JP2001122875A - New wk-6326 material and method for preparing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new WK-6326 material having activities for inhibiting the activity of interleukin 4, and further having antibacterial activities against Gram positive and Gram negative bacteria, and further to provide a method for preparing the material. SOLUTION: This WK-6326 material is obtained by culturing a microorganism belonging to actinomyces, and having abilities for producing the WK-6326 material represented by formula [I] in a medium to accumulate the WK-6326 material in the cultured material, and collecting the WK-6326 material from the cultured material. The material is useful for the therapy of allergic disease such as asthma, atopic dermatitis and pollinosis, and many infectious diseases because the material has an extreme inhibiting activities against the activities of the interleukin 4.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel WK-632.
It relates to a method for producing six substances. More specifically, the present invention is a novel antibacterial agent which is produced by culturing an antibiotic-producing bacterium belonging to the genus Streptomyces, has a remarkable inhibitory effect on interleukin-4 activity, and has an antibacterial activity against Gram-positive and negative bacteria. The present invention relates to a WK-6326 substance and a method for producing the same.

[0002]

2. Description of the Related Art Some preventive and remedies for allergic diseases such as asthma, atopic dermatitis and hay fever are known. For example, steroid hormones are used in many allergic or inflammatory diseases due to their strong anti-inflammatory effects. However, at the same time, it has many side effects due to disruption of the hormone balance in the living body. Therefore, development of a new antiallergic drug without side effects is desired.

On the other hand, a large number of antibacterial substances produced by microorganisms have been discovered so far, and it is well known that some of them have been put to practical use in the fields of pharmaceuticals, veterinary drugs and agricultural chemicals. It is on the street. However, on the other hand, there are problems in various points such as toxicity, side effects, and resistance, and at present, there is a strong demand for the development of new antibacterial substances that can solve these problems.

[0004]

In recent years, allergic diseases such as asthma, atopic dermatitis and hay fever have increased remarkably and are regarded as a social problem as a modern disease. These diseases involve I, an immunoglobulin produced from B cells.
When gE binds to a receptor present on the surface of mast cells or basophil cells, various chemical mediators such as histamine are released and induced.

B cells are stimulated by interleukin-4, one of the cytokines, to cause Janus kinase (Janus kinase).
kinase) is activated, followed by Signal Transducer and Activator of Transcription (Signal transduc)
er and activator of trans
Activation of the script 6; STAT 6) initiates transcription of the nuclear gene. (K. Takeda et al.,
Journal of Molecular Medicine, 75
317-326, 1997).

[0006] By this signal transduction, B cells express CD23, which is a low-affinity IgE receptor, and class switch to IgE antibody-producing cells (Class switch).
It is known to cause (DH. Conrad
Et al., Journal of Immunology, 141, 10
Pp. 91-1097, 1988; Messner
Et al., Journal of Immunology, 159, 33
30-3337, 1997).

[0007] Therefore, a substance that inhibits interleukin 4 activity suppresses IgE production and is presumed to be effective for such diseases. Under such circumstances, providing a substance having an interleukin-4 activity inhibitory action is extremely useful in treating allergic diseases such as asthma, atopic dermatitis and hay fever. In addition, providing a novel antibacterial substance having antibacterial activity against Gram-positive and negative bacteria is considered to be extremely important as a solution to human medical or social problems.

[0008]

DISCLOSURE OF THE INVENTION The present inventors have isolated bacterial strains from various soils for the purpose of searching for a novel physiologically active substance, and have continued to study metabolites produced by the same. It was found that a substance having a remarkable inhibitory effect on interleukin 4 activity and having an antibacterial activity against Gram-positive and negative bacteria was produced in the isolated culture of WK-6326 strain. Next, as a result of separating and purifying the active substance from the culture, no substance having such a chemical structure has been known at all. Therefore, this substance was designated as WK-6326 substance. The present invention has been completed based on such findings, and has the following formula [I]

[0009]

Embedded image WK-632, which is a compound represented by the formula:
6 substances. The present invention further provides the following formula [I] belonging to actinomycetes:

[0010]

Embedded image A microorganism having the ability to produce the WK-6326 substance represented by the following formula is cultured in a medium, and WK-6326 is added to the culture.
The present invention relates to a method for producing a WK-6326 substance, which comprises accumulating the substance and collecting the WK-6326 substance from the culture.

