JPH03216192A - Wf-19849 substance and preparation thereof - Google Patents

Abstract

NEW MATERIAL:A WF-19849 substance having the following physicochemical properties. Color and property: white powder; melting point: 218-229 deg.C (decomposition); specific rotation: [alpha] =+54.1 deg. (C=1.0, H2O); molecular weight: C16H23N7O5; mass spectrum (FABMS): 394 (M+H)<+>: solubility: easily soluble in water, slightly soluble in methanol and ethanol, insoluble in chloroform and ethyl acetate; coloring reaction: positive to cerium sulfate and ninhydrin; amphoteric, etc. USE:An antifungal agent. PREPARATION:A strain belonging to the Kernia strain such as Kernia.sp- F-19848 strain (FERM P-11107) is cultured preferably at 23-26 deg.C for 50-300 hours.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] This invention provides novel WF-1984 having antifungal activity.
It concerns nine substances and is used in the medical field.

[Purpose of the invention]

This invention relates to a novel WF-19849 substance.

More specifically, the present invention provides novel WF-19849 substances and salts thereof that exhibit antifungal activity, and methods for producing them.

[Structure of the invention]

This invention has the following physical and chemical properties: WF=198
It consists of 49 substances, their salts, and their production methods.

Physical and chemical properties of W F-19849 substance ■ Color and properties: white powder ■ Melting point = 218-229°C (decomposition) ■ Specific optical rotation. [α] ”,'= +54.1° (C
=1.0H2O) ■Molecular formula: C,. H. ．． N , O , ■ Mass spectrum (FABMS): ■ Ultraviolet absorption spectrum: λH2° = 260 nm Sugar & X λH, O゜OOINHC1 = 258 nm &ν;3;= 3330.3180,2960,+635
．． 1595.1470.1385, +325.1290
,1240,1205,1168,1065,955,
820,795,715,640 cm■'H nuclear magnetic resonance absorption spectrum δ(D,O): 8. 67 (IH, S), 8.
19 (IH, S), 6. 19 (IH, d, J
= 3. 0Hz), 4. 75 (2H, o+)
, 4. 54 (IH, d, J= 7.3Hz
), 4. 08 (IH, d, J-5.0ce).
2. 14 (IH, m), l, 57 (IH, m),
1. 38 (IH, m), 1. 05 (3H,
d, J=7. lse), 1. 02(3H, t,
J=7. 4th),■+30-Nuclear magnetic resonance absorption spectrum δ(D,O): 178.6, 172.6,1
57.9, 155.1, 150.9, 142. 7,
121. 1, 91. 9, 83. 6, 76.
4, 60.2, 57. 2, 39. 0, 2
7. 9, 15. 6, 13. 5, [Phase] Solubility: Easily soluble: Slightly soluble in water: Insoluble in methanol, ethanol: Chloroform, ethyl acetate ■ Color reaction: Positive: Cerium sulfate, ninhydrin @ Thin dust chromatography: Rf = 0.6 [Carrier: Nuri force Gel (Kiesel Gel 60
F'-254: Trade name, manufactured by Merck & Co.) Developing solvent: Isobanol: Water = 7:3 Rf = 0.3 [Carrier: Silica gel (Kieselgel 60F)
-254: Trade name, Merck & Co., Ltd.) developing solvent. n-butanol:acetic acid:water=4:1:2 ■Acidic, basic/amphoteric The WF-19849 substance of the present invention can be used to produce WF-19849 substances, such as WF-14Hl49 substance-producing bacteria belonging to the genus Cernia. It can be produced by culturing bacteria in a medium and separating and collecting the WF-19849 substance from the resulting culture.

Among the WF-19849 substance-producing bacteria used in this invention, the strain newly isolated by the inventors from a soil sample collected in Morioka City, Iwate Prefecture (numbered as strain F-19849) is shown below. It has similar mycological properties.

41 on Seed Ichiku medium This strain grew suppressed on various media such as malt extract agar medium and potato glucose agar medium, and formed orange-white to pale orange colonies. Also,
By culturing on oatmeal agar, cornmeal agar, and YpS agar for 2 to 4 weeks,
Closed ascus shell (teleomorph) and unmouthed conidia (
The formation of anamorphs was observed. The morphology, culture and physiological properties of this strain are shown below.

Culture characteristics Table 1 shows the culture characteristics on various media. When it was inoculated onto the center of a potato glucose agar medium and cultured at 25°C for 2 weeks, growth was extremely suppressed and it spread to a diameter of 1.5 cm. The surface of the settlement was raised in the form of wrinkles and was white to pale orange in color.

