JPH11124397A - Proteasome inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new compound comprising a specific epoxy-peptide derivative, having inhibiting actions on a proteasome and useful as a therapeutic agent or the like for autoimmune disease, inflammatory enteritis, asthma, Alzheimer disease or the like caused by hyperactions of the proteasome. SOLUTION: This new compound is represented by formula I [R<11> is an amino acid residue having an amino group acylated with a (substituted)acyl group or an amino acid residue in which the amino group may be protected; R<12> is N or a 1-6C alkyl; R13 and R14 are each H or a 1-6C alkyl or R<13> and R<14> together denote methylene] [e.g. 2-(2-carbamoyl-propionyl-Thr-Thr-Leu)-1- hydrpxy-2,3-epoxypropane] and is useful as a proteasome inhibitor or the like. The compound is obtained by carrying out the condensation reaction of a preepoxyamino acid derivative represented by formula II (TBDMS is tert butyldimethylsilyl) with an N-terminal modified amino acid derivative represented by formula III and then conducting the epoxidation reaction.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a proteasome inhibitor. More specifically, the present invention relates to a proteasome inhibitor comprising an epoxy-peptide derivative, a therapeutic agent for an autoimmune disease, an inflammatory bowel disease, asthma, and Alzheimer's disease, and a novel epoxy-peptide derivative containing such a compound as an active ingredient.

[0002]

BACKGROUND OF THE INVENTION Proteasomes initially have a sedimentation coefficient of 20%.
It was discovered as a catalytic unit of S and was called a multifunctional protease, but the name of the proteasome has now become established. The structure of the proteasome is a large multi-component complex with a characteristic structure. For example, the 26S proteasome is a dumbbell-shaped molecule having a molecular weight of about 2 million in which a U-shaped regulatory unit is associated with both ends of a 20S proteasome (cylindrical molecule) as a catalytic unit, and is an eukaryotic ATP-dependent protease. (Tissue Culture, 22, 75-105 (1996)).

[0003] In recent years, the proteasome has become known as TNF-α, I
Transcriptional regulator NF-κ of inflammatory cytokines such as L-1
It has been clarified that it plays an important role in the activation of B (Vito J. Palombella, et al., Cell, 7
8, 773-785 (1994), DimitrisThanos, et al., Cell, 8
0, 529-532 (1995)). In addition, it has been revealed that proteasome plays an important role in processing amyloid accumulated in the brain of Alzheimer's disease patients (Shin-ichi Kojima, et al., FEBS, 304, 57-60).
(1992)).

[0004] Thus, it has become clear that the proteasome plays an important role in controlling the degradation of many proteins that are closely related to cell proliferation and life phenomena including the immune system.

[0005] On the other hand, research on inhibitors for this proteasome is also in progress. For example, Z-Ile is an effective peptidylaldehyde-type proteasome inhibitor.
^{-Glu (OBu t) -Ala-Leu} -H ( aldehyde) have been synthesized and found to inhibit the activation of NF-κB (EBMTraenckner et al. , EMBO J., 1
3,5433-5441,1994). Also, Z-Leu-Leu-L
Eu-H (aldehyde) is synthesized and inhibits the processing of endogenous antigens and the conversion of precursor NF-κB to the mature form, and has a neurite outgrowth effect equal to or higher than that of NGF (nerve growth factor). Recognized (Y.Sato
et al., Neurosci. Lett., 120, 1-4, 1990).

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel peptidyl epoxy-type proteasome inhibitor. Another object of the present invention is to provide a therapeutic agent for an autoimmune disease, inflammatory bowel disease, asthma, Alzheimer's disease and the like, comprising the compound of the present invention used as a proteasome inhibitor as an active ingredient. Still another object of the present invention is to provide a novel compound having a proteasome inhibitory action.

[0007]

That is, the gist of the present invention is as follows.
[1] General formula (1)

[0008]

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(Wherein, R ¹ is a substituted or unsubstituted acyl group, an amino acid residue having an amino group acylated with a substituted or unsubstituted acyl group, or a compound in which the amino group is protected by a protecting group. also shows an amino acid residue, R ² represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, R ³ and R ⁴ represents hydrogen atom or an alkyl group having 1 to 6 carbon atoms, or R ³ And R ⁴ together represent a methylene group) [2] In the general formula (1), R ¹ is a substituted or unsubstituted acyl group, and R ² is A proteasome inhibitor according to the above [1], wherein R ³ and R ⁴ together represent a methylene group;
In the general formula (1), R ¹ is an amino acid residue having an amino group acylated with a substituted or unsubstituted acyl group, or an amino acid residue in which the amino group may be protected with a protecting group; ² is a methyl group; R ³ and R ⁴
Is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, the proteasome inhibitor according to the above [1], [4] the compound represented by the general formula (1) according to any one of the above [1] to [3]. [5] The therapeutic agent for a disease caused by enhanced proteasome action, comprising: [5] the disease is selected from the group consisting of autoimmune disease, inflammatory bowel disease, asthma, and Alzheimer's disease. Therapeutic agent, [6] The therapeutic agent according to [5], wherein the autoimmune disease is rheumatoid arthritis, [7] The general formula (2)

[0010]

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(Wherein R ¹¹ represents an amino acid residue having an amino group acylated by a substituted or unsubstituted acyl group, or an amino acid residue in which the amino group may be protected by a protecting group; R ¹² represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, R ¹³ and R ¹⁴ represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, or R ¹³ and R ¹⁴ are taken together Wherein R ¹¹ is an amino acid residue having an amino group acylated by a substituted or unsubstituted acyl group, or a protective group represented by the formula: Is an amino acid residue which may be protected, R ¹² is a methyl group,
The compound according to the above [7], wherein ¹³ and R ¹⁴ are a hydrogen atom or an alkyl group having 1 to 6 carbon atoms,