[0011] The present invention further relates to a microorganism belonging to the genus Streptomyces and capable of producing a WK-6326 substance, wherein the microorganism is Streptomyces sp.
myces sp. ) WK-6326 (FERM P-
17592). The present invention further belongs to the genus Streptomyces,
The present invention relates to a microorganism capable of producing a K-6326 substance. In the present invention, the microorganism may further be Streptomyces sp. WK-6.
326 (FERM P-17592).

[0012] The WK-6326 substance-producing bacterium of the present invention is not particularly limited as long as it is an actinomycete and has a WK-6326 substance-producing ability. A preferred example of the strain used for producing the WK-6326 substance of the present invention is an actinomycete newly isolated from the soil by the present inventors, Streptomyces sp.
treomyces sp. ) WK-6326 strain. The bacteriological properties of this strain are as follows.

1. Morphological properties The vegetative mycelium develops well on various agar media and no fragmentation is observed. Aerial mycelium is made of starch inorganic salt agar, glycerol
It grows abundantly on asparagine agar, etc., and exhibits a gray color tone. Observation under a microscope shows a chain of 20 or more spores on the aerial hyphae, the shape of which is linear or spiral, and the size of the spores is 1.0 × 0.6 μm and cylindrical. The spore surface is smooth. Sclerotia, sporangia and zoospores are not found.

2. Various properties on various media EB Shirring (EB Shirlin)
g) and the method of D. Gottlieb (International Journal of Sistimatic Bacteriology, 16, 313,
1966).
And Table 3 below. The color tone was determined using the Color Harmony Manual 4th Edition (Container Corporation of America, Chicago, 1958) as a standard color, and the code was written in parentheses along with the color chart name. The following are the results of observation of the culture properties in each medium at 27 ° C. for two weeks unless otherwise specified.

[0015]

[Table 1]

[0016]

[Table 2]

[0017]

[Table 3]

[0018] 3. Physiological properties (1) Melanin pigment formation (a) Tyrosine agar negative (b) Peptone yeast iron agar positive (c) Trypton yeast solution positive (2) Nitrate reduction negative (3) Gelatin liquefaction (21- (23 ° C) Negative (glucose / peptone / gelatin medium) (4) Starch hydrolysis positive (5) Coagulation of skim milk (27 ° C) Negative

(6) Peptone conversion of skim milk (27 ° C.) positive (7) Growth temperature range 5 to 32 ° C. (8) Utilization of carbon source (Priedam Götrib agar medium) Use: glucose, use slightly: Fructose, not used: Arabinose, xylose, sucrose, mannitol, rhamnose, melibiose, inositol, raffinose (9) Degradation of cellulose negative

4. Cell wall composition The diaminopimelic acid of the cell wall is of the LL type. The bacteriological properties of this strain are summarized as follows. Diaminopimelic acid in the cell wall is of the LL type. The form of the spore chain is linear or spiral, forming long spore chains,
The spore surface is smooth. The vegetative mycelium has a pale yellow color, and the aerial mycelium has a gray color as the culture-like properties. Produces a small amount of brown soluble pigment.

From these results, this strain is a strain belonging to the genus Streptomyces, and is classified into Pridham and Tresner (Virgins Manual of Determinative Bacteriology, 8th edition, pp. 748-829, 1
974). The strain was Streptomyces sp. WK-63.
No. 26 has been deposited at the Institute of Biotechnology and Industrial Technology, located at 1-3 1-3 Higashi, Tsukuba, Ibaraki Prefecture. Deposit date is October 4, 1999, deposit number is FER
MP-17592.

A preferred strain of the present invention is WK-63.
The description of the 26 substance-producing bacteria has been given, but as a general property of the bacteria, the mycological properties are extremely susceptible to variation, are not constant, and are naturally or normally performed by ultraviolet irradiation, X-ray irradiation or a mutant derivative agent, For example, N-methyl-
It is a well-known fact that mutations are made by artificial mutation means using N-nitro-N-nitrosoguanidine, ethyl methanesulfonate, etc., and such artificial mutants as well as natural mutants are of the genus Streptomyces. Belongs to
Any strain capable of producing WK-6326 substance can be used in the present invention. In addition, strains mutated by cell engineering such as cell fusion and gene manipulation are also available in WK-6.
326 substance producing bacteria.

