JPS6097997A - Physiologically active substance of4949, its preparation, and drug - Google Patents

Abstract

NEW MATERIAL:OF4949- I shown by the formula I , and OF4949-II shown by the formula II. USE:Having immunologically regulating action, raising immunological ability of organism, an immunological immunotherapy drug, and an anti-inflammatory drug for inhibiting formation of bradykinin by anti-aminopeptidase B activity. PREPARATION:A fungus such as Penicillium rugulosum OF4949(FERM-P203), belonging to the genus Penicillium, capable of producing OF4949, is cultivated at 4-7pH at 25-30 deg.C.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel physiologically active substance 0P4949.

The present inventors conducted a search for new physiologically active substances produced by microorganisms, and found that Penicillium (Pencilium), which was newly isolated from soil,
According to the present inventor, there is a physiologically active substance that strongly inhibits the activity of aminopeptidase B in microorganisms belonging to the genus Icilium.

It was found that a water-soluble amphoteric peptide substance named 0F4949 was accumulated.

As a result of more detailed research 1. The present inventors found two substances 0F4949-r and 0F4949 with similar properties to 0F4949.
- Succeeded in isolating and purifying U. From physicochemical and biological properties 0F4949-1 and 0F4949-
The present invention was completed by confirming that II is a new physiologically active substance having the same structure except for the presence of a different substituent at one position.

Herein KO4949-T and/or 0F4949
- TI is designated as 0F4949.

In recent years, immunomodulatory therapy has been attempted for autoimmune diseases such as nephritis, rheumatoid arthritis, systemic lupus erythematosus, and malignant tumors, and nine different drugs have been developed. For example, it has been reported that levamisole is effective to some extent when applied as an immunotherapeutic agent for cancer or a therapeutic agent for autoimmune diseases such as rheumatoid arthritis. Various other immunomodulators have been developed, but many of these drugs have problems such as side effects that occur when used for a long period of time, and there is currently no satisfactory therapeutic agent.

0F4949 exhibits a remarkable enhancing effect on cell-mediated immunity, as indicated by delayed hypersensitivity obtained by inoculating sheep red blood cells as an antigen into mouse paws.

That is, since 0F4949 has an immunomodulating effect and enhances the immune capacity of the living body, it can be used as an immunotherapeutic agent for malignant tumors and the like.

Furthermore, since 0F4949 has anti-aminopeptidase B activity, it inhibits the production of bradykinin and exhibits anti-inflammatory effects, and can be used as a therapeutic agent for various diseases.

A representative strain producing 0P4949 was isolated from soil in Kyoto Prefecture by the present inventors, and its mycological properties are as follows.

(1) Growth status in various media (24°C, 2 weeks) ■
Malt extract agar Growth is slow and the diameter of the colony is 2.0 to 2.5 cm in two weeks. The surface of the village is dark green or blue-green, and the center of the village is slightly raised. The area around the colony is flat and forms a velvety mycelium. The edge of the colony is 1-2 mm long and white. The underside becomes partially yellow, but no pigment is secreted into the agar.

■ Potato, glucose medium Growth is slow, colony diameter is 2.5-3.0 cm in 2 weeks
.

The center of the village is white or gray-green. The surrounding area is yellow to yellow-green and forms a velvety mycelium. Radial grooves can be seen on the surface of the village. The underside is pale yellow to yellow-orange, slightly folded, and no pigment is secreted into the agar.

■ Growth on Tuapek agar is extremely slow and the settlement of monoconidia is also poor. The diameter of the colony is 1.5 to 2.0 cm in two weeks, and the surface forms pale yellow to yellow-orange velvety mycelium. It may be partially white or gray. The back side is not colored and no pigment is secreted into the agar.

■Zapek agar with addition of Constaplica Growth is slow and the diameter of the colony reaches 2.0 to 2.5 cm in two weeks, forming yellow-green to dark green velvety mycelium on the surface.

The center of the village may form folds. The edge of the village is 1-2 mm wide and white. The back side is not colored and no pigment is secreted into the agar.

