JPH0365341B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention discloses novel physiologically active substances p-23924A, B, C, which have protocollagen/prolyl hydroxylase inhibitory effects, collagen biosynthesis inhibitory effects, etc.
D, E, F, regarding their manufacturing method and formulation. Protocollagen proline hydroxylase is an enzyme that specifically hydroxylates proline in protocollagen synthesized by ribosomes in animal cells, and is one of the important rate-limiting factors for collagen biosynthesis. Conventionally, substances that inhibit this enzyme activity include iron chelators (e.g., α, α′.dipyridyl, etc.), SH enzyme inhibitors (e.g., p-chloromercury benzoate, etc.), and certain heavy metals (e.g., Cu, Zn, etc.). ), but all of these substances non-specifically inhibit the biosynthesis of collagen and non-collagen proteins, resulting in significant side effects and could not be used as medicines. Does not inhibit biosynthesis of non-collagen proteins,
If a substance that specifically inhibits collagen biosynthesis is found, it can be used to treat arteriosclerosis, liver cirrhosis, scleroderma, keloids, rheumatoid arthritis,
It can be used to prevent and treat diseases including organ fibrosis that involves excessive accumulation of collagen, such as pulmonary fibrosis. The present inventors searched for a substance that inhibits collagen proline hydroxylase activity in microbial metabolic products, and as a result of intensive searches, discovered a novel physiologically active substance P that specifically inhibits collagen biosynthesis.
−23924A, B, C, D, E and F found;
Based on this, we conducted further research and completed the present invention. The present invention provides (1) physiologically active substances P-23924A, B,
C, D, E or F, (2) Physiologically active substance belonging to the genus Streptomyces P-
A microorganism capable of producing 23924A, B, C, D, E and/or F is cultured in a medium, and the physiologically active substance P-23924A, B, C, D,
Physiologically active substance P- characterized in that it produces and accumulates E and/or F and collects it.
23924A, B, C, D, E and/or F production method and (3) physiologically active substance P-23924A, B, C,
This is a preventive/therapeutic agent for organ fibrosis containing D, E and/or F. In this specification, the physiologically active substance P-
23924A, B, C, D, E and/or F as "bioactive substance P-23924" or simply "P-
23924". In addition, the physiologically active substance P-23924A is referred to as "P-23924A," the physiologically active substance P-23924B is referred to as "P-23924B," the physiologically active substance P-23924C is referred to as "P-23924C," and the physiologically active substance P-23924D is referred to as "P-23924C." may be abbreviated as "P-23924D", physiologically active substance P-23924E as "P-23924E", and physiologically active substance P-23924F as "P-23924F". The microorganism used in the method of the present invention belongs to the genus Streptomyces and has the ability to produce the physiologically active substance P-23924 (hereinafter sometimes abbreviated as "P-23924 producing microorganism"). Either is fine. A specific example is Streptomyces sp., which was isolated from the soil of Ishigaki Island, Okinawa Prefecture.
No. 23924 stock (hereinafter sometimes abbreviated as "No. 23924 stock") is mentioned. Regarding strain No. 23924, International Journal of Systematic Bacteriology (International Journal of Systematic Bacteriology)
Bactriology), Vol. 16, No. 3, pp. 313-340 (1966
The properties examined according to the method described in 2007 are as follows. Note that the findings on the culture medium were observed after culturing at 28°C for 14 days unless otherwise specified. () Morphological characteristics Short spore chain-forming hyphae extend from the basal hyphae in a spiral, sometimes incomplete spiral, open spiral, or hook shape in a simple branched form, and no paraphytic branches are observed. do not have. Mature spore chains generally consist of 5 to 10 spores. The spores are cylindrical or oval in shape, and the size is 0.4-0.7 x 0.7-1.2μ.
Its surface is smooth. () Growth status on various media Growth level (G) on various media, growth and color tone of aerial mycelium (AM), soluble pigment (SP)
The presence/absence and color tone are listed below. Regarding color descriptions, the standard color tone symbols shown in parentheses are Container Corporation
Container Corporation of America
The Color Harmony Manual, 4th edition, published in 1958. (a) Seuucrose/nitrate agar medium (G): Poor (AM): Poor, light gray brown (3ge) (SP): None (B) Glucose/asparagine agar medium (G): Poor (AM): None (SP ): None (c) Glycerin/asparagine agar (G): None (d) Starch/inorganic salt agar (G): Abundant (AM): Abundant, powdery, gray (3ih) (SP): Yellowish brown ( (4gc) (e) Tyrosine agar (G): Poor (AM): Poor, gray (3ba) (SP): Pale yellow red (4ea) (F) Nutrient agar (G): Moderate (AM): None (SP): None (K) Yeast/malt agar medium (G): Medium (AM): Poor, gray (3fe) (SP): Pale yellowish brown (4ne) (Q) Oatmeal agar medium (G): Medium Degree (AM): Poor, light grayish brown (3ge) (SP): None () Physiological properties (a) Growth temperature range: 115-35℃ (b) Liquefaction of gelatin (glucose, peptone,
Gelatin medium, 24℃, 3 weeks): Positive (weak) (C) Hydrolysis of starch: Positive (D) Coagulation and peptonization of skim milk: Both negative (E) Reducibility of nitrate: Negative (F) Melanin Formation of similar pigments: Tyrosine agar: positive Peptone yeast iron agar: negative () Assimilation of carbon sources (Pridham-Gottlieb agar) L-arabianose ± inositol - P-xylose L-rhamnose - D-glucose Raffinose - D-Fructose ± D-Mannitrate - Sucrose ± Control - (Note): Abundant growth, ±: Slight growth, -:
