JPH01299280A - Novel antibiotic substance h9 and production thereof - Google Patents

Abstract

NEW MATERIAL:An antibiotic substance H9 expressed by the formula. USE:Useful as a remedy for mycosis with low toxicity and great antimicrobial activity against fungi of the Candida, etc. PREPARATION:A strain, belonging to the genus Streptomyces and having the ability to produce the antibiotic substance H9 is inoculated into a nutrient source-containing culture medium and aerobically cultured to afford the compound expressed by the formula. For example, glucose or fructose, is used as a carbon source and, e.g., soybean flour or cottonseed flour, is used as a nitrogen source. For example, a sodium or iron salt, is used as an inorganic nitrogen compound. A method for shaking or submerged spinner culture with aeration is especially optimum for the culture method. The culture temperature is preferably 22-32 deg.C and culture pH is preferably 6-8, especially about 7. The production of the H9 in the resultant culture is sufficiently enhanced by culturing for 2-6 days.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel antibiotic H9 useful as a therapeutic agent for fungal infections and a method for producing the same.

[Conventional technology]

Conventionally, amphotericin B1 has been used as a treatment for fungal infections.
There are about 20 types, including nystatin, tryfmycin, griseofulvin, virolnidoline, clotrimazole, and micosazole nitrate, but they have problems in terms of efficacy and toxicity.

[Problem to be solved by the invention]

The object of the present invention is to provide a novel antibiotic with high activity and low potency as a therapeutic agent for fungal infections.

[Means to solve the problem]

The present inventors isolated a large number of microorganisms from soil for the purpose of searching for new antibiotics, isolated the antibiotics they produced, and investigated their biological properties. We have discovered that a novel antibiotic, which has not yet been described in any literature, is produced in culture that exhibits antibacterial activity against Candida albicans and other pathogenic fungi.

This antibiotic was isolated from the culture and its physicochemical properties were investigated. As a result, it was confirmed that it was a new substance having the structural formula of formula (2) described below, and this antibiotic was named H9.

That is, the present invention provides antibiotic H9 represented by the following formula and a method for producing the same.

First, the microorganism used in the present invention is a species belonging to the genus Streptomyces that has the ability to produce the novel antibiotic H9.

For example, Streptomyces sp, PH317
A new strain of microorganism called . Stove) 1 cess Bp, FH3
17C (hereinafter simply referred to as strain IFH317) was submitted to the Institute of Microbiology, Agency of Industrial Science and Technology for deposit on April 30, 1986.
(Japan), and was entrusted with the Microtechnical Research Institute No. 10012.

Strain FH317 has the following mycological properties.

1. Morphological properties Aerial mycelium grows well on yeast/malt agar, starch/inorganic salt agar, tyrosine agar, etc. The branching method is simple branching, and axle branching is not allowed. The shape of the aerial mycelium tip is straight or bent, and short chains of 3 to 10 spores are observed. The surface structure of the spore is smooth, the shape is oval or cylindrical, and the size is 0.6 to 0.7 × O,
It is 9 to 1.0 μm. Structures such as sporangia, sclerotia, and hyphal bundles are not observed.

[Properties on various media (28°C, observation after 2 weeks of culture)] (1) Yeast/malt agar medium (Technical SP medium A2)
) Growth is normal. Aerial mycelial growth is good and gray in color. The underside of the growth is yellowish brown to dark brown. No soluble dyes are observed.

(2 Oatmeal agar medium (according to engineering sp medium A3) Growth is rather poor. Aerial mycelial attachment is somewhat poor and white to gray in color. The underside of growth is pale yellow with no characteristics.

No soluble dyes are observed.

(According to J starch/inorganic salt agar medium CISP medium Gataka 4) Growth is normal. Aerial mycelial growth is good and gray in color. The underside of the growth is featureless and pale yellow or yellowish brown. No soluble dyes are observed.

(→Glycerin/asparagine agar medium (according to Engineering SP Medium Store 5) Growth is poor.6 Aerial mycelium is absent or very poor. No soluble pigments are observed.

