JP2002145849A - Alkynylamino acid derivative and method for manufacturing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new immunotype proteasome-inhibiting substance, and thereby to provide an excellent immunosuppressant, an antiinflammatory agent, an antiallergic agent, a treating agent for an autoimmune disease, an antitumor agent and a treating agent for a neurodegenerative disease. SOLUTION: A new alkynylamino acid derivative expressed by the general formula (1), and a pharmacologically permissible salt and a hydrate of the compound inhibits selectively an immunotype proteasome.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an immunosuppressive agent, an anti-inflammatory agent, an anti-allergic agent, a therapeutic agent for an autoimmune disease, an anti-cancer agent, a nervous system, which selectively inhibits an immunological proteasome without inhibiting a constitutive proteasome. The present invention relates to alkynyl amino acid derivatives useful as therapeutic agents for degenerative diseases and methods for producing them.

[0002]

2. Description of the Related Art Proteasome is a huge soluble high molecular weight protein complex having a very complicated molecular structure, mainly present in the nucleus and cytoplasm of eukaryotic cells.
There are two molecular species, ATP-independent type and ATP-dependent type with a sedimentation coefficient of 26S. The 20S proteasome itself has chymotrypsin-like activity, trypsin-like activity and peptidylglutamyl peptide hydrolysis activity, and can degrade short-chain peptides. The 26S proteasome is formed by binding the regulatory subunit complex to the 20S proteasome, which has a catalytic function, and degrades ubiquitinated proteins into peptides and amino acids (Experimental Medicine, vol. 15, 2056, 1997, Science vol.26
8, 533, 1995, Nature 357, 375, 1992).
In addition, studies on the catalytic mechanism of the purified archaeal proteasome have revealed that the proteasome is a threonine protease (Science vol.268, p.579, 1995).
Year).

[0003] More recently, unlike proteasomes normally found in eukaryotes (constitutive proteasomes), IFN-γ
Immunoproteasome was discovered as a proteasome strongly induced by Adv. Immunol., Vol. 64, 1
P. 1997).

The physiological functions of the proteasome include T
Inflammatory cytokines such as NF-α, IL-1, IL-2 and ICAM-
1. Plays an important role in the activation of inflammatory transcription factor NF-κB, which is important for the expression of adhesion molecules such as VCAM-1 (Cell
vol.78, p.773, 1994, Cell vol.80, p.529, 199
5 years). It also functions as an endogenous antigen processing enzyme and plays an important role in the immune response. Furthermore, it is involved in cell cycle control and is also involved in the degradation of tumor suppressor proteins and apoptosis of nerve cells (Nature vol. 349, p. 132, 1991, Cell vol. 75,
495, 1993, FEBS vol. 304, 57, 1992, Tissue Culture 22, 106, 1996, EMBO Journal vol. 15, 3845
P. 1996).

[0005] As described above, it has become clear that the proteasome is deeply involved in various life phenomena such as cell proliferation and control of the immune system. Therefore, drugs that inhibit the proteasome having such various physiological actions are immunosuppressants effective for rejection in organ transplantation, or autoimmune diseases such as rheumatoid arthritis, nephritis, knee osteoarthritis, and systemic lupus erythematosus. It is considered to be effective as a therapeutic agent for chronic inflammatory diseases such as inflammatory bowel disease, allergic diseases such as asthma and dermatitis, a therapeutic agent for cancer and a neurodegenerative disease.

[0006] As a proteasome inhibitor reported to date, lactacystin (J. Antibiotic vol.4)
0, page 113, 1991, protein nucleic acid enzyme 41, 327, 199
6 years, Cell Engineering 15, Vol. 929, 1996), natural products such as TMC-95 (JP-A-11-29595), and peptide aldehydes (WO
95/24914, J. Med.Chem., Vol. 38, p. 2276, 1995, B
ioorganic & Med. Chem. Lett., vol. 6, p. 287, 1996.
Years, JP-A-10-36289, JP-A-11-292833), peptidic ketoamides and ketoaldehydes (Bioorganic & Med. Che.
m. Lett., vol. 9, p. 2603, 1999, Bioorganic & Me
d. Chem. Lett., vol. 8, p. 373, 1998), peptidic epoxy ketone (Tetrahedron Lett., vol. 39, 1343)
Page, 1996, JP-A-11-124397, Chemistry & Biology,
vol.6, 811, 1999, Bioorganic & Med. Chem. Let
t., vol. 9, p. 2283, 1999, Bioorganic & Med. Che
m. Lett., vol. 9, p. 3335, 1999), peptide boric acid (WO96 / 13266, Bioorganic & Med. Chem. Lett., vol.
8, p. 333, 1998). No difference in the inhibitory activity of these inhibitors on constitutive proteasome and immune proteasome has been reported. Furthermore, there are no reports on compounds that selectively inhibit the immune proteasome.

