21 (Example 20), -22 (Example 22), -23 (Example 23),- 24 (Example 26), -25 (Example 27), -26 (Example 28)) were newly synthesized and developed. NPR-56 (Ishibashi et al. (2016). EBioMedicine, Vol. 9, p238-249) purchased from ASINEX and GN8 (2-pyrrolidin-1-yl-N- [4- [4-[ 4- (2-pyrrolidin-1-yl-acetylamino) -benzyl] -phenyl] -acetamide, molecular weight 420) was used as a positive control for antiprion drugs (Kuwata et al. (2007). Proc Natl Acad Sci USA. 104, 11921-11926). These compounds were completely dissolved in 100% dimethyl sulfoxide (DMSO) and prepared as a stock solution to 10 mM. The stock solution of the compound was diluted with sterile water, medium, or phosphate buffered saline (PBS) and used in the assay. PrP-specific antibodies (Santa Cruz Biotechnology, M20; SPI-Bio, SAF32, SAF61, SAF83), Iba-1 antibodies (WAKO, 019-19741 (IHC)) and GFAP antibodies (DAKO, Z033429) are commercially available. I used the one. Horseradish peroxidase-bound anti-goat (Jackson ImmunoResearch) and anti-mouse (GE Healthcare Life Sciences) IgG antibodies were also used for immunoblotting.
The chemical structures of NPR-130, -162 and -56 are shown below.

2. Surface plasmon resonance (SPR) analysis Interactions between recombinant PrP and compounds were evaluated using the Biacore T200 system (GE Healthcare) (Nakagaki et al. (2013) Autophagy 9, 1386-1394). Human or mouse PrP (23-231) was synthesized by E. coli in a protein expression system using a pET vector, and the protein was purified by imidazole in an NTA column (Atarashi et al. (2011) Nat Med 17, 175-178). ). The recombinant PrP solution was diluted with running buffer (containing 10 mM HEPES, pH 7.4, 150 mM NaCl, 0.05% Tween 20 (Sigma-Aldrich) and 5% DMSO) to 10 μg / ml to give the ligand. It was fixed on a CM5 sensor chip (GE, BR-100530) using an amine coupling kit (GE, BR-1000-50). PrP immobilization was performed using an average value of 2000 RU. Compounds of various concentrations were evaluated by diluting with the same running buffer and injecting sequentially for 2 minutes at a flow rate of 30 mL / min, then further at the same flow rate to wash out compounds bound only with running buffer. Infused for 20 minutes. The data was corrected using a blank sensor chip as a control. Each compound was dissolved in DMSO and diluted to 5% with running buffer buffer.

3. 3. Cell culture mouse neuroblastoma neuro 2a cells were obtained from the American Type Culture Collection (CCL 131). N2a-58 cells are ^{established from N2a cells that overexpress PrP C} and integrate the mouse PRNP gene in neuro 2a cells. N2a-FK cells established from N2a-58 cells were infected with the Fukuoka-1 (mouse-adapted Gerstmann-Straussler-Scheinker strain) strain. Dulbecco's modified Eagle's medium (Wako) (4,500 mg / mL glucose, 10% heat-inactivated fetal bovine serum, 100 units / mL penicillin and 100 μg / mL) of the above cells at 37 ° C in 5% CO _2. It was grown in streptomycin (including Nakalaitesk). Cell viability was determined by counting viable cells using Luna automatic cell counters (Logos Biosystems), and cell morphology was visualized under a microscope. Immunoblotting was performed by the method of Homma, T. (Sci Rep 4, 4504 (2014)). Cytolysis was digested with 20 μg / mL proteinase K (PK) at 37 ° C for 30 minutes to detect PrP ^Sc. After adding SDS-sample buffer, apply the sample to a 15% SDS-PAGE gel and transfer to a PVDF membrane. Subsequently, ^{in order to detect PrP Sc} , anti-goat IgG-HRP was supplied as a primary antibody (1: 1000) and as a secondary antibody (1: 5000). Bands were visualized using the Clarity Western ECL Substrate Western Blotting Detection Kit (Bio-Rad). Band intensity was quantified using Image J software (National Institutes of Health).

