WO2008053764A1 - Anti-prion active compound, anti-prion active agent, and method for inhibition of the production of abnormal prion protein - Google Patents

Abstract

[PROBLEMS] To provide an anti-prion active compound having an excellent effect of preventing the increase in the level of an abnormal prion protein, and also provide an anti-prion active agent and a method for inhibiting the production of an abnormal prion protein, both utilizing the anti-prion active compound. [MEANS FOR SOLVING PROBLEMS] The anti-prion active compound is a pyrazolone derivative represented by the general formula (1). (1) wherein R1 represents an aryl group which may have a substituent or an alkyl group (including a cycloalkyl group) which may have a substituent; R2 represents a hydrogen atom, an aryl group which may have a substituent, a nitrogenated heterocyclic functional group which may have a substituent, an alkyl group (including a cycloalkyl group) which may have a substituent or an acyl group ; and R3 represents an aryl group which may have a substituent, an alkyl group (including a cycloalkyl group) which may have a substituent, RCONH-, RNHCO-, ROCONH- or RNHCONH- [wherein R represents an aryl group or an alkyl group (including a cycloalkyl group)].

Description

 Specification

 Anti-prion active compound, anti-prion activator and method for inhibiting abnormal prion protein production

 Technical field

 The present invention relates to an anti-prion active compound having a pyrazolone skeleton that suppresses an increase in abnormal prion protein, an anti-prion activator and a method for inhibiting the production of abnormal prion protein using the same.

 Background art

 [0002] Prion disease represented by Creutzfeld's Jacob's disease is a general term for a group of neurodegenerative diseases in which prion protein is involved, and is characterized by the accumulation of degenerated abnormal prion protein in tissues such as the brain. To do. The infectious agent of prion disease is considered to be this abnormal type prion protein, and abnormal type prion protein proliferates by converting prion protein expressed in normal tissues into abnormal type prion protein. For this reason, prion diseases also have an aspect like infectious diseases.

 [0003] As described above, if the cause of prion disease is the conversion of a prion protein expressed in normal tissue by abnormal prion protein to an abnormal prion protein, if a substance that inhibits this conversion is found, It should be possible to stop the progression of prion disease. From such a view, a compound that suppresses the growth of abnormal prion protein (hereinafter referred to as “anti-prion active compound”) has attracted attention as a therapeutic agent for prion disease.

 [0004] For example, in Patent Document 1, lysosomal affinity drugs such as pentosan polysulfate, quinine, quinacrine and chloroquine, and reactive dyes such as reactive green and reactive red are effective as anti-prion active compounds. It is stated that there is! / In addition, quinoline derivatives and naphthyl derivatives (Patent Document 2) and chlorophyll derivatives (Patent Document 3) are also known to have similar effects.

[0005] Patent Document 1: Japanese Patent Laid-Open No. 2003-40778

 Patent Document 2: JP 2004-99553 A

Patent Document 3: Japanese Patent Publication No. 2003-155239 Disclosure of the invention

 Problems to be solved by the invention

[0006] However, none of the conventional anti-prion active compounds described above is sufficiently effective as a therapeutic agent, and there has not yet been established an effective treatment method for this disease that results in death when illness occurs. is the current situation. The present invention has been made in view of the above-described conventional circumstances, and it is an object to be solved to provide an anti-prion active compound that exhibits an excellent effect in suppressing the proliferation of abnormal prion protein! .

 Furthermore, the present invention also provides an anti-prion activator and a method for inhibiting the production of an abnormal prion protein, which are effective in suppressing the proliferation of abnormal prion protein, using the anti-prion active compound of the present invention. It is a problem to be solved.

 Means for solving the problem

[0007] The inventors noted that there are compounds known as free radical scavengers that act as scavengers for reactive oxygen species among compounds that act as anti-prion active compounds. The various compounds having properties were investigated for their anti-prion activity. As a result, we found that edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one), which is used as a brain protective agent in clinical practice, has anti-prion activity.

[Chemical 1

 Edaravone

 Furthermore, using this edaravone as a lead compound, we synthesized compounds with different types of substituents attached to the pyrazolone skeleton, and conducted extensive research on their anti-prion activity! As a result, a series of derivatives having a pyrazolone skeleton was found to have excellent anti-prion activity, and the present invention was completed.

 That is, the anti-prion active compound of the present invention is

An anti-prion active compound that inhibits the growth of abnormal prion protein, It is a pyrazolone derivative represented by the following general formula (1).

 [Chemical 2]

In the formula, R ¹ may have a substituent! /, May have an aryl group or a substituent! /, Represents an alkyl group (including a cycloalkyl group), R ² represents hydrogen, a substituent. It may have an aryl group, may have a substituent! /, A nitrogen-containing heterocyclic functional group, may have a substituent, an alkyl group (including a cycloalkyl group) or an acyl group. , R ³ represents an aryl group which may have a substituent, an alkyl group (including a cycloalkyl group) which may have a substituent, RCONH—, RNHCO—, ROCONH or RNHCONH (where R is an aryl) Groups or alkyl groups (including cycloalkyl groups) Note that the carbonyl group of the above formula (1) is actually interconverted with an enol-type tautomer, but for convenience, the above general formula (1) Expressed in

 [0009] The inventors examined the anti-prion activity of the pyrazolone derivative having the above structure using prion persistently infected cells, and confirmed that it exhibited excellent anti-prion activity!

[0010] In the general formula (1), R ¹ can be a phenyl group or a cycloalkyl group which may have a substituent. R ² may be hydrogen, a phenyl group, a pyridinole group, an alkyl group (including a cycloalkyl group) or a benzoyl group. Furthermore, R ³ represents an alkyl group, an optionally substituted phenyl group, RCONH—, RNHCO—, ROCONH or R NHCONH— (where R represents an aryl group or an alkyl group (including a cycloalkyl group)). ) The power S

As such a pyrazolone derivative, R ¹ is a phenyl group or a cycloalkyl group which may have a substituent, and R ² is hydrogen, a phenyl group, a pyridyl group, an alkyl group (a cycloalkyl group). Or R ³ is an alkyl group, an optionally substituted phenenore group, RCONH—, RNHCO—, ROCONH or RNHCONH (where R is an aryl group or an alkyl group (including a cycloalkyl group) ) Indicates the power S.

R²及び R³の組み合わせが（R¹:フエニル基， R² = H, R³ = CH ) 、（R¹:シクロへキシル基， R² = H, R³ = CH )、 （R¹:フエニル基， R² = H, R³ = 4-N [0011] The inventors

The combination of R ² and R ³ is (R ¹ : phenyl group, R ² = H, R ³ = CH) , (R ¹ : cyclohexyl group, R ² = H, R ³ = CH), (R ¹ : phenyl group, R ² = H, R ³ = 4-N

 Three

O Ph), (R ¹ : Phenol group, R ² = H, R ³ = CF), (R ¹ : Phenol group, R ² = H, R ³ = CH

2 3 3

(1 ^ = 4— ClPh, R ² = H, R ³ = CH), (R ¹ : phenyl group, R ² = phenyl group,

 Three

R ³ = CH), phenyl group, R ² = H, R ³ = isopropenyl group), phenyl group,

Three

R ² = isobutyl, R ³ = CH), (R ¹ : phenyl, R ² = benzoyl, R ³ = CH), (

 3 3

R = phenyl group, R ² = H, R ³ = PhCONH), (R ¹ : phenyl group, R ² = 2_pyridyl group, R ³ = CH), (R ¹ : phenyl group, R ² = H, R ³ = PhNHCO), (R ¹ = 2-Pyridyl-, R ² = H, R ³

 Three

= CH), (1 ^ = 2-pyridyl group, R ² = H, R ³ = CH), (R ¹ : phenyl group, R ² = H, R ³ = I ^?

 3 3

4- (1- CH) Py +-), (1 ^ = 4- nC H Ph, R ² = H, R ³ = 4- NO Ph), (1 ^ = 4- nC HP

3 12 25 2 12 25 h, R ² = H, R ³ = 4-CH OPh) and (1 ^ = 2-pyridyl group, R ² = n_C H, R ³ = CH)

 3 13 27 3 If it is a deviation, make sure that it has anti-prion activity!

[0012] An anti-prion active agent can be produced by using the anti-prion active compound of the present invention. That is, the anti-prion active agent of the present invention contains the anti-prion active compound according to any one of claims 1 to 5 as an active ingredient.

