JPH11286486A - New physiologically active substance and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new compound having immunosuppression activity, antiallergic activity, antitumor activity and multidrug resistance overcoming action and useful as an inhibitor for immunorejection due to organ transplantation, etc., a therapeutic agent for allergic diseases such as atopic dermatitis and a therapeutic agent, etc., for autoimmune diseases such as chronic rheumatoid arthritis. SOLUTION: This compound is represented by the formula (R is H or a 1-5C lower alkyl). The compound of the formula is obtained by culturing a microorganism, preferably Streptomyces sp.-SNA15896 strain (FERM P-16667), preferably at pH of culture medium of 4-8 and 25-30 deg.C culture temperature for 96-144 hr culturing time by a method of shaking culture, deep submerged culture, etc., using a medium comprising carbon source such as glucose or soluble starch, a nitrogen source such as soybean flour or ammonium sulfate, an inorganic salt such as magnesium sulfate or potassium primary phosphate to produce a compound of the formula in the culture solution and collecting the compound by utilizing usual separation means.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel neoantimycin-based physiologically active substance, a microorganism producing the same, a method for producing a novel physiologically active substance using the microorganism, and an effective method for producing the novel physiologically active substance. It relates to a medicine as an ingredient. The novel physiologically active substance of the present invention is an agent for suppressing immune rejection by organ transplantation, a therapeutic agent for allergic diseases such as atopic dermatitis, a therapeutic agent for autoimmune diseases such as rheumatoid arthritis, and an antitumor agent in cancer chemotherapy Or, it is useful as an agent for overcoming multidrug resistance.

[0002]

2. Description of the Related Art Immunosuppressants are used to suppress diseases such as allergic diseases such as atopic dermatitis and autoimmune diseases such as rheumatism, which are also called modern diseases, and to suppress immune rejection by organ transplantation. Effective for treatment. Although drugs such as cyclosporin A and FK506 have been put to practical use, development of drugs with different action mechanisms and new drugs with weak side effects has been desired. On the other hand, in cancer chemotherapy, many compounds such as mitomycin C, 5-fluorouracil, and adriamycin have already been put into practical use as pharmaceuticals. However, its effect on various tumors is not always sufficient, and the resistance of tumor cells to these drugs is also a major obstacle, and the development of antitumor substances with a new mechanism of action is always required. I have.

[0003]

SUMMARY OF THE INVENTION The present inventors have conducted intensive searches in view of the above situation, and have newly found a strain of the genus Streptomyces ( Streptomyces sp. S.
A novel bioactive substance having an immunosuppressive activity, an antiallergic activity, an antitumor activity and a multidrug resistance overcoming effect has been found in a culture solution of NA15896). That is, the present invention provides a novel physiologically active substance having an immunosuppressive activity, an antiallergic activity, an antitumor activity, and a multidrug resistance overcoming action, a microorganism producing the same, a method for producing the substance by culturing the microorganism, and It is an object to provide a medicine containing the substance as an active ingredient.

[0004]

SUMMARY OF THE INVENTION The present invention relates to a novel physiologically active substance represented by the following general formula: and a pharmacologically acceptable salt thereof.

[0005]

Embedded image (Here, R represents a lower alkyl group of which a hydrogen or a C ₁ ~C _5.)

More specifically, the present invention relates to a novel physiologically active substance represented by the following structural formulas: chemical formula 2 (substance A) and chemical formula 3 (substance B) and a pharmacologically acceptable salt thereof.

[0007]

Embedded image

[0008]