Four. Immunofluorescence analysis Immunofluorescence staining of prion-infected cells was performed as follows. Cells were cultured in chamber slides (BD Falcon) and treated with DMSO as a control, each drug NPR-130, -162, and 23 NPRS drugs at concentrations up to 10 μM for 48 hours. The cells were then washed twice with PBS and then fixed with 4% formaldehyde for 30 minutes at room temperature. After permeabilization with 0.5% Triton X-100, the slides were treated with 5% skim milk and reacted at room temperature for 1 hour. Slides were reacted with 3M guanidine thiocyanate for 5 minutes at room temperature for detection of PrP ^Sc. The cells were then ^{subjected to SAF61 (1: 200) as the primary antibody overnight at 4 ° C. for detection of PrP Sc} , followed by Alexa Fluor® 488-conjugated anti-mouse IgG (Invitrogen) (1: 500). , Reacted at 37 ° C for 1 hour. The nuclei were stained with Vectashield mounting medium containing DAPI (Vector Laboratories). Aggresomes were detected using the ProteoStat® agresome detection kit (Enzo Life Science) and then detected at a fluorescence wavelength of 600 nm with an excitation wavelength of 500 nm. All images were visualized using a confocal laser scanning microscope LSM700 (Carl Zeiss).

Five. In vivo injection experiment
4-week-old male ddY mice were purchased from SLC (Hamamatsu, Japan). In the brains of the mice were inoculated with 10 ^-1 dilution 20μL brain homogenate prepared from mice with end-stage infected with FK-1 strain. Various compounds (NPR-130 and -162) were dissolved in 0.25% DMSO-containing saline, and mice were intraperitoneally administered 2.0 mg of compound / kg / day every other day. Mice inoculated intraperitoneally with physiological saline containing 0.25% DMSO were used as controls. Mice were monitored every other day until the end of the disease or until sacrifice. Clinical onset was defined as the presence of two or more symptoms such as greasy or yellowish hair, hunchback, weight loss, yellow pubic hair, ataxic gait and non-parallel hindlimbs. Mice were sacrificed at clinical onset (100 dpi) or at the end of life and the brain and spleen were removed. Right hemisphere and spleen sections of the brain were immediately frozen and homogenized in 20% (weight / volume) in PBS. For immunoblotting analysis, the total protein was mixed with an equivalent of 2x lysis buffer (300 mM NaCl, 1% Triton-X-100, 1% sodium deoxycholate and 100 mM Tris-HCl; pH 7.5). Extracted. The remaining brain and spleen were fixed with 10% neutral buffered formalin for pathological analysis.

6. Pathological analysis Fixative tissue was embedded in paraffin and cut into sections 3-5 μm thick. Sections were stained with hematoxylin (WAKO, 131-09665) and eosin (WAKO, 056-06722) to assess spongy changes (degree of vacuolar degeneration) in brain tissue. After deparaffinization and rehydration for Iba-1 staining, sections were boiled in Target Retrieval Solution, Citrate pH 6 (DAKO, S2369) for 20 minutes to activate the antigen. The sections were then treated with 0.3% hydrogen peroxide solution (WAKO, 086-07445) and methanol (Hayashi Junyaku, 130-02069) for 30 minutes to inactivate endogenous peroxidase and then 3% defatted milk powder. It was treated with Tris-buffered saline (TBST) containing (Megmilk Snow Brand, FA-08) and reacted at room temperature for 60 minutes. TBST is a Tris buffered saline solution containing 0.1% Tween-20. Anti-IBA-1 antibody (WAKO, 019-19741) and anti-GFAP antibody (DAKO, Z033429) were used as primary antibodies overnight at room temperature, followed by EnVision® Polymer Horseradish Peroxidase (HRP) -conjugated anti-rabbit IgG. The reaction was carried out at room temperature for 60 minutes using (DAKO, K4002). Visualization was performed using 3,3-diaminobenzidine (DAB; Dojin Chemical Research Institute, D006) as a color-developing substrate for immunostaining. Treatment of the autoclave formic acid method for PrP ^Sc staining was performed according to the method described in Ishibashi, D. et al., (2012) J Virol 86, 4947-4955, and SAF32 (1: 200) for the primary antibody. Was reacted using the above-mentioned method and analyzed according to the above-mentioned method.

