JPH05244972A - New pharmaceutically active substance concanamycin d, concanamycin e, concanamycin g and their production - Google Patents

Abstract

PURPOSE:To provide concanamycin D, E and G hopeful as active ingredients for new medicines for arteriosclerosis. CONSTITUTION:Concanamycin-productive bacteria belonging to Streptomyces are cultured to accumulate concanamycin D, E and G in the culture solution followed by collecting the concanamycin D, E and G from the solution and then purifying them, thus obtaining the objective concanamycin D, E and G.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to concanamicin D,
E, G, a method for producing the substance, and a strain producing the substance. Concanamycin D, E, and G are oxidized LDs by inhibiting acidification of lysosomes and endosomes.
It inhibits the foaming of macrophages by L and is expected as an arteriosclerosis preventive or therapeutic drug.

[0002]

2. Description of the Related Art Conventionally, HMG has been used as a therapeutic agent for arteriosclerosis.
-Mevalotin, which is a CoA reductase inhibitor, and probucol, which is an antioxidant, are used.

[0003]

The above-mentioned therapeutic agents are excellent in efficacy and toxicity, but there is a long-awaited discovery of new compounds suitable for therapeutic agents for arteriosclerosis based on a new mechanism of action.

[0004]

Therefore, as a result of various searches for metabolites of microorganisms, the present inventors have found that one strain belonging to the genus streptomyces has the following formula (1) having a foaming and suppressing effect on macrophages. ),
It was found that the novel substances conkanamycin D, E and G shown in (2) and (3) were produced.

[0005]

[Chemical 3]

[0006]

[Chemical 4]

[0007]

[Chemical 5]

The present invention has been completed based on the above findings. The above-mentioned pharmacologically active substances conkanamycin D, E,
G is a concanamicin D, E, G-producing bacterium belonging to the genus Streptomyces,
Obtained by collecting G. A typical example of concanamicin D, E and G producing bacteria has the following mycological properties.

1. Morphological properties As a result of observing after 2 weeks at 27 ° C, the aerial hyphae are simply branched and their tips are helical. No formation of sporangia or crusty silk is observed. The spore surface is smooth, the spores are cylindrical, and the size is 0.5-0.7 x 1.0-1.2μ.
m. In addition, 20 or more chains are formed to form spores.

2. Growth in various media Table 1 below shows the growth conditions after 2 weeks at 27 ° C on various media.

[0011]

3. Physiological properties Appropriate growth range: 24-37 ° C Nitrate reduction: Positive gelatin liquefaction (on glucose medium, peptone gelatin medium, 20 ° C): Positive starch hydrolysis (starch / inorganic salt agar medium): positive Degreasing Coagulation of milk: Anonymous defatted milk peptone: Positive melanin-like pigment formation: Positive 4. Utilization of carbon source (on Pridham Godleybe agar medium) L-arabinose + D-xylose + D-glycose + D-fructose + sucrose + inositol + L-rhamnose + raffinose + D-mannitol +

5. Diaminopimelic acid in the cell wall LL-diaminopimelic acid. To summarize the above,
This strain has a cell wall of LL-diaminopimelic acid, and according to the method of the International Streptomyces project (abbreviated as ISP), the morphology of sporulating hypha belongs to section spirals (Spirales) and the spore surface is smooth. , The color of mature hyphae is gray (Gray col
or series) produces melanin-like pigments and brown pigments in the medium. The color of the basal hyphae is from pale yellow to pale brown. Examples of carbon sources include L-arabinose, D-glucose, D-fructose, sucrose, inositol, L-rhamnose, raffinose, D-mannose,
D-mannitol and D-xylose are used. Based on the above properties, ER Buuffanan and N. Gibbons, Vergis Manual of
Determinating Bacteriology (Bergey 'S Manu
al. Determinative Bacteriology) 8th edition, 1974. As a result of the search, the strain A1509 was found to belong to the genus Streptomyces. Therefore, this strain was named Streptomyces sp. A1509. The strain has been deposited at the Institute of Microbial Technology, National Institute of Industrial Science and Technology, as Micromachine Research Institute No. 12809.

