JPS61176531A - Novel substance kt5556 and preparation thereof - Google Patents

Abstract

NEW MATERIAL:KT5556 having the following characteristics. Appearance, pale yellow powder; melting point, 262-266 deg.C (decomposition); specific rotation, [alpha]<20>D=97.1 deg. (C=0.56, DMF), solubility,soluble easily in alkaline water, DMSO and DMO, soluble in mathanol, etc., and insoluble in chloroform, etc.; color reactions, positive to iodine reaction and negative to ferric chloride reaction and Reyden-Smith reaction; elemental analysis, H4.35, C68.71, N9.01 (C26H19N3O5)]. USE:Agent for suppressing the isolation of histamine. PREPARATION:A microbial strain belonging to Nocardiopsis genus and capable of producing KT5556 (e.g. Nocardiopsis sp. K-290, FERM BP-696) is cultured in a medium (containing proper amounts of carbon source, nitrogen source, inorganic components, etc.) at 20-40 deg.C for 3-15 days under nearly neutral condition under shaking. The microbial cells are removed from the culture liquid by filtration, centrifugal separation, etc., and the objective KT5556 is isolated by the adsorption treatment, etc., of the liquid and collected.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application 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.

Problems to be Solved by the Prior Art and the Invention Many compounds such as disodium cromoglycate are known as compounds that have the effect of inhibiting histamine release.
No substance has been found to have an inhibitory effect on histamine release from microbial cultures.

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 the culture of newly isolated microorganisms has the effect of inhibiting histamine release. We have discovered the fact that physiologically active substances are produced.

Subsequently, the substance was isolated and purified from the culture, and its physicochemical properties were investigated, and it was found to be a new substance. Hereinafter, this substance will be referred to as KT5556.

Means to solve problems The physicochemical properties of KT5556 are as shown below.

Properties: Pale yellow powder Melting point: 262-266℃ [decomposition] Specific rotation: [
α]”=+97.1° (C=0.56. DMF) Solubility: Easy Solubility: Alkaline water, DMSO (dimethyl sulfoxide), DMF (N, N dimethylformamide) Poor Solubility: Methanol, ethanol Insolubility: 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.
740 cl' Mass Spectrum The mass spectrum (SI-MS) of two substances gives the following molecular ions.

m/z 453 (Ma Elemental analysis: H4,35, C68,71, N 9.01
(Actual measurement value) H4,22 as C*sH+sNs○5
,C68,87,N 9.27 (calculated value)')I-
NMR (DMSO-da) δ9.24 (d, 1H, J=7.7Hz), 8.
1-7.85 (m.

3H), 7.55-7.25 (n, 4)1),
7.12 (dd, 1H, J=4, 9.7.4Hz)
, 5.04 (d, 1H, J=17.5Hz).

4,99 (d, 1)1. J=17.5Hz),
3.38・(dd, E, J=7.4, 13.8Hz
), 2.23 (s,, H), 2.00 (dd
, L.H.

J=4,9. 13.8Hz) I3C-NMR (DMSO-da) δ 174, 0. 171.8. 139.9. 13
6.8. 132.9°128.3. 125.6.
125.3. 124, 9. 124, 1. 123.
9°122.6. 121.1. 120J, 11
9.5. 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. Detection was performed by irradiating 2537 people with ultraviolet light.

Table 1 Silica gel thin layer chromatography of KT5556 1
Methanol:water=8:2 (V/V)
0.552 Me9) -f&: Water: Ryou
*&A=63:27:10 (V/V)
0.6 1 thin layer: Reversed phase silica gel plate RP1
8 (Merck & Co. Art 15685) Development: Room temperature, ascending method, 1 hour Since KT5556 is a weakly acidic substance, it can form water-soluble base addition salts. As such salt,
Ammonium salts, lithium, sodium.

Alkali metal salts, such as potassium salts, calcium.

Included are alkaline earth metal salts such as magnesium salts, salts with organic bases such as triethylamine, morpholine, piperidine, dicyclohexylamine, and salts with amino acids such as arginine and lysine. Non-toxic, pharmacologically acceptable salts such as those exemplified above are preferred;
Other salts are also useful in purification.

KT5556 has an inhibitory effect on histamine release.