7. 7. The results of the statistical analysis graph represent the mean ± SD from at least three independent experiments. Statistical analysis of all data was performed using Statcel 2 from Excel and GraphPad Prism software. Student's t-test was performed for comparison between the two groups, and ANOVA with Tukey-Kramer test was used for multiple comparisons. The Log-rank test was used to analyze the mortality rate of prion-infected mice.

FIG. 1 shows the direct binding of the compound to recombinant PrP using SPR analysis. Specifically, FIG. 1 shows sensorgrams of NPR-130 and NPR-162 for binding to mouse or human PrP23-231. The concentrations of the compounds are 0, 0.2, 0.4, 0.8, 1.6, 3.1, 6.3, 12.5, 25 and 50 μM from the bottom.

FIG. 2 shows the effect of NPR-130 and NPR-162 on cytotoxicity in non-prion infected cells (N2a-58). N2a-58 cells were treated with the solvents DMSO, NPR-130, and -162 at 10 μM, and the number of cells 48 hours later was examined. The upper part of the figure shows the cell viability. The lower part shows a photograph under a phase-contrast microscope. The bar is 100 μm.

FIG. 3 shows the dose response of anti-prion compounds (NPR-130 and NPR-162) to ^{aberrant prion protein (PrP Sc} ) in prion-infected cells (N2a-FK). Specifically, FIG. 2 shows N2a-FK cells treated with NPR-130, -162 compounds at different concentrations (0, 0.1, 0.5, 1, 5 and 10 μM) for 48 hours and PrP by Western blotting. The expression of ^{Sc is shown.}

FIG. 4 shows images of the effects of anti-prion compounds (NPR-130 and NPR-162) on prion-infected cells (N2a-FK) observed under a microscope. The upper row shows the localization ^{of PrP Sc} in N2a-FK cells treated with 10 μM NPR-130 or -162 for 48 hours using immunofluorescent staining. PrP ^Sc and nuclei were detected using SAF61 antibody (green) and DAPI (blue), respectively. In the lower row, the expression of agresomes after compound treatment was observed. Intracellular agresomes are shown in red. Each stained cell was visualized by a confocal laser scanning microscope. The scale bar represents 10 micrometers.

FIG. 5 shows the evaluation of NPR-130 and -162 using a prion bioassay system. Specifically, FIG. 5 shows NPR-130 or -162 from the second day (dpi) after inoculation of 10% Fukuoka-1 (FK-1) prion strain-infected brain emulsion into 4-week-old ddy mice. The survival curve of the prion-infected mouse which ingested any of the above compounds is shown. The solvent-administered group (vehicle) (circle: N = 9), NPR-130 (triangle: N = 10) and NPR-162 (square: N = 11) were compared. Statistical significance was analyzed using the Log-rank test.

FIG. 6 shows the expression ^{of PrP Sc} at 100 dpi in prion-infected mice treated with NPR-130 and -162 and in the brain and spleen at the end of infection. The upper row shows the results of sacrificing each prion-infected mouse at 110 dpi ^{and measuring PrP Sc levels in the brain and spleen.} PrP ^Sc was analyzed by Western blotting using M20 antibody as the primary antibody. The accumulated amount of PrP ^Sc was graphed by measuring the band intensity. The lower row shows the results of sacrificing each prion-infected mouse at the end of infection (after onset) ^{and measuring PrP Sc levels in the brain and spleen as described above.} Two-way ANOVA with Chuky-Kramer test for multiple comparisons was performed. * P <0.05 and ** P <0.01 are shown.

FIG. 7 shows pathological changes in the 100 dpi brain and spleen of prion-infected mice treated with NPR-130 and -162. From the top, the degree of vacuolar degeneration, microglial activation, astrocyte activation, and PrP ^Sc accumulation were observed under a microscope. The lower row shows the accumulation ^{of PrP Sc in the spleen.} The degree of vacuole formation in the thalamic region of the mouse brain was compared by HE staining, and the occupied area of the vacuole in each section was calculated. Microglial activation in the thalamic region of the mouse brain was analyzed by analyzing Iba-1 expression by immunohistochemical staining and quantifying IBA-1 positive cells. Astrocyte activation in the thalamic region of the mouse brain was analyzed by analyzing GFAP expression by immunohistochemical staining and quantifying GFAP-positive cells. ^{Accumulation of PrP Sc} in the thalamic region of the mouse brain and in the spleen was analyzed for SAF32 antibody-positive reactions. In histological analysis, all scale bars represent 50 micrometers. The graph on the right is a graph of each staining positive rate. In the bar of the graph, white indicates the solvent administration group (vehicle), gray indicates the NPR-130 administration group, and black indicates the NPR-162 administration group. Statistical significance was used in a histological analysis in the brain region thalamus (Thalamus) by performing a two-way ANOVA with the Chuky-Kramer test for multiple comparisons. * P <0.05 and ** P <0.01.

