JPH1129595A - New proteasome inhibitory substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new substance having proteasome inhibitory activity, and useful for preventing/treating chronic arthrorheumatism, by culturing microorganisms having the ability to produce the new compound TMC-95A and belonging to the genus Apiospora or Arthrinium. SOLUTION: This substance, which is a new compound TMC-95A (salt) of the formula, has proteasome inhibitory activity, and is useful as a proteasome inhibitor or e.g. an active ingredient of pharmaceutical compositions such as preventive and/or therapeutic agents for chronic arthrorheumatism. This new substance is obtained by culturing microorganisms having the ability to produce the compound TMC-95A and belonging to the genus Apiospora or Arthrinium [e.g. Apiospora montagnei Sacc. F2067 strain (FERM P-16153)] and collecting the product from the resulting cultured product or a treated product thereof.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel compound TMC-95A having proteasome inhibitory activity or a pharmaceutically acceptable salt thereof, a process for producing the same, and a pharmaceutical composition containing the same as an active ingredient.

[0002]

2. Description of the Related Art The 20S proteasome is a giant protein complex having a molecular weight of about 700,000 composed of about 15 subunits each having a molecular weight of 20,000 to 30,000. The 20S proteasome exhibits three types of protease activities, and is called chymotrypsin-like activity, trypsin-like activity or peptidylglutamyl peptide hydrolyzing activity depending on whether the C-terminal side of the cleavage site is a hydrophobic, basic or acidic amino acid. (Nature [35]
7, 375-379, 1992); European Journal of Biochemistry [Europe
ean Journal of Biochemistr
y], Vol. 203, pp. 9-23, 1992; Biochemistry, Vol. 29,
10289-10297, 1990; Archives of Biochemistry and Biophysics [Archives of Biochemistry]
yand Biophysics], Vol.
8, 1989; Journal of Biological Chemistry [Journal of Biology
Ial Chemistry], Vol. 264, 122
15-12219, 1989). The association of the regulatory subunit complex with both ends of the 20S proteasome forms a characteristic dumbbell-shaped 26S proteasome. The 26S proteasome degrades ubiquitinated intracellular proteins into peptides and amino acids in an ATP-dependent manner (Annual Review of Biochemistry [Annual Review of Biochemistry].
chemistry], Vol. 61, pp. 761-807,
1992; European Journal of Biochemistry [European Journal of Biochemistry]
Biochemistry], Volume 203, 9-23
Page 1992; Nature, 35
7, 375-379, 1992). Ubiquitin-
The proteasome degradation system is a system that recognizes and degrades a target protein very quickly, and is usually involved in the degradation of a protein having a short half-life.

[0003] The ubiquitin-proteasome system is involved in the activation of the inflammatory transcription factor NF-κB. That is, NF-κB is its inhibitor, I-
Although it exists as an inactive form by binding to κB,
I-κB is degraded by the stimulation of cytokines such as -α, and NF-κB translocates into the nucleus and promotes the transcription of various genes. Here, it is known that the degradation of I-κB is caused by the proteasome (international publication number: WO95 / 2).
5533). In addition, the proteasome is involved in the formation of NF-κB. That is, NF-κB is composed of two subunits, p65 and p50,
50 is formed by processing from p105. It is known that the proteasome is involved in this processing (International Publication Number: WO9
5/25533). Therefore, proteasome inhibitors are promising as therapeutic agents for inflammatory / immune diseases such as rheumatoid arthritis, in which NF-κB is involved in pathogenesis.

The ubiquitin-proteasome system is an MHC
-I is involved in antigen presentation (International publication number: WO94
/ 17816). That is, in antigen presenting cells, the proteasome degrades the antigen protein into an antigen peptide that binds to MHC-I. Therefore, proteasome inhibitors may be effective therapeutics for autoimmune diseases and rejection in tissue transplantation.

The ubiquitin-proteasome system is involved in the degradation of cyclins, which play an important role in cell cycle control (Nature, 34).
9, 132-138, 1991). Therefore,
Proteasome inhibitors have a function of arresting the cell cycle, and are expected to be useful for treating diseases associated with cell proliferation, such as cancer, psoriasis, and restenosis.

