JPH0881431A - New compound b1371e or b1371f and its production - Google Patents

Abstract

PURPOSE: To obtain a new compound B1371E or B1371F, having inhibiting activities against calpain which is one of cysteine proteases and useful as an anticerebral infarction agent, an anticardiac infarction agent, an antithrombotic agent, etc., by culturing a specific marine bacterium and collecting the compound from the resultant cultured product. CONSTITUTION: This compound of formula I or II. Furthermore, this method for producing the compound of formula I or II is to culture a marine bacterium SANK70992 (FERM BP-4158) (e.g. at 23-30 deg.C and pH 5.0-8.5 for 1-3 days by spinner culture with aeration) and collect the compound from the resultant cultured product. This compound of formula I has the following physical properties: property: neutral fat-soluble white and hygroscopic powder unstable in an alkaline solution at ambient temperature; solubility: readily soluble in methanol and sparingly soluble in chloroform and hexane; color reaction: negative to the ninhydrin reaction; molecular formula: C32 H49 N3 O8 ; molecular weight: 604.35925 (found) and 604.35979 (calculated); specific rotatory power: [α]<25> D-27 deg. (C 1.4. methanol); circular dichroic spectrum: λnm (θ) 238.4 (-2.92 deg.×10<3> ), 206.6 (-1.95 deg.×10<4> ), etc.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel peptide compound B137 showing enzyme inhibitory activity of cysteine protease.
1E or B1371F, a process for producing the same, a producing bacterium, and an application.

[0002]

2. Description of the Related Art Calpain, a kind of cysteine protease, is an enzyme normally existing in cells and activated by Ca ²⁺ under neutral conditions. Calpain is two molecular species (I type (μ calpain), I
There is type I (m calpain). Usually type I, II
Both types exist as inactive precursors composed of a subunit having a molecular weight of approximately 80K and a common 30K, but when intracellular Ca ²⁺ concentration increases, the precursor binds to Ca ²⁺ and is converted into an active form. However, when Ca ²⁺ excessively flows into cells due to some cause such as ischemia, calpain is randomly activated. As a result, processing of proteins in the cytoplasm may be unnecessarily enhanced, cell function and
It irreversibly cleaves and decomposes enzymes and proteins essential for maintaining the structure. Diseases based on such uncontrolled activation of calpain include muscular dystrophy, cerebral infarction, myocardial infarction, cataract, thrombus, inflammation and the like (Kawashima, S.
Et al., Protein Nucleic Acid Enzyme, 37, 2144-2160,
(1992)).

On the other hand, a large amount of cysteine protease, cathepsin B, is contained in the lysosome of phagocytic cells such as neutrophils and macrophages, and plays a role of decomposing and digesting foreign substances inside and outside the cells. However, if the activity of cathepsin B is increased more than necessary, not only foreign substances but also self-organs are decomposed and digested. Examples of such diseases based on the enhancement of cathepsin B activity include inflammation, muscular dystrophy, osteoporosis, cancer metastasis and the like (K-atsunuma, N. et al., Experimental Medicine, 5).
Vol., 926-930, (1987)). Therefore, it is possible to prevent or treat the progress of pathological conditions against these diseases by using substances that inhibit the enzymatic activity of cysteine proteases such as calpain and cathepsin B.

Leopeptin (Aoyagi, T. et al., J. Antibiot., Vol.
58-568, (1969)), E-64 (Hanada, K.
Et al Agric. Biol. Chem., Vol. 42 (3), 523-5.
28 (1978)), Calpeptin (Tsujinaka, T.
Biochem. Biophys. Res. Commun., Vol.153,12
01-1208 (1988)), Strepin (Ogura,
K. et al., Agric. Biol. Chem., Vol. 49, 799-80.
5, (1985)), stucco pins (Saito, M. et al., Ag.
ric. Bilo. Chem, vol. 51 (3), 861-86
8, (1987)) and the like, and all have an aldehyde group at the C-terminus.

E-64 related compounds include E-64
d (Tamai, M. et al., J. Pharmacobio-Dyn., vol. 9, 67)
2-677, (1986)), NCO-700 (Takan
o, H. et al., Biochem. Med. Metab. Biol., vol.45.
(1), 41 to 47, (1991)) and the like are known, and all have epoxysuccinic acid as an active center. Although these compounds do not inhibit serine protease, their inhibitory activity against cysteine protease is not necessarily strong.

From this point of view, a physiologically active substance having a novel structure for cysteine protease, not a leupetine derivative or an E-64 analog, and having a strong inhibitory activity specific to cysteine protease is desired. There is.

[0007]

DISCLOSURE OF INVENTION Problems to be Solved by the Invention The present inventors have conducted an earnest search to find a physiologically active substance having a novel structure and exhibiting an enzyme inhibitory activity of calpain, a kind of cysteine protease, from microbial metabolites. As a result, it was found that new physiologically active substances B1371E and B1371F exhibiting enzyme inhibitory activity against cysteine protease are produced from the culture solution of the marine bacterium SANK70992 strain isolated from the sea urchin collected on the coast of Oshika Peninsula, Miyagi Prefecture, Japan. The present invention has been completed.