■Grows well on wort agar and the diameter of the colony reaches 3.0-4.0 cm in two weeks. The center of the village is slightly raised and has a dark green or blue-green velvety color, and the surrounding area is yellow or yellow-green. surface.

Many folds are formed on the underside, and the underside is yellow-orange to yellowish brown, but no pigment is secreted into the agar.

(2) Morphology The hyphae are colorless and have septate walls, and most of Benicillas have a regular biwhorled shape, but occasionally they have a single whorled shape. Conidia are oval in shape, 3.0.17 m to 4.0 μm long.
2.5 μm to 3.0 μm in m, and the size of a conidial chain ranges from 50 μm to 80 μm. The cyclids are torpedo-shaped or spear-shaped, and grow in clusters of 4 to 8 in parallel.

Length 10.0 μm to 13.0 μm, medium 2.0 μm.
17 m to 2.5 μm. The basal zygotes grow in clusters of 2 to 8 in a compact and somewhat open state, and are 1 in length.
0 μm to 13 μm, medium 2.5 μm to 3.0 μm
It is. Conidiophores are 50 μm to 80 μm long and 2.5 μm to 3.0 μm in length, and most protrude from aerial or vegetative hyphae without branching, but mono-branched ones are also seen.

(3) Growth conditions pH: Grows in the range of 2 to 12, and the optimum growth pH is.

3~6° It can grow in the temperature range of 26°C to 33°C, and the optimum range is.

The temperature is 18°C to 28°C.

As a result of the above observations, one bacterium was identified as Penicillium rugulosum.

The mycological properties of Penicillium rugulosum are as follows:
Co-authored by Raper & Thom.

In 1968, published by Haffner Publishers.
“A Man of the Benicilia”
ual of the Penicilia), co-authored by Domsh & Gam5 1
In 1972, Longman's “Fungy in Agricultural Soils” (Fu
ngi in Agricultural 5 oils
)” and “Journal of General and Applied Microbiology” by Abe (Jo
Urnal of Generaland Appli
ed Microbiology)” Volume 2, Page 1
It is described in detail in 956.

The present inventors identified this bacterium as “Penicillium rugrosum 0F4949 (Penicillium)” based on 0F4949 productivity.
ium rugulosum OF4949)”.The nine bacteria were submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, as part of the Microbiological Research Institute No. 203 (PERM BP-
It has been deposited under the accession number 203).

As for the bacteria used in the present invention, the deposited bacteria mentioned above are representative strains, and in addition to these, any strain belonging to the genus Penicillium that produces 0F4949 can be used. In addition, 0F4949 is also present in naturally occurring mutant strains and artificial mutant strains obtained by irradiating these strains with ultraviolet rays and treating them with mutagenic agents used for mutagenesis of microorganisms such as nitrosoguanidine. These mutant strains can also be used in the present invention.

In the culture used in the present invention, the medium may be either liquid or solid, but shaking culture is usually performed using a liquid medium.

Alternatively, aerated agitation culture is used. Any medium may be used as long as it is suitable for mold growth and can produce 0F4949. That is, glucose is used as a carbon source.

Sugars such as fructose, maltose, sucrose, lactose, dextrin, starch, glycerin, and sorbitol, and vegetable oils and fats such as soybean oil are used.

Examples of nitrogen sources include peptone and yeast extract.

Meat extract, soybean flour, cottonseed flour, cornstarch.

Malt extract, amino acids such as glutamic acid, aspartic acid, tyrosine, phenylalanine, and their salts.

Urea ammonium salts, nitrates, etc. are used. In addition, inorganic salts such as magnesium phosphate, calcium, sodium, potassium 1 iron, manganese, and vitamin Bl.

Micronutrients such as vitamins such as calcium pantothenate can be added as appropriate.

Liquid culture is preferred for large-scale culture.

Culture conditions such as medium PR + culture temperature are 0F4949.
In the case of liquid shaking or aerated agitation culture, p) 14 to 7. Culture temperature 25℃
Cultivation at ~30°C for a culture period of 2 to 10 days is appropriate.

