JPS5974992A - Physiologically active substance p-23924, its preparation and pharmaceutical - Google Patents

Abstract

PURPOSE:To prepare a novel physiologically active substance P-23924A, B, C, D and/or E capable of inhibiting specifically the biosynthesis of collagen, by cultivating a microorganism of the genus Streptomyces. CONSTITUTION:A microorganism, e.g. Streptomyces sp. No.23924 strain (FERM BP-338), belonging to the genus Streptomyces, and having the ability to produce a physiologically active substance P-23924A, B, C, D and/or E is inoculated into a nutrient culture medium and cultivated at about 20-40 deg.C temperature and about 4-9pH for 24-240hr under aerobic conditions to collect the aimed physiologically active substance P-23924A, B, C, D and/or E from the resultant culture fluid.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides novel physiologically active substances P-23924A and B that have an inhibitory effect on collagen prolyl hydroxylase and an inhibitory effect on collagen biosynthesis.

C, D, E, regarding its Na method and formulation.

Collagen proline hydroxylase is an enzyme that specifically hydroxylates proline in protocollagen synthesized in ribosomes in animal cells, and is one of the important factors that determine the rate of collagen biosynthesis. Conventionally, tree (i'I
Substances that inhibit primary active gold 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''+, ZII). However, 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.

If a substance is found that specifically damages collagen biosynthesis without inhibiting non-collagen protein biosynthesis, that substance may be used to treat arteriosclerosis, liver cirrhosis, scleroderma, keloids, rheumatoid arthritis, For the prevention and treatment of diseases including organ fibrosis that involves excessive accumulation of collagen such as pulmonary fibrosis.
can be used.

The present inventors searched for a substance that inhibits protocollagen prolyl hydroxylase activity in microorganisms, and as a result of intensive searches, discovered a novel physiologically active substance that specifically inhibits collagen biosynthesis. P-23924A, B, C
.

B, C, D or E, (2) Nutrebutomyces (Str
eptomyce8) Physiologically active substance belonging to Jg P-2
r producing 3924A, B, C, D and/or E
A microorganism having jH activity was cultured in a medium, and physiologically active substances P-23924A, B, and C were present in the culture.

Physiologically active substances P-23924A and B, which are characterized by producing and accumulating Ei and collecting the same.
, C, D and/or E, and (3) a fibrosis inhibitor containing physiologically active substances P-23924A', B, C, D and/or E. Active substances P-23924A, B, C, D and/or
Or E is sometimes collectively referred to as ``Physiologically Active Substance P-23924j'' or simply rP-23924J.In addition, the physiologically active substance P-2351-4A is
924Aj and the physiologically active substance P-23924B as rP-
23924Bj and the physiologically active substance P-23924C
p-23924CJ and physiologically active substance NP-23924D
rP-23924DJ and physiologically active substance P-2392
4E is sometimes referred to as r p-23924bJ, respectively.

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 rp-23924-producing bacteria). A specific example of which may be used is Nutrebutomyces sp. 23924 strain (hereinafter sometimes abbreviated as "Nagi 23924 strain"), which was isolated from the soil of Ishigaki Island, Okinawa Prefecture.
is mentioned.

Regarding strain 1423924, the International Journal of Systematics ``Bacteriology'' (International, Tournq)
, 1 of Systematic Bacteri
), Volume 6, Issue 3.

The properties investigated according to the method described on pages 313 to 340 (1966) are as follows. Note that the findings on the ξ medium were clearly observed after culturing at 28'C for 14 days unless otherwise stated.

(I) Morphological characteristics Substrate hyphae, spiral, sometimes incompletely 1.) Short spore chain-forming hyphae extending in a prone, open spiral, or hook shape in a simple branched manner; Whorled branches are not allowed. 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.
X 0.7-1.2μ, its surface is smooth.

(11) 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 description of corners, the standard color tone symbols shown in parentheses are those of Container Corporation of America (Container Corporation of America).
tainer Corporation afA
merica ) (7,) Color Harmony Manual/I/(Tbe Ca]-or Ha,
rmony Ma, nua], ) 4th edition, 1958
Depends on the year.

(7) Sucrose/nitrate agar medium (G): Poor CAM): Poor, light grayish brown (3ge) (SP): None i) Glucose/Asparagine agar medium (G): Poor (AM): None SP ): Nashikutsu] Glycerin/asparagine agar medium (G): Nashiku ni) Nutarch/inorganic salt agar medium (G): Toyo'g (AM): Utomi, powder, gray (3ih) (S:P) : Yellowish color (4gc) H tyrosine agar medium (G): poor (AM): poor, gray (3bFL) (SP), : light yellowish red (4e Fl, ) nutrient agar medium (G): moderate (AM): None SP): None) Yeast/Malt Agar (G): Medium (AM): Poor, gray (3fe) (SP): Pale yellowish brown (4ne) Shio) Metomi - (G): Moderate (AM): Poor, pale gray (3t<e) (SP): None (m) Physiological properties (a) Growth temperature range = 15~ 35°C (a) Liquefaction of gelatin (glucose/vegetone/gelatin medium, 24°C, 3 cells): Positive (weak) (2) Hydrolysis of starch (2 positive) Coagulation/peptonization of skim milk: Both Negative C nitrate reducibility: Negative (SoJ) Production of melanin-like pigment: Dilozone base medium: False positive Peptone Yeast iron occlusion medium: Negative (IV) Assimilation of carbon sources (Breedham-Boll-Leap Agar%) L-afvinone Inositol -D-xylone L-Rhamnose -D-Glucose Raffinose 11]-Fructose ± D-Mannitol -Sucrose Comparison - (Note>: 1+ -: Abundant growth, ±: Slight growth, - No growth. Judging from the descriptive characteristics, it is clear that this strain belongs to Streptomyces K.