No growth Judging from the above morphological characteristics, it is clear that this strain belongs to the genus Streptomyces. The above Streptomyces sp. No. 23924 strain is
Institute of Fermentation (IFO) on September 20, 1981
Deposited with accession number IFO 14205. In addition, this microorganism was submitted to the Microbial Research Institute (FRI), Agency of Industrial Science and Technology, Ministry of International Trade and Industry on October 1, 1981, with the accession number
The deposit was deposited as FERM P-6739, and the deposit was converted into a deposit under the Budapest Treaty and is kept at the same research institute (FRI) under accession number FERM BP-338. In general, the properties of Streptomyces bacteria are easily changeable, and can be easily mutated by, for example, X-ray irradiation, ultraviolet irradiation, radiation irradiation, or artificial mutagenesis means using an artificial mutagenic agent. Even among such mutant strains, any strain capable of producing P-23924 can be used in the method of the present invention. When culturing P-23924 producing bacteria, the medium may be liquid or solid, but shaking culture or aerated agitation culture using a liquid medium is usually convenient. Any medium may be used as long as it is suitable for the growth of actinobacteria and can produce P-23924. That is, carbon sources include, for example, glucose, lactose, glycerin, starch, sucrose, dextrin, molasses, organic acids (eg, acetic acid, tartaric acid, etc.), and nitrogen sources include, for example, peptone, casamino acid (manufactured by Difco), N -Protein hydrolysates such as Z Amine A (manufactured by SIE Field), yeast extract, malt extract, soybean meal, corn staple liquor, amino acids (e.g., aspartic acid, glutamic acid, etc.), various ammonium salts (e.g., Ammonium sulfate, ammonium chloride, etc.) are used. Inorganic salts include various phosphates (e.g., monobasic sodium phosphate, dibasic potassium phosphate, etc.), metal salts such as magnesium sulfate, sodium chloride, ferrous sulfate, and heavy metal salts (e.g., manganese sulfate, zinc sulfate, etc.) ) may be added, and vitamins (e.g., vitamin B ₁ ,
Calcium pantothenate, etc.), nucleic acid-related compounds (eg, adenine, uracil, etc.), etc. may also be added. Depending on the culture method and culture conditions, P-23924 may be added to the culture medium by adding antifoaming agents such as silicone, polypropylene glycol derivatives (e.g., Actocol (manufactured by Takeda Pharmaceutical Co., Ltd.), etc.), soybean oil, etc. may be effective in increasing production. Culture conditions such as culture temperature, culture time, and liquid nature of the medium will vary depending on the type of microorganism used and the composition of the medium, but should be adjusted appropriately so that the final production amount of P-23924 is maximized. In most cases, the culture is carried out under aerobic conditions at approximately 20 to 40°C for approximately 24 to 240 hours, during which time the liquid quality of the medium is maintained.
It is best to keep the pH around 4-9. The physiologically active substance P-23924 can be easily collected from the culture fluid thus obtained by appropriately combining various methods based on the properties of the substance. That is, for example, extraction with neutral to slightly acidic and water-immiscible organic solvents such as ethyl acetate, butyl acetate, chloroform, butanol, benzene, toluene, diethyl ether, methylene chloride, methyl isobutyl ketone, activated carbon, silica gel,
Adsorption chromatography using alumina etc.
These include gel filtration using a Sephadex column and ion exchange chromatography using an ion exchange resin. By using a suitable combination of these means, the physiologically active substance P-23924 can be isolated from the culture as crystals or crystalline powder. Physiologically active substance P- obtained in Example 2 described below
The properties of 23924 are shown below. (1) Physical and chemical properties (a) Melting point: A 186-188℃; B 200-202℃;
187-190℃; D205-207℃; E174-176℃;
FF1916195℃ (b) Elemental analysis value (actual value):

[Table] (c) Molecular weight (based on mass spectrum) and molecular formula:

【table】 (d) Specific rotation:

[Table] (e) Solubility: P-23924A, B, D, and F are all easily soluble in dimethyl sulfoxide, dimethyl formamide, pyridine, and 5% sodium bicarbonate water. Soluble in methanol, dioxane and acetic acid. water, acetone, chloroform, n
- Slightly soluble or insoluble in hexane and petroleum ether. P-23924C is dimethyl sulfoxide,
Easily soluble in dimethylformamide, pyridine, and 5% sodium bicarbonate water. Soluble in methanol, dioxane, acetone, and acetic acid. Slightly soluble or insoluble in water, chloroform, n-hexane, and petroleum ether. P-23924E is dimethyl sulfoxide, dimethyl formamide, pyridine, 5
% Sodium bicarbonate water, easily soluble in methanol. Soluble in dioxane and acetic acid. Slightly soluble or insoluble in water, chloroform, n-hexane, and petroleum ether. (f) Ultraviolet and visible absorption spectra: 1st
~ Shown in Figure 6. The wavelength (nm) showing the maximum absorption immediately after being dissolved in each solvent and the E1% 1cm value are shown in Table 1 below. In Figures 1 to 6, - indicates the measurement results in methanol, --- indicates the measurement results in 0.1N HCl-90% methanol water, and --- indicates 0.1N.
