JPH0259839B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel physiologically active substance OF4949 and a method for producing the same. 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 was discovered that a physiologically active substance that strongly inhibits aminopeptidase B activity, that is, a water-soluble amphoteric peptide substance named OF4949 by the present inventors, is accumulated. 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. From the physicochemical and biological properties, it was confirmed that OF4949- and OF4949- are new physiologically active substances that have the same structure except for the presence of different substituents. Among these, the present invention relates to OF4949. OF4949—and/or OF4949—
is called OF4949. 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. Furthermore, OF4949 exhibits an effect of enhancing cell-mediated immunity, which can be obtained by inoculating sheep red blood cells as an antigen into mouse footpads, using delayed-type hypersensitivity as an indicator, and can be used as an immune cancer suppressant by increasing the immune capacity of the body. 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 agar extract medium Growth is slow and colony diameter is 2.0 to 2.5 in 2 weeks.
cm. 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 village is 1-2 mm wide 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 to 3.0 in 2 weeks
cm. 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 in 2 weeks
cm. 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 is 3.0 to 4.0 cm in 2 weeks.
reach. 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.No pigment is secreted into the agar. (2) Morphology The hyphae are colorless and have septate walls, and most penicillas are biwhorled, but occasionally they are monowhorled. Conidia are oval in shape, length
It is 3.0 μm to 4.0 μm, the width is 2.5 μm to 3.0 μm, and the size of the conidial chain ranges from 50 μm to 80 μm. The stalks are spiky or spear-shaped, growing in clusters of 4 to 8 in parallel, 10.0 to 13.0 μm in length, and 2.0 to 2.5 μm in width. The basal sycamores grow in clusters of 2 to 8 in a compact or somewhat open state, 10 to 13 μm long, and wide.
It is 2.5 μm to 3.0 μm. conidiophore, length
They are 50 μm to 80 μm wide and 2.5 μm to 3.0 μm wide, and protrude from many aerial or vegetative mycelia without branching, but branched mycelium 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 mycelium was found to be Penicillium.
It was identified as Penicillium rugulosum. The mycological properties of Penicillium rugulosum can be found in Raper & Thom, 1968, “A.
Manual of the Penicilia (A
Manual of the Penicillia, Domsh & Gams, 1972, Longman's Fungi in Agricultural Soils.
Agricultural Soils)” and “Journal of General and Applied Soils” by Abe.
Microbiology (Journal of General
and Applied Microbiology), Vol. 2, p. 1, 1956. The present inventors have identified this bacterium as “Penicillium rugulosum OF4949 (Penicillium rugrosum)” based on OF4949 productivity.
rugulosum OF4949)". This bacterium has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, under the accession 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.In addition, these strains can be irradiated with ultraviolet light. Alternatively, OF4949-producing strains have been found among artificial mutant strains obtained by treatment with mutagenic agents used for microbial mutation such as nitrosoguanidine, and among naturally occurring mutant strains. These mutant strains can also be used in the present invention. For the culture used in the present invention, the medium may be liquid or solid, but shaking culture or aerated agitation culture using a liquid medium 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, glycerin, sorbitol, etc. sugars,
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 its salts, urea ammonium salts, nitrates, etc. are used. In addition, trace nutrients such as inorganic salts such as magnesium phosphate, 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 culture filtrate and 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 or nonionic adsorption resin, ion exchange chromatography using ion exchange resin, Appropriate combinations of ordinary means for separating and purifying organic substances from microbial cultures are used, such as partition chromatography using cellulose, 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 strong base 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 thus obtained, Diamondion HP-
20 or the like, and then desalted by eluting with a solvent, and then concentrated 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- according to the present invention will be explained in detail while comparing them with the properties of OF4949-, which has similar properties. [Physical and chemical properties] Molecular formula OF4949-: C ₂₃ H ₂₆ O ₈ N ₄ OF4949-: C ₂₂ H ₂₄ O ₈ N ₄ -element analysis value OF4949-: Actual value Carbon 52.30%, Hydrogen 5.46%, Nitrogen 10.64% Theoretical value Carbon 52.87%, hydrogen 5.79%, nitrogen 10.72% (as C ₂₃ H ₂₆ O ₈ N ₄ 2H ₂ O) OF4949: Actual value Carbon 51.60%, Hydrogen 5.48%, Nitrogen 11.6% Theoretical value Carbon 51.97%, Hydrogen 5.55%, nitrogen 11.02% (as C ₂₂ H ₂ B ₄ O ₈ N ₄ ×2H ₂ O) Molecular weight SIMS (Secondary lon Mass
OF4949 measured by spectrometry method