[9] 2- (2-carbamoyl-propionyl-Thr-Thr-Leu)
-1-hydroxy-2,3-epoxypropane.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The proteasome inhibitor of the present invention has the general formula (1):

[0013]

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(In the formula, R ¹ is a substituted or unsubstituted acyl group, an amino acid residue having an amino group acylated by a substituted or unsubstituted acyl group, or a compound in which the amino group is protected by a protecting group. also shows an amino acid residue, R ² represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, R ³ and R ⁴ represents hydrogen atom or an alkyl group having 1 to 6 carbon atoms, or R ³ And R ⁴ together represent a methylene group). That is, the proteasome inhibitor of the present invention comprises a compound having a structural feature of a peptide derivative having an epoxy group (epoxy-peptide derivative).

In the present invention, the proteasome inhibitory action of the proteasome inhibitor refers to the proteasome protein (high molecular weight) decomposition activity and peptide (low molecular weight) activity.
It refers to inhibiting degradation activity.

In R ¹ , the unsubstituted acyl group includes, for example, an aliphatic acyl group or an aromatic acyl group. Examples of the aliphatic acyl group include straight-chain, branched or cyclic ones having 2 to 30 carbon atoms, and specifically,
Acetyl group, propionyl group, butyryl group, isobutyryl group, valeryl group, isovaleryl group, dodecanoyl group,
Examples include a pentadecanoyl group, a docosanoyl group, and a nonacosanoyl group. As the aromatic acyl group, for example,
Examples include a benzoyl group, a benzylcarbonyl group, a phenethylcarbonyl group, a naphthylcarbonyl group, a naphthylmethylcarbonyl group, and the like. The substituted acyl group for R ¹ includes, for example, those in which a hydrogen atom of the unsubstituted acyl group is substituted with a substituent. The number of substituents in the substituted acyl group is not particularly limited, and may be one or two or more. Examples of the substituent include a hydroxyl group, an amino group, a nitro group, a fluorine atom, a chlorine atom, a bromine atom, an alkoxy group such as a methoxy group or an ethoxy group, a substituted aminocarbonyl group such as a carbamoyl group, a methylcarbamoyl group or a dimethylcarbamoyl group. And the like.

In R ¹ , the amino acid in “an amino acid residue having an amino group acylated with a substituted or unsubstituted acyl group or an amino acid residue in which the amino group may be protected with a protecting group” Is not particularly limited and means various commonly used amino acids, and preferably includes serine, threonine, isoleucine, leucine, normal valine and the like.

In the above-mentioned "amino acid residue having an amino group acylated by a substituted or unsubstituted acyl group", the substituted or unsubstituted acyl group substituted for the amino group of the amino acid is the same as the above substituted R ¹ Or the same thing as an unsubstituted acyl group is mentioned. As the unsubstituted acyl group, an aliphatic acyl group such as an acetyl group, a propionyl group, a butyryl group, an isobutyryl group, a valeryl group and an isovaleryl group is preferable. Preferred examples of the substituted acyl group include a substituted aminocarbonyl group such as a hydroxyl group, a carbamoyl group, a methylcarbamoyl group, and a dimethylcarbamoyl group.

The protecting group for the amino group may be any commonly used one, for example, Protective Gro.
up in Organic Synthesis (TW Greene, 1981)
And the like can be used. Preferably, a benzyloxycarbonyl group, a t-butyloxycarbonyl group, a p-nitrobenzyloxycarbonyl group and the like are mentioned.

In the above R ² , the alkyl group having 1 to 6 carbon atoms includes methyl, ethyl, propyl, n-
Examples thereof include a butyl group, an n-pentyl group, and an n-hexyl group, and a methyl group is preferable.

R ³ and R ^{4 each} have 1 to 6 carbon atoms.
Examples of the alkyl group include a methyl group, an ethyl group, a propyl group, an n-butyl group, an n-pentyl group, and an n-hexyl group, and a methyl group is preferable.

[0022] In the general formula (1), suitable compounds as proteasome inhibitors, 1) R ¹ is a substituted or unsubstituted acyl group, R ²
Is a hydrogen atom, and R ³ and R ⁴ together are a methylene group; 2) an amino acid residue having an amino group in which R ¹ is acylated by a substituted or unsubstituted acyl group; have group is protected with a protecting group is also optionally amino acid residue, R ²
Is a methyl group, and R ³ and R ⁴ are a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. More specifically, the following compounds are mentioned as preferable compounds, but the present invention is not limited thereto.

That is, 2- {6-methylheptanoyl-S
er- (3,4-dehydro-Leu)}-1-hydroxy-2,3-epoxypropane, 2- (6-methylheptanoyl-Ser-Leu) -1-hydroxy-2,3
-Epoxypropane, 2- {6-methylheptanoyl-
Thr- (3,4-dehydro-Leu)}-1-hydroxy-2,3-epoxypropane, 2- (6-methylheptanoyl-Thr-Leu) -1-hydroxy-2,
3-epoxypropane, 2- (2-carbamoyl-propionyl-Thr-Thr-Leu) -1-hydroxy-2,3-epoxypropane, 2- {2-carbamoyl-propionyl-Thr-Thr- (3,4- Dehydro-Leu)}-1-hydroxy-2,3-epoxypropane, 2- (2-carbamoyl-propionyl-Thr
-Ser-Leu) -1-hydroxy-2,3-epoxypropane, 2- {2-carbamoyl-propionyl-
Thr-Ser- (3,4-dehydro-Leu)｝-1
-Hydroxy-2,3-epoxypropane, 2- (benzyloxycarbonyl-Ile-Thr-Leu) -1
-Hydroxy-2,3-epoxypropane, 2- (benzyloxycarbonyl-Leu-Thr-Leu) -1
-Hydroxy-2,3-epoxypropane, 2- (benzyloxycarbonyl-n-Val-Thr-Leu)
-1-hydroxy-2,3-epoxypropane and the like.

