JPS6034185A - Physiologically active substance p-23924, its preparation, and pharmaceuitical preparation - Google Patents

Abstract

PURPOSE:To obtain novel physiologically active substance P-23924 having an inhibitory action on hydroxylase of collagen proline, and an inhibitory action on collagen biosynthesis, etc., by cultivating a bacterium belonging to the genus Streptomyces. CONSTITUTION:Streptomyces sp. No23924 strain(FERM-P6739) is separated from soil. When the bacterium is cultivated in an ordinary medium under aerobic condition at 20-40 deg.C for 24-240hr, novel physiologically active substances A, B, C, D, E and F are produced. The physiologically active substances have the following properties. Melting point: A186-188 deg.C, B200-202 deg.C, C187-190 deg.C, D205-207 deg.C, E174-176 deg.C, and F191-195 deg.C. Molecular weight: A393, B423, C409, D409, E409, and F439.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides novel physiologically active substances P-23924A and B that have glo[collagen/proline hydroxylase inhibitory action, collagen biosynthesis inhibitory action, etc.].

C, D, E, F, and their manufacturing methods and formulations.

Protocollagen 10-phosphorus hydroxylase is an enzyme that specifically hydroxylates proline in protocollagen synthesized in ribosomes in animal cells, and is one of the important factors that limit the rate of collagen biosynthesis. Conventionally, iron chelators (e.g. α, α
'-dipyridyl, etc.), 8H enzyme inhibitors (e.g., p-chloromercury benzoate, etc.), and certain heavy metals (e.g., Cu r Zn+, etc.), but all of these substances are known to inhibit collagen and non-collagen. Because it nonspecifically inhibits the biosynthesis of sexual proteins, it has serious side effects and cannot be used as a medicine. If a substance is found that specifically inhibits collagen biosynthesis without inhibiting the biosynthesis of non-collagen proteins, that substance can be used to treat arteriosclerosis, liver cirrhosis, scleroderma, and keloids. It can be used for the prevention and treatment of diseases including organ fibrosis associated with excessive collagen accumulation, such as rheumatoid arthritis and pulmonary fibrosis.

The present inventors have incorporated a substance that inhibits protocollagen proline hydroxylase activity into microbial metabolic products, and have worked diligently to achieve t!
As a result of 1jlA, new physiologically active substances P-23924A, B+ that specifically inhibit collagen biosynthesis
As a result of discovering c, n, E and F and further research based on this, the present invention was completed.

The present invention provides (1) physiologically active substance P-23924A.

B, C, D, B or F, (2) Streptomyces (S
Physiologically active substance P-2 belonging to the genus Treptomyces
Microorganisms having the ability to produce 3924A, B, C, D, E and/or F are cultured in a medium, and the physiologically active substance ffP-23924A,:s is added to the culture.

Physiologically active substance P-23924 characterized by producing and accumulating C, D, E and/or F and collecting them
A, B, C, D, E and/or F manufacturing method and (
3) A fibrosis inhibitor containing physiologically active substances p-23924A, B, C, D, E and/or F.

In this specification, the physiologically active substance P-23924A,
B, C, D, E and/or F are “bioactive substances p-
23924J or simply rp-23924J. In addition, the physiologically active substance p-23924A was used as rp-23924Aj, and the physiologically active substance P-23924
B is rp-23924BJ and physiologically active substance P-239
24c [P-23924CJ and physiologically active substance P-2
3924D with rP-23924DJ and physiologically active substance P
-23924E may be abbreviated as rp-23924EJ, and the physiologically active substance p-2392tr may be abbreviated as rP-23924]i'J.

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 1p-23924 (hereinafter sometimes abbreviated as "P-23924 producing microorganism"). Either is fine.

A specific example of this is Streptomyces sp. 23924 strain (hereinafter sometimes abbreviated as "Tama 23924 strain"), which was isolated from the soil of Ishigaki Island, Okinawa Prefecture.
can be mentioned.

Regarding the m23924 strain, the International Journal of Systematic Bacteriology (International Journal of Systematic Bacteriology)
Stematic Bacteriology ) +
The properties investigated according to the method described in Vol. 16, No. 3, pp. 313-340 (1966) are as follows. Note that the findings on the culture medium were observed after culturing at 28° C. for 14 days, unless otherwise specified.

(I) Morphological characteristics Insect-trapping hyphae, spiral-shaped, sometimes completely spiral-shaped,
Short spore chain-forming hyphae in an open spiral or hook shape extend in a simple branching pattern, with no whorled branches. Mature spore chains generally consist of 5 to 10 spores. The spores are cylindrical or oval in shape, and the size is 0.4 to 0.7
O, 7-1.2μ, its surface is smooth.

(II) Growth status on various media The degree of growth (G) on various media, the growth and color tone of aerial mycelium (AM), the presence or absence of soluble pigment (SP), color tone, etc. are listed below.

Regarding the color description, the standard color tone symbol indicated by 0 is Container Corporation Ogu America (Cont.
ainer Corporation of Amer
ica) Nosa Color Harmony Manu 7 /l/
(The Calor Harmony Manual
) 4th edition, 1958.

(7) Sucrose/nitrate agar medium (G): poor (AM), poor, light gray brown (3ge) (SP): None U) Glucose/asparagine agar medium (G): Poor (AM): None SP ): Nasakutsu] Glycerin/asparagine agar medium (G): Nashiku d) Starch/inorganic salt agar @ ground (G): Abundant (AM): Abundant, powdery, gray (3ih) (SP): Yellowish brown ( 4gc) 2) Tyrosine agar medium (G): Poor CAM): Poor, gray (3ha) (SP) Two pale yellow red (4ea) (B) Nutrient agar medium (G): Medium (/M): None SP): None) Yeast/malt agar medium (G): Medium (AM): Poor, gray (3fe) (SP): Pale yellowish brown (4ne) Shio] Oatme μ agar medium (G): Medium (AM), poor, light grayish brown (3ge) (SP): None III) Physiological properties) Growth temperature range: 15-35°C B) Liquefaction of gelatin (glucose pegtone gelatin medium, 24°C, 3 week): Positive (weak) @ Hydrolysis of starch: Positive) Coagulation and peptonization of skim milk: Both negative (3) Reducibility of nitrate: Negative Q] Production of melanin-like pigment: Tyrosine agar medium: Mediastinum Peptone Iron Agar: Assimilation of Negative (IV) Selective Source (Breedham Boratory 1 Agar) L-arabinose Inosyto-μ-D-xylose + L-rhamnose-D-glucose Fuphynose -D-fructose ± D-mannitol
- Sucrose control - (Note): +: Abundant growth, ±: Slight growth, -: No growth Judging from the above morphological characteristics, this strain is Streptomyces (
It is clear that it belongs to the genus Streptomyces.