Next, a method for manufacturing KT5556 will be explained. KT55
56 belongs to the genus Nocardiopsis, and a microorganism capable of producing KT5556 is cultured in a medium, and KT5 is added to the culture.
KT5556 is produced and accumulated, and KT5556 is collected from the culture.

KT5556-producing microorganisms belong to the genus Nocardiopsis. Any microorganism may be used as long as it has the ability to produce KT5556. As a specific preferred example, Nocardiopsis sp. (NOCardiOpSiSsp) isolated from the soil of a grassland in Tama City, Tokyo.
-290 stocks are listed. The strain was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, as deposit number 696 (deposit address: 19).
It has been deposited as (January 19, 1985).

Observing the morphological characteristics and physiological properties of the K-290 strain
The test results are as follows.

Internuclear cells generally show fair or good growth on a variety of natural and synthetic media used for the growth of actinomycetes. Aerial hyphae colonization is relatively good on synthetic media, and the color is white or page two. The color of long-growing hyphae is yellowish-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.

The spores form a chain of 20 or more at the tip, which is simply branched from the aerial hyphae, and are curved. The shape of the spores is spherical to cylindrical, and the size is 0.6-0.8 x 0.7-1.0μ.
m, and the spore surface observed by electron microscopy is smooth (sm
ooth) or warty.

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 Co1or
)larmony Manual (Contain
erCdrporation of America
According to the color classification according to ), no pigment was produced in any of the media.

(1) Synicrose/Nitrate Agar Medium Growth: Normal Aerial Hyphae: Medium/Natural (2dc) Long Mycelia Color: Pearl (3ba) (2) Glucose/Asparagine Agar Medium Growth:
Good, raised aerial hyphae: Moderate to good, pearl (3ba) to beige (3gc) Color of long hyphae: nirite/aibo! J-(,2ca)(3
)Glycerol-asparagine agar medium Growth: Normal aerial mycelium 2 Good, Veil Blue (14c a)
Color of long-growing hyphae: birchmen) (lcb) to dark cobalt gray (2ih) (4) Normal aerial hyphae grown on starch agar medium: moderate to good, pearl (3ba) Color of long-growing hyphae: biscef) (2ec) ) (5) Growth on tyrosine agar medium: Good Aerial mycelium: Good, Color of long-growing mycelia of Bistu (3ec):
Nude tan (4gc) (6) Nutrient agar medium Growth: Normal Aerial hyphae: Poor, Bistu (3ec) Long hyphae color:
Nude Tan (4gc) (7) Yeast/malt agar medium Growth: Good, pleated aerial hyphae: Medium, pearl (2ba) Color of long hyphae:
Amber (3ic) ~ Clove Brown (3Pjri (8) Growth on oatmeal agar medium: Poor 9 granular aerial hyphae: None Color of long hyphae Nirite Aibo'J- (26a) (9)
Peptone yeast on iron agar medium Growth: Good, pleated aerial hyphae: None Color of long hyphae: Nicamel (3ie) Various physiological properties The physiological properties of strain -290 are shown below. Regarding the growth temperature range, the results after 2 days are skimmed milk.

Regarding the effect on gelatin and cellulin, 28t, 1
Observation results were shown after 3 weeks at 28° C. for the others.

(1) Assimilation of carbon sources D-glucose, i-inositol, D-fructose, and L-rhamnose are assimilated, but L-arabinose and D
- Xylose, D-mannitol, sinicrose and raffinose are not assimilated.

(2) Liquefaction of gelatin: Not liquefied (3) Coagulation and peptonization of skim milk: Peptonization, but only slight coagulation.

(4) Decomposition of cellulose: None 5) Hydrolysis of starch: Yes (6) Growth temperature range: 10-42℃ (7) Production of melanin-like pigment: None -290 strain grows at medium temperature It is a fungus that forms aerial hyphae on agar media. The spores are formed as a curved chain of 20 or more from the sporophyte, which is simply branched from the aerial hyphae, and no motility is observed. In long-growing hyphae, branching was frequently observed, but no clear division was observed.

In hydrolytic analysis of whole bacterial cells, men-type diaminopimelic acid was detected, and characteristic sugars such as majurose were not detected. Therefore, the strain was classified as Nocardiopsis.
It was named 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 bacteria.

As carbon S sources, carbohydrates such as glucose, fructose, stahyrose, starch, textrin, mannose, 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. .