Embedded image

[0009] The present invention also relates to a microorganism capable of producing the novel physiologically active substance of the present invention belonging to Streptomyces. Specifically, the present inventors include Streptomyces sp. SNA15896 strain isolated and collected from soil in Yuki City, Ibaraki Prefecture. The present invention also relates to a method for producing a novel bioactive substance, comprising culturing the microorganism, producing the bioactive substance in a culture, and collecting the bioactive substance. The present invention also provides
The present invention relates to a medicament comprising the physiologically active substance and a pharmacologically acceptable salt thereof as an active ingredient. The medicament of the present invention is an agent for suppressing immune rejection by organ transplantation and the like, a therapeutic agent for allergic diseases such as atopic dermatitis, a therapeutic agent for autoimmune diseases such as rheumatoid arthritis, or an antitumor agent in cancer chemotherapy,
It is useful as an agent for overcoming multidrug resistance.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The novel physiologically active substance of the present invention can be obtained by culturing a microorganism. When this is not particularly limited as long as bacterium producing the compound of the present invention is a microorganism to be used, preferably Streptomyces (Strept
omyces ), particularly preferably Streptomyces sp. SNA15896 strain. This Streptomyces sp SNA1589
The six strains were isolated by the present inventors from soil collected from Yuki City, Ibaraki Prefecture, and were submitted to the Institute of Biotechnology, Ministry of International Trade and Industry as RIKEN No. 16667 (FERM P-16667). Deposited on February 25, 2009. Streptomyces sp. Strain SNA15896 can be isolated from the soil in Yuki city, Ibaraki prefecture by a method commonly used for the isolation of actinomycetes, for example, the method of Goodfellow M. (Actinomycetologica, Vo.
l.2, No. 1, pp13-29 (1988)). The properties of the Streptomyces sp. SNA15896 strain thus isolated are as follows.

1. Morphology Forms base and aerial mycelia that elongate and diverge well. The aerial hyphae has a white color tone, and a spiral chain of 10 or more spores is formed. Spore surface is rough, 0.6μ in size
It is about m × 0.8 μm. No special organs such as sporangia, sclerotium, zoospores, etc. are found. 2. Growth state in various media Table 1 shows the results of culturing at 27 ° C for 14 days on various agar plate media. The colors are described in JIS Color Name Book (JIS Z 8102 compliant, Japan Standards Association, April 15, 1993).
1st edition, 3rd printing issue).

[0012]

[Table 1]

3. Physiological properties (1) Growth temperature range Using yeast / malt agar medium, 7 ℃, 12 ℃, 17 ℃, 22 ℃
As a result of testing at each temperature of 27 ° C., 27 ° C., 32 ° C., 37 ° C., 42 ° C., and 47 ° C., it grew at any temperature except 7 ° C., 12 ° C., 42 ° C. and 47 ° C. The optimal growth temperature seems to be around 27 ° C to 32 ° C. (2) Liquefaction of gelatin In a glucose / peptone / gelatin medium at 27 ° C. or a simple gelatin medium or 20 ° C., liquefaction starts about 10 days after cultivation in any medium, and the liquefaction effect in the observation after 21 days is medium. It was about. (3) Hydrolysis of starch Culture on starch / inorganic salt agar medium at 27 ° C.
Water degradability has been observed since around the day, and its effect is relatively strong. (4) Coagulation / Peptone Formation of Skim Milk In the defatted milk medium, cultured at 37 ° C., no coagulation was observed, but peptone formation was observed around 10 days after the culture. (5) Production of melanin-like pigment No melanin-like pigment was observed in any of the cultures at 27 ° C. using a tryptone yeast broth medium, a peptone yeast iron agar medium, or a tyrosine agar medium. (6) Utilization of carbon source In Prideham-Godlieve agar medium, cultured at 27 ° C,
D-glucose, D-xylose, D-fructose,
Using sucrose, raffinose, inositol, mannose, D-mannitol, D-galactose,
-Arabinose and L-rhamnose are not used. 4. Chemical taxonomic properties (1) The optical isomer of diaminopimelic acid in bacterial cells 2,6-diaminopimelic acid is described in the Journal of General Applied Microbiology (Journa
l of General Applied Microbiology), Vol. 29, p. 319 (1983), and was found to be LL-type (cell wall type I). (2) Cell Menaquinone The main menaquinone was analyzed by MK-9 (H6), MK-9 ( H8).

[0014] or more morphological characteristics, the physiological properties and chemical properties, the SNA15896 strain Streptomyces (Streptom
yces ). "Bergey's Manual of Determinative Bacteriology"
Eighth Edition, and "International Journal of Systematic Bacteriology (International
Journal of Systematic Bacteriology), Vol. 18, p. 69,
279 pages (1968), Vol. 19, p. 391 (1969), Vol. 22, 26
As a result of a search on page 5 (1972), this strain was identified as Streptomyces melanosporofaciens ( Streptomyces).
melanosporofaciens ). The microorganism used in the present invention may be obtained by subjecting the aforementioned microorganism of the present invention to irradiation treatment with X-rays, ultraviolet rays, or the like, or nitrite, N-methyl-N′-nitro-N
-A microorganism which has been mutated by a commonly used bacterial species conversion treatment method such as treatment with a mutagenic agent such as nitrosoguanine (NTG), transformation, transduction or fusion.

