JPH0146518B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel physiologically active substance OF4949. The present inventors conducted a search for new physiologically active substances produced by microorganisms, and discovered that microorganisms belonging to the genus Penicillium newly isolated from soil contained
It has been found that a physiologically active substance that strongly inhibits aminopeptidase B activity, that is, a water-soluble amphoteric peptide substance named OF4949, is accumulated according to the present invention. As a result of further detailed research, the present inventors found that OF4949
Two substances with more similar properties OF4949 and OF4949
was successfully isolated and purified. The present invention was completed by confirming from physicochemical and biological properties that OF4949- and OF4949- are novel physiologically active substances having the same structure except for the presence of a different substituent at one position. OF4949—and/or OF4949—
is referred to as OF4949. In recent years, immunomodulatory therapy has been attempted for autoimmune diseases such as nephritis, rheumatoid arthritis, and systemic lupus erythematosus, or malignant tumors, and various 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. OF4949 exhibits a remarkable enhancing effect on cell-mediated immunity, as indicated by delayed-type hypersensitivity obtained by inoculating sheep red blood cells as an antigen into mouse footpads. That is, OF4949 has an immunomodulatory effect and enhances the immune capacity of the living body, so it can be used as an immunotherapeutic agent for malignant tumors and the like. Furthermore, OF4949 has anti-aminopeptidase B activity, inhibits the production of bradykinin, exhibits anti-inflammatory effects, and can be used as a therapeutic agent for various diseases. The representative strain producing OF4949 was isolated by the present inventors from soil in Kyoto Prefecture, and its mycological properties are as follows. (1) Growth status on various media (24℃, 2 weeks) Malt extract agar Growth is slow and the diameter of colonies is 2.0 to 2.5 cm in 2 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 greenery in the village is 1 to 2 mm wide.
And white. The underside becomes partially yellow, but no pigment is secreted into the agar. Potato, glucose agar Growth is slow, and the diameter of the colony is 2.5 to 3.0 cm in two 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. Tuapetsk agar Growth is extremely slow and conidia do not adhere well. The diameter of the colony is 1.5 to 2.0 cm in two weeks. 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. Tuapetsk agar with added Constaplica Growth is slow and the diameter of the colony is 2.0 to 2.5 cm in two weeks. The surface forms yellow-green to dark green velvety mycelium, and the center of the colony may form folds.
The greenery in the village is 1-2 mm wide and white. The back side is not colored and no pigment is secreted into the agar. Wort agar Growth is good 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. It forms many folds on both the front and back sides, and the back side 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 penicillas are biwhorled, but occasionally there are monowhorled ones. The conidia are oval in shape, 3.0 μm to 4.0 μm long, 2.5 μm to 3.0 μm wide, and the size of the conidial chain ranges from 50 μm to 80 μm. The stalks are comb-shaped or spear-shaped, growing in parallel clusters of 4 to 8 pieces, length 10.0 μm to 13.0 μm, width.
It is 2.0 μm to 2.5 μm. The basal sycamores grow in clusters of 2 to 8 in a compact state or in a somewhat open state,
The length is 10 μm to 13 μm, and the width is 2.5 μm to 3.0 μm. Conidiophores are 50 μm to 80 μm long and wide.
Most of them are 2.5μm to 3.0μm and protrude from aerial or vegetative hyphae without branching, but branched ones can also be seen. (3) Growth conditions PH: Grows in the range of 2 to 12, and the optimal growth PH is 3.
~6. Temperature: Can grow in the range of 6°C to 33°C, and the optimum range is 18°C to 28°C. As a result of the above observations, this bacterium was identified as Penicillium rugulosum. The mycological properties of Penicillium rugulosum can be found in “A Manual of the Penicillium” by Raper & Thom, 1968, published by Hafner Publishers.
"Manual of the Penicillia", Domsh & Gams, 1972; Longman's "Fungi in Agricultural Soils", Domsh & Gams, 1972;
Soils)” and “Journal of General and Applied Microbiology” by Abe.