The above-mentioned Streptomyces sp. strain 23924 was deposited with the Fermentation Research Institute (Engineering FO) on September 20, 1980 under the accession number IFO14205, and this microorganism was deposited with the Agency of Industrial Science and Technology of the Ministry of International Trade and Industry on October 1, 1980. Accession number FEBM P-6739 to the Microbial Technology Institute (FBI)
Both have been deposited as .

In general, the properties of Streptomyces bacteria are easily changeable, and they can be easily mutated by, for example, X-ray irradiation, ultraviolet irradiation, artificial mutagenesis means using radiation irradiation 1 artificial mutagenic agents, etc.

Even among such mutant strains, any strain capable of producing p-23924 can be used in the method of the present invention.

In 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 actinomycetes and can produce P-23924.

In other words, as a secondary element source, for example, glucose, rough)
Su, glycerin, IWII powder, sucrose.

Dextrins, molasses, organic acids (eg acetic acid, tartaric acid, etc.), nitrogen sources such as peptone.

Protein hydrolysates such as casamino acids (manufactured by Difco), NZ amine A (manufactured by Chef Yield), yeast extract, malt extract, soybean meal, corn staple liquor, amino acids (eg, anupawogic acid).

glutamic acid, etc.), and various ammonium salts (eg, ammonium sulfate, ammonium chloride, etc.). Various phosphates as inorganic salts (e.g. monobasic sodium phosphate, dibasic potassium phosphate, etc.).

Metal salts such as magnesium sulfate, sodium chloride, ferrous sulfate, etc.1 heavy metal salts (e.g. manganese sulfate, zinc sulfate, etc.)
Vitamins (eg, vitamin Bl) may be added for the purpose of promoting the growth of bacteria.

pantothenic acid, p-sium, etc.), nucleic acid-related compounds (e.g.
Adenine, uranium μ, etc.) may be added. In addition, the culture method and culture conditions include adding an antifoaming agent such as silicone, polypropylene glyco-1V sulfate (e.g., Actol (manufactured by Takeda Pharmaceutical Company Limited), etc.), soybean oil, etc. to the culture medium. It may be effective in increasing the production of Niyori and P-23924.

Culture conditions such as culture temperature, culture time, and liquid nature of the medium vary depending on the type of microorganism used and the composition of the medium.
In the end, it is only necessary to appropriately select and adjust the amount of P-23924 to maximize production, and in most cases, culturing is carried out under aerobic conditions at about 20 to 40'C for about 24 to 240 hours. During this time, it is preferable to maintain the pH of the medium at around pH 4 to 9.

The physiologically active substance p-2 was extracted from the culture solution thus obtained.
3924 can be easily collected by appropriately combining various methods based on the properties of the substance. That is, for example, extraction with a neutral to acidic organic solvent that is immiscible with water, such as ethyl acetate, butyl acetate, chloroform, butanol, benzene, toluene, diethyl ether/L/, methylene chloride, methyl quintimethyl ketone, etc. , activity, silikage/
L', adsorption chromatography using alumina, etc.
Examples include Ge/L/filtration using a Sephadex column and ion exchange chromatography using an ion exchange resin.

By using an appropriate combination of these methods, the physiologically active substance P-23924 can be isolated from the culture as crystals or crystalline powder. The properties are shown below.

(1) Physical and chemical properties (a) Melting point: A 186-188℃iB 200-20
2°C; C187-190°C; D 205-207°C; E 1γ4-176°C; F 191
-195℃ Can) Elemental analysis value (actual value): (C) Molecular weight (based on mass spectrum) and molecular formula: % Formula %: (e) Solubility: P-23924A, B, D and F are all dimethyl sulfoxide , dimethylformamide,
Pyridine, 5% reconstituted sodium hydrogen acetate, easily soluble in water. Methanol
/l/, dioxane.

Soluble in acetic acid. Water, acetone, chloroform μ.

Slightly soluble or insoluble in n-hexane and petroleum ether. P-2
3924c is easily soluble in dimethyl sulfoxide, dimethyl formamide, pyridine, and 5% sodium hydrogen ash water. methanol.

Soluble in dioxane, acetone and acetic acid. Slightly soluble or insoluble in water, chlorophom, 11-hexane, and petroleum ether. P-
23,924 g is easily soluble in dimethyl sulfoxide, dimethyl μhopamide, pyridine, 5% aqueous sodium hydrogen ashate, and methanol. Soluble in dioxane and acetic acid. water, chlorophore μ
Slightly soluble or insoluble in rubber, n-hexane, and petroleum ether.

(f) Ultraviolet and visible absorption spectrum /L/: 1st to
It is shown in FIG. The wavelengths (nm) and El values showing maximum 1% absorption immediately after being dissolved in each solvent are shown in Table 1 below. In Figures 1 to 6, □ indicates the measurement results in methanol μ, and -111 indicates the measurement results in 0.1 N MCl.
- Measurement results in 90% methanol-μ water,
-- indicates the measurement results in Q and 1N NaOH-90% methanol-μ water, respectively.

(gl infrared absorption spectrum) A/: The infrared absorption spectra μ measured with potassium bromide tablets are shown in Figures t to 12. The main wave numbers that exhibit maximum absorption are as follows.