0F4949 is accumulated in the culture fluid thus obtained.

The substance according to the present invention exists in both the culture filtrate and the bacterial cells, and can be collected from either. To extract the target product from the culture filtrate and bacterial cells, 1. Based on the properties of the substance according to the present invention, 9. For example, adsorption chromatography using activated carbon, nonionic adsorption resin, ion exchange chromatography using ion exchange resin, cellulose, etc. Partition chromatography, alkyl group-bonded silica gel,
reversed phase partition chromatography.

A suitable combination of means for separating and purifying organic substances from a culture of microorganisms, such as gel filtration using a gel filtration carrier, is used. More specifically, the culture filtrate, aqueous acetone extract of bacterial cells, or this substance was added to a column packed with an adsorbent such as activated carbon (manufactured by Wako Pure Chemical Industries, Ltd.) or Diaion IIP-20 (manufactured by Mitsubishi Kasei Corporation). After adsorption through the containing liquid.

Elution is performed using a mixed solvent such as acid, alkali, water, methanol, ethanol, acetone, etc. alone or in combination. Furthermore, this eluate was treated with a strongly acidic cation exchange resin such as DOWEX 50W (H" type) (manufactured by Dow Chemical Company) or DOWEX 1X2 (OH-type) (Dow Chemical Company).
It is adsorbed onto a column packed with a strongly basic anion exchange resin such as (manufactured by Chemical Co., Ltd.), and eluted using an acid, alkali, or salt solution.

Further, impurities can also be removed by passing through DOWEX IX2 (CI type). . The solution thus obtained was desalted by adsorption using Diaion IP-20 or the like and then elution with a solvent as described above.

Concentration under reduced pressure yields the desired product as a yellowish-brown crude substance. Next, this crude material was applied to a column such as crystalline cellulose Avicel (manufactured by Funakoshi), alkyl group-bonded silica gel paste Kurobletsubu RP-8 (manufactured by Merck & Co.), and cartridge column 018 (manufactured by Waters).

The target product can be obtained as a single product by developing with a mixed solvent such as acid, alkali, water, ifi liquid, methanol, acetonitrile, propatool, n-butanol, etc. alone or in combination.

(See next page) 1 Next, 0F4949-1 and 0P4949-I of the present invention
The properties of I will be explained in detail.

The structure of 0F4949-1 is represented by the following formula (n),
0P4949-11 is represented by the subject expression (III). In this specification, a compound represented by the general formula [1] 0NH2 2 (in the formula, R represents hydrogen or a methyl group) including [■] and (1) is referred to as 0P4949.

[Physical and chemical properties]

■Molecular formula 0F4949-I: C23H2eOe
N40F4949-n: C22H240B N4
■ Elemental analysis value 0F4949-1: Actual value Carbon 52.30%, Hydrogen 5.46%, Nitrogen 10
．． 64% theoretical value Carbon 52.87%, Hydrogen 5.79%,
Nitrogen 10.72% (C23H260e N 4 ・2H
20) OP4949-It: Actual value Carbon 51.60%, Hydrogen 5.48%, Nitrogen 11
．． 06% theoretical value carbon 51.97%, hydrogen 5.55%,
Nitrogen 11.02% (C22H240e N 4 ・2H
20) ■ Molecular weight S I M S (Secondary Ion Ma
The molecular weights of 0F4949-I and 01'4949-It measured by the SS Spectrometry method are as follows.

0F4949-I n 486 0F4949-U: 472 ■ Melting point 0F4949-1. Both 0F4949-n decompose at around 280°C.

■ Specific optical rotation 0F4949-I: (α] Amase-64.8° (C-
1,0, H20) OF4949-II: (α1ffl = −42,
6° (C=1.0, H20) (1) Ultraviolet Absorption Spectrum The wavelength and Ej4 value at which maximum absorption occurs when 0F4949-1 and 0F4949-II are dissolved in the following solvents are as follows.

(See next page) ① Infrared absorption spectra The wave numbers showing the maximum absorption measured with potassium bromide tablets of 0F4949-1 and 0F4949-II are as follows.

