WO1997043434A1

WO1997043434A1 - Novel physiologically active substance and process for producing the same

Info

Publication number: WO1997043434A1
Application number: PCT/JP1997/001612
Authority: WO
Inventors: Kazuhiko Kurosawa; Kousaku Takahashi; Kyouko Matsubara; Masayuki Okue; Naohiro Washida; Kanji Higashio
Original assignee: Snow Brand Milk Products Co., Ltd.
Priority date: 1996-05-14
Filing date: 1997-05-14
Publication date: 1997-11-20
Also published as: JP3851661B2

Abstract

A novel physiologically active substance; a microorganism to be used for producing the same; and medicines containing the same as the active ingredient. A novel physiologically active substance SNF4435 represented by formula (I); a strain Streptomyces spectabilis SNF4435 isolated from the soil in the main island of Okinawa; a process comprising culturing the microorganism in a medium and separating the substance from the culture; and a process for producing the substance via chemical synthesis from its analog produced in the culture. The above substance is usable as medicines such as an immunosuppressant, an antibacterial agent for autoimmune diseases, allergic diseases and infectious diseases, and an antagonist against tolerance to anticancer drugs.

Description

Description New physiologically active substance and method for producing the same

The present invention relates to novel nitrophenyl pyrone system of the physiologically active substance, _c further relates to a method for producing a microorganism and novel physiologically active substance that is used to produce and manufacture the same, the present invention provides such novel physiologically Pharmaceuticals containing an active substance as an active ingredient, in particular, inhibitors of immune rejection due to organ transplantation, etc., therapeutic agents for autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, uveitis, allergies such as atopic dermatitis —Regarding therapeutic agents for diseases, antibacterial agents for infections caused by various microorganisms, or agents for overcoming anticancer drug resistance. Background art

Immunosuppressants are effective in the prevention and treatment of diseases and conditions that require a reduced immune response. Prevent transplant rejection, for example, kidney, liver, bone marrow, heart and corneal transplantation, or treat autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, uveitis, or allergic diseases such as atopic dermatitis It is indicated as a therapeutic agent for many immune diseases. Since the US Food and Drug Administration (USFDA) approved cyclosporine in 1983, immunosuppressants have been actively developed, and drugs such as Prograf and Spanidine have been put into practical use. It has a high reputation in the field. However, new problems such as frequent occurrence of renal damage and the generation of autoreactive lymphocytes associated with the use of existing drugs have been pointed out, and the use of existing drugs has been reduced for autoimmune diseases caused by the generation of autoantibodies. The efficacy was not always satisfactory c Disclosure of the invention

In view of the above situation, the present inventors have sought extensively in nature a substance having excellent immunosuppressive activity and low side effects, and as a result of intensive search, have found that the activity of the strain in a culture of a strain belonging to the genus Streptomyces sp. A novel nitrophenylpyrone-based physiologically active substance having the following features: Accordingly, an object of the present invention is to provide a novel nitrophenylpyrone-based physiologically active substance, a microorganism used for producing and producing the novel physiologically active substance, and a method for producing the novel physiologically active substance. And

More specifically, a novel bioactive substance having an immunosuppressive activity, an antiallergic activity, an antibacterial activity and an effect of overcoming anticancer drug resistance and having few side effects, a microorganism used for producing and producing the bioactive substance, and a culture of the microorganism It is another object of the present invention to provide a method for producing the physiologically active substance by obtaining the physiologically active substance.

Furthermore, an object of the present invention is to provide a medicament for preventing or treating various diseases by utilizing the above-mentioned physiological activities.

That is, the present invention relates to a novel physiologically active substance represented by the following formula (1) and a pharmacologically acceptable salt thereof.

The present invention also relates to a microorganism belonging to Streptomyces, which can be used for producing and producing a novel physiologically active substance represented by the formula (I).

Such microorganisms include, specifically, the soil of Okinawa Island by the present inventors. And S. streptomyces spectabil (s) SN F4435 strain (FERM BP-5915).

Further, the present invention relates to a method for producing a novel bioactive substance, comprising culturing the microorganism, producing the novel bioactive substance in a culture, and collecting this. In addition, the present invention relates to a method for producing a novel bioactive substance by synthetic chemistry by culturing the microorganism, producing an analog Spectinabin in the culture, and closing the tetraene moiety.

Furthermore, the present invention relates to a medicament comprising a novel physiologically active substance represented by the above formula (I) or a pharmacologically acceptable salt thereof as an active ingredient.

The medicament of the present invention is used as a therapeutic agent for immune rejection or autoimmune disease due to organ transplantation, a therapeutic agent for allergic disease, an antibacterial agent for infectious disease, or an agent for overcoming anticancer drug resistance.