The above-mentioned Streptomyces NB&23924 strain was acquired by the Fermentation Research Institute (IFO) on September 20, 1981.
), and this microorganism was submitted to the Institute of Microbial Technology (FR Engineering), Ministry of International Trade and Industry, Institute of Technology, Ministry of International Trade and Industry, with accession number 5jFERM in July 1982.
Both have been deposited as p-Otsu 737.

In general, Streptomyces bacteria are mutant strains that can easily change their properties and can be easily mutated by, for example, X-ray irradiation, ultraviolet irradiation, artificial mutagenesis means using artificial mutagenic agents, etc. However, all those capable of producing P-23924 can be used in the method of the present invention.

In culturing P-23924 bovine 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.

That is, carbon sources include, for example, gluco-7, lactose, glycerin 1 powder 1 sucrose.

Examples of nitrogen sources include dextrose, molasses, and organic acids (eg, alcoholic acid, tartaric acid, etc.).

Protein hydrolysates such as casamino acids (Difco IH), p+-Z amine A (Chef Yield), fermented fυ Equine, malt extract, soybean meal, corn staple liquor,
Amino acids (e.g. aspartic acid.

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

(ik magnesium acid, sodium chloride, sulfuric acid 1-4
Metal salts such as car metal salts (e.g. manganese sulfate, zinc sulfate, etc.)? : Vitamins (e.g., vitamin Bl + calcium pantothenate, etc.), nucleic acid-related compounds (e.g., adenine, uracil, etc.) may be added for the purpose of promoting bacterial growth. Depending on the method and culture conditions, silicone, polypropylene,
Adding antifoaming agents such as Glyco/V derivatives [e.g., Actco-V (manufactured by Takeda Pharmaceutical Company Limited)] and soybean oil to the culture medium is effective in increasing the total production of P-23924. In some cases.

11 - Cultivation conditions such as cultivation temperature, cultivation time, and liquid properties of the medium will vary depending on the type of organism used, medium composition, etc., but in the end, it will be necessary to maximize the production of P-23924. In most cases, culture is carried out in an aerobic environment at about 20 to 40°C for about 24 to 240 hours, during which time the liquid pH of the medium is about 4 to 4.
9 It is best to keep the culture Y obtained in this way close to 9
Collecting the physiologically active substance P-23924 from C () can be easily carried out by appropriately combining various methods based on the properties of the substance. That is, for example, with neutral to slightly acidic Organic solvents that are immiscible with water, such as ethyl acetate, butyl acetate, chloroform,
Getanol, benzene, toluene, diethyl ether,
Extraction with methylene crophyte, methyl imbutyl ketone, etc., adsorption chromatography using live charcoal, silicage A], alumina, etc., gel using Sephadec column: 1r3j, and ion exchange chromatography using ion exchange resin. Fees, etc. These means k
The physiologically active substance P-23924 can be isolated from the culture as crystals or crystalline powder by combining with Jl and extracting the same amount.

Physiologically active substance P- obtained in Example 2 of Example 1 after '37I<
The properties of 23924 are shown below.

(1) Gon Chemical 1 Go 1: Quality (R,) F, 11 (Point: A 186-+88″c;
B 200-202'C; c 187-190°C;
■〕205-207'CIE 174-176℃(b
) Elemental analysis value 2 (c1 molecule Fi ((by mass spectrum) and fraction 2 r formula: % formula %: (C=) Molten ship 1 raw: P-23924A, B and 1]
All are easily soluble in dimethyl sulfoxide, dimethyl formamide, pyridine, and 5% sodium bicarbonate water. Soluble in methanol, dioxane and acetic acid. Slightly soluble or insoluble in water, acetone, chloroform, n-20 Bequirif 2 petroleum ether.

P-239240 is dimethyl sulfoxide.

Tsume 1A/Formamide, pyridine, 5% sodium hydrogen acetate, easily soluble in water. methanol, dioxane, acetone,
Soluble in acetic acid. Slightly soluble or insoluble in water, chloroform, di-1-hexane, and petroleum ether.

P-23924E is dimethyl sulfoxide.

Easily soluble in dimethylformamide, pyridine, 5% sodium bicarbonate, methanol, and soluble in dioxane and acetic acid.

Dissolvable or insoluble in water, chloroform, 11-hexane, and petroleum ether.

(f) Ultraviolet and visible absorption vectors: 1st to 5th
As shown in the figure. Immediately after dissolving in each dissolved All (°) values are shown in Table 1. In addition, in Figures 1 to 5, 111 indicates the 7J!!I constant results in methanol. -1'
j: Measurement results in 0.1N HCl-90% methanol water, ---111---t'', O, I N NaO
The results of H-90% methanol suspended in water are shown below.+g) Infrared absorption spectrum: Infrared absorption spectra measured using potassium bromide tablets are shown in FIGS. The main wave numbers showing maximum absorption are as follows.