Miorobiology), Volume 2, Page 1, 1956. The present inventors have identified this bacterium as "Penicillium rugulosum OF4949 (Penicillium rugrosum)" based on OF4949 productivity.
rugulosum OF4949)”.This bacterium was entrusted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, under the acceptance number FERM BP-203. As for the bacteria used in the present invention, the deposited bacteria mentioned above are representative strains, and in addition to these, all strains belonging to the genus Penicillium that produce OF4949 can be used. Artificial mutant strains obtained by irradiation or treatment with mutagenic agents used for mutagenesis of microorganisms such as nitrosoguanidine, as well as naturally occurring mutant strains, also produce OF4949. These mutant strains can also be used in the present invention. For the culture used in the present invention, the culture medium may be liquid or solid, but shaking culture in a liquid medium or aeration agitation culture is usually used. The medium may be any medium as long as it is suitable for mold growth and can produce OF4949.In other words, carbon sources include glucose, fructose, maltose, sucrose, lactose, dextrin, starch, and glycerin. , sugars such as sorbitol,
and vegetable oils and fats such as soybean oil. Examples of nitrogen sources include peptone, yeast extract, meat extract, soybean flour, cottonseed flour, cornstarch, malt extract, amino acids such as glutamic acid, aspartic acid, tyrosine, and phenylalanine, and their salts, and ammonium urea. Salts, nitrates, etc. are used. Others: magnesium phosphate,
Micronutrients such as inorganic salts such as calcium, sodium, potassium, iron, and manganese, and vitamins such as vitamin B ₁ and calcium pantothenate can be added as appropriate. Liquid culture is preferred for large-scale culture. Culture conditions such as medium PH and culture temperature are as follows:
It may be changed as appropriate within the scope of producing OF4949, but
In case of liquid shaking or aeration stirring culture, pH4~
7. Cultivation at a culture temperature of 25°C to 30°C and a culture period of 2 to 10 days is appropriate. OF4949 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, based on the properties of the substance according to the present invention, for example, adsorption chromatography using activated carbon, nonionic adsorption resin, ion exchange chromatography using ion exchange resin, cellulose can be used. Conventional means for separating and purifying organic substances from microbial cultures are used in appropriate combinations, such as partition chromatography using silica gel, reverse phase partition chromatography using alkyl group-bonded silica gel, and gel filtration using a gel filtration carrier. More specifically, activated carbon (manufactured by Wako Pure Chemical Industries, Ltd.), Diaion HP-20 (manufactured by Mitsubishi Chemical Corporation),
After passing the culture filtrate, aqueous acetone extract of bacterial cells, or a liquid containing this substance through a column packed with an adsorbent such as Elute using a mixed solvent. Furthermore, this eluate was treated with a strongly acidic cation exchange resin such as Dowex 50W (H+ type) (manufactured by Dow Chemical Company) or a strongly basic cation exchange resin such as Dowex 1×2 (OH ^- type) (manufactured by Dow Chemical Company). It is adsorbed onto a column packed with anion exchange resin and eluted using an acid, alkali, or salt solution. Further, impurities can also be removed by passing through a Dowex 1×2 (C1 ^- type). From the solution obtained in this way, Diamondion HP was prepared as described above.
-20 etc., followed by desalting by elution with a solvent, and then concentration under reduced pressure to obtain the target product as a yellowish brown crude substance. Next, this crude material was transferred to crystalline cellulose Avicel (manufactured by Funakoshi Co., Ltd.), alkyl group-bonded silica gel Ricroprep RP-8 (manufactured by Merck & Co., Ltd.), and cartridge column C ₁₈ .