The compound of the present invention is produced in its culture by inoculating a nutrient-containing medium which can be used by ordinary microorganisms with a microorganism producing the compound of the present invention and growing it. Any nutrient source may be used as long as it is used as a nutrient source for actinomycetes, and any of a synthetic medium, a semi-synthetic medium, and a natural medium can be used. For example, as a carbon source, glucose, glycerol, maltose, starch, sucrose, molasses, syrup, dextrin or the like is used alone or as a mixture. Nitrogen sources include organic nitrogen sources such as soy flour, corn gluten meal, corn steep liquor, meat extract, yeast extract, cottonseed meal, peptone, wheat germ, fish meal, urea, and inorganic nitrogen such as ammonium sulfate and sodium nitrate. The sources are used alone or as a mixture. As the inorganic salt, calcium carbonate, sodium chloride, potassium chloride, magnesium sulfate or various phosphates can be used, and if necessary, iron,
Trace amounts of heavy metal salts such as copper, cobalt, molybdenum, manganese or zinc can also be added. During culturing, when foaming is remarkable, various antifoaming agents known as antifoaming agents may be appropriately added to the medium. In addition, organic and inorganic substances which are used by the producing bacteria and are useful for producing the compound of the present invention can be appropriately used.

The method for culturing the strain may be the same as the method for producing general microbial metabolites, and may be either solid culture or liquid culture. In the case of liquid culture, any of stationary culture, stirring culture, shaking culture or aeration culture may be performed, but when culturing Streptomyces sp.SNA 15896 strain, in particular, shaking culture or deep aeration. Agitation culture is preferred. Although it depends on the culturing conditions, the pH of the medium is preferably in the range of 4 to 8, and the culturing temperature is suitably 22 to 37 ° C, preferably 25 to 30 ° C. The culture time is 48-168
Hours, preferably 96-144 hours.

In order to isolate the desired novel physiologically active substance of the present invention from the culture, a separation means usually used for isolating a metabolite produced by a microorganism may be appropriately used.
Since the novel physiologically active substance of the present invention produced by culturing is often accumulated both inside and outside the cells in the culture, it is separated into the culture filtrate or cells by means such as centrifugation and filtration. Normal separation means such as dialysis, solvent extraction, a method utilizing the solubility difference with impurities, ion exchange resin method or adsorption or partition chromatography, and gel filtration. Alternatively, they can be separated and purified by appropriately combining them and, if necessary, repeatedly using them. The physicochemical properties of the compound of the present invention thus obtained are shown below.

Physics of the novel physiologically active substance (substance A) of the present invention
Chemical properties (1) Color and properties; white powder (2) Molecular _{formula; C 34 H 42 N 2 O} 12 (3) Mass spectrum (FAB-MS); m / z 671
(M + H) ⁺ (4) Specific rotation: [α] _D ²⁷ + 47.8 ° (c 0.20, C
HCl ₃ ) (5) Ultraviolet absorption spectrum; shown in FIG. λ _max ^MeOH nm (ε); 223 (37000), 341 (7700) (6) Infrared absorption spectrum; FIG. ν _max ^KBr cm ^-1 ; 3400,2970,1760,1700,1650,1520,120
0,1130 (7) ¹ H-nuclear magnetic resonance spectrum; FIG. (500M
Hz, CD ₃ OD) δ (ppm); 8.35 (s, 1H), 8.26 (dd, J = 8.1,1.5Hz, 1H), 7.74 (d
d, J = 8.1,1.5Hz, 1H), 7.29-7.18 (m, 5H), 6.91 (t, J = 8.1H
z, 1H), 5.78 (dq, J = 6.7,2.8Hz, 1H), 5.49 (dd, J = 10.1,5.2H
z, 1H), 5.48 (q, J = 6.9Hz, 1H), 5.23 (d, J = 2.8,1H), 4.72 (d, J
= 8.0Hz, 1H), 3.29 (s, 1H), 3.15 (dd, J = 10.1,14.0Hz, 1H), 2.
96 (dd, J = 5.2,14.0Hz, 1H), 1.91 (m, 1H), 1.56 (m, 1H), 1.40
(s, 3H), 1.32 (s, 3H), 1.28 (d, J = 6.7Hz, 3H), 1.23 (m, 1H), 1.
02 (d, J = 6.9Hz, 3H), 0.93 (d, J = 7.0Hz, 3H), 0.90 (t, J = 7.5H
(8) ¹³ C-nuclear magnetic resonance spectrum; FIG. (125M
Hz, CD ₃ OD) δ (ppm); 177.4 (s), 171.9 (s), 171.5 (s), 170.1 (s), 169.0
(s), 162.2 (d), 152.5 (s), 138.7 (s), 130.4 (d), 129.5 (d),
128.2 (s), 127.7 (d), 126.5 (d), 124.2 (d), 119.5 (d), 116.
0 (s), 80.0 (d), 76.6 (d), 73.4 (d), 72.7 (d), 70.6 (d), 56.8
(d), 46.8 (s), 40.6 (t), 37.4 (d), 27.2 (q), 25.9 (t), 22.5
(q), 17.8 (q), 16.6 (q), 14.7 (q), 10.9 (q) (9) Solubility; applicable to methanol, ethanol, dimethyl sulfoxide, acetone, pyridine, ethyl acetate, chloroform, diethyl ether, etc. Dissolution. Hexane, insoluble in water. (10) Color reaction; positive for 50% sulfuric acid, iodine, torence reagent and phenol reagent. (11) HPLC: Retention time 8.32 minutes Column: TSKOH, TSK gel ODS-80TM 4.6 mm dia. X 2
50 mm solvent: 55% acetonitrile-water, flow rate 1.0 ml / min, UV 2
20nm (12) Acid, neutral, basic distinction; neutral