5 ■ Solubility 0F4949-1. Both 0F4949-II are soluble in water, alkaline water, and dimethyl sulfoxide. Slightly soluble in methanol and ethanol. Insoluble in n-propanol, n-butanol, acetone, ethyl acetate, chloroform, ether, benzene, hexane.

(2) Color reaction 0F4949-I, 0F4949-I [both positive for ninhydrin, potassium permanganate, Lydon Smith, and iodine reactions. Sakaro, Prokatiki, Harness, Anthrone test negative.

0F4949-1 was negative for ferric chloride reaction. 0F4949
-II is positive for ferric chloride reaction.

[Phase] Distinction between neutral, acidic, and basic 0F4949-1. Both 0F4949-11 are amphoteric substances.

0 The Rf value on a thin layer chromatography silica gel plate (manufactured by Merck & Co.) is as follows.

6. High performance liquid chromatography The retention time of 0F4949-I and 0F4949-II using Nucleosil 5C+e (manufactured by M Nagel) is as follows.

Column size: φ4. Omm M 150.0inm
Filler: Nucleosil 5c18 Solvent: 0. IM phosphoric acid mild fifi (pH5
, 7) Niacetonitrile = 90:10 Flow rate: 0.5ml/min Detection: UV 275nm ■ Color of substance OF4'114Q-I, OF4'349-I [both white powder.

■ Proton nuclear magnetic resonance spectra 0.06 of 0F4949-1 and 0F4949-I[
The chemical shift, number of protons, and multiplicity measured in N-heavy ammonia water are as follows.

Unit: Chemical shift ppm Internal standard: DSS 1.41 tNl, d(ln [phase] C-13 nuclear magnetic resonance spectrum 0F4949-
The measured chemical shifts of 1 and 0F4949-II in 0.06N deuterium ammonia water are as follows.

Unit: Chemical shift ppm Internal standard: p-dioxane (67.4ppm)
As above, 0F4949-1 and 0F4949-I
I is a property class (closely related to 0F4949-It)
has one phenolic hydroxyl group, but 0F4949
-1 is a novel substance having the same structure except that it is substituted with one methoxy group.

The present invention includes 0F4949-1 and 0F4949-I
Of course, carboxylic acid metal salts of I as well as salts with mineral or organic acids are included.

9 [Biological properties] 0F4949 has physiological activity that significantly inhibits aminopeptidase B activity of Ehrlich cancer cells.

The method for measuring aminopeptidase B is described by Hops et al. (V.

K., Hops, K., Makinen, G.G.
, Glenner, Archives of Bioc
hemistry and biophysics +
144+557 (1966)]. Namely, 3IIIM arginine-β-naphthylamide (manufactured by Sigma) dissolved in Hank's solution (manufactured by Ssui Pharmaceutical Co., Ltd.). 0.7ml of Hank's solution containing the sample in 1ml
After heating the mixture at 37° C. for 3 minutes, 0.2 ml of Ehrlich cancer cell suspension prepared at a concentration of 2.5×10 7 cells/ml was added to Hank's solution.

After reacting at 37°C for 30 minutes, Garnet GBC (orthoaminoazotoluene diazonium salt) (manufactured by Sigma) was added at a concentration of +0.3 mg/cc, and Tween 20 (Wako Pure Chemical Industries, Ltd.) was added at a concentration of 3%. Add 3 ml of 1.0 M acetate buffer (pH 4, 2) containing 1.0 M acetate buffer (pH 4, 2) and leave it at room temperature for 15 minutes.
The absorbance (a) at m was measured.