That is, the physiologically active substance of the present invention is a novel nitrophenylpyrone-based compound, and in view of its activity, an inhibitor of immune rejection by organ transplantation or the like, systemic erythematosus, rheumatoid arthritis, uveitis, etc. It is useful as a therapeutic agent for autoimmune diseases, a therapeutic agent for allergic diseases such as atopic dermatitis, an antibacterial agent for infectious diseases caused by various microorganisms, or a drug for overcoming anticancer drug resistance. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an ultraviolet absorption spectrum of compound SNF4435C of the present invention in methanol (1S gZml).

FIG. 2 shows an infrared absorption spectrum of the compound of the present invention SNF4435C with a potassium bromide tablet.

FIG. 3 shows a 500 MHz NMR spectrum (TMS standard) of a compound of the present invention SNF4435C in a double-mouthed form solution. FIG. 4 shows a 125 MHz ¹³ C NMR spectrum of the compound of the present invention SNF 4435C in a heavy-mouthed form solution (based on heavy-mouthed form).

FIG. 5 shows an ultraviolet absorption spectrum of compound SNF4435D of the present invention in methanol (18.0 g Zml).

FIG. 6 shows an infrared absorption spectrum of a compound of the present invention SNF4435D with a lithium bromide tablet.

FIG. 7 shows a 500 MHz NMR spectrum (based on TMS) of the compound of the present invention SNF4435D in a double-mouthed form solution.

FIG. 8 shows a 125 MHz ¹³ C NMR spectrum (based on double-mouthed form) of a compound of the present invention S NF4435D in a heavy chloroform solution. BEST MODE FOR CARRYING OUT THE INVENTION

The novel physiologically active substance of the present invention (hereinafter, referred to as the compound of the present invention) can be obtained by culturing a microorganism. At this time, the microorganism to be used is not particularly limited as long as it is a bacterium that produces the compound of the present invention, but is preferably a bacterium belonging to the genus Streptomyces (Streptomyces sp.), Particularly preferably Streptomyces ′ (Strep tomyces_spectabi 1 is) Strain SN F4435 can be mentioned. This Streptomyces scutabilis SNF4435 strain is a strain isolated from soil collected from Oki Jiphonjima Island by the present inventors, and has a deposit number of FERM P.R. -Deposited on February 27, 1996 as 15476, then transferred from this original deposit to a deposit under the Budapest Treaty, and given the accession number FERM BP-5915.

Isolation of Streptomyces spectabili ^ SNF 4435 strain is carried out by the method usually used for the isolation of actinomycetes from the soil of Okijima Island, for example, Goodfellow, M .; Actinomycetologica Vol. 2, No. .1 13-29 (1988) It can be easily implemented by the method.

The properties of the Streptomyces' Scuctabilis SNF4435 strain thus isolated are as follows.

1. Form

The aerial hyphae settle down in the stomach, and pseudo-ringy branching is slightly observed, but no division of the hyphae is observed. Aerial hyphae have a reddish or rarely white hue, and a chain of more than 10 spores is formed, forming a flexible linear shape. The surface of the spores is almost smooth and cylindrical with a size of about 0.4X0.8 mm. No special organs such as sclerotia, spores, zoospores, etc. are found.

2. Growth conditions in various media

Table 1 shows the results of culturing at 27 ° C for 14 days on various agar plate media. The description of the colors was based on the JIS Color Name Book (JIS Z 8102 compliant, Japan Standards Association, April 15, 1993, 1st edition, 3rd printing).

Table 1

Agar medium Growth bacteria fungi Bottom surface Soluble dyesIast malt Good good Salmon pink Gold Red

(8R 7.5 / 7.5), None

(ISP2) Diffusible powder, thin (9R 5.5 / 14)

Medium pink (2.5R 7/7), vivid yellowish red

Diffusible Powdery, faint (7.5R 5/15) None Starch ・ Inorganic salt Medium Bright yellowish red Bright red

(7.5R 7/9), None

(ISP4) Sensitivity powdery, light (5R 4/14)

Glycerin, moderately bright yellow-red yellow-red

Asparagine (5YR 7/14), None (ISP5) Diffusible powder, light (5YR 6/12)

Peptone-yeast-bad brown color dark brown black brown (2.5 iron (1GY 4.5 / 3.5),

(ISP6) Diffusion Powdery, faint (5YR 3/2) YR 2 / 1.5 Tyrosine Medium Vivid yellow-red Yellow-red

(5YR 7/14) None (ISP7) Diffusible powdery, smoky (5YR 6/12) (1) Growth temperature range

6. Use yeast 'malt agar medium'. C, 12 ° C, 17 ° C, 22 ° C, 27 ° C, 32 ° C, 37. As a result of testing at each temperature of C, 42 ° C and 47 ° C, it grew at any temperature except 7 ° (, 42 ° C and 47 ° C. The optimum growth temperature was 27 ° C to 32 ° C. It seems to be near.

(2) Liquefaction of gelatin

In the glucose-peptone-gelatin medium, 27 ° C culture or simple gelatin medium or 20 ° C culture, liquefaction started about 10 days after cultivation in any medium, and the liquefaction effect was moderate after 21 days of observation.