[0007]

DISCLOSURE OF THE INVENTION The present invention provides an excellent immunosuppressant, anti-inflammatory, anti-allergic, or autoimmune disease therapeutic agent with less side effects by providing a substance that selectively inhibits the immunoproteasome. And an anticancer agent and a therapeutic agent for a neurodegenerative disease.

[0008]

Means for Solving the Problems The present inventors have conducted intensive studies on compounds having an immunoproteasome-selective inhibitory activity. As a result, the present inventors have developed a novel proteasome inhibitor having a different structure from the known proteasome inhibitors. The present inventors have found that a novel alkynyl amino acid derivative selectively inhibits the immunoproteasome, and completed the present invention.

That is, the present invention provides a compound represented by the general formula (1) [In the formula, A represents a hydrogen atom, a benzyloxycarbonyl group (Z group), a t-butoxycarbonyl group (Boc group), a trityl group, a benzyl group, a trifluoroacetyl group, or R—
CO—, wherein R is an optionally substituted benzene ring, pyrazine ring, naphthalene ring, styryl group, phenethyl group,
A phenylbutyl group or a benzylamino group), R1
Represents a hydrogen atom, a lower alkyl group having 1 to 4 carbon atoms or a benzyl group; R2, R3 and R4 are the same or different and are a hydrogen atom, a lower alkyl group having 1 to 4 carbon atoms which may be substituted, cyclohexyl; A methyl group, an optionally substituted benzyl group, a naphthylmethyl group, an indolylmethyl group, a pyridylmethyl group, a thenyl group or a thiazolemethyl group, and R5 represents a lower alkoxy group having 1 to 4 carbon atoms, a hydroxy group, Lower alkylamino groups of the formulas 1-4, N,
An O-dimethylhydroxylamino group, a methoxyamino group, a lower alkyl group having 1 to 4 carbon atoms, a phenyl group or a morpholino group, and m represents 0 or 1.] It is a physiologically acceptable salt and an immunoproteasome selective inhibitor containing at least one or more of them as an active ingredient.

The pharmacologically acceptable salts of the compound represented by the general formula (1) in the present invention include hydrochloride, hydrobromide, methanesulfonate, citrate, and tartrate. Suitable acid addition salts and alkali salts include metal salts such as sodium, potassium and magnesium.

In the general formula (1) of the present invention, the "lower alkyl group" such as a lower alkyl group, a lower alkoxy group and a lower alkylamino group means a linear or branched group such as methyl, ethyl and isopropyl. Carbon number 1
Represents an optionally substituted lower alkyl group having 1 to 4 carbon atoms, and represents a methylthio group, a methylsulfinyl group, a methylsulfonyl group, a hydroxy group at any position of the lower alkyl group. A carboxyl group, a lower alkoxycarbonyl group having 1 to 4 carbon atoms, an amino group, a carbamoyl group, a benzyloxycarbonylamino group, t
-Butoxycarbonylamino group and those having a 2,4-dichlorobenzoylamino group. The “optionally substituted benzene ring, optionally substituted benzyl group” means a lower alkoxy group having 1 to 4 carbon atoms, a phenoxy group, a hydroxy group, Cl, B at any position on the ring.
Those having a halogen atom such as r, I, F, etc., a phenyl group, a sulfonamide group, a nitro group, a t-butoxycarbonylaminomethyl group, and a trifluoroacetylaminomethyl group are exemplified.

According to the present invention, the compound represented by the above general formula (1) can be produced by the following route. That is, the general formula (2) Wherein A, R1, R2, R3 and m are as described above.
And a compound represented by the general formula (3) [Wherein R4 and R5 are as described above].

The reaction can be produced by a mixed acid anhydride method or an active ester method used in a usual peptide bond formation reaction, and a method using a condensing agent is preferable. Dicyclohexylcarbodiimide (DCC),
In the presence of a condensing agent such as diisopropylcarbodiimide (DIPC), diphenylphosphoryl azide (DPPA), diethylphosphoryl cyanide (DEPC), 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (WSC), 4-Dimethylaminopyridine (DMAP) is added as a catalyst, and THF, methylene chloride, DMSO, DMF and the like are used as a reaction solvent, and the reaction can be carried out at 0 ° C. to room temperature.

Among the compounds represented by the general formula (3),
A compound in which R5 is a lower alkylamino group having 1 to 4 carbon atoms, an N, O-dimethylhydroxylamino group, a methoxyamino group, a lower alkyl group having 1 to 4 carbon atoms, a phenyl group or a morphonyl group, ) [Wherein, R 9 is a lower alkylamino group having 1 to 4 carbon atoms,
A N, O-dimethylhydroxylamino group, a methoxyamino group, a lower alkyl group having 1 to 4 carbon atoms, a phenyl group or a morphonyl group, and R4 is as described above.] The compound represented by the general formula (15) [Wherein R4 and R9 are as described above].