FIG. 8 shows the effect of NPRS compounds (26 types) on ^{PrP Sc} in cells continuously infected with prions (N2a-FK). Specifically, FIG. 8 shows screening of antiprion drug candidates in N2a-FK cells. All compounds were added to cells at 10 μM and treated for 48 hours. PrP ^Sc was detected by Western blotting. DMSO-treated cells were used as a negative control. The molecular weight (kDa) is shown on the left side of each panel. The lane number of each panel indicates an individual candidate compound of the NPRS series. Statistical significance is a two-way ANOVA with the Chuky-Kramer test for multiple comparisons, with red bars _{indicating IC 50} measurable compounds. * P <0.05 and ** P <0.01.

Figure 9 shows anti-prion compounds (NPRS-3, -6, -7, -9, -11, -17, -19, -20) against ^{aberrant prion protein (PrP Sc} ) in prion-infected cells (N2a-FK). , -21, -23, -24, -25) dose response. Specifically, FIG. 9 shows N2a-FK cells NPRS-3, -6, -7, -9, -11, -17, -19, -20, -21, -23, -24, -25. Was treated at different concentrations (0, 0.1, 0.5, 1, 5 and 10 μM) for 48 hours and Western blotting showed expression of ^{PrP Sc.}

FIG. 10 shows the binding affinity screening of the compound NPRS group for PrP using SPR analysis as a bar graph quantifying the binding ability of each compound to human and mouse PrP23-231. Specifically, FIG. 10 shows the relative response (RU) obtained from various NPRS compounds when binding to human (top) and mouse (bottom) PrP using a Biacore T200 instrument. The results as a primary screening of prion compounds are shown. The concentration of the compound is 10 μM.

FIG. 11 shows the direct binding of the compound to recombinant PrP using SPR analysis. Specifically, FIG. 11 shows a sensorgram of each NPRS group for binding to human PrP23-231. The concentrations of the compounds are 0, 0.2, 0.4, 0.8, 1.6, 3.1, 6.3, 12.5, 25 and 50 μM from the bottom.

FIG. 12 shows the direct binding of the compound to recombinant PrP using SPR analysis. Specifically, FIG. 12 shows a sensorgram of each NPRS group for binding to mouse PrP23-231. The concentrations of the compounds are 0, 0.2, 0.4, 0.8, 1.6, 3.1, 6.3, 12.5, 25 and 50 μM from the bottom.

<Result>
1. 1. ^{Assessment of binding affinity between PrP C} and NPR-130, -162 using surface plasmon resonance (SPR) analysis
NPR-130, in order to evaluate the interaction between -162 and PrP ^C, was analyzed direct bond. Dynamic analysis showing the gradient of the sensorgram for each compound showed a dose-dependent response (Fig. 1). The KD value shown as the dissociation constant was 1.02 ± 0.9 mM for human PrP and 0.12 ± 0.14 mM for mouse PrP in NPR-130. In NPR-162, it was 2.6 ± 2.3 mM for human PrP and 0.058 ± 0.058 mM for mouse PrP, and NPR-130 and NPR-162 had binding ability for each PrP ( Figure 1, Table 1).

2. Toxicity study of NPR-130, -162 in cultured cells
NPR-130, in order to evaluate the interaction between -162 and PrP ^C, was examined for cell viability after compound addition in N2a-58 cells derived from mouse neuroblastoma. No change was observed in the cell viability 48 hours after the addition of the 10 μM compound, and there was no change in the cell morphology under a phase-contrast microscope, and there was no manifestation of cytotoxicity (Fig. 2). ..