Among these compounds, 2- {6-methylheptanoyl-Ser- in which R ¹ in the general formula (1) is a compound having a 6-methylheptanoyl group.
(3,4-dehydro-Leu)｝-1-hydroxy-
2,3-Epoxypropane is a known antibiotic with antitumor activity, eponemycin (Journal of Antibiotics, 43, 8-18 (1990).
Year); Synthesis, 300-304, 1994). The present inventors have isolated the same compound as eponemycin from a novel microorganism and newly found that it has proteasome inhibitory activity.

Thus, in the general formula (1), R ¹
Is a compound having a 6-methylheptanoyl group, which includes known substances, wherein R ¹ is an amino acid residue having an amino group acylated by a substituted or unsubstituted acyl group, or the amino group is protected by a protecting group. Compounds having the structural feature of being an amino acid residue which may be present are novel compounds. In the present specification, these new compounds are particularly represented by the general formula (2).

General formula (2)

[0027]

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(Wherein R ¹¹ represents an amino acid residue having an amino group acylated by a substituted or unsubstituted acyl group, or an amino acid residue in which the amino group may be protected by a protecting group; R ¹² represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, R ¹³ and R ¹⁴ represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, or R ¹³ and R ¹⁴ are taken together Represents a methylene group).

In the general formula (2), R¹¹Is replaced
Has an amino group acylated by an unsubstituted acyl group
Amino acid residue or amino group is protected with a protecting group
Examples of the amino acid residue include those represented by R in general formula (1).
¹Are the same as those described for¹², R¹³as well as
R¹⁴For R in general formula (1)^Two, R^Three, R ^Four
And respectively.

In the general formula (2), R ¹¹ is an amino acid residue having an amino group acylated by a substituted or unsubstituted acyl group, or an amino acid residue wherein the amino group may be protected by a protecting group. R ¹² is a methyl group, R ¹³ is a hydrogen atom and R ¹⁴ is a methyl group, or
Compounds in which ¹³ and R ¹⁴ together are a methylene group are preferred.

In order to produce the compound represented by the general formula (1) or the general formula (2) of the present invention, the compound can be easily synthesized according to a series of known synthetic reaction steps such as a step of synthesizing eponemycin. (Tetrahedron Lett., 34,1047-1050 (1
993), Synthesis, 300-304 (March 1994)).

For example, taking the synthesis of SPA-1344 as an example, according to the procedure described in Synthesis, 300-304 (March 1994), a pre-epoxy-amino acid derivative [(3RS, 4S) -4- Amino-2-tert-butyldimethyls
ilyloxymethyl-3-hydroxy-6-methyl-1,6-heptadiene)
(10) and N-terminal modified amino acid derivative [(S) -N- (6-Methy
lheptanoyl) serine] (11), and then the desired compound (SPA-1344) can be synthesized by condensation reaction and epoxidation reaction of these compounds.

[0033]

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A specific synthesis route is described in the aforementioned reference (Synthe
sis) in Scheme 1 and Scheme 2. Further, detailed reaction conditions and the like are described on pages 301 to 303 of the reference, and the reaction may be performed in accordance with these conditions. Further, in order to synthesize the compound represented by the general formula (1) or the general formula (2) other than SPA-1344, a starting material is appropriately selected, and a pre-epoxy resin corresponding to a structure of a target compound is appropriately selected. It can be easily performed by preparing an amino acid derivative and an N-terminal modified amino acid derivative, respectively, and performing the same condensation reaction and epoxidation reaction as described above. The solvent used in each reaction and the reaction conditions such as the reaction temperature and time are not particularly limited, and may be appropriately selected according to the conditions described in the above-mentioned literature (Synthesis) or according to ordinary organic synthesis procedures. Just do it.

Alternatively, it can also be produced by culturing a microorganism capable of producing these compounds and recovering the microorganism from the culture. The following is an example of a method for culturing microorganisms. The microorganism used in the present invention is not particularly limited as long as it has an ability to produce the compound of the general formula (1) or (2), but a microorganism belonging to the genus Streptomyces is exemplified as a preferable microorganism. You. For example, 2- {6-methylheptanoyl-Ser- (3,4-dehydro-Leu)}-1-hydroxy-2,3-epoxypropane and 2- (2-carbamoyl-propionyl-Thr-Thr-Leu)
-1-Hydroxy-2,3-epoxypropane was obtained from Streptomyces sp. SPA-1344 and Streptom, respectively.
yces sp. SPA-1761.
It can be obtained by purification. In this specification, each compound is designated as SPA-1344 and SPA-1761. Each structure is shown below. In addition, SPA-
There are two types of isomers in 1761, which are designated as SPA-1761A and SPA-1761B in this specification, respectively.

[0036]

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[0037]

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The above Streptomyces sp. SPA-1344
And Streptomyces sp. SPA-1761 were isolated from a soil sample collected in Japan, and were sent to the Institute of Biotechnology and Industrial Technology of the Ministry of International Trade and Industry, July 1, 1997.
On day 8, FERMP-16340 and FERM respectively
Deposited as P-16341.