Physics of the novel physiologically active substance (substance B) of the present invention
Chemical properties (1) Color and properties; white powder (2) Molecular _{formula; C 33 H 40 N 2 O} 12 (3) Mass spectrum (FAB-MS); m / z 657
(M + H) ⁺ (4) Specific rotation; [α] _D ²⁷ + 49.1 ° (c 0.20, C
HCl ₃ ) (5) Ultraviolet absorption spectrum; FIG. λ _max ^MeOH nm (ε); 223 (34700), 341 (6900) (6) Infrared absorption spectrum; FIG. ν _max ^KBr cm ^-1 ; 3400,2970,1760,1700,1650,1530,120
0,1130 (7) ¹ H-nuclear magnetic resonance spectrum; FIG. (500M
Hz, CD ₃ OD) δ (ppm); 8.35 (s, 1H), 8.27 (dd, J = 8.0,1.5Hz, 1H), 7.75 (d
d, J = 8.0,1.5Hz, 1H), 7.29-7.19 (m, 5H), 6.93 (t, J = 8.0H
z, 1H), 5.79 (dq, J = 6.4,3.1Hz, 1H), 5.50 (dd, J = 10.3,5.2H
z, 1H), 5.48 (q, J = 6.7Hz, 1H), 5.23 (d, J = 3.1,1H), 4.64 (d, J
= 7.3Hz, 1H), 3.29 (s, 1H), 3.15 (dd, J = 10.3,13.9Hz, 1H), 2.
97 (dd, J = 13.9,5.2Hz, 1H), 2.08 (m, 1H), 1.41 (s, 3H), 1.33
(s, 3H), 1.28 (d, J = 6.4Hz, 3H), 1.03 (d, J = 6.7Hz, 3H), 0.99
(d, J = 6.7 Hz, 3H), 0.96 (t, J = 6.7 Hz, 3H) (8) ¹³ C-nuclear magnetic resonance spectrum; FIG. (125M
Hz, CD ₃ OD) δ (ppm); 177.4 (s), 171.9 (s), 171.6 (s), 170.0 (s), 169.0
(s), 162.1 (d), 153.1 (s), 138.7 (s), 130.4 (d), 129.5 (d),
128.3 (s), 127.7 (d), 126.3 (d), 124.4 (d), 119.0 (d), 116.2
(s), 79.9 (d), 77.9 (d), 73.4 (d), 72.6 (d), 70.5 (d), 56.8
(d), 46.9 (s), 40.5 (t), 31.3 (d), 27.1 (q), 22.5 (q), 18.5
(q), 18.3 (q), 17.7 (q), 16.6 (q) (9) Solubility; soluble in methanol, ethanol, dimethylsulfoxide, acetone, pyridine, ethyl acetate, chloroform, diethyl ether and the like. Hexane, insoluble in water. (10) Color reaction; positive for 50% sulfuric acid, iodine, torence reagent and phenol reagent. (11) HPLC: retention time 6.00 min Column: TSK gel ODS-80TM 4.6 mm dia. X 2
50mm solvent: 55% acetonitrile-water, flow rate 1.0ml / min, UV 2
20nm (12) Acid, neutral, basic distinction; neutral