The reaction is carried out using methanol, ethanol, ethyl acetate or 1,4-dioxane in which hydrochloric acid gas is dissolved, using trifluoroacetic acid with methylene chloride as a solvent, or using trifluoroacetic acid as a solvent. Can be carried out at 0 ° C. to room temperature.

Among the compounds represented by the general formula (15), those wherein R9 is a lower alkyl group having 1 to 4 carbon atoms or a phenyl group, ie, a compound represented by the general formula (14) [Wherein, R 8 represents a lower alkyl group having 1 to 4 carbon atoms or a phenyl group, and R 4 is as described above], represented by the general formula (13) [Wherein R 4 is as defined above] and a compound represented by the general formula (9) [Wherein M represents Li, MgI, MgBr or MgCl, and R8 is as described above], and can be produced by reacting the compound.

The reaction is carried out in an atmosphere of an inert gas such as argon, using THF or the like as a solvent, and the reaction temperature is-
It can be carried out at 78 ° C to room temperature.

In the compounds represented by the general formula (15), including the compound represented by the general formula (13), R9 is a lower alkylamino group having 1 to 4 carbon atoms, N, O-dimethylhydroxyamino Group, a methoxyamino group or a morphonyl group, that is, a compound represented by the general formula (12) [Wherein, R6 represents a hydrogen atom or a lower alkyl group having 1 to 4 carbon atoms, and R7 represents a lower alkyl group having 1 to 4 carbon atoms or a methoxy group, or R6 and R7 are bonded to form a cyclic structure. A morpholine ring, and R4 is as described above], a compound represented by the general formula (11) [Wherein R 4 is as defined above] and a compound represented by the general formula (6): [Wherein R6 and R7 are as described above].

The reaction can be produced by a mixed acid anhydride method or an active ester method used in a usual peptide bond formation reaction, and a method using a condensing agent is preferable. DCC, DIPC, DPPA, DEPC, WS
C. In the presence of a condensing agent such as C, DMAP is optionally added as a catalyst, and THF, methylene chloride, DMSO, DMF, or the like is used as a reaction solvent, and the reaction temperature is 0.
C. to room temperature.

Further, among the compounds represented by the above general formula (1), compounds wherein R5 is a hydroxy group, ie, a compound represented by the general formula (5) [Wherein A, R1, R2, R3, R4 and m are as described above], the compound represented by the general formula (4) [Wherein, R10 represents a lower alkyl group having 1 to 4 carbon atoms, and A, R1, R2, R3, R4 and m are as described above]
Can be produced by hydrolyzing the compound represented by

The reaction is carried out in the presence of an aqueous alkali solution such as sodium hydroxide, potassium hydroxide or lithium hydroxide in the presence of methanol, ethanol, 1,4-dioxane, DMF,
The reaction can be performed at 0 ° C. to room temperature using a solvent such as THF and DMSO.

Further, among the compounds represented by the general formula (1), R5 is a lower alkylamino group having 1 to 4 carbon atoms,
A compound which is an N, O-dimethylhydroxyamino group, a methoxyamino group or a morphonyl group, that is, a compound represented by the general formula (7): [Wherein A, R1, R2, R3, R4, R6 and R7 are as described above], are represented by the general formula (5) Wherein A, R1, R2, R3, R4 and m are as described above, and a compound represented by the general formula (6): [Wherein R6 and R7 are as described above].

The reaction can be produced by a mixed acid anhydride method or an active ester method used in a usual peptide bond formation reaction, and a method using a condensing agent is preferable. DCC, DIPC, DPPA, DEPC, WS
C. In the presence of a condensing agent such as C, DMAP is optionally added as a catalyst, and THF, methylene chloride, DMSO, DMF, or the like is used as a reaction solvent, and the reaction temperature is 0.
C. to room temperature.

Among the compounds represented by the general formula (1), R
A compound in which 5 is a lower alkyl group having 1 to 4 carbon atoms or a phenyl group, that is, a compound represented by the general formula (10): [Wherein A, R1, R2, R3, R4, R8 and m are as described above], are represented by the general formula (8) Wherein A, R1, R2, R3, R4 and m are as described above, and a compound represented by the general formula (9) [Wherein R8 and M are the same as those described above].

The reaction is carried out in an atmosphere of an inert gas such as argon, using THF or the like as a solvent, and the reaction temperature is-
It can be carried out at 78 ° C to room temperature.

[0026]

Next, the present invention will be described with reference to specific examples, but the present invention is not limited to these examples. Further, although the compound of the present invention has an asymmetric carbon, the present invention also includes optical isomers and racemates thereof.