The measurement results in NaOH-90% methanol water are shown.

【table】

[Table] (g) Infrared absorption spectrum: Infrared absorption spectrum measured with potassium bromide tablets
It is shown in FIG. The main wave numbers showing maximum absorption are as follows. (i) P-23924A 3300, 2930, 1710, 1665, 1640, 1620,
1570, 1540, 1500, 1460, 1420, 1380,
1335, 1280, 1240, 1200, 1180, 1110,
1040, 1000, 970, 900, 860, 800 See Figure 7 (ii) P-23924B 3300, 2950, 1725, 1705, 1660, 1630,
1590, 1540, 1460, 1420, 1370, 1330,
1300, 1270, 1210, 1190, 1170, 1130,
1100, 1060, 1020, 980, 950, 890, 860,
760, 700, see Figure 8 (iii) P-23924C 3400, 3070, 2940, 1720, 1680, 1635,
1600, 1540, 1490, 1460, 1420, 1380,
1350, 1310, 1250, 1220, 1180, 1120,
1100, 1040, 990, 930, 880, 820, 800,
705 See Figure 9 (iv) P-23924D 3400, 3300, 2960, 1720, 1670, 1635,
1610, 1540, 1440, 1380, 1340, 1300,
1260, 1220, 1205, 1130, 1100, 1060,
1000, 900, 870, 820, 800, 780, 690 See Figure 10 (v) P-23924E 3550, 3300, 3080, 2950, 1725, 1660,
1640, 1610, 1570, 1540, 1490, 1455,
1420, 1380, 1325, 1300, 1240, 1210,
1180, 1120, 1080, 1040, 1010, 940,
920, 860, 820, 800, 770, 700 See Figure 11 (vi) P-23924F 3300, 1720, 1680, 1660, 1630, 1600,
1530, 1490, 1460, 1420, 1380, 1335,
1310, 1260, 1220, 1190, 1130, 1100,
1030, 1000, 980, 955, 870, 700 See Figure 12 (h) Color reaction: P-23924 was positive for ferric chloride reaction, alcoholic magnesium acetate reaction, and Lydon-Smith reaction. Ninhydrin reaction and Ehrlichi reaction were negative. (i) Properties: All P-23924s are acidic and fat-soluble and exhibit yellow to yellow-orange or orange-yellow crystals, crystalline powder, or powder. (j) Nuclear magnetic resonance spectra: Spectra measured at 400 MHz in dimethyl- _d6 sulfoxide are shown in Figures 13-18. Characteristic peaks are shown below. (i) P-23924A δDMSO-d ₆ ppm: 1.86 (3H, s), 2.09 (3H,
d, J = 1.6Hz), 2.71 (1H, dd, J = 8.8,
13.6Hz), 2.91 (1H, dd, J=5.0, 13.6Hz),
3.66 (1H, d, J = 12.9Hz), 3.78 (1H, d,
J=12.9Hz), 3.96 (3H, s), 4.47 (1H,
dtlike, J = 5.0, 8.3, 8.8Hz), 6.89 (1H,
q, J=1.6Hz), 7.09 (1H, s), 8.18 (1H,
d, J=8.3Hz), 12.40 (1H, s) See Figure 13 (ii) P-23924B δDMSO-d ₆ ppm: 1.86 (3H, s), 1.95 (3H,
s), 2.70 (1H, dd, J=8.8, 13.7Hz), 2.89
(1H, dd, J = 4.9, 13.7Hz), 3.65 (1H, d,
J = 13.1Hz), 3.77 (1H, d, J = 13.1Hz),
3.95 (3H, s), 4.03 (3H, s) 4.47 (1H,
dtlike, J=4.9, 8.1, 8.8Hz), 7.11 (1H,
s), 8.18 (1H, d, J = 8.1Hz), 12.59 (1H,
s) See Figure 14 (iii) P23924C δDMSO−d ₆ ppm: 1.85 (3H, s), 2.71 (1H,
dd, J = 8.8, 13.7Hz), 2.91 (1H, dd, J =
4.9, 13.7Hz), 3.66 (1H, d, J = 12.9Hz),
3.77 (1H, d, J = 12.9Hz), 3.88 (3H, s),
3.95 (3H, s), 4.47 (1H, dtlike, J=4.9,
8.1, 8.8Hz), 6.26 (1H, s), 7.15 (1H, s),
8.18 (1H, d, J = 8.1Hz), 12.73 (1H, s) See Figure 15 (iv) P-23924D δDMSO-d ₆ ppm: 1.85 (3H, s), 1.94
(3H, s), 2.73 (1H, dd, J=8.8, 13.5
Hz), 2.92 (1H, dd, J=4.8, 13.5Hz), 3.64
(1H, d, J = 12.8Hz), 3.76 (1H, d, J =
12.8Hz), 4.00 (3H, s), 4.48 (1H, dtlike,
J=4.8, 8.1, 8.8Hz), 7.03 (1H, s) 8.17
(1H, d, J=8.1Hz), 12.79 (1H, s) See Figure 16 (v) P-23924E δDMSO-d ₆ ppm: 1.85 (3H, s), 2.71 (1H,
dd, J = 8.5, 13.7.Hz), 2.90 (1H, dd, J =
4.9, 13.7Hz), 3.67 (1H, d, J = 13.1Hz),
3.79 (1H, d, J = 13.1Hz), 3.98 (3H, s),
4.47 (2H, d, J = 2.2Hz), 4.47 (1H,
dtlike, J = 4.9, 8.1, 8.5Hz), 5.47 (1H,
s), 6.82 (1H, t, J=2.2Hz), 7.15 (1H,
s), 8.18 (1H, d, J = 8.1Hz), 12.29 (1H,
s) See Figure 17 (vi) P-23924F δDMSO-d ₆ ppm: 1.85 (3H, s), 2.70 (1H,
dd, J = 8.8, 13.7Hz), 2.90 (1H, dd, J =
4.9, 13.7Hz), 3.67 (1H, d, J = 13.1Hz),
3.80 (1H, d, J = 13.1Hz), 3.97 (3H, s),
4.10 (3H, s) 4.36 (2H, d, J = 3.7Hz),
4.47 (1H, dtlike, J=4.9, 8.3, 8.8Hz),
4.93 (1HH, brt), 7.16 (1H, s), 8.20 (1H,
d, J = 8.3Hz), 12.70 (1H, s) See Figure 18 (k) Rf value for thin layer chromatography: silica gel plate (manufactured by Merck & Co., product number 5729) and reversed-phase HPTLC plate, RP -8 (manufactured by Merck & Co., product number 13725), the Rf values are as follows.