[0006] The ubiquitin-proteasome system is involved in the degradation of the tumor suppressor protein p53 (Cell).
75, 495-505, 1993). When cells are treated with drugs or radiation that damage DNA, p53
Is induced, and the cell cycle is stopped in the G1 phase. Many of the cancer cells express mutant p53, and the above-described treatment does not cause cell cycle arrest, and cancer cells are easily damaged. Administering the proteasome inhibitor systemically,
If the p53 content of normal cells is increased, it is expected that the proliferation of normal cells can be stopped, and that the side effects of anticancer drugs can be reduced.

[0007] It is known that the enhancement of proteolysis associated with fasting, denervation, steroid therapy and febrile infection is due to non-lysosomal ATP-dependent proteolysis, that is, proteolytic activity (US Patent No. : No. 5693617). Therefore, the proteasome inhibitor is expected to be useful for alleviating the symptoms based on the enhanced proteolysis.

[0008] A variety of proteasome inhibitors have been reported (Nature,
357, 375-379, 1992; Biochemistry, Volume 29,
10289-10297, 1990; Archives of Biochemistry and Biophysics [Archives of Biochemistry]
y and Biophysics], Vol. 268, 1
-8, 1989; Journal of Biological Chemistry [Journal of Biolo
chemical Chemistry], Vol. 264, 12
215-12219, 1989; European Journal of Biochemistry [Europea
n Journal of Biochemistr
y], 203, 9-23, 1992; U.S. Pat. No. 5,693,617; International Publication No .: WO95 /
25533; International publication number: WO94 / 17816; International publication number: WO96 / 13266; International publication number: W
O96 / 32105). However, there are few proteasome-specific inhibitors.

[0009]

DISCLOSURE OF THE INVENTION The present invention relates to a compound having excellent proteasome inhibitory activity or a pharmaceutically acceptable salt, a method for producing the same, and prevention and / or treatment of a disease involving the proteasome as an active ingredient thereof. Provide the agent.

[0010]

Means for Solving the Problems The present inventors examined a culture of microorganisms mainly isolated from soil, and found that a compound having a proteasome inhibitory activity was produced in a culture of microorganisms of the genus Apiospora. Found to be. Further, the present compound was collected from the culture solution, and its physicochemical properties were examined to determine the chemical structure. As a result, this compound was found to be a novel compound, and was therefore named TMC-95A.

The present invention provides a compound of the formula (I):

[0012]

Embedded image

The present invention provides a compound TMC-95A or a pharmaceutically acceptable salt thereof.

Further, the present invention provides a method for culturing a microorganism belonging to the genus Apiospora or the genus Arthrinium having the ability to produce the compound TMC-95A, and culturing the microorganism from the culture or a processed product of the culture to obtain the compound TMC-95A. It is intended to provide a method for producing a compound TMC-95A, which comprises collecting 95A.

Further, the present invention relates to a compound TMC-95A
Or a pharmaceutical composition comprising a pharmacologically acceptable salt thereof as an active ingredient.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION The compound TMC-95A of the present invention and a pharmaceutically acceptable salt thereof can be used as a proteasome inhibitor because they selectively inhibit the proteasome at a low concentration. The present invention is useful as a prophylactic / therapeutic agent for diseases related to the disease, in particular, a prophylactic and / or therapeutic agent for rheumatoid arthritis.

In the present invention, the microorganism used for producing the compound TMC-95A is, for example, a genus Apiospora or Arthurinium (Art).
(Hrinium) and any compound capable of producing the compound TMC-95A. An example of such a microorganism is F2067 strain isolated from soil. The bacteriological properties of this strain are as follows.

The number of colonies on Miura medium (LCA) is 25.
It spreads thinly in 7 days at 70 ° C. and reaches 70 mm. Irradiation with light improves conidial colonization and improves olive tea (Tawny
Olive, brownish olive 5Y5
/ 4). The back is almost colorless or light brown. No soluble dye is produced.