Reference Example 1 (4S) -4- (t-butoxycarbonyl) amino-5
-Phenyl-2-pentinoic acid Boc- (L) -Phe-H was used as a starting material and described in the literature (Chem.Commun., 73
According to the method of 3-734 (1996), Tetrahedron Lett., 3769 (1972)), the target compound was obtained as a pale yellow powder. Less than,
The following Reference Examples 2 and 3 were synthesized in the same manner using various aminoaldehydes.

Reference Example 2 (4S) -4- (t-butoxycarbonyl) amino-6
-Methyl-2-heptic acid The desired product was obtained as a yellow oil.

Reference Example 3 (4S) -4- (t-butoxycarbonyl) amino-5
-(3-Indolyl) -2-pentinic acid The desired product was obtained as a pale green amorphous.

Reference Example 4 (4S) -4- (t-butoxycarbonyl) amino-5
-Ethyl phenyl-2-pentynate The compound of Reference Example 1 (590 mg) was dissolved in DMF (10 mL), potassium carbonate (564 mg) and iodoethane (326 mL) were added, and the mixture was stirred at room temperature for 3 hours. The mixture was poured into ice water, extracted with ethyl acetate, and the organic layer was washed successively with saturated aqueous sodium hydrogen carbonate, water (twice) and saturated brine, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (hexane: ethyl acetate = 4: 1) to give the desired compound (572 mg) as a yellow oil. Hereinafter, using Reference Examples 2 and 3, the following Reference Examples 5 and 6 were synthesized in the same manner.

Reference Example 5 (4S) -4- (t-butoxycarbonyl) amino-6
-Ethyl methyl-2-heptinate The desired product was obtained as a colorless oil.

Reference Example 6 (4S) -4- (t-butoxycarbonyl) amino-5
-(3-Indolyl) -2-ethyl pentate The desired product was obtained as a yellow amorphous.

Example 1 N-methoxy-N-methyl- (4S) -4- (t-butoxycarbonyl) amino-5-phenyl-2-pentynamide The compound of Reference Example 1 (450 mg) and N, O-dimethylhydroxyamine hydrochloride (152 mg) were dissolved in methylene chloride (15 mL), and triethylamine (261 μL) and WSC (449 mg) were added, followed by stirring at room temperature for 3 hours. . Ethyl acetate (100m
L), washed sequentially with diluted hydrochloric acid, water, saturated aqueous sodium hydrogen carbonate, water and saturated saline, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to obtain the desired product (377 mg) as a yellow oil. . Hereinafter, the compound of Reference Example and various amines were condensed in the same manner as in Example 1 to obtain the compounds of Table 1. Unless otherwise specified, amino acids in the table indicate L-form.

[0034]

[Table 1]

Example 6 N- [N- (benzyloxycarbonyl)-(L) -leucyl- (L) -phenylalanyl]-(4S) -4
-Ethyl amino-5-phenyl-2-pentynate The compound of Reference Example 1 (550 mg) was dissolved in methylene chloride (3 mL), and trifluoroacetic acid (2 mL) was added under ice-cooling.
Stirred for 0 minutes. The solvent was distilled off under reduced pressure, the residue was dissolved in a small amount of methylene chloride, a large amount of hexane was added, and the precipitated crystals were collected by filtration and dried, and the trifluoroacetic acid (TFA) salt of a de-Boc form was removed.
(496 mg) was obtained as light brown powdery crystals. Z- (L) -Leu- (L) -Phe
-OH was dissolved (122 mg) in DMF (1mL), HOBt · H 2 O (45.2mg),
The above-mentioned TFA salt (97.6 mg) and triethylamine (41 μL) were sequentially added, cooled to 0 ° C., 10 minutes later, added with WSC (84.9 mg), and stirred at the same temperature for 2 hours. Pour the reaction solution into ice water,
Extracted with ethyl acetate, washed sequentially with diluted hydrochloric acid, water, saturated aqueous sodium hydrogen carbonate, water and saturated saline, dried over anhydrous sodium sulfate,
The solvent was distilled off under reduced pressure. The residue was purified by silica gel chromatography (hexane: ethyl acetate = 3: 1) to give the desired product (135 mg) as colorless powdery crystals. mp 118-120 ℃ Hereinafter, using the same procedure as in Example 6, the compounds of Reference Examples and Examples were condensed with various amino acids and carboxylic acids to obtain the compounds shown in Tables 2 and 3.

[0036]

[Table 2]

[0037]

[Table 3]

Example 79 N- [N- (benzyloxycarbonyl)-(L) -phenylalanyl]-(5S) -5-amino-6-phenyl-3-hexyn-2-one Under an argon stream, the compound of Example 70 (321 mg) in THF (5 mL)
And cooled to -78 ° C, and 1.05 mol / L of methyllithium
An ether solution (1.79 ml) was added dropwise, followed by stirring at the same temperature for 30 minutes. The mixture was quenched with a 10% aqueous citric acid solution (1 mL), further added with water (50 mL), returned to room temperature, and extracted with ethyl acetate. The organic layer was washed successively with water and saturated saline, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to obtain pale yellow crystals. This was dissolved in a small amount of ethyl acetate, and a large amount of hexane was added for crystallization.The precipitated crystal was collected by filtration, dried, and dried.
(209 mg) was obtained as colorless powdery crystals.