(3) Starch hydrolysis

In starch / inorganic salt agar medium, 27 ° C culture, water degradability was observed from about 3 days after culture, and the effect was relatively strong.

(4) Coagulation of defatted milkPeptone

No coagulation was observed in the defatted milk medium at 37 ° C, but peptone formation was observed around 7 days after the culture.

(5) Melanin-like pigment formation

In a 27 ° C culture using tryptone 'yeast' broth medium, peptone yeast 'iron agar medium or tyrosine agar medium, only peptone yeast' iron agar medium 'was cultured. Was.

(6) Utilization of carbon source

Prideham-Godlieb agar medium, 27 ° C culture, using D-glucose, D-xylose, D-fructose, raffinose, inositol, D-mannitol, mannose, D-galactose , L-arabinose, sucrose and L-rhamnose are not used.

To summarize the above properties, the mycological properties of SNF 4435 strain are that the aerial hyphae are linear and do not form spores, but there are slight pseudoringy branches. At the tip of the aerial mycelium It links about 10 spores and its surface is smooth. In various media, the color of the aerial mycelium changes from pink to red. As a soluble pigment, melanin-like pigment is produced on peptone, yeast, and iron agar medium, and starch has a moderate hydrolytic property and moderate protein degradation ability o

The 2,6-diaminopimelic acid contained in the cell wall was of the LL-type. Based on these properties, the SNF4435 strain is considered to belong to the genus Streptomyces, and is described in "Bergey's Manual of Determinative Bacteriology", 8th ed., And ISP Report "International Journal of Systematic Bacteriology", Volume 18, 69, 279 (1968), 19, 391 (1969) 22 and 265 (1972), Streptomyces and Streptomyces spectabilis are closely related.Therefore, the SNF4435 strain and Streptomyces sp. (Streptomyces spectabilis IFO 13424) The results are shown in Table 2.

Table 2

The results indicate that there are slight differences in the color tone and growth temperature range of the aerial hyphae, as well as the morphology of the aerial hyphae, the surface of the spores, the state of growth, the point of producing melanin-like pigments, and the availability of carbon sources. And both agree well. Therefore, the SNF4435 strain was identified as Streptomyces spectavilis (Streptomyces spectabi 1 is). The microorganism used in the present invention may be irradiated with X-rays, ultraviolet rays, or the like, or treated with a mutagenic agent such as nitrite, N-methyl-N, -nitro-N-nitrosoguanine (NTG), transformation, transduction, or the like. It may be a microorganism mutated by a commonly used bacterial species conversion treatment method such as fusion.

The compound of the present invention is produced in a culture obtained by inoculating a nutrient source-containing medium that can be used by ordinary microorganisms with a bacterium producing the compound of the present invention and growing it, or producing the compound. The target compound of the present invention can be produced by organic synthesis using a culture obtained by inoculating and growing the microorganisms used in the culture of the present invention. Any medium such as 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, starch syrup or dextrin are used alone or as a mixture. Nitrogen sources include soy flour, corn gluten meal, corn steep liquor, meat extract, yeast extract, cottonseed kashiwa, peptone, wheat germ, fish meal, urea and other organic nitrogen sources, ammonium sulfate, sodium nitrate, etc. The inorganic nitrogen 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.If necessary, heavy metals such as iron, copper, cobalt, molybdenum, manganese or zinc can be used. A trace amount of salt can also be added. When foaming during culture is remarkable, various antifoaming agents known as antifoaming agents may be appropriately added to the medium. In addition, organic and inorganic substances useful for the production of the compound of the present invention, which are utilized by the producing bacteria, 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, and aeration culture may be performed.However, when culturing Streptomyces sucker evening bill SNF 4435 strain, in particular, Shaking culture or deep aeration stirring culture Nutrition is preferred. Although it depends on the culture conditions, the pH of the culture medium is preferably in the range of 4 to 8, and the culture temperature is 22 to 37 ° C, preferably 25 to 30 ° C. The culturing time is 48 to 168 hours, preferably 96 to 144 hours.

In order to isolate and produce 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. If necessary, synthetic chemistry means may be used as appropriate.

Since the compound of the present invention produced by culturing is usually accumulated both inside and outside the cells in the culture, it is often separated into cultured liquid and cells by means such as centrifugation and filtration. Conventional separation methods from culture filtrates and cells, such as dialysis, solvent extraction, methods utilizing the difference in solubility with impurities, ion-exchange resin or adsorption or partition chromatography, and gel filtration. Separation and purification can be performed alone or in an appropriate combination, and in some cases, repeated use.