<l) P-23924A 3300.2930, 1710.1665.1640° + 620.1570.1540, 1500.1460° + 420.1380.1335.1280.1240° 1200.1180.1110.1040. +000°970, 900. 860. 800 See Figure 7 (ii) P-23924B 3300.2950.1?25. +705.1660°1630.1590.1540. +460.1420°13γ0.1330. +300.1270.1210°1190.1+70.1130.1+00.1060°1020, 980. 950. 890. 860°790, 760. 700 See Figure 8 (Ill) P-23924C 3400, 3070, 2940, 1720, 1680° 1635, +600. 1540. 1490. 14
60°+420. 1380. 1350. 1310
．． +250°1220, +180. 1120. +
100. 1040°990, 930. 880. 8
20. 800゜05 See Figure 9 (IV) P-23924D 3400.3300.2960, 1720.1670゜1635.1610.1540.1440.1380゜+340.1300.1260, 1220.1205゜1130.1100.106 0.1000 ．． 900°870, 820. 800. 780. 690 1st
See figure 0 (v) p-23924g 3550.3300.3080.2950.1γ25°1660, T640,16+0.1570.1540°1490, +455.1420. Ta2O, 1325°1300.1240.1210,1180. +120°1080, +040. l0T0. 940. 920°860, b2o, 800. 770. 700 See Figure 11 (Vl) P-23924F 3300.1720.16g0.1660.1630°+600.1530.1490,1460,1420°1380.1335.13+0.1260.1220°1190, 1130. 1100. 1030. +0
00°980, 955. 870. 700 See Figure 12 (bl color reaction: P-23924 is positive for ferric chloride reaction, alcoholic magnesium acetate reaction, and Lydon-Smith reaction.

Ninhydrin reaction was negative.

(1) Characteristics: All p-23924 is acidic and fat-soluble, and shows yellow to yellow-orange or orange-yellow crystals, crystalline powder, or powder.

(j) Nuclear magnetic resonance spectrum/L': 400 MHz,
dimethic μmd & Nubec measured in sulfoxide)/
v is shown in FIGS. 13-18. Characteristic peaks are shown below.

(+)P-23924A δ DMSO-d6ppm: 1.86 (3H, s)
, 2, 09 (3H.

d+J=1.6Hz)+2.71(IH, dd, J-
8,8°13.6Hz), 2.91 (IH2dd, J=
5.0+13.6Hz), 3.66(IH,d,,T
= 12.9Hz).

3-78 (IHld, J=12-9Hz) + 3.96
(3H.

s), 4.47 (IH, d-t, 1ike, J-5,0
,8,3°8.8H2),6.89(IHl(1,J=
1.6Hz).

7.09 (IH, s), 8.18 (IH, d, J-8-
3Hz) +12.40 (IH, s) See Figure 13 (ii) P-23924B II DISO-Tun ppm: 1.86 (3H, s),
1.95 (3H, s), 2. rO(IH, dd,, T-
8, 8, 13, IHz), 2.89 (IH, dd, J
= 4.9, 13.7Hz).

3.65 (IH, d, J=I3.IHz), 3.77
(IH.

d, J=13.1Hz), 3.95 (3H, s), 4.
03 (3H, 8), 4-47 (lH, a tlik
e + J = 4.9 + 8.1, 8.8Hz), 7.1
1 (IH, s), 8. i8(iH,d,,r=8.1
Hz), 12.59 (IH, s) See Figure 14 (III) P-23924G δ DISO-d6ppm: 1.85 (3H, s)
, 2.71 (JR, dd, , T-8, 8, 13, 7Hz
), 2.91 (IH2dd, J=4-9+1'3-7H
z), 3.66 (IH.

d + J = 12.911 z ) + 3.77
(I H+ d, J = 12-9Hz), 3.88
(31(,s), 3.95(3H,s).

4.47 (IH, d like, J=4.9.8.1
,8.8Hz), 6.26(IH,s), 7.15(I
H, a).

8.18 (11i, d, J=8.1Hz), 12.73
(IH.

8) See Figure 15 (IV) P-23924D I DMSO-da ppm: 1-85 (:3H, s
), I, 94(3H,s), 2.73(in,
dd, J=8.8, 13.5Hz), 2.92 (IH,
dd, J-4, 8, 13, 5Hz).

3.64 (IH, d, J-12,8Hz), 3.76
(IHld, J=12.8Hz), 4.00 (3H, s
), 4.48 (IH, dtlikel, v-4, 8+8
-1.8.8Hz) +7.03(IIIs), 8.
17 (1'H, dIJ"8.IH2).

12.79 (IH, s) See Figure 16 (v) p-23924E δ DMSO-6,6ppm: 1.85 (3H, s
), 2.71 (tu, aa, J=8.5, 13.7H
z), 2.90 (IHldd, J=4.9, 13.7
Hz), 3.67 (IH, d, J=13.1Hz), 3
．． 79 (IH, d, , T = 13.1 Hz).

3.98 (3H, s), 4.47 (2H, d, J-2,
2Hz).

4.47 (IH, dtlike, J=4.9.8.1,
8.5Hz), 5.47 (lH, s), 6.82 (IH
, t, J-2, 2Hz), 7.15 (IH; s), 8.
18 (IH, d.

J=8.1Hz), 12.29 (IH, s) See Figure 17 <vl> p -23924F δ DMSO-d6ppm: 1.85 (3H, s)
, 2-70 (lH, dd, J=8.8, 13.7Hz
), 2.90 (IH9dd, J=4.9, 13.7H
z>, 3.67 (IH, d, J=13, 1Hz), 3
．． 80(lu+a+J=IZ, 11(z).

3.97 (3H, s), 4.10 (31, s), 4.3
6 (2H1d, J=3.7Hz) 14.47 (IH
, dt 1ike, J=4.9,8.3.8.8'H
z), 4.93' (IH, br t).

7.16(1,H,s), 8.20(IH,d,,T
=8.3Hz).

12.70 (IH, s) See Figure 18 (k) Rf value of thin layer chromatography: silica μ・
I? The f value is as follows.

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 estimated structural formula of p-23924 is shown below.

Since p-23924 has a free carboxy group, it can form a salt. Methods known per se are used to form the salts. Examples of such salts include psium salts, magnesium salts, barium salts, sodium salts, potassium salts, manganese salts, iron salts, copper salts, and zinc salts.

The biological properties of P-23924 are shown below.