At the same time, the absorbance (b) of a control 0 using only Hank's solution containing no specimen was measured, and the inhibition rate of aminopeptidase B was calculated using b - a 100 . Ehrlich cancer cells used above.

ddY system This strain is maintained in the peritoneal cavity of 4- to 6-week-old (male) mice, and 2×10B cells were collected from ascites fluid 7 to 10 days after intraperitoneal transplantation of the mouse. Cell suspension was prepared using Tris-ascites fluid containing Ehrlich cancer cells.
The cells were treated with ammonium chloride solution (pF17.2 >) to remove red blood cells, washed three times with Hank's salt solution, and adjusted to the specified cell number with Hank's salt solution. It is a homogeneous cell population of 96% or more. Based on the inhibition rates at several concentrations of 0F4949-I and 0F4949-11 according to the above test method, a 50% inhibitory concentration was assigned to each. The results are shown in Table 1. It was shown to.

Table 1: This substance also induces delayed type hypersensitivity (delayed type hypersensitivity) obtained by inoculating sheep red blood cells as an antigen into the feet of mice.
It showed an effect of enhancing cell-mediated immunity using hyper-sensitivity (abbreviated as DTH) as an index. That is, using sheep red blood cells as an antigen, 1 x 105 CDF, system
10 week old female mice (8 mice per group) were sensitized by intravenous injection, and immediately after sensitization, 0F4949-I or 0F4949-II was administered.
Dissolved in sterile water, 5.50. 500μg/
4 days later, 108 sheep red blood cells were injected subcutaneously into the left hind paw, and 24 hours later, the degree of swelling observed in the paw (step thickness) The determination was made by measuring the The results are shown in Table 2.

In mice administered with 0F4949-1 or 0F4949-If, the degree of swelling was significantly enhanced compared to the control.

This indicates that 0F4949-T and 0F4949-U have an enhancing effect on the establishment of cellular immunity.

In addition, 0F4949-1 and 0F4949-]I are
As a result of an acute toxicity test using 300 mg/kg administered intraperitoneally to ICR mice, no toxicity was observed.

(See next page) 3 When the compound of the present invention is administered as a pharmaceutical 9 The compound of the present invention may be administered as is or in a pharmaceutically acceptable non-toxic and inert carrier 9 For example, 0.1% to 99.5%, preferably 9 is 0
．． It is administered to animals, including two people, as a pharmaceutical composition containing 5% to 90%.

As carriers, one or more solid, semisolid, or liquid diluents, fillers, and other formulation auxiliaries are used.

Preferably, the pharmaceutical composition is administered in dosage unit form.

The pharmaceutical composition of the present invention can be administered orally, intratissueally, or locally.

(transdermal administration, etc.) or rectally.

Of course, it is administered in a dosage form suitable for these administration methods. For example, injections are particularly preferred.

It is desirable to adjust the dose of the immunomodulator after taking into consideration the condition of the patient (3 years old, 9 body weight, etc.), the route of administration, the nature and severity of the disease, etc. (1) Normally, the effectiveness of the present invention for adults. Ingredient amount: 0.1-1000mg per day
range is common. In some cases, a lower dose than this may be sufficient, and in other cases, a higher dose may be required. When administering a large amount, it is desirable to divide the dose into several times a day.

4 For oral administration, solid or liquid dosage units 1 such as powders are used.

It can be carried out in the form of a single powder tablet, sugar-coated tablet, capsule, granule, suspension, liquid, syrup, drop, sublingual tablet, or other dosage form.

Powders are prepared by comminuting the active substance to a suitable fineness. Powders are prepared by mixing the active substance to a suitable fineness and then a similarly finely divided pharmaceutical carrier such as starch, an edible carbohydrate such as mannitol, or the like. Flavoring agent, preservative 3 dispersing agent 1 coloring agent as required.

Flavors and other substances may be mixed.

Capsules are manufactured by first filling powdered powders or powders as described above, or granules as described in the section on tablets, into capsule shells such as gelatin capsules. Ru. Lubricating agent or fluidizing agent 1 For example, colloidal silica, talc, magnesium stearate, calcium stearate, solid polyethylene glycol, etc. are mixed into a powder.

A filling operation can also be carried out afterwards. Addition of disintegrants and solubilizers 2, such as carboxymethylcellulose, calcium carboxymethylcellulose, low-substituted hydroxypropylcellulose, calcium carbonate, and sodium carbonate, can improve the effectiveness of the drug when the capsule is ingested. can.