(Glucose-asparagine agar medium growth is poor. Aerial mycelium is absent or very poor. No soluble pigments are observed.

(e) Growth on tyrosine agar medium (according to SF medium A7) is normal. Aerial mycelial attachment is good and gray. The underside of growth is dark brown.

No soluble dyes are observed.

(Kashucrose/nitrate agar medium Growth is poor. Aerial mycelial growth is poor and the color ranges from white to light gray.

No soluble dyes are observed.

(8) Growth on nutrient agar medium is normal. Aerial mycelial growth is poor and white to gray in color. The underside of the growth is pale yellow with no characteristics. No soluble dyes are observed.

2. Physiological properties (1) Growth temperature range (tryptone yeast liquid medium,
(Using temperature gradient device, 7 days) Possible growth temperature: 12°C to 40°C Suitable growth temperature: 22°C
~32℃ (2) Liquefaction of gelatin (Glutose No. peptone gelatin medium, 28℃, 2 weeks) Positive (3) Coagulation of skim milk, peptonization (10% skim milk,
28℃, 2 weeks) Peptonization: Positive Coagulation: Negative (→ melanoid production・Gotlieb agar medium, carbon source 1%, 28°C, 2 weeks) Positive: Glutose,
Fructose, xylose, mannitol, raffinose False positive: Arabinose negative: Rhamnose, inositol, sucrose (6) Salt tolerance (addition of N&OA' to yeast/malt agar medium, 28°C, 2 weeks) Na()/l up to 9% Growth, NILOA' 13%
It won't grow.

In addition to the morphological and physiological properties described above, LL-diaminopimelic acid was detected in the hydrolyzate of whole bacterial cells.
Strain FH317 is clearly recognized as a strain belonging to the genus Streptomyces.

The properties of the IPH317 strain are described in Purging Aids Manual-Op Determinative Bacteriology (8th edition, 1974); The Actinomycetes Volume 2 (Waxman, 1962) and E.B. Shearing et al. Report (International Journal of Systematic Bacteriology Vol. 18, 1)
968, p. 69, vol. 18, 1968, p. 279, vol. 19, 1969, p. 391, vol. 22, 1972, p. 265). When comparing Streptomyces species and their characteristics, one species that is relatively similar is Streptomyces halstedi (Streptomyeshalst).
edii) and Streptomyces catenulae (S
2 of treptomycsa oatenulaa)
Examples include bacterial species. The above two bacterial species are similar in that they do not produce melanoid pigments, have short spore chains (10 or less), have smooth spore surfaces, and have straight or curved spore chain morphology. Streptomyces halsteadei is similar to this fungus in that its aerial hyphae are gray in color and the underside of its growth is a featureless gray-yellow to yellowish-brown color, but its spore chain morphology is mainly curved, and its salt tolerance is NhO1. ≧7% (10%>)
This bacterium differs from this bacterium in that it does not use mannitol and raffinose as carbon sources.

Streptomyces catenulae has m resistance of Naat≧1
It is similar to this bacterium in that it is 0% (13%>) and that the availability of carbon sources is the same except for xylase. However, it differs from this fungus in that the color of the aerial mycelium ranges from green to gray-green, and the underside of growth ranges from gray-yellow to gray-green-brown.

As described above, although the IFH317 strain is similar to Streptomyces halsteadii and Streptomyces catenulae, there are also differences, and it is not appropriate to assign this bacterium to either of the above two bacterial species. Streptomyces ip, 1FH317 (St
reptomyaes sp, IFH317).

Antibiotic H9 of the present invention is produced by inoculating the above strain into a nutrient-containing medium and culturing it aerobically. The antibiotic H9-producing bacteria are not limited to the above-mentioned strains, but any strain that belongs to the genus Streptomyces and has the ability to produce antibiotic H9 can be used in the present invention.

Next, culturing of bacterial strains in the production of antibiotic H9 will be explained.