3. Effect of NPR-130, -162 on PrP ^{Sc in prion-infected cells}
To evaluate the effect of NPR-130, -162 on aberrant prion protein (PrP ^Sc ), after prion infection in N2a-58 cells, 0.1, 0.5, 1, 5, 10 μM of established prion persistently infected cells The cells were treated at a concentration of 48 hours, and changes in the expression level of ^{PrP Sc in the cell lysate were examined by Western blotting. A marked decrease in PrP Sc} expression was observed in the high concentration treatment of 10 μM (Fig. 3). Similar experiments were performed three or more times _{to examine IC 50} (μM). _{The IC 50 of the} NPR-130 was 6.7 ± 3.9 μM, and the IC _{50 of the} NPR-162 was 5.0 ± 2.3 μM.

^{4. Evaluation of PrP Sc} after treatment with prion-infected cells using immunofluorescent antibody method An immunofluorescent antibody method was used to visualize ^{PrP Sc in prion-infected cells.} To selectively detect PrP ^Sc , persistent prion-infected cells were incubated with an anti-PrP monoclonal antibody and treated with guanidine thiocyanate ^{to reveal aggregated PrP Sc} (green) in the cells. ^{As a result, the expression of PrP Sc} in N2a-FK cells treated with NPR-130 and -162 for 48 hours was significantly reduced (upper part of FIG. 4). In addition, cells were stained with Proteostat, which detects agglutinating proteins containing intracellular agresomes. Aggresomes are intracellular inclusion bodies that are established by aggregation of various unnecessary proteins and coexist with phosphorylated tau accumulation and α-synuclein in pathological tissues of conformational diseases and cell culture models. Aggresomes are large complexes of aberrant proteins that are resistant to degradation and are therefore isolated as inclusion bodies. It is believed that sequestered agresomes can be degraded by the autophagy system. Recent reports indicate that in persistently infected cells, P62, a major factor in agresome formation ^{, interacts closely with PrP Sc} and is important in promoting degradation via the autophagy system. It has been reported. Aggresome detection probes (Proteostat) were used to investigate whether NPR-130 and NPR-162 affect agresome levels in prion persistently infected cells. Many agresomes were detected in the cytoplasm of N2a-FK cells treated with DMSO alone (red in the lower part of FIG. 4). Treatment of the antiprion compounds NPR-130 and NPR-162 at 10 μM for 48 hours not ^{only suppressed PrP Sc} in N2a-FK cells, but also significantly reduced the number of intracellular agresomes (lower part of FIG. 4). These results indicate that NPR-130 and NPR-162 may promote the removal of dysfunctional and unwanted proteins in ^{PrP Sc and cytoplasm.}

5. Efficacy Evaluation of Compounds in Bioassay Using Prion Disease Model Mice To evaluate the efficacy of NPR-130 and NPR-162 in animal models of prion disease, 20 μL of Fukuoka-1 strain prion-infected mouse brain homogenate was intracerebral. To CD-1 (ddy) mice inoculated with either NPR-130 or NPR-162, 2 mg / kg / day of 2 mg / kg / day was administered from 2 days (dpi) to 3 times a week after prion inoculation until killing. bottom. The survival time of mice was 189 ± 23 days (n = 9) in the solvent-administered (vehicle) group (circle) and 291 ± 134 days (n = 10; p = 0.0381 vs solvent group, Log) in the NPR-130-administered group (triangle). -Rank test), 258 ± 93 days (n = 11; p = 0.0074 vs solvent group, Log-rank test) in the NPR-162 administration group (square), and the compound administration group significantly prolonged the survival time (-rank test). Figure 5). This suggests that NPR-130 and NPR-162 compounds are useful as therapeutic drug candidates for prolonging survival in prion infections.