(a) Protocollagen proline hydroxylase inhibitory effect; measurement of inhibitory activity was carried out by the method of Ni, T., Kivirriko et al., and Halme et al. CJournal-o
f Biological Chemistry ■、4
007 (1967) and Biochimica e
t Biophysica Acta 19B +46
(Pro-Pro-Gly)5 using a partially purified enzyme preparation prepared from chicken embryos according to 0 (1967)).
・CMethod by R, E, Rhoa, ds et al. using 4H20 (Protein Research Foundation, Osaka) as a substrate
s inKnzymology X%'ll B, 3
06 (1971)]. Kenpo ni p
The activity of 100 μg (as protein) of Partial M enzyme was 50
The concentrations of P-23924 required for % inhibition were as follows.

P-23924A: 6.7X10 MP-2392
4B: 9.5xl OMP-23924C: 5.7X
10 M P-23924D: 2.7X10 M P-23924E: 2.1xlOM P-23924F: 2.110 M As is clear from the above results, p-23924 has a significant protocollagen prolyl hydroxylase inhibitory effect. have

(b) Collagen biosynthesis inhibitory effect: T, Ishimar
U et al.'s method CBiochemical Pharmac
According to ology 31 +915 N982)),
P-23924 was administered once a day for 6 days to SD-strain rats (
It was administered intraperitoneally to female females (3 weeks old), and the amount of collagen and non-collagen protein in the uterus was compared with the control group. As shown in Table 2 below, p-23924 selectively and significantly inhibited collagen biosynthesis.

(Left below) ※※ Amount of non-collagen protein = total protein amount - amount of collagen ※※※ Estofudiol lv-17β was dissolved in physiological saline containing 5% ethanol.

In addition, 10 rats per group were used in the experiment.

The acute toxicity CI, I) 50 value (rat, intraperitoneal administration) of P-23924 is shown below.

P-23924A: 100-200Mg/#.

P-23924B: 50-100 Da/phantom, P-23
924C: 100-200V/kq, p-239
24D: 400111/# or more, P-23924E:
Approximately 50q/#, P-23924F: Approximately 50q/kq 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 can be used as a preventive/therapeutic agent for organ fibrosis in mammals (rabbits, rats, mice, dogs, cats, humans, etc.), such as pulmonary fibrosis.

It is used for the prevention and treatment of liver cirrhosis, nephrosclerosis, arteriosclerosis 9 scleroderma, myelofibrosis, T44 arthritis, etc.

The dosage of P-23924 depends on the target disease, symptoms, administration target,
Although it varies depending on the administration method, when administered as a prophylactic/therapeutic agent for organ fibrosis, approximately 10 to 100 doses per adult per day.
09 will be administered in 1 to 3 divided doses.

When administering P-23924, it can be administered by itself or mixed with appropriate pharmacologically acceptable carriers, excipients, and diluents, and administered in dosage forms such as powders, granules, tablets, turnip tablets, and injections. It can be administered orally or parenterally.

When manufacturing the above oral preparations, such as tablets, a binder (
(e.g., hydroxypropyl-seμlose, hydroxypropylmethylcellulose, macrogol, etc.), disintegrants (e.g., starch, carboxymethylcellulose calcium, etc.), excipients (e.g., lactose, starch, etc.), lubricants (e.g., Magnesium stearate, riμri, etc.) can be blended as appropriate.

In addition, when manufacturing parenteral preparations, such as injections,
Isotonic agents (e.g., glucose, D-subito'-/I/, D
- mannitol, sodium chloride, etc.).

Preservatives (e.g., benzene-acor/I/, chlorobutano-
μ, methi/L/baraoxybenzoate, propyl baraoxybenzoate, etc.) 1M! A buffer (eg, phosphate buffer, sodium acetate buffer, etc.) can be added as appropriate.

P-23924 can be used as a biochemical reagent, for example, as an inhibitor of collagen biosynthesis in cultured animal cells.
Alternatively, it can be effectively used 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. m23924 (IFO14
205, FERM p-6739) to glucose 2.0
%, glycerin 1.0%, raw soybean flour 0.5%, corn/
Stape liquor 0.5%, polypeptone 0.3%,
Sodium chloride 0.3%, calcium legate 0.5%, p
The mixture was inoculated into five 2e Sakaro flasks containing 400 gt of a liquid medium consisting of H7,0, and cultured with reciprocal shaking at 28° C. for 2 days to obtain a culture solution of about 21 g. This culture solution was transferred to a 200 g tank containing the same medium 1001 as above, and cultured with aeration at 28° C. for 2 days. 60g of the obtained culture solution was mixed with 4% soluble starch, 2% defatted soybean flour, and 0.1% sodium thiosulfate.
%, magnesium sulfate 0.05%, dipotassium phosphate 0
．． The cells were transferred to a 2000e tank containing Kanal liquid medium 12001 containing 0.5% potassium chloride and 0.03% (pH 6.0), and cultured with aeration at 24° C. for 5 days. About 11501 of the obtained culture solution was filtered to remove bacterial cells and solid matter using a filter press pre-coated with Togekobarai) #34 (Toko Perlite Industries Co., Ltd. % Formula % (manufactured by Johns Manville, USA > 34 kQ). The bacterial cells and solid matter were washed with water 1501 and filtered again to obtain a washing liquid 1507?.

Combine this p solution and washing solution, adjust the pH to 3.0 with sulfuric acid,
Countercurrent extraction was carried out using 2 volumes of ethyl acetate in an 8150ff Bodbyl-Niak extractor (Podopi-Niak, USA). The obtained extracted compound 4304 was transferred to 1% reconstituted sodium hydrogen oxide aqueous solution using the same equipment,
I got 5e. The resulting solution was adjusted to pH 3.0 with sulfuric acid, extracted again with 2 volumes of ethyl acetate, washed twice with 2 volumes of water, and then concentrated under reduced pressure. The concentrated solution (400 txl) was transferred to a silica gel 1L/(manufactured by Merli, West Germany) column (4,
The mixture was run through a 6X65 filter and eluted with 1500 ml of ethyl acetate/L followed by 3e of ethyl acetate containing 1% sulfuric acid.

The active fraction (approximately 3 g) was collected, washed twice with 500 d of water, and then concentrated to dryness under reduced pressure, and the resulting solid was dissolved in approximately 300 g J of dimethyl sulfoxide.