When the novel physiologically active substance of the present invention is used as a medicine, it may be a pharmacologically acceptable salt. As pharmacologically acceptable salts, alkali metal salts such as sodium and potassium, or alkaline earth metal salts such as magnesium and calcium, aluminum and other metal salts,
And organic amine salts such as alkylamine salts and pyridine salts. This compound or a pharmacologically acceptable salt thereof is safely administered orally and parenterally as a medicament to humans and animals. Parenteral administration includes, for example, intravenous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, transdermal administration, pulmonary administration, nasal administration, enteral administration, buccal administration, transmucosal administration, and the like. These formulations are administered. For example, injections, suppositories, aerosols, transdermal absorption tapes and the like can be mentioned. Examples of oral administration preparations include tablets (including sugar-coated tablets, coated tablets, and buccal tablets), powders, capsules (including soft capsules), granules (coated, pills, troches, liquids, and preparations thereof). Sustained-release preparations which are acceptable in general. Liquid preparations for oral administration include suspensions, emulsions, syrups (including dry syrups), elixirs and the like. These preparations are administered as a pharmaceutical composition together with pharmacologically acceptable carriers, excipients, disintegrants, lubricants, coloring agents, etc., according to known pharmaceutical manufacturing methods. As carriers and excipients used in these preparations, for example, lactose, glucose, sucrose, mannitol, potato starch, corn starch, calcium carbonate, calcium phosphate, calcium sulfate, crystalline cellulose, licorice powder, gentian powder, etc. For example, starch, tragacanth, gelatin, syrup, polyvinyl alcohol, polyvinyl ether, polyvinylpyrrolidone, hydroxypropylcellulose, methylcellulose, ethylcellulose, carboxymethylcellulose, etc. Lubricants such as crystalline cellulose, calcium carbonate, sodium bicarbonate, and sodium alginate Magnesium stearate, talc, hydrogenated vegetable oil, macrogol, those which are allowed to added to pharmaceuticals as coloring agents, can be used respectively. Tablets and granules are sucrose, gelatin, hydroxypropylcellulose, purified shellac, gelatin, glycerin, sorbitol, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, cellulose phthalate acetate, hydroxypropylmethylcellulose phthalate, methyl methacrylate as required. And a film of methacrylic acid polymer or two or more layers. Further, capsules made of a substance such as ethyl cellulose or gelatin may be used. In addition, when preparing an injection, a pH adjusting agent, a buffer, a stabilizer,
Each injection is prepared by adding a solubilizing agent or the like in a conventional manner.

The form of the external preparation may be solid, semi-solid, transdermal or transmucosal for oral or nasal administration.
Semi-solid or liquid preparations are included. Examples of the liquid preparation include pharmaceutically acceptable emulsions or emulsions such as lotions, tinctures for external use, and liquid preparations for transmucosal administration. This formulation is commonly used diluent,
For example, it contains ethanol, oil, emulsifier and the like. Examples of semisolid preparations include ointments such as oily ointments and hydrophilic ointments. The formulation contains commonly used carriers such as water, petrolatum, polyethylene glycol, oils, surfactants and the like. Examples of semisolid or solid preparations include patches for transdermal administration such as plasters (rubber plaster, plaster, etc.), films, tapes, and cataplasms, or for transmucosal (intraoral, nasal) administration. No. This formulation is a commonly used carrier, such as natural rubber,
Rubber-based polymers such as butadiene rubber, synthetic rubber such as SBR and SIS, mud-forming agents such as gelatin, kaolin, and zinc oxide; hydrophilic polymers such as sodium carboxymethylcellulose and sodium polyacrylate; acrylic resins; liquid paraffin And tackifiers, water, other oils, and surfactants. These preparations may further use adjuvants such as stabilizers, solubilizers, and transdermal absorption enhancers, or additives such as fragrances and preservatives.
When the novel physiologically active substance of the present invention is administered to a patient, it depends on conditions such as the degree of symptoms, age of the patient, body weight, and health condition, and is not particularly limited.
It suffices to administer 10 g orally or parenterally once a day or more. When the pharmaceutical composition uses an external preparation, for example, an ointment in which the active ingredient has a concentration of 0.01 to 1%,
It may be applied at least once a day.