[0013] Further, by using the anti-prion active compound of the present invention, the proliferation of abnormal prion protein is inhibited. That is, the method for inhibiting abnormal prion protein production according to the present invention is characterized in that the anti-prion active compound according to any one of claims 1 to 5 is administered to inhibit the proliferation of abnormal prion protein. .

 BEST MODE FOR CARRYING OUT THE INVENTION

[0014] Hereinafter, Examples further embodying the anti-prion active compounds of the present invention;! To 19 will be described in detail in comparison with Comparative Examples 1 to 4.

 Example 1 is edaravone, Examples 2 to 19 are a phenyl group present in edaborane. A methyl group at the 3-position of the pyrazolone ring is converted, or a substituent is introduced at the 4-position of the pyrazolone ring. (Specific structures are shown in Table 1 below).

Comparative Examples 1 to 4 are compounds described in Patent Document 1 or Patent Document 2 as anti-prion activation compounds. Comparative Example 1 is quinine, Comparative Example 2 is quinidine, Comparative Example 3 Is MQAA (structure is shown in Table 1 below) and Comparative Example 4 is 4- (4-dimethylaminostyryl) quinoline. Reagents used in the synthesis of anti-prion active compounds The physical property data measuring apparatus is as follows.

 (Melting point)

 Melting points were measured using Yanagimoto's micro melting point measuring device or Buchi547 melting point measuring device. (Proton NMR)

 Proton NMR was recorded on a JEOL JNM-LA500 spectrometer using CDC1 or DMSO-d as the solvent. The chemical shift (δ) was in ppm and tetramethylsilane was the internal standard.

 (Elemental analysis)

 Elemental analysis was performed using a Yanaco CHN CORDER NT-5 analyzer, and all values agreed with the calculated values within 0.4%.

 (MS)

 The high resolution mass spectrum was measured using a JEOL JMS-SX102A mass spectrometer. GC-MS analysis was measured with Shimadzu GCMS-QP2010.

 (Reagent)

 As the anti-prion active compound of Example 10, a reagent from Tokyo Chemical Industry was used as it was. For all other reagents and solvents, commercially available reagents were purchased and used as they were. For flash column chromatography, silica gel 60 (particle size: 0.046-0.063 mm) manufactured by Merck & Co. was used.

[0015] (Example 1)

 The anti-prion active compound of Example 1 was edaravone, and a commercially available reagent (manufactured by Tokyo Chemical Industry Co., Ltd.) was used as it was.

[Example 2]

Add trimethylamine (3.04 mL, 22.0 mmol) to a suspension of ethyl-3-oxobutanoate (2.60 g, 20.0 mmol) and cyclohexylhydrazine hydrochloride (20.0 mmol) in acetic acid (30 mL). The mixture is then refluxed for 17 hours. The solvent was distilled off with an evaporator, the residue was extracted with ethyl acetate, the organic layer was washed with saturated NaHCO, water and brine, dehydrated with anhydrous sodium sulfate, filtered and concentrated in vacuo. By performing silica gel flash chromatography (developing solvent n-hexane / ethyl acetate = 1/1), 1-cyclohexyl-3-methyl-5-methyl, an anti-prion active compound of Example 1 as a yellow solid, was obtained. Pyrazolone (Yield 42%). The crude product was purified by recrystallization from ethanol. The physical properties are shown below.

 mp 152-153 ° C;

^: H-NMR (CDC1, 400ΜΗζ, δ ppm) 3.99 (1Η, m), 3.20 (2Η, s), 2.10 (3Η, s), 1.50 (

 Three

 10Η, m);

MS (EI) m / z: 180 (M ⁺ );

 Elemental analysis (C H N O) calculated C, 66.63; H, 8.95; N, 15.54. Measured C, 66.56; H, 8.8

 10 16 2

 1; N, 15.58

[0017] (Example 3)

 To a solution of 4-nitrobenzoyl acetate ethyl ester (10.0 mmol) in 10 ml of ethanol, add phenylhydrazine (10.0 mmol). The mixture was refluxed for 15 hours, and the precipitate formed after cooling was collected by filtration. 85% of the anti-prion active compound 3_ (4-nitrophenyl) -1-phenyl-5-pyrazolone of Example 2 was collected. The yield was obtained. Further, the crude product was purified by recrystallization from a mixed solvent of THF / ethanol. The physical properties are shown below. mp 190-191 ° C;

 'H-NMR (CDCl, 400ΜΗζ, δ; ppm) 8.32 (1H, d, J = 9.1 Hz), 7.95 (2H, d, J = 8.7H

 Three

 z),

 7.46 (2H, d, J = 8.7 Hz), 7.46 (2H, t, J = 7.6 Hz), 3.91 (2H, s);

MS (EI) m / z: 291 (M ⁺ );

 Elemental analysis (C H N O) Calculated value C, 64.05; H, 3.94; N, 14.94. Measured value C, 63.77; H, 4.0

 15 11 3 3

 2; N, 15.12.

 [0018] (Example 4)

The anti-prion active compound of Example 4 was 1-phenyl-3-trifluoromethyl-5-pyrazolone and was synthesized by the same method as in Example 2. That is, add phenylhydrazine (10.0 mmol) to a solution of 4, 4, 4-trifluoroacetoacetic acid ethyl ester (10.0 mmol) in 10 ml of ethanol. The mixture was refluxed for 15 hours, and the precipitate formed after cooling was collected by filtration. 85% of 1-phenyl-3-trifluoromethyl-5-pyrazolone, which is the anti-prion active compound of Example 3, was collected. Obtained in yield. Furthermore, the crude product is ethyl acetate / n -Purified by recrystallization from a mixed solvent of -hexane. The physical properties are shown below. mp 195-196. C;

^: H-NMR (CDC1, 400ΜΗζ, δ ppm) 7.80 (2Η, d, J = 7.6 Hz), 7.66 (2H, d, J = 7.8 H

 Three

 z), 6.61 (1H, s), 5.91 (1H, s), 3.71 (2H, s);

MS (EI) m / z: 228 (M ⁺ );

 Elemental analysis (C H F N O) Calculated: C, 52.64; H, 3.09; N, 12.28. Measured C, 52.76; H, 3

 10 7 3 2

 .17; N, 12.65.

 [Example 5]

 To a solution of 3-amino-1-phenyl-5-pyrazolone (1.75 g, 10.0 mmol) in 15 ml of pyridine, add 0 ° C. methyl chloroformate (0.95 g, 10.0 mmol) dropwise. The mixed solution is stirred at 0 ° C. for 1 hour, and further stirred at 50 ° C. for 2 hours. The reaction solution was poured into water, and the precipitated solid was collected by filtration, washed with water and ethanol, recrystallized with a mixed solvent of methanol / chloroform, and methyl 5-oxo-1-phenyl-4,5. 545 mg (yield 23%) of crystals of -dihydro-1 H-pyrazole-3-ylcarbamate were obtained. The physical properties are shown below.

 mp 231-232. C;

 'H-NMR (CDCl, 400ΜΗζ, δ; ppm) 7.82 (2H, d, J = 7.6 Hz), 7.38 (2H, t, J = 8.0

 Three

 Hz),

 7.20 (2H, t, J = 7.3 Hz), 4.00 (2H, s), 3.82 (3H, s);

MS (EI) m / z: 233 (M ⁺ );

 Elemental analysis. Calculated (C H N O :) C, 56.65; H, 4.75; N, 18.02. Measurements: C, 56.84; H,

 11 11 3 3

 4.85; N, 17.89

[0020] (Example 6)

To a solution of 3-amino-1-phenyl-5-pyrazolone (1.75 g, 10.0 mmol) in 15 ml of pyridine, add 0 ° C. phenyl formic acid phenyl ester (1.57 g, 10.0 mmol) dropwise. The mixed solution is stirred at 0 ° C. for 1 hour, and further stirred at 50 ° C. for 2 hours. The reaction solution was poured into water, and the precipitated solid was collected by filtration, washed with water and ethanol, purified by silica gel flash chromatography, and phenyl 5-oxo-1-phenyl-4,5-dihydro- -Pyrazole-3-yl carbonate 627mg was obtained (yield 21%).

Yes

mp 225-226. C;

^: H-NMR (CDC1, 400ΜΗζ, δ; ppm) 7.84 (2H, d, J = 8.5 Hz), 7.69 (1H, s), 7.42 (2

 Three

 H, t, J = 8.5 Hz), 7.40 (2H, t, J = 7.5 Hz), 7.29 (1H, t, J = 7.3 Hz), 7.19 (3H, t, J = 8.3 Hz);

MS (EI) m / z: 295 (M ⁺ );

Elemental analysis. Calculated (C H NO :) C, 65.08; H, 4.44; N, 14.23. Measured values: C, 65.17; H,

 11 11 3

 4.71; N, 14.40.

