JPH0462318B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD The present invention relates to a novel substance produced by a microorganism belonging to the genus Nocardiopsis and having an effect of inhibiting histamine release, and a method for producing the same. PRIOR ART AND PROBLEMS TO BE SOLVED BY THE INVENTION Many compounds such as disodium chromoglycate are known as compounds that inhibit histamine release, but substances that inhibit histamine release from microbial cultures are No examples have been found. With the aim of providing useful new physiologically active substances, we conducted research on the products of numerous microorganisms obtained from the natural world, and as a result, we discovered that cultures of newly isolated microorganisms have the effect of inhibiting histamine release. We have discovered the fact that physiologically active substances are produced. Then, the substance is isolated and purified from the culture,
After examining its physical and chemical properties, it was discovered that it is a new substance. Hereinafter, this substance will be referred to as KT5556. Means for solving the problem The new substance KT5556 is represented by the following formula (1). The physical and chemical properties of KT5556 are as shown below. Properties: Pale yellow powder Melting point: 262-266℃ (decomposed) Specific rotation: [α] ²⁰ _D = +97.1° (c = 0.56, DMF) Solubility: Easy to dissolve: alkaline water, DMSO (dimethyl sulfoxide), DMF (N,N dimethylformamide) Soluble: methanol, ethanol Insoluble: chloroform Color reaction: Positive for iodine, negative for ferric chloride and Lydon Smith reactions Ultraviolet absorption spectrum (aqueous solution) Figure 1 Infrared absorption Spectrum (KBr) 3430, 1720, 1665, 1632, 1591, 1460, 1313,
1279, 1235, 1133, 740cm ^-1 mass spectrum: Mass spectrum of this substance (SI
-MS) gives the following molecular ion. m/z 453 (M ⁺ ) Elemental analysis: H4.35, C68.87, N9.01 (actual value) H4.22, C68.87, N9.27 (calculated value) as C ₂₆ H ₁₉ N ₃ O ₅ ^1H -NMR (DMSO- _d6 ) δ9.24 (d, 1H, J=7.7Hz), 8.1-7.85 (m,
3H), 7.55-7.25 (m, 4H), 7.12 (dd, 1H, J=
4.9, 7.4Hz), 5.04 (d, 1H, J=17.5Hz), 4.99
(d, 1H, J=17.5Hz), 3.38 (dd, 1H, J=7.4,
13.8kHz), 2.23 (S, H), 2.00 (dd, 1H, J=
4.9, 13.8Hz) ^13C -NMR (DMSO- _d6 ) δ174.0, 171.8, 139.9, 136.8, 132.9, 128.3,
125.6, 125.3, 124.9, 124.1, 123.9, 122.6,
121.1, 120.3, 119.5, 119.4, 115.7, 114.8,
114.5, 109.0, 99.2, 85.0, 84.4, 45.4, 42.5,
22.8 Based on the above data, KT5556 was found to be a new compound. Next, Table 1 shows the Rf values of KT5556 in thin layer chromatography using various developing agents. The detection is
This was done by irradiating ultraviolet light at 2537 Å.

[Table] Development: room temperature, ascending method, 1 hour
Since KT5556 is a weakly acidic substance, it can form a water-soluble base addition temperature. Such salts include ammonium salts, alkali metal salts such as lithium, sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases such as triethylamine, morpholine, piperidine and dicyclohexylamine. Includes salts and salts with amino acids such as arginine and lysine. Although non-toxic pharmacologically acceptable salts such as those exemplified above are preferred, other salts are also useful in the isolation and purification of the product. KT5556 has an inhibitory effect on histamine release. Next, the manufacturing method of KT5556 will be explained.
KT5556 belongs to the genus Nocardiopsis,
Cultivate microorganisms capable of producing KT5556 in a medium,
It is produced by producing and accumulating KT5556 in a culture and collecting KT5556 from the culture. The KT5556-producing microorganism may be any microorganism that belongs to Nocardiopsis and has the ability to produce KT5556. A specific preferred example is Nocardiopsis sp. K-290 strain isolated from grassland soil in Tama City, Tokyo. The strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as FAIKEN Article No. 696 (deposit date: January 19, 1985). The results of observing and testing the morphological characteristics and physiological properties of the K-290 strain are as follows. The fungus exhibits fair or good growth on a variety of natural and synthetic media generally used for the growth of actinomycetes. Aerial mycelial growth is relatively good on synthetic media, and the color is white or beige. The color tone of the basal hyphae is yellow-gray to beige, but becomes reddish-brown on tyrosine agar, yeast/malt agar, and peptone/yeast iron agar. No soluble dye formation is observed. There is also no production of melanin-like pigments. Spores are simply branched from aerial hyphae, with 20
It forms a chain of more than one piece and has a bent shape. The morphology of the spores is spherical to cylindrical, and the size is 0.6 to 0.8 x 0.7.