[0022]

The present invention will be described in more detail with reference to the following examples, which are merely illustrative and do not limit the present invention in any way.

[0023]

[Example 1] (1) Isolation of microorganisms Soil collected in Yuki City, Ibaraki Prefecture is heat treated (60 ° C, 10 minutes)
The suspension obtained by suspending 1 g of the dried soil obtained in the above step was suspended in 10 ml of sterilized water, and the suspension was diluted 10 ^-4 times. The agar plate medium (humic acid 0.12%, glucose 0.005%, L-asparagine)
0.005%, soluble starch 0.005%, dipotassium phosphate
0.01%, magnesium sulfate 0.005%, sodium chloride
0.05%, ferrous sulfate 0.001%, calcium chloride 0.001%
, Agar 1.8%, pH 7.0) and culture (27 ° C, 10 days)
Was performed. The emerged colonies are streaked on the agar plate medium described above, purely separated, and the actinomycetes Streptomyces sp. SNA 15896 strain ( Stre
ptomyces sp. SNA 15896). This strain has been deposited with the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry on February 25, 1998 under the name of Life Science Bacteria No. 16667 (FERM P-16667).

(2) Cultivation of Microorganism A 1 cm square of each mat was cut out from a slope medium (glucose / starch / asparagine agar medium) of Streptomyces sp. SNA 15896 obtained in Example 1- (1), and pre-cultured in 70 ml. Medium (0.14% ammonium sulfate, 0.2% monopotassium phosphate, 0.03% calcium chloride, 0.03% magnesium sulfate, 0.03% urea, 0.5% polypeptone, 0.1% yeast extract, 3% soy flour, 1% glucose, 0.5% soluble starch, Ferrous sulfate 0.0005%, manganese sulfate
0.00016%, zinc sulfate heptahydrate 0.00014%, cobalt (II) chloride 0.0002%, pH adjusted) were inoculated into two 500 ml Erlenmeyer flasks, and cultured on a rotary shaker at 27 ° C. for 6 days. To obtain a preculture. 140 ml of this preculture solution was inoculated into a 10 L tank containing 5 L of the main culture medium having the same composition as the preculture medium, and cultured at 27 ° C. for 5 days under aeration and agitation (aeration rate of 120 L / min, agitation of 300 revolutions / minute, internal pressure of 0.1 L / min). kg / cm ² ).

[0025]

Example 2 Purification of Culture 5 L of the culture was centrifuged (5000 rpm, 15 minutes), and the obtained cells were extracted with 5 L of acetone. The acetone extract was concentrated under reduced pressure to remove acetone, and then extracted twice with an equal volume of ethyl acetate. The ethyl acetate layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 3 g of a crude extract. This was dissolved in a small amount of chloroform and applied to a 500 ml silica gel column equilibrated with chloroform. Change this column to 1.
After elution with 5 L of chloroform, the compound of the present invention was eluted with hexane-ethyl acetate (7: 3) to obtain 52 mg of a crude product. The crude product was dissolved in a small amount of methanol and passed through a HPLC column (TOSOH, TSK gel ODS-80TM Φ20mm x 250mm) equilibrated with 70% acetonitrile aqueous solution at a flow rate of 10ml / min and eluted with the same solvent. did. The absorption at 254 nm was detected, and the detection peak of the compound of the present invention was collected. The fraction with the largest absorption (retention time 23 minutes) is designated as substance B, and the fraction with the largest absorption (retention time 28 minutes) is subsequently designated as A.
Substance. These fractions were concentrated under reduced pressure, and
g and 8 mg of substance B were obtained as white powders. It was confirmed that these powders were A and B by measuring the specific rotation of each, and these substances exhibited the above-mentioned physicochemical properties.