The compound of the present invention, which is collected using synthetic chemistry means, uses the above-described separation means for analogs (nitrophenylpyrone-based) of the compound of the present invention produced in a culture solution by culturing a microorganism. In this way, the compound can be separated and purified, and can be produced by synthetic chemistry means, for example, a ring closure reaction using a catalyst. Examples of the organic solvent used in the reaction include methanol, ethanol, tertiary butyl alcohol, tetrahydrofuran, dimethyl ether, ethylene glycol, dimethyl ether, dimethylformamide, dimethyl sulfoxide, benzene, toluene, xylene, dioxane, and the like. Examples include methylene chloride, chloroform, dichloroethane, and acetonitrile. When the ring closure reaction is performed using light, 200ηπ! Light with a wavelength of up to 800 nm is used, but wavelengths higher or lower can be selected as necessary. The reaction temperature for the ring-closure reaction is usually from 20 to 400 ° C, and a higher or lower temperature can be selected as necessary. The pressure during the ring closure reaction is usually 1 to 10 atm, and a higher or lower pressure can be selected as necessary. Catalyst used for ring closure reaction Examples thereof include aluminum chloride, tin chloride, boron fluoride, copper fluoborate and iodine. The reaction time of the ring closure reaction is usually in the range of 30 minutes to 2 days, but a longer or shorter time can be selected as necessary.

Fig. 2 shows a reaction formula for closing the tetraene moiety of spectinapyrine in the present invention.

After performing the ring closure reaction under the above reaction conditions, SNF4435C and SNF4435D are separated and purified by a method known in the field of synthetic organic chemistry, for example, a method using solvent extraction, recrystallization, chromatography, ion exchange resin, HP LC, etc. be able to.

The properties of the obtained compound of the present invention are shown. Note that SNF4435 has the same planar structure (see formula (I)) but has optical isomers, each of which is designated as SNF4435C or D. These optical isomers can be separated into their respective optical isomers by a conventional separation means such as liquid chromatography. When used as a medicament, the optical isomer may be used alone or in a racemic form.

Physicochemical properties of S N F 4435 C

(1) Color and properties: milky white powder (2) Molecular formula: C ₂₈ H _3, N0 ₆

(3) Mass spectrum (FAB—MS): m / z 478 (Μ + Η) ⁺

(4) Specific _{^{rotation: [a] D 26 - 105.6}} ° (C 0.10, CHC1 3)

(5) Ultraviolet absorption spectrum: shown in Figure 1.

A ^Me0H nm (ε): 271 (19322)

(6) Infrared absorption spectrum: shown in Figure 2.

Re: ^KBr cm— ¹ : 2950, 2850, 1670, 1600, 1520, 1350, 1255, 1165,

1040

(7) 'Η-nuclear magnetic resonance spectrum: shown in Fig. 3. (500MHz, CDC1 ₃₎

δ (ppm): 8.19 (d, J = 8.7H _Z .2H), 7.55 (d, J = 8.7H _Z .2H), 5.58 (d, J = l.4H _Z .1H),

4.95 (S, 1H), 4.78 (t, J = 8.2H _Z .1H), 4.32 (d, J = 9.8H _Z .1H),

3.97 (d, J = 9.8H _Z .1H), 3.96 (S, 3H), 3.64 (S, 1H), 2.84 (S, 1H), 2.43 (d, J = 8.2H _Z .2H), 1.89 (S , 3H), 1.84 (S, 3H), 1.74 (S, 3H), 1.72 (d, J = 1.4Hz.3H), 1.30 (S, 3H)

(8) ¹³ C—nuclear magnetic resonance spectrum: shown in FIG. (125MHz, CDC)

(ppm): 180.5 (s), 162.2 (s), 155.2 (s), 146.9 (s), 145.l (s), 131.0 (s),

130.4 (s), 129.1 (d), 123.8 (d), 123.6 (d), 122.0 (d), 119.6 (s), 100.3 (s), 73.5 (d), 70.6 (t), 63.7 (d), 55.6 (q), 51.8 (s), 51.1 (d), 46.4 (t), 43.0 (s), 30.4 (q), 23.0 (q), 22.2 (q), 9.4 (q), 7.0 (q)

(9) Solubility: Soluble in methanol, chloroform, ethanol, acetone, pyridine, ethyl acetate, getyl ether, dimethyl sulfoxide, etc. Hexane, insoluble in water

(10) Color reaction: positive for 50% sulfuric acid and iodine

(11) HP LC: retention time 14.6 minutes Column: Inatosyl ODS-2 (ø4.6X250, GL Sciences) Solvent: 80% methanol / water, flow rate 1 mlZmin, UV 220nm

(12) Distinguishing between acidic, neutral and basic substances: neutral

Physicochemical properties of S NF4435D

(1) Color and properties: milky white powder

(2) Molecular formula: C ₂₈ H ₃₁ N0 ₆

(3) Mass spectrum (FAB-MS): m / z 478 (M + H) ⁺

(4) Specific rotation: [] ⁶ + 84.8. (C 0.10, CHCh)

(5) UV absorption spectrum: shown in Fig. 5.

A _max ^Me0H nm (ε): 270 (21462)

(6) Infrared absorption spectrum: shown in Figure 6.