(manufactured by Waters), acid,
By developing with a mixed solvent such as alkali, water, buffer, methanol, acetonitrile, propanol, n-butanol, etc. alone or in combination, the desired product can be obtained as a single product. Next, the properties of OF4949 and OF4949 of the present invention will be explained in detail. The structure of OF4949- is represented by the following formula [] [formula] [formula], and OF4949- is represented by the formula []. In this specification, the general formula [] which includes [] and [] The compound represented by (in the formula, R represents hydrogen or a methyl group) is called OF4949. [Physical and chemical properties] Molecular formula OF4949: C ₂₃ H ₂₆ O ₈ N ₄ OF4949: C ₂₂ H ₂₄ O ₈ N ₄ elemental analysis OF4949: Actual carbon 52.30%, hydrogen 5.46%, nitrogen 10.64% Theoretical carbon 52.87 %, Hydrogen _5.79 %, Nitrogen 10.72% (as _C23H26O8N4・_{2H2O )} OF4949: Actual value Carbon 51.60% _, Hydrogen 5.48%, Nitrogen 11.06% Theoretical value Carbon 51.97%, Hydrogen 5.55%, Nitrogen 11.02% ( _{as C22H24O8N4}・_2H2O ) _Molecular weight _SIMS (Secondary lon Mass Spectrometry)
The molecular weights of OF4949 and OF4949 measured by the method are as follows. OF4949―: 486 OF4949―: 472 Melting point Both OF4949― and OF4949― decompose at around 280℃. Specific optical rotation OF4949-: [α] ²⁵ _D = -64.8゜ (C = 1.0, H ₂ O) OF4949-: [α] ²⁵ _D -42.6゜ (C = 1.0, H ₂ O) Ultraviolet absorption spectrum OF4949- and The wavelength showing the maximum absorption and the E1% 1cm value when OF4949 is dissolved in the following solvent are as follows. [Table] Infrared absorption spectrum The wave numbers showing the maximum absorption measured with potassium bromide tablets of OF4949 and OF4949 are as follows. [Table] Solubility Both OF4949 and OF4949 are soluble in water, alkaline water, and dimethyl sulfoxide. Slightly soluble in methanol and ethanol. n-propanol, n
-Insoluble in butanol, acetone, ethyl acetate, chloroform, ether, benzene, hexane. Color reaction OF4949-, OF4949-both ninhydrin,
Potassium permanganate, Lydon Smith, iodine reaction positive. Sakaguchi, Prokatka, Harness, Anthrone test negative. OF4949- had a negative ferric chloride reaction. OF4949―
is positive for ferric chloride reaction. Distinction between neutral, acidic, and basic OF4949 and OF4949 are both amphoteric substances. Thin layer chromatography Rf on silica gel plate (Merck)
The values are as follows. [Table] The retention capacity and retention time of OF4949 and OF4949 using high performance liquid chromatography Nucleosil 5C ₁₈ (manufactured by M Nagel) are as follows. Column size: φ4.0mm x 150.0mm Packing material: Nucleosil 5C ₁₈ Solvent: 0.1M citrate buffer (PH5.7): Acetonitrile = 90:10 Flow rate: 0.5ml/min Detection: UV275nm [Table ] Color of the substance Both OF4949 and OF4949 are white powders. Proton nuclear magnetic resonance spectra The chemical shifts, proton numbers, and multiplicities of OF4949 and OF4949 measured in 0.06N deuterium ammonia water are as follows. Unit: Chemical shift ppm Internal standard: DSS OF4949: 2.66 (1H, t), 2.75-3.00 (2H,
m) 3.37 (1H, dd), 3.70 (1H, dd) 3.93 (3H, s), 4.42 (1H, d) 4.48 (1H, dd), 5.83 (1H, d) 6.87 (2H, m), 7.07 ( 2H, m) 7.26 (1H, dd), 7.46 (1H, dd) OF4949―: 2.65 (1H, t), 2.70~2.95 (2H,
m) 3.36 (1H, dd), 3.67 (1H, dd) 4.40 (1H, d), 4.46 (1H, dd) 5.78 (1H, d), 6.67 (1H, dd) 6.74 (1H, d), 6.85 ( 1H, dd) 7.05 (1H, dd), 7.22 (1H, dd) 7.41 (1H, dd) C-13 nuclear magnetic resonance spectra The chemical shifts of OF4949 and OF4949 when measured in 0.06N deuterium ammonia water are as follows: It is. Unit: Chemical shift ppm Internal standard: p-dioxane (67.4ppm) OF4949: 178.7, 176.0, 174.9, 168.3 153.4, 149.4, 148.0, 136.5 132.9, 131.5, 128.2, 125.0 123.1, 122.1, 116.2, 113 .0 72.9，57.9， 56.7, 55.1 53.8, 39.7, 38.9, OF4949: 178.8, 176.0, 175.8, 168.3 154.1, 149.0, 145.6, 136.1 132.8, 131.4, 127.4, 125.2 123.1, 122.0, 117.0, 1 16.8 73.0, 57.9, 55.1, 54.0 39.7, 39.1 As mentioned above, OF4949- and OF4949- are closely related substances with similar properties, and OF4949- has one phenolic hydroxyl group, except that OF4949- has this substituted with one methoxy group. , both are new substances with the same structure. Of course, the present invention includes OF4949 and OF4949 metal carboxylate salts and salts with mineral or organic acids. [Biological Properties] OF4949 has a physiological activity that significantly inhibits aminopeptidase B activity of Ehrlichi cancer cells. The method for measuring aminopeptidase B is described by Hops et al. [VKHops, KKMakinen, GGGlenner,
Archives of Biochemistry and Biophysics,
144, 557 (1966)]. That is, it was dissolved in Hank's solution (manufactured by Nitsusui Pharmaceutical Co., Ltd.).