The bacteriological properties of this strain are described below. 1. Streptomyces sp. SPA-1344 (hereinafter abbreviated as SPA-1344 strain) SPA-1344 strain is an actinomycete isolated from a soil sample collected in Hirado City, Nagasaki Prefecture, and its mycological properties are as follows. It is.

(A) Morphological properties The SPA-1344 strain grows well on a medium such as yeast / malt agar medium, starch / inorganic salt agar medium, glycerin / asparagine agar medium, and the underlying mycelium does not form a simple branch. Forms an aerial mycelium. A long chain of spores (consisting of about 20 to 50 spores) is formed on the aerial hyphae, and the spore chain shape is spiral. Spores are 0.4-0.6 μm in diameter,
It has a cylindrical shape with a length of 0.7 to 1.2 μm, and the spore connection part rises like a bamboo node. The spore surface is wrinkled. No special structures such as disruption of the base hyphae or aerial hyphae, sporangia, flagella spores, sclerotia, etc. are observed.

(B) Cultural properties SPA-1344 cultured on various media at 27 ° C. for 3 weeks
The culture characteristics of the strain are as follows.

(1) Yeast / malt agar medium (ISP medium N
o.2) Growth and aerial mycelium formation are good. The aerial mycelium tone is gray. The color of the underlying mycelium is yellow. There is no diffusible dye into the medium.

(2) Oatmeal agar medium (ISP medium No.
3) Growth is good. The aerial hyphae settle, but become mucus clumps as the culture proceeds. The aerial mycelium tone is gray or black. The color of the underlying mycelium is yellow. There is no diffusible dye into the medium.

(3) Starch / inorganic salt agar medium (ISP medium)
No.4) Growth and aerial mycelium formation are good. The aerial mycelium tone is gray. The color of the underlying mycelium is yellow. There is no diffusible dye into the medium.

(4) Glycerin-asparagine agar medium (ISP medium No. 5) The growth and aerial hyphae formation are good. The aerial mycelium tone is gray. The color of the underlying mycelium is yellow. There is no diffusible dye into the medium.

(C) Physiological properties (1) Growth temperature range 14-37 ° C (yeast / malt agar medium, culture for 2 weeks) (2) Production of melanin-like pigment Peptone / yeast / iron agar medium (ISP medium No. 6) Negative Tyrosine agar medium (ISP medium No. 7) Negative (3) Availability of carbon source L-arabinose positive D-fructose positive D-glucose positive Inositol positive D-mannitol positive Raffinose positive L-rhamnose positive Sucrose positive D -Xylose positive

(D) Chemical taxonomic and other properties (1) Analysis of cell wall diamino acids LL-Diaminopimelic acid detected (2) Starch hydrolysis negative

From the above results, it was found that the SPA-1344 strain was
Identified as a strain belonging to the genus reptomyces, Streptomyces sp.
It was named SPA-1344.

[0049] 2. Streptomyces sp. SPA-1761 (hereinafter abbreviated as SPA-1761) SPA-1761 is an actinomycete isolated from a soil sample collected in Kurume City, Fukuoka Prefecture, and its bacteriological properties are as follows. It is. (a) Morphological properties The SPA-1761 strain grows well on a medium such as yeast / malt agar medium, oatmeal agar medium, or starch / inorganic salt agar medium, and the underlying mycelium forms a simple branch to form aerial mycelia. I do. A long chain of spores (composed of about 10 to 50 spores) is formed on aerial hyphae, and the shape of the spore chain is circular or loose spiral. Spores are 0.4-0.7 μm in diameter and length
It is 0.8-1.4 μm elliptical and the spore surface is smooth. No special structures such as disruption of the base hyphae or aerial hyphae, sporangia, flagella spores, sclerotia, etc. are observed.

(B) Cultural properties The cultural properties of the SPA-1761 strain cultured on various media at 27 ° C. for 3 weeks are as follows.

(1) Yeast / malt agar medium (ISP medium N
o.2) Growth and aerial mycelium formation are good. The aerial mycelium tone is gray. The color of the underlying mycelium is brown. The diffusible dye into the medium is brown.

(2) Oatmeal agar medium (ISP medium No.
3) Growth and aerial mycelium formation are good. The aerial mycelium tone is gray. The color of the underlying mycelium is brown. The diffusible dye into the medium is brown.

(3) Starch / inorganic salt agar medium (ISP medium)
No.4) Growth and aerial mycelium formation are good. The aerial mycelium tone is gray. The color of the underlying mycelium is yellow. There is no diffusible dye into the medium.

(4) Glycerin-asparagine agar medium (ISP medium No. 5) The growth and aerial mycelium formation are good. The aerial mycelium tone is gray. The color of the underlying mycelium is brown. The diffusible dye into the medium is brown.

(C) Physiological properties (1) Growth temperature range 11 to 46 ° C. (yeast / malt agar medium, culture for 2 weeks) (2) Production of melanin-like pigment Peptone yeast / iron agar medium (ISP medium No. 6) Positive Tyrosine agar medium (ISP medium No. 7) Negative (3) Use of carbon source L-arabinose positive D-fructose positive D-glucose positive Inositol positive D-mannitol positive Raffinose positive L-rhamnose positive Sucrose negative D-xylose positive

(D) Chemical taxonomic and other properties (1) Analysis of cell wall diamino acids LL-Diaminopimelic acid detected (2) Starch hydrolysis positive

From the above results, the SPA-1761 strain was transformed into St.
Identified as a strain belonging to the genus reptomyces, Streptomyces sp.
It was named SPA-1761.