Furthermore, a solution of this phenyl 5-oxo-1-phenyl-4,5-dihydro-1H-pyrazole-3-yl carbonate (295 mg, 1.0 mmol) in xylene was added to cyclopentylamine (85.2 mg, 1.0 mmol). Add to reflux with stirring for 3 hours. The reaction solution was poured into ethanol, and the resulting precipitate was washed with water and ethanol, collected by filtration, and 1-cyclopentyl-3- (5_oxo-1-, which is the anti-prion active compound of Example 6. 119 mg (42% yield) of phenyl-4,5-dihydro-1H-pyrazol-3-yl) urea was obtained. Furthermore, physical property data of this crude product purified by recrystallization from THF / _n monohexane mixed solvent are shown below. mp 184-185 ° C;

 'H-NMR (CDCl, 400ΜΗζ, δ ppm) 8.48 (1Η, s), 7.74 (2Η, d, J = 8.2 Hz), 7.41 (2H,

 Three

 t, J = 8.1 Hz), 7.19 (2H, t, J = 7.4 Hz), 4.17 (1H, q, J = 6.0 Hz), 3.64 (1H, s), 2.03 (2H, m), 1.74 (4H, m);

MS (EI) m / z: 286 (M ⁺ );

Elemental analysis. Calculated (C H N O :) C, 62.92; H, 6.34; N, 19.57. Measurements: C, 62.58; H,

 15 18 4 2

 6.36;

N, 19.60.

 (Example 7)

In Example 7, 4-cyclohexyl hydrazine hydrochloride was used in place of cyclohexyl hydrazine hydrochloride in Example 2 (but ethanol was used in place of acetic acid as the solvent). Performing the same operation, it is an anti-prion active compound of Example 1 1 -(4-Chlorophenyl) -3-methyl-5-pyrazolone was synthesized. The crude product thus obtained was purified by recrystallization using ethanol as a solvent. The physical property data of this purified product is shown below.

mp 173-174. C;

^: H-NMR (CDC1, 400ΜΗζ, δ; ppm) 7.85 (2H, d, J = 8.8 Hz), 7.35 (2H, d, J = 8.8

 Three

 Hz), 3.44 (2H, s), 2.20 (3H, s);

MS (EI) m / z: 208 (M ⁺ );

Elemental analysis. Calculated (C H C1N 0 :) C, 57.57; H, 4.35; N, 13.43. Measured C, 57.52; H,

 10 9 2

 4.40;

N, 13.58.

 (Example 8)

 The anti-prion active compound of Example 8 is 3-methyl-1,4-diphenyl-5-pyrazolone and was synthesized in the following two steps.

<First stage>

 A suspension of sodium hydride (60%, 1.50 g, 37.5 mmol) in THF (30 mL) is cooled to 0 ° C, and a solution of phenylacetic acid ethyl ester (5.00 g, 30.5 mmol) in THF (10 mL) is added dropwise. . Then, stir at room temperature for 1 hour, add a solution of acetic anhydride (3.20 mL, 33.9 mmol) in THF (10 mL) at 0 ° C and stir for 1 hour. The reaction solution is poured into ice water and extracted with ethyl acetate. The ethyl acetate layer is separated, washed with water, saturated aqueous sodium hydrogen carbonate solution and brine, and then dried over anhydrous sodium sulfate. After filtration, concentration under reduced pressure, and purification by silica gel flash chromatography (developing solvent: n-hexane / ethyl acetate = 10: 1), yellow oily 3-oxo-2-phenylbutyric acid ethyl ester 3.23 g (Yield 51%) was obtained. The physical properties are shown below. 'H-NMR (CDC1, 400 MHz, δ ppm) 7.52-7.10 (5H

 Three

 , M), 4.15 (2H, q, J = 7.1 Hz), 3.57 (0.5H, s), 2.24 and 1.90 (3H, each s), 1.22 and 1.20 (3H, each t, J = 7.1 Hz).

 <Second stage>

3-Oxo-2-phenylbutyric acid ethyl ester (2.22 g, 10.0 mmol) obtained in the first step above is dissolved in 10 mL of ethanol, and phenylhydrazine (1.08 g, 10.0 mmol) is added dropwise thereto. The mixture was refluxed for 15 hours and then cooled, and the resulting precipitate was collected by filtration, and 0.4 g (yield: 13%) of 3-methyl-1,4-diphenyl-5-pyrazolone in the form of a yellow oil was collected. Obtained. The crude product thus obtained was purified by recrystallization from ethanol. The physical property data of this purified product is shown below.

mp 203-205 ° C;

^: H-NMR (DMSO, 400 MHz, δ ppm) 11.35 (1H, br), 7.75 (2H, d, J = 8.0 Hz), 7.65-7.50 (2H, m), 7.48 (2H, t, J = 7.7 Hz), 7.39 (2H, t, J = 7.6 Hz), 7.25-7.20 (2H, m), 2.30 (3H, s);

MS (EI) m / z: 250 (M ⁺ );

Elemental analysis. Calculated value (C H N 0 :) C, 76.78; H, 5.64; N, 11.19.Measured value C, 76.56; H,

 13 14 2

 5.73; N, 11.18.

 (Example 9)

 The anti-prion active compound of Example 9 is 3-isopropenyl-1-phenyl-5-pyrazolone and was synthesized in the following two steps.

<First stage>

 Monoethyl potassium malonate (9.80 g, 57.6 mmol), MgCl (6.00 g, 63.0 mmol) and triethylamine (17.50 mL, 126.0 mmol) were dissolved in acetonitrile (100 mL), and metacryloyl chloride was cooled with ice. Mouth ride (3.00 g, 28.7 mmol) is added dropwise. After stirring for 30 minutes, add 2 M hydrochloric acid and stir for another 30 minutes. The mixture is poured into ice water and extracted with ethyl acetate. The organic layer is separated, washed with water, saturated aqueous sodium hydrogen carbonate solution and brine, and dried over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, the product was purified by silica gel flash chromatography (developing solvent: n-hexane / ethyl acetate = 5: 1), and colorless oily 4-methyl-3-oxopent-4- 2.52 g of enoic acid ethyl ester (43% yield) was obtained. The physical properties are shown below.

 'H-NMR (CDC1, 400MHz, δ; ppm) 5.96 (1H, s), 5.93 (1H, s), 5.90 (1H, s), 5.89 (

 Three

 1H, s),

5.35 (0.3H, s), 4.23 (2H, q, J = 7.1 Hz), 4.21 (2H, q, J = 7.1 Hz), 3.74 (1.4H, s), 1.90 (3H, s), 1.31 (3H, t, J = 7.3 Hz), 1.27 (3H, t, J = 7.3 Hz).

 <Second stage>

 Using the 4-methyl-3-oxo-pent-4-enoic acid ethyl ester obtained in the first step above, the same procedure as in the second step of Example 8 was performed to give 3-isopropenyl-1-phenyl-5- Pyrazolone was obtained in 11% yield. The physical properties are shown below.

 mp 56 ~ 5;

 'H-NMR (CDC1, 400MHz, δ; ppm) 7.90 (2H, d, J = 7.8 Hz), 7.41 (2H, t, J = 7.6

 Three

 Hz),

 7.20 (1H, t, J = 7.3 Hz), 5.43 (1H, s), 5.34 (1H, s), 3.64 (2H, s), 2.11 (3H, s); HRMS calculation CHNO: 200.095, measurement 200.094 ;

 12 12 2

Elemental analysis. Calculated (C H N O -0.2H 0 :) C, 70.71; H, 6.13; N, 13.74. Found: C, 70.

 12 12 2 2

 97; H, 5.99;

N, 13.62.

 (Example 10)

 The anti-prion active compound of Example 9 was 4-isobutyl-3-methyl-1-phenyl-5-pyrazophane, which was synthesized in the following two steps.

<First stage>

Ethyl _3_oxobutanoate (3.00 g, 23.1 mmol) is added all at once to a solution of sodium ethoxide in ethanol (20%, 8.70 g, 25.6 mmol), and the mixture is warmed to 80 ° C. Then, a solution of isobutyl iodide (5.10 g 27.71 mmol) in THF (5.0 mL) was added dropwise over 2 hours, and the reaction solution was kept at 80 ° C. for another 2 hours. After cooling, pour in ice-cooled 2M hydrochloric acid and extract with ethyl acetate. The organic layer is separated, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel flash chromatography (developing solvent: _n- hexane / ethyl acetate = 20: 1), colorless. As a result, 1.83 g (yield 43%) of oily 2-acetyl-4-ethylpentanoic acid ethyl ester was obtained. The physical properties are shown below.