[Table] No compound exhibiting the above-mentioned physicochemical properties is known, and P-23924 is considered to be a new substance. Based on the above physical and chemical properties, the structural formula of P-23924 is shown below.

[Table] Since P-23924 has a dielectric carboxy group,
Can form salts. Methods known per se are used to form the salts. Examples of the salts include calcium salts, magnesium salts, barium salts, sodium salts, potassium salts, manganese salts,
Examples include iron salts, copper salts, zinc salts, etc. The biological properties of P-23924 are shown below. (a) Protocollagen proline hydroxylase inhibitory effect: The inhibitory activity was measured by the method of KIKivirriko et al. and J. Halme et al. [Journal of Biological
Chemistry 242 , 4007 (1967) and
Bicohimica et Biophysica Acta198, 460
(1967)], using a partially purified enzyme preparation prepared from chicken embryos, (Pro−Pro−Gly) ₅・4H ₂ O
(Protein Research Foundation, Osaka) As a substrate, R.
Methods in Enzymology
B, 306 (1971)]. 100 μg of partially purified enzyme (as protein) using this method
The concentration of P-23924 required to inhibit 50% of the activity of P-23924 was as follows. P-23924A: 6.7×10 ^-5 M P-23924B: 9.5×10 ^-5 M P-23924C: 5.7×10 ^-5 M P-23924D: 2.7×10 ^-4 M P-23924E: 2.1×10 ^-5 M P-23924F: 2.1×10 ⁻⁵ M As is clear from the above results, P-23924 has a remarkable protocollagen proline hydroxylase inhibitory effect. (b) Collagen biosynthesis inhibitory effect: T. Ishimaru et al.'s method [Biochemical Pharmacology 31 , 915
(1982)], P-23924 was administered once a day for 6 days.
The drug was administered intraperitoneally to SD-strain rats (female, 3 weeks old) for days, and the amount of collagen and non-collagen protein in the uterus was compared with a control group. Second below
As shown in the table, P-23924 selectively and significantly inhibited collagen biosynthesis.

[Table] (B)−(A)
** Amount of non-collagen protein = total protein amount - amount of collagen *** Estradiol-17β was dissolved in physiological saline containing 5% ethanol.
The experiment used 10 rats per group. The acute toxicity [ _LD50 value (rat, intraperitoneal administration)] of P-23924 is shown below. P-23924A: 100-200mg/Kg, P-23924B:
50~100mg/Kg, P-23924C: 100~20mg/Kg,
p-23924D: 400mg/Kg or more, P-23924E: Approx.
50mg/Kg, P-232924F: Approximately 50mg/Kg or more. As mentioned above, P-23924 has a protocollagen/prolyl hydroxylase inhibitory effect and a selective collagen biosynthesis inhibitory effect, so it is useful as, for example, a biochemical reagent or an animal tissue fibrosis inhibitor. . That is, P-23924 is a mammal (rabbit,
For example, as a preventive/therapeutic agent for organ fibrosis in rats, mice, dogs, cats, humans, etc.