Ammonium chloride, ammonium sulfate,
Ammonium salts such as ammonium nitrate and ammonium phosphate, amino acids such as aspartic acid, glutamine, cystine, and alanine, urea, heptone, meat extract, yeast extract, dried yeast, corn steep liquor, soybean flour, cottonseed meal, soybean casein , casamino acids, Pharmamedia, 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, common salt, etc. are used.

Other substances that promote the growth of bacterial cells or the production of KT5556, 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 using shaking culture method, aeration agitation culture method, etc.
It is carried out at a temperature of 0°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, bacterial cells are removed by filtration, centrifugation, etc., and active substances are removed by column chromatography or thin layer chromatography using adsorption resins, silica gel, silanized silicage nope aluminum, cellulose, diatomaceous earth, magnesium silicate, gel filtration agents, etc. KT by adsorption/desorption treatment etc.
5556 is isolated.

One example of isolating KT5556 from culture fluid is as follows.

The bacterial cells are removed by filtering or centrifuging the culture solution. The obtained filtrate or supernatant is treated with an adsorption resin, Diaion HP-10 (manufactured by Mitsubishi Chemical Corporation), 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, this aqueous solution is treated with Diaion HP-2033 (manufactured by Mitsubishi Chemical Industries, Ltd.) to adsorb the active substance. Then, elution is performed with a suitable solvent such as methanol, the eluate is concentrated under reduced pressure, and chromatography is performed using LH-20 (manufactured by Pharmacia) (developing solvent: methanol). The resulting eluate was then concentrated under reduced pressure and subjected to HP-20SS column chromatography again at a concentration of 40% (V/V) to 60% (V/V).
This is carried out by elution using a linear concentration gradient of methanol up to /V). Fractions containing KT5556 are collected and concentrated under reduced pressure, and then subjected to reverse phase silica gel chromatography (Merck Rover Column RP-8, Art N (L11804)) using a developing solvent and 1 to 50% (V/V) methanol. The resulting eluate was 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 5I.
W17m? L I8 w 7- −”'++1'?
5 + Fl y'y Shinto Bunma 1-) 2537 people carried out the ultraviolet irradiation method.

Examples are shown below.

Example 1 Nocardiopsis sp. -290 is used as the inoculum. Glucose 1.0g with internuclear space as first type medium
/dl, soluble starch 1.0g/! 17. Meat extract 0
．． 3 g/dl, yeast extract 0.5 g/dl, batato tryptone (manufactured by Difco) 0.5 g #12, calcium carbonate 0.2 g/JS pH 7.2-7.4 in a 300 ml Erlenmeyer flask. Inoculate Qml seed medium. Then, the culture is carried out at 30° C. with shaking until the bacteria grow sufficiently. Inoculate 4 ml of this seed culture into 4 Q ml of the following fermentation medium in a 300 ml Erlenmeyer flask.

Fermentation medium; glucose 0.5g/d1, soluble tenbun 3.0g/dl, soybean flour 3.0g/dt! , Corn Steep Liquor 0.5g/I2!1! , yeast extract 0
．． 5 g/J, calcium carbonate 0.3 g/J, pH 7
, 2-7.4°Culture is carried out at 30°C for 9 days with aeration and agitation. After culturing,
The culture solution is continuously centrifuged (15,00 Orpm). 8.41 of the supernatant liquid was passed through column 11 packed with Diaion HP-10 (manufactured by Mitsubishi Chemical Industries, Ltd.), and 11 (D water,
21 (7) 50% (V/V) Meta/-Nob.