[0008]

Means for Solving the Problems The present invention comprises: (1) novel compound B1371E, (2) novel compound B1371F, (3) marine bacterium SANK70992, and culturing the novel compound B1371E or B1371F from the culture. And a method for producing B1371E or B1371F.

Compounds of the invention B1371E and B13
71F culture, purification and isolation, structure determination is related compound B13
Data of 71A culture, purification and isolation method, and structural analysis were referred to.

[B1371A] (1) Structural formula

[0011]

[Chemical 5]

(2) Physical properties 1) Properties; neutral fat-soluble white, hygroscopic powder. Unstable at room temperature in alkaline solutions. 2) Solubility; easily soluble in methanol, poorly soluble in water, chloroform, and hexane.

3) Color test: Ninhydrin reaction, Sakaguchi reaction and Fluorescamine reaction are negative.

4) Molecular formula; C ₄₆ H ₆₇ N ₅ O ₁₁ 5) Molecular weight; Determined by the LSI-MASS method (M + H)
⁺ 866.4918 (measured value) 866.4915 (calculated value) 6) Specific optical rotation (measured value); [α] _D ²⁵ -26.4 ° (C
0.27, methanol) 7) Ultraviolet absorption spectrum; λ max nm (ε) The ultraviolet absorption spectrum measured in methanol is as shown below. 227.4 (3.45 × 10 ³ ) 223.6 (1.91 × 10 ⁴ ) 8) Infrared absorption spectrum; νmax (KBr) cm ^-1 KBr The infrared absorption spectrum measured with a disk shows the following maximum absorption. 1516.939,1536.626,1668.284,1747.731,2931.559,2962.
582,3315.963 9) ¹ H-nuclear magnetic resonance spectrum; (δ: PPM) ¹ H-nuclear magnetic resonance spectrum (400 MHz) measured by using TMS (tetramethylsilane) as an internal standard in deuterated methanol is It is as shown below.

0.90 (3H, t), 0.95 (6H, d), 1.02 (3H, d), 1.07
(3H, d), 1.2-1.50 (12H, m), 1.98 (1H, m), 2.14 (1H, m), 2.25-
2.50 (4H, m), 2.73 (1H, dd), 2.86 (1H, dd), 2.93 (3H, s), 3.21
(2H, m), 3.80-4.10 (5H, m), 4.20 (1H, d), 4.40 (1H, d), 4.52
(1H, dd), 4.79 (1H, m), 6.25 (1H, d), 6.45 (1H, dd), 6.70 (2H,
d), 6.75 (2H, d), 7.02 (2H, d), 7.08 (2H, d) 10) ¹³ C-nuclear magnetic resonance spectrum; (δ: PPM) in deuterated methanol using TMS as internal standard It was measured
^{The 13} C-nuclear magnetic resonance spectrum (400 MHz) is as shown below.

14.4 (1C, q), 16.5 (1C, q), 18.6 (1C, q), 19.
9 (1C, q), 20.0 (1C, q), 23.7 (1C, t), 26.7 (1C, t), 30.0
(1C, d), 30.4 (1C, t), 30.7 (1C, t), 31.3 (1C, d), 33.0 (1
C, t), 34.6 (1C, t), 37.6 (1C, t), 38.3 (1C, t), 40.2 (1C,
q), 42.0 (1C, t), 44.6 (1C, t), 50.4 (1C, d), 56.4 (1C,
d), 59.4 (1C, d), 60.4 (1C, d), 64.3 (1C, t), 68.2 (1C,
d), 69.8 (1C, d), 70.0 (1C, d), 116.2 (2C, d), 116.6 (2C,
d), 126.1 (1C, d), 129.9 (1C, s), 130.5 (1C, s), 131.2 (4C,
d), 141.1 (1C, d), 155.5 (1C, s), 156.0 (1C, s), 169.0 (1C,
s), 170.8 (1C, s) 171.5 (1C, s), 171.7 (1C, s), 173.2 (1C, s
s), 173.6 (1C, s ) 11) High performance liquid chromatographic separation columns; μBondasphere 5μC ₁₈ -100A (column size, Φ3.9 × 150mm, Waters Corp.) solvent: 37% acetonitrile for 16 minutes 45% acetonitrile gradient Flow rate: 1.0 ml / min Detection; UV220 nm retention time; 8.43 min B1371E or B1371F of the present invention has the following structural formula and properties.

[B1371E] (1) Structural formula

[0018]

[Chemical 6]

(2) Physical properties 1) Properties; neutral fat-soluble white, hygroscopic powder. Unstable at room temperature in alkaline solutions.

2) Solubility; easily soluble in methanol, sparingly soluble in water, chloroform and hexane.

3) Color test; negative for ninhydrin reaction.