After adding 30Id of water to this solution, Prep, PA
Prep LC/ equipped with K-5001018 column
System 500A type preparative liquid chromatograph (manufactured by Waters, USA) was introduced into a methanol-μ-water (1:1) mixture, followed by methanol-water (3:2).
) 5 g of the mixture was finally eluted using Mepnor 1v211, and the eluate was collected in 500 sl portions. Mainly P-23
Fractions containing 924A, C, D and E (7 lacs,
2~a > 3.511 and mainly P-23924
It was divided into two parts containing B6 (furanone, y9-13) 2.5e. When the P-23924B-containing fraction was allowed to stand at room temperature, approximately 2.31 yellow-orange P-23924B crystals were precipitated, which were separated in a furnace, and the mother liquor was concentrated under reduced pressure to a volume of approximately 1 g. After extracting the extract three times with 50 f of anhydrous sodium sulfate and dehydrating it, it was concentrated to about 30 g Z, and when 200 ml of n-hexane was added, about 7.1 g of yellow-orange crystalline P-23924B coarse powder was obtained. Ta. This crude powder was recrystallized from a methanol-μ solution and P-23924
Crystalline 31 of B was obtained. Fractions containing P-23924A, C, D, and E were concentrated to approximately 10 logs, loaded onto a silica gel column (4,0X42cII), and eluted with ethyl acetate/I/-24 containing 1% oxalic acid.

The eluate was collected in 100+wt portions and was treated as raw P-.

Both parts containing 23924A and D (7 lactylon 2
~7) 600 ssl and a fraction containing mainly P-23924C and E (7 lac Vyo 78-14) 700 ssl
Divided into +w/. Mainly P-23924A and D
After washing both sections (600m+?) containing 2! l,t
rl hard L, ip no 1v50s+j? Dissolved in L, water 50%
After diluting by adding m1, it was applied to the same preparative liquid chromatograph apparatus as above and eluted with 5.511 g of a methanol-μ-water (1:1) mixture. The eluate was collected in 500 sl portions, collected in sections 13 to 15, and concentrated under reduced pressure to approximately 7 QOmt.
Extraction was repeated three times by adding ethyl acetate (D). The extracts were combined and dehydrated by adding 30 ff7 of anhydrous sodium sulfate, and then concentrated under reduced pressure.
00g/ was added, yielding 1.7f2>f of yellow-orange coarse powder containing P-23924A and p. This crude powder was dissolved in 100 g of a chloroform-acetic acid (4:1) mixture, passed through a silica gel column (3,5X52cII), and 400 liters of a chloroform-acetic acid (7:3) mixture was dissolved. ) Elution was performed using mixture 11.

Eluate 10gt each preparative L1 mainly p-23924
A-containing sections (7 units of S/Yon 25-29) and sections mainly containing P-23924D (7 units of VW-3)
6 to 136). p-23924A-containing fraction (5
0g/) was concentrated to dryness under reduced pressure and methanol-yv200
200 liters of water was added and chromatography was performed again using the preparative liquid chromatograph described above to collect the p-23924A fraction. This fraction was concentrated under reduced pressure, extracted with ethyl acetate, dehydrated, and concentrated to obtain 2501v of p-23924A in the form of a crude powder. When this crude powder was recrystallized from a methanol-μ solution, about 1001q of yellow-orange P-23924A crystals were obtained.

Section 11 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 repeatedly with 200 g of water three times.

After dehydrating the washed ethyl acetate/L/M with 50f of anhydrous sodium sulfate, ethyl acetate was distilled off under reduced pressure, and the concentrated liquid was concentrated at 30tx.
When 200 tl of n-hexane is added to t, yellow-orange P-2
3924: o Approximately 0.7f of coarse powder was observed.

This crude powder was dissolved in 25 g of a chloroform-acetic acid (4:1) mixture and applied to a silica gel column (3X451).
Elute with chloroform-acetic acid (421) mixture, P-23
Collected 924D classification. 400 gt of this fraction was concentrated under reduced pressure, 100 g of water was added to 80 tsl of the concentrate, and the mixture was repeatedly extracted three times with 2 volumes of ethyl acetate. The extracts were combined, washed with water, and dehydrated. After distilling off the ethyl acetate, 500 q of the resulting crude powder was dissolved in methanol-μm ethyl acetate/' (1:5).
Recrystallization from the mixed solution yielded about 330 da of yellow-orange crystalline powder of P-23924D. Next, mainly P-2392 was observed by silica gel chromatography.
4c, segment containing E (7 raxions 8-14) 7
00g? After washing with water and dehydrating, ethyl acetate/L' was distilled off, and the residue was dissolved in methanol-IV 50 d. After diluting this solution twice with water, it was applied to a preparative liquid chromatography device (same column as above), and methanol-μm water (1:1
) was eluted in the mixed solution. The eluate was collected in 200 sgJ portions,
It was divided into a fraction containing mainly P-23924E (fractions 10-13) 80 (Ig/) and a fraction containing P-23924c (fractions 14-18) IJ.

The P-23924E-containing fraction was concentrated under reduced pressure, extracted three times with 2 volumes of ethyl acetate, dehydrated, and concentrated to about 20 gt.
When 200g/n-hexane is added, yellow-orange P-23
12,011g of coarse powder of 924E was returned. This coarse powder was mixed with 30 g of chloroform-methanol-μ (3:2) mixture/
Dissolved in V-Rikage 7 column (3X45ffi), eluted with chloroform-methanol-μ (3:2),
The fractions containing p-23924F were collected and concentrated to obtain a concentrated solution of 10
g/ethyl acetate/' 10 ml, n-hex 750
When 11 t was added, about 30 da of yellow-orange crystalline P-239241 powder was returned. 1 g of both portions containing P-23924C was concentrated to dryness under reduced pressure, the dried product was dissolved in 15 ml of chloroform, and the silica gel column (3, OX45
aI) and eluted with a chloroform-acetic acid (4:1) mixture, and the fraction containing P-23924C was collected.

When this fraction was concentrated to dryness, a crude powder of P-23924c (1504F) was obtained. This coarse powder was mixed with ethyl acetate/S'
After adding 70 d of water and dissolving it, washing it three times with 20 m+/ of water, adding 2.5 f of anhydrous sodium sulfate to dehydrate it, and concentrating the ethyl acetate solution under reduced pressure to about 10 gt.
- P-23924 becomes yellow-orange when 25ml of hexane is added.
Approximately 85 crystals of c were observed.