A mixture of 0.1 ml of 3mM arginine-β-naphthylamide (manufactured by Sigma) and 0.7 ml of Hank's solution containing the specimen was heated at 37°C for 3 minutes, and then added to Hank's solution at a concentration of 2.5 × 10 ⁷ cells/ml. Add 0.2 ml of Ehrlichi cancer cell suspension prepared as follows, and incubate at 37°C for 30
After reacting for a minute, the garnet was added to a concentration of 0.3 mg/ml.
Contains GBC (ortho-aminoazotoluene diazonium salt) (manufactured by Sigma) and 1.0M containing Tween20 (manufactured by Wako Pure Chemical Industries, Ltd.) at a concentration of 3%.
After adding 3 ml of acetate buffer (PH4.2) and leaving it at room temperature for 15 minutes, absorbance of the supernatant at 525 nm (a)
was measured. At the same time, the absorbance (b) of a control using only Hank's solution containing no specimen was measured, and aminopeptidase B
The inhibition rate was calculated by b-a/b×100. The Ehrlichi cancer cells used above have been maintained in the peritoneal cavity of ddY 4- to 6-week-old (male) mice, and 2 x 10 ⁶ cells were transplanted into the peritoneal cavity of the mouse for 7 days. It was collected from ascites fluid on the 10th day. The cell suspension was prepared by mixing ascites fluid containing Ehrlichi cancer cells with a Tris-ammonium chloride solution (PH
7.2) to remove red blood cells, wash three times with Hank's solution, and adjust the number of cells to a predetermined level with Hank's solution. This is a homogeneous cell population with a living cell count of 96% or more of Ehrlichi cancer cells. OF4949- and OF4949- according to the above test method
The inhibition rates at several concentrations were determined, and a 50% inhibitory concentration was assigned to each. The result is the first
Shown in the table. [Table] This substance also produces delayed-type hypersensitivity, which can be obtained by inoculating sheep red blood cells as an antigen into the footpads of mice.
It showed an effect of enhancing cell-mediated immunity using Type Hypersensitivity (abbreviated as DTH) as an indicator. That is, 1×10 ⁵ sheep red blood cells were used as an antigen to be sensitized by intravenously injecting ₁ group of 8 CDF 1 female mice aged 10 weeks, and immediately after sensitization, OF4949- or OF4949- was dissolved in sterile water. was injected intraperitoneally at concentrations of 5, 50, and 500 μg/Kg, and 4 days later, ¹⁰⁸ sheep red blood cells were subcutaneously injected into the footpad of the left hind paw, and 24 hours later, the results were found in the footpad. The degree of swelling (thickness of the footpad) was determined by measuring with a caliper. The results are shown in Table 2. In mice administered with OF4949- or OF4949-, the degree of swelling was significantly enhanced compared to controls. This means that OF4949 and OF4949
- shows that it has an enhancing effect on the establishment of cell-mediated immunity. [Table] In addition, OF4949- and OF4949- showed no toxicity as a result of an acute toxicity test when administered intraperitoneally to ICR mice at 300 mg/Kg. When the compound of the present invention is administered as a medicament, the compound of the present invention may be administered as is or in a pharmaceutically acceptable non-toxic and inert carrier, for example, from 0.1% to 99.5%.
It is preferably administered to animals including humans as a pharmaceutical composition containing 0.5% 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, locally, (transdermally, etc.)