In the present invention, by culturing a microorganism belonging to the genus Streptomyces as described above, 2-
{6-Methylheptanoyl-Ser- (3,4-dehydro-Leu)}-1-hydroxy-2,3-epoxypropane (SPA-1344) or 2- (2-carbamoyl-propionyl-Thr-Thr- Leu)-
1-hydroxy-2,3-epoxypropane (SPA-
1761) can be produced.

In the production method of the present invention, the culturing of the microorganism is carried out by the usual cultivation and recovery method.
It can be purified. That is, the medium used for culturing the microorganism may be liquid or solid, but usually, shaking culture or aeration and stirring culture using a liquid medium is advantageous. The medium composition is not particularly limited as long as it contains nutrients used by the producing bacterium, and is not particularly limited.Examples of the carbon source include glucose, sucrose, lactose,
Glycerin, starch, dextrin, molasses and the like can be mentioned. As a nitrogen source, for example, peptone, protein hydrolyzate such as casamino acid, meat extract, yeast extract, soybean meal,
Organic nitrogen sources such as cottonseed meal, corn steep liquor and amino acids, and inorganic nitrogen sources such as ammonium salts and nitrates. In addition, inorganic salts such as various phosphates, magnesium nitrate, sodium chloride, and calcium carbonate can be added for osmotic pressure adjustment, pH adjustment, replenishment of trace components, and the like. In order to further promote the growth of bacteria,
Various vitamins, nucleic acid-related compounds and the like may be added to the medium. During the culture period, it is also possible to add an antifoaming agent such as silicone oil, a polypropylene glycol derivative, and soybean oil. The culture temperature is preferably 20 to 3
A temperature in the range of 7 ° C, more preferably in the range of 25 to 30 ° C. The culture period is, for example, 3 to 7 days. The pH of the medium may be, for example, in the vicinity of neutrality.

From the culture, SPA-1344 or SP
In order to collect A-1761, a commonly used isolation means can be used from a culture solution of a metabolite produced by a microorganism.
As a method for isolating from the culture supernatant, a usual isolation method from a culture filtrate, for example, a solvent extraction method, an ion exchange resin method, adsorption or partition chromatography, and gel filtration chromatography can be mentioned. These isolation methods can be performed alone or in combination. In addition, isolation and purification by high performance liquid chromatography (HPLC), thin layer chromatography and the like are also possible. When the target substance is isolated from the cultured cells, it can be directly extracted from the cells collected by means such as filtration or centrifugation using an organic solvent. Examples of the organic solvent used for the extraction include acetone, methanol and the like. The desired product can be obtained from the extract in the same manner as in the isolation and purification method from the culture supernatant. The method for producing SPA-1761 using the SPA-1761 strain is specifically described in Production Example 1 described below and also in SPA-1761.
The method for producing SPA-1344 using the 1344 strain is specifically described in Production Example 2 described later.

The compound thus obtained is useful as a proteasome inhibitor because it has a proteasome inhibitory action, and serves as a therapeutic agent for diseases caused by enhanced proteasome action. In addition, since the proteasome inhibitor of the present invention also has an inhibitory activity on TNF-α, it is considered that the proteasome inhibitor also functions to suppress an autoimmune disease. Examples of various diseases considered to be caused by enhanced action of the proteasome include autoimmune diseases, inflammation, neuronal degenerative diseases, and more specifically, autoimmune diseases such as rheumatoid arthritis, and inflammatory diseases. Enteritis, asthma, Alzheimer's disease and the like.

Accordingly, the present invention provides a therapeutic agent for an autoimmune disease comprising the compound of the present invention as an active ingredient. As the therapeutic agent for an autoimmune disease, a therapeutic agent for rheumatoid arthritis is preferable. The present invention also provides a therapeutic agent for inflammatory bowel disease, a therapeutic agent for asthma, and a therapeutic agent for Alzheimer's disease, comprising the compound of the present invention as an active ingredient.

The efficacy of the therapeutic agent of the present invention in autoimmune diseases, inflammation, neuronal degenerative diseases and the like can be confirmed not only by its proteasome inhibitory activity but also by its inhibitory activity on TNF-α. The method for measuring such inhibitory activity is as follows:
This is described in Examples 1 and 2.

The therapeutic agents of the present invention, such as therapeutic agents for autoimmune diseases such as rheumatoid arthritis disease, therapeutic agents for inflammatory bowel disease, asthma and Alzheimer's disease, can be used orally or therapeutically for such diseases. It can be administered parenterally. That is, it can be orally administered as a commonly used dosage form, for example, tablets, capsules, granules, powders and the like, or can be injected intramuscularly or subcutaneously as a liquid, emulsion, suspension, liposome and the like. ,
It can also be administered rectally as a suppository. Such dosage forms include the usual pharmaceutically acceptable carriers, excipients,
It can be produced by blending the active ingredient of the present invention with a binder, a stabilizer, a buffer, a solubilizing agent, an isotonic agent and the like.

The dose and the number of times of administration depend on the patient's symptoms, medical history,
Although it varies depending on the age, body weight, administration form, etc., for example, when orally administered to an adult, it is usually 5 to 500 mg per day,
Preferably, it can be administered in one or several doses, suitably adjusted in the range of 10 to 100 mg.

[0066]

EXAMPLES The present invention will be described in more detail with reference to Production Examples and Examples, which should not be construed as limiting the present invention.