<l) P-23924A 3300.2930,1710. +665.1640°1620.1570.1540.150 (1, +460
゜1420.1380, 1335.1280.1240
゜1200.1180,1110,1 (140,100
0°970, 900. 860. 800 Kiotsu 1 piece M, (ii) P-23924B 3300', 2950, 1725, 1705, 1660
°+630.1590.1540.1460.1420
゜1370.1330,1300,1270.12+0
゜1190.1+70.1130. +100.1060
゜1020, 980. 950. 890. 860
゜790, 760. 700 纂7趺三郎(III) P-23924C 3400, 30 了. 2940. 1720. 1
680°1G35,1600,1540,1490,1
460°1420.1380.1350,1310,1
250°1220.1180.1120. +100
．． 1040°990, 930. 880. 8
20. Boo.

05 & 42-9 Wing, (IV) P-23924D 3400, 3300. 2960. 1720.
+670°1635, 1610. 1540. 14
40. 13B0°+340. 1300. 1260
．． 1220. 1205°N30. 1100. 1
060. 1000. 900°870, 820
．． 80[), 7B0. 690 Ju Z] Habi J Kai, (V) P-23924E 3550, 3300. 30B0. 2950. 1
725°+660. 1640. 1610. +5
70. 1540°1490, 1455. 1420
．． 13B0. 1325°1300, +240.
1210. 1180. 1120°10B0.
+040. 1010. 940. ' 920° (1
60,820,800,770,70011θ1fl Kosan, o-1) Color reaction: P-23924 is positive for ferric chloride reaction and alcoholic magnesium lactate reaction 9 Negative for ninhydrin reaction and Ehrlich reaction.

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

l fj) M Magnetic resonance mepectol = 400 MHz, spectra measured in dimethyl-d6 nulphoxide are shown in Figures 11-15. The characteristic peak is shown below.

(1) P-23924A 1 a DMSO-d6 ppnn: 1.86 (3
H,q) ,2.09(3H,cl,,,T=1.6
Hz), 2.71 (IH, r(d,, T=8.8°13
．． 6Hz), 2.91 (IH, dd, J=5.0,13
．． 6Hz).

3.66 (IH, d, J = 12.9Hz), 3.78 (
1) J, d.

J=12.9Hz), 3.96(3H output, 4.47(
IH, d beat 2' J=5. [l, 8.3, 8.8Hz
), 6.89 (IHjq,, T = L6H7).

7.09 (IH, s), 8.18 (lIi, d, J=8
．． 3Hz).

12.40 (IH, s) Saw //] A Kiho, (ii) P-23924B δT) MSO- (16ppm + 1.86 (3H, s)
), 1.95 (311, s), 2.70 (IH, dd,
J=8.8, 13.71(z).

2.89 (ILdd, J=4.9, 13.7Hz)
, 3.65 (IH, d, J=I3.1Hz), 3.
77 (IH, d, J = (IH, s), 8.18 (IH,
d, J=8. IT (Z), 12.59 (HT, r3) Should/λ diagram + group, (III) P-23924C δ']) MSO-%, ppm: 1.85
< 3H, 8), 2.71(I H, (1(1,
J = 8.8 z I 3.7 N7 ) + 2.
91 (IH.

dd,, J=4.9, 13.7Hz), 3.66 (IH
, d, J = 12.9H7), 3.77 (IH, d, J
= 12.9Hz), a, da people (3H, s), 3.95 (3H, s), 4.47 (IH
, dt-; r=4.9, 8.1.8.8Hz), 6.2
6 (IH, n), 7.15 (+H1 mill), 8.18 (I
H, cl,, T = 8.1 Hz).

12.73 (IH, e) 1 Ju 73 Kokusanban (lv) P-23924D δDMSO-cl, 6 ppm: 1.85 (3H
, rl), 1.94(3H-) + 2-73 (
l H, dd, J-8-8, l 3.5 Hz)
.

2.92. (IH, dd,, J=4.8, 13.5Hz
), 3.64 (IH, (1, J=12.8H2), 3.
76 (IH, d, J=, uu 12.8Hz), 4.00 (3H, q), 4.48 (I
H, dE-J=4.8, 8.1.8.8Hz), 7.0
3(IH,I:I), 8.17<IH,d, J=8.
IHz), +2.79 (+H, θ) i 岑/薖や1winter- (v) p-23924E J 1) MS+J-d6pp+n: l, Q5 (3H,
O), 2.71 (IH, da, J=8.5, 13.
7H1), 2.90 (IH.

dd, J = 4.9 + l 3.7 HZ), 3
．． +37 (I H, d, J = 13.1 Hz),
3.79 (IH, d, 1'=13.1Hz), 3.
98R), 6.82 (IH, t, J=2.2Hz), 7
．． 15 (IH, s).

0.18 (IH, d, J=Q, IHz), 12.29 (
II, s) 薯/ to Kuninaka λ ((kJ 7・, 'j Rf value of II chromatography: Silica gel play 1 (manufactured by Merck & Co., product number')
729) Rf on ↓ and reversed phase HP TLC plate, ■Map-B (manufactured by Merck & Co., product R913725)
The values are as follows.No compound exhibiting the above-mentioned physicochemical properties is known, and P-23924 is considered to be a new substance.

From the above physical and chemical properties, estimation of P-23924 +1
・The hexadecimal system is shown in F below.

Since P-23924 has a free carboxy group, it 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, iron salts, copper salts, zinc salts, and the like.