The back side of the village is pale orange. No conidial structure or asci were formed. Growth when similar cultures were grown on oatmeal agar was similarly suppressive (1.
5-2.0cm). The surface of the colony was slightly raised and orange-white to pale (storm orange). The underside of the colony was pale orange. Conidial structures and ascus shells were formed.

Morphological Characteristics Observation of morphological characteristics was mainly based on growth on cornmeal agar medium. The asci can be buried or superficial, singly, without an opening (cleistocele), spherical or subglobular, light brown to orange in color, and 7 mm in diameter.
0-150(-200)μ-.

At the early stage of culture, the ascus shell is covered with lateral hair-like hyphae, but when it matures, the hyphae disappear and it becomes bare. The ascus wall is 4-6
It consists of layers of cells, with ascus scattered within the ascus shell. Asci are single-walled, effaced, subglobose to obovate, 9-diameter.
It has a 15 (-18) μ intestine and produces 8 ascospores. Ascospores are pale to light brown, reddish-brown when aggregated, smooth, 1-celled, prominently kidney-shaped, 4-6(-7)X
3. It is 5-5 μpeng. Each end of the ascospore has one germination hole. The anamorph of strain F19849 consists of broom-shaped conidiophores and Annelo-type conidia. Conidiophores are colorless, smooth, and septate, erecting singly or in short conidiophore bundles. These techniques are done 2 to 4 times,
Length 30-50 (-60) μm 1 Width 10-20 (-30
) μm broom-like shape.

The size of the branches is 8-10 x 3-5 μm, each giving rise to 3-5 further branches. The apical cell of the conidiophore is
Colorless, smooth, cylindrical to rod-shaped, 8-17(-25)Xi
-3 (-3.5) μm anthrophore. Conidia are colorless, smooth, 1 cell, obovate to obovate pear-shaped, base truncated, 4-8XI. 5-3. The size is 5 μm. These are not linked and form a conidial soul with a diameter of 30-70 (-100) μm at the tip of the conidiophore (bundle).

The vegetative hyphae are septate, colorless, smooth and branched. Hyphal cells are cylindrical to barrel-shaped or oval, 2-5 (-7) wide.
It is mu fat.

Chlamydospores were abundantly formed, singly or in series, subglobose to broadly ellipsoidal, and 8-12 μm in diameter.

Physiological Characteristics Strain F-19849 can grow in the range of 2-34°C, and the optimum temperature for growth was 23-26°C (measured on potato glucose agar).

The above characteristics and Alux (^rx. J.^. v
The General of Funghi 11 - Svolrating in Pure Carcinia (The g
enera or Fungi--Sporulati
ng in Pure Culture), 3rd edition, page 315, Mailoch and Kane (Mailoch,
D. , and R. F. Cain)
The genus Kern
ia) [Canadian 3 Journal of Botany (Can. J. Bot.), Volume 49, No. 8
55-867, 1971], Udagawa and Furuya, Emeryseropsis sphaerosvora and Cernia velpiana, Tou 221 Ninoleponorefu Thaleistotesianore Ascomycetes (
Emericellopsis sphaerospo
ra and Kernia peruviana. T
vo new soil-borne cleisto
thecial AscoIIlycetes) [Mycotaxon, Vol. 18, No. 291
-Page 301, 1988], P-198
49 strains belong to the ascomycete genus Kernia.
uvland). Therefore, this production strain was used as Kernia sp.
rnia sp. F-19849).

Table 1 Culture characteristics of the F 9849 strain Samurai 11 above, 14 days at 25°C Seki 1a#il:fl
Inspection k0 color name description 1j, mezuen handbook-1
MeLhuen Handbook
of Color)! 3mIz52N+& and CI
, line 7, &0 This Kernia sp. strain F-19849 has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Microtechnology Research Grant No. 11107 on November 14, 1989.

The IP-19849 substance producing bacteria used in this invention can be treated with irradiation such as X-rays or ultraviolet rays, such as nitrogen mustard, azaserine, nitrous acid, 2-7 minoprine, N-methyl-N-nitro-N-nitrosoguanidine. (
The production ability of the YF-19849 substance can be increased by commonly used bacterial species mutation treatment methods such as treatment with a mutagenic agent such as NTG, phage contact, transformation, transduction, and conjugation.