It is used for the prevention and treatment of liver cirrhosis, nephrosclerosis, arteriosclerosis, scleroderma, myelofibrosis, chronic arthritis, etc. The dosage of P-23924 varies depending on the target disease, symptoms, recipient, and administration method, but when administered as a prophylactic/therapeutic agent for organ fibrosis, approximately 10 to 1000 mg per adult per day. It will be administered in three divided doses. When administering P-23924, it can be administered by itself or mixed with appropriate pharmacologically acceptable carriers, excipients, diluents, etc., into powders, granules, tablets, capsules, injections, etc. It can be administered orally or parenterally. When manufacturing the above oral preparations, such as tablets,
Binders (e.g., hydroxypropylcellulose, hydroxypropylmethylcellulose, macrogol, etc.), disintegrants (e.g., starch, carboxymethylcellulose calcium, etc.), excipients (e.g.,
Lactose, starch, etc.), lubricants (eg, magnesium stearate, talc, etc.), etc. can be added as appropriate. In addition, when manufacturing parenteral preparations, such as injections, tonicity agents (e.g., glucose, D-sorbitol, D-mannitol, sodium chloride, etc.),
Preservatives (eg, benzyl alcohol, chlorobutanol, methyl paraoxybenzoate, propyl paraoxybenzoate, etc.), buffers (eg, phosphate buffer, sodium acetate buffer, etc.), etc. can be added as appropriate. P-23924 can be effectively used as a biochemical reagent, for example, as an inhibitor of collagen biosynthesis in cultured animal cells or as a specific inhibitor of protocollagen proline hydroxylase activity. The present invention will be explained in more detail below using Examples. Percentage of medium indicates weight/volume percentage. Example 1 Streptomyces sp. No. 23924 (IFO
14205, FERM BP-338) with glucose 2.0%,
Inoculate five 2-volume Sakaguchi flasks containing 400 ml of liquid medium consisting of 1.0% glycerin, 0.5% raw soybean flour, 0.5% corn stave liquor, 0.3% polypeptone, 0.3% sodium chloride, 0.5% calcium carbonate, and pH 7.0. Then, culture was carried out at 28°C for 2 days with reciprocal shaking to obtain about 2 volumes of culture fluid. This culture solution was transferred to a 200-volume tank containing 100% of the same medium as above, and cultured with aeration at 28°C for 2 days. The obtained culture solution 60 was mixed into a liquid medium 1200 containing 4% soluble starch, 2% defatted soybean flour, 0.1% sodium thiosulfate, 0.05% magnesium sulfate, 0.05% dipotassium phosphate, and 0.03% potassium chloride (PH6.0). The cells were transferred to a 2000 tank containing a 2000-liter tank, and cultured with aeration and agitation at 24°C for 5 days. Add Topcoperlite #34 to the obtained culture solution of about 1150
(manufactured by Toko Perlite Kogyo Co., Ltd.) 15Kg was added,
Cells and solid matter were filtered using a filter press pre-coated with 34 kg of Hyflo Super Cell (manufactured by Johns Manville, USA), and the liquid was evaporated.
Got 1000. The bacterial cells and solid matter were washed with 150% water and filtered again to obtain a 150% washing solution. This solution and the washing solution were combined, the pH was adjusted to 3.0 with sulfuric acid, and countercurrent extraction was performed using 1/2 volume of ethyl acetate using a 6150 Podbylniak extractor (manufactured by Podbylniak, USA). Approximately 430 of the obtained extract was dissolved in 1/2 volume of 1% sodium bicarbonate water using the same device to obtain a solution of 215. After adjusting the pH of the inverted solution to 3.0 with sulfuric acid, it was extracted again with 1/2 volume of ethyl acetate, washed twice with 1/2 volume of water, and then concentrated under reduced pressure. Pour the concentrate (400ml) into silica gel (Merck, West Germany)
Pour into a column (4.6 x 65 cm), add 500 ml of ethyl acetate,
Subsequently, elution was performed with ethyl acetate 3 containing 1% oxalic acid. Collect the active fraction (approximately 3) and add 2 with 500ml of water.
After washing twice, it was concentrated and dried under reduced pressure, and the obtained solid was dissolved in about 300 ml of dimethyl sulfoxide.
After adding 30ml of water to this solution, Prep PAK-
PreP LC with 500/C ₁₈ column/
Introduced into a System 500A preparative liquid chromatograph (Waters Inc., USA), 3 methanol-water (1:1) mixture, 5 methanol-water (3:2) mixture, and finally 2 methanol
was used for elution, and the eluate was collected in 500 ml portions.
It was divided into 3.5 fractions containing mainly P-23924A, C, D, and E (fractions 2-8) and 2.5 fractions containing mainly P-23924B (fractions 9-13). When the P-23924B-containing fraction is left at room temperature, approximately 2.3 g of yellow-orange P-23924B is produced.
After separating the precipitated crystals, the mother liquor was concentrated under reduced pressure to a volume of about 1, extracted three times with 1/3 volume of ethyl acetate, and after dehydration by adding 50 g of anhydrous sodium sulfate to the extract, about 30 ml was obtained. When 200 ml of n-hexane was added, about 7.1 g of yellow-orange crystalline P-23924B crude powder was obtained. This coarse powder was recrystallized from a methanol solution, and approximately 3 g of P-23924B crystals were obtained.
I got it. The fractions containing P-23924A, C, D, and E were concentrated to approximately 100 ml and placed on a silica gel column (4.0
x 42 cm) and eluted with ethyl acetate 2 containing 1% oxalic acid. The eluate was separated into 100 ml portions: 600 ml fractions containing mainly P-23924A and D (fractions 2 to 7), and 700 ml fractions containing mainly P-23924C and E (fractions 8 to 14).
Divided into ml. The fraction (600 ml) mainly containing P-23924A and D was washed with water, concentrated to dryness,
Dissolve in 50 ml of methanol, dilute with 50 ml of water, apply to the same preparative liquid chromatography device as above, and add 5.5 ml of methanol-water (1:1) mixture.
It was eluted. Separate the eluate in 500 ml portions and collect fractions 1.5 from fractions 13 to 15, approximately 700 ml.