The Streptomyces sp. A1509 strain used in the present invention is, for example, irradiated with ultraviolet rays, ⁶⁰ Co, etc., nitrogen mustard, nitrous acid, N-methyl-
N'-nitro-N-nitrosoguanidine (NTG), 2
-The production capacity of conkanamycins D, E, and G can be increased by commonly used mutation treatment means such as mutation treatment with a mutagenic agent such as aminopurine, transduction, transformation, and cell fusion.

To produce the concanamycins D, E and G of the present invention, a microorganism belonging to the genus Streptomyces and capable of producing conkanamycins D, E and G is cultivated in a medium and the Kanamycin D, E, G can be produced and accumulated, and then collected. The culturing method is basically similar to the culturing method of actinomycetes, but the deep culturing method by liquid culturing is usually advantageous. The medium used for the culture may be any medium containing a nutrient source utilized by the strain Streptomyces sp.

As the nutrient source, known ones conventionally used for culturing actinomycetes can be used. For example, glucose, galactose, D-xylose, carbon sources can be used.
D-fructose, sucrose, lactose, glycerol, dextrin, starch, starch syrup, soybean oil and the like can be used alone or in combination. As the inorganic and organic nitrogen sources, ammonium chloride, ammonium sulfate, ammonium nitrate, urea, sodium nitrate, peptone, meat extract, yeast extract, dry yeast, corn steep liquor, soybean powder,
Pharmamedia, oatmeal, casamino acid, pact soyton, solible vegetable protein, etc. can be used alone or in combination.

Other inorganic salts such as sodium chloride, magnesium sulfate, copper sulfate, zinc sulfate, manganese chloride, calcium carbonate, and phosphate can be added, if necessary.
Organic substances such as nucleic acids, amino acids, vitamins and inorganic substances that promote the growth of the strain and the production of chrysosporin can be appropriately added. When foaming is remarkable during culture, vegetable oils such as soybean oil and flaxseed oil and pronal 1
(Toho Chemical Co., Ltd.), Silicon KM-70 (manufactured by Shin-Etsu Chemical Co., Ltd.) or the like may be added as appropriate. The culture temperature is preferably 25 to 30 ° C., and the pH is preferably neutral to slightly acidic. In liquid culture, when the culture is usually performed for 3 to 6 days, conkanamycins D, E, and G are produced and accumulated in the cells. When the production amount in the cells reaches the maximum, the culture is stopped and the cells are separated by filtration. The desired product is purified and isolated from the obtained bacterial cells.

For the purification and isolation of this substance from the bacterial cells, the separation and purification methods generally used for isolating microbial metabolites from the bacterial cells are used. Concanamycin D,
E and G are substances that are soluble in organic solvents such as methanol, acetone, ethyl acetate, and ethanol, but are hardly soluble in water. The purification is performed by the method used for the purification of fat-soluble substances. That is, extraction with various organic solvents, silica gel chromatography, preparative high performance liquid chromatography and the like can be appropriately combined and used.

For example, the culture solution is filtered, the bacterial cells are collected and extracted with acetone. After the acetone solution was concentrated under reduced pressure, the pH was adjusted to 8
Extract with dichloromethane. The dichloromethane solution is dried over anhydrous sodium sulfate and then concentrated under reduced pressure to dryness. The oil obtained is dissolved in dichloromethane and purified by silica gel chromatography. Hexane-acetone was used as the developing solvent, and the solvent was changed stepwise (80: 20 → 60: 40) and eluted. Collect the active fractions, concentrate under reduced pressure to dryness, and then
% Dissolved in methanol, purified by preparative high performance liquid chromatography using a reversed phase column, and purified from the active fraction to obtain 3 types of colorless crystals.

The titers of conkanamycin D, E, and G in the culture and crude fractions were determined by measuring the ability of macrophage J774 to inhibit the production of cholesterol ester by the radioactivity value using [ ¹⁴ C] oleic acid. The physicochemical properties of Conkanamycin D, E, and G obtained as described above are shown below.