The growth of colonies on oatmeal agar (OA) is vigorous, reaching 70 mm within 7 days at 25 ° C. Hyphae have a fluffy or rope-like shape, and when conidia are formed, they become gray (Battleship Gray, mediu).
m gray N6 / 0). The color of the back is Oyster (yellowish gray 5)
Y7 / 2).

Growth on malt extract agar (ME) is OA
Almost as above, filamentous hyphae are prominent.

The growth pH is 3 to 11, and the optimum pH is 4 to 9.
Met.

The growth temperature range was 4 ° C to 32 ° C, and the optimum temperature was 15 ° C to 25 ° C.

The conidiophore mother cells are flask-shaped, 5-9.5
× 2.5-4.0 μm, conidiophores with width 1-2 μm protrude from the tip. The conidium is bassoic and grows densely on the side of the conidiophore. It typically has a convex lens-shaped, dark brown, light-colored germination zone on the equatorial plane. 6.0-7.5 μm diameter
(Average 6.5 to 7.0 μm, standard deviation 0.42), and the thickness of the side surface is 4.5 to 5.5 μm. Elliptical,
There are many spindle-shaped and club-shaped conidia, and 7.0-13.
It becomes 5 × 4.5 to 6.5 μm.

The conidia of this bacterium had a dark convex lens shape and had a light-colored germination hole on the equatorial plane, so they could be easily distinguished from other genera, and were determined to belong to the genus Arthrinium. About 20 species of this genus have been described so far, and they are classified according to the shape, size, conidia morphology, etc. of conidia (by Ellis, DeMatiaceias hyphomycetes [MB Ellis, Dematiaceou].
s Hypomycetes] 1971). F20
In the 67 strain, the conidium is in the form of a convex lens, so that the conidium is Arthrinium.
euphorbiae); Saccharic Cola (As
accharicola), A.I. A. phaeospermum, A. A. sacchari, Arisulinium State of Apiospora Montagne (Arthrin)
ium state of Apispora mo
ntagnei). Of these, A. A. euphorbiae
Has a conidium size as small as 4.0 to 5.5 μm in diameter,
A. Saccharicola (A. saccharicola)
And A. Phaeospermum (A. phaeospermu)
m) was different from the present bacterium in that the diameter was as large as 9 to 10 μm.
A. A. sacchari has a number of brown septa in the conidiophore. On the other hand, Apiospora montagne
In (i), the conidial stalk was as thin as 0.5 μm, but coincided with the present bacterium in important points such as the size of the conidium and the partition walls of the conidial stalk were colorless.

From the above characteristics, the F2067 strain was transformed into Apiospora montagne sac (Apisporam).
ontagnei Sacc. ). The genus Apiospora is a sexual generation (teleom) of the genus Arthrinium.
orph). This strain was deposited on March 26, 1997 with the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology, under the accession number 16153.
No. (FERM P-16153).

The F2067 strain is a sexual generation (teleomo).
rph) is the ascomycetes Apiospor
a) Since the genus, the anamorph, belongs to the genus Imperfect Fungus Arthrinium, any strain of either genus has the compound TMC-95.
A may be produced. Further, the F2067 strain has the property of being easily mutated, like general filamentous fungi. For example, the F2067 strain or a mutant strain derived therefrom, a transgenic strain,
Any strain that produces the compound TMC-95A, even if it is a recombinant, can be used in the present invention.

In order to produce the compound TMC-95A according to the present invention, a microorganism belonging to the genus Apiospora or the genus Arthrinium having the ability to produce the compound TMC-95A is cultured, and its culture or its culture is cultured. From compound TM
What is necessary is just to collect C-95A. Culture was performed using the compound TMC-
It can be carried out by inoculating a nutrient source-containing medium with a 95A-producing bacterium and growing it aerobically. The medium contains the compound TMC
For the purpose of producing -95A, a liquid medium is preferred. In this case, the pH of the medium is 4-8, especially 5-7.
It is preferable to set the degree. Desired compound TMC-95A
Is produced in culture.