After concentration under reduced pressure, 200 ml of ethyl acetate was added and extracted three times. The combined extracts were dehydrated by adding 30 g of anhydrous sodium sulfate, and then concentrated under reduced pressure. When 200 ml of n-hexane was added to about 30 ml of the concentrated liquid, 1.7 g of yellow-orange coarse powder containing P-23924A and D was obtained. Ta. This crude powder was dissolved in 100 ml of a chloroform-acetic acid (4:1) mixture and poured into a silica gel column (3.5 x 52 cm), followed by 400 ml of a chloroform-acetic acid (4:1) mixture and 1 1 ml of a chloroform-acetic acid (7:3) mixture. It was eluted using The eluate was collected in 10 ml portions and divided into fractions containing mainly P-23924A (fractions 25 to 29) and fractions containing mainly P-23924A (fractions 25 to 29).
Classification containing 23924D (fractions 36 to 136)
Divided into. The P-23924A-containing fraction (50 ml) was concentrated to dryness under reduced pressure, dissolved in 200 ml of methanol, 200 ml of water was added, and chromatography was performed again using the preparative liquid chromatograph described above to obtain P-23924A. Collected 23924A classification. This fraction was concentrated under reduced pressure, extracted with ethyl acetate, dehydrated, and concentrated to obtain about 250 mg of P-23924A crude powder. This crude powder was recrystallized from a methanol solution to obtain about 100 mg of yellow-orange P-23924A crystals.
Section 1 containing P-23924D was then concentrated to dryness under reduced pressure, and the dried product was dissolved in about 500 ml of ethyl acetate and washed three times with 200 ml of water. After dehydrating the washed ethyl acetate layer with 50 g of anhydrous sodium sulfate,
Ethyl acetate was distilled off under reduced pressure, and about 30 ml of the concentrated solution was
- P-23924D becomes yellow-orange when 200ml of hexane is added.
Approximately 0.7 g of coarse powder was obtained. This crude powder was dissolved in 25 ml of a chloroform-acetic acid (4:1) mixture, applied to a silica gel column (3 x 45 cm), and eluted with a chloroform-acetic acid (4:1) mixture.
Collected classifications. 400 ml of this fraction was concentrated under reduced pressure, 100 ml of water was added to 80 ml of the concentrated solution, and the mixture was repeatedly extracted three times with 1/3 volume of ethyl acetate. The extracts were combined, washed with water, dehydrated, and the ethyl acetate was distilled off. Approximately 500 mg of the resulting crude powder was recrystallized from a methanol-ethyl acetate (1:5) mixture to give a yellow-orange P-
Approximately 330 mg of crystalline powder of 23924D was obtained. next,
After washing and dehydrating 700 ml of fractions containing mainly P-23924C and E (fractions 8 to 14) obtained by silica gel chromatography, ethyl acetate was distilled off, and the residue was dissolved in 50 ml of methanol. This solution was diluted twice with water, applied to a preparative liquid chromatograph (the same column as above), and eluted with a methanol-water (1:1) mixture. The eluate was collected in 200 ml portions, mainly containing P-
Classification containing 23924E (fractions 10 to 13)
It was divided into 800 ml and 1 fraction (fractions 14 to 18) containing P-23924c. The fraction containing P-23924E was concentrated under reduced pressure, extracted three times with 1/3 volume of ethyl acetate, dried, concentrated to about 20 ml, and extracted with n-hexane.
When 200 ml was added, about 120 mg of yellow-orange P-23924E crude powder was obtained. This crude powder was dissolved in 30 ml of a chloroform-methanol (3:2) mixture, applied to a silica gel column (3 x 45 cm), eluted with chloroform-methanol (3:2), and P-
The fractions containing 23924E were collected and concentrated, and 10 ml of ethyl acetate and 50 ml of n-hexane were added to 10 ml of the concentrated solution to obtain about 30 mg of yellow-orange crystalline powder of P-23924E. Fraction 1 containing P-23924C was concentrated to dryness under reduced pressure, and the dried product was dissolved in 15 ml of chloroform and applied to a silica gel column (3.0 x 45 cm).
Elute with chloroform-acetic acid (4:1) mixture, P
Sections containing −23924C were collected. This fraction was concentrated to dryness to obtain about 150 mg of P-23924C crude powder. This crude powder was dissolved by adding 70 ml of ethyl acetate, washed 3 times with 20 ml of water, dehydrated by adding 2.5 g of anhydrous sodium sulfate, the ethyl acetate solution was concentrated under reduced pressure to about 10 ml, and 25 ml of n-hexane was added.
When ml is added, about 85 mg of yellow-orange P-23924C crystals are obtained.
It was raised. Next, in exactly the same manner as above, the culture solution was extracted with ethyl acetate, transferred with 1% sodium bicarbonate water, extracted with ethyl acetate on the acidic side, washed with water, and concentrated, resulting in a concentrated solution ( 200ml)
was applied to a silica gel (Merck, West Germany) column (4.6 x 65 cm) and eluted with ethyl acetate 2 and then with ethyl acetate 7 containing 1% oxalic acid. Collect Category 2, which mainly contains P-23924F, and collect 300ml.
After washing twice with water, the mixture was concentrated to dryness under reduced pressure, and the resulting solid content was dissolved in 80 ml of a chloroform-acetic acid (9:1) mixture. This solution was poured into a silica gel column (3.4 x 54 cm), and chloroform:acetic acid (9:
1) Eluted with mixture 1.2, (8:2) mixture 1.2, and (7:3) mixture 1.2, mainly P-
The fractions containing 23924F (approximately 1.5) were collected and concentrated to dryness under reduced pressure, and the resulting residue was dissolved in 400 ml of a methanol-water (1:1) mixture, followed by Prep PAK-
Prep LC/ with 500/C ₁₈ column
The mixture was introduced into a System 500A preparative liquid chromatograph and eluted using methanol-water (3:2) mixture 5. When the fraction containing mainly p-23924F is collected and concentrated to dryness under reduced pressure, P-
1.3 g of coarse powder of 23924F was obtained. This was recrystallized from a methanol:water (8:2) mixture to obtain about 500 mg of yellow-orange P-23924F crystals. Example 2 The same seed culture solution as in Example 1 was mixed with 4% soluble starch, 1% dextrin, 2% defatted soybean flour, 0.1% sodium thiosulfate, and ferrous sulfate/7 crystal water.