: H-NMR (CDC1, 400MHz, δ; ppm) 12.91 (0.2H, s), 4.20 and 4.19 (2H, each q, J =

 Three

 7.1 Hz),

3.50 (0.8H, dd, J = 8.3, 6.6 Hz), 2.23 and 2.00 (3H, each s), 2.05 (0.4H, d, J = 7.1 Hz),

 1.80-1.76 (0.8H, m), 1.72—1.66 (0.8H, m), 1.56-1.51 (1H, m), 1.31 and 1.27 (3H, e ach t, J = 7.1 Hz), 0.92, 0.91 and 0.87 (6H, each d, J = 6.7 Hz).

 <Second stage>

 Using the 2-acetyl-3-ethylpentanoic acid ethyl ester obtained in the first step, 4-isobutyl-3-methyl-1-phenyl-5-pyrazolone was obtained in the same manner as in the second step of Example 8. Obtained in 70% yield. However, acetic acid was used instead of ethanol. The crude product thus obtained was purified by recrystallization from n-hexane / ethyl acetate. The physical properties are shown below.

mp 117-119. C;

 'H-NMR (CDCl, 400ΜΗζ, δ; ppm) 7.89 and 7.72 (2H, each d, J = 7.8 Hz), 7.39 (2

 Three

 H, t, J = 7.6 Hz), 7.17 and 7.15 (1H, each t, J = 7.3 Hz), 6.80 (0.3H, br), 3.26 (0.7 H, t, J = 6.3 Hz),

 2.18-2.15 (3.6H, m), 2.01—1.94 (1H, m), 1.78 (1.4H, t, J = 7.3 Hz), 0.962, 0.958, and nd 0.920 (6H, each d, J = 6.6 Hz) ;

MS (EI) m / z: 230 (M ⁺ );

Elemental analysis. Calculated (C H N O -0.2H O) C, 73.01; H, 7.88; N, 12.16. Found: C, 73.

 14 18 2 2

 26; H, 7.93; N, 12.38.

(Example 1 1)

 To a suspension of 3-methyl_1-phenyl_2-pyrazolin-5-one (349 mg, 2.00 mmol) in dioxane (2.5 mL) was added calcium hydroxide (221 mg, 2.98 mmol) and benzoyl chloride (0.25 mL, 2.2 mmol). And stir the mixture at 100 ° C. for 1 hour. Pour the reaction mixture into 2 M hydrochloric acid (8.0 mL) and extract with ethyl acetate. The organic layer is separated, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel flash chromatography (developing solvent: n-hexane / ethyl acetate = 9: 1). Thus, yellow oily 4-benzoyl-3-methyl-1-phenyl-1H-pyrazol-5 (4H) _one 440 mg (yield 79%) was obtained. The physical properties are shown below.

mp 90-91 ° C; Ή-NMR (CDC1, 500MHz,?; Ppm) 7.88 (2H, d, J = 8.1 Hz), 7.65 (2H, d, J = 7.6 H

 Three

z), 7.58 (1H, t, J = 7.5 Hz), 7.52 (2H, t, J = 7.5 Hz), 7.47 (2H, t, J = 8.1 Hz), 7.31 (1H, t, J = 7.3 Hz) , 2.10 (3H, s); MS (EI) m / z: 278 (M ⁺ );

 Elemental analysis. Calculated (C H N O :) C, 73.37; H, 5.07; N, 10.07. Found: C, 73.39; H, 4

 17 14 2 2

 .84; N, 10.02.

 [Example 12]

 Add benzoyl chloride (0.25 mL, 2.17 mmol) to a suspension of 3-amino-1-phenyl-2-pyrazolin-5-one (351 mg, 2.00 mmol) in dioxane (5.5 mL) and stir at room temperature for 3.5 hours. The The reaction mixture is then poured into 2 M hydrochloric acid (8.0 mL) and extracted with ethyl acetate. The organic layer is separated, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel flash chromatography (developing solvent: n-hexane / ethyl acetate = 3: 1), It was obtained with 83 mg (yield 15%) of N- (5-oxo-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl) benzamide as a light brown solid. The physical properties are shown below.

 mp 218-219 ° C;

 'H-NMR (CDCl, 500MHz, δ; ppm) 8.58 (1H, s), 7.89 (2H, d, J = 7.3 Hz), 7.85 (2

 Three

 H, d, J = 7.5 Hz), 7.64 (1H, t, J = 7.5 Hz), 7.54 (2H, t, J = 7.7 Hz), 7.41 (2H, t, J = 8.1 Hz), 7.19 (1H, t, J = 7.5 Hz), 4.23 (2H, s);

MS (EI) m / z: 279 (M ⁺ );

 Elemental analysis (C H N O :) C, 68.81; H, 4.69; N, 15.05. Found: C, 68.93; H, 4.89; N,

 16 13 3 2

 14.87.

 [0027] (Example 13)

 The anti-prion active compound of Example 13 was 3-methyl-1-phenyl-4- (pyridin-2-yl) -1H-5 (4H) -one and was synthesized in the following two steps.

 <First stage>

Sodium hydride (60%, 600 mg, 15.0 mmol) in THF (8.0 mU in 2_pyridylacetic acid ethyl ester (0.45 mL, 3.0 mmol) was added dropwise with cooling in an ice-water bath and the mixture was added for 35 minutes. 60. Stir at C, add a solution of acetic anhydride (1.25 m, 13.2 mmol) in THF (3.0 mL) at 0 ° C., and stir at room temperature for 16 h. 2M salt Neutralize with acid and extract with ethyl acetate. The organic layer is separated, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel flash chromatography (developing solvent: n-hexane / ethyl acetate = 5: 1), yellow oil 639 mg of ethyl 3-oxo-2- (pyridin-2-yl) butanoate was obtained.

[0028] <Second stage>

 Ethyl 3-oxo-2- (pyridin-2-yl) butanoate (50%, 6 39 mg, 1.50 mmol) obtained in the first step above was dissolved in ethanol (7.0 mL), and phenylhydrazine (162 mg, 1.50 mmol) was added and the mixture was stirred at 82 ° C. for 2 hours. The reaction solution was poured into ice water, ethanol was distilled off with an evaporator and extracted with ethyl acetate. The organic layer is separated, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, purified by silica gel flash chromatography (developing solvent: ethyl acetate), and yellow solid 3-methyl-1- Phenyl-4- (pyridin-2-yl) -1H-5 (4H) _one 200 mg (8% overall yield over 2 steps) was obtained. The physical properties are shown below.

 mp 184-185 ° C;

 'H-NMR (CDCl, 500MHz, δ; ppm) 8.04 (2H, d, J = 7.6 Hz), 7.91 (1H, d, J = 5.8

 Three

 Hz),

 7.77 (1H, t, J = 7.9 Hz), 7.47 (1H, d, J = 8.8 Hz), 7.41 (2H, t, J = 7.9 Hz), 7.16 (1H, t, J = 7.5 Hz), 6.90 ( 1H, t, J = 6.7 Hz), 2.50 (3H, s);

MS (EI) m / z: 251 (M ⁺ ); elemental analysis. Calculated (CHN 0 :) C, 71.70; H, 5.21; N, 16.72

 15 13 3

 • Measured values: C, 71.93; H, 5.29; N, 16.53

[0029] (Example 14)

 The anti-prion active compound of Example 14 was 5-oxo-N, l-diphenyl-4,5-dihydro-1H-pyrazole-3-carboxamide and was synthesized in the following two steps.

 <First stage>

To a solution of 5-oxo-1-phenyl-2-pyrazoline-3-carboxylic acid (818 mg, 4.01 mmol) in dichloromethane (15 mL), oxalyl chloride (0.70 mL, 8.2 mmol) and a catalytic amount of DMF And dripping. Thereafter, the mixture was stirred at room temperature for 35 minutes, and the reaction mixture was concentrated with an evaporator, washed with dichloromethane three times, and then a brown solid of 5-oxo-1-phenyl-4,5-dihydro-1 H-pyrazole-3-carbourek mouthride was obtained.