4) Molecular formula; C ₃₂ H ₄₉ N ₃ O ₈ 5) Molecular weight; Determined by FAB-MASS method (M + H)
⁺ 604.35925 (measured) 604.35979 (calculated) 6) Specific optical rotation (measured value); [α] _D ²⁵ -27 ° (C
1.4, Methanol) 7) Circular dichroism spectrum; λnm (θ) 238.4 (-2.92 ° x 10 ³ ) 206.6 (-1.95 ° x 10 ⁴ ) 8) ¹ H-nuclear magnetic resonance spectrum; (δ: PPM ) The main peaks of the ¹ H-nuclear magnetic resonance spectrum (400 MHz) showing the B1371E structure measured by using TMS as an internal standard in deuterated methanol are as follows.

2.96 (2H, d), 3.70 (3H, s), 5.41 (1H, m), 5.54
(1H, m), 6.02 (1H, d), 6.64 (2H, d), 6.93 (1H, dd), 7.00 (2H,
d) 9) High performance liquid chromatographic separation columns; μBondasphere 5μC ₁₈ -100A (column size, Φ3.9 × 150mm, manufactured by Waters Co.) Solvent: 37% acetonitrile for 16 minutes 45% acetonitrile gradient flow rate; 1.0 ml / min Detection; UV220nm retention time; 13.29 minutes [B1371F] 1) Structural formula

[0024]

[Chemical 7]

2) Properties; neutral fat-soluble white, hygroscopic powder. Unstable at room temperature in alkaline solutions.

3) Solubility; easily soluble in methanol, sparingly soluble in water, chloroform and hexane.

4) Color test: Ninhydrin reaction is negative.

5) Molecular formula; C ₄₆ H ₆₅ N ₅ O ₁₀ 6) Molecular weight; Determined by FAB-MASS method (M + H)
⁺ 848.48115 (measured) 848.48097 (calculated) 7) Specific optical rotation (measured value); [α] _D ²⁵ -21.5 ° (C
4.2, Methanol) 8) Ultraviolet absorption spectrum; λ max nm (ε) The ultraviolet absorption spectrum measured in methanol is as shown below. 278.2 (2.31 × 10 ³ ) 223.7 (1.41 × 10 ⁴ ) 9) Circular dichroism spectrum; λnm (θ) 226.2 (-1.25 ° × 10 ⁴ ) 208.6 (+3.6 ° × 10 ³ ) 10) ¹ H- nucleus Magnetic resonance spectrum; (δ: PPM) The main ¹ H-nuclear magnetic resonance spectrum (400 MHz) peaks showing the B1371F structure measured by using TMS as an internal standard in deuterated methanol are as follows.

2.88 (3H, s), 2.96 (2H, d), 5.44 (1H, m), 5.54
(1H, m), 6.24 (1H, d), 6.45 (1H, dd), 6.67 (2H, d), 6.72 (2H,
d), 7.01 (2H, d ), 7.03 (2H, d) 11) High performance liquid chromatographic separation columns; μBondasphere 5μC ₁₈ -100A (column size, Φ3.9 × 150mm, manufactured by Waters Co.) Solvent: 37% acetonitrile 16 Minute 45% acetonitrile gradient Flow rate; 1.0 ml / min Detection; UV220 nm retention time; 14.34 minutes Peptide compound B1371A or B137 of the present invention
The above-mentioned SANK 70992 strain that produces 1B is a marine bacterium isolated from Aonori collected on the coast of the Oshika Peninsula in Miyagi Prefecture. The mycological properties of the SANK 70992 strain, which is a B1371A or B1371B producing bacterium, are as follows.

1. Morphological Properties When observed after culturing on Marine Agar (manufactured by Difco) at 23 ° C. for 24 hours, cells have a diameter of 0.3 to 0.4 μm, a length of 1.5 to 2.0 μm, and a spiral shape. It has monopolar hairs and is active. Does not form large spherical cells (coccoid body).
It does not sporulate and Gram stain is negative.

2. Growth on Marine Agar The colonies cultured at 23 ° C. for 48 hours are somewhat pinkish-white, grey-white, opaque, circular, full-edged. Does not form water soluble dyes.

3. Physiological properties 1) Seawater requirement (Proteose Peptone No. 3 (Difco) 0.1%, yeast extract (Difco)
0.1%, Phyton Peptone (manufactured by BBL) 0.1%
When using a medium of composition): Seawater is required for growth.

2) OF (oxidative-fermentative) test [Hugh Leifso (Hugh Leifso
n) Method, adding yeast extract (Difco) 0.05%,
Adjusted with 75% artificial seawater]: Decomposes glucose oxidatively.

3) Catalase: + 4) Behavior to oxygen: aerobic 5) Reduction of nitrate: -6) Hydrolysis of starch: -7) Decomposition of agar: -8) Liquefaction of gelatin: -9) Growth temperature : Grows slightly at 8 ° C, 12 ° C to 40 ° C
Shows good growth. Does not grow at 50 ° C.