Production Example 1 Production of 2- (2-carbamoyl-propionyl-L-threonyl-L-threonyl-L-leucyl) -1-hydroxy-2,3-epoxypropane (SPA-1761A and B) (1) SPA-1761 strain was prepared using glucose 2%, sucrose 5%, cottonseed meal 2%, sodium nitrate 0.1%, dipotassium phosphate 0.05%, potassium chloride 0.07%,
Two 0.5-liter Sakaguchi flasks containing 100 ml of a liquid culture medium having a composition of L-histidine 0.1% and magnesium sulfate heptahydrate 0.0014% were inoculated into two flasks.
Seed mothers were prepared by shaking culture at 7 ° C for 5 days. Next, glucose 2%, sucrose 5%, cottonseed meal 2%, sodium nitrate 0.1%, dipotassium phosphate 0.05%, potassium chloride 0.07%, L-histidine 0.1%, magnesium sulfate. 100 ml of each of the above seeds was inoculated into a 5-liter tank containing 3 liters of a liquid medium having a composition of heptahydrate of 0.0014%, and inoculated at a flow rate of 1.5 liter / min with stirring at 600 rpm at 27 ° C. The cells were cultured with aeration and stirring for days.

(2) 6 liters of the obtained culture solution was placed at 4 ° C.
4. High-speed centrifugation at 8000 rpm for 5 minutes,
5 liters were obtained. 5.5 L of the supernatant was passed through a column filled with 0.5 L of Diaion HP-20 (manufactured by Mitsubishi Chemical Corporation) and adsorbed. An aqueous methanol solution was used as an eluent, and the methanol concentration was increased stepwise for elution. The fraction eluted with 60% methanol was collected, the eluate was concentrated under reduced pressure to 1/2 volume, and extracted with 1.5 liter of n-butanol. This n-butanol extract was concentrated under reduced pressure to obtain 2.6 g of a yellow powder.

This powder was dissolved in 25 ml of methanol, adsorbed on a column packed with 1 liter of a reversed-phase ODS (manufactured by Nacalai Tesque) equilibrated with 10% methanol, and adsorbed on 4 liters of 10% methanol and 25% methanol. Elution was performed sequentially with 4 liters. The obtained fraction eluted with 25% methanol was concentrated under reduced pressure to obtain 0.6 g of a yellow powder. This powder was dissolved in a small amount of methanol, and passed through a reverse phase high performance liquid chromatography ODS column (manufactured by Water Corporation, diameter 50 mm, length 300 mm) equilibrated with 20% methanol. The methanol concentration was increased in the min. The proteasome inhibiting activity fractions were collected and concentrated under reduced pressure to obtain 70 mg of a yellow powder. This powder was dissolved in a small amount of methanol, and passed through a reverse phase high performance liquid chromatography ODS column (Yamamura Chemical Co., Ltd., diameter 20 mm, length 250 mm) equilibrated with 25% methanol. / Min to elute with increasing methanol concentration. Similarly, proteasome inhibitory fractions were collected and concentrated under reduced pressure to obtain 20 mg of a white powder. This powder was dissolved in a small amount of methanol and equilibrated with 10% acetonitrile.
DS column (Yamamura Chemical Co., Ltd., diameter 20 mm, length 250
mm). Elution was performed after increasing the acetonitrile concentration from 10% to 40% after 20 minutes at a flow rate of 10 ml / min. The peak at 31.2 min showing the proteasome inhibitory activity was collected and concentrated under reduced pressure to obtain a white powder 6 m
g (SPA-1761A) was obtained. The peak at 33.0 min was collected, concentrated under reduced pressure, and then 6 mg of white powder (SPA-1)
761B).

The proteasome inhibitory activity fraction in this experiment was obtained by detecting the proteasome inhibitory activity by the method described in Example 1.

The structural analysis data of SPA-1761A obtained is shown below. (1) Mass spectrometry value: positive ion FABMS spectrum: m / z 489 (M +
H) ⁺ anion FABMS spectrum: m / z 487 (M-
H) ^- (2) High-resolution FABMS: (M + H) ⁺ m / z Observed: 489.2613 Calculated as C ₂₁ H ₃₇ N ₄ O ₉ : 489.2561 (3) [α] _D ²⁰ + 4.3 ° (C = 0.03, methanol solution) (4) Ultraviolet absorption spectrum: In the methanol solution, only terminal absorption was observed. (5) Infrared absorption spectrum: FIG. 1 shows a spectrum measured with a potassium bromide tablet. (6) ¹ H-NMR spectrum: FIG. 2 shows a spectrum measured at 500 MHz in deuterated dimethyl sulfoxide (d ₆ -DMSO). (7) ¹³ C-NMR spectrum: FIG. 3 shows a spectrum measured at 125 MHz in deuterated dimethyl sulfoxide (d ₆ -DMSO).

The structural analysis data of SPA-1761B obtained is shown below. (1) Mass spectrometry value: positive ion FABMS spectrum: m / z 489 (M +
H) ⁺ anion FABMS spectrum: m / z 487 (M-
^H) - (2) [α] ^{_{D 20 + 3.8 ° (c =}} 0.03, methanol) (3) Ultraviolet absorption spectrum: methanol solution, was observed only end absorption. (4) ¹ H-NMR spectrum: FIG. 4 shows a spectrum measured at 500 MHz in deuterated dimethyl sulfoxide (d ₆ -DMSO). (5) ¹³ C-NMR spectrum: FIG. 5 shows a spectrum measured at 125 MHz in deuterated dimethyl sulfoxide (d ₆ -DMSO).

As a result of the structural analysis study, SPA-1761
A, SPA-1761B was found to be a structural isomer.