Next, in exactly the same manner as above, extract the culture solution with ethyl acetate, transfer with 1% aqueous sodium hydrogen hypooxide, extract with ethyl acetate on the acidic side, and wash with water.

The concentrated liquid (200+wl) obtained through operations such as concentration was passed through a silica gel (manufactured by Merck, West Germany) column (4.6x
65m) K flow, acetic acid x CH/l12B, followed by 1%
It was eluted with ethyl acetate/I/71 containing oxalic acid. Mainly P-
Section 21 containing 23924F was collected, washed twice with 300 grams of water, concentrated to dryness under reduced pressure, and the resulting solid was dissolved in 80 rxl of a chloroform-acetic acid (9:1) mixture. Pour this solution into a gel column (3,4X 54
z) K sink, chloroform:acetic acid (9:1) mixture 1
．． 2e, same (8:2) mixture 1.21, same (7:3)
Mixture 1.2m" elution area, mainly P-239241i'
The fractions containing (approximately 1.5 It) were collected and concentrated to dryness under reduced pressure, and the resulting residue was diluted with methanol-water' (1:
1') Prep P after dissolving in 400ml of mixed solution.
AK-500/c Yu. Prep LC with column
/ System 500 A-type preparative liquid chromatography device, and 5 g of methanol-water (3:2) mixture was added.
It was eluted using When the fractions containing mainly p-23924F are collected and concentrated to dryness under reduced pressure, P-23924
Coarse powder of F1. '3 f was obtained. Add this to methano/
L/: When recrystallized from a water (8:2) mixture, a yellow-orange P-
Approximately 500 Da of crystals of 23924F were obtained.

Example 2 60 g of the same seed culture as in Example 1 was mixed with 4% soluble starch,
Dextrin 1%, defatted soy flour 2%, sodium thiosulfate 0.1%, ferrous sulfate/7 crystal water 0.05%, dipotassium phosphate 0.05%, potassium chloride 0.03% pH 6
, 0 to a 200 ON capacity tank containing 1200 # of liquid medium, and cultured with aeration and stirring at 24° C. for 6 days. Approximately 1,100 # of the resulting culture solution was filtered, extracted with ethyl acetate, dissolved in sodium hydrogen sulfate solution, re-extracted with ethyl acetate, and then concentrated to obtain a concentrated solution 7 in the same manner as in Example 1.
00IIl was obtained. Transfer this concentrated solution to a silica gel column (
Pour into ethyl acetate Iv3#, followed by ethyl acetate containing 1% oxalic acid. Section 2.64 eluted at
and mainly contains P-23924c and El Category 4.24? ,! :,EE,! : and p-23924F'
Category 3 containing Q. It was divided into Og and Og. P-23924A
, B and D-containing fractions were washed with water and dehydrated, and the residue obtained by distilling off ethyl acetate was dissolved in 200st methanol-μ. Add 200 d of dimethys μ oxide and 200 ml of water to this solution, and prepare PrepPAK-500/C,B.
Prap LC/System 5 with column
It was introduced into a liquid chromatography device made by A for separation, and eluted with a methanol-μ-water (1:1) mixture, mainly containing P-2.
Segment 2.5E containing 3924A and D and Segment 3.1g containing primarily P-23924B were collected.

The fractions containing P-23924A and D were rechromatographed using the liquid chromatograph described above, and then subjected to concentration, ethyl acetate/ethyl acetate extraction, dehydration, and concentration operations to obtain 50 g of a concentrated solution. This is a silicage rho column (4,5X70tfi
＞, dichloromethane-acetic acid (9:1) mixture 1.5
#, same (8:2) mixed solution 5. Oe, (7:3) mixed i 2
g +7)ll@lc elution L, mainly p-23924
Section 11 containing A and section 1.21 containing P-23924D were collected. P-23924A containing category 30
After washing three times with 0st water, the dichloromethane layer was separated and concentrated to dryness, and the dried product was dissolved in methanol-Iv200al? 200a+t of water was added thereto, and the mixture was applied to a preparative liquid chromatograph under the same conditions as above. P-2392
4A-containing fractions were collected and concentrated to remove methanol and
After repeating extraction three times with 2 volumes of ethyl acetate, the extracts were combined, dehydrated, and concentrated to dryness. When the dried product was dissolved in methanol-μ and left to stand, 300q of crystals of P-23924A appeared. ffKP-23924D containing category (1,24
? ) was concentrated under reduced pressure, and 30 g of the concentrated solution was passed through a silicage μ column (3.5 x 50 cM), and 1 e each of dichloromethane-acetic acid (9:1) mixture, 1 e of dichloromethane-acetic acid (9:1) mixture, 1 e of dichloromethane-acetic acid (8:2) mixture, and 1 e of (T:3) mixture were added. It was eluted sequentially. 1.4 g of the fraction containing mainly P-23924D was collected and concentrated to dryness under reduced pressure, and 200 g of methano-A' was added and dissolved in the dried product. Add 200 g of water to this solution and perform preparative liquid chromatography under the same conditions as above.
．． Collected 71.

Concentrate 1 obtained by distilling off methanol from this fraction under reduced pressure
．． After adding 5#KZ volume of ethyl acetate and extracting it three times, dehydrating it and leaving it to concentrate to about 25ml, P-2392
Approximately 11 4D crystals were changed. Fractions (3, 11) containing P-23924B become P-23924 when left at room temperature.
Crystals of B were precipitated.