Example 80 N- [N- (benzyloxycarbonyl)-(L) -leucyl- (L) -phenylalanyl]-(5S) -5
-Amino-6-phenyl-3-hexyn-2-one The same procedures as in Example 79 were carried out except for using the compound of Example 7 to give a desired product as a pale yellow amorphous. FABMS: 582 ([M + H] ⁺ ) Hereinafter, the compound of Example was changed to MeLi, EtMgB in the same manner as in Example 79.
Reaction with organometallic compounds such as r and PhLi yielded the compounds shown in Table 4.

[0040]

[Table 4]

Example 89 N- [N- (benzoyl)-(L) -phenylalanyl]-(4S) -4-amino-5-phenyl-2-pentynoic acid The compound of Example 21 (115 mg) was dissolved in a mixed solvent of DMF (0.5 mL) and water (0.5 mL), cooled to 0 ° C., and lithium hydroxide (50 mg)
Was added and stirred at the same temperature for 3 hours. The reaction solution was diluted with water (50 mL), neutralized with dilute hydrochloric acid, and the precipitated crystals were collected by filtration and dried to obtain the desired product (103 mg) as pale yellow powdery crystals. FABMS: 441 ([M + H] ⁺ )

The compound of Example 89 was reacted with various amines in the same manner as in Example 1 to obtain the compounds shown in Table 5 below.

[Table 5]

Example 92 N- [N- (benzoyl)-(dl) -α-glutamyl]
-Ethyl (4S) -4-amino-5-phenyl-2-pentynate The compound of Example 65 (32 mg) was dissolved in methylene chloride (1 mL), and after cooling to 0 ° C., a mixture of methylene chloride (1 mL) and trifluoroacetic acid (1 mL) was added, and the mixture was stirred at the same temperature for 15 minutes. . The reaction solution was concentrated and dried to obtain the desired product (32 mg) as a pale brown amorphous. MS: 450 (M ⁺ )

Example 93 N- [N- (benzoyl)-(dl) -α-lysyl] −
Ethyl (4S) -4-amino-5-phenyl-2-pentynate Dissolve the compound of Example 53 (22 mg) in methylene chloride (1 mL), cool to 0 ° C., add a mixture of methylene chloride (1 mL) and trifluoroacetic acid (1 mL), return to room temperature and stir for 1 hour did. The reaction solution was concentrated and dried to obtain the desired product (26 mg) as a pale brown amorphous. FABMS: 450 ([M + H] ⁺ )

Next, the results supporting the usefulness of the compound of the present invention are shown by experimental examples.

<Experimental Example 1> Proteasome Inhibition Test It has been reported by Eleuteri et al. That the proteasome contained in bovine spleen is an immune proteasome (Journal of Biological Chemistry, Vol. 272, No. 18). , 11824-1183
1, p. 1997). Therefore, the immunoproteasome was purified from bovine spleen and used. The constitutive proteasome was purified from bovine kidney and used. Purification of proteasome and measurement of its activity are described in Tanaka et al. (Journal of Biological Chemistry, 263, 31, 31)
209-16217, 1988).
Specifically, a centrifugal supernatant obtained by adding Tris buffer (pH 7.5) to bovine spleen or kidney and homogenizing the resulting mixture was subjected to ammonium sulfate precipitation, ion exchange chromatography (Q Sepharose FF), gel filtration chromatography (high-load chromatography).・ Proteasome was purified by fractionation using Superdex 200). Proteasome activity is Suc-L
The measurement was performed using the degradation of the eu-Leu-Val-Tyr-MCA substrate as an index. Test compound D was added to a 96-well plate.
1 μL of the MSO solution and 89 μL of a proteasome solution of about 1 U / mL were added and mixed, and incubated at 37 ° C. for 30 minutes. Subsequently, 200 μmol / L of Suc-Le
10 μL of u-Leu-Val-Tyr-MCA substrate was added and mixed, and incubated at 37 ° C. for 1 hour. 10
After stopping the reaction by adding 100 μL of a% SDS solution, the fluorescence intensity of aminomethylcoumarin released from the substrate was measured at an excitation wavelength of 355 nm and a fluorescence wavelength of 460 nm, and the value was defined as proteasome activity. Based on the proteasome activity when the test compound was not added, the concentration (IC ₅₀ ) of the compound that inhibited the activity by 50% was calculated. It is shown in Table 6.