[0026]

Example 3 Test Example 1: Mouse lymphocyte mixed reaction (ML
R) male C57BL / 6 mouse spleen cells were treated with mitomycin C (manufactured by Wako Pure Chemical Industries, Ltd.) at 37 ° C. for 30 minutes as inhibitory stimulator cells against R) , and male BALB / c mouse spleen cells were used as reaction cells. The cells were suspended in RPMI 1640 medium (GIBCO BRL) containing 10% fetal bovine serum, and the novel physiologically active substances (substances A and B) of the present invention were added at various concentrations, and the suspension was added at 37 ° C., 5 ° C.
The cells were cultured for 72 hours under the condition of a concentration of CO 2 (J. Antibio
tics, Vol.XL, No.9, 1256, 1987). Harvest 4
An hour before, the cells were pulse-labeled with ³ H-thymidine, and after the culture was completed, the cells were collected with a cell harvester, and the amount of radiation incorporated into the cells was measured to calculate a 50% inhibitory activity (IC ₅₀ ). Table 2 shows the results. As a result, it was revealed that the physiologically active substances of the present invention both inhibit the lymphocyte mixed reaction.

[0027]

[Table 2]

[0028]

[Example 4] Test example 2: For primary cultured rat hepatocytes
Cytotoxicity 10% fetal bovine serum, Willia, including 10 ^-8 M dexamethasone
Hepatocytes prepared from adult rat liver in m's E medium (GIBCO BRL) (Experimental Medicine, Vol.7 No.131989)
Was suspended at a concentration of 1 × 10 ⁴ cells / ml and cultured at 37 ° C. and 5% CO 2 for 24 hours, and then the novel physiologically active substances of the present invention (substances A and B) were added at various concentrations. The culture was continued under the same conditions for 48 hours. Four hours before harvesting, the cells were pulse-labeled with ³ H-thymidine, and after completion of the culture, the cells were collected with a cell harvester, and the amount of radiation incorporated into the cells was measured to calculate a 50% inhibitory activity (IC ₅₀ ). .
Table 3 shows the results. As a result, it was revealed that all of the physiologically active substances of the present invention had a lower inhibitory activity on rat primary cultured hepatocytes than MLR, and thus had lower cytotoxicity.

[0029]

[Table 3]

[0030]

Example 5 Test Example 3: For cytokine production of lymphocytes
Effect of splenocytes from male BALB / c mice RPMI 1 containing 10% fetal calf serum
Suspension in 640 medium (GIBCO BRL), 50 ng of the novel physiologically active substances (substances A and B) of the present invention, mitogen (concanavalin A, 1 μg / ml, Sigma) were added, and the mixture was added at 37 ° C. The cells were cultured for 24 hours under the condition of 5% carbon dioxide concentration. IL-2, IL-4, IL-5, IL-6, contained in the culture supernatant
IFN-γ was measured by a sandwich ELISA method (kit for cytokine quantification: manufactured by ENDOGEN). Table 4 shows the results. As a result, it was clarified that both of the present physiologically active substances suppress cytokine production of lymphocytes.

[0031]

[Table 4]

[0032]

Example 6 Test Example 4: Action of activated C lymphocytes on activated lymphocytes Male C57BL / 6 mouse spleen cells were treated with mitomycin C (manufactured by Wako Pure Chemical Industries, Ltd.) at 37 ° C. for 30 minutes, and as reaction cells. Using male BALB / c mouse splenocytes, the cells were transformed with RPMI 1640 containing 10% fetal calf serum.
Suspended in a medium (GIBCO BRL) (J. Antibiotics,
Vol.XL, No.9, 1256, 1987), 0, 24, 48
On the 68th hour, the novel physiologically active substances of the present invention (substances A and B) were added at 0.5 μg / ml, respectively. Culture at 37 ° C., 5% CO 2 for 72 hours, pulse labeling with ³ H-thymidine 4 hours before harvesting, and after completion of the culture, collect the cells with a cell harvester and incorporate the radiation into the cells. The amount was measured to calculate the inhibitory activity. Table 5 shows the results. As a result, it was revealed that the physiologically active substances of the present invention both inhibit activated lymphocytes.

[0033]

[Table 5]

[0034]

Example 7 Test Example 5: Antitumor against human cultured cancer cells
In RPMI 1640 medium containing cell activity 10% fetal bovine serum (GIBCO BRL) human colon carcinoma cells HCT-116, human lung cancer cell DMS1
14, or human gastric cancer cells MKN74 were suspended at a concentration of 1 × 10 ⁴ cells / ml and cultured at 37 ° C. under 5% CO 2 concentration. After 24 hours, the novel physiologically active substances of the present invention (substances A and B) were added at various concentrations, and culturing was continued under the same conditions for 48 hours. The antitumor cell activity was determined by measuring the proliferation rate by a colorimetric method using the protein-binding dye sulforhodamine B (SRB) (J. Natl. Cancer Inst. 82: 1113, 1990).
% Inhibitory activity (IC ₅₀ ) was calculated and shown. Table 6 shows the results. As a result, the physiologically active substances of the present invention
It showed antitumor activity against CT-116, DMS114 or MKN74 human cultured cancer cells.