10) Nutritional Requirements (Jounal of Bacteriology, 107
Vol., 268-294 (1971) with basal medium): Yeast extract required.

11) Availability of carbon compounds (Journal
Jounal of Bacteriology,
107, 268-294 (1971), basal medium containing 0.02% of yeast extract and static culture for 7 days): L-arabinose-, sucrose-, D-
3. Xylose −, sodium succinate +, D-glucose +, sodium acetate +, maltose −, glycerol + 4. Chemotaxonomic properties 1) G + C (guanine + cytosine) content of DNA: 4
9.8 mol% (HPLC method) 2) Quinone system: Ubiquinone Q-10 The SANK70992 strain having the above-mentioned mycological properties is a Bergey's Manual of Systematic Bacteriolog.
According to the classification of y), Volume 1 (1984), it is separated from the marine environment, requires seawater for growth, cells are spiral, D
Since the G + C content of NA is 49.8%, it is considered to be the closest to the genus Oceanospirillum. However, the flagella of SANK70992 are monopolar hairs and only one pole of the cell is settled, whereas the flagella of Oceanospirillum bacteria are multipolar hairs, rarely monopolar hairs, but attached to both poles of the cell. Be born Also, SANK70992
Strains produce acid aerobically from sugar, whereas Oceanospirillem bacteria do not. In addition, SANK
In the 70992 strain, large spherical cells observed in Oceanospirillem bacteria are not found. Therefore, the present inventors have identified the present bacterium as a new genus close to the genus Oceanospirillum, a new species of marine bacterium SANK70992 strain (depositor, Institute of Biotechnology, Institute of Industrial Science: Deposit No. 4158
(FERM BP-4158): Deposit date, January 5, 1993.

Although the SANK 70992 strain has been described above, it is well known that the various properties of marine bacteria are not constant and can easily be changed naturally or artificially. B13 belonging to marine bacteria
It includes all strains producing 71A or B1371B.

Compounds of the invention B1371E or B13
An efficient method for producing 71F is a method in which the SANK70992 strain capable of producing the compound is cultured in a suitable medium and separated from the culture.

The medium used for producing the substance of the present invention is most preferably, but not limited to, shaking culture or aeration-agitation culture in a liquid medium. Medium is B1371
It is preferable that the E or B1371F producing bacterium grows and accumulates the compound in the medium. For example, glucose, glycerin, molasses, organic acids and the like can be used as the carbon source. As the nitrogen source, for example, bactotryptone,
Various organic or inorganic nitrogen compounds such as yeast extract, peptone, amino acids, ammonium salts, nitrates and the like are used. As the inorganic salt, sodium chloride, calcium chloride, magnesium chloride, various phosphates or the like may be added. Also, bacterial growth and B1371E
Alternatively, vitamins, coenzymes, etc. that promote the production of B1371F may be added.

In the culture of the B1371E or B1371F producing bacterium, the culture temperature, the culture period, the stirring speed, the aeration amount,
Conditions such as pH of the culture solution are B1371E or B13
It is appropriately selected and adjusted so that the accumulated amount of 71F is maximized. For example, in the case of ordinary aeration stirring culture, the culture temperature is 2
Culturing at 3 ° C. to 30 ° C. for 1 to 3 days is preferable, and the pH of the culture solution is preferably adjusted to pH 5.0 to 8.5.

The change with time of the amount of B1371E or B1371F accumulated in the culture medium with the progress of culture can be measured by the inhibitory activity or reverse phase HPLC described later. Usually, the maximum production is reached after 48 to 72 hours of culture. After the culturing, B1371E or B1371F, which mainly accumulates in the liquid portion, is preferably separated from the filtrate or supernatant liquid by removing the bacterial cells and other solid portions by filtration or centrifugation, but if necessary, It is also possible to separate the compound from the culture broth without removing the cells. B1 from culture
Various methods based on the physicochemical characteristics can be used for the separation and purification of 371E or B1371F. For example, B1371E present in the filtrate or supernatant
Alternatively, B1371F can be extracted and purified with an organic solvent immiscible with water under neutral pH conditions, for example, ethyl acetate, n-butanol, dichloromethane, chloroform, ethylene chloride, methylene chloride, etc., alone or in combination thereof. Alternatively, as the adsorbent, for example, Diaion HP-20 (manufactured by Mitsubishi Corp.) or the like is used.
The fraction containing B1371E or B1371F is passed through the layer of the above adsorbent to adsorb and remove impurities, or after adsorbing B1371E or B1371F, it is eluted with methanol water, acetone water, etc. The compound can be obtained by Further, adsorption column chromatography using a carrier such as silica gel or Florisil, Sephadex LH-20 (manufactured by Pharmacia), Toyopearl HW-40 (Tosoh Corporation).
B1371E by partition column chromatography using, for example, Sephadex G25 (manufactured by Pharmacia), gel filtration chromatography, and high performance liquid chromatography using normal phase and reverse phase columns.
Alternatively, B1371F can be purified.