In the present invention, first, a WK-6326 substance producing bacterium belonging to the genus Streptomyces is cultured in an appropriate medium. In culturing this strain, ordinary methods for culturing fungi are generally applied. As the medium, a nutrient medium containing a carbon source that can be assimilated by microorganisms, a nitrogen source that can be digested, and, if necessary, inorganic salts and the like is used. As the assimilable carbon source, glucose, sucrose, molasses, starch, dextrin, cellulose, glycerin, organic acids and the like are used alone or in combination.

Examples of digestible nitrogen sources include organic nitrogen such as peptone, meat extract, yeast extract, dried yeast, soy flour, corn steep liquor, cottonseed meal, casein, soy protein hydrolyzate, amino acids, and urea. Sources, inorganic nitrogen compounds such as nitrates and ammonium salts are used alone or in combination. In addition, if necessary, inorganic salts such as sodium salt, potassium salt, calcium salt, magnesium salt and phosphate, and heavy metal salts are added. Furthermore, micronutrients, growth promoting substances, and precursors that promote the growth of the strain and the production of the WK-6326 substance may be appropriately added to the medium, if necessary.

The cultivation is usually carried out under aerobic conditions such as shaking or aeration and stirring. Industrially, deep aeration stirring culture is preferred. It is preferable to culture the medium at a pH around neutrality. The cultivation temperature may be in the range of 20 to 37 ° C, but is usually 24 to 30 ° C, preferably 27 ° C.
It is better to keep it near. In the case of liquid culture, culturing is usually performed for 3 to 6 days, so that the WK-6326 substance of the present invention is produced and accumulated. do it.

The culture conditions, such as the culture composition, the liquid properties of the medium, the culture temperature, the stirring speed, and the aeration rate, are appropriately adjusted and selected so as to obtain preferable results according to the type of the strain used and external conditions. Needless to say. When foaming occurs in the liquid culture, an antifoaming agent such as silicone oil, vegetable oil, or a surfactant may be appropriately used. WK-6 accumulated in the culture obtained as described above
Since the 326 substance is contained in the culture filtrate or cultured cells, the culture filtrate may be filtered, if necessary, by adding a filter aid, for example, celite, high flow supercell, or the like, or centrifuged. It is advantageous to separate the WK-6326 substance of the present invention from the filtrate separated from the culture filtrate and the cells, and the organic solvent extract of the culture filtrate and the cells concentrated.

The culture filtrate can be used to prepare the WK-632 of the present invention.
To collect 6 substances, first, the culture filtrate was extracted with a non-hydrophilic organic solvent such as ethyl acetate, butyl acetate, and benzene.
The extract is concentrated under reduced pressure to obtain a crude substance, WK-6326 substance. The crude substance can be further separated and purified from the WK-6326 substance of the present invention by a known method usually used for purifying a fat-soluble substance, for example, column chromatography using a carrier such as silica gel or alumina.

In order to collect the WK-6326 substance of the present invention from the cells, the cells are extracted with a water-containing hydrophilic organic solvent such as water-containing acetone and water-containing methanol, and the obtained extract is concentrated under reduced pressure. The extract is extracted with a non-hydrophilic organic solvent such as ethyl acetate, butyl acetate, benzene, etc., and the obtained extract is separated and purified together with the extract obtained from the culture solution, or the same method as described above. By the WK- of the present invention
6326 substances can be separated and purified.

Next, the physicochemical properties of the WK-6326 substance of the present invention will be described. (1) Molecular formula: C ₂₈ H ₃₁ NO ₈ (m / z 510, 212 in high-resolution FAB mass spectrum (positive))
6 (M + H) observed) (calculated 510, 212)
8) (2) Molecular weight: 509 (FAB mass spectrum (pos
m / z 510 (M + H) ⁺ , 532
(M + Na) ⁺ was observed)

(3) Specific rotation: [α] _D ²⁵ −21.7 °
(C = 0.03, methanol: chloroform 1: 1) (4) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in methanol is as shown in FIG.
8, 244, 266, 275, 322, and 382 nm. (5) Infrared absorption spectrum: The infrared absorption spectrum measured by the potassium bromide tablet method is as shown in FIG.
λmax ^KBR cm ^-1 : 3492, 1716, 1616,
1591, 1454, 1371, 1338, 1299,
1247 and 1133 have characteristic absorption bands.