[0035]

[Table 6]

[0036]

[Example 8] Test example 6: Multidrug resistance overcoming effect 2780AD cells (Rogan AM et al., Science, v.) In RPMI 1640 medium (GIBCO BRL) containing 10% fetal bovine serum.
224, p994, 1984) were suspended at 1 × 10 ⁶ cells / ml, and cultured at 37 ° C. for 24 hours under the conditions of 5% carbon dioxide gas concentration. ³ H-Vincristine (Amersham) R
After the medium was replaced with PMI 1640, the novel physiologically active substances (substances A and B) of the present invention were added at various concentrations, and culturing was continued for 2 hours.
ation system (Beckman). Table 7 shows the results. As a result, it was revealed that both the present physiologically active substance enhances the uptake of vincristine into cells, and has an action of overcoming multidrug resistance.

[0037]

[Table 7]

[0038]

Industrial Applicability The present invention relates to a novel neoantimycin-based physiologically active substance, a microorganism producing the same, a method for producing a novel physiologically active substance using the microorganism, and a method for preparing the novel physiologically active substance as an active ingredient. Related to medicine. The novel physiologically active substance of the present invention is an agent for suppressing immune rejection by organ transplantation, a therapeutic agent for allergic diseases such as atopic dermatitis, a therapeutic agent for autoimmune diseases such as rheumatoid arthritis, and an antitumor agent in cancer chemotherapy Or, it is useful as an agent for overcoming multidrug resistance.

[Brief description of the drawings]

FIG. 1 shows an ultraviolet absorption spectrum of a compound of the present invention (substance A) in methanol (20 μg / ml).

FIG. 2 shows an infrared absorption spectrum of a compound of the present invention (substance A) with a potassium bromide tablet.

FIG. 3 shows a 500 MHz _Z ¹ H-NMR spectrum (based on TMS) of a compound of the present invention (substance A) in a heavy methanol solution.

FIG. 4 shows a 125 MHz _Z ¹³ C-NMR spectrum (based on heavy methanol) of a compound of the present invention (substance A) in a heavy methanol solution.

FIG. 5 shows an ultraviolet absorption spectrum of the compound of the present invention (substance B) in methanol (20 μg / ml).

FIG. 6 shows an infrared absorption spectrum of the compound of the present invention (substance B) using a potassium bromide tablet.

FIG. 7 shows a 500 MHz _Z ¹ H-NMR spectrum (TMS standard) of a compound of the present invention (substance B) in a heavy methanol solution.

FIG. 8 shows a 125 MHz _Z ¹³ C-NMR spectrum (based on heavy methanol) of a compound of the present invention (substance B) in a heavy methanol solution.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 31/335 ADU A61K 31/335 ADU ADZ ADZ C12N 1/20 C12N 1/20 A C12P 17/08 C12P 17/08 // ( C12N 1/20 C12R 1: 465) (C12P 17/08 C12R 1: 465) (72) Inventor Kanji Higashio 1769-10 Yamada, Kawagoe-shi, Saitama

Claims (5)

[Claims] 1. A novel physiologically active substance represented by the following general formula: and a pharmacologically acceptable salt thereof. Embedded image (Here, R represents a lower alkyl group of which a hydrogen or a C ₁ ~C _5.) 2. A microorganism which is classified into the genus Streptomyces and produces the novel physiologically active substance according to claim 1. 3. A Streptomyces sp. (Streptom
3. The microorganism according to claim 2, which is a strain SNA15896 (FERM P-16667) or a mutant thereof. 4. Culturing the microorganism according to claim 2 or 3,
A method for producing a novel physiologically active substance, comprising producing the novel physiologically active substance according to claim 1 in a culture solution and collecting the produced substance. 5. A medicament comprising the novel physiologically active substance according to claim 1 or a pharmacologically acceptable salt thereof as an active ingredient.