Les _{^{^{max KBr cm "1: 2950,}}} 2850, 1670, 1600, 1520, 1350, 1255, 1165,

1040

(7) 'H-nuclear magnetic resonance spectrum: shown in Fig. 7. (500MHz, CDCh)

δ (ppm): 8.15 (d, J = 8.7H _Z .2H), 7.47 (d, J = 8.7H _Z .2H), 5.70 (S, 1H),

4.94 (dd, J = 7.0, 9.5H Z, 1H), 4.89 (S, 1H), 4.18 (d, J = 9.2H Z, 1H), 3.84 (d, 2Hz, 1H), 3.74 (S, 1H) , 3.54 (S, 3H), 2.74 (S, 1H), 2.48 (dd, J = 13.1, 9.5H Z, 1H), 2.28 (dd, J two 13.1, 7.0H _Z .1H),

1.97 (S, 3H), 1.84 (S, 3H), 1.80 (S, 3H), 1.74 (d, J = 1.5H _Z .3H), 1.3KS, 3H

(8) ' ³ C—nuclear magnetic resonance spectrum: shown in Figure 8. (125MHz, CDC)

5 (ppm): 180.5 (s), 161.9 (s), 154.9 (s), 146.9 (s), 144.0 (s), 131.3 (s),

131.2 (s), 129.9 (d), 124.4 (d), 123.2 (d), 121.9 (d), 119.7 (s), 100.0 (s), 72.5 (d), 70.6 (t), 61.1 (d), 55.2 (d), 54.8 (q), 51.1 (s), 45.5 (t), 43.0 (s), 30.7 (q), 22.2 (q), 22.l (q), 9.4 (q), 6.8 (q)

(9) Solubility: soluble in methanol, chloroform, ethanol, acetone, pyridine, ethyl acetate, dimethyl ether, dimethyl sulfoxide, etc. Hexane, insoluble in water

(10) Color reaction: positive for 50% sulfuric acid and iodine

(11) HPLC: retention time 16.6 minutes

Column: Intosyl ODS-2 (ø4.6 x 250mm, GL Sciences Inc.)

Solvent: 80% methanol · water, flow rate 1 ml / min, UV 220nm

(12) Distinguishing between acidic, neutral and basic substances: neutral

When the novel physiologically active substance of the present invention is used as a medicine, it may be a pharmacologically acceptable salt. Pharmaceutically acceptable salts include alkali metal salts such as sodium and potassium, alkaline earth metal salts such as magnesium and calcium, aluminum and other metal salts, and organic amines such as alkylamine salts and pyridine salts. Salt. This compound or a pharmacologically acceptable salt thereof is safely orally and parenterally administered to humans and animals as a medicament. Parenteral administration includes, for example, intravenous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, transdermal administration, pulmonary administration, transmucosal administration, enteral administration, buccal administration, transmucosal administration, etc. These formulations are administered. For example, injections, suppositories, aerosols, transdermal absorption tapes and the like can be mentioned. Examples of oral preparations include tablets (including sugar-coated tablets, coated tablets, and buccal tablets), powders, capsules (including soft capsules), granules (coated products, pills, troches, and liquids). , Or a pharmaceutically acceptable sustained release preparation thereof). Liquid preparations for oral administration include suspensions, emulsions, syrups (including dry syrups), elixirs and the like.

These preparations can be pharmacologically acceptable as preparations according to known pharmaceutical manufacturing methods. Carriers, excipients, disintegrants, lubricants, coloring agents, and the like, which are administered as pharmaceutical compositions. Examples of the carriers used in these preparations include lactose, glucose, sucrose, mannitol, potato starch. , Maize starch, calcium carbonate, calcium phosphate, calcium sulfate, calcium cellulose, crystalline cellulose, canzo powder, gentian powder, etc. Examples of binders include starch, tragacanth gum, gelatin, syrup, polyvinyl alcohol, polyvinyl ether, polyvinyl pyrrolidone, hydroxypro. Disintegrators such as pill cellulose, methyl cellulose, ethyl cellulose, carboxymethyl cellulose and the like include, for example, starch, agar, gelatin powder, sodium carboxymethyl cellulose, carboxymethyl cellulose calcium Lubricants such as crystalline cellulose, calcium carbonate, sodium bicarbonate, sodium alginate, etc .; lubricating agents such as magnesium stearate, talc, hydrogenated vegetable oils, McGall, etc .; Can be used, respectively. For tablets and granules, sucrose, gelatin, hydroquinpropyl cellulose, purified shellac, gelatin, glycerin, sorbitol, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, if necessary It may be coated with cellulose phthalate acetate, hydroxypropyl methylcellulose phthalate, methyl methacrylate, methacrylic acid polymer or the like, or may be coated with two or more layers. Further, capsules made of a substance such as ethyl cellulose or gelatin may be used. When preparing injections, add a pH adjuster, buffer, stabilizer, solubilizer, etc. to the main drug as needed, and make each injection by a conventional method.

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, health and weight of the patient, and is not particularly limited. Alternatively, it may be administered parenterally once or more times a day. Acute toxicity was hardly observed as shown in Example 6.

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.