As the culture in the present invention, for example,
Examples include a culture solution or viable cells of the microorganism,
Examples of the processed product of the culture include a processed product of a culture solution (such as a culture supernatant) and a processed product of cells.

The nutrient source may be any one that can be used by the compound TMC-95A producing bacterium and is useful for the production of the compound TMC-95A. It is possible to use carbon sources and nitrogen sources that can be used as nutrient sources for microorganisms. it can. Examples of such nutrients include, for example, peptone, meat extract, corn steep liquor, cottonseed flour, peanut flour, soy flour, yeast extract, NZ-amine, casein hydrolyzate, nitrate or ammonium nitrogen And carbohydrates such as starch, glycerin, sucrose, glucose, galactose, mannose and molasses, and carbon sources such as fats. In addition, salts or inorganic salts containing metal ions may be appropriately added. The culturing temperature is not particularly limited as long as the producing bacteria grow and a desired substance is produced, but the culturing can be suitably carried out usually at 20 ° C to 35 ° C.

The compound TMC-95A can be collected from the culture by a method usually used for collecting a physiologically active substance from the culture. For example, the culture supernatant from which the cells have been removed can be subjected to silica gel chromatography, ion exchange. The treatment can be performed by a chromatographic method such as chromatography, partition chromatography, reverse phase chromatography, gel filtration chromatography, or a combination thereof.

The compounds of the present invention can be used for pharmaceutical use in free form or in the form of a pharmaceutically acceptable salt. Such pharmacologically acceptable salts may be any conventional non-toxic salts, for example, inorganic acid salts such as hydrochloride, hydrobromide, sulfate or phosphate, formic acid Organic salts such as salts, acetates, trifluoroacetates, oxalates, maleates, fumarates, tartrates, metasulfonates, benzenesulfonates or toluenesulfonates, sodium salts, potassium salts And alkaline earth metal salts such as calcium salts and salts with amino acids such as arginine salts, aspartates and glutamates.

The compound TMC-95A of the present invention and its pharmacologically acceptable salts are understood to include any of inner salts, adducts, solvates and hydrates thereof. It should be.

Further, the compound TMC-95A has an asymmetric carbon in its skeleton, and thus may exist as isomers. However, each of these isomers, a mixture thereof, and a pharmaceutically acceptable salt thereof are also included. Included in the present invention.

When a pharmaceutical composition containing the compound TMC-95A of the present invention and a pharmaceutically acceptable salt thereof as an active ingredient is used as a drug, an aerosol, tablet, pill, powder, granule, troche, Oral or parenteral in the form of conventional pharmaceutical preparations such as solutions, suspensions, emulsions, capsules, microcapsules, suppositories, injections, ointments, syrups, cataplasms, liniments, lotions and the like ( Intravenous,
Intramuscular, intradermal, subcutaneous, intraperitoneal or rectal).

[0035] Additives for these drugs may be used as long as they do not impair the therapeutic effect of each drug and are harmless in the dose of the drug. Any conventional additives can be used. For example, stabilizers, buffers, flavoring agents, suspending agents, emulsifiers, fragrances, preservatives, solubilizers, excipients, coloring agents, binders, disintegrants, sweeteners, thickeners, wetting Agents, solvents and the like can be used.

The amount of the active ingredient in the pharmaceutical preparation may be an amount sufficient to produce a desired therapeutic effect, and is, for example, 0.01 mg / kg to 100 mg / kg orally or parenterally, preferably 0.1 mg / kg or less. / Kg to 30 mg / kg.

[0037]

EXAMPLES The present invention will be specifically described with reference to the following experimental examples and examples, but the present invention is not limited to these examples.

Experimental Example 1 (Proteasome Inhibitory Activity of Compound TMC-95A) Proteasome was purified from human monocyte-derived THP-1 cells. Purification of the proteasome and measurement of its activity were performed according to the Biochemistry Experiment Method 30 Protease I (Society Press Center, 170
-223, 1993) and Meyer [Meyer,
S. ], Etc. (FEBS Letters [FEBS Le
ters], Vol. 413, pp. 354-358, 199
7 years).