The present invention relates to B1371E or B1371.
The present invention relates to an anti-cerebral infarction agent, an anti-myocardial infarction agent, an anti-thrombotic agent, an anti-cataract agent, an anti-muscular dystrophy agent, an anti-inflammatory agent, an anti-osteoporosis agent or a cancer metastasis preventive agent containing F as an active ingredient.

B1371E or B1371F of the present invention
When used as an anti-cerebral infarction agent, anti-myocardial infarction agent, anti-thrombotic agent, anti-cataract agent, anti-muscular dystrophy agent, anti-inflammatory agent, anti-osteoporosis agent or cancer metastasis preventive agent, it is administered in various forms. Examples of the dosage form include oral administration by tablets, capsules, granules, syrups, etc., and parenteral administration by injections (intravenous, intramuscular, subcutaneous), eye drops, suppositories, etc.

These various preparations are usually used in the conventional pharmaceutical preparation technical field such as excipients, binders, disintegrating agents, lubricants, flavoring agents, solubilizing agents, suspending agents, coating agents, etc. It can be formulated with known auxiliary agents that can be used. The amount used varies depending on symptoms, age, weight, administration method, dosage form and the like, but usually 50 mg to 1000 mg can be administered to an adult daily.

[0045]

【Example】

(Reference Example) Purification of B1371A (A) Culture 5 liter Erlenmeyer flask (seed flask) containing 1 liter of medium having the composition described below, which was obtained by sterilizing the SANK 70992 strain.
Was inoculated. This was then heated at 26 ° C. for 3 days at 200 rp
m pre-cultured on a rotary shaker. Furthermore, the same medium 100
One liter of this seed culture solution was put into a 200 liter tank containing liters, and stirred and cultured at 26 ° C. for 47 hours with an aeration rate of 1.0 vvm and a stirring speed of 110 rpm.

[0046]

[Table 1] Medium composition Bactryptone (manufactured by Difco) 16 g Yeast extract (manufactured by Difco) 10 g ………………………………………………………………………… ……………………… Artificial seawater Jamarin S (manufactured by Jarmarin Laboratory) 1000 ml pH 6.8 13) Isolation Method by Ishiguro, H. et al. (Biochemistry, vol.26, 28)
63-2870 (1987)), B1371A was isolated and purified by measuring the inhibitory activity against type II calpain prepared from bovine kidney.

The activity of type II calpain was determined by the method of Yoshimura, N. et al. (J. Biol. Chem., Vol. 258, 8883-88).
89 (1983)) according to the following method.

0.25% casein, 5 mM cysteine,
5 mM CaCl ₂ , 100 mM MImidazol-H
The enzyme preparation was added to the solution containing Cl (pH 7.5) to start the reaction. The final volume was 500 μl. 30 ℃,
After reacting for 30 minutes, 10% trichloroacetic acid 500μ
1 was added to stop the reaction, the mixture was allowed to stand at 4 ° C. for 30 minutes and then centrifuged, and the A ₂₈₀ of the supernatant was measured. Instead of 5 mM CaCl ₂ , EDTA was allowed to react at 5 mM, which was subtracted as a blank to obtain the type II calpain activity. The type II calpain activity that increases A ₂₈₀ by 1 by this method was defined as 1 enzyme unit.

The inhibitory activity of B1371A was obtained by adding the B1371A DMSO solution 5 to the above reaction solution containing 0.1 enzyme units.
The same measurement was carried out by adding μl, and it was determined by comparison with a control (DMSO).

Culture liquid 2 for two 200 liter tanks
The supernatant obtained by centrifuging 00 liters was 190
After adjusting the liter to pH 7.0 with hydrochloric acid,
The extraction operation was performed by adding liter of ethyl acetate. The ethyl acetate layer was dehydrated with anhydrous sodium sulfate and then concentrated to obtain 107 g of a black syrup-like substance. Silica gel equilibrated with dichloromethane was filled in a 400 ml glass column, and a black syrup-like substance dissolved in dichloromethane was donated, followed by washing with dichloromethane. Elution with a mixture of dichloromethane / methanol (75/25: v / v). The calpain-inhibiting activity fractions were collected, extracted with ethyl acetate and concentrated to obtain 43 g of a black syrup-like substance. After dissolving this black syrup-like substance in methanol,
% Methanol solution, equilibrated with a solvent having the same composition as Cosmosil 140C ₁₈ -OPN (manufactured by Nacalai Tesque, Inc.) column, and washed with the same solvent.
Elution with 60% methanol. The calpain-inhibiting activity fractions were collected, extracted with ethyl acetate and concentrated to obtain 23.6 g of a brown syrupy substance. After dissolving this brown syrup-like substance in acetonitrile, it was prepared to be a 30% acetonitrile-0.1% trifluoroacetic acid solution and equilibrated with a solvent having the same composition as Cosmosil 140C ₁₈ -OPN (Nacalai Tesque Corp.). This column was applied to this column. After washing with the same solvent, elution was performed with a 50% acetonitrile-0.1% trifluoroacetic acid solution. The obtained inhibitory activity fractions were collected, extracted with ethyl acetate and then concentrated to remove brown syrup-like substances by 1
7.6 g was obtained. This was further purified by preparative reverse phase HPLC. That is, after dissolving about 2 g of the above brown syrupy substance in methanol in one operation, reverse phase preparative HPLC (TSKgel ODS-120T, diameter 21.5 m) was used.
m × length 300 mm, manufactured by Toso Co., Ltd.), room temperature, flow rate 9.9 ml / min, 30% acetonitrile-0.
After washing with 1% trifluoroacetic acid, it was eluted with 37% acetonitrile-0.1% trifluoroacetic acid, and fractions with a retention time of about 54 minutes were collected. The same operation was performed on the remaining brown syrupy substances, and the collected eluates were pooled.
The eluate was concentrated under reduced pressure and freeze-dried to obtain 9.1 mg of white powder B1371A.