Or it can be administered 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 an immunomodulator after considering the patient's condition such as age, weight, route of administration, nature and severity of the disease, etc., but usually,
The amount of the active ingredient of the present invention for adults is generally in the range of 0.1 to 1000 mg per day. 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. Oral administration is carried out in solid or liquid dosage units, such as powders, powders, tablets, dragees, capsules, granules, suspensions, solutions, syrups, drops, sublingual tablets, and other dosage forms. be able to. Powders are prepared by comminuting the active substance to a suitable fineness. Powders are prepared by bringing the active substance to a suitable fineness and then mixing it with similarly finely divided pharmaceutical carriers, such as starch, edible carbohydrates such as mannitol, and the like. Flavoring agents, preservatives, dispersants, coloring agents, fragrances, and other substances may be mixed as necessary. 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. Lubricants and flow agents, such as colloidal silica, talc, magnesium stearate,
Mix something like calcium stearate and solid polyethylene glycol into a powder,
A filling operation can also be carried out afterwards. Addition of disintegrants or solubilizers, such as carboxymethylcellulose, calcium carboxymethylcellulose, low-substituted hydroxypropylcellulose, calcium carbonate, sodium carbonate, can improve the effectiveness of the drug when the capsule is ingested. . Alternatively, the fine powder of this product can be suspended and dispersed in vegetable oil, polyethylene glycol, glycerin, or a surfactant, and then wrapped in a gelatin sheet to form a soft capsule. Tablets are manufactured by preparing a powder mixture, granulating or slugging it, adding a disintegrant or lubricant, and then tableting. 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, dicalcium phosphate, etc.) may also be used in combination. The powder mixture can be first moistened with a binder such as syrup, starch paste, acacia, cellulose solution or polymeric substance solution and then forced through a sieve to form granules. 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 sticking to each other by adding stearic acid, stearate salts, talc, mineral oil, etc. as lubricants. 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 such as occlusive coatings of silica, coatings of sugar or polymeric materials, and polishing coatings of wax may also be used. Other oral dosage forms, such as solutions, syrups,
Elixirs and the like can also be in dosage unit form so that a fixed amount of the drug 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 (e.g. ethoxylated isostearyl alcohols,
polyoxyethylene sorbitol esters),
Preservatives, flavoring agents (eg, peppermint oil, saccharin), and others can also be added as desired. If desired, dosage unit formulations for oral administration may be microencapsulated. The formulation can also be coated or embedded in polymers, waxes, etc. to prolong the action time and provide 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 an 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. Additional vials or carriers may be provided 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. Rectal administration is accomplished by using suppositories containing low melting point water soluble or insoluble solids such as polyethylene glycol, cocoa butter, higher esters (e.g. myristyl palmitate), and mixtures thereof. sell. In addition to the active ingredient according to the present invention, other drugs such as cytosine arabinoside, adriamycin, or mitomycin may be added to the preparation of the compound of the present invention, or may be used in combination. 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 OF4949 (Ministry of International Trade and Industry, Agency of Industrial Science and Technology, Institute of Microbial Technology, accession number FAIKEN JOKYO NO. 203) was cultured on a malt extract agar slant medium for 7 days. From this slant culture, 500 ml of a medium containing 2% glucose, 0.5% polypeptone, 0.1% yeast extract, 0.05% potassium phosphate, and 0.05% magnesium sulfate was dispensed into 2-volume Erlenmeyer flasks. Sterilized at 120℃ for 20 minutes, inoculated and incubated at 27℃ with shaking at 190 revolutions per minute.
Cultured for 1 day. This culture was placed in 15 stainless steel fermentation vessels of 30 containing 20 pre-sterilized medium containing 2% glucose, 0.5% polypeptone, 0.1% yeast extract, 0.05% potassium phosphate, and 0.05% magnesium sulfate. Culture was started by inoculating one seed per plant. The culture was carried out at 27° C. with an aeration rate of 20 per minute and a rotational speed of 300 revolutions per minute, and an antifoaming agent was added as necessary. After culturing for 4 days, take out the culture, filter the bacterial cells, separate the culture filtrate, and compress the volume.
6.5 bacterial cells and 300 culture filtrate were obtained. Example 2 The bacterial cells obtained in Example 1 were added to 50% acetone water24
The acetone was removed by extracting the extract 72 three times and concentrating the extract 72 to a volume of 30% under reduced pressure. Concentrate 30
It was adsorbed on an activated carbon column together with 300ml of the culture filtrate obtained in Example 1, and washed thoroughly with 80ml of water.
It was eluted with a PH2 hydrochloric acid aqueous solution of 150 containing 50% acetone. Aminopeptidase inhibitory activity fraction 80
After concentrating to 30% under reduced pressure and removing acetone,
The concentrated solution 30 was applied to a 1.2 column of strongly basic anion exchange resin Dowex 1×2 (C1 ^- type) and eluted with water. Next, correct the eluate 80 to pH 3 and use strong acidic cation exchange resin DOWEX 50W (H ⁺ type).
After washing with water adsorbed on a 2.0 column,
It was eluted with 100% of 1.0N ammonia water. active area
60 was neutralized with 2N hydrochloric acid and then adsorbed on nonionic adsorption resin Diaion HP-20 2. After washing with 20 ml of water, it was eluted with 50% methanol water 20 ml. The obtained active area was concentrated under reduced pressure to obtain 28.8 g of crude substance.