[0030] <Second stage>

 5-Oxo-1-phenyl-4,5-dihydro-1H-pyrazole-3-carbonyl chloride obtained in the first step above is dissolved in dichloromethane (16 mL), and aniline (2.0 mL, 22 mmol) is dissolved. In addition, the mixture is stirred for 19 hours at room temperature. The reaction mixture is then poured into 2M hydrochloric acid (15 mL) under ice-cooling and extracted with ethyl acetate. The organic layer is separated, washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel flash chromatography (developing solvent: n-hexane / ethyl acetate = 4). : 1, then 1: 1), and 825 mg of light brown solid 5-oxo-N, l-diphenyl-4,5-dihydro-1H-pyrazole-3-carboxamide (74% overall yield in 2 steps) Obtained. The physical properties are shown below.

 mp 214-215 ° C;

 'H-NMR (DMSO, 500MHZ, δ; ppm) 12.1 (1H, br), 9.93 (1H, br), 7.85 (2H, d, J = 8.5 Hz),

 7.80 (2H, t, J = 7.6 Hz), 7.53 (2H, t, J = 8.1 Hz), 7.37 (1H, t, J = 7.5 Hz), 7.34 (2H, t, J = 7.9 Hz), 7.08 ( 1H, t, J = 7.5 Hz), 6.02 (1H, s);

MS (EI) m / z: 279 (M ⁺ ); Anal. Calcd for CHNO: C, 68.81; H, 4.69; N, 15.05. Fo

 16 13 3 2

 und: C, 69.04; H, 4.75; N, 15.01

[0031] (Example 15)

The anti-prion active compound of Example 15 was 3-methyl-1- (pyridin-2-yl) -5-pyrazophane and was synthesized as follows. That is, 2-hydrazinoviridine (2.10 g, 20.0 mmoL) was added to acetic acid (20 mU) with 3-oxobutanoic acid ethyl ester (2 · 60 g, 20. OmmoL, 1 · 0 equivalent), and the mixture was added. The mixture was stirred at reflux temperature for 3 hours, the solvent was distilled off, the residue was extracted with ethyl acetate, the organic layer was washed with saturated aqueous sodium hydrogen carbonate solution, water and saturated brine, and dried over anhydrous sodium sulfate. , Vacuum concentration, and silica gel flash chromatography (acetic acid ester / n-hexane = 2/1), 2.72 g of white solid 3-methyl-1- (pyridin-2-yl) -5-pyrazolone The solid (l.OOg) was recrystallized from n-hexane and ethyl acetate and filtered to obtain 507 mg (yield 57%) of a white solid, the melting point of which was as follows. — NMR data, MS data, HRMS data, elemental content The analysis value is shown.

 m.p .: 112.4-113.5 ° C;

'H-NMRCCDCl, 500MHZ, δ: ppm) Enoreno type: 12.7 (lH, br), 8.24 (lH, d, J = 3.7Hz),

 Three

 7.84 (lH, m), 7.12 (lH, m), 5.42 (lH, s) 2.26 (3H, s);

MS (EI) m / z: 175 (M ⁺ );

 HRMS: calculated value (C H N 0) 175.075, measured value 175.075;

 9 9 3

Elemental analysis: Calculated value (C H N O) C, 61.70; H, 5.18; N, 23.99. Measured value C, 61.94; H, 4.99; N, 23.7

 9 9 3

 7.

 (Example 16)

 The anti-prion active compound of Example 16 is 1-methyl-4- (5-oxo-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl) pyridinium iodide, and the following three steps Was synthesized.

Stage 1>

To a suspension of malonate monoethyl ester potassium salt (3.40 g, 20.0 mmoL), magnesium chloride (3 · 81 g, 40.0 mmoL) and triethylamine (7.46 mL, 53.7 mmoL) in acetonitrinole (lOOmL) Isonicotinoyl hydrochloride (1.87 g, 10 mmoL) was gradually added under ice cooling. The mixture was stirred at 0 ° C. and stirred for 4 hours, and then 2N hydrochloric acid (50 mU was added to the reaction mixture and stirred at room temperature for 1 hour. The reaction mixture was poured into ice-cold water and extracted with ethyl acetate. The layers were separated, washed with saturated aqueous sodium bicarbonate solution, then saturated brine, dried over anhydrous magnesium sulfate, filtered, concentrated in vacuo, and then flashed on silica gel (n-hexane / ethyl acetate = 1/2). To obtain 1 · 47 g (yield 76%) of 3-oxo-3- (pyridine 4-yl) propionic acid ethyl ester as a white solid, and its ¹ H-NMR data and MS data are shown below. .

'H-NMRCCDCl, 500MHZ, δ: pm)

 Three

Ennore type: 12.4 (lH, s), 8.71 (2H, d, J = 6.1Hz), 7.60 (2H, d, J = 4.4Hz),

5.77 (lH, s), 4.29 (2H, q, J = 7.3Hz), 1.35 (3H, t, J = 7.0Hz); Keto type: 8.84 (0.5H, d, J = 6.1Hz), 7.72 (0.5 H, d, J = 6.1Hz), 4.21 (0.5H, q, J = 7.0Hz), 3.99 (0.5H, s), 1.25 (lH, t, J = 7.0Hz); MS (EI) m / z : 193 (M ⁺ ).

Stage 2> Using 3-oxo-3- (pyridin-4-yl) propionic acid ethyl ester obtained in the first step, 1-phenyl-3- (pyridin-4-yl) -5-pyrazolone Was synthesized. In other words, 3-hydroxy-3- (pyridin-4-yl) propionic acid ethyl ester (1.63 g, 8. 45 mMol) was added to ethyl alcohol (20 mU in a solution of phenylhydrazine (912 mg, 1. The mixture was heated at reflux for 4 hours, the solvent was distilled off and extracted with ethyl acetate, the organic layer was separated, washed with water and saturated brine, and anhydrous magnesium sulfate After filtration and concentration in vacuo, the residue was purified by silica gel flash chromatography (acetic acid ester / methanol = 10/1) to give a pale yellow solid of 1-phenyl-3- (pyridine-4-yl). As a result, 1.21 g (yield 60%) of -5-pyrazolone was obtained, and its ¹ H-NMR data and MS data are shown below.

'H-NMRCCDCl, 500MHZ, δ: pm)

 Three

 : 8.74 (2H, d, J = 6.1Hz), 7.94 (2H, d, J = 7.6Hz), 7.63 (2H, d, J = 6.1Hz), 7.45 (2H, t, J = 7.6Hz), 7 .

25-7.30 (lH, m), 3.86 (2H, s);

MS (EI) m / z: 237 (M ⁺ ).

Stage 3>

 Using 1-phenyl-3- (pyridin-4-yl) -5-pyrazolone obtained in the second step, 1-methyl-4- (5-oxo-1-phenyl-iodide- 4,5-Dihydro-1H-pyrazol-3-yl) pyridinium was synthesized. That is, methyl iodide (3.0 mL, 38.0 equivalents) was added to a solution in which 1-phenyl-3- (pyridine-4-yl) -5-pyrazolone (300 mg, 1.26 mmoL) was added to black mouth form (10 mL). It was. The mixture was stirred at 60 ° C. for 18 hours. The solvent was distilled off and jetino ether was added. The precipitated solid was collected by filtration and washed with jetyl ether and n-hexane to obtain 443 mg (yield 92%) of a brown solid. The crude product (200 mg) was recrystallized with ethyl alcohol and n-hexane to give a brown solid of 1-methyl-4- (5_oxo-1-phenyl-4,5-dihydro- -pyrazole- 133 mg (yield 66%) of 3-yl) -pyridinium were obtained. The melting point, ¾-NMR data, and MS data are shown below.

m.p .: 269.8 -271.5 ° C;

'H-NMRCDMSO-d, 500MHz, δ: pm):

 6

8.91 (2H, d, J = 6.7Hz), 8.45 (2H, d, J = 7.0Hz), 7.84 (2H, d, J = 7.6Hz), 7.54 (2H, t, J = 7.6Hz), 7.40 (lH, t, J = 7.3Hz), 6.50 (2H, s), 4.30 (3H, s);

MS (FAB) m / z: 252 (M ⁺ -l).

 (Example 17)

The anti-prion active compound of Example 17 is 1- [4- (n-dodecyl) phenyl] -3_ (4-nitrophenyl) -5-pyrazolone, and was synthesized in the following four steps.