Alternatively, one product can be made into soft capsules by suspending and dispersing the fine powder in vegetable oil, polyethylene glycol, glycerin, and a surfactant, and wrapping this in a gelatin sheet.

Tablets are made by making a powder mixture and granulating or slugging it.

It is then manufactured by adding a disintegrant or a lubricant and then compressing into tablets.

Powder mixtures are prepared by mixing suitably powdered materials with the diluents and bases mentioned above, and optionally binders (e.g. sodium carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, polyvinyl alcohol, etc.) and dissolution retarders. (e.g. paraffin), reabsorbents (e.g. quaternary salts) and/or adsorbents (e.g. bentonite, kaolin, calcium phosphate, etc.) may also be used in combination. The powder mixture can be granulated by first being moistened with a binder such as syrup, starch paste, gum arabic, cellulose solution or polymeric substance solution and then forced through a sieve. Instead of granulating the powder in this way, it is also possible to first run it through a tablet press and then crush the resulting imperfectly formed slugs into granules.

The granules thus produced can be prevented from adhering to each other by adding stearic acid, stearate, talc, mineral oil, or the like as a blanching agent. The mixture thus lubricated is then compressed into tablets. Alternatively, the drug may be mixed with a fluid inert carrier and then directly compressed into tablets without going through the granulation or slugging steps as described above. Transparent or translucent protective coatings consisting of an occlusive coating of shellac, coatings of sugar or polymeric materials, and overcoatings consisting of wax may also be used.

Other oral dosage forms, such as solutions, syrups, elixirs, etc., can also be made into dosage units, a fixed amount of which contains a fixed amount of the drug. Syrups are prepared by dissolving the compound in a suitable flavored aqueous solution, and elixirs are prepared by using a non-toxic alcoholic carrier. Suspensions are formulated by dispersing the compound in a non-toxic carrier. Solubilizers and emulsifiers (eg, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol esters), preservatives, flavoring agents (eg, peppermint oil, saccharin), and others may also be added as required.

If desired, dosage unit formulations for oral administration may be microencapsulated. The formulation may also be coated.

By embedding it in polymers, wax, etc., it is possible to prolong the action time and achieve sustained release.

Parenteral administration may be carried out using liquid dosage unit forms, such as solutions or suspensions, intended for subcutaneous, intramuscular or intravenous injection. These are prepared by suspending or dissolving a given amount of the compound in a non-toxic liquid carrier compatible with the purpose of injection, such as an aqueous or oily vehicle, and then sterilizing the suspension or solution. Ru.

Alternatively, a fixed amount of the compound may be placed in a vial, and then the vial and its contents may be sterilized and sealed. Preliminary vials or carriers may be prepared with the powdered or lyophilized active ingredient for dissolution or mixing immediately prior to administration. Nontoxic salts or salt solutions may be added to make the injection solution isotonic. Furthermore, stabilizers, preservatives, emulsifiers, and the like can also be used in combination.

For rectal administration, the compound may be administered as a solid substance with a low melting point, soluble or insoluble in water, such as polyethylene glycol, cocoa butter, and higher esters (such as myristyl palmitate).
and mixtures thereof by using mixed sentencing.

In addition to the active ingredient according to the present invention, the preparation of the compound of the present invention may contain other drugs such as cytosine arabinoside, adriamycin, or mitomycin.

Or they may be used together.

(Next page below) Examples of the manufacturing method according to the present invention will be specifically described below, but the present invention is not limited thereto.

Example 1 Penicillium rugulosum 0F4949 (Ministry of International Trade and Industry, Agency of Industrial Science and Technology, Institute of Microbial Technology, accession number FEIKEN JOKYO NO. 203) was cultured on a malt extract agar slant medium for 7 days. From this slant culture - the platinum loop was treated with 2% glucose;
27 500ml media containing 0.5% polypeptone, 0.1% yeast extract, 0.05% potassium phosphate, 0.05% magnesium sulfate! Dispense into Erlenmeyer flasks, sterilize at 120°C for 20 minutes, and inoculate.
The cells were cultured for 6 days at 7°C with shaking at 190 revolutions per minute.