^{6. Examination of PrP Sc} expression in prion disease model mice treated with NPR-130 and NPR-162 To evaluate the efficacy of NPR-130 and NPR-162 in prion disease animal models, Fukuoka-1 strain prion-infected mice CD-1 (ddy) mice infused with 20 μL of brain homogenate were given either NPR-130 or NPR-162 2 mg / kg / day 3 times a week from 2 days (dpi) after prion inoculation. Was administered. ^{The expression of PrP Sc} in the brain and spleen of mice at 100 dpi after prion infection (before the onset of mice) and at the terminal stage of infection was evaluated using Western blotting.
^{The expression of PrP Sc} in the brain 100 dpi after prion infection tended to decrease in the NPR-130 and NPR-162 administration groups compared to the solvent-administered (vehicle) group (upper left in Fig. 6), and PrP ^{Sc in} the spleen. The expression tended to decrease in the NPR-130-administered group and significantly decreased in the NPR-162-administered group compared to the solvent-administered (vehicle) group (upper right in FIG. 6). ^{The expression of PrP Sc} in the brain at the end of prion infection was significantly decreased in the NPR-162 group compared with the solvent-administered (vehicle) group (lower left in Fig. 6), and the expression ^{of PrP Sc in the spleen was} Compared with the solvent-administered (vehicle) group, the NPR-162-administered group had a significant decrease (lower right in Fig. 6). The expression level of the NPR-130 administration group was similar to that of the solvent administration (vehicle) group. This means that the NPR-130 and NPR-162 compounds can suppress the accumulation ^{of PrP Sc in vivo in prion infection.}

7. Examination of histological pathological changes in prion disease model mice treated with NPR-130 and NPR-162 To evaluate the efficacy of NPR-130 and NPR-162 in animal models of prion disease, Fukuoka-1 strain prion Infected mouse CD-1 (ddy) mice infused with 20 μL of brain homogenate were given either NPR-130 or NPR-162 at 2 mg / kg 3 times a week from 2 days (dpi) after prion inoculation. A dose of / day was administered. Histological changes were analyzed for the degree of vacuolar degeneration in the thalamic region of the mouse brain and the expression of microglia and astrocytes and the expression of PrP ^{Sc at 100 dpi after prion infection (before the onset of mice).} Regarding the expression of PrP ^Sc , the degree of accumulation in the spleen was also examined. The degree of vacuolar degeneration by HE staining was significantly reduced in the NPR-130 and NPR-162 administration groups as compared with the solvent administration (vehicle) group (top row of FIG. 7). Microglial expression is EF hand protein and IBA-1 protein, a marker of activated microglia (also known as allograft inflammatory factor 1: AIF1) (Ito, D. et al., (1998) Brain Res Mol. It was examined in Brain Res 57, 1-9, etc.). The number of IBA-1 positive cells was significantly reduced in the NPR-130 and NPR-162 administration groups as compared with the solvent administration (vehicle) group (second row from the top of FIG. 7). The expression of astrocytes was significantly decreased in the NPR-130 and NPR-162 administration groups as compared with the solvent administration (vehicle) group (third row from the top of FIG. 7). The expression of PrP ^Sc was significantly decreased in the NPR-130 and NPR-162 administration groups as compared with the solvent administration (vehicle) group (4th row from the top of FIG. 7). The expression of PrP ^{Sc in} the spleen was significantly reduced in the NPR-130 and NPR-162 administration groups as compared with the solvent administration (vehicle) group (bottom of FIG. 7). The above results show that NPR-130 and NPR-162 compounds ^{suppress the accumulation of PrP Sc} in vivo in prion infection and suppress the expression of vacuolar degeneration and gliosis associated with neuronal cell death, which are the major pathological changes. It means that you can.

8. Effect of newly synthesized NPRS compounds on prP ^Sc in prion-infected cells NPRS-1 to -26 (26), a group of newly synthesized compounds using NPR-130 and NPR-162, which are effective as anti-prion drugs, as lead compounds. The drug efficacy evaluation was examined using the type). To evaluate the effect on aberrant prion protein (PrP ^Sc ), N2a-58 cells were infected with prion, and established prion persistently infected cells were treated at a concentration of 10 μM for 48 hours, and PrP ^Sc in the cell lysate was treated. The change in the expression level was examined by the Western blot method. The same experiment was performed three or more times, and ^{the expression level of PrP Sc} was quantified and made into a bar graph. NPRS-3, -4, -6, -7, -8, -9, -10, -11, -17, -19, -20, -21, -23, -24 compared to the solvent (DMSO) group , -25 compounds (15 in total) treatment group significantly ^{reduced PrP Sc} expression at high concentration treatment of 10 μM (Fig. 8). ^{Among them, the compounds in which the expression level of PrP Sc} was reduced by 50% or more were NPRS-3, -6, -7, -9, -11, -17, -19, -20, -21, -23,-. There were a total of 12 pieces, 24 and -25.