1) Concanamycin D (1) Appearance; colorless crystals (2) Molecular weight; FAB-MS m / z; 808 (M
⁺ ) (3) Molecular formula; C ₄₄ H ₇₂ O ₁₃ (4) Solubility; Soluble in dichloromethane, ethyl acetate, acetonitrile, acetone and lower alcohols Insoluble in water (5) Ultraviolet absorption spectrum; UVλ max nm (ε) 245 (30800 ), 285 (16000) (6) Infrared absorption spectrum; Infrared absorption spectrum measured in chloroform is shown in FIG. The maximum absorption value (wave number cm ^-1 ) is shown below. 3448, 2966, 29
33, 2878, 1718, 1686, 1618, 14
58, 1438, 1389, 1363, 1279, 12
51, 1167, 1106, 1067, 1029, 96
7,918

(7) Hydrogen nuclear magnetic resonance spectrum: measured in deuterated chloroform. Proton number Concanamycin D C2-OCH ₃ 3.52 s H3 6.39 S C4-CH ₃ 2.00 brs H5 5.63 brd (10.0) H6 2.58 m C6-CH ₃ 1.01 d (7.0) H7 4.01 brd H8 2.87 m C8-H ₂ − − C8-CH ₃ 1.10 d (7.0) C8-CH ₂ CH ₃ − C8-CH ₂ CH ₃ − H9 3.06 brd H10 2.34 m C10-CH ₃ 1.04 d (7.0) C11-H ₂ 1.77 m C12-CH ₃ 1.85 brs H13 5.82 brd (10.5) H14 6.54 dd (15.0, 10.5) H15 5.16 dd (15.0, 9.5) H16 3.82 brt (9.5) C16-OCH ₃ 3.24 s H17 5.03 brd (9.5) H18 2.14 m C18-CH ₃ 0.80 d ( 6.6) H19 3.98 brd (10.5) H20 1.72 m C20-CH ₃ 1.04 d (7.0) H22 − C22-H ₂ 1.10 m 2.33 dd (11.0, 4.0) H23 3.73 m H24 1.22 m C24-CH ₃ 0.86 d (6.2) H25 3.94 brt H26 5.22 m (15.0, 7.8) H27 5.51 dq (15.0, 6.6) C28-H ₃ 1.57 d (8.6) H1 '4.55 dd (9.0, 1.5) H2'ax 1.60 dd H2'eq 2.13 dd H3' 3.60 m H4 '3.08 brt (9.0) H5' 3.20 dq (9.0, 6.0) C6'-CH ₃ 1.27 d (6.0) OCO-NH ₂ −

2) Concanamycin E (1) Appearance; colorless crystals (2) Molecular weight; FAB-MS m / z; 837 (M
⁺ ) (3) Molecular formula: C ₄₄ H ₇₁ NO ₁₄ (4) Solubility; Soluble in dichloromethane, ethyl acetate, acetonitrile, acetone, lower alcohol Insoluble in water (5) UV absorption spectrum; UVλ max nm (ε) 245 (21300) ), 285 (14100) (6) Infrared absorption spectrum; Infrared absorption spectrum measured in chloroform is shown in FIG. The maximum absorption value (wave number cm ^-1 ) is shown below. 3448, 2967, 29
35, 2879, 1718, 1688, 1618, 14
58, 1376, 1364, 1251, 1163, 11
08, 1068, 1029, 989

(7) Hydrogen nuclear magnetic resonance spectrum: measured in deuterated chloroform. Proton number Concanamycin E C2-OCH ₃ 3.55 s H3 6.37 S C4-CH ₃ 2.05 brs H5 5.89 brd (10.0) H6 2.66 m C6-CH ₃ 1.05 d (6.2) H7 4.02 m H8 − C8-H ₂ 1.50 m 2.97 m C8-CH ₃ − C8-CH ₂ CH ₃ − C8-CH ₂ CH ₃ − H9 3.51 m H10 2.19 m C10-CH ₃ 0.80 d (7.0) C11-H ₂ 1.96 m C12-CH ₃ 1.70 d H13 5.94 brd (10.5) H14 6.36 dd (15.0, 10.5) H15 5.18 dd (15.0, 9.2) H16 3.76 t (9.2) C16-OCH ₃ 3.22 s H17 4.95 brd (9.2) H18 2.19 m C18-CH ₃ 0.77 d (6.9) H19 3.95 m H20 1.75 m C20-CH ₃ 1.03 d (7.0) H22 − C22-H ₂ 1.13 m 2.30 dd (11.0, 4.0) H23 3.74 m H24 1.24 m C24-CH ₃ 0.85 d (6.5) H25 3.93 dd (10.8, 8.0) H26 5.24 m (15.0, 8.0) H27 5.52 dq (15.0, 6.6) C28-H ₃ 1.55 d (6.6) H1 '4.53 brd (9.0) H2'ax 1.63 dd H2'eq 2.18 dd H3' 3.72 m H4 ' 4.26 t (9.0) H5 '3.34 m (9.0, 6.0) C6'-CH ₃ 1.22 d (6.0) OCO-NH ₂ 4.78 s