This was collected and re-solidified from methano-μ, yielding about 31 crystals of P-23924B. Next, mainly P-239
Both fractions (4, 24) containing 24C and E were washed three times with water from step 11, dehydrated with 30f of anhydrous sodium sulfate, and the residue obtained by distilling off ethyl acetate μ was purified with methanol. It was dissolved in 7L/290g/. Add 200 d of water to this and repeat the liquid chroma grab under the same conditions as above.
Category 1.84 and P-239 containing P-23924F
Segment 2.41 containing 240 was collected. P-2392
The 4F-containing fraction 1.84 was concentrated under reduced pressure to about 50 gt, suspended in methanol-μ, and packed in a Sephadex Lu-20 column (3,5 x 52 W) (manufactured by Sia, Sweden). eluted with methanol μ,
Segment 200d containing only P-23924 was collected. Both parts were concentrated under reduced pressure, and ethyl acetate/L was added to 20d of the concentrated solution.
' 20 *t and 200 g/n-hexane were added to obtain yellow-orange P-239241 as a crystalline powder 70011I. Next, Section 2.44 containing mainly P-239240 was concentrated under reduced pressure, 2 volumes of ethyl acetate was added to the concentrated liquid 1.24 and extracted three times, and the ethyl acetate layer was extracted using anhydrous sodium sulfate 20F. After dehydration and ethyl acetate was distilled off, the resulting residue was dissolved in methanol-μ and allowed to stand to precipitate crude crystals of P-23924c. When this is recrystallized from methanol-μ, p-239240 is crystallized! ,
5 f was obtained.

Next, the section mainly containing P-23924F (
3, Oβ) was washed with water and dehydrated, and the residue obtained by distilling off the ethyl acetate was mixed with a mixture of chloroform and acetic acid (9:1) 100-
dissolved in. This solution was applied to a silica gel column (3,4X5
4ff) g, chloroform-acetic acid (9:1) mixture 1
g, same (8:2) mixture 171! , (7:3') mixture 16[K elution L, fraction 1.81 containing mainly P-23924F was collected and concentrated to dryness. Dissolve the dry matter in Megno μm water (1:1) 400xt and prep
Prep equipped with PAK-500/”18 column
The mixture was introduced into a LC/System 500 A-type preparative liquid chromatograph and eluted with a methanol-p-water (3:2) mixture to extract the fraction (2) containing mainly P-23924F.
, 41) t-collected.

When this was concentrated under reduced pressure, crude crystals of p-23924F were precipitated. This was recrystallized from methanol-1v:water (3:2) to obtain 2.51 crystals of P-239241'.

Example 3 Tablet (1) P-23924A IoO da (2) Lactose 47 # (3) Corn starch 40 da (4) Hydroxybucypyr 7 μlose-112 MI (
5) Magnesium stearate 1 paddle The above ingredients are compressed into tablets according to a conventional method (wet method).

Example 4 Tablet (1) P-23924B l 00Q (2) Lactose 47q (3) Cornstarch 4 (lq) (4) Hydroxoproxycetate, lu'ulose-L 12
Each tablet costs 200 Da. The above ingredients are compressed into tablets according to a conventional method (wet method).

Example 5 Capsule (1) P-23924C100 (2) Lactose 135q (3) Cornstarch 60q (4) Magnesium stearate 5 da Above ingredients (1)
), (2), (3) and (4) are mixed and then granulated according to a conventional method. To this, ingredient (4)
Add the remainder of and add No. 1 gelatin kaguse according to the usual method.
(Japanese Pharmacopoeia, 10th edition) to form capsules.

Example 6 Capsule (1) P-23924D 300q (2) Lactose 135q (3) Cornstarch 60q (4) Magnesium stearate 5 Ingredients of Dahi (
1), (2), (3) and (4) are mixed together and then granulated according to a conventional method. Add to this the ingredients (4
) and add the rest of OO gelatin capsules according to the usual method.
I/ (10th edition Japanese Pharmacopoeia) to form a capsule.

Example 7 Tablet (1) P-23924E 10 (1 (2) Lactose 47q (3) Corn starch 4 (11) (4) Hydroxypropyl cemu-lose-L 12"!
(5) Magnesium stearate 1q The above ingredients are compressed into tablets according to a conventional method (wet method).

Example 8 Tablet (1)]? -239241i” 100~(2) Lactose
47 da (3) Cornstarch 40 li (4) Hydroxyderobicella p-su I, 12m
g The above ingredients are riveted according to a conventional method (method a) to form tablets.

[Brief explanation of the drawing]

Figures 1 to 6 show the physiologically active substance P-23924A,
Figures 7 to 12 show the ultraviolet and visible absorption spectra of B, c, D, E, and y of the physiologically active substance P-239.
24A, B, C, D. The infrared absorption spectra of E and F are shown in Figures 13 to 1.
Figure 8 shows physiologically active substances P-23924A and B. C, D, E, F nuclear magnetic resonance subek)/1/'i each hemme bright binding Rizu light belt Ri Tomoe Koi reed quality the Σ Tomoe ml gutter Ri Tomoe 0t? Stomach Expo + Z Type Wari 2 (Ten) Shrimp @-? Procedural amendment (voluntary) 1. Indication of the case 1982 Patent Application No. 142347 2 Title of the invention Physiologically active substance P-23924, its manufacturing method and formulation 3
, Relationship with the case of the person making the amendment Patent applicant address 2-27 Doshomachi, Higashi-ku, Osaka Name (29
3) Takeda Pharmaceutical Co., Ltd. Representative: Iku Kurabayashi, Department 4, Agent address: 2-17-85 Jusohonmachi, Yodoyo-ku, Osaka, Tokyo Contact information (Patent Laws and Regulations Division) 11, 27g-2219-221
86. Contents of the amendment (1) "te," in line 10 of page 15 of the specification is corrected to "deposited." (2) On page 15, lines 12 and 13 of the same book, ``respectively deposited'' was replaced with ``respectively deposited,'' and the deposit was changed to a deposit based on the Budavenut Treaty, with accession number FERM BP-
It is kept at the same research institute (1'RI) as No. 338. ” is corrected. (3) “P-6739” on page 38, line 2 of the same book [BP
Corrected to -3384. Notification of change in accession number January/Date/Indication of incident 1982 Patent application No. 142347 λ Name of invention Physiologically active substance P-23924, its manufacturing method and formulation 3
Relationship with the person who filed the procedure Patent applicant address 2-27 Doshomachi, Higashi-ku, Osaka Name (293)
) Takeda Pharmaceutical Co., Ltd. Representative Iku Shibu Kurabayashi Agent Address 2-17-85, Jusanhonmachi, Yodoyo-ku, Osaka City] Name of former depository institution: Agency of Industrial Science and Technology Institute of Microbial Technology 6 Formerly entrusted Number: FERM P-6739 Z Name of new depositary institution: Institute of Microbial Technology, Agency of Industrial Science and Technology and new deposit number: FL! RM EP-338 ta List of attached documents (1) One or more documents certifying the new accession number