This culture was mixed with pre-sterilized glucose 2%, polypeptone 0.5%, yeast extract 0.1%, and phosphoric acid.
Medium 207 containing 0.05% potassium and 0.05% magnesium sulfate! 307 with ! stainless steel fermenter 1
Five plants were inoculated with IR per plant and culture was started. The culture was carried out at an air flow rate of 201 degrees per minute and a rotation speed of 300 revolutions per minute.
The reaction was carried out at 0.degree. C., and an antifoaming agent was added if necessary. After culturing for 4 days, the culture was taken out, the bacterial cells were filtered, and the culture filtrate was separated, resulting in a compressed volume of 6.5J! of bacterial cells and a culture filtrate of 300β were obtained.

Example 2 The bacterial cells obtained in Example 1 were extracted three times with 50% acetone water 24β, and the extract 721 was concentrated under reduced pressure to 3OA to remove acetone. Concentrate 307I and culture filtrate 3007 obtained in Example 1! and adsorbed on an activated carbon column.

After thorough washing with 801 water, p1 containing 50% acetone.
It was eluted with 150j2 aqueous hydrochloric acid solution. The aminopeptidase inhibitory activity fraction 80β was concentrated under reduced pressure to 301, and after removing acetone, the concentrated solution was 307! 1.2 of strongly basic anion exchange resin DOWEX 1X2 (CI-type)
1 column and eluted with water. Next, the eluate 804 was corrected to pH 3, and 2. 1 adsorbed on an Ol column
07! After washing with water, 1. ON ammonia water 1
It eluted at 001. After neutralizing active area 60 Il with 2N hydrochloric acid, use nonionic adsorption resin Diaion HP-202i.
was adsorbed to. After washing with 20 ffi of water, it was eluted with 50% methanol water 201. The obtained active area was concentrated under reduced pressure to obtain 28.8 g of crude material.

Example 3 28.8 g of the crude material obtained in Example 2 was sprinkled on 30 g of Suavicel at the opening of the crystal cell, and after sufficiently drying under reduced pressure, it was packed in the upper part of Avicel column 11. Developing solvent IN ammonia water: n-butanol-15:85 127! When developed, an active area mainly containing 0F4949-I was separated, followed by an active area mainly containing 0F4949-n. Neutralize each active area with 1NllCl.

After concentration under reduced pressure, redissolve in water and use Diaion IIP-20.
It was adsorbed to 500ml. After washing with water, elute with 2β 50% methanol water.

By concentrating the active area under reduced pressure, 0F4949-I
3.1 g of coarse powder of 0F4949-If and 4.1 g of coarse powder of 0F4949-If were obtained.

Example 4 0F4949-1 coarse powder obtained in Example 33.
1 g was dissolved in 15n+1 water, and using a high-performance liquid chromatography (system 500.Waters) for large-scale preparative separation, column: Cartli-Zzi column (CI8.
Waters), developing solvent: 0. Reversed phase partition chromatography was performed under the conditions of IM citrate buffer and niacetonitrile = 90:10, and the aminopeptidase B inhibitory activity was assayed. The active area is Diaion HP-202
After adsorption with 00ml and washing with water, it was eluted with 1z of 50% methanol water. The active fraction was collected, concentrated under reduced pressure, and then freeze-dried to obtain 455 mg of 0F4949-1.

2 Coarse powder of 0F4949-II obtained in Example 3
4.1g was similarly subjected to 018 reverse phase partition chromatography using the developing solvent 0.1M citric acid buffer solution Niacetonitrile-95:5.
Perform column chromatography on 0F4949
-n 671 mg was obtained.