I got it. Example 3 28.8 g of the crude material obtained in Example 2 was sprinkled on 30 g of crystalline cellulose Avicel, and after sufficiently drying under reduced pressure, it was packed in the upper part of Avicel column 1. Developing solvent 1N ammonia water: n-butanol = 15:85
When developed with 12, an active area mainly containing OF4949-I was separated, and then an active area mainly containing OF4949- was separated. Neutralize each active area with 1NHCl, concentrate under reduced pressure, redissolve in water, and add diamond ion.
It was adsorbed onto 500ml of HP-20. 50% of 2 after washing with water
By eluting with methanol water and concentrating the active area under reduced pressure, 3.1g of coarse powder of OF4949 and
4.1 g of coarse powder of OF4949 was obtained. Example 4 OF4949-coarse powder obtained in Example 3
Dissolve 3.1 g in 15 ml of water and use high-performance liquid chromatography (System 500, Waters) for large-scale preparative separation, column: cartridge column (C ₁₈ , Waters), developing solvent: 0.1M citrate buffer. Reversed phase partition chromatography was performed under conditions of liquid:acetonitrile=90:10, and the aminopeptidase B inhibitory activity was assayed. The active area was adsorbed on 200ml of Diaion HP-20, and after washing with water,
It was eluted with 50% methanol water. By collecting the active area, concentrating it under reduced pressure, and freeze-drying it,
OF4949- 455 mg was obtained. OF4949 coarse powder obtained in Example 3 4.1
Similarly, for g, _C18 reverse phase partition chromatography was performed using a developing solvent of 0.1M citrate buffer: acetonitrile = 95:5.
Perform HP-20 column chromatography,
OF4949- 671 mg was obtained.

[Brief explanation of drawings]

Figure 1 shows the ultraviolet absorption spectrum of OF4949. - indicates when the solvent is H ₂ O, ... indicates when the solvent is
In the case of 0.05NH Cl, -・- means that the solvent is 0.05N
The case of NaOH is shown respectively. Figure 2 is OF4949
- shows the infrared absorption spectrum measured in potassium bromide. Figure 3 shows the proton nuclear magnetic resonance spectrum of OF4949- measured in 0.06N deuterium ammonia water (internal standard: DSS). Figure 4 shows
This figure shows the C-13 nuclear magnetic resonance spectrum of OF4949 measured in 0.06N deuterium ammonia water (internal standard: p-dioxane 67.4ppm). Figure 5 shows OF4949-
The ultraviolet absorption spectrum of - is the solvent
. _. . indicates the case where the solvent is 0.05NH Cl, and -.- indicates the case where the solvent is 0.05N NaOH. Figure 6 shows the infrared absorption spectrum of OF4949 measured in potassium bromide. Figure 7 shows the proton nuclear magnetic resonance spectrum of OF4949 measured in 0.06N deuterium ammonia water (internal standard: DSS). FIG. 8 shows the C-13 nuclear magnetic resonance spectrum of OF4949 measured in 0.06N deuterium ammonia water (internal standard: p-dioxane 67.4ppm).

Claims (1)

[Claims] First-order general formula [] (In the formula, R represents hydrogen or a methyl group.) A compound called OF4949, or a salt thereof. 2. The compound according to claim 1, referred to as OF4949-, in which R is a methyl group, or a salt thereof. 3. The compound according to claim 1, designated as OF4949-, in which R is hydrogen, or a salt thereof. 4 Belongs to the genus Penicillium
OF4949-producing bacteria are cultured in a medium, and the following general formula [] is obtained from the culture. A compound called OF4949- represented by (in the formula,
when R is a methyl group) and/or OF4949-
(wherein R is hydrogen), characterized in that OF4949 and/or
Or the manufacturing method of OF4949. 5 Belongs to the genus Penicillium
The OF4949-producing bacterium is Penicillium lugrosum.
The method for producing OF4949 (Penicillium rugulosum OF4949) according to claim 4. 6 The following general formula [] (In the formula, R represents hydrogen or a methyl group.) An immunomodulator whose main component is a compound called OF4949 or a salt thereof. 7. The immunomodulator according to claim 6, wherein R is a methyl group. 8. The immunomodulator according to claim 6, wherein R is hydrogen.