Claims (1)

[Claims] [1] Physiologically active substance P-23 having the following physical and chemical properties
924A, B, C, D, E or F: (1) Physiologically active substance P-23924A (a) Melting point: 186-188°C i'b) Molecular formula: Cl8H, N07SO85, methanol/L/) (d) Ultraviolet and visible absorption spectrum, corrugated plate showing maximum absorption (nm): 220±2.263±2 (shoulder), 270±2°420±
10 (in methanol); 221±2. 264±2 (shoulder), 270±2.420±10 (0.1M HCl-9
0% methanol in water) i216±2.238±2°27
7±2,540±10 (0, IN NaOH-90% methanol in water) (e) Infrared absorption spectrum μ, potassium bromide tablet, principal wave number showing maximum absorption (m'): 3300.2930.1710.1665 ．． +640°1620.1570.1540,1500.1460°1420.1380,1335.1280.1240°1200, 11B0. 1110. 1040. 1
000°970, 900. 860. 800 (2) Physiologically active substance P-23924B (a) Melting point: 200-202℃ (b) Molecular formula 200, ■3, N08S O,51, methano-/I/) (ω ultraviolet and visible absorption spectra μ, wavelength showing maximum absorption (nm): 220±2,262±2 (shoulder), 268±2°308±
2.420±10 (in methanol); 220±2. 2
61±2 (shoulder), 268 soil 2° 308 soil 2. 420±
10 (0,1N MCl-90% methanol-μ in water)i
216 soil 2.237±2°284±2.540±10(
(e) Infrared absorption spectrum, potassium bromide tablet, wave number showing maximum absorption (CIII): 3300.2950.1γ25.1705.1660°+630.1590,1540. 1460,1420°1370.1330.1300.1270.1210°1190.1170.1130.1100,1060°+020. 980. 950. 890. 860°790, 760. 700 (3) Physiologically active substance P-239240 (a) Melting point = 187-190°C (b) Molecular formula: C. H, N08S (c) Specific rotation: [α123-93° ±10' (c=
D 0.5+, methanol/L/) (d) Ultraviolet and visible absorption spectra, wavelength showing maximum absorption (nm): 221±2.260±2 (shoulder), 265±2°306±
2. 420±10 (in methanol)■221±2,2
60±2 (shoulder), 266±2゜306-t: 2.420
±10 (0,1NHCI-90% methanol-p water) +2
16±2,236±2°286±2. 540±10(
(e) Infrared absorption spectrum, potassium bromide tablet, principal wavenumber showing maximum absorption (ml): 3400.3070, 2940, 1720.1680° 1635.1600.1540 ,1490.1460°1420.1380.1350. +310.1250°+220.1180.1+20.1100.1040°990, 930. 880. 820. 800°05 (4) Physiologically active substance P-23924D (ω melting point: 205 to 207°C (b) Molecular formula: Cl8H engineering. N08S O, 51, methano-/L/) (a) Ultraviolet and visible absorption spectra, Wavelength showing maximum absorption (nm): 220±2.271±2,306±2.420±10(
Methanol-μ medium>; 220±2.271±2°306
±2.420±10 (0,IN acl-90% methanol in water), 216±2,234±2°299±2.
540±20 (0,1N NaOH-9Q% in methanol) (a) Optical external absorption Nubec)/L', potassium bromide tablet, principal wave number showing maximum absorption (cat-'): 3400,
3300. 2960. +720. 1670°1
635, 1610. 1540. 1440. 13
80°+340. 1300. 1260. 1220
．． 1205°+130. 1100. 1060.
1000. 900°870, 820. 800. 7
80. 690(5) Physiologically active substance P-23924F (a) Melting point = 174-176°C (b) Molecular formula: C08■0. N08S (c) Specific optical rotation: [α)'3-86° ±10° (c
-0,51 Tutanoh/L/) (d) Ultraviolet and visible absorption spectra, wavelength showing maximum absorption (nm): 221±2.263±2 (shoulder) + 271±2°420
±10 (in methanol) +221±2°264±2 (shoulder), 271±2.420±10 (0,1uHCI -9
0% methanol in water) s 216±2°238±2.
287±2. 540±10(0,1NNaOH-90
% methanol-μ water) (e) Optical external absorption spectrum, potassium bromide tablet, principal wave number showing maximum absorption (1): 3550.3300.30B0,2950. +725°1660.1640,1610.1570,1540°1490.1455,1420.13B0. +325°1300.1240.1210.1+80.1120°1080.1040.1010. 940. 920°860, 820. 800. 770. 700 (6
] Physiologically active substance P-23924F (a) Melting point = 191-195°C (b) Molecular formula: C19■21 NO98 (c) Specific optical rotation: [α] -91° ± 10° (c = 0.51, methano -/'V) (dJ ultraviolet and visible absorption spectrum, wavelength showing maximum absorption (am): 222±2.262±2 (shoulder), 268±2°308±
2.420±15 (in methano-p); 221±2. 2
62±2 (shoulder>, 267±2゜307 soil 2. 415±
15 (0,1N HCI-90% methanol-1V in water)
; 222±2 (shoulder), 236±2°270±2. 5
42±20 (0, IN NaOH-90% methanol in water) (e) Infrared absorption spectrum ~, potassium bromide tablet, wave number showing maximum absorption (il) H 3300, +720.1680.1660.1630° +600.1530 .1490.1460.1420°1380.1335.1310.1260.1220°1!90,1130.1+00.1030.1000°980, 955. 870. 700 [2] Physiologically active substance belonging to the genus Streztomycenu P-23924A, B
, C, D, E and/or P are cultured in a medium, and the physiologically active substance p-2 is added to the culture.
3924A,B. Physiologically active substance P-23924 characterized by producing and accumulating C, D, E and /i or F and collecting them
Method for producing A, B, C, D, E and/or F. [3] Physiologically active substances P-23924A, B, and C. A fibrosis inhibitor containing D, E and/or F.