Production Example 2 2- {6-methylheptanoyl-L-seryl- (3,4
-Dehydro-L-leucyl)}-1-hydroxy-2,
Production of 3-epoxypropane (SPA-1344) (1) SPA-1344 strain was prepared from glucose 2%, sucrose 5%, cottonseed meal 2%, sodium nitrate 0.1%, dipotassium phosphate 0.05%, 0.07% potassium chloride,
Inoculate 30 2 liter Sakaguchi flasks containing 300 ml of a liquid medium having a composition of 0.1% L-histidine and 0.0014% magnesium sulfate heptahydrate.
The cells were cultured with shaking at 7 ° C. for 7 days.

(2) 9 liters of the obtained culture solution was placed at 4 ° C.
High-speed centrifugation was performed at 8,000 rpm for 5 minutes to obtain 8 liters of the culture supernatant. The supernatant was extracted with 8 liters of ethyl acetate. The ethyl acetate extract was concentrated under reduced pressure to obtain 30 g of a yellow oil. This oily substance was dissolved in 25 ml of acetone, and adsorbed on a column packed with 1 liter of silica gel (manufactured by Merck) equilibrated with a mixed solvent of acetone-n-hexane (25:75). (25:75, 2 liters, 40:60, 2 liters and 60:40, 4 liters). The obtained fraction eluted with acetone-n-hexane (60:40) was concentrated under reduced pressure to obtain 3 g of a yellow oil. This oil was dissolved in 10 ml of chloroform and adsorbed on a column packed with 300 ml of silica gel (manufactured by Merck) equilibrated with a mixed solvent of chloroform-methanol (40: 1). (0.5 liter, 20: 1, 1 liter). The obtained chloroform-methanol (20:
1) The eluted fraction was concentrated under reduced pressure to obtain 1.5 g of a yellow oil. This oily substance was dissolved in a small amount of dimethyl sulfoxide and equilibrated with 30% acetonitrile.
(mm, length 300 mm).
Elution was carried out with increasing acetonitrile concentration at 0 ml / min. Collect the proteasome inhibitory activity fraction, concentrate under reduced pressure,
30 mg of a yellow powder were obtained. This powder was dissolved in a small amount of dimethyl sulfoxide, and passed through a reverse-phase high-performance liquid chromatography ODS column (Yamamura Chemical Co., Ltd., diameter 20 mm, length 250 mm) equilibrated with 40% acetonitrile. The acetonitrile concentration was increased at a flow rate of 10 ml / min for elution. The proteasome inhibitory fractions were collected and concentrated under reduced pressure to obtain 5 mg of a white powder. This powder was dissolved in a small amount of dimethyl sulfoxide, and equilibrated with 35% acetonitrile. An ODS column for reversed-phase high performance liquid chromatography (Yamamura Chemical Co., Ltd., 4.6 mm in diameter, 250 mm in length)
Then, the mixture was eluted with 35% acetonitrile at a flow rate of 1 ml / min. The proteasome inhibitory fractions were collected and concentrated under reduced pressure to obtain 1.2 mg of white powder (SPA-13).
44) was obtained.

The proteasome inhibitory activity fraction in this experiment was obtained by detecting the proteasome inhibitory activity by the method described in Example 1.

The structural analysis data of SPA-1344 obtained is shown below. (1) Mass spectrometry value: positive ion FABMS spectrum: m / z 399 (M +
H) ⁺ anion FABMS spectrum: m / z 397 (M-z
H) ^- (2) ¹ H-NMR spectrum FIG. 6 shows a spectrum measured at 500 MHz in heavy acetone (d ₆ -acetone). (3) ¹³ C-NMR spectrum FIG. 7 shows a spectrum measured at 125 MHz in heavy acetone (d ₆ -acetone).

SPA-1344 is the result of a structural analysis study, Journal of Antibiotics, 43
Vol. 8, pp. 18-18 (1990), which is the same compound as the antitumor peptide (eponemycin).

Example 1 Proteasome Inhibitory Activity of SPA-1344 and SPA-1761B Proteasome can be obtained from rat brain tissue by a known method (Kojima et al., FEBS, Vol. 304, pp. 57-60, 1992).
Was used. Specifically, a 5-fold amount of 0.1 M HEPES buffer (pH 7.25) was added to rat brain tissue, and the homogenized extraction solution was ice-cooled, and the solution was subjected to high-speed centrifugation at 40,000 G for 30 minutes under cooling.
The supernatant was obtained as a crude enzyme solution. Enzyme crude purified solution 130m
was passed through a column packed with an anion exchange resin EMD-DEAE equilibrated with 20 mM Tris-HCl buffer (pH 7.5) to adsorb the proteasome. The proteasome adsorbed on the resin was eluted by increasing the concentration of sodium chloride. The proteasome was eluted at a sodium chloride concentration of about 0.5M. Suc-Leu-Leu as a substrate for measuring proteasome inhibitory activity
-Val-Tyr-MCA (manufactured by Peptide Research Laboratories) was used.

As a test compound, S obtained in Production Example 1 was used.
PA-1761B and SPA-1 obtained in Production Example 2
344 was used. Each compound dissolved in 1 μl of DMSO was added to a 0.1 M HEPES buffer (pH 7.25).
20 μM Suc-Leu-Leu-Val prepared in
After adding 50 μl of the Tyr-MCA solution, 50 μl of the proteasome solution was added, and the mixture was incubated at 37 ° C. for 2 hours. The fluorescence intensity of the released aminomethyl coumarin is
It was measured at x 355 nm and Em 460 nm. The amount of the compound that inhibits the activity of the proteasome by 50% (IC ₅₀ ) was calculated based on the fluorescence intensity without adding the test compound as 100. Table 1 shows the results.

[0081]

[Table 1]

Example 2 TNF-α Inhibitory Activity of SPA-1344 Using Human Vascular Endothelial Cell HUVEC Cells (Kurabo Co., Ltd.), E-Selectin Expression Level Expressed When Treated with TNF-α was Measured Assayed TNF-α inhibitory activity.