[0047]

[Table 6]

<Experimental Example 2> Luciferase reporter gene assay The NF-κB activation inhibitory action of the test compound based on the proteasome inhibitory action was measured by a reporter gene assay using firefly luciferase as a reporter gene. Thymidine kinase (TK) promoter upstream of the firefly luciferase (Luc) gene, which is a reporter gene, and
Further, a plasmid (pNFkB-LUC: Stratagene) having a κB sequence repeated 5 times further upstream was used as a reporter vector. Also, for internal standard,
A plasmid (pRL-TK: Stratagene) in which a reporter gene, Renilla luciferase gene (RL), was linked upstream of a TK promoter was used. Gaczyns
According to the method of ka et al. (Nature, 1993, 365, 264), human IFN-γ (human interferon-γ, Boehringer Mannheim) was added to induce immunoproteasomes in human histiocytic lymphoma cells U937 (ATCC No. CRL159
3) 100 units / mL of hIFN-γ and 10% fetal bovine serum (FB
The cells were maintained in an RPMI-1640 medium (Gibco) containing S).
The U937 cells were co-transfected with the above plasmids simultaneously using Lipofectamine reagent (Gibco). The cells transiently transfected with the gene are suspended in the above medium, and dispensed into a 24-well plate at 7 × 10 ⁵ cells / well.
Cultured overnight. The test compound is diluted in the above medium, and
Added to 24-well plates and pre-treated for 45 minutes. Thereafter, hTNF-α (human tumor necrosis factor, Boehringer Mannheim) was added to a final concentration of 3 ng / mL, and further added.
The cells were cultured for 5 hours to stimulate the cells. After completion of the culture, the cells were collected to obtain a cell lysate. The firefly luciferase activity in the cell lysate was measured using a dual luciferase reporter assay system (Promega).
At the same time, Renilla luciferase activity was measured using the same assay kit. The NF-κB transcription activity detected by firefly luciferase activity was normalized from the internal standard Renilla luciferase activity. The NF-κB activation inhibitory effect of the test compound was observed by a decrease in NF-κB transcription activity, and the inhibition rate (%) was calculated by the following formula. Inhibition rate (%) = (1-(Test-Blank) / (Control-B
lank)) × 100 (Test: Transcriptional activity of cells stimulated with TNF-α with addition of compound, Blank: Transcriptional activity of cells without stimulation with TNF-α without addition of compound, Control: Addition of compound Without, T
Transcriptional activity of cells stimulated with NF-α)

[0049]

[Table 7] As described above, the compound of the present invention represented by the general formula (1) was confirmed to have an immunoproteasome-selective inhibitory effect, and was also confirmed to suppress NF-κB transcriptional activity.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07C 275/24 C07C 275/24 4C204 311/16 311/16 4C206 317/50 317/50 4H006 323/60 323 / 60 4H045 C07D 209/20 C07D 209/20 213/56 213/56 241/24 241/24 277/30 277/30 277/58 277/58 295/16 295/16 Z 333/24 333/24 C07K 5 / 062 C07K 5/062 // A61K 31/27 A61K 31/27 31/381 31/381 31/405 31/405 31/426 31/426 31/4402 31/4402 31/4406 31/4406 31/495 31 / 495 31/5375 31/5375 38/00 A61P 25/00 A61P 25/00 29/00 29/00 35/00 35/00 37/00 37/00 37/06 37/06 37/08 37/08 43 / 00 111 43/00 111 A61K 37/02 C07D 277/30 (72) Inventor Kazuhiko Kuriyama 1-7-16 Otome, Koyama City, Tochigi Prefecture (72) Inventor Satoshi Iwanami Shimotsuga, Tochigi Prefecture 67-1 Nojimacho Junjima F-term (Reference) 4C023 EA11 4C033 AD08 AD17 4C055 AA01 BA01 BA02 BA34 BB02 BB19 CA01 CA02 CA34 CB02 CB19 DA01 4C084 AA07 BA01 BA14 CA59 NA14 ZA012 ZB082 ZB112 ZB132 ZB026 BC02 BC02 ABC023 BC82 NA14 ZA01 ZB08 ZB11 ZB13 ZB26 ZC20 4C204 BB01 CB02 DB13 DB22 EB02 FB01 GB01 4C206 AA03 HA22 HA24 NA14 ZA01 ZB08 ZB11 ZB13 ZB26 ZC20 4H006 AA01 AB20 AB22 AB29 BJ50 BM30 BM30 A30 AE30

Claims (10)