(Example 1)

(1) Separation of microorganisms

The dried soil lg obtained by heat-treating the soil collected at Okijima Island (60 ° C, 10 minutes) was suspended in 10 ml of sterilized water. The suspension was diluted 10- to ^3- fold and agar plates (ammonium sulfate 0.05%, monopotassium phosphate 0.05%, calcium chloride 0.01%, magnesium sulfate 0.01%, urea 0.01%, glucose 0.05%, soluble starch) 0.1%, chloramphenicol 0.008%, sulfuric acid first failure 0.0005%, manganese sulfate 0.000 16%, zinc sulfate heptahydrate 0.00014%, cobalt (II) chloride 0.0002%, agar 1.8%, PH6.0) (27, 10 days). The emerged colonies were streaked on the agar plate medium described above and subjected to pure separation to obtain an actinomycete Streptomyces sputapiris SNF4435 strain (FERM BP-5915) which produces a physiologically active substance SNF4435.

(2) Microbial culture

A 1 cm square of each mat was cut out from a slant medium (glucose starch-asparagine agar medium) of Streptomyces sp. Species sp. NF4435 strain (FERM BP-5915), and 70 ml of a preculture medium (ammonium sulfate 0.14%, phosphorus Monopotassium acid 0.2%, calcium chloride 0.03%, magnesium sulfate 0.03%, urea 0.03%, polypeptone 0.5%, yeast extract 0.1%, soybean flour 3%, glucose 1%, soluble starch 0.5%, ferrous sulfate 0.0005%, Inoculate 10 500 ml Erlenmeyer flasks containing 0.00016% manganese sulfate, 0.00014% zinc sulfate heptahydrate, 0.00014%, cobalt chloride (II) 0.0002%, no pH adjustment) and incubate on a rotary shaker at 27 ° C for 4 days To obtain a preculture solution. 700 ml of this preculture was added to the main culture medium (soluble starch 1%, glucose 2%, soybean flour 2.5% Inoculate a 200 L tank containing 0.4% dried yeast, 0.1% meat extract, 0.005% dibasic phosphate, 0.2% sodium chloride, and 20L PH7.2M, and incubate at 27 ° C for 5 days with aeration and stirring (aeration rate of 120L / Min, stirring 200 revolutions / min, internal pressure 0.1 kg / cm ² ).

(Example 2)

(1) Method for producing the compound of the present invention by purifying a culture

120 L of the culture obtained in Example 1 (2) was centrifuged by continuous centrifugation (17,000 rpm, 20 L / h), and the obtained cells were extracted with 36 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 70 g of a crude extract. This was dissolved in a small amount of hexane monoethyl acetate (7: 3) solvent, and applied to a 2 L silica gel column equilibrated with the same solvent. The column was eluted with 6 L of hexane monoacetate (7: 3), then the compound of the present invention was eluted with hexane monoacetate (1: 1), and concentrated under reduced pressure to obtain 2.2 g of a yellow powder. Was. Furthermore, this powder was dissolved in a small amount of methanol and passed through an HPLC column (capsule pack C18—SG120, ø20 X 250 mm. Shiseido) equilibrated with 80% methanol aqueous solution at a flow rate of lOml / min. Eluted with the same solvent. Absorption at 220 nm was detected, and a detection peak of the compound SNF4435 of the present invention was collected. The fraction with the largest absorption (retention time about 27 minutes) was designated SNF4435C, and the fraction with the largest absorption (retention time about 29 minutes) was designated SNF4435D. These fractions were concentrated under reduced pressure to obtain 18 mg of the compound SNF4435C and 10 mg of the SNF4435D, each as a milky white powder. It was confirmed that the powders were SNF4435C and SNF4435D by measuring the specific rotation of each powder, and showed the above-mentioned physicochemical properties.

(2) Method for producing the compound of the present invention by synthesis

4 L of the culture obtained in Example 1 (2) was centrifuged (5000 rpm). Extracted with 2 L of acetone. The acetone extract was concentrated under reduced pressure to remove acetone, extracted twice with an equal amount of ethyl acetate, the ethyl acetate layer was dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 2.5 g of a crude extract. This was dissolved in a small amount of chloroform, applied to a 100 ml silica gel column equilibrated with the same solvent, eluted with 300 ml of chloroform, concentrated under reduced pressure to dryness, and added with 50 ml of methanol.ー晚 Stand still, 49.5 mg of Spectinavirin was precipitated and precipitated. This was dissolved in 5 ml of chloroform, 2. Omg of iodine was added, and the mixture was stirred at room temperature for 4.5 hours. Water was added to the reaction solution, extracted with ethyl acetate, washed successively with a 10% aqueous sodium thiosulfate solution and a saturated aqueous sodium chloride solution, and the organic layer was dried. The oily residue obtained by distilling off the solvent was subjected to silica gel gel chromatography, treated with a mixture of hexane and ethyl acetate (1: 1) to elute the compound of the present invention, and concentrated under reduced pressure. 30.5 mg of a yellow powder were obtained. Furthermore, this powder was dissolved in a small amount of methanol and passed through an HP LC column (Power Cell Pack C18—S G120, ø20 X 250 mm, Shiseido) equilibrated with 80% methanol aqueous solution at a flow rate of 10 ml / min. Elution was performed, and the absorption at 220 nm was detected. The peak of the compound of the present invention, SNF4435, was collected. The fraction with the largest absorption (retention time about 27 minutes) was designated SNF4435C, and the fraction with the next largest absorption (retention time about 29 minutes) was designated SNF4435D. These fractions were concentrated under reduced pressure to obtain 19.8 mg of compound SNF4435C and 2.2 mg of SNF4435D, each of which was obtained as milky white powder. It was confirmed that the powders were SNF4435C and SNF4435D by measuring the specific rotation of each powder, and showed the above-mentioned physical and chemical properties.