The diameter of the spores is 1.0 μm, and the spore surface is smooth or warty when observed using an electron microscope. No flagella are observed and no sporangia are found. Growth status on various media The characteristics of growth and color when cultured on various media at 28°C for 20 days are shown below. Color display is
Color Harmony Manual (Container
Corporation of America). Note that no pigment was produced in any of the media. (1) Syuclor/Nitrate agar medium Growth: Normal Aerial hyphae: Medium/Natural (2dc) Base hyphae color: Pearl (3ba) (2) Glucose/Asparagine agar medium Growth: Good, raised Aerial hyphae: Moderate to good, pearl (3ba) to beige (3ge) Base mycelium color: light ivory (2ca) (3) Glycerol/asparagine agar medium Growth: normal Aerial mycelium: good, pale blue (14ca) Base Color of live mycelia: Parchment (1cb) to dark cobalt gray (2ih) (4) Starch agar medium Growth: Normal Aerial mycelium: Moderate to good, pearl (3ba) Color of base mycelium: Biscuit (2ec ) (5) Tyrosine agar medium Growth: Good Aerial mycelium: Good, bisque (3ec) Base mycelial color: Nude tan (4gc) (6) Nutrient agar medium Growth: Average Aerial mycelium: Poor, bisque (3ec) ) Base mycelia color: nude tan (4gc) (7) Yeast/malt agar medium Growth: good, pleated Aerial mycelia: medium, pearl (2ba) Base mycelia color: amber (3lc) to clove・Brown (3pl) (8) Oatmeal agar medium Growth: Poor, granular Aerial hyphae: None Base hyphae color: Light wheat (2ea) (9) Peptone yeast Iron agar medium Growth: Good, pleated Aerial Hyphae: None Color of basal mycelium: Camel (3ie) Various physiological properties The physiological properties of strain K-290 are shown below. Regarding the growth temperature range, the results after 2 days, skim milk,
For effects on gelatin and cellulose, see 28
℃, after 1 month, and others after 3 weeks at 28℃. (1) Assimilation of carbon sources D-glucose, i-inositol, D-fructose, and L-rhamnose are assimilated, but L-rhamnose is assimilated.
- Arabinose, D-xylose, D-mannitol, sucrose and raffinose are not assimilated. (2) Liquefaction of gelatin: Not liquefied (3) Coagulation and peptonization of skim milk: Peptonization occurs, but coagulation is slight. (4) Cellulose decomposition effect: None (5) Starch hydrolysis effect: Yes (6) Growth temperature range: 10-42℃ (7) Melanin-like pigment production: None Strain K-290 is a mesophilic bacterium. Yes, and forms aerial mycelium on agar medium. Spores are formed as a curved chain of 20 or more from sporophores that simply branch from aerial hyphae, and are not motile. In the basal hyphae, branching was frequently observed, but no clear division was observed. In the hydrolytic analysis of the whole bacterial cell, meso-type diaminopimelic acid was detected, and characteristic sugars such as majulose were not detected in the sugar chamber. Therefore, the strain was named Nocardiopsis sp.K-290. When culturing the microorganism, the usual culture method used for culturing actinomycetes is applied. The medium to be used may be either a natural medium or a synthetic medium, as long as it contains appropriate amounts of carbon sources, nitrogen sources, inorganic substances, etc. that can be assimilated by the bacteria. Carbon sources include glucose, fructose, stabilose, starch, dextrin, mannose,
Carbohydrates such as maltose and molasses, organic acids such as citric acid, malic acid, acetic acid, and fumaric acid, amino acids such as glutamic acid, or glycerol are used. Nitrogen sources include ammonium salts such as ammonium chloride, ammonium sulfate, ammonium nitrate, and ammonium phosphate, amino acids such as aspartic acid, glutamine, cystine, and alanine, urea, peptone, meat extract, yeast extract, dried yeast, corn steep liquor, Soybean flour, cottonseed meal, soybean casein, casamino acids, firma media, etc. are used. Inorganic substances include potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, ferrous sulfate, manganese sulfate, cobalt sulfate, zinc sulfate, calcium pantothenate, ammonium molybdate, potassium aluminum sulfate, barium carbonate,
Calcium carbonate, cobalt chloride, salt, etc. are used. Other substances that promote bacterial growth or KT5556 production, such as vitamins and thiamine, can be added to the medium as necessary. If the microorganism used requires specific substances for growth, it is of course necessary to add substances necessary for growth. Culture is carried out by shaking culture method, aeration stirring culture method, etc.