[Claims] 1. General formula (1) [In the formula, A represents a hydrogen atom, a benzyloxycarbonyl group (Z group), a t-butoxycarbonyl group (Boc group), a trityl group, a benzyl group, a trifluoroacetyl group, or R—
CO—, wherein R is an optionally substituted benzene ring, pyrazine ring, naphthalene ring, styryl group, phenethyl group,
A phenylbutyl group or a benzylamino group), R1
Represents a hydrogen atom, a lower alkyl group having 1 to 4 carbon atoms or a benzyl group; R2, R3 and R4 are the same or different and are a hydrogen atom, a lower alkyl group having 1 to 4 carbon atoms which may be substituted, cyclohexyl; A methyl group, an optionally substituted benzyl group, a naphthylmethyl group, an indolylmethyl group, a pyridylmethyl group, a thenyl group or a thiazolemethyl group, and R5 represents a lower alkoxy group having 1 to 4 carbon atoms, a hydroxy group, Lower alkylamino groups of the formulas 1-4, N,
An alkynyl amino acid derivative, which represents an O-dimethylhydroxylamino group, a methoxyamino group, a lower alkyl group having 1 to 4 carbon atoms, a phenyl group or a morpholino group, and m represents 0 or 1. Pharmacologically acceptable salts and hydrates. 2. General formula (1) [In the formula, A represents a hydrogen atom, a benzyloxycarbonyl group (Z group), a t-butoxycarbonyl group (Boc group), a trityl group, a benzyl group, a trifluoroacetyl group, or R—
CO—, wherein R is an optionally substituted benzene ring, pyrazine ring, naphthalene ring, styryl group, phenethyl group,
A phenylbutyl group or a benzylamino group), R1
Represents a hydrogen atom, a lower alkyl group having 1 to 4 carbon atoms or a benzyl group; R2, R3 and R4 are the same or different and are a hydrogen atom, a lower alkyl group having 1 to 4 carbon atoms which may be substituted, cyclohexyl; A methyl group, an optionally substituted benzyl group, a naphthylmethyl group, an indolylmethyl group, a pyridylmethyl group, a thenyl group or a thiazolemethyl group, and R5 represents a lower alkoxy group having 1 to 4 carbon atoms, a hydroxy group, Lower alkylamino groups of the formulas 1-4, N,
An alkynyl amino acid derivative, which represents an O-dimethylhydroxylamino group, a methoxyamino group, a lower alkyl group having 1 to 4 carbon atoms, a phenyl group or a morpholino group, and m represents 0 or 1. An immunoproteasome-selective inhibitor comprising at least one of pharmacologically acceptable salts and hydrates as an active ingredient. 3. General formula (2) [In the formula, A is a hydrogen atom, a Z group, a Boc group, a trityl group, a benzyl group, a trifluoroacetyl group or R—CO— (R is an optionally substituted benzene ring, a pyrazine ring,
A naphthalene ring, a styryl group, a phenethyl group, a phenylbutyl group or a benzylamino group), R1 represents a hydrogen atom, a lower alkyl group having 1 to 4 carbon atoms or a benzyl group, and R2 and R3 represent the same or different hydrogen atoms. Atom, optionally substituted lower alkyl group having 1 to 4 carbon atoms, cyclohexylmethyl group, optionally substituted benzyl group, naphthylmethyl group, indolylmethyl group, pyridylmethyl group, thenyl group or thiazolemethyl group And m
Represents 0 or 1] and a compound represented by the general formula (3) [Wherein, R4 represents a hydrogen atom, an optionally substituted lower alkyl group having 1 to 4 carbon atoms, a cyclohexylmethyl group, an optionally substituted benzyl group, a naphthylmethyl group, an indolylmethyl group, a pyridylmethyl group. R5 represents a lower alkoxy group having 1 to 4 carbon atoms, a hydroxy group, a lower alkylamino group having 1 to 4 carbon atoms, an N, O-dimethylhydroxylamino group, a methoxyamino group, Represents a lower alkyl group, a phenyl group or a morpholino group represented by the following formulas (1) to (4)]. The method according to claim 1, wherein A, R1, R2, R3, R4, R5 and m are as described above. 4. General formula (4) Wherein A is a hydrogen atom, a Z group, a Boc group, a trityl group, a benzyl group, a trifluoroacetyl group or R—CO— (R is an optionally substituted benzene ring, a pyrazine ring,
A naphthalene ring, a styryl group, a phenethyl group, a phenylbutyl group or a benzylamino group), R1 represents a hydrogen atom, a lower alkyl group having 1 to 4 carbon atoms or a benzyl group, and R2, R3 and R4 are the same or different. Hydrogen atom, optionally substituted lower alkyl group having 1 to 4 carbon atoms, cyclohexylmethyl group, optionally substituted benzyl group, naphthylmethyl group, indolylmethyl group, pyridylmethyl group, thenyl group or thiazole A methyl group; R10 represents a lower alkyl group having 1 to 4 carbon atoms;
Represents 0 or 1], wherein the compound represented by the general formula (5) is hydrolyzed: The method of claim 1, wherein A, R1, R2, R3, R4 and m are as described above. 5. General formula (5) Wherein A is a hydrogen atom, a Z group, a Boc group, a trityl group, a benzyl group, a trifluoroacetyl group or R—CO— (R is an optionally substituted benzene ring, a pyrazine ring,