(Example 3)

Inhibitory activity of mitogen on the proliferation of splenocytes

The spleen cells of male BALB / c mice were suspended in RPMI 1640 medium containing 10% fetal calf serum, and the compounds of the present invention, SNF 4435 C and SNF 4435D, were added at various concentrations, and mitogen (concanapalin A or (LPS) with or without 37 ° C, 5% 48 hours incubation under the conditions of 1 theta carbon dioxide concentration was determined intracellular up Write-rate of cell proliferation ³ .eta. thymidine as a reference. The results are shown in Table 3 (SNF4435C) and Table 4 (SNF4435D). In Tables 3 and 4, TZC represents the cell growth rate ((Mean soil SD) / Control).

Table 3 Test compounds Mitogen Mean soil S.D.T / C ICso concentration (ng / ml) (i / ml) (cpm) (%) (ng / ml)

533 Sat 29 100

50 ― 480 64 90

100 442 ± 52 83

500 256 Sat 11 48 500

1000 55 Sat 19 10

5000 15 5 3 One Con A 1 36794 Sat 5245 100

50 35 593 Sat 4 392 97

100 31 271 6421 85

500 16 285 ± 2883 44 400

1000 10401 Sat 470 28

5000 1796 Sat 310 5 P S 5 11 261 951 100

50 5 10 309 911 92

100 5 7 121 Sat 475 63

500 5 3151 Sat 223 28 200

1000 5 1326 Sat 31 12

5000 5 89 11 1 Table 4 Test compounds Mitogen Mean soil S, D. T / C ICsn concentration (ng / ml) (// g / ml) (cpm) (%) (ng / ml)

_ 533 priests 29 100

50 1 480 Sat 64 90

100 ― 442 Sat 52 83

500 1 256 Sat 11 48 500

1000 ― 55 Sat 19 10

5000 ― 15 warriors 5 3

Con A 1 39631 Sat 2501 100

5 ι 29 107 1265 73

10 ι 24902 Sat 2654 63

50 ι 12629 2692 32 20

100 1 12082 Sat 1813 30

500 ¹ 5569 k 696 14

L P S 5 11 647 833 100

5 5 7730 Sat 257 66

10 5 6083 Sat 611 52

50 5 2683 132 23 10

100 5 1969 Sat 100 17

500 5 566 Sat 25 5

(Example 4)

Cytotoxicity

(1) in 10% fetal bovine serum RPM I 1640 medium with, K562 (human leukemia) cells were inoculated 1 X 10 ⁵ cells / ml, 37 ° C, 5 % C0 2 incubator for 24 hours After culturing, the compounds of the present invention S NF4435C and S NF4435D were added thereto at a concentration of 1 mg / ml, respectively, and further culturing was continued. 4 After 8 hours, culture the K562 cells Observation with a mirror revealed that no growth inhibition was observed when any of the compounds of the present invention, SNF 4435 C and SNF 4435 D, was added.

(2) 10% fetal bovine serum in R PM I 1640 medium supplemented, KB (human nasopharyngeal cancer) cells were inoculated 1 X10 ⁵ cells / ml, 37 ° C, 5 % C0 2 incubator After the cells were cultured for 24 hours, the compounds of the present invention SNF4435C and SNF4435D were added to each to a concentration of 1 mgZml, and the culture was further continued. After 48 hours, the cultured KB cells were observed under a microscope, and no growth inhibition was observed when any of the compounds of the present invention, SNF 4435 C and SNF 4435 D, was added.

(Example 5)

Antimicrobial activity

The antimicrobial activity of the compounds of the present invention SNF 4435 C and SNF 4435 D against various microorganisms was observed by the size (diameter) of an inhibition circle by a paper disk method (diameter 8 mm). The bioactive substances S NF4435C and S NF4435D were each measured by dissolving 100 g of methanol dissolved in methanol into a paper disk. Table 5 shows the results.