9. Calculated _{IC 50} for the inhibitory effect of NPRS on prP ^Sc of prion-infected cells (N2a-FK)
NPRS-3, -6, -7, -9, -11, -17, -19, -20, -21, -23, -24, -25, which reduces the expression level of ^{PrP Sc} by 50% or more, 12 in total to evaluate an IC ₅₀ concentration of effects on PrP ^Sc in number of compounds, 48 hours in N2a-58 cells after prion infection, at a concentration of 0.1,0.5,1,5,10 [mu] M in the established prion persistently infected cells ^{Then, changes in the expression level of PrP Sc} in the cell lysate were examined by Western blot method. ^{For all compounds, a significant decrease in PrP Sc} expression was observed in a concentration-dependent manner and at a high concentration treatment of 10 μM (Fig. 9). Similar experiments were performed three or more times _{to examine IC 50} (μM). Table 2 shows the IC ₅₀ of each NPRS. Each compound has an already reported IC ₅₀ of GN8 of 6.7 ± 1.1 (Ishibashi D, et al. EBioMedicine 9 (2016) 238-249), which is comparable to or higher than that of the compound. Conceivable. It is suggested that it is a highly effective compound as a novel therapeutic agent for prion disease.

a represents the experimental result in Ishibashi D, et al. EBioMedicine 9 (2016) 238-249.

10. Surface plasmon resonance (SPR) NPRS-3 having a binding affinity for evaluation antiprion effect between the NPRS compound having PrP ^C and anti-prion effect by high-throughput screening using the analysis, -6, -7, Human and mouse to evaluate the interaction between ^{PrP C} and a total of 12 compounds of -9, -11, -17, -19, -20, -21, -23, -24, -25. The direct binding affinity of NPRS for PrP was analyzed. Using high throughput screening in Biacore (TM), using a sensor chip immobilized with human or mouse PrP ^C, the compound of 10μM were sequentially subjected to SPR analysis. The binding affinity of each compound was measured, and NPRS-3, -6, -7, -9, -11, -17, -19, -20, -21, -23, -24, -25, total 12 number of compounds were confirmed to have an ability to bind to human and murine PrP ^C. Its binding relative response (RU) was previously reported in GN8, NPR-56 (Ishibashi D, et al. EBioMedicine 9 (2016) 238-249), and NPR-130, -162, which had effects on prion-infected mice. The binding ability was equal to or higher than that of the above. In particular, NPRS-19 and NPRS-23 had several times higher binding ability (Fig. 10). From these results, the NPRS compound can be expected as a novel compound useful as an anti-prion drug, using NPR-130 and NPR-162 as lead compounds without impairing its anti-prion effect and ability to bind to PrPC. It suggests.

11. Evaluation of concentration-dependent binding affinity between the surface plasmon resonance (SPR) human PrP ^C and NPRS compound using analysis
Between a total of 12 compounds of NPRS-3, -6, -7, -9, -11, -17, -19, -20, -21, -23, -24, -25 and human PrP ^C Direct binding was analyzed to assess the interaction. Dynamic analysis showing the gradient of the sensorgram for each compound showed a dose-dependent response (Fig. 11). The values of KD (mM) shown as each dissociation constant are shown in Table 3. Each NPRS had a binding ability to human PrP (Fig. 11, Table 3).

12. Evaluation of concentration-dependent binding affinity between the surface plasmon resonance (SPR) mouse PrP ^C and NPRS compound using analysis
Between a total of 12 compounds of NPRS-3, -6, -7, -9, -11, -17, -19, -20, -21, -23, -24, -25 and mouse PrP ^C Direct binding was analyzed to assess the interaction. Dynamic analysis showing the gradient of the sensorgram for each compound showed a dose-dependent response (Fig. 12). The values of KD (mM) shown as each dissociation constant are shown in Table 3. Each NPRS had a binding ability to mouse PrP (Fig. 12, Table 3).

The compound of the present invention has an effect of suppressing the production of abnormal prions and is useful as a therapeutic agent for prion diseases and the like.
This application is based on Japanese Patent Application No. 2018-177224, the contents of which are incorporated herein by reference in its entirety.