0.05%, dipotassium phosphate 0.05%, potassium chloride
2000 containing liquid medium 1200 consisting of 0.03% PH6.0
The cells were transferred to a large tank and cultured with aeration and stirring at 24°C for 6 days. About 1100 of the obtained culture solution was used in Example 1.
In the same manner as above, the mixture was filtered, extracted with ethyl acetate, transferred to a sodium bicarbonate solution, extracted again with ethyl acetate, and then concentrated to obtain 700 ml of a concentrated solution. This concentrated solution was poured into a silica gel column (5.4 x 62 cm),
Elution with ethyl acetate 3 followed by ethyl acetate 10 containing 1% oxalic acid resulted in segment 2.6 containing mainly P-23924A, B and D, and segment 2.6 containing mainly P-23924A, B and D.
It was divided into Category 4.2, which contains 23924C and E, and Category 3.0, which mainly contains P-23924F.
The fractions containing P-23924A, B and D were washed with water, dehydrated, and 200ml of the residue obtained by distilling off ethyl acetate.
of methanol. Add 200 ml of dimethyl sulfoxide and 200 ml of water to this solution and prepare
Prep LC/ equipped with PAK-500/C ₁₈ column
Introduced into a System 500A type preparative liquid chromatography device and eluted with a methanol-water (1:1) mixture. Section 2.5 mainly contains P-23924A and D, and Section 3.1 mainly contains P-23924B.
Collected. The fractions containing P-23924A and D were rechromatographed using the liquid chromatograph described above, and then subjected to concentration, ethyl acetate extraction, dehydration, and concentration operations to obtain 50 ml of a concentrated solution. This was applied to a silica gel column (4.5 x 70 cm), and 1.5 of dichloromethane-acetic acid (9:1) mixture, 5.0 of dichloromethane-acetic acid (8:2) mixture, and 2 (7:3) mixture were eluted in this order, mainly to detect P-23924A. Containing category 1 and P-
Category 1.2 containing 23924D was collected. P-
After washing the 23924A-containing section three times with 300 ml of water, separate the dichloromethane layer and concentrate to dryness, dissolve the dried product in 200 ml of methanol, add 200 ml of water,
It was applied to a preparative liquid chromatograph under the same conditions as above. The fraction containing P-23924A was collected, concentrated to remove methanol, and diluted with 1/3 volume of ethyl acetate.
After repeated extraction, the extracts were combined, dehydrated, and concentrated to dryness. The dried product was dissolved in methanol and allowed to stand, yielding 300 mg of crystals of P-23924A. Next, the P-23924D-containing section (1.2) was concentrated under reduced pressure, and 30 ml of the concentrated solution was transferred to a silica gel column (3.5
x 50cm) and dichloromethane-acetic acid (9:
1) Elution was carried out sequentially with 1 part of the mixture, 1 part each of the same (8:2) mixture, and 1 part each of the same (7:3) mixture. Mainly P-23924D
The fraction 1.4 containing was collected and concentrated to dryness under reduced pressure, and 200 ml of methanol was added to the dried product to dissolve it.
Add 200 ml of water to this solution and perform preparative liquid chromatography under the same conditions as above.
Collected 23924D Division 2.7. Methanol was distilled off from this fraction under reduced pressure. 1/3 volume of ethyl acetate was added to 1.5 of the concentrated solution obtained, and 1/3 volume of ethyl acetate was added, extracted three times, and then dehydrated and concentrated to approximately 25 ml. When left to stand, approximately 1 g of P-23924D crystals were obtained. It was raised. When the fraction (3.1) containing P-23924B was allowed to stand at room temperature, crystals of P-23924B precipitated. This was collected and reconstituted from methanol to obtain about 3 g of crystals of P-23924B. Next, the fraction (4.2) mainly containing P-23924C and E was washed three times with water from step 1, dehydrated with 30 g of anhydrous sodium sulfate, and ethyl acetate was distilled off. The residue was dissolved in 200 ml of methanol. Add 200 ml of water to this and repeat the liquid chromatography under the same conditions as above.