[Brief explanation of drawings]

Figure 1 shows the ultraviolet absorption spectrum of 0F4949-I. - indicates when the solvent is H20(7), -・- indicates when the solvent is 0.0
In the case of 5N HCl, --- means that the solvent is 0.05N Na
The case of 011 is shown respectively. Figure 2 shows 0F4949-
1 shows an infrared absorption spectrum measured in potassium bromide of No. 1. FIG. 3 shows a proton nuclear magnetic resonance spectrum of 0F4949-I measured in 0.06 N deuterium ammonia water (internal standard: DSS). Figure 4 shows 0F4949
-1 of 0.06 C-1 measured in N heavy ammonia water
3 nuclear magnetic resonance spectrum (internal standard: p-di
oxane 67.4 ppm). FIG. 5 shows the ultraviolet absorption spectrum of 0F4949-11. - means 1 when the solvent is H2O
．． In the case of 05NHCI, -m- means that the solvent is 0.05N
The case of Na0tl is shown respectively. Figure 6 is QF494
9 shows an infrared absorption spectrum of TI measured in potassium bromide. Figure 7 shows 0.0 of 0F4949-IT.
The figure shows a proton nuclear magnetic resonance spectrum measured in 6N deuterium ammonia water (internal standard: DSS). Figure 8 is 0F4
949-II measured in 0.06N deuterium ammonia water (internal standard:
p-dioxane 67.4 ppm). Applicant: Takara Shuzo Co., Ltd. 5 Figure 1 Patent application filed on November 2, 1982 (No. III notification not received) 20 Name of invention Physiologically active substance 0F4949. Its manufacturing method and medicine 3,
Relationship with the person making the amendment Patent applicant address 609 Takenaka-cho, Fushimi-ku, Kyoto Name Takara Sake Brewery Co., Ltd. (1 person) Representative
Takashi Omiya 4, Agent Address: 14, Kisshoin Nishinosho Monguchi-cho, Minami-ku, Kyoto City, 601 Column 6 of the detailed description of the invention in the specification, Contents of amendment (1) Chemical structural formula listed on page 13 of the specification In middle (1), correct ``[ 0ro'' to ``[ . Written amendment to the above procedure (voluntary) 20 Name of invention Physiologically active substance op4949. Its manufacturing method and medicine 3,
Relationship with the person making the amendment Patent applicant address 609 Takenaka-cho, Fushimi-ku, Kyoto Name Takara Shuzo Co., Ltd. (1 other person) Representative Takashi Omiya 4, agent residence Address 601 Kisshoin Nishinoshyo Monguchi-cho, Minami-ku, Kyoto Column 6 of the detailed description of the invention in the specification at No. 14, Contents of amendment (1) In the fourth line from the bottom of page 5 of the specification, the words "glucose medium" are corrected to "glucose agar." (2) In the fourth line from the bottom of page 11 of the specification, the text "rcI type" is corrected to "CL type." (3) r 0.3mg/cc on page 21, line 13 of the specification
Correct J to r 0.3 mg/mlJ. that's all

Claims (8)

[Claims] (1) A compound called 0F4949 represented by the following general formula (I) and l-10t-1 CONl-12 (in the formula, R represents hydrogen or a methyl group), or a salt thereof. (2) Referred to as 0F4949-1. The compound according to claim 1, or a salt thereof, wherein R is a methyl group. (3) Referred to as 0F4949-II. The compound according to claim 1, or a salt thereof, wherein R is hydrogen. (4) 0F4949-producing bacteria belonging to the genus Penicillium are cultured in a medium, and from the culture, a compound called 0F4949-1 represented by the following general formula (1) (in the formula,
When R is a methyl group) and/or 0F4949-n (
wherein R is hydrogen). 0F4949-1 and/or 0F
4949- Method for producing IF. (5) The 0F4949-producing bacterium belonging to the genus Penicillium is Penicillium rugulosum 0P4949 (Penicilli++ m rugu
losum 0F494'J). (6) An immunomodulator containing a compound called 0F4949 or a salt thereof represented by the following general formula CI) and) 10H oNH2 (in the formula, R represents hydrogen or a methyl group) as a main component. (7) The immunomodulator according to claim 6, wherein R is a methyl group. (8) The immunomodulator according to claim 6, wherein R is hydrogen.