The HUVEC cells were detached from the flask by trypsin treatment, and the detached cells were diluted to about 40,000 cells / ml with a low serum liquid medium for human vascular endothelial cell growth (Kurabo). The cell diluent was spread into a 96-well culture plate for tissue culture (manufactured by Falcon) at 100 μl / well, and SPA-
The solution of 1344 was added to a final concentration of 0 to 20 μg / ml and placed in a CO ₂ incubator (5% CO ₂ ).
At 37 ° C. One hour later, recombinant human T
NF-α (manufactured by Gibco) was added to a concentration of 30 pg / ml and cultured for 4 hours, and the cells were fixed with a 0.1% glutaraldehyde solution. PB with 0.05% Tween20
After washing once with S (-) (hereinafter abbreviated as T-PBS),
E-selectin antibody (manufactured by Serotec) diluted 5000-fold with BS (-) was added at 50 μl / well, and the reaction was carried out at 37 ° C. for 1.5 hours. After the reaction, the plate was washed once with T-PBS, and then washed with PBS (-).
Labeled with anti-mouse IgG antibody (Kirkegaard & Perry Laboratories) diluted 1000 times with
Was added at 50 μl / well, and the reaction was carried out at 37 ° C. for 1 hour. After the reaction, the plate was washed twice with T-PBS, and 100 µl of 3,3 ′, 5,5′-tetramethylbenzidine solution (manufactured by Kirkegaard & Perry Laboratories) was added.
1 / well and allowed to react for 10 minutes in the dark, after which 1M phosphoric acid solution was added to 100 μl / well to stop the reaction. Each absorbance was measured at a wavelength of 570 nm using a microplate reader. The TNF-α inhibitory activity was defined as the expression amount of E-selectin when the test compound was not added as 100.
The amount of compound that inhibits the activity of TNF-α by 50% (I
_C50 ) was calculated. IC ₅₀ of the SPA-1344 0.3
μg / ml.

[0084]

Industrial Applicability The present invention provides a proteasome inhibitor useful for treating autoimmune diseases, inflammatory bowel diseases, asthma, Alzheimer's disease and the like.

[Brief description of the drawings]

FIG. 1 is a diagram showing an infrared absorption spectrum of SPA-1761A.

FIG. 2 is a chart showing a ¹ H-NMR spectrum of SPA-1761A.

FIG. 3 is a diagram showing a ¹³ C-NMR spectrum of SPA-1761A.

FIG. 4 is a chart showing ¹ H-NMR spectrum of SPA-1761B.

FIG. 5 is a chart showing ¹³ C-NMR spectrum of SPA-1761B.

FIG. 6 is a chart showing a ¹ H-NMR spectrum of SPA-1344.

FIG. 7 is a chart showing a ¹³ C-NMR spectrum of SPA-1344.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 38/55 ACJ A61K 37/64 ACD AED ACJ C07K 5/083 AED (72) Inventor Kazuo Kumagai 4-chome Takashi Takarazuka-shi, Hyogo No. 1 Sumitomo Pharmaceutical Co., Ltd. (72) Inventor Yoshio Hosoya 4-2-1 Takashi Takarazuka-shi, Hyogo Sumitomo Pharmaceutical Co., Ltd.

Claims (9)

[Claims] 1. A compound of the general formula (1) (Wherein R ¹ is a substituted or unsubstituted acyl group, an amino acid residue having an amino group acylated with a substituted or unsubstituted acyl group, or an amino acid in which the amino group may be protected with a protecting group. R ² represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; R ³ and R ^{4 each} represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; or R ³ and R ⁴ Together represent a methylene group). 2. In the general formula (1), R ¹ is a substituted or unsubstituted acyl group, R ² is a hydrogen atom,
The proteasome inhibitor according to claim 1, wherein R ³ and R ⁴ together represent a methylene group. 3. In the general formula (1), R ¹ is an amino acid residue having an amino group acylated by a substituted or unsubstituted acyl group, or an amino acid residue wherein the amino group may be protected by a protecting group. R ² is a methyl group;
R ³ and R ⁴ is an alkyl group having 1 to 6 hydrogen atoms or carbon atoms, the proteasome inhibitor of claim 1. 4. A therapeutic agent for a disease caused by enhanced proteasome action, comprising a compound represented by the general formula (1) according to claim 1 as an active ingredient. 5. The therapeutic agent according to claim 4, wherein the disease is selected from the group consisting of an autoimmune disease, an inflammatory bowel disease, asthma, and Alzheimer's disease. 6. The therapeutic agent according to claim 5, wherein the autoimmune disease is rheumatoid arthritis. 7. A compound of the general formula (2) (Wherein, R ¹¹ is an amino acid residue with an acylated amino group with a substituted or unsubstituted acyl group, or an amino group represents an amino acid residue which may be protected by a protecting group, R ¹² is R ¹³ and R ¹⁴ represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, or R ¹³ and R ¹⁴ represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, or R ¹³ and R ¹⁴ are taken together to form a methylene group The compound represented by the following. 8. In the formula (2), R ¹¹ is an amino acid residue having an amino group acylated by a substituted or unsubstituted acyl group, or an amino acid residue in which the amino group may be protected by a protecting group. R ¹² is a methyl group;
R ¹³ and R ¹⁴ is an alkyl group having 1 to 6 hydrogen atoms or carbon atoms, claim 7 compound according. 9. 2- (2-carbamoyl-propionyl-Thr-Thr-Leu) -1-hydroxy-2,3
-Epoxy propane.