The molecular weights of OF4949 and OF4949 are as follows. OF4949-: 486 OF4949-: 472 Melting point Both OF4949- and OF4949- decompose 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- Wavelength showing maximum absorption and E ¹ % _1cn when OF4949 and OF4949 are dissolved in the following solvents
is as follows. [Table] [Table] Infrared absorption spectrum The maximum absorption measured with potassium bromide tablets of OF4949 and OF4949 is as follows. [Table] [Table] Solubility Both OF4949 and OF4949 are soluble in water, alkaline water, and dimethyl sulfoxide. Slightly soluble in methanol and ethanol. n-propanol, n-butanol, acetone, ethyl acetate,
Insoluble in chloroform, ether, benzene, hexane. Color reaction OF4949-, OF4949-both ninhydrin, potassium permanganate, Lydon Smith iodine reaction positive, Sakaguchi Prokatka harness, Anthrone reaction 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 on silica gel plate (Merck)
The Rf values are as follows. [Table] [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 (PH
5.7): Acetonitrile = 90:
10 Flow rate: 0.5ml/min Detection: UV275nm OF4949-: Retention time 9.9 minutes Retention volume 5.0ml OF4949- Retention time 4.2 minutes Retention volume 2.1ml Color of substance Both OF4949- and OF4949- are white powder. 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) 6.8 (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 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, 179.0, 145.6, 136.1 132.8, 131.4, 127.4, 125.2 123.1, 122.0, 117.0, 11 6.8 73.0, 57.9, 55.1, 54.0 39.7, 39.1 Above As shown, OF4949- and OF4949- are closely related substances with similar properties, and OF4949- is 1
OF4949 has phenolic hydroxyl groups.
- is a new substance that has the same structure, except that it is substituted with one methoxy group. Of course, the present invention includes OF4949-carboxylic acid metal salts as well as salts with mineral or organic acids. [Biological Properties] OF4949 has a physiological activity that significantly inhibits aminopeptidase B activity of Ehrlichi cancer cells. Below are the biological properties of OF4949.
- will be explained in detail while comparing with. 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 ^0.71 ml of Hank's solution containing the specimen was heated at 37°C for 3 minutes. Add 0.2 ml of Ehrlichi cancer cell suspension prepared as follows, and incubate at 37°C.
After reacting for 30 minutes, the concentration of garnet was reduced to 0.3mg/cc.
1.0M containing GBC (orthoaminoazotoluene diazonium salt) (manufactured by Sigma) and Tween 20 (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, the 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 were maintained in the peritoneal cavity of ddY 4-6 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, washed three times with Hank's solution, and adjusted to the desired cell number with Hank's solution. This is a homogeneous cell population with more than 96% of living Ehrlichi cancer cells. OF4949 by the above test method and OF4949
The inhibition rates at several concentrations of - were determined, and a 50% inhibitory concentration was assigned to each. The results are shown in Table 1. [Table] This substance can also be used for delayed-type hypersensitivity, which can be obtained by inoculating sheep red blood cells as an antigen into mouse footpads.
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 injected subcutaneously 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 administered as a medicament, the compounds of the present invention may be administered neat or in a pharmaceutically acceptable non-toxic and inert carrier, e.g. 0.1% to 99.5%, preferably 0.5%.
It is administered to animals, including humans, as a pharmaceutical composition containing 90% 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, intracellularly, locally (transdermally, etc.), or rectally. Of course, it is administered in a dosage form suitable for these administration methods. For example, injections are particularly preferred. Although it is desirable to adjust the dose by considering the patient's condition such as age, weight, route of administration, nature of the disease, etc., the amount of the active ingredient of the present invention for adults is usually 1. 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. 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. Clean formulations are prepared by comminution of the active substance to an appropriate size. 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 powders or powders that have been turned into powder as described above, or granules as described in the section on tablets, into capsule shells such as gelatin capsules. be done. Lubricants and fluidizing agents, such as colloidal silica, talc, magnesium stearate, calcium stearate, solid polyethylene glycol, can also be mixed into the powder prior to the filling operation. .
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, and a surfactant, and then wrapped in a gelatin sheet to form a soft capsule. tablets make a powder mixture;
It is manufactured by granulating or slugging, 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, gum arabic, 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 a quantity of the compound in a non-toxic liquid carrier, such as an aqueous or oily vehicle, for injection purposes and then sterilizing the suspension or solution. . Alternatively, a fixed amount of the compound may be placed in a vial, and the vial and its contents may then be sterilized and sealed.