3) Conkanamycin G (1) Appearance; colorless crystals (2) Molecular weight; FAB-MS m / z; 660 (M
⁺ ) (3) Molecular formula; C ₃₈ H ₆₀ O ₉ (4) Solubility; Soluble in dichloromethane, ethyl acetate, acetonitrile, acetone and lower alcohols Insoluble in water (5) Ultraviolet absorption spectrum; UVλ max nm (ε) 245 (32800 ), 285 (14500) (6) Infrared absorption spectrum; Infrared absorption spectrum measured in chloroform is shown in FIG. The maximum absorption value (wave number cm ^-1 ) is shown below. 3448, 2970, 28
77, 1936, 1686, 1618, 1458, 13
76, 1356, 1248, 1105, 969

(7) Hydrogen nuclear magnetic resonance spectrum: measured in deuterated chloroform. Proton number Concanamycin G C2-OCH ₃ 3.53 s H3 6.41 s C4-CH ₃ 1.98 brs H5 5.80 brd (9.0) H6 3.60 m C6-CH ₃ 1.00 d (6.8) H7 3.70 brd. H8 2.89 m C8-H ₂ − − C8-CH ₃ 1.16 d (6.6) C8-CH ₂ CH ₃ − C8-CH ₂ CH ₃ − H9 3.08 brd (10.0) H10 2.36 m C10-CH ₃ 1.00 d (6.7) C11-H ₂ 1.49 m C12- CH ₃ 1.60 brs H13 5.84 brd (10.8) H14 6.49 brq (15.8, 10.8) H15 5.19 dd (15.8, 9.6) H16 3.73 t (9.6) C16-OCH ₃ 3.22 s H17 5.09 brd (9.6) H18 2.09 m C18-CH ₃ 0.92 d (6.0) H19 3.72 dd H20 2.86 m C20-CH ₃ 1.10 d (6.7) H22 6.26 brd (15.8) C22-H ₂ − − H23 6.85 dd (15.8, 8.0) H24 2.36 m (8.0, 6.7) C24 -CH ₃ 0.99 d (6.7) H25 3.87 t (7.2) H26 5.41 brd (15.5, 7.2) H27 5.66 dq (15.5, 6.2) C28-H ₃ 1.69 brd (6.2) H1'-H2'ax-H2'eq- H3 '- H4' - H5 ' - C6'-CH 3 - OCO-NH 2 -

The compound of the present invention is expected as a drug such as a therapeutic agent for arteriosclerosis based on the inhibitory effect on the formation of cholesterol ester associated with the incorporation of oxidized LDL which causes foaming of macrophages as described below. When the compound of the present invention is used as a medicine, it is administered alone or as an excipient, such as an injection, an oral preparation, a suppository and the like. Any excipient may be used as long as it is pharmacologically acceptable, and its type and composition depend on the administration route and administration method.
For example, water, alcohol or soybean oil, peanut oil, mineral oil or other animal or vegetable oil or synthetic oil can be used as the liquid excipient. As the solid excipient, saccharides such as maltose and sucrose, various amino acids, cellulose derivatives such as hydroxypropyl cellulose, organic acid salts such as magnesium stearate and the like can be used.

In the case of injectable preparations, physiological saline, various buffer solutions, sugar solutions such as glucose, inositol and mannitol, and glycol solutions such as ethylene glycol and polyethylene glycol are desirable as excipients. In addition, lyophilized with inositol, mannitol, glucose, mannose, maltose, sucrose and other excipients of amino acids such as phenylalanine, a suitable solvent for injection at the time of administration, for example, sterile water, glucose solution. Alternatively, it may be dissolved in an electrolyte solution, an amino acid solution or the like and administered intravenously or intramuscularly.