Claims (3)

Equation (I):

[During the ceremony,
Ring A represents a C _5-10 hydrocarbon ring or a 5- to 10-membered heterocycle;
R ¹ represents a hydrogen atom, a C _1-6 alkyl group or an optionally substituted 5- to 6-membered monocyclic aromatic heterocyclic group;
R ² indicates a hydrogen atom or an oxo group;
Alternatively, R ¹ and R ² combine to form a C _4-8 hydrocarbon ring;
R ³ indicates a hydrogen atom, a C _1-6 alkyl group or an optionally substituted amino group;
Y is -NR ⁴ R ⁵ or

Show;
R ⁴ and R ⁵ each independently represent a hydrogen atom or an optionally substituted C _1-6 alkyl group;
X indicates O or NR ⁶ ;
Ring B represents a 4- to 8-membered nitrogen-containing non-aromatic heterocycle;
n indicates 0 or 1;
R ⁶ represents a C _1-6 alkyl group, a C _3-10 cycloalkyl group or an optionally substituted C _1-6 alkyl-carbonyl group. ]
Compounds represented by (However, the following compounds:

except for. ) Or its salt. Ring A is a C _6-10 arene ring or a 5- to 10-membered non-aromatic heterocycle;
R ¹ is substituted with 1 to 3 substituents selected from (1) hydrogen atom, (2) C _1-6 alkyl group or (3) (a) 5 to 6 member monocyclic aromatic heterocyclic group. A 5- to 6-membered monocyclic aromatic heterocycle optionally substituted with 1 to 3 substituents selected from the C _1-6 alkyl group and (b) C _{3-10 cycloalkyl group.} Is the basis;
R ² is hydrogen atom or an oxo group; or,
R ¹ and R ² combine to form a C _4-8 cycloalkene ring;
R ³ is 1 to 3 substituents selected from (1) hydrogen atom, (2) C _1-6 alkyl group or (3) (a) 5 to 6 member monocyclic non-aromatic heterocyclic group. optionally substituted with 1-3 substituents selected from optionally substituted C _1-6 alkyl group and (b) 5 to 6-membered monocyclic non-aromatic heterocyclic group C _1-6 An amino group optionally substituted with one or two substituents selected from alkyl-carbonyl groups;
Y is -NR ⁴ R ⁵ or

And;
R ⁴ and R ⁵ are independently composed of (1) hydrogen atom or (2) (a) mono- or di-C _1-6 alkylamino group and (b) 5- to 6-membered monocyclic non-aromatic group. _{A C 1-6} alkyl group optionally substituted with 1-3 substituents selected from the heterocyclic group;
Ring B is a 4- to 8-membered nitrogen-containing non-aromatic heterocycle;
X is O or NR ⁶ ;
n is 0 or 1;
R ⁶ is 1 to 3 substitutions selected from (1) C _1-6 alkyl group, (2) C _3-10 cycloalkyl group or (3) 5 to 6 member monocyclic non-aromatic heterocyclic group. _{A C 1-6} alkyl-carbonyl group that may be substituted with a group;
The compound according to claim 1 or a salt thereof. Equation (I):

[During the ceremony,
Ring A represents a C _5-10 hydrocarbon ring or a 5- to 10-membered heterocycle;
R ¹ represents a hydrogen atom, a C _1-6 alkyl group or an optionally substituted 5- to 6-membered monocyclic aromatic heterocyclic group;
R ² indicates a hydrogen atom or an oxo group;
Alternatively, R ¹ and R ² combine to form a C _4-8 hydrocarbon ring;
R ³ indicates a hydrogen atom, a C _1-6 alkyl group or an optionally substituted amino group;
Y is -NR ⁴ R ⁵ or

Show;
R ⁴ and R ⁵ each independently represent a hydrogen atom or an optionally substituted C _1-6 alkyl group;
X indicates O or NR ⁶ ;
Ring B represents a 4- to 8-membered nitrogen-containing non-aromatic heterocycle;
n indicates 0 or 1;
R ⁶ represents a C _1-6 alkyl group, a C _3-10 cycloalkyl group or an optionally substituted C _1-6 alkyl-carbonyl group. ]
A drug for treating prion disease, which contains the compound represented by (1) or a salt thereof as an active ingredient.

Images