Stage 1>

 4-iodonitrobenzene (25.0g, 100mmoL), 1-dodecine (19.0g, 117mmoL), tetrakis (triphenylphosphine) palladium (3.1g, 2.8mmoL), copper iodide (3.8g, 20.0mmoL), A mixture of triethylamine (50 mL) and acetonitrile (200 mL) was stirred at 100 ° C. for 13 hours. The mixture was concentrated and poured into water and the whole was extracted with ethyl acetate. The organic layer was washed with saturated aqueous sodium hydrogen carbonate solution, water, 2N hydrochloric acid and saturated brine, and dried over anhydrous sodium sulfate. Purification by filtration, vacuum concentration, and silica gel flash chromatography (n-hexane / ethyl acetate = 1/5) yielded 23.9 g of l- (n-dodec-1-ynyl) -4-nitrobenzene as a brown oil. (Yield 83%). The ifi-NMR data and MS data are shown below.

'H-NMRCCDCl, 500MHZ, δ: pm):

 Three

 8.15 (2H, d, J = 7.0Hz), 7.51 (2H, d, J = 7.0Hz), 2.44 (2H, t, J = 7.3Hz), 1.63 (2H, quin, J = 7.3Hz), 1.44 ( 2H, quin, J = 7.0Hz), 1.30-1.21 (12H, m), 0.88 (3H, t, J = 7.3Hz);

MS (EI) m / z: 287 (M ⁺ ).

Stage 2>

Using l- (n-dodec-1-ynyl) -4-nitrobenzene obtained in the first step, 4-dodecylphenylamine was synthesized as follows. That is, a mixture of l_ (n-dodec-1-ynyl) -4-nitrobenzene (7.81 g, 27.2 mmol) and palladium carbon (780 mg) in ethyl alcohol (80 mL) at room temperature for 7 days under a hydrogen atmosphere. Stir. The mixture was filtered through a celite layer, the organic layer was separated and dried over anhydrous sodium sulfate. Purification by filtration, vacuum concentration and silica gel flash chromatography (n-hexane / ethyl acetate = 5/1) gave 3.09 g of colorless oily 4- (n-dodecyl) phenylamine (yield 44%) Obtained. The ¹ H-NMR data and MS data are shown below. H-NMR (CDC1, 500MHz, δ: pm):

 6.96 (2H, d, J = 8.5Hz), 6.62 (2H, d, J = 8.2Hz), 3.53 (2H, s), 2.48 (2H, t, J = 7.9Hz), 1.30-1.20 (l 8H, m),

 0.87 (3H, t, J = 7.3Hz);

MS (EI) m / z: 261 (M ⁺ ).

Stage 3>

Using 4- (n-dodecyl) phenylamine obtained in the second step, 4- (n-dodecyl) phenylhydrazine hydrochloride was synthesized as follows. That is, a solution of sodium nitrite (1.04 g, 15 mmoL, 3 equivalents) in water (10 mU) was added to 6N hydrochloric acid (15 mU, 4 (n dodecyl) phenylamine (1.30 g, 5. OmmoU was added under ice cooling). To the stirred suspension was added over 15 minutes.After another 30 minutes in the ice bath, stannous chloride (2.65 g, 14. OmmoL, 2.8 eq) was slowly added to 6N hydrochloric acid. The resulting suspension was stirred for 3 hours, and the resulting solid was collected by filtration and dissolved in a 40% potassium hydroxide solution (lOOmU and acetic acid ethyl ester (lOOmL) mixture. The organic layer was separated. The mixture was shaken with 4N hydrochloric acid and ethyl ester, and the precipitate was collected by filtration and washed with hexane to obtain 1-28 g (82% yield) of 4- (n-dodecyl) phenylhydrazine hydrochloride as a white solid. The ¹ H-NMR data and MS data are shown below.

'H-NMRCDMSO-d, 500MHz, δ: pm):

 10.l (2H, br), 8.l (lH, br), 7.10 (2H, d, J = 8.5Hz), 6.90 (2H, d, J = 8.5Hz)

1.52- 1.48 (2H, m), 1.30-1.20 (20H, m), 0.85 (3H, t, J = 7.0Hz); MS (EI) m / z: 277 (M ⁺ ).

Stage 4>

Using 4- (n-dodecyl) phenylhydrazine hydrochloride obtained in the third step, 1- [4- (n-dodecyl) phenyl] -3_ (4-nitrophenyl) -5-pyrazolone Was synthesized. In other words, 4_ (n-dodecyl) phenylhydrazine hydrochloride (312 mg, 1.0 mmoL) was added to acetic acid (10 mL), and 4-nitrobenzoyl acetic acid ethyl ester (237 mg, 1.0 mmoL, 1 equivalent), triethylamine. (0.08 mL, 1.2 equivalents) was added, and the mixture was stirred at reflux temperature for 7.5 hours. The reaction mixture was extracted with ethyl acetate, washed with saturated aqueous sodium hydrogen carbonate solution, water and saturated brine, and dried over anhydrous sodium sulfate. Filtration, vacuum concentration and silica gel 1- [4- (n-dodecyl) phenyl] -3_ (4-nitrophenyl) -5-pyrazolone was purified by rush chromatography (n-hexane / ethyl acetate = 1/4). 151.8 mg (yield 34%) was obtained. The solid was recrystallized from n-hexane and collected by filtration to give a yellow solid 1- [4- (n-dodecinole) phenol] -3- (4-nitrophenol) -5 -Pyrazolone (129.5 mg, yield 85%) was obtained. The melting point, ifi-NMR data, ¹³ C-NMR, HRMS data, and elemental analysis values are shown below.

m.p .: 116.2-117.2 ° C;

'H-NMRCCDCl, 500MHZ, δ: pm):

 8.31 (2H, d, J = 8.8Hz), 7.93 (2H, d, J = 8.8Hz), 7.81 (2H, d, J = 8.5Hz), 7.25 (2H, d, J = 8.5Hz), 3.8 8 (2H,),

 2.63 (lH, t, J = 7.6Hz), 1.70-1.55 (2H, m), 1.45-1.30 (18H, m), 0.88 (3H, t, J = 7.0Hz);

1 ³ C-NMR (CDC1, 600 MHz, δ: pm):

 14.1,22.7,29.2,29.4,29.5,29.6,29.7,31.4,31.9,35.5,39.2,119.4,124.3, 126.6,128.9,13 5.4,136.6,140.9, 151.9,169.7;

 HRMS: Calculated value (C H N O) 449.268, measured value 449.268;

Elemental analysis: Calculated value (C H N O) C, 72.13,; H, 7.85; N, 9.35; measured value C, 72.15; H, 7.84; N, 9.2

1.

 (Example 18)

The anti-prion active compound of Example 18 is 1- [4- (n-dodecyl) phenyl] -3_ (4-methoxyphenyl) -5-pyrazolone, and 4- (n-dodecyl) phenyl of Example 17 Using hydrazine hydrochloride, it was synthesized as follows. That is, 4- (n-dodecyl) phenylhydrazine hydrochloride (31 2 mg, 1.0 mmoL) was added to acetic acid (10 mL) to a suspension of 4-methoxybenzoyl acetate ethyl ester (222 mg, 1.0 mmoL, 1.0 equivalent). ) And triethylamine (0.08 mL, 1.2 equivalents) were added, and the mixture was stirred at reflux temperature for 7.5 hours. The reaction mixture was extracted with ethyl acetate, washed with saturated aqueous sodium hydrogen carbonate solution, water and saturated brine, and dried over anhydrous sodium sulfate. Purification by filtration, vacuum concentration and silica gel flash chromatography (n-hexane / ethyl acetate = 1/3) gave a crude yellow product. The crude product was recrystallized from n-hexane and collected by filtration to give 1_ [4- (η-dodecyl) phenyl] -3_ (4-methoxy ΉΙ / VN—H _T O) ^ Sat r :. ¾ (% 9 Χ | Τ) Ζ · 8 ^^ e ^^ e ^ ^

 Λ (^ Ά-Χ.-Ζα) ^ ^ 1 ί, 舔) (1/01 = / d / ^ e ¾ / be 4 ^^^^ ^ Y ^ ^-^^^ ^ m

View οζ ^ αο8 呦 ^^ Summary Earthen οε 缀 Co _ 缀)

^-w ^, ^ ΰϊ 土 ¾ 氺 Γΐοιυιυ _ε · ₈₉ ' ₉ · Α) / d / ^

ε κ 罾 sect rri ^omm rW ^s T (r) 缀 / — π: / / ^ e, ^ 4 Emma <^ 4% 0

, ^ ΊΗ-u)--

 Qi m [seoo]