When culturing the bacteria that produce H9, assimilated organic carbon compounds such as glucose, fructose, starch, dextrin, glycerin, molasses, starch syrup, fats and oils, and organic acids are used as the carbon source, and soybean flour as the nitrogen source. organic and inorganic nitrogen compounds such as cottonseed flour, corn steep liquor, casein, peptone, yeast extract, meat extract, germ, urea, amino acids, ammonium salts, and salts such as sodium salts, potassium salts,
Inorganic salts such as calcium salts, magnesium salts, and phosphates may be used alone or in appropriate combinations. Furthermore, if necessary, heavy metals such as iron salts, copper salts, zinc salts, and cobalt salts, vitamins such as biotin and vitamin B1, and other organic and inorganic substances that support the growth of bacteria and promote H9 production are added as appropriate. May be added. Furthermore, antifoaming agents and surfactants such as silicone oil and polyalkylene glycol ether may be added to the medium.

As the culture method, methods used in the production of general antibiotics are employed, but liquid culture methods, particularly shaking or deep aeration agitation culture, are most suitable.

Aerobic conditions are suitable for culturing, and the culture temperature is usually 22-32°C.
°C is preferred, and the culture pH is 6 to 8, preferably around 7. Live M of H9 in culture! Ik becomes sufficiently high after 2 to 6 days of culture.

In order to collect H9 accumulated in the culture as described above from the culture, it is advantageous to take advantage of the physical and chemical properties of the present antibiotic described below.

That is, since n9 is contained in the culture solution F, the bacterial cells are first removed by adding a filter aid to the culture and performing filtration or centrifugation. Advantageously, a method is used in which the obtained culture medium GlP liquid is brought into contact with a carrier to adsorb the active ingredient in the liquid, and then the active substance is desorbed with a solvent and then fractionated and collected.

As carriers for chromatography, active carbon, powdered cellulose, adsorbent resins, and other carriers that utilize differences in adsorption of compounds are effectively used. In order to elute the target compound from these carriers, the combination will differ depending on the type and nature of the carrier, but for example, an appropriate combination of aqueous solutions of water-soluble organic solvents, such as aqueous acetone, aqueous alcohols, etc. can be used.

In addition, a purified product can also be obtained by subjecting the crude substance containing antibiotics obtained by these methods to preparative high-performance liquid chromatography.

In more detail, if an adsorbent resin such as Diaion HP 20 or 5P207 (manufactured by Mitsubishi Chemical Industries, Ltd.) or Amparite XAD-2 (manufactured by Rohm and Haas, USA) is used as a carrier, the Antibacterial substances are adsorbed and eluted with aqueous acetone or aqueous alcohols. The fractionated eluted fraction can be concentrated.

High performance liquid chromatography (HPI+C) can be effectively used to further purify the obtained crude material.

Examples of carriers that can be used include TSK gel (manufactured by Toso Corporation) and ypAo gel (manufactured by Yamamura Kagaku Kenkyusho), and as a mobile phase, water-containing methanol or water-containing acetonitrile can be used.

The antibiotic H9 obtained as described above has the following physicochemical properties.

■Appearance: Colorless viscous liquid ■Specific optical rotation: (α)''-461,0° (cO, 17tonitrile) ■Elemental analysis (as 0uHuQn) 0% H% Actual value 63 .63 8.03 Theoretical value 63.70 8.02 ■Molecular fi (according to IFAB mass spectrum) 319
(MH + Glycerin) ■ Ultraviolet (trv) absorption spectrum: Acetonitrile-water (see Figure 1) 1st - λ, aX 255 nm (11377) 1st country - ■ Infrared (trv) absorption spectrum: KBr H (second
(See figure) Shows main absorption (wave Wl) 3450.2970,1845,1775°1275
, 920 cm-' ■1m□Nuclear magnetic resonance (NMR) size: 50
MHzsδPT1m The following signal is observed in % deuterated chloroform (see Figure 3) 165.6
(s), 165.2(11), 143. ．． 6(s)
-142,1k), 72.2(Tsukasa, 41.f(t
), 36.8<a).