Further, add 30% (V/V) acetone* to
Elute with 11 60% (V/V) acetone water. All the eluate is collected and concentrated to dryness to obtain 1.6 g of oil. This was suspended in water and filled with water.84m
No. 1 Diaion HP-20SS (manufactured by Mitsubishi Chemical Corporation)
Feed at the top of the column. 200ml of water, plus 20
After washing with Qml of 40% (V/V) methanol, 140
ml+7) 60% (V/V) Me9/-lute elute. All the eluted fractions are collected and concentrated to dryness under reduced pressure to obtain about 379 mg of an oily substance. 50% of 5ml (v
/V) 10 Qml of Sephadex L dissolved in methanol and filled with 50% (V/V) methanol
It is supplied to the upper end of a H20 (manufactured by Pharmacia) column, and eluted using a solvent having the same composition as the filling solvent as a developing solvent. Fraction number 7 is obtained when the eluted fraction is separated into 3g portions.
KT5556 is eluted from 7 to 91. The fractions were collected and concentrated to dryness under reduced pressure to obtain about 47.4 mg of brown powder. This powder is dissolved in 5 ml of 40% (V/V) methanol and adsorbed onto a 33 ml HP-20SS column packed with 40% (V/V) methanol. The developing solvent is 40% (V/V) y! Evening/-le 15
Elute with a linear gradient of methanol from 40% (V/V) to 60% (V/V) using h+1 and 15 Qml of 60% (V/V) methanol. When the eluted fractions are collected in 3 g portions, KT5556 is eluted in fraction numbers 10 to 15. Both components are collected and concentrated to dryness under reduced pressure, yielding about 40 cm of brown powder. Dissolve this powder in approximately 5 ml of 50% (V/V) methanol,
Silanized silica gel RP-8 (size B, manufactured by Merck & Co., Ltd., A
rt11804) and eluted using a solvent having the same composition as the solvent used for equilibration as a developing solvent.

Fraction number 33~
KT5556 is eluted at 40. The fractions were collected and concentrated to dryness to obtain about 29.6 mg of pale yellow powder.

In addition, detection of KT5556 during the above powder-making process was performed by silica gel thin layer chromatography, followed by iodine reaction or
It was conducted using the ultraviolet irradiation method on 537 people.

Example 2 Formation of Sodium Salt of KT5556 1.2 g of KT5556 was mixed with 0. I N N a H
COs was dissolved in 40 Qml and passed through a 45 ml column filled with HP-20SS (manufactured by Mitsubishi Chemical Industries, Ltd.) using water.
/V) Elute with methanol. The eluted fractions are collected and freeze-dried to obtain about 162 g of the sodium salt of KT5556. The obtained sodium salt of KT5556 is 1
It is soluble at a rate of about 1.1 mg per ml of water.

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 cell suspension and release of histamine Rats weighing 150 to 180 g were killed by exsanguination under dry ether anesthesia, and the method of 5ullivan et al.
Immunol, 114, 1473. (197,5
) Mast cell culture medium (mast c
ell medium) (abbreviated as MCM, composition: 150
mM NaCj! .

3.7mM KCl, 3mM Na, HP○.

3.5mM KH2P○, 1mM CaCj22
5, 6 mM glucose, 0.1% bovine serum albumin.

10U/lT11 heparin), 5m1/anima
1 was injected intraperitoneally. After the abdomen was massaged for 2 minutes, the abdomen was opened and the peritoneal exudate was collected. After centrifuging the exudate collected from 6 mice at 4°C and 1100X for 5 minutes, add an appropriate amount of water-cooled MCM to the sediment and wash it 3 times, and the final mast cell count will be approximately 3 x 10' cells/ml. cell suspension (peritonealexudate c)
cells (abbreviated as PEC) were prepared.

Note that mast cells were identified by staining intracellular granules with 0.05% toluidine blue.

After 1 ml of PEC obtained in this way was brain-incubated at 37°C for 100 minutes, test drug solutions Q and 1 of various concentrations were added.
Add 100x/ml of phosphatidyl, serine and concanavalin A and incubate for 10 minutes.
0.1 ml each of 1000 clots/ml was added and incubated for an additional 15 minutes. 3 m of ice-cold saline
After stopping the reaction, the mixture was centrifuged at 4° C. and 1 liter at 100× 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 (197
8)] by a fluorescence method. The histamine release rate was expressed as a percentage of the amount of histamine in the cell relative to the total amount of histamine in the cells. In addition, the histamine release inhibition rate of the test drug solution was calculated using the following formula.

2) Experimental Results Table 2 As shown in Table 2, KT5556 inhibited histamine release in a concentration-dependent manner.

Effect of the invention KT5556 has an inhibitory effect on histamine release.