Table 5 Blocking circle (mm)

Test bacteria SNF4435C S NF4435D

Escherichia coli BE 1186 0 0

Salmonella typhimurium TV 119 0 0

Pseudomonas aeruginosa IFO 13 130 0 0

Xanthomonas oryzae IFO 3312 0 0

Xant omonas ci tri IFO 3781 0 0

Erwinia carotovora IFO 12380 0 0

Staphylococcus aureus 209P 0 9

Baci Uus subti 1 is H17 Rec ⁺ 0 10

Bacillus subti 1 is M45 Rec ― 0 10

Micrococcus luteus IFO 12708 0 0

Mycobacterium phlei IFO 3158 14 18

Alternaria mali IFO 8984 0 0

Botryotinia fuckel iana IFO 5365 0 0

Glomerella lagenaria IFO 7513 0 0

Pyricularia oryzae IFO 5994 18 16

Fusarium oxvsporum IFO 9761 0 0

Trichophyton rubrum IFO 6203 0 27

Aspergi 1 lus fumigatus IFO 9733 0 0

Candida albicans IFO 1594 9 9

Schizosaccharomvces _pombe IFO 0638 0 0

(Example 6)

Effect of overcoming anticancer drug resistance

5% fetal bovine serum was added RPM I 1640 medium (Gibco), 2780AD cells (Rogan AM et al., Science , vol.224, pp994-996 (1984)) so that the the IX 10 ⁶ cells Zml and cultured for 24 hours was suspended in 37 ° C, 5% C0 ₂ incubator within one.

After replacing the medium with RPMI 1640 containing 3H-vink listin (Amersham), After adding the compound of the present invention SNF4435C or SNF4435D at various concentrations and continuing culturing for 2 hours, the uptake of vincristine into cells was measured using a scintillation system USER No. 10 (Beckman). The results are shown in Table 6.

Table 6

[Example Ί]

Acute toxicity

When the compounds of the present invention S NF4435C and S NF4435D were intravenously administered to 6-week-old male BAL BZc mice, respectively, and their toxicity was examined, no abnormalities were found at 25 mg / kg. Industrial applicability

From the above results, the present invention provides a novel physiologically active substance, a microorganism used for producing and producing the physiologically active substance, and a method for producing the same. More specifically, a novel bioactive substance having an immunosuppressive activity, an antibacterial activity, and an effect of overcoming anticancer drug resistance, a microorganism used for producing the bioactive substance, and a bioactive substance obtained by using the microorganism to collect the bioactive substance A method for producing an active substance is provided. The novel physiologically active substance according to the present invention is an inhibitor of immune rejection due to organ transplantation, etc. Useful as a therapeutic agent for autoimmune diseases such as lupus erythematosus, rheumatoid arthritis, uveitis, a therapeutic agent for allergic diseases such as atopic dermatitis I *, an antibacterial agent for infections caused by various microorganisms, or a drug for overcoming anticancer drug resistance It is. Reference to microorganisms

Depositary organization: Institute of Biotechnology, Industrial Technology Institute, Ministry of International Trade and Industry

Address: 1-3-1, 1-3 Higashi, Tsukuba, Ibaraki, Japan

Deposit date: February 27, 1996

(Originally deposited on February 27, 1996, transferred to international deposit based on the Budapest Treaty on April 14, 1997)

Accession number: FERM B P-5 9 1 5

Claims

The scope of the claims

1. A novel physiologically active substance represented by the following formula (1), SNF4435 and a pharmacologically acceptable salt thereof.

0

0 ₂

2. Specific luminosity with sodium D line at 26 ° C is -105.6. The novel physiologically active substance according to claim 1, which is (C 0.10, CHC), SNF4435C and a pharmacologically acceptable salt thereof.

3. Specific luminous intensity by sodium D line at + 26 ° C is +84.8. The novel physiologically active substance, SNF4435D and a pharmacologically acceptable salt thereof according to claim 1, which is (C 0.10, CHCh).

4. A microorganism belonging to the genus Streptomyces and capable of producing the novel physiologically active substance according to any one of claims 1 to 3.

5. The microorganism according to claim 4, wherein the microorganism belonging to the genus Streptomyces is Streptomyces spectabilis SNF4435 strain (FERM BP-5915) or a mutant thereof.

6. The microorganism according to claim 4 or 5 belonging to the genus Streptomyces is cultured, and the novel physiologically active substance according to any one of claims 1 to 3 is produced in the culture, and collected. The method for producing a novel physiologically active substance according to any one of claims 1 to 3, characterized in that:

7. The method for producing a novel physiologically active substance according to any one of claims 1 to 3, wherein the tetraene portion of scutinapyrine is closed.

8. A medicament comprising the novel physiologically active substance according to any one of claims 1 to 3 or a pharmacologically acceptable salt thereof as an active ingredient.

9. The medicament according to claim 8, which is an immunosuppressant.

10. The medicament according to claim 8, which is a therapeutic agent for allergic diseases.

11. The medicament according to claim 8, which is a therapeutic agent for infectious diseases.

12. The medicament according to claim 8, which is a therapeutic agent for an autoimmune disease.

13. The medicament according to claim 8, which is an agent for overcoming anticancer drug resistance.