• 0

9'Ν: 68 · 8'Η: · Α Γΐί¾: ^ 9'Ν: ΐ8 · 8'Η: '8ε ·' (Ο Ν Η 0) ^疆窵: draft

 ■ 6Z- ^ W ^ Q ^ H ON H D) M SVi} iH

 : Γ0Αΐ'9 · ΐ9ΐ'ε · ΐ'Γ0Μ'6 · 9εΐ

· ΑΖΐ'8 · εζΐ'ζ · 6ΐΐ'ε · π ^ · 99 '6ε'9 · 9ε'6 · ΐε' · ΐε'Γ6ζ'9 · 6ζ'9 · 6 ^ 6 ε · 6ζ Έζ'ΐ · ΐ

 (: g '2 009' so-called N-

• ( ^Z H

^{8 · ε '(Ζ Η9 ·} 8 = ί>'Ρ'Η 96 · 9 '(Ζ ΗΖ · 8 = ί>'Ρ'Η ΖΖ · (Ζ Η9 · 8 = ί>'Ρ'Η ΐΑ · Α' ( ^Ζ ΗΖ · 8 = ί ^> 'Ρ'Η 8 · Α

: (: G 'ZH) 0S' ^£ IG) So-called N- Η _Τ

· Ο _ο 9Έ6-0Έ6: · ^ · ω

° draft-^ SM H H-Ο _一 , one ^ 一 / V

NH _T , ^ Whip O) ^ Sat «. _ ¾ (% ZS X | T) ^Sm S'8ST Be α /-S- (/-Engineering ^ ^:

8 L0L0 / L00ZdT / 13d VZ 179.CS0 / 800Z OAV Data and MS data are shown. H-NMR (CDC1, 500MHz, δ: pm)

 Three

 4.15 (2H, q, J = 7.3Hz), 3.35 (lH, t, J = 7.6Hz), 2.18 (3H, s), 1.86-1.74 (2H, m), 1.32-1.23 (25H, m),

 0.84 (3H, t, J = 7.0Hz);

MS (EI) m / z: 312 (M ⁺ ).

Stage 2>

Using 2-acetyl-pentadecanoic acid ethyl ester obtained in the first step, 3-methyl-1- (pyridin-2-yl) -4- (n-tridecyl) -5-pyrazolone was obtained as follows. Synthesized. That is, 2-hydrazinopyridine (3530¾, 3.201010 1.0 equivalent) was added to 2-acetyl-pentadecanoic acid ethyl ester (1.01 g, 3.2 mmoL) in acetic acid (10111) and refluxed for 7 hours. The reaction mixture was extracted with ethyl acetate, washed with saturated aqueous sodium bicarbonate, water and brine, dried over anhydrous sodium sulfate, filtered, concentrated in vacuo and silica gel flash chromatography (n-hexane). / Ethyl acetate = 2/1, then eluted with ethyl acetate) to give 3-methyl-1- (pyridine-2-yl) -4- (n-tridecyl) -5- 965 mg (yield 84%) of pyrazolone was obtained, and the solid was recrystallized from n-hexane and collected by filtration to give 3-methyl-1- (pyridine-2-inole) -4- (n-tridecyl). Obtained 712 mg of colorless crystals of -5-pyrazolone (yield 34%) . Shown below its melting point, ¹ H- NMR data, MS data, HRMS data, the elemental analysis mp: 56.2-56.8 ° C;

'H-NMRCCDCl, 500MHz,: ppm):

 Three

 12.5 (lH, br), 8.22 (2H, d, J = 5.2Hz), 7.84-7.79 (2H, m), 7.10-7.05 (lH, m),

 2.33 (2H, d, J = 7.6Hz), 2.22 (3H, s), 1.55-1.46 (2H, m), 1.40-1.30 (20H, m),

0.88 (3H, t, J = 7.0Hz); MS (EI) m / z: 357 (M ⁺ );

 HRMS: Calculation direct (C H N 0) 357.278, Measurement direct 357.279;

 22 35 3

Elemental analysis: calculated (C H N 0) C, 73.91; H, 9.87; N, 11.75; measured C, 73.69; H, 9.89; N, 11.

 22 35 3

 77.

(Comparative Example 1)

Comparative Example 1 was quinine, and a commercially available reagent was used as it was. This compound is patented 1. The anti-prion active compound according to 1.

[0037] (Comparative Example 2)

 Comparative Example 2 was quinidine, and a commercially available reagent was used as it was. This compound is an anti-prion active compound described in Patent Document 2.

[0038] (Comparative Example 3)

 Comparative Example 3 was MQAA (see the chemical formula below), and a commercially available reagent was used as it was. This compound is an anti-prion active compound described in Patent Document 2.

 [Chemical 3]

 MQAA

(Comparative Example 4)

 Comparative Example 4 was 4- (4-dimethylaminostyryl) quinoline (see the chemical formula below), and a commercially available reagent was used as it was. This compound is an anti-prion active compound described in Patent Document 2.

 [Chemical 4]

 4- (4-Dimethylaminostyryl) quinonne

[0040] <Evaluation>

The anti-prion activity compounds of Examples 1 to 19 and the anti-prion activity compounds of Comparative Examples 1 to 4 obtained as described above were evaluated for anti-prion activity by the following method. Fi.

 [0041] The evaluation was performed using two types of TSE (Transmissible Spongiform Encephalopathy) -derived prion-infected mouse neuroblastoma cell line (N2a). That is, N2a cells infected with RML strain (ScN2a) and N2a # 58 cells infected with Fukuoka-red (F3). N2a # 58 cells are known to have 5 times more normal prion protein (PrP) expression than N2a cells. ScN2a cells and F3 cells were grown in an Opti-MEM (Invitrogen) culture medium supplemented with 10% ushi fetal serum using 6-well culture plates. These cells were cultured until they became dense in the culture vessel, and when subcultured using 5% of these cells, anti-prion active compounds were added to the culture solution at various concentrations. When DMSO or ethanol was used to dissolve the anti-prion active compound, their concentrations were added to 0.2% or less with respect to the medium. Thus, a TSE-derived prion-infected mouse neuroblastoma cell line (N2a) to which an anti-prion active compound was added was grown until it became dense (3-4 days) in the culture vessel.

 [0042] Measurement of anti-prion activity

 The anti-prion activity of the anti-prion active compound was measured by measuring the concentration of ScN2a! /, Which inhibits the formation of F3 abnormal prion by 50% (hereinafter referred to as “IC50”). The method is as follows.

That is, when a culture solution containing an anti-prion active compound containing an anti-prion active compound added to a desired concentration is prepared and subcultured to a new plate at a density of 10% in a dense state, The cells were cultured in the culture solution containing the anti-prion active compound. After the TSE-derived prion-infected mouse neuroblastoma cell line (N2a) grew to a dense state, the cells were lysed with a lysis buffer (0.5% deoxycholate sodium, 0.5% Nonidet P_40, phosphate buffered saline). And dissolved. The cell lysate is digested with proteinase K (a protein-degrading enzyme) at a concentration of 10 mg / ml for 30 minutes (abnormal prion protein remains because it is not digested by proteinase K), and then GLASSFOG ( (Fine glass beads) and centrifuged at 15,000 X g for 5 minutes at 24 ° C. The precipitate obtained by centrifugation was resuspended in a sample loading buffer (sample solution for electrophoresis) and boiled. The sample thus obtained was separated by electrophoresis on a 15% Tris-glycine-SDS-polyacrylamide gel and transferred to a membrane for Western blotting. Abnormal prion protein is SAF8 Detection was performed by binding 3 antibodies (stock solution applied at 1: 5000 dilution, available from France's SPI-Bio) and secondary antibody bound with alkaline phosphatase. The signal detected by immunological reaction (that is, the abnormal prion protein band detected on the membrane) is visualized using CDP-Star detection reagent (Amersham Biosciences, USA) The concentration was determined by a densitometer, which is a density measurement of the protein band. In the measurement, the concentration of the anti-prion active compound at 50% inhibition of the production of abnormal prion protein relative to the control group was determined from at least three independent experiments. <Result>

 Table 1 shows the results of measuring the anti-prion activity as described above.