That is, 10 μl of an aminomethylcoumarin (hereinafter referred to as AMC) substrate (2 mM, dimethyl sulfoxide solution) and 0.2% SDS were placed in a 96-well plate.
20 μl of the sample solution and 20 μl of the sample solution were added thereto, and the purified proteasome (1 to 5 pmole of A per minute) was added thereto.
100 mM Tris- containing the amount of enzyme that releases MC)
HCl (pH 8.0) and 150 μl of a 1 mM dithiothreitol solution were added, and the reaction was started at 37 ° C. The reaction rate from 5 minutes to 15 minutes after the start of the reaction was determined by measuring the released AMC over time with a fluorometer. As a substrate, Suc-Leu-Leu-Val-Tyr-A was used for measurement of chymotrypsin-like activity.
MC was used to measure trypsin-like activity using Boc-Leu-
Arg-Arg-AMC and Z-Leu-Leu-Gl were used for measuring peptidylglutamyl peptide hydrolysis activity.
u-AMC was used respectively.

The compound TMC-95A inhibited the chymotrypsin-like activity, trypsin-like activity and peptidylglutamyl peptide hydrolysis activity of the proteasome in a concentration-dependent manner, and their IC50 values were 7.6 nM, 160 nM and 95 nM, respectively.

As a result of examining the action of compound TMC-95A on trypsin, cathepsin L, m-calpain and granzyme B, it was found that the compound did not inhibit these proteases and was a highly specific compound.

Experimental Example 2 (Therapeutic effect of compound TMC-95A on rat adjuvant-induced arthritis) Heat-killed M. tuberculosis (M. tuberculosis) suspended in liquid paraffin at the ridge of Lewis rats (6 weeks old, female) M. tuberculosis] H37Ra [Di
fco]) to induce arthritis by administering 1.1 mg / kg. Compound TMC-95A was dissolved in physiological saline, and administered subcutaneously once a day for 21 days from the day of heat-killed Mycobacterium tuberculosis administration to the end of the test. The single dose was 0.2 mg / kg.

FIG. 1 shows the degree of swelling of the footpad. The volume of the footpad was measured using the foot volume measurement device Plety.
It was measured using a smometer (Unicom).
As is clear from FIG. 1, the compound TMC-95A suppressed the onset and progress of adjuvant-induced arthritis.

From the above results, the compound TMC-95A
Shows that proteasomes such as rheumatoid arthritis have a preventive / therapeutic effect on diseases associated with pathogenesis.

Example 1 Apiospora Montagne F2067 (Apios
pora montagnei F2067; FERM
P-16153) conidia in 70 ml of seed medium (1% glucose, 2.5% lactose, 1% defatted wheat germ, 0.5% corn steep liquor, 0.5% sodium chloride, 0.4% calcium carbonate) %) Was inoculated into a 500 ml Erlenmeyer flask and cultured on a rotary shaker at 27 ° C. and 220 rpm for 5 days. The obtained seed culture was inoculated into 50 500 ml Erlenmeyer flasks each containing 70 ml of the main culture medium having the same composition as the seed medium at 27 ° C. and 220 rpm.
m. After 5 days of culture, compound T
A culture solution containing MC-95A was obtained.

Example 2 A seed culture prepared in the same manner as in Example 1 was used as a seed for jar culture. The jar culture is performed using the main culture medium 1
8L (glucose 1.0%, dextrin 5.0%, defatted wheat germ 1.0%, corn steep liquor 0.5%,
180 ml of a seed mother was inoculated into a 30 L jar fermenter containing 0.5% of sodium chloride, 0.4% of calcium carbonate, and 0.1% of an antifoaming agent [CC-438, manufactured by NOF CORPORATION]. ° C, aeration rate 9 L / min (0.5 VVM),
The cells were cultured at a pressure of 0.5 kg / cm ² · G and 350 rpm. Five days after the start of the culture, a culture solution containing the compound TMC-95A was obtained.