(6) Solubility in solvent: soluble in dimethylsulfoxide, methanol, ethyl acetate, chloroform, insoluble in water (7) Basic, acidic, neutral: neutral (8) Color and shape of substance : Yellow-brown powder (9) Proton nuclear magnetic resonance spectrum: Varian
The proton nuclear magnetic resonance spectrum (measured in deuterated dimethyl sulfoxide, 400 MHz) measured using a nuclear magnetic resonance spectrometer manufactured by Japan Co. is shown in FIG. 3 (10) Carbon nuclear magnetic resonance spectrum: Varian
The carbon nuclear magnetic resonance spectrum (measured in deuterated dimethyl sulfoxide, 100 MHz) measured using a nuclear magnetic resonance spectrometer manufactured by Japan Co., Ltd. is shown in FIG. 4 (11) Chemical structure: as shown in the following formula [I]

[0032]

Embedded image

Next, the biological properties of the WK-6326 substance of the present invention will be described in detail. (1) Interleukin 4 activity inhibitory action Foumie
r and M.R. The method of Sartati et al. (Journal of
Clinical Investigation, Volume 89, 131
2-1321, 1992).

That is, U9 which is a human-derived monocyte-like cell
After adjusting 37 cells to a concentration of 1 × 10 ⁶ cells / ml, interleukin 4 was added to a final concentration of 10 ng / ml, and the cells were cultured under 5% CO ₂ for 72 hours. After 72 hours, the cells were collected, and anti-human CD2 fluorescently labeled with FITC was used.
Reaction was performed with 3 antibodies (Cosmo Bio) for 60 minutes. Next, the cells were washed with a phosphate buffer containing 2% bovine serum albumin, CD23-positive cells were detected with a flow cytometer (manufactured by Coulter, USA), and the fluorescence intensity was defined as interleukin-4 activity. By this method, the WK-6326 substance of the present invention inhibited the expression of CD23 by 50% at a drug concentration of 0.1 μg / ml.

(2) Anti-Gram positive and negative bacteria
Bacterial activity Minimum inhibitory concentration (MIC) for each test strain and 50
% Growth inhibitory concentration (IC ₅₀) Is the micro broth dilution method
(M. Suzanne et al., Journal of Violoncello
Zical Chemistry, Vol. 272, 32709-327
14 (1997)).

That is, 2 × 10 ⁴ -1 × 10 ^4
It was adjusted to a concentration of ⁵ cells / ml and cultured with the WK-6326 substance at a concentration of 0-125 µg / ml. The growth of each test bacterium was determined using the measured value at OD 600 nm as an index. The culture temperature was Xanthomonas aeruginosa, P
yricularia oryzae, Aspergi
llucs niger, Mucor racemosu
s, Candidaalbicans, Sacchar
27 ° C for Omyces cerevisiae
The temperature of other test bacteria was 37 ° C. Culture time is Pyri
cularia oryzae and Aspergil
rus niger was set to 48 hours, and the other test bacteria were set to 24 hours. The MIC (concentration of the drug that completely inhibits the growth of the test bacterium) and IC ₅₀ (the concentration of the drug that inhibits the growth of the test bacterium by 50%) for each test bacterium are as shown in Table 4 below.

[0037]

[Table 4]

Next, the present invention will be described specifically with reference to examples, but the present invention is not limited to these examples.

EXAMPLE 2.4 Starch 2.4 in a 500 ml Erlenmeyer flask
%, Yeast extract 0.5%, glucose 0.
1%, peptone 0.3%, meat extract 0.3% and Ca
Medium containing 0.4% CO ₃ (adjusted to pH 7.0) 100
ml of Streptomyces sp., which had been cotton-plugged, steam-sterilized, and grown on an agar medium.
myces sp. ) WK-6326 (FERM P-
17592) aseptically with a platinum loop, and 7
The cells were cultured with shaking for 2 hours.

Then, using it as a seed culture solution,
4.0% high quality toasted meal 2.0%
0.1 N sodium thiosulfate 32 μl / l, FeSO_Four
・ 7H _TwoO 0.05%, K_TwoHPO_Four0.05%, K
1 medium containing 0.03% Cl (adjusted to pH 6.5)
100 bottles of 500 ml Erlenmeyer flask
After steam sterilization, 1 ml of the culture solution
Aseptically transplant into a square flask and shake at 27 ° C for 6 days.
Nourished.