In the case of oral preparations, it is preferable to form tablets, capsules, powders, granules, liquids, dry syrups, etc. together with the liquid or solid excipients.
In addition, as a pellet, a transdermal agent, a topical agent such as a mucosal agent may be used. The content of the present compound in the preparation is usually 0.001 to 1% by weight, preferably 0.01 to 1% by weight.
It is 0.1% by weight. For example, in the case of an injection, it is usually 0.01 to 0.05% by weight. In the case of an oral preparation, it is 0.005 to 1% by weight, preferably 0.05 to 0.5% by weight, and the balance is an excipient. The dose is determined according to the patient's age, body weight, symptom, therapeutic purpose, etc. In general, parenteral administration is 0.1 to 5 μg / Kg / day, and oral administration is 0.5 to 30 μg / Kg / day.

[0030]

[Action]

Test 1 Measurement of [ ¹⁴ C] oleic acid uptake inhibitory activity into cholesterol ester Mouse macrophage J774 was incubated at 37 ° C. for 5 minutes in 0.3 ml of a medium having each of the following compositions.
Incubate in air containing% CO ₂ . The basic medium composition is 100 units / ml of penicillin in Dulbecco's medium.
0 μg / ml streptomycin, 10% fetal bovine lipoprotein serum and 100 μM [ ¹⁴ C] oleic acid (1 ×
10 ⁴ dpm / nmol) -albumin complex. It is assumed that 1) 100 μg protein / ml oxidized LDL 2) 100 μg protein / ml oxidized LDL and inhibitor sample 3) inhibitor sample were added to this. After culturing, the cells were collected by centrifugation, washed with physiological phosphate buffer, and the collected cells were washed with 0.5
Extract twice with ml of n-hexane-isopropanol (3: 2) solution. Collect the extracts and concentrate under reduced pressure to dryness.
After dissolving in 1 n-hexane, TLC analysis is performed. The cholesterol ester forming ability is measured from the radioactivity of the fraction corresponding to cholesterol ester. The inhibitory activity is calculated by the rate of production inhibition of [ ¹⁴ C] oleic acid on cholesterol ester (Methoclsin Enzynology 9
8 , 241-260, 1983). The results of inhibitory activity are shown in Table 2.

Table 2 Inhibitor of Cholesterol Esterification by Conkanamycin D, E, G [ ¹⁴ C] Oleic Acid Uptake Inhibition (IC ₅₀ ) Conkanamycin D 8 nM Conkanamycin E 17 nM Conkanamycin G 720 nM

Test 2 Method for measuring inhibitory activity of lysosomal acidification 1) Method for measuring lysosomal acidification of macrophage J774 The lysosome preparation method for macrophages is performed as follows. 100 units / ml penicillin, 100μ
5 ml of FITC-dextran was added to Dulbecco's medium containing g / ml streptomycin, 10% serum to a concentration of 2 mg / ml, and the mixture was incubated at 37 ° C. for 5 minutes, and immediately replaced with the above medium containing no FITC-dextran. Incubate at 30 ° C for 30 minutes. After washing the cells 3 times with PBS, 10 mM triethanolamine, 10 mM acetic acid, 1
Buffer consisting of mM EDTA and 250 mM sugar (pH
Scrape in 7.4) and collect. FIT after treatment by the method of Marsh et al. (J. Cell Biol, 104 , 875-886, 1987)
Collect the C-labeled lysosomal fraction. 20 mM
Tris-trimethylamine (pH 7.4), 100 mM
20 with buffer containing salt, 50 mM KCl, 5 mM MgCl ₂
Dispense to 0 μg protein. 2m after leaving at room temperature for 10 minutes
Add M adenosine triphosphate (potassium salt). FIT
The acidification ability of lysosomes labeled with C-dextran is measured according to the method of Schmidt et al. (Cell 52 , 73-83, 1988).

2) Method for measuring acidification ability of rat liver lysosome The preparation of lysosome from rat liver is performed according to the method of Schneider et al. (Methods in Enzy-mology 157 , 591-594 1988), and the measurement is performed by Moriyama et al. (J. Bioche-mistry
95 , 995-1007 1984).