[Brief explanation of drawings]

FIG. 1 shows the ultraviolet absorption spectrum of KT5556. The solid line is 0.1N HCJ! Medium, dotted line is underwater (neutral),
The dashed line is 0. It represents the results measured in IN NaOH. The concentration of KT5556 in each of the above media was 4,3
tail/ml. Procedural amendment (2)1 1. Display of the case 1985 Patent Application No. 17531 2, Title of the invention: New substance KT5556 and its manufacturing method 3, Person making the amendment Relationship to the case Patent applicant Postal code: 100 Address: 6-chome, Otemachi, Chiyoda-ku, Tokyo Number 1 name
(102) In the claims section of the Kyowa Hakko Kogyo Co., Ltd. specification and the details of the invention, page 1, line 6, “difficult” was replaced with “soluble”
Correct. On the bottom 8th line of page 3, after "developing solvent", "(1) A new substance KT5556 having the following physical and chemical properties"
and its salts: Properties: Pale yellow powder ° Melting point = 262-266 °C (decomposition) Specific optical rotation: [α] 20D = +97.1 ° (c = 0
．． 56. DMF) Solubility: Easy: Possible in alkaline water, dimethyl sulfoxide, dimethylformamide Soluble: Insoluble in methanol, ethanol Solubility: Chloroform Color reaction: Positive for iodine, negative for ferric chloride and Lydon Smith reactions Ultraviolet absorption spectrum (Aqueous solution) Fig. 1 Infrared absorption spectrum (KBr) 3430, 1720, 1665.1632.1591.
1460.1313°1279, 1235.1133.
740 cm-'Mass spectrum: The mass spectrum (SI-MS) of this substance gives the following molecular ions. m/z 453 (M elemental analysis: H4,35, C68,71, N 9.01
(Actual measurement value) H4,22 as C25H+sNa○
, C6g, 87. N 9.27 (calculated value) Otori H
-NMR (original MSO-ds) 89.24 (d, 11 (, J = 7.7Hz),
8.1~? , 85 (m. 3) 1), 7.55-7.25 (m, 4) 1
), 7.12 (dd, 1) 1°J=4, 9.
7.4Hz), 5.04 (d, 1)1.
J=17.5Hz); 4,99 (d, 1H, J
=1? , 5Hz), 3.38 (dd, 1H,
J=7.4, 13.81(z), 2.23(s
, 3H), 2.00 (dd, 1M. J=4, 9.13.8Hz) I3C-NMR (DNS low dS) δ 174, 0. 1?1.8. 139.9. 13
6.8. 132.9゜128.3.125.6.12
5.3.124, 9.124, 1.123. Skin 122.
6. 121.1. 120.3. 119.5. 1
19.4, 115.7°114, 8. 114, 5.
109.0. 99.2. 85.0. 84, 4°45.4, 42.5. 22.8 (2) Belongs to the genus Nocardiopsis. A microorganism capable of producing KT5556 is cultured in a medium, and KT55 is added to the culture.
A method for producing KT5556, which comprises producing and accumulating KT5556 and collecting KT5556 from the culture. (3) The microorganism is Nocardiopsis sp. K-29
The manufacturing method according to claim 2, characterized in that the method is manufactured by 0Feikoken Joyori No. 69.6.

Claims (3)

[Claims] (1) KT5556, a new substance with the following physical and chemical properties
and its salts: Properties: Pale yellow powder Melting point: 262-266°C (decomposed) Specific rotation: [α]^2^0_D=+97.1° (c=0
．． 56, DMF) Solubility: Easily soluble: Alkaline water, DMSO, DMF Slightly 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: The mass spectrum (SI-MS) of this substance gives the following molecular ions. m/z453 (M^+) Elemental analysis: H4.35, C68.71, N9.01 (actual value) H4.6H_1_9N_3O_5 as H4.
22, C68.87, N9.27 (calculated value) ^1H-N
MR (DMSO-d_6) 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, 13.8Hz), 2.23(s, 3H)
, 2.00 (dd, 1H, J=4.9, 13.8Hz) ^1^3C-NMR (DMSO-d_6) δ174.0, 171.8, 139.9, 136.8,
132.9, 128.3, 125.6, 125.3, 1
24.9, 124.1, 123.9, 122.6, 12
1.1, 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 (2) A microorganism belonging to the genus Nocardiopsis and capable of producing KT5556 is cultured in a medium, and KT5555 is added to the culture.
A method for producing KT5556, which comprises producing and accumulating KT5556 and collecting KT5556 from the culture. (3) The microorganism is Nocardiopsis sp. K-29
The manufacturing method according to claim 2, characterized in that the manufacturing method is manufactured by 0Feikoken Joyori No. 696.