25.2(d), 24.0(t), 21.3(q)
, 15.7(q).

10, 1 (q) 8 doublet, d: doublet, t: triplet, q: quartet ■High performance liquid chromatography (HPI, O) column:
Capsuleback 0ts (4.6φ x 250m, manufactured by Shiseido) Mobile phase: 0HsON-HzO (1:1), flow rate: 0.8m 11 minutes, Rt = 15.5 C minutes) Color reaction: Negative: Ninhydrin, Dragendorff, Sakaguchi, Lydon Smith, Resorcin - Sulfuric acid, potassium permanganate O Solubility: Soluble: methanol, acetonitrile, chloroform, ethyl acetate Poorly soluble: Water O Distinction between acidic, neutral and basic: Neutral As a result of examining various spectra, H9 was determined to have the structure of the above formula (2). Since no known substance matching this structure has been reported, H9 was determined to be a new substance.

Next, the antibacterial spectrum of H9 against each FJI organism was determined as follows.
Table 1 (based on liquid medium dilution method)
100 Escherichia Free N Engineering HJ > 100 Sarah, acute toxicity test using H9 mice showed 200q/
No abnormalities were observed after intraperitoneal administration of Kp.

〔Example〕

Next, the present invention will be explained in more detail with reference to Examples.

In addition, unless otherwise specified, 7-cent numbers in the culture medium are shown as weight/volume %.

Example 1 One platinum loop was collected from a slant culture of pa317 strain.
Seed medium (glucose 1.0%, polypeptone 0.2%
, beef extract 0.1%, yeast extract 0.1%) into a 500 rsl Erlenmeyer flask, and
A seed culture solution was obtained by culturing with shaking at 7°C for 2 days. 1400 mj of this seed culture solution was mixed with 140 mj of production medium (Botetosu 0.7%, glucose 0.7%, SBM 1.0
%, yeast extract 0.35%, sodium chloride 0.2
%, calcium carbonate 0.3%) into a 200/vol jar 7-armentor, and cultured with aeration at 27-30°C for 66 hours. (Airflow amount 80~l 101/I!Iin
, stirring 20 Or, p, m). Adjust the culture solution to pH 3
After adjustment, 1.5k of Celite was added and filtered with a filter press to remove bacterial cells to obtain Liquid F 110j.

This F liquid was added to 41 adsorption type synthetic resin Diamond Ion HP20.
(manufactured by Mitsubishi Kasei Corporation), the column was washed with water, and then eluted with 50% methanol water.

The eluate was concentrated under reduced pressure to obtain a residue 24. I got SP. This residue was extracted with methanol, and the extract was concentrated under reduced pressure to obtain 17.5 F of crude material. The crude substance was subjected to preparative reverse-phase high performance liquid chromatography [column: YMOS-343 (manufactured by Yamamura Kagaku Kenkyusho), mobile phase 50% aqueous a7tonitrile] to obtain an active fraction. Both portions were concentrated under reduced pressure to obtain a colorless viscous liquid antibiotic u9 2.05P.

[Effects of the Invention] H9 of the present invention is a new antibiotic produced by actinomycetes, and has low toxicity and antibacterial activity against Candida, so it is useful as a clinical drug, such as a therapeutic agent for fungal diseases.

[Brief explanation of the drawing]

Figure 1 is the ultraviolet absorption spectrum of antibiotic H9, Figure 2 is the infrared absorption spectrum of the same antibiotic, and Figure 3 is 11C.
This is a nuclear magnetic resonance (NMR) spectrum.

Claims (1)

[Claims] 1. A novel antibiotic H9 represented by the following formula (I) ▲ Numerical formulas, chemical formulas, tables, etc. are available▼. 2. A strain belonging to the genus Streptomyces that produces the antibiotic H9 is cultured in a nutrient medium, and the antibiotic H9 is extracted from the culture.
A method for producing a novel antibiotic H9, which comprises collecting .