Collected 2.4. P-23924E containing fraction 1.8 is approximately 50
Concentrate to ml under reduced pressure, apply it to a Sephadex LH-20 (manufactured by Pharmacia, Sweden) column (3.5 x 52 cm) that was pre-suspended in methanol and packed, elute with methanol, and collect 200 ml of the fraction containing only P-23924E. Ta. This fraction was concentrated under reduced pressure, and 20 ml of ethyl acetate and 200 ml of n-hexane were added to 20 ml of the concentrated solution to obtain about 700 mg of yellow-orange crystalline powder of P-23924E. Next, mainly P-
Section 2.4 containing 23924C was concentrated under reduced pressure, and 1/3 volume of ethyl acetate was added to concentrated solution 1.2 and extracted three times. The ethyl acetate layer was dehydrated using 20 g of anhydrous sodium sulfate, and the ethyl acetate was distilled off. When the residue obtained is dissolved in methanol and left to stand, P-
Crude crystals of 23924C were precipitated. When this was recrystallized from methanol, 1.5 g of crystals of P-23924C were obtained. Next, the section (3.0) mainly containing P-23924F was washed with water, dehydrated, and the residue obtained by distilling off ethyl acetate was mixed with chloroform-acetic acid (9:
1) Dissolved in 100ml of mixed solution. Pour this solution onto a silica gel column (3.4 x 54 cm) and chloroform-
1 part of acetic acid (9:1) mixture, 1 part of acetic acid (8:2) mixture
, (7:3) mixture 1 was eluted in this order, and fraction 1.8 containing mainly P-23924F was collected and concentrated to dryness. The dry matter was mixed with methanol-water (1:1) 400
ml of methanol- _water (3:
2) The mixture was eluted and the fraction (2.4) containing mainly p-23924F was collected. When this was concentrated under reduced pressure, crude crystals of P-23924F were precipitated. When this is recrystallized from methanol:water (3:2), P
2.5 g of -23924F crystals were obtained. Example 3 Tablets (1) p-23924A 100mg (2) Lactose 47mg (3) Corn starch 40mg (4) Hydroxypropyl cellulose-L 12mg (5) Magnesium stearate 1mg 200mg per tablet Compress the tablets into tablets according to the Act. Example 4 Tablets (1) P-23924B 100mg (2) Lactose 47mg (3) Corn starch 40mg (4) Hydroxypropylcellulose-L 12mg (5) Magnesium stearate 1mg 200mg per tablet Compress the tablets into tablets according to the Act. Example 5 Capsule (1) P-23924C 100mg (2) Lactose 135mg (3) Cornstarch 60mg (4) Magnesium stearate 5g 300mg per capsule Above ingredients (1), (2), (3) and (4) ) and then granulate it according to the usual method. In this, the ingredients
Add the remainder of (4) and fill in No. 1 gelatin capsules (Japanese Pharmacopoeia, 10th edition) according to the usual method to prepare capsules. Example 6 Capsule (1) P-23924D 300mg (2) Lactose 135mg (3) Corn starch 60mg (4) Magnesium stearate 5mg 500mg per capsule Above ingredients (1), (2), (3) and (4) ) and then granulate it according to the usual method. In this, the ingredients
Add the remainder of (4) and fill in No. 00 gelatin capsules (10th edition Japanese Pharmacopoeia) according to the usual method to prepare capsules. Example 7 Tablets (1) P-23924E 100mg (2) Lactose 47mg (3) Corn starch 40mg (4) Hydroxypropylcellulose-L 12mg (5) Magnesium stearate 1mg 200mg per tablet Compress the tablets into tablets according to the Act. Example 8 Tablets (1) p-23924F 100mg (2) Lactose 47mg (3) Corn starch 40mg (4) Hydroxypropylcellulose-L 12mg (5) Magnesium stearate 1mg 200mg per tablet Compress the tablets into tablets according to the Act.

[Brief explanation of drawings]

Figures 1 to 6 show the physiologically active substance P-
Figures 7 to 12 show the ultraviolet and visible absorption spectra of 23924A, B, C, and DEF. Figures to Figure 18 show physiologically active substances P-23924A, B, C, D, E, F.
represents the nuclear magnetic resonance spectra of

Claims (1)

[Claims] 1. General formula [In the formula, R ₁ is hydrogen, methyl group or hydroxymethyl group, R ₂ is hydrogen or methoxy group,
R ₃ represents a hydrogen group or a methoxy group. however,
When R ₂ and R ₃ are methoxy groups, R ₁ is hydrogen, methyl or hydroxymethyl; when R ₂ is hydrogen and R ₃ is methoxy, R ₁ is methyl or hydroxymethyl; R ₂ When is a methoxy group and R ₃ is a hydroxyl group, R ₁ represents a methyl group. ] A compound represented by. 2 Belongs to the genus Streptomyces and has the general formula [In the formula, R ₁ is hydrogen, methyl group or hydroxymethyl group, R ₂ is hydrogen or methoxy group,
R ₃ represents a hydroxyl group or a methoxy group. however,
When R ₂ and R ₃ are methoxy groups, R ₁ is hydrogen, methyl or hydroxymethyl; when R ₂ is hydrogen and R ₃ is methoxy, R ₁ is methyl or hydroxymethyl; R ₂ When is a methoxy group and R ₃ is a hydroxyl group, R ₁ represents a methyl group. ] A microorganism having the ability to produce at least one compound represented by the above formula is cultured in a medium, the compound is produced and accumulated in the culture, and the compound is collected. A method for producing a compound. 3 General formula [In the formula, R ₁ is hydrogen, methyl group or hydroxymethyl group, R ₂ is hydrogen or methoxy group,
R ₃ represents a hydroxyl group or a methoxy group. however,
When R ₂ and R ₃ are methoxy groups, R ₁ is hydrogen, methyl or hydroxymethyl; when R ₂ is hydrogen and R ₃ is methoxy, R ₁ is methyl or hydroxymethyl; R ₂ When is a methoxy group and R ₃ is a hydroxyl group, R ₁ represents a methyl group. ] A prophylactic/therapeutic agent for organ fibrosis containing a compound represented by.