Next, the present invention will be described in more detail with reference to examples and formulation examples, but the present invention is not limited thereto.

(Example 1) B1371E and B1371
Purification of F (A) Culture 5 liter Erlenmeyer flask (seed flask) containing 1 liter of medium having the composition described below, which is obtained by sterilizing the SANK 70992 strain
Was inoculated. This was then heated at 26 ° C. for 3 days at 200 rp
m pre-cultured on a rotary shaker. Furthermore, the same medium 100
One liter of this seed culture solution was put into a 200 liter tank containing liters, and stirred and cultured at 26 ° C. for 47 hours with an aeration rate of 1.0 vvm and a stirring speed of 110 rpm.

[0053]

[Table 2] Medium composition Bactryptone (manufactured by Difco) 16 g Yeast extract (manufactured by Difco) 10 g ………………………………………………………………………… ……………………… Artificial seawater Jamarin S (manufactured by Jarmarin Laboratory) 1000 ml pH 6.8 (B) Isolation Method by Ishiguro, H. et al. (Biochemistry, vol.26, 28)
63-2870 (1987)), B1371E or B1371F was isolated and purified by measuring the inhibitory activity against type II calpain prepared from bovine kidney.

The activity of type II calpain was determined by the method of Yoshimura, N. et al. (J. Biol. Chem., Vol. 258, 8883-88).
89 (1983)) according to the following method.

0.25% casein, 5 mM cysteine,
5 mM CaCl ₂ , 100 mM MImidazol-H
The enzyme preparation was added to the solution containing Cl (pH 7.5) to start the reaction. The final volume was 500 μl. 30 ℃,
After reacting for 30 minutes, 10% trichloroacetic acid 500μ
1 was added to stop the reaction, the mixture was allowed to stand at 4 ° C. for 30 minutes and then centrifuged, and the A ₂₈₀ of the supernatant was measured. Instead of 5 mM CaCl ₂ , EDTA was allowed to react at 5 mM, which was subtracted as a blank to obtain the type II calpain activity. The type II calpain activity that increases A ₂₈₀ by 1 by this method was defined as 1 enzyme unit.

The inhibitory activity of B1371E or B1371F was found in the above reaction mixture containing 0.1 enzyme units.
5 μl of a DMSO solution of E or B1371F was added, the same measurement was performed, and the value was determined by comparison with a control (DMSO).

B1371E or B required for 50% inhibition
The concentration of 1371F was calculated and used as the IC ₅₀ (nM) value.