It is carried out at a temperature of 20-40°C and a pH around neutrality. After 3 to 15 days of culture, the accumulation of KT5556 reaches its maximum and the culture is completed. When KT5556 accumulated in a culture is isolated and collected from the culture solution, a conventional method for collecting physiologically active substances from the culture solution is applied. That is, removal of bacterial cells by filtration, centrifugation, etc., removal of active substances by column chromatography or thin layer chromatography using adsorption resins, silica gel, silanized silica gel, aluminum, cellulose, diatomaceous earth, magnesium silicate, gel filtration agents, etc. KT5556 is isolated by adsorption/desorption treatment. An example of isolating KT5556 from culture fluid is as follows. The bacterial cells are removed by filtering or centrifuging the culture solution. The resulting filtrate or supernatant is treated with an adsorption resin, Diaion HP-10 (manufactured by Mitsubishi Chemical Industries, Ltd.), to adsorb the active substance to the resin.
Then, it is eluted with a suitable solvent such as acetone, and the eluate is concentrated under reduced pressure to remove the solvent and form an aqueous solution. Next, add this aqueous solution to Diamond Ion.
Treat with HP-20SS (manufactured by Mitsubishi Chemical Industries, Ltd.) to adsorb active substances. Then, elute with a suitable solvent such as methanol, concentrate the eluate under reduced pressure, and transfer to LH-20.
(manufactured by Pharmacia) chromatography (developing solvent: methanol). The eluate obtained was then concentrated under reduced pressure, and the HP-20SS column chromatography was performed again from 40% (V/V) to 60% (V/V).
Elution is performed using a linear concentration gradient of methanol up to V). Collect the fractions containing KT5556, concentrate under reduced pressure, and use 1-50% as a developing solvent.
(V/V) Reversed phase silica gel using methanol (Merck Rover Column RP-8, Art No.
11804) Perform chromatography. The resulting eluate is then concentrated under reduced pressure to obtain a pale yellow powder of KT5556. Detection of KT5556 during the above purification process was performed using silica gel thin layer chromatography, followed by iodine reaction or
This was carried out using a 2537 Å ultraviolet irradiation method. Examples are shown below. Example 1 Nocardiopsis sp. K- as a seed fungus
Use 290. The bacteria were used as a first-class medium, glucose 1.0 g/dl, soluble starch 1.0 g/dl, meat extract 0.3 g/dl, yeast extract 0.5 g/dl, Bactotryptone (manufactured by Difco) 0.5 g/dl, and carbonic acid. 300 with a composition of calcium 0.2g/dl and PH7.2-7.4
Inoculate 40 ml of seed medium in a ml Erlenmeyer flask. Then, culture with shaking at 30°C until the bacteria grow sufficiently.
Inoculate 4 ml of this seed culture into 40 ml of the following fermentation medium in a 300 ml Erlenmeyer flask. Fermentation medium: glucose 0.5g/dl, soluble starch 3.0g/dl, soy flour 3.0g/dl, corn steep liquor 0.5g/dl, yeast extract 0.5g/dl
dl, calcium carbonate 0.3g/dl, PH7.2-7.4. Cultivation is carried out at 30°C for 9 days with aeration and agitation. After culturing, the culture solution is continuously centrifuged (15,000 rpm).
8.4 of the supernatant liquid was passed through column No. 1 packed with Diaion HP-10 (manufactured by Mitsubishi Chemical Industries, Ltd.).
After washing with water, 2 with 50% (V/V) methanol, and 1 with 30% (V/V) acetone water, 1
Elute with 60% (V/V) acetone water. All the eluate was collected and concentrated to dryness to obtain 1.6 g of oil. This was suspended in water and filled with 84ml of Diaion HP-20SS (Mitsubishi Chemical Industries, Ltd.)
Co., Ltd.) is supplied to the top of the column. After washing with 200 ml of water and an additional 200 ml of 40% (V/V) methanol,
Elute with 140 ml of 60% (V/V) methanol.
All the eluted fractions were collected and concentrated to dryness under reduced pressure to give approx.
379 mg of oily substance are obtained. 50% of 5ml (V/
V) 100 ml of Sephadex dissolved in methanol and filled with 50% (V/V) methanol
It is supplied to the top of an LH20 (manufactured by Pharmacia) column and eluted using a solvent with the same composition as the filling solvent as a developing solvent. When the eluted fraction is collected in 3 g portions, KT5556 is eluted in fraction numbers 77 to 91. These fractions were collected and concentrated to dryness under reduced pressure, resulting in approx.