The biological properties of P-23924 are shown in F below.

(a) Measurement of collagen prolyl hydroxylase inhibitory effect/inhibitory activity was carried out using the method of ■, K., Kivirriko et al. and J., Halme et al. (Journal of
Biology-cal Chemistry
242.4007 (1967) and Biochi
mica et BiophysicFL
Ac I, t 198.

460 (1967)), using a partially purified enzyme preparation prepared from chicken embryos, (Pro-Pro-G], y)
The method of R, E., Rhoads et al.
p inEnzymology XMI B,
The concentration of P-23924 necessary to inhibit 50% of the activity of 100 tlg (as a protein) of the illegally purified partially purified enzyme was as follows.

P-23924A: 6.7XIOM P-23924B: '9.5xlOM P-23924C: 5.7X10 MP-23924
D: 2.7X10 MP-23924E: 2.1XI
OM As is clear from the above results, P-23924 has a significant protocollagen proline hydroxylase inhibitory effect.

(b) Collagen biosynthesis inhibitory effect: T, IphJ, m
Aru et al.'s method CBiochemical Phar
macolap;y 3 l.

915 (1982)) P-23924 was administered once a day to 6F (174'J, SD-strain rats (female, 3
The amount of cofugen and non-cofugen protein rR in the uterus were compared with the control group. The second below
As shown in the table, P-23924 selectively and significantly inhibited collagen biosynthesis [7].

(hereinafter referred to as 2t-shir) 1) Kaisho 59-74992 (9) *Ilu harm level < %) = ,C"!2""'--X
+ 00 (Fl) - (A) ※※ Non-collagen 1 raw protein amount = total protein amount - collagen measure ※※※ Estofudior/''-17β was dissolved in physiological saline containing 5% ethanol.

The experiment was conducted using 10 animals per group.

P-23924 wins, 1st life it<e (L D50 value (
[rat, intracavitary administration]] as shown below: P-23924A: 100-200'y/A:q.

P-23924B: 5O-100q/#q, p-239
24C: too-201Jln9. /kq, P-23
924D: 400wy/kqJa, J=, P-2
3924E: Approximately 50 t/kg As mentioned above, p-23924 has protocofugen prolyl hydroxylase inhibitory action and selective collagen biosynthesis inhibition f'F, so it can be used as a biochemical reagent or for animals. It is useful as a 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.

The dosage of P-23924, which may be used for the prevention and treatment of liver cirrhosis, +i1 sclerosis, arteriosclerosis 2 scleroderma, myelofibrosis, and 1ρL'lE arthritis, depends on the disease, symptoms, and administration target. ,
Although it varies depending on the administration method, when administered as a prophylactic/therapeutic agent for organ fibrosis, it is approximately 10 to +000 mg per adult per day.
0 My is 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 the form of powder, granules, two tablets, capsules, injections, etc. Oral or parenteral (e.g., hydroxy 7" lopylcellulose, hydroxybrovir methylcellulose, macrogol).

M #/rlj <e.g., starch, carboxymethyl cell ■-1-sucalcium, etc.), excipients (e.g., lactose, starch, etc.), lubricants (e.g., magnesium stearate, talc, etc.), etc. They can be blended as appropriate.

Also, when administering parenteral preparations, such as injections, tonicity agents (eg, glucose, D-sorbitol, D-mannide-μ, sodium chloride, etc.) are used.

Buffers (e.g., phosphate buffer, sodium acetate M buffer, etc.) may be added as appropriate. can.

p-23924 can be used as a biochemical reagent, for example as an inhibitor of collagen biosynthesis in cultured animal cells, or as an active agent for protocrine proline hydroxylase. +1 Can be effectively used as a harmful agent.

The present invention will be explained in more detail below using examples.

Percentage of medium indicates nail JK/volume percent.

Example 1 Nutreptomyces sp. L23924 (F0 1
4205. FaRM p-73) to glucose 2.
0%, glycerin 1.0%, raw soy flour 0.5%, corn staple liquor 0.5%, polypeptone 0.3%
, sodium chloride 0.3%, acid strength/I/sium 0.5
%, pT (7.0) in five 2e Sakaro flasks containing 400 ml of liquid medium, and cultured at 28'C for 21'' with reciprocating shaking elbows to obtain a culture solution of approximately 2e. The culture solution 60e was transferred to a 200e tank containing the same medium 100e as above, and cultured with aeration at 28°C for 2 days. 1%, magnesium sulfate 0.05%,
Dipotassium phosphate 0.05%, potassium chloride 0.03%
Liquid medium consisting of (pH 6,0) + 20 containing 200#
Transplanted into a 0-l tank and aerated (W agitation culture was performed at 24°C for 5 days. Approximately 1150 g of the obtained culture solution was mixed with Topco Perphy) '34 (manufactured by Takko Perfit Kogyo Co., Ltd. 'A).
15 kg was added, and the solids were filtered using a filter press containing 34 Ag of Hyflo Nupers (Johns Manville, USA).
Bacterial cells and solid matter to obtain a filtrate of about + 000 e are 1 part water
After washing at 50e, it was filtered again to obtain a washing liquid of 150e. This F
The three solutions and the washing solution were combined, adjusted to pH 3.0 with sulfuric acid, and countercurrently extracted with 2 volumes of ethyl acetate using a 6150 Bodbyl Niack extractor (manufactured by Podvy Niack, USA). The resulting inverted 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. Concentrate (400/ml) was mixed with silica gel (Merck, West Germany) by Kawoto (4,6X
65 cM) to flow L2, ethyl acetate/l 1500 py/.