After the culture, the culture was extracted with 10 liters of ethyl acetate, and the extract was concentrated under reduced pressure to obtain 2.5 g of a crude product. This crude product was suspended in 10 ml of chloroform, charged into a column of silica gel (300 ml, key cell gel 60), and subjected to column chromatography eluting with chloroform and methanol. Each fraction is 50
Fractions containing 0 ml each were collected, and the fractions containing the active ingredient were collected and dried under reduced pressure to obtain 629 mg of a crude active substance.
This crude active substance was suspended in 10 ml of methanol, and
(200ml, Senshu Kagaku SSC-ODS-
7515-12) and subjected to column chromatography eluting with water and acetonitrile. Each fraction was fractionated by 50 ml, the fraction containing the active ingredient was collected, and after removing acetonitrile,
The aqueous layer fraction was extracted with ethyl acetate and dried under reduced pressure to obtain 284 mg of a crude active substance.

The 284 mg of the crude active substance was separated and purified by high performance liquid chromatography. The device is PU-
980 (manufactured by JASCO Corporation), and the column was YMC-Pa
ckODS-AM (ODS resin, manufactured by Yamamura Chemical Laboratory)
Was used, the solvent system was 50% acetonitrile water, and the detection was performed at UV 220 nm at a flow rate of 6 ml / min. As a result, 18 mg of the WK-6326 substance of the present invention was isolated.

[0042]

As described above, a microorganism having the ability to produce a WK-6326 substance belonging to the genus Streptomyces is cultured, the WK-6326 substance is accumulated in the culture, and the WK-6326 substance is recovered from the culture. By collecting 6326 substances, a substance having a remarkable inhibitory effect on interleukin 4 activity and having antibacterial activity against Gram-positive and negative bacteria can be obtained. This substance is expected to be effective in treating allergic diseases such as asthma, atopic dermatitis and hay fever, as well as many infectious diseases.

[Brief description of the drawings]

FIG. 1 is an ultraviolet absorption spectrum of a WK-6326 substance according to the present invention.

FIG. 2 is an infrared absorption spectrum of a WK-6326 substance according to the present invention.

FIG. 3 is a proton nuclear magnetic resonance spectrum of a WK-6326 substance according to the present invention.

FIG. 4 is a carbon nuclear magnetic resonance spectrum of a WK-6326 substance according to the present invention.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 37/08 A61P 37/08 43/00 111 43/00 111 C12N 1/20 C12N 1/20 A C12P 17 / 06 C12P 17/06 // (C12N 1/20 (C12N 1/20 C12R 1: 465) C12R 1: 465) (C12P 17/06 (C12P 17/06 C12R 1: 465) C12R 1: 465) (72 ) Inventor Masayoshi Arai 5-9-1, Shirogane, Minato-ku, Tokyo Inside the Kitasato Institute (72) Inventor Yoko Takahashi 5-9-1, Shirogane, Minato-ku, Tokyo F-term in the Kitasato Institute (reference) 4B064 AE46 BA06 CA04 DA01 DA03 4B065 AA50X CA18 CA34 CA44 4C063 AA01 BB01 CC79 DD78 EE01 4C086 AA02 AA03 AA04 BA08 GA02 NA14 ZA59 ZA89 ZB13 ZB35 ZC02 ZC13 ZC20

Claims (5)

[Claims] [Claim 1] The following formula [I] A novel WK-6, which is a compound represented by the formula:
326 substances. 2. The following formula [I] belonging to actinomycetes: A microorganism having the ability to produce the WK-6326 substance represented by the following formula is cultured in a medium, and WK-632 is added to the culture.
A method for producing a novel WK-6326 substance, comprising accumulating six substances and collecting WK-6326 substance from the culture. 3. WK-6 belonging to the genus Streptomyces
Microorganisms capable of producing 326 substances have been identified as Streptomyces sp.
p. ) WK-6326 (FERM P-17592). 4. WK-6, belonging to the genus Streptomyces.
A microorganism capable of producing 326 substances. 5. The method according to claim 5, wherein the microorganism is Streptomyces sp. WK-632.
The microorganism according to claim 4, which is 6 (FERM P-17592).