Culture liquid 2 for two 200 liter tanks
200 liters of supernatant obtained by continuous centrifugation of 10 liters
After adjusting the pH of the liter to 7.0 with hydrochloric acid, 250
The extraction operation was performed by adding liter of ethyl acetate. The ethyl acetate layer was dehydrated with anhydrous sodium sulfate and then concentrated to obtain 30.4 g of a black syrup-like substance. Silica gel equilibrated with dichloromethane was filled in a 400 ml glass column, and a black syrup-like substance dissolved in dichloromethane was donated, followed by washing with dichloromethane. After elution fractionation with a mixed solution of dichloromethane / methanol (75/25: v / v), elution was performed with 100% methanol. The calpain inhibitory activity fractions were collected, extracted with ethyl acetate, concentrated, and
0.5 g of black syrupy substance was obtained. After dissolving this black syrup-like substance in methanol, it was prepared to be a 30% methanol solution and equilibrated with a solvent of the same composition.
Cosmosil 140C ₁₈ -OPN (Nacalai Tesque, Inc.)
Column), washed with the same solvent, and eluted with 60% methanol. After fractionation, it was further eluted with 100% methanol. The calpain-inhibiting activity fractions were collected, extracted with ethyl acetate and concentrated to obtain 20.2 g of a brown syrup-like substance.
After dissolving this brown syrup-like substance in acetonitrile, it was prepared to be a 30% acetonitrile solution and equilibrated with a solvent having the same composition as Cosmosil 140C ₁₈ -OP.
This fraction was applied to an N (Nacalai Tesque, Inc.) column, washed with the same solvent, and then eluted with a 50% acetonitrile solution. The obtained inhibitory activity fractions were collected, extracted with ethyl acetate and concentrated to obtain 3.2 g of a brown syrupy substance. This was prepared to be a 30% acetonitrile-0.1% trifluoroacetic acid solution and Cosm equilibrated with a solvent of the same composition
osil 140C _18- OPN (manufactured by Nacalai Tesque, Inc.)
It was applied to the column, washed with the same solvent, and then eluted with 50% acetonitrile-0.1% trifluoroacetic acid. Approximately 300 ml of eluate was used as one fraction, and B1371E, F
Fraction 3 containing was obtained. This active fraction was extracted with ethyl acetate and then concentrated, and 1.07 g of the obtained substance was further purified by preparative reverse phase HPLC. That is, about 50 mg of the above-mentioned brown syrup-like substance was dissolved in methanol in one operation, and then preparative reverse phase HPLC (TSKgel ODS-
120T, diameter 21.5mm x length 300mm, Toso
Co., Ltd., room temperature, flow rate 9.9 ml / min, 37
After washing with 37% acetonitrile, 37% acetonitrile, 7
Elute with a 45% acetonitrile gradient for 0 minutes, retention time 46 minutes (B1371E eluted), and 53 minutes (B137).
Fractions before and after (1F was eluted) were collected. The same operation was performed on the remaining brown syrupy substances, and the collected eluates were pooled. The acetonitrile of each eluate was removed by an evaporator, and the remaining aqueous layer was freeze-dried. B1371E 20 mg, B1371F2
3 mg of white powder was obtained.

(Formulation Example 1) Oral capsule

[0060]

[Table 3] The powders having the above formulation were mixed and passed through a 30-mesh sieve, and 350 mg of this powder was put into a gelatin capsule to obtain a capsule.

[0061]

The effects of the present invention will be described with reference to test examples.

(Test Example 1) B1371E or B137
Inhibitory activity of 1F against type II calpain The inhibitory activity against type II calpain of B1371E or B1371F obtained in Example 1 was determined in Example 1
It was measured by the method described in.

0.25% casein, 5 mM cysteine,
5 mM CaCl ₂ , 100 mM MImidazol-H
0.1 enzyme unit type calpain was added to the solution containing Cl (pH 7.5) to start the reaction. Final volume is 500
μl. After reacting at 30 ° C. for 30 minutes, 500 μl of 10% trichloroacetic acid was added to stop the reaction, the mixture was allowed to stand at 4 ° C. for 30 minutes and then centrifuged, and the A ₂₈₀ of the supernatant was measured. 5m
What was reacted with 5 mM of EDTA instead of M CaCl ₂ was subtracted as a blank to obtain type II calpain activity.

The inhibitory activity of B1371E or B1371F was determined by adding 5 μl of a DMSO solution of B1371E or B1371F and setting the final liquid volume to 500 μl in the same manner, and comparing it with a control (DMSO). The inhibitory activities of leupeptin and E-64 were also measured at the same time. The results (expressed as IC ₅₀ values) are shown below.

[0065]

Table 4 Type II Calpain Inhibitory Activity (IC ₅₀ ; nM) B1371E 7257.0 B1371F 38.9 Leupeptin 336.4 E-64 1035.2 B1371F Inhibits Type II Calpain Stronger than Leupeptin and E-64. It showed activity. B1371
E also had an inhibitory activity against type II calpain.

(Test Example 2) B1371E and B1371
Inhibitory activity of B on type I calpain Type I calpain was isolated from bovine kidney by the method of Inomata, M. et al. (J. Biochem., Vol. 93, 291-294 (19).
83)).

The activity of type I calpain was determined by the method of Yoshimura, N. et al. (J. Biol. Chem., Vol. 258, 8883-888).
9 (1983)) according to the following method.

0.25% casein, 5 mM cysteine,
5 mM CaCl ₂ , 100 mM MImidazol-H
0.1 enzyme unit type I calpain was added to a solution containing Cl (pH 7.5) to start the reaction. Final volume is 500
μl. After reacting at 30 ° C. for 30 minutes, 500 μl of 10% trichloroacetic acid was added to stop the reaction, the mixture was allowed to stand at 4 ° C. for 30 minutes and then centrifuged, and the A ₂₈₀ of the supernatant was measured. 5m
What was reacted with 5 mM EDTA instead of M CaCl ₂ was used as a blank and subtracted to obtain type I calpain activity.

The inhibitory activity of B1371E or B1371F was determined by adding 5 μl of a DMSO solution of B1371E or B1371F and setting the final liquid volume to 500 μl in the same manner, and comparing it with a control (DMSO). The inhibitory activities of leupeptin and E-64 were also measured at the same time.
The results (expressed as IC ₅₀ values) are shown below.