Subsequently, elution was carried out with 3 g of ethyl acetate containing 1% oxalic acid.

Collect the 1st particulate fraction (approximately 3e), wash twice with 500 ml of water, reduce EE'F and concentrate to dryness, and add the resulting solid to a solution of approximately 300 ml of dimethyl nulphoxide. After adding 30M of water, Prep PAK-
Prep LC/S equipped with 500/C□8 column
System2050 OA type preparative liquid chromatograph (Tars Inc., USA) was introduced into a methanol-water (1:I) mixture, followed by a methanol-water (3:1) mixture.
: 2) ,,i-type tei! 5 e, finally jll/-)
P-23924A, C, D and Ef were eluted using V2i'r and the eluate was collected in 500 yl portions.
: Containing both (Hook John 2 to 8) 3.5e and mainly containing P-23924B [01 minute (Hook John 9 to 13) 2.5 eVc division 7'ttP-
When the 2392'4B-containing fraction is released in Ura for 1 rt, approximately 2
．． 3f yellow-orange)'-23924T (crystals were precipitated, so this was separated, and the mother (α) was concentrated under reduced pressure to about 1e, extracted three times with 2 volumes of ethyl acetate. After adding 50 g of anhydrous sodium sulfate to the liquid and dehydrating it, approximately 3
After concentrating to 0M/1 and adding 7,200 yi of 11-hex, approximately 31% of IN crystals of seedling P-23924B were obtained. P-
23924A, C, II) and E containing approximately 1
The column was washed with an OA column up to 0,100 t, VC flowed through a silica gel column (4.0 x 42 cnr), and eluted with ethyl acetate TV2E containing 1% crushed acid. The eluate was divided into 100 stomachs each, and 600 ml was divided into both portions (Fukjohn 2 to 7) containing mainly P-23924A and D, and 600 ml containing mainly P-23924A and D.
-23924 It was divided into two parts containing C and E (23924) and 700*/ (23924) in sections 8-14. Mainly P-23
Both fractions (600*/) containing 924 people and D were washed with water, concentrated to dryness, dissolved in methanol/1150 crocodile lK, diluted by adding 50M/water, and then mixed with the same preparative liquid as above. Methanol-water (1:1) was applied to a chromatographic device.
) It was eluted with mixture 5 and 54. The eluate was fractionated into 500 mL portions, and 1.51 fractions of Hook John 13 to 15 were collected, concentrated under reduced pressure to approximately 7 ml, and then 200 ml of eluate was collected.
ml of ethyl acetate/l/f: was added and extracted three times.

The combined extracts were dehydrated by adding 309f of anhydrous sodium sulfate, and then 200ml of n-hexane was added to about 30al of the concentrated liquid under reduced pressure to obtain a yellow-orange color containing P-2,3924A and D. 1°7f of powder was obtained. Dissolve the crude powder in 100 ml of chloroform-acetic acid (4:I) mixture] and place on a silica gel column (3.5X52rJI).
Pour into I), add 11 colors of chloroform-10F acid (4:1), 400 we of chloroform-acetic acid (7:3) mixture, 14
11) Collect the eluate into Kl portions,
The fractions containing mainly P-23924AI (fractions 25 to 29) and mainly p-23924T)'f
: Divided into containing fractions (fractions 36 to 136). A portion (513 ml) containing P-23924A was concentrated to dryness under reduced pressure and dissolved in 1v200 ml of methanol, then 200 tons of water was added and chromatographed again using the preparative liquid chromatograph described above. Do P-
Collected 23924A section-1 After concentrating this section under reduced pressure,
Extract with ethyl acetate, dehydrate, and concentrate to obtain P-
When the coarse powder of 23924A was recrystallized from a methanol solution, a yellow-orange P-23 was obtained.
924A crystal was changed to about 1100 rI.

Next, Section 1a containing p-239-24D was concentrated to dryness under reduced pressure, and the dried product was dissolved in about 500 MIK of ethyl fluoride.
Dissolved and washed repeatedly three times with 200 ml of water.

After dehydrating the washed ethyl acetate layer with 5Of anhydrous sodium sulfate, ethyl acetate/L' was distilled off under reduced pressure F to obtain approximately 30*I sap.
! When 20 [lKl of n-hexane is added to the mixture, a yellow-orange P-
23924I) Coarse powder 0. I was satisfied. −
This crude powder was mixed with chloroform-acetic acid (4:l) mixture 25*
I! Ic melt angle γ, silica gel ° column (3X45α
) and eluted with a chloroform-acetic acid (4:I) mixture to collect P-23924D molecules. This category 400
ml was concentrated under reduced pressure, and 7-condensate was added to 80 liters of water to 1
After adding Oml, the mixture was extracted three times with 2 volumes of ethyl acetate. After combining all the extracts, washing with water, and dehydrating, ethyl acetate was distilled off, and the resulting crude powder of 500'f was recrystallized from methanol-ethyl acetate/L/(1:5)ff\X liquid to give a yellow color. Orange p-23924D crystalline powder about 330M
g was changed. Next, 700 liters of the fraction containing mainly P-23924C and E (fractions 8 to +4) obtained by silica gel chromatography were washed with water.
After dehydration, ethyl acetate was distilled off, and the residue was purified with methanol (v5).
Dissolved in Qme. After diluting this solution twice with water, it was applied to a preparative liquid chromatography device (the same column as above) and eluted with a methanol-water (1:I) mixture.