47.4 mg of brown powder is obtained. 5ml of this powder
Dissolved in 40% (V/V) methanol, 40% (V/V)
V) Adsorb onto a 33 ml HP-column packed with methanol. The developing solvent is 40% (V/V) methanol 150ml and 60% (V/V) methanol 150ml.
Elute with a linear gradient of methanol from 40% (V/V) to 60% (V/V). When the eluted fractions are collected in 3 g portions, KT5556 is eluted in fraction numbers 10 to 15. The fractions are collected and concentrated to dryness under reduced pressure to obtain about 40 mg of brown powder. Approximately 5 ml of this powder at 50% (V/V)
Silanized silica gel RP dissolved in methanol and equilibrated with 50% (V/V) methanol
-8 (size B, manufactured by Merck & Co., Ltd., Art 11804) and eluted using a solvent having the same composition as the solvent used for equilibration as a developing solvent. When the eluted fraction is separated into 5g portions, fraction numbers 33 to 40 are KT5556.
is eluted. When these fractions were collected and concentrated to dryness, approx.
29.6 mg of pale yellow powder is obtained. Note that detection of KT5556 during the above powder-making process was performed by silica gel thin layer chromatography, followed by iodine reaction or 2537 Å ultraviolet irradiation. Example 2 Method for forming the sodium salt of KT5556 1.2 g of KT5556 was dissolved in 400 ml of 0.1N NaHCO ₃ and passed through a 46 ml column filled with HP-20SS (manufactured by Mitsubishi Chemical Industries, Ltd.) using water. . After washing with 200 ml of water, elute with 60% (V/V) methanol. When the elution fractions are collected and freeze-dried, the amount is approximately 1.2 g.
The sodium salt of KT5556 is obtained. obtained
The sodium salt of KT5556 is approximately 1.1mg per 1ml of water.
It is soluble in a proportion of Next, the histamine release inhibiting effect of KT5556 will be explained using experimental examples. Experimental example Effect on histamine release from rat peritoneal exudate cells (1) Preparation of rat peritoneal exudate cell suspension and histamine release Rats weighing 150 to 180 g were sacrificed by exsanguination under dry ether anesthesia using the method of Sullivan et al. [J.
Mast cell medium prepared according to Immunol. 114 , 1473, (1975)]
(abbreviated as MCM, composition: 150mM NaCl, 3.7mM
KCl, 3mM _Na2HPO4 , _{3.5mM KH2PO4 ,}
1mM CaCl ₂ , 5.6mM glucose, 0.1% bovine serum albumin, 10U/ml heparin), 6ml/
The animal was injected intraperitoneally. After 2 minutes of abdominal surgery, the abdomen was opened and intraperitoneal exudate was collected. After centrifuging the exudate collected from 6 animals at 4°C and 100 x g for 5 minutes, an appropriate amount of water-cooled MCM was added to the sediment and washed three times, and the final mast cell count was approximately 3 x 10 ⁴ cells/ml. A cell suspension (peritoneal exudate cells, abbreviated as PEC) was prepared. Note that mast cells were identified by staining intracellular granules with 0.05% toluidine blue. 1 ml of PEC obtained in this way was heated at 37°C.
After pre-incubating for 10 minutes, 0.1 ml of test drug solutions of various concentrations were added and incubated for 10 minutes.
0.1 ml each of concanavalin A and 1000 μg/ml were added and incubated for an additional 15 minutes. After stopping the reaction by adding 3 ml of ice-cold physiological saline, the mixture was centrifuged at 1100 xg at 4°C for 10 minutes to obtain a supernatant and a precipitate. The amount of histamine in the supernatant and sediment was determined by Komatsu's method [Allergy 27 , 67 (1978)]
It was measured by fluorescence method according to the following. Histamine release rate was expressed as a percentage of the amount of histamine in the supernatant relative to the total amount of histamine. In addition, the histamine release inhibition rate of the test drug solution was calculated using the following formula. Release inhibition rate (%) = (1 - histamine release in the presence of drug - spontaneous release / histamine release in the absence of drug - spontaneous release) x 100 (2) Experimental results

[Table] As shown in Table 2, KT5556 inhibited histamine release in a concentration-dependent manner. Effects of the Invention KT5556 has an inhibitory effect on histamine release.

[Brief explanation of drawings]

Figure 1 shows the ultraviolet absorption spectrum of KT5556. The solid line is in 0.1N HCl, the dotted line is in water (neutral),
The dashed-dotted line represents the results measured in 0.1N NaOH. The concentration of KT5556 in each of the above media is 4.3μ
g/ml.

Claims (1)

[Claims] 1. A novel substance KT5556 represented by the following formula (1) and its salt