[0070]

Table 5 Type I Calpain Inhibitory Activity (IC ₅₀ ; nM) B1371E 5106.0 B1371F 30.6 Leupeptin 149.2 E-64 881.3 B1371F Inhibits Type I Calpain Stronger than Leupeptin and E-64. It showed activity. B1371E
Also had inhibitory activity against type I calpain.

(Test Example 3) B1371E or B137
Inhibitory activity of 1F against cathepsin Cathepsin B activity was determined by the method of Barrett, AJ et al.
in Enzymology, vol.80, 535-561 (198
It was measured according to 1)).

1 mM EDTA, 75 mM phosphate buffer (pH 6.0), 0.045% Brij35, 2 mM
To a solution containing DTT, 1 μg / ml cathepsin B, 10
μM Carbobenzoxy-L-Arginyl-L-Argine-4-Methyl-Couma
Add ryl-7-Amide (Cbz-Arg-Arg-MCA) to make the final volume 1
The reaction was started as 000 μl. After reacting at 30 ° C. for 30 minutes, 100 μl of 50% trichloroacetic acid was added to stop the reaction. The released aminomethylcoumarin was measured with a fluorometer (Excitation: 370 nm, Emmi
ssion: 460 nm).

The inhibitory activity of B1371E or B1371F was obtained by removing the Cbz-Arg-Arg-MCA from the above reaction solution, and adding it to DMSO solution 5 of B1371E or B1371F.
After adding μl and incubating at 30 ℃ for 5 minutes, Cb
z-Arg-Arg-MCA was added, the same measurement was performed, and the control (DMS
It was determined by comparison with O). The inhibitory activities of leupeptin and E-64 were also measured at the same time. The results (expressed as IC ₅₀ values) are shown below.

[0074]

Table 6 Cathepsin B inhibitory activity (IC ₅₀ ; nM) B1371E 1142.3 B1371F 7.5 leupeptin 16.8 E-64 47.6 B1371F has a stronger inhibitory activity against cathepsin B than leupeptin and E-64. Indicated.

(Test Example 4) B1371E or B137
Inhibitory activity of 1F against trypsin The activity of trypsin was determined by the method of Ogura, K. et al. (Agric. Biol. C
hem, vol.49, 799-805 (1985)).

0.25% casein, 50 mM Tris
To the solution containing -HCl (pH 8.0), 0.1 enzyme unit trypsin was added, and the final liquid volume was 500 μl to start the reaction. After reacting at 37 ° C for 10 minutes, 500 µl of 10% trichloroacetic acid is added to stop the reaction, and then 4 ° C.
After leaving it for 30 minutes, it was centrifuged and the A ₂₈₀ of the supernatant was measured. The trypsin activity for increasing A ₂₈₀ by 1 was defined as 1 enzyme unit.

The inhibitory activity of B1371E or B1371F was determined by adding 5 μl of a DMSO solution of B1371E or B1371F and setting the final liquid volume to 500 μl in the same manner, and comparing it with a control (DMSO). The inhibitory activities of leupeptin and E-64 were also measured at the same time. The results (expressed as IC ₅₀ values) are shown below.

[0078]

Table 7 Trypsin inhibitory activity (IC ₅₀ ; nM) B1371E> 70000 B1371F> 12000 leupeptin 1300 E-64> 22000 B1371E or B1371F showed no inhibitory activity against trypsin unlike leupeptin.

From the above results, B1371E or B1371F of the present invention has a strong inhibitory activity against type I calpain, type II calpain and cathepsin B. Therefore, B1371E or B1371F is useful as a prophylactic / therapeutic drug for diseases such as muscular dystrophy, cerebral infarction, myocardial infarction, cataract, thrombosis, inflammation, osteoporosis, and cancer metastasis.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location // A61K 31/22 9455-4C 38/55 ABE ABL ABN ABS ACB ADF ADU C12N 9/99 (C12P 21/02 C12R 1:01) A61K 37/64 ABN ABS ACB ADF ADU (72) Inventor Yasuyuki Takamatsu 389-4 Daisaku, Shimokawa, Izumi-cho, Iwaki-shi, Fukushima Sankyo Co., Ltd. (72) Yasuko Maruyama, Shinagawa, Tokyo Sankyo Co., Ltd. 1-25-2 Hiromachi, Ku (72) Inventor Takeshi Kinoshita 1-25-28 Hiromachi, Shinagawa-ku, Tokyo Sankyo Co., Ltd.

Claims (3)

[Claims] 1. A novel compound B1371 represented by the formula (I).
E: [Chemical 1] 2. A novel compound B137 represented by the formula (II).
1F: [Chemical formula 2] 3. A marine bacterium SANK70992 is cultured,
From the culture, the following formula (I) or (II) [Chemical 4] B1371E or B1371 characterized by collecting the compound B1371E or B1371F represented by
Manufacturing method of F.