The eluate was collected in 200 ml portions, mainly containing P-239.
Section containing 24F (fractions 10 to 13)
It was divided into +300 shoulder t and 1# fractions (fractions 14 to 18) containing P-23924C.

The aliquot containing P-23924E was concentrated under reduced pressure, extracted three times with 2 volumes of conityl acetate, dehydrated, concentrated at about 20 volumes/L, and added with 200 volumes of II-hexane to give a yellow-orange P.
A crude powder of 120"I of -2392.4E was obtained. This crude powder was mixed with chloroform-methanol (3:2).
Dissolve in 30 ml of the mixed solution and apply to a silica gel column (3-4
5 t'm) and chloroform-methanol/l/
(3:2) Ifr) J, l L, P-23924F
Concentrate the containing fraction in step 4) and add ethyl acetate joN/, n-hexane 50% to concentrated solution 1 (lr/) to obtain about 30q of yellow-orange crystalline powder of P-23924E.
Fraction 14 containing -23924C was evaporated to dryness under reduced pressure, and the dried product was dissolved in 15 liters of chloroform.
P-2
Category Y containing 3924c6 was collected.

This fraction was concentrated to dryness to obtain 150~ of P-23924G as a coarse powder. Coarse powder with ethyl acetate/'70
ml to dissolve, wash repeatedly with 20wt water 3 times, add anhydrous sodium sulfate 2.5F to dehydrate,
The ethyl acetate/L' solution was concentrated under reduced pressure to about 10-, and n-
P-23924C becomes yellow-orange when 25% of hexane is added.
Approximately 85 crystals were obtained.

Example 2 The same seed culture solution 60e as in Example 1 was mixed with 4% soluble powder,
Dextrin 1%, defatted soy flour 2%, sodium thiosulfate Ao, 1%, ferrous sulfate/T crystal water 0.05%, dipotassium phosphate 0.05%, UK potassium chloride 0.03
2 containing a liquid medium 1200e consisting of 610 pH 6.0
The cells were transplanted into a 000e capacity tank and cultured with aeration at 7.24°C for 6 days. Approximately 11,006 of the obtained culture solution was subjected to filtration, ethyl acetate extraction, and sodium bicarbonate solution in the same manner as in Example 1. - After transferring the solution to night and re-extracting it with ethyl acetate, 71! +! It was contracted to obtain ) 700 #I/.

This gamma sap was poured into a silica gel column (5.4x (i5c
ys) and 1.1 tonic acid Edge/I/31, followed by 1
% oxalic acid in ethyl acetate/I/3 (l), mainly P
Category 2.64 containing -23924A, B and D
and mainly P-23924C and Ef, containing sulfur [
Divided into lK and l categories. P-23924A, B and D
Both components were washed with water and dehydrated, and the ethyl acetate/I/ was distilled off, and the resulting residue was dissolved in 200 methanol. Add dimethyl fV7 to this solution. Add 1L'Hoxide 200* (and 200% water, PrepPAK-500/C
Pre7yLC/eyestem attached to I8 column 1
500A preparative liquid chromatograph, and eluted with a methanol-water (1:I) mixture, mainly P-2.
Section 2.56 and main and I containing 3924A and D;
-Category 3.1 containing cp-23924Bk? Collection f4. , P-23924A, T, and D were rechromatographed using a liquid chromatography apparatus, followed by 11 operations including concentration, ethyl acetate extraction, dehydration, and drying to obtain a concentrated solution with a resistance of 50. Transfer this to a silica gel column (4
, 5X 7 Q an ) and dichloromethane-acetic acid (9.1) UR+,! M, same (8:2) mixture 5. Oe
, (7:3) mixture elutes in the order of P-2e, mainly P-2.
Section i4H containing 3924A: P-23924r+
We collected the segment 1.24 containing -j + f÷. P-23
After washing the 924A-containing section three times with 300 g of water, separate the dichloromethane layer and concentrate to dryness, dissolve the dried product in 200 methanol, add 200 *t of water,
It was applied to a preparative liquid chromatograph under the same conditions as above. The P-23924A-containing fractions were collected, concentrated to remove methanol, extracted three times with 2 volumes of ethyl acetate, and the extracts were combined, dehydrated, and concentrated to dryness. When the dry solid is dissolved in methanol and left to stand, p-23924A
300q of crystals were obtained. Next VCp-23924B
Concentrate the content category < 1.2e) under reduced pressure and make 30 liters of concentrated liquid.
was applied to a silica gel column (3,5×50cyN),
Dichloromethane-acetic acid (9:I) mixture 1e, dichloromethane-acetic acid (8:2)
) mixture and (7:3) mixture were sequentially eluted 14 times each. Section 1.4e containing mainly P-23924D was collected and concentrated to dryness under reduced pressure, and the dried product was mixed with 1 L/200 m of methanol.
1 was added and dissolved. 200 wl of water was added to this solution, and preparative facepiece chromatography was performed under the same conditions as described in O1J, and 2.74 pieces of P-23924B were collected.