A vial or carrier may be prepared in advance with the powdered or lyophilized active ingredient for dissolution or mixing immediately before 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 two Erlenmeyer flasks. , sterilized at 120°C for 20 minutes, and cultured at 27°C with shaking at 190 revolutions per minute for 6 days. This culture was placed in a pre-sterilized medium containing 2% glucose, 0.5% polypeptone, 0.1% yeast extract, 0.05% potassium phosphate, and 0.05% magnesium sulfate.
Cultivation was started by inoculating one inoculation into 15 stainless steel fermentation tanks. Culture is carried out at an aeration rate of 20 per minute.
, at 300 revolutions per minute and 27°C, and an antifoaming agent was added as necessary. After culturing for 4 days, the culture was taken out, and the cells were filtered to separate the culture filtrate, yielding a compressed capacity of 6.5 cells and 300 cells of the culture filtrate. 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
After neutralizing 60 with 2N hydrochloric acid, it was adsorbed onto 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 (IN ammonia water: n-butanol = 15:
85) When developed in 12, an active area mainly containing OF4949- was separated, and then an active area mainly containing OF4949- was separated. 1NHCl for each active area
After neutralizing with water and concentrating under reduced pressure, it was redissolved in water and adsorbed on 500 ml of Diaion HP-20. After washing 2
By eluting with 50% methanol water and concentrating the active area under reduced pressure, 3.1 g 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. Adsorb the active area in 200ml of Diaion HP-20, wash with water,
It was eluted with 50% methanol water of 1. By collecting the active area, concentrating it under reduced pressure, and freeze-drying it,
OF4949- 455 mg was obtained.

[Brief explanation of drawings]

Figure 1 shows the ultraviolet absorption spectrum of OF4949. ―――― is the case when the solvent is H ₂ O, …… is the case when the solvent is 0.05N HCl, ―――― is when the solvent is
The case of 0.05N NaOH is shown. Figure 2 is
This shows the infrared absorption spectrum of OF4949 measured in potassium bromide. Figure 3 shows OF4949-
This figure shows a proton nuclear magnetic resonance spectrum measured in 0.06N deuterium ammonia water (internal standard: DSS). Figure 4 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] 1. A physiologically active substance OF4949-I characterized by having the following physicochemical properties. Molecular formula: C ₂₃ H ₂₆ O ₆ N ₄ elemental analysis values Actual values Carbon 52.30%, Hydrogen 5.46%, Nitrogen 10.64% Theoretical values Carbon 52.87%, Hydrogen 5.79%, Nitrogen 10.72% (C ₂₃ H ₂₆ O ₈ N ₄・2H ₂ O) Molecular weight SIMS (Secondary Ion Mass
The molecular weight measured by the method (Spectrometry) is as follows. :486 Decomposes at melting point around 280℃. Specific optical rotation: [α] ²⁵ _D = -64.8゜ (C = 1.0, H ₂ O) Ultraviolet absorption spectrum The wavelength showing the maximum absorption when dissolved in the following solvent and the E ¹ % _1cn value are as follows. . [Table] Infrared absorption spectrum The wave numbers showing the maximum absorption measured with potassium bromide tablets are as follows. [Table] Solubility Soluble in water, alkaline water, dimethyl sulfoxide. Slightly soluble in methanol and ethanol.
Insoluble in n-propanol, n-butanol, acetone, ethyl acetate, chloroform, ether, benzene, hexane. Color reaction: Ninhydrin, potassium permanganate, Lydon Smith iodine reaction positive, Sakaguchi Prokatka harness, Anthrone reaction negative. Ferric chloride reaction negative. Distinction between neutral, acidic, and basic It is an amphoteric substance. Thin layer chromatography on silica gel plate (Merck)
The Rf values are as follows. [Table] The retention capacity and retention time 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 (PH
5.7): Acetonitrile = 90:
10 Flow rate: 0.5ml/min Detection: UV275nm Retention time 9.9min Retention volume 5.0ml Color of substance White powder. Proton nuclear magnetic resonance spectrum The chemical shift, number of protons, and multiplicity measured in 0.06N deuterium oxide water are as follows. Unit: Chemical shift ppm Internal standard: DSS: 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) C-13 nuclear magnetic resonance spectrum 0.06N The chemical shifts measured in ammonia water are as follows. Unit: Chemical shift ppm Internal standard: p-dioxane (67.4ppm): 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 2 A method for producing the physiologically active substance OF4949, which comprises culturing OF4949-producing bacteria belonging to the genus Penicillium in a medium and collecting OF4949 from the culture. 3. The production method according to claim 2, wherein the OF4949-producing bacterium belonging to the genus Penicillium is Penicillium rugulosum OF4949.