Example 3 29.5 L of the culture solution obtained in Example 2 was diluted with 17.5 L of water, followed by centrifugation (3000 rpm) to separate cells into an aqueous layer. The obtained aqueous layer (40 L) was placed in an 800 ml column of Diaion HP-20 (manufactured by Mitsubishi Chemical Corporation) suspended in water, washed with 3 L of 10% acetone aqueous solution, and then washed with 60% of water.
Elution was performed with 3 L of an acetone aqueous solution. The obtained eluate was used for 0.1 ml.
After concentration under reduced pressure to 9 L, extraction was performed twice with 0.45 L of n-butanol. After concentrating the obtained extract under reduced pressure to 10 ml, n-
The precipitate was added dropwise to 0.5 L of hexane, and the resulting precipitate was dried under reduced pressure to obtain 2.76 g of a black-brown solid.

The obtained black-brown solid was suspended in a small amount of a mixture of methylene chloride and methanol (1: 1) and then suspended in methylene chloride using silica gel (Wakog, manufactured by Wako Pure Chemical Industries, Ltd.).
(elC-200, 44 mm × 430 mm). After washing the column with 1.0 L of methylene chloride, the column was eluted with 2.5 L of a mixture of methylene chloride and methanol (9: 1). The eluate was silica gel thin layer chromatography (TL
C), the fractions containing the compound TMC-95A were collected and concentrated to dryness under reduced pressure to give a light yellow powder 438.
mg was obtained.

The obtained pale yellow powder was dissolved in a small amount of dimethylsulfoxide, and this solution was suspended in an aqueous solution containing 28% acetonitrile (YMC-GEL ODS (A60) manufactured by YMC, 22 mm).
× 230 mm) column. Use the same solvent for the column.
The eluate was monitored by analytical reversed-phase high-performance liquid chromatography (HPLC) to give Compound T.
The fraction containing MC-95A was separated and concentrated to dryness under reduced pressure to obtain 314 mg of a pale yellow powder.

After dissolving the obtained pale yellow powder in a small amount of dimethyl sulfoxide, preparative high performance liquid chromatography (column: YMCPack D-OD manufactured by YMC)
S-5-B 30 mm x 250 mm, solvent: aqueous solution containing 28% acetonitrile, flow rate: 25 ml / min). Active fractions containing the compound TMC-95A were collected, concentrated under reduced pressure to dryness. Compound TMC-95A
Is dissolved in a small amount of a mixture of methylene chloride and methanol (1: 1), and Sephadex LH previously equilibrated with a mixture of methylene chloride and methanol (1: 1)
The mixture was applied to a -20 (Pharmacia, 22 mm × 440 mm) column, subjected to gel filtration with the same solvent, and the active fraction was concentrated under reduced pressure to obtain 78.7 mg of compound TMC-95A as a white powder.

The physicochemical properties of the compound TMC-95A of the present invention are as follows.