This quarter castle! T: After adding 1.5eicZ volume of ethyl acetate to the concentrated solution obtained by distilling off methanol/L/ to the water and extracting it three times, it was dehydrated and concentrated to about 25ml.
Approximately 11 -23924D crystals were recovered. P-23924
Fractions (3, 14) containing 13 were exposed to Nt at room temperature, and crystals of P-23924I3 were precipitated.

This was collected and re-condensed from methanol, yielding about 3 g of crystals of P-23924B. Next, the above-mentioned mainly P-2,
3924c and Ef: fraction (4,2e) containing 1
After washing 3 times with water from e, anhydrous sodium sulfate 30f
The residue obtained by distilling off ethyl acetate/L' was dissolved in methanol/L/200 ml. Add 200 w/e of water to this and repeat the fi1 body chromatography under the same conditions as above, containing P-23924E-containing section 1,84 and P-23924C.
? : Collection meta. P-23924B4 1.84 is approximately 5
Concentrate under reduced pressure to 0W/p1v in methanol in advance.
H-20 (Faμ)
Macia tLM, Nueden) column (3,5X52ff
i), eluted with methanol, and collected 200 vtl of fraction containing only P-23924F. This fraction was concentrated under reduced pressure, and when 200 ml of ethyl acetate/v20 gastric acid and n-hexane were added to the concentrated solution 20F/20F/20F/20, a yellow-orange P-23 was obtained.
Approximately 7001 Hg of 924F crystalline powder was obtained. Next, segment 2.46' containing mainly P-239240
Concentrate fL under reduced pressure and add 1.2 NK% volume of ethyl acetate.
After repeated extraction three times with the addition of 50% ethyl acetate, the ethyl acetate/1' layer was dehydrated using 20F total anhydrous sodium sulfate, and the residue obtained by distilling off ethyl acetate was dissolved in methanol and allowed to stand, resulting in the crude P-239240. Crystals precipitated. When this is recrystallized from methanol, P-23924C crystals 1.
Example 3 Tablet (1) P-23924A I 00H
9(2) Lactose 47~
, (3) Corn starch 40 liters (4) Hydroxypropylcellulose-L +
2. The above ingredients are compressed into tablets according to a conventional method (wet method).

Example 4 Tablet (1) P-2392411100PV (2) Lactose 47”y
(3) Cornstarch 40g (4
) Hydroxy 10 Bill 7/I/Loose-L +
2. Compress the above ingredients into tablets according to the conventional method (wet method),
Tablet Example 5 Capsule (1) P-23924Cj00tsu (2) Lacto-7135~ (3) Cornstarch 60~(
4) Magnesium stearate 5q After mixing the above components (1), (2), (3) and (4), it is granulated according to a conventional method. Add to this the ingredients (4
) and add No. 1 gelatin capsule tv according to the usual method.
Enclosed in <10th revised Japanese Pharmacopoeia> and combined with capsules Example cuff 9 Cell formulation (1) r-23924Ll 30
0~(2) Rough 1-s +35f
Ingredients (1), (2), (3) and (
After mixing the - in 4), the mixture is made into 1M particles according to a conventional method.

The rest of ingredient (4) is added to this, and the mixture is sealed in No. 00 gelatin capsules (Japanese Pharmacopoeia, 10th edition) according to a conventional method to form capsules.

Example 7 Tablet (1) p-239241': 10
0Q+(2) Lactose 4
71'fl (3) Cornstarch
40-(4) Hydated xypropylcellulose-L
12-(5) Magnesium stearate
1 - The above ingredients are ladleped into tablets according to a conventional method (wet method).

4. 11+1 simple h) υjll of the drawings Figures 1 to 5 show the physiologically active substance P-23924A,
Figures 6 to 10 show the ultraviolet and visible absorption numbers of B, C, D, and E for the physiologically active substance P-23924.
Infrared absorption of A, B, C, D, and E 1 Spec.
4A, B, C, T), ELy) Nuclear Magnetic Resonance Svec)/L', respectively.

Samurai Yai-shi” Y Misaki Y Rimibi Hikari--immersion eyebrows? Darkness←? Notification of Change of Entrustment Number October 1980 Director General of the Japan Patent Office/Sign of the Case + I/I Japanese Patent No. 185043, 7! Name of the invention) JJi active substance 7τP-23924, its manufacturing method and formulation 3. Lock 1 series with the case of the person who filed the procedure Patent applicant address: 2-27 Doshomachi, Higashi-ku, Osaka Name (293)
) Takeda Pharmaceutical Co., Ltd. Representative: Iku Kurabayashi 4 Branch Agent Address: 2-17 B5fS, Jusanhonmachi, Kawayo-ku, Osaka City
Name of the old depositary institution: Agency of Industrial Science and Technology BP-338 9, Attachment 1 List of documents (1) One or more documents certifying the new accession number Procedural amendment (voluntary) October 1, 1980 Commissioner of the Japan Patent Office 2. Name and physiology of the invention Active Substance P-2: 3924. Its manufacturing method and formulation 3, Person making the amendment f'l Patent applicant's office Address: 2-27 Doshomachi, Higashi-ku, Osaka Name (
293) Take+11 Yakuhin Kogyo Co., Ltd. Representative Kura
Iku Hayashi Part 4 Part 6 Contents of the amendment (1) Deposited with "te" in line 20 of shell number 13 of the specification. ” is corrected.