[table 1]

 Ph: Phenyl

表 1に示すように、 ScN2a細胞については、実施例 7 (1^ = 4—クロ口フエニル、 R² =H、 R³ =メチル）、実施例 19 (1^ = 2—ピリジル、 R² = n—トリデシル、 R³ =メチル) 実施例 3 (R¹ =フエニル、 R² = H、 R³ = 4—二トロフエニル）、実施例 17 (R¹ = 4—（n ードデシル）フエニル、 R² = H、 R³ = 4—二トロフエ二ル）、実施例 5 (1^ =フエニル、 R² =H、 R³ =メトキシカルボニルァミノ）および実施例 11 (R^{1 =}フエニル、 R² =ベンゾィ ノレ、 R³ =メチノレ）の IC (nM)カそれぞれ 0. 50、 1、 3. 16、 5. 01、 6. 31および 6.

As shown in Table 1, for ScN2a cells, Example 7 (1 ^ = 4—black mouth phenyl, R ² = H, R ³ = methyl), Example 19 (1 ^ = 2—pyridyl, R ² = n—tridecyl, R ³ = methyl) Example 3 (R ¹ = phenyl, R ² = H, R ³ = 4—ditropenyl), Example 17 (R ¹ = 4— (n Dodecyl) phenyl, R ² = H, R ³ = 4-nitrophenyl), Example 5 (1 ^ = phenyl, R ² = H, R ³ = methoxycarbonylamino) and Example 11 (R ^{1 =} IC (nM) of phenyl, R ² = benzoinole, R ³ = methinole) 0.50, 1, 3.16, 5.01, 6.31 and 6.

 50

It was 31 and showed high anti-prion activity compared with Comparative Examples 1-4. In addition, Example 2 showed almost the same anti-prion activity as Comparative Example 4, and Example 8, Example 13, Example 6, Example 9, Example 18, Example 16, Example 4, and Example 14 Showed higher anti-prion activity than Comparative Example 3, and Example 1 and Example 12 showed higher anti-prion activity than Comparative Example 2. On the other hand, for F3 cells, Example 8 (1 ^ = phenyl, R ² = phenyl, R ³ = methyl) and Example 3 (1 ^ = phenyl group, R ² = H, R ³ = 4-nitrotropenyl) IC) (nM)

 50

These were 1.26 and 3.16, and showed high anti-prion activity in Examples 1 to 19; In addition, Example 10, Example 2, Example 6, Example 19 and Example 5 showed relatively high anti-prion activity, and Example 4, Example 13, Example 9, Example 15, Example 15 11, Example 12 and Example 14 also showed anti-prion activity.

As described above, Example 7, Example 19, Example 3, Example 17, Example 5, Example 11, and Example 8 have high anti-prion activity for at least one of F3 cells and ScN2a cells. It became clear to show. In addition, Example 3 (1 ^ = phenyl, R ² = H, R ³ = 4-nitrophenyl) was effective in both F3 cells and ScN2a cells! Therefore, a broad spectrum of anti-prion activity can be expected for the pyrazolone derivative.

As shown in Table 1, or introduce Bound phenyl group Ya cycloalkyl Le group Hue alkenyl group Ya substituent as R ^1, or by introducing a cycloalkyl group or Ashiru group R ^2, phenylene in R ³ Examples of the pyrazolone derivatives of Examples 1 to 14 in which a substituent, a phenyl group to which a substituent is bonded, a cycloalkyl group, RCONH—, RNHCO—, ROC ONH—, and RNHCONH— are introduced. It was found to have anti-prion activity. From these results, R ¹ may have a substituent! /, An aryl group or a substituent! /, An alkyl group (including a cycloalkyl group), R ² may be hydrogen, An aryl group which may have a substituent, an alkyl group (including a cycloalkyl group) or an acyl group which may have a substituent, and R ³ may have a substituent! /, An aryl group , Substituents, alkyl groups (including cycloalkyl groups) ), RCONH—, RNHCO—, ROCONH— or RNHCONH— (wherein R is an aryl group or an alkyl group (including a cycloalkyl group), it is obvious that those skilled in the art have an anti-prion activity). This is a range that can be inferred.

Among these, those having particularly excellent anti-prion activity are Example 2 (1 ^ = cyclohexyl group, R ² = H, R ³ = methyl group), Example 3 (1 ^ = phenyl group, R ² = H, R ³ = 4—diphenyl group), Example 8 (R ^{1 =} phenyl group, R ² = phenyl group, R ³ = methyl group), Example 10 phenyl group, R ² = isobutyl group R ³ = methyl group).

The present invention is not limited to the description of the embodiments of the above invention. Various modifications are also included in the present invention as long as those skilled in the art can easily conceive without departing from the scope of the claims.

 Industrial applicability

[0047] The anti-prion active compound of the present invention can be used as a therapeutic drug for human prion diseases such as Creutzfeldt-Jakob disease, for which there is no effective treatment at present, and for veterinary drugs as a countermeasure for mad cow disease. In addition, since systematic compound groups can be used, it is also useful as a research and development reagent for elucidating the infection / onset mechanism of prion diseases and developing therapeutic methods.

Claims

The scope of the claims  [1] An anti-prion active compound that inhibits the growth of abnormal prion protein,  An anti-prion active compound, which is a pyrazolone derivative represented by the following general formula (1):  [Chemical 1

In the formula, R ¹ may have a substituent! /, May have an aryl group or a substituent! /, Represents an alkyl group (including a cycloalkyl group), R ² represents hydrogen, a substituent May have an aryl group, may have a substituent! /, A nitrogen-containing heterocyclic functional group, may have a substituent, an alkyl group (including a cycloalkyl group) or an acyl group. R ³ represents an aryl group which may have a substituent, an alkyl group (including a cycloalkyl group) which may have a substituent, RCONH—RNHCO—R OCONH or RNHCONH— (where R Is an aryl group or an alkyl group (including an alkyl group). [2] The anti-prion active compound according to [1], wherein R ¹ is a substituent, a phenyl group or a cycloalkyl group. 3. The anti-prion active compound according to claim 1 or 2, wherein R ² is hydrogen, a phenyl group, a pyridyl group, an alkyl group (including a cycloalkyl group) or a benzoyl group. [4] R ³ may have an alkyl group, an alkenyl group, or a substituent! /, A phenyl group, RCONH  4. The method according to claim 1, wherein RNHCO—ROCONH— or RNHCONH— (wherein R represents an aryl group or an alkyl group (including a cycloalkyl group)). Anti-prion active compound. [5] R ¹ is a phenyl group or a cycloalkyl group which may have a substituent, and R ² is hydrogen, a phenyl group, a pyridyl group, an alkyl group (including a cycloalkyl group) or a benzoyl group, R ³ represents an alkyl group, an alkenyl group, an optionally substituted phenyl group, RCONH— , RNHCO—, ROCONH—, or RNHCONH— (wherein R represents an aryl group or an alkyl group (including a cycloalkyl group))! The anti-prion active compound according to Item. [6] The combination of R ² and R ³ is (R ¹ : phenyl group, R ² = H, R ³ = CH), (R ^x = 3 cyclohexyl group, R ² = H, R ³ = CH), (R ¹ : phenyl group, R ² = H, R ³ = 4-NO Ph),  3 2 (R ¹ : Phenol group, R ² = H, R ³ = CF), (R ¹ : Phenol group, R ² = H, R ³ = CH OCONH  3 3 ), (R ^^ ClPh, R "= H, R ³ = CH), (R ¹ : phenyl group, R ² = phenyl group, R ³ = CH  3 3 ), (R ¹ : Pheninole group, R ² = H, R ³ = Isopropeninole group), (R ¹ : Pheninole group, R ² = Isobutinole group, R ³ = CH), (i = Pheninole group, R ² = benzoinole group, R ³ = CH), (R ¹ : Phenol  3 3 Ninole group, R ² = H, R ³ = PhCONH), (R ¹ : phenyl group, R ² = 2_pyridyl group, R ³ = CH)  Three , (R ¹ : phenyl group, R ² = H, R "= PhNHCO), (1 ^ = 2-pyridyl group, R ² = H, R ³ = CH  Three ), (R ¹ : phenyl group, R ² = H, R ³ = I—4- (1- CH) Py +-), (1 ^ = 4- nC H Ph, R ² = H,  3 12 25 R ³ = 4-NO Ph), (^ = 4-11-0 H Ph, R ² = H, R ³ = 4_CH OPh) and (1 ^ = 2-pyrid  2 12 25 3 6), wherein R ² = nC H, R ³ = CH).  13 27 3  2. Anti-prion active compound according to claim 1, [7] An anti-prion active agent comprising the anti-prion active compound according to any one of claims 1 to 6 as an active ingredient. [8] A method for inhibiting the production of abnormal prion protein, comprising administering the anti-prion active compound according to any one of claims 1 to 6 to inhibit proliferation of the abnormal prion protein.