The production of YF-19849 substance, which is carried out by culturing WF-19849 substance-producing bacteria belonging to the genus Cernia in a medium, is in principle based on the culture method of general microorganisms.
Deep culture methods using liquid media are usually advantageous. The medium used for culture is WF-1, which belongs to the genus Kernia.
Any medium may be used as long as it contains a nutrient source used by the 9849 substance producing bacteria. That is, a synthetic medium, a semi-synthetic medium or a natural medium is used, and the medium composition includes, for example, glucose, euculose, maltose, glycerin, starch, liquefied starch, etc. as a carbon source, and as a nitrogen source,
For example, meat extract, casein hydrolyzate, bebutone, gluten meal, corn meal, cottonseed flour, soybean flour, corn steep liquor, peanut powder, wheat germ, dried yeast, yeast extract, urea, ammonium phosphate, etc. are used. In addition, examples include disodium hydrogen phosphate, potassium dihydrogen phosphate, magnesium chloride, magnesium sulfate, calcium carbonate, sodium iodide, cobalt 6 chloride.
Inorganic salts such as aqueous salts are also added to the medium as necessary.

In addition, when foaming is significant during culturing, for example, vegetable oils such as soybean oil, linseed oil, octadecanol, tetradecanol, etc.
Higher alcohols such as hebutanol and antifoaming agents such as silicon compounds may be added as appropriate.

The appropriate culture temperature is around 23 to 26°C, and good results are often obtained if seed culture is carried out as appropriate as the culture volume increases. The appropriate culture time for the main culture is about 50 to 300 hours, and the culture time may be further extended as the medium becomes more concentrated.

The culture conditions described above are selected and used depending on the characteristics of the production strain used.

Next, the WFl9849 substance produced by culture is usually
Since it often accumulates within the bacterial cells in culture, it is generally separated into bacterial cells and filtrate (supernatant liquid) by means such as centrifugation or filtration, and then used in the production of general antibiotics from the empty cells. It is separated, purified and collected by means. That is, after dissolving the bacterial cells in a solvent and extracting the target substance with the solvent,
Liquid conversion, such as treatment with resins such as anion exchange resins, cation exchange resins, and nonionic adsorption resins, treatment with adsorbents such as activated carbon, silicic acid, sori gel, alumina, and cellulose, crystallization, and reuse. By combining or repeatedly applying means such as crystallization in any order, the target substance WF-19849 substance can be separated and purified.

The 11F-19849 substance obtained in free form can be converted into a desired salt by reacting with a base such as sodium hydroxide or potassium hydroxide.

The substance WF-19849, which is the object substance of the present invention, can be mixed with an organic or inorganic solid or liquid carrier suitable for oral, parenteral or external use, and prepared into a conventional pharmaceutical formulation, such as a capsule. It may be used in the form of solid preparations such as tablets, granules or suppositories, semi-solid preparations such as ointments or liquid preparations such as solutions or emulsions.

In addition, the above-mentioned formulation may contain appropriate amounts of conventional additives such as stabilizers, wetting agents, and emulsifiers.

The dose of the target compound [1] depends on the patient's age, weight, symptoms, etc., but is usually administered in the range of about 0.1 to 1000 μ at a time.

〔Effect of the invention〕

Substance IIIP-19849 and its pharmaceutically acceptable salts, which are the target substances of this invention, have antifungal activity and are useful as antifungal agents for humans and animals, as is clear from the following test results. .

(1) Antibacterial activity test Antibacterial activity The antibacterial activity of the WF-19849 substance was measured by the conventional micro-broth dilution method using a 96-concave multi-tray as described below.

Test bacterial suspensions were prepared by culturing the bacteria overnight in Eagle's minimum essential medium (E.MEM) and diluting the culture broth.
(2×10' pieces/m1) was adjusted.

E, MEM of WF-19849 substance (100μ!)
Medium serial concentration serially diluted solutions were added to the wells of the multi-tray.

100μ of test suspension in four parts! After adding , the multi-tray was heated at 30°C for 40 hours in a 5% carbon dioxide atmosphere, and then the minimum inhibitory concentration (ffilc) was measured.

The results are shown in Table 2.

Table 2 Antibacterial activity test of IP-19849 Animals 4 weeks old, ICR mice weighing 18-21 g each, 1
Groups consisted of 5 mice.

Test method The pathogenic bacteria Candida T.
Candida albicans) FI'633 1
The mice were injected into the lateral tail vein at a viable count of 2×10 cells per mouse. One hour after infection (injection into the lateral tail vein), a saline solution of the WF-19849 substance was administered to the mice by subcutaneous injection. This subcutaneous injection was repeated once a day for 3 days starting the day after infection. The number of surviving mice was observed on day 11 after infection, and the results are shown in Table 3.

Table 3 (2) Toxicity test The acute toxicity of YF-19849 substance was measured by intravenous injection once in 4-week-old female mice, and the dose was 320 m
No deaths were observed at 100 g/kg.

〔Example〕

The present invention will be explained below with reference to Examples.