(2) Ibid., pages 14, No. 1 of 2 to 30, respectively, have been deposited. ' has been deposited, and the deposit has been deposited with the deposit of the prefecture in Pudabes, accession number raRMBP-3.
It is kept at the same research institute (F'RI) as No. 38. ”
Correct.

(3) The same book, page 35, line 1 [p-6739J [T3
Corrected to P-3381.

that's all

Claims (1)

[Claims] [1] Physiologically active substance P-23 having the following physical and chemical properties
924A, n, c, Dt or E: (1) Physiologically active substance P
-23924A (a) Melting point: 186-188°C 2 (b) Molecular formula: al131119 NOF5O95,
(dJ ultraviolet and visible absorption Nuvector, wavelength showing large absorption (nm): 220 ± 2,263 ± 2 (published), 270 ± 2 ° 420 ±
10 (in methanol); 221±2,264±2 (1
ri). 2γ0±2. 420±10(0,1N HCl-9
0% methanol in water); 216±2. 238±2
．． 277±2゜540±10 (0,11 pieces NF3
(0H-90% methanol in water) (e) Red eye 11ζ1 flash-harvesting Nuvector, potassium bromide l-tablets, principal wave number showing maximum absorption (cm): 3300.2930. +710.1665.1640°1620.1570,1540. 1500, 1460
゜1420.1380. 1335.1281), 12
40°1200.1180. N1(1,1040,+0
00°970, 900. 860. 800 (2) Physiologically active substance P-23924B (a) Melting point: 200-202"C (b) Molecular formula: C19H21NOB 5fcJ Specific optical rotation: [α)) 23-90° ±10° (cm0.51
．． Methanol/L/) (()) Ultraviolet and visible absorption 11.7 Nuvec/L/
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±2°308±2,420±I
O (0,IN HCl-90% methanol in water):
216±2,237±2,284±2゜540±10<
0.1HmaoH-90% methanol/water) (ρ) Optical external absorption Nuvector, 11 tablets of potassium bromide, wave number showing large absorption (cm'-'): 3300, 2950. +725,1705,1660°1630.1590. +540.1460,1420°1370.1330. +300.1270.1210°1190.1170.1130,1100,1060°1020, 980. 950. 890. 860°790, 760. 700 (3) Physiologically active substance P-23924C Ia,) Melting point: 187-190'c (b) Molecular formula: cIB H19NOB 5fcl Specific optical rotation: [αi3-93 ±10 (C=0.51.methanol/ L/) Cd) Ultraviolet and visible absorption spectrum, wavelength showing maximum absorption (nm): 221 ± 2, 260 ± 2 (shoulder), 265 ± 2° 306 ±
2,420±10 (in methanol): 221±2,26
0±2 (shoulder), 266±2, sat, z2゜420
±1o (0,1 N HCl-90% methanol in water)
;216±2,236±2,286±2. 540±1
0(Q, lN Na, 0H-90% methanol in water) (e) Optical external absorption spectrum, potassium bromide tablet, principal wavenumber showing strong absorption ((7)). 3400.3070,2940,1720.1680°1635.1600,1540,1490,1460°1420.1380,1350.1310.1250°1220.1180. N20. +100.1040°9
90, 930. 880. 820. 800゜05 (4) Physiologically active substance P-23924D (a) Melting point: 205-207℃ (b) Molecular formula: CIBH□9NO8S (c) Specific optical rotation: [α] 3-62° ±10° (c
=0.51. methanol/L/) fd) Ultraviolet and visible absorption spectra, wavelength showing maximum absorption (nu): 220 ± 2,271 ± 2,306 ± 2,420 ± 10 (
(in methanol); 220±2,271±2゜306-1
-2. 420+l0(0,1NHc1-90%jri/
-) L' in ice water, 216±2,234±2,299±2
゜540±20(0,IN NaOH-90% methanol-
μ water) (el infrared absorption spectrum, potassium bromide tablet, principal wave number showing maximum absorption (°C): 3400.3300, 2960.+720, 1670°1635, +610.1540, 1440.1380°+340.1300.H60 , +220.1205°1
130.1100.1060,1000. 900°8
70, 820. 800. 780. 690 (5)
Raw 11i Active Substance P-23924Ffa,) Melting Point: 17
4-176°C (b) Molecular formula: cl, 8 H19NoB 5 (cJ Specific optical rotation: [α]'i3-86° ±10° (c=0
．． 5. Methanol) (dJ ultraviolet and visible absorption spectrum, 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 (0,1NHCI-90%
(in methanol water); 216±2°238±2,287±
2,540±10 (,0,1N NaOH-90% methanol in water) (e) Infrared absorption subek) / potassium bromide tablet, principal wave number showing maximum absorption ((7)-1): 3550.330
0,3080,2950,1725°1660,+64
0.1610.1570. +540°1490,145
5,1420,1380,1325°1300.124
0,1210.1180.1120°1080, +04
0. +010. 940. 920°860, 82
0. 800. 770. 700 [2] Physiologically active substances belonging to the genus Streptomyces P-23924A, B
, C, D and/or E are cultured in a medium, and the physiologically active substance P-239 is added to the culture.
24A, B, C. Physiologically active substances P-23924A, B, which are characterized by producing and accumulating D and/or E and collecting them;
Method for producing C, D and/or E. 3] Physiologically active substances P-23924A, B, and C. A fibrosis inhibitor containing D and/or E.