The inhibitory activity was found to correlate substantially with the acidification ability of macrophage lysosomes and rat liver lysosomes, so that concanamicin D, E and G were replaced with rat liver lysosomal acidification ability inhibitory activity. .. The results of inhibitory activity are shown in Table 3.

Table 3 Substance Name Lysosome Acidification Inhibitory Activity (IC ₅₀ ) Conkanamycin D 0.085 nM Conkanamycin E 0.038 nM Conkanamycin G 1.500 nM

[0036]

As is clear from the above results, the concanamycins D and E of the present invention strongly inhibit the acidification of lysosomes, strongly inhibit the formation of cholesterol ester in macrophages, and concanamycin G is weak. However, since it has the same action, it can be expected as a novel therapeutic agent for arteriosclerosis whose mechanism of action is to suppress foaming of macrophages. Examples of the present invention will be shown below, but this is merely an example and does not limit the present invention in any way, and various modifications are possible.

Example 1 1) Fermentation In a 500 ml Sakaguchi flask, glucose 1%, soluble starch 3%, peptone 1%, defatted soybean flour 1%, yeast extract 0.5%, calcium carbonate 0.2%, CB442.
0.02% 80 ml dispensed 3 pieces 120
Autoclave sterilization was performed at 20 ° C for 20 minutes. One platinum loop of Streptomyces sp. A1509 was inoculated into this and cultured at 200 strokes / min at 28 ° C for 4 days on a reciprocal shaker to obtain a seed culture solution. On the other hand, 15 liters of the above-described medium having the same composition is charged into a 30 liter jar and sterilized at 120 ° C. for 20 minutes to prepare two groups. To this, 240 seed cultures were added, 250 per minute
The cells were cultivated at 10 ° C. and aerated at 28 ° C. for 4 days under deep agitation. The obtained culture solution (36.5 liters) is filtered to collect bacterial cells.

2) Purification After washing the bacterial cells with water and extracting with 7.5 liters of acetone,
Concentrate under reduced pressure to 4.1 liters. After adjusting the pH to 8.0, the mixture was extracted with 10.3 liters of dichloromethane, the organic solvent layer was dried over anhydrous sulfuric acid, and then concentrated to dryness under reduced pressure to give 21.7 g.
Of oily substance was obtained. The oily substance was dissolved in dichloromethane, charged on a silica gel column (56 × 360 mm), washed with hexane, and then hexane-acetone (90:
1), hexane-acetone (80:20) and hexane-acetone (60:40). Collect the active fractions in the eluate of hexane-acetone (60:40),
Inertosil PREP-ODS column for adjustment (30 x 2
50 mm) HPLC is performed. Acetonitrile-water (5
5:45) to develop concanamycin D
3.6 mg, conkanamycin E 1.6 mg and conkanamycin G 4.1 mg were each isolated.

[Brief description of drawings]

FIG. 1 shows an infrared absorption spectrum of concanamycin D (measured in chloroform).

FIG. 2 shows an infrared absorption spectrum of concanamycin E (measured in chloroform).

FIG. 3 shows an infrared absorption spectrum of concanamycin G (measured in chloroform).

Continuation of front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location C07D 407/14 313 8829-4C C07H 17/04 C12P 19/62 7432-4B // (C12P 17/08 C12R 1 : 465) (C12P 19/62 C12R 1: 465) 7804-4B

Claims (4)

[Claims] 1. Formulas (1) and (2): 2. Formula (3): Concanamicin G, a new pharmacologically active substance represented by 3. A microorganism belonging to the genus Streptomyces and having the ability to produce the pharmacologically active substances conkanamycin D, conkanamycin E and conkanamycin G is cultured in a medium, and the pharmacologically active substance conkanamycin D, A method for producing the pharmacologically active substances conkanamycin D, conkanamycin E and conkanamycin G, which comprises producing and accumulating conkanamycin E and conkanamycin G, and then collecting these 4. A therapeutic agent for arteriosclerosis containing a pharmacologically active substance, concanamycin D, concana machine E or concanamycin G as an active ingredient.