4% sucrose, 2% cottonseed flour, 1% moratin, 1% bebutone, 0.2% potassium phosphate. Carbonate: Pour seed medium (+6 [1 ml) containing 02% lucium] into two 500 ml X Lullenmeyer flasks at 121°C.
, 30/AIifllll&bacteria. One platinum loop of a slant culture of Kernia SB strain F9849 (Feikoken Bacteria No. 11107) was inoculated into each medium, and cultured with shaking at 25°C for 4 days.

Pine de Ix #3 3%, Nucleose 1%, Sorghum flour 1%, Moratin 1%, Sodium nitrate 02%
, potassium phosphate 0.1%. Magunium sulfate decahydrate 0. Production medium (2 o1 to 3 (Jl
Pour into a jar/fermenter and sterilize at 121°C for 30 minutes. The seed culture (320 ml) obtained above was inoculated into the production medium, stirred at 200 rpm, and stirred at 2 (ac) per minute.
Incubate for 5 days at 25°C under aeration.

The culture thus obtained (5Ql) was added to the diatom ±(
Ikg) and filter. The filtrate (36N) was treated with anion exchange resin Dawenox IX 2 (OH-g, trade name,
The mixture was applied to a column (6 g) manufactured by Dow Chemical Co., USA. The column was washed with water (]2β) and 0. Elute with 5M sodium chloride. The eluate (101) is applied to a column (21) using activated carbon (manufactured by Wako Pure Chemical Industries, Ltd.). Dip the column into water (
Wash with 41) and elute with 50% acetone water. After concentrating the eluate (61) under reduced pressure to remove acetone, adsorption resin SP-207 (trade name, manufactured by Mitsubishi Chemical Industries, Ltd.) was applied.
column (21). Wash the column with water (4 g) and elute with 50% methanol/water. The eluate (61) is concentrated under reduced pressure to remove methanol, and then applied to a column (0.5N) of cation exchange CM Sephadix C-25 (trade name, manufactured by Pharmacia). Wash the column with water (1!) and elute with 0.08M sodium chloride. The eluate (1 g) was applied to an SP-207 column (1 g).
00ml), washed with water (3oov) and diluted with 50ml
Elute with % methanol water. The eluate (2DDd) was concentrated under reduced pressure to remove methanol, and then lyophilized to give WF19f! 149 substance (620 mg) is obtained as a white powder.

Claims (1)

[Scope of Claims] [1] WF-19849 substance and its salts having the following physical and chemical properties. (1) Color and properties: white powder (2) Melting point: 218-229°C (decomposed) (3) Specific rotation: [α]^2^0_D=+54.1°(
C=1.0H_2O) (4) Molecular formula: C_1_6H_2_3N_7O_5(5
) Mass spectrum (FABMS): 394 (M+H)^
+ (6) Ultraviolet absorption spectrum: ▲Mathematical formulas, chemical formulas, tables, etc.▼ (7) Infrared absorption spectrum ν^K^B^r_m_a_x=3330, 3180, 2
960, 1635, 1595, 1470, 1385, 1
325, 1290, 1240, 1205, 1168, 1
065, 955, 820, 795, 715, 640cm
^-^1(8)^1H-Nuclear magnetic resonance absorption spectrum δ(D_2O): 8.67(1H,S), 8.19(1
H, S), 6.19 (1H, d, J = 3.0Hz), 4
．． 75 (2H, m), 4.54 (1H, d, J = 7.3
Hz), 4.08 (1H, d, J=5.0Hz), 2.
14 (1H, m), 1.57 (1H, m), 1.38 (
1H, m), 1.05 (3H, d, j=7.1Hz),
1.02 (3H, t, J=7.4Hz), (9)^1^
3C-Nuclear magnetic resonance absorption spectrum δ(D_2O): 17
8.6, 172.6.157.9, 155.1, 150
．． 9, 142.7, 121.1, 91.9, 83.6,
76.4, 60.2, 57.2, 39.0, 27.9,
15.6.13.5, (10) Solubility: Easily soluble: Slightly soluble in water: Methanol, ethanol Insoluble: Chloroform, ethyl acetate (11) Color reaction: Positive: Cerium sulfate, ninhydrin (12) Thin layer chromatography: Rf=0.6 [Carrier: Silica gel (Kiesel gel 60F
-254: Trade name, manufactured by Merck & Co.) Developing solvent: Isopropanol: Water = 7:3 Rf = 0.3 [Carrier: Silica gel (Kieselgel 60F
-254: Product name, Merck & Co.) Developing solvent: n-butanol: acetic acid: water = 4:1:2 (13) Acid, basicity: amphoteric [2] Cultivate WF-19849 substance-producing bacteria belonging to the genus Kernia. and collecting the WF-19849 substance from the culture. [3] The method for producing the WF-19849 substance according to claim 2, wherein the WF-19849 substance-producing bacterium belonging to the genus Cernia is Cernia sp.