A naphthalene ring, a styryl group, a phenethyl group, a phenylbutyl group or a benzylamino group), R1 represents a hydrogen atom, a lower alkyl group having 1 to 4 carbon atoms or a benzyl group, and R2, R3 and R4 are the same or different. Hydrogen atom, optionally substituted lower alkyl group having 1 to 4 carbon atoms, cyclohexylmethyl group, optionally substituted benzyl group, naphthylmethyl group, indolylmethyl group, pyridylmethyl group, thenyl group or thiazole Represents a methyl group, m represents 0 or 1], and a compound represented by the general formula (6) [Wherein, R6 represents a hydrogen atom or a lower alkyl group having 1 to 4 carbon atoms, and R7 represents a lower alkyl group having 1 to 4 carbon atoms or a methoxy group, or R6 and R7 are bonded to form a cyclic structure. Which represents a morpholine ring]. The method for producing a compound according to claim 1, wherein A, R1, R2, R3, R4, R6, R7 and m are as described above. 6. The general formula (8) Wherein A is a hydrogen atom, a Z group, a Boc group, a trityl group, a benzyl group, a trifluoroacetyl group or R—CO— (R is an optionally substituted benzene ring, a pyrazine ring,
A naphthalene ring, a styryl group, a phenethyl group, a phenylbutyl group or a benzylamino group), R1 represents a hydrogen atom, a lower alkyl group having 1 to 4 carbon atoms or a benzyl group, and R2, R3 and R4 are the same or different. Hydrogen atom, optionally substituted lower alkyl group having 1 to 4 carbon atoms, cyclohexylmethyl group, optionally substituted benzyl group, naphthylmethyl group, indolylmethyl group, pyridylmethyl group, thenyl group or thiazole A methyl group, m represents 0 or 1] and a compound represented by the general formula (9): [Wherein R8 represents a lower alkyl group having 1 to 4 carbon atoms or a phenyl group, and M represents Li, MgI, MgBr or MgCl]. The method of claim 1, wherein A, R1, R2, R3, R4, R8 and m are as described above. 7. General formula (3) [Wherein, R4 represents a hydrogen atom, an optionally substituted lower alkyl group having 1 to 4 carbon atoms, a cyclohexylmethyl group, an optionally substituted benzyl group, a naphthylmethyl group, an indolylmethyl group, a pyridylmethyl group. R5 represents a lower alkoxy group having 1 to 4 carbon atoms, a hydroxy group, a lower alkylamino group having 1 to 4 carbon atoms, an N, O-dimethylhydroxylamino group, a methoxyamino group, A lower alkyl group, a phenyl group or a morpholino group represented by Formulas 1 to 4], and the alkynyl amino acid derivative according to claim 1, and a pharmacologically acceptable salt or hydrate. 8. The general formula (11) [Wherein, R4 represents a hydrogen atom, an optionally substituted lower alkyl group having 1 to 4 carbon atoms, a cyclohexylmethyl group, an optionally substituted benzyl group, a naphthylmethyl group, an indolylmethyl group, a pyridylmethyl group. , A thenyl group or a thiazolemethyl group] to the compound represented by the general formula (6) [Wherein, R6 represents a hydrogen atom or a lower alkyl group having 1 to 4 carbon atoms, and R7 represents a lower alkyl group having 1 to 4 carbon atoms or a methoxy group, or R6 and R7 are bonded to form a cyclic structure. Which represents a morpholine ring]. The method for producing a production intermediate according to claim 7, wherein a compound represented by the formula: wherein R4, R6 and R7 are as described above is obtained, and the compound is subjected to acid hydrolysis. 9. General formula (13) [Wherein, R4 represents a hydrogen atom, an optionally substituted lower alkyl group having 1 to 4 carbon atoms, a cyclohexylmethyl group, an optionally substituted benzyl group, a naphthylmethyl group, an indolylmethyl group, a pyridylmethyl group. , A thenyl group or a thiazolemethyl group], and a compound represented by the general formula (9) Wherein R 8 represents a lower alkyl group having 1 to 4 carbon atoms or a phenyl group, and M represents Li, MgI, MgBr or MgCl. [Wherein R4 and R8 are as defined above], and the compound is subjected to acid hydrolysis.
A method for producing the production intermediate described above. 10. The general formula (15) [Wherein, R4 represents a hydrogen atom, an optionally substituted lower alkyl group having 1 to 4 carbon atoms, a cyclohexylmethyl group, an optionally substituted benzyl group, a naphthylmethyl group, an indolylmethyl group, a pyridylmethyl group. R9 represents a lower alkoxy group having 1 to 4 carbon atoms, a hydroxy group, a lower alkylamino group having 1 to 4 carbon atoms, an N, O-dimethylhydroxylamino group, a methoxyamino group, A lower alkyl group, a phenyl group or a morpholino group of formulas 1 to 4], and subjecting the compound to acid hydrolysis.