(I) Property of substance: white powder (2) Molecular weight: m / z 679 (M + H, according to FAB mass spectrum) (3) Molecular formula: C ₃₃ H ₃₈ N ₆ O ₁₀ (4) Specific rotation: [Α] ²³ _D = + 102 ゜ (c =
(0.54, methanol) (5) UV absorption spectrum (methanol solution) λm
ax, nm (logε): 206 (4.59), 22
2 (4.63), 294 (3.74) (see FIG. 2) (6) Infrared absorption spectrum (KBr tableting) cm ^-1 :
3380, 3060, 2970, 2935, 287
5, 1720, 1660, 1515, 1445, 142
0, 1265, 1230, 1200, 1140, 112
0, 1075, 760 (see FIG. 3) (7) ¹ H nuclear magnetic resonance spectrum (400 MHz, DM
SO-d ₆ , internal standard; tetramethylsilane) δ pp
m: 0.87 (3H, t), 1.01 (3H,
d), 1.44 (1H, m), 1.60 (3H, d
d) 1.69 (1H, m), 2.38 (1H, d
d), 2.45 (1H, dd), 2.95 (1H, d)
d) 3.06 (1H, Brd), 3.38 (1H,
m), 3.92 (1H, dd), 4.17 (1H, d
d) 4.64 (1H, m), 4.66 (1H, m),
4.66 (1H, m), 5.78 (1H, d), 6.1
9 (1H, s), 6.46 (1H, m), 6.66 (1
H, dd), 6.77 (1H, d), 6.95 (1H,
brs), 6.97 (1H, t), 7.17 (1H,
d), 7.23 (1H, brd), 7.24 (1H, d
d), 7.29 (1H, brs), 7.35 (1H, d
d), 8.09 (1H, s), 8.20 (1H, d),
9.01 (1H, d), 9.02 (1H, d), 9.3
5 (1H, s) (see FIG. 4) (8) ¹³ C nuclear magnetic resonance spectrum (400 MHz, DM
SO-d ₆ , internal standard; tetramethylsilane) δ pp
m: 11.0 (q), 11.2 (q), 14.5
(Q), 24.8 (t), 36.5 (t), 36.6
(T), 39.7 (d), 49.5 (d), 51.6
(D), 54.4 (d), 75.1 (d), 77.5
(S), 105.0 (d), 115.0 (d), 12
0.4 (d), 120.4 (s), 122.3 (d),
122.9 (s), 124.9 (d), 125.0
(S), 129.1 (s), 129.8 (d), 13
0.7 (d), 132.9 (d), 139.9 (s),
153.1 (s), 158.8 (s), 167.2
(S), 170.1 (s), 170.1 (s), 17
0.3 (s), 177.9 (s), 201.3 (s)
(See FIG. 5) (9) Solubility: Easily soluble in alcohols such as methanol, ethanol, and butanol, acetone, dimethyl sulfoxide, acetic acid, and pyridine. Soluble in ethyl acetate and water. Insoluble in chloroform, diethyl ether and n-hexane.

(10) Color reaction: positive for iodine and sulfuric acid. Negative for ninhydrin.

(11) Thin layer chromatography: adsorbent; silica gel (manufactured by Merck, using Kieselgel 60); developing solvent; methylene chloride: methanol (5:
1), Rf = 0.36.

[0055]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 4 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Leu Leu Val Tyr 14

[Brief description of the drawings]

FIG. 1 shows the inhibitory effect of compound TMC-95A on rat adjuvant-induced arthritis. Control (-): Control group to which tuberculosis heat-killed bacteria were not administered Control (+): Control group to which tuberculosis heat-killed bacteria were administered

FIG. 2 shows an ultraviolet absorption spectrum of compound TMC-95A.

FIG. 3 shows an infrared absorption spectrum of compound TMC-95A.

FIG. 4 shows the ¹ H nuclear magnetic resonance spectrum (400 MHz, measured in DMSO-d ₆ ) of compound TMC-95A.

FIG. 5 shows the ¹³ C nuclear magnetic resonance spectrum (100 MHz, measured in DMSO-d ₆ ) of compound TMC-95A.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 38/00 ACS A61K 37/02 ABX ADA ABY ADP ACD AED ACS C07K 1/16 ADA C12N 9/99 ADP C12P 21/02 AED / / (C12P 21/02 C12R 1: 645) (72) Inventor Takaaki Murakami 3-13-31 Kashima Yodogawa-ku, Osaka-shi, Osaka Tanabe Pharmaceutical Kashima Dormitory (72) Inventor Maki Nishio 2--10 Nishikamata, Ota-ku, Tokyo No. 2

Claims (5)

[Claims] 1. A compound of formula (I): Or a pharmacologically acceptable salt thereof. 2. A microorganism belonging to the genus Apiospora or the genus Arthrinium having the ability to produce the compound TMC-95A according to claim 1 is cultured, and the compound TMC is obtained from the culture or a processed product of the culture. A method for producing compound TMC-95A, comprising collecting -95A. 3. A compound of formula (I): A pharmaceutical composition comprising, as an active ingredient, the compound TMC-95A or a pharmaceutically acceptable salt thereof represented by the following formula: 4. The pharmaceutical composition according to claim 3, which is a proteasome inhibitor. 5. The pharmaceutical composition according to claim 3, which is an agent for preventing and / or treating rheumatoid arthritis.