JPH03197489A - Antibiotic tan-1171 and its production - Google Patents

Abstract

NEW MATERIAL:Antibiotic TAN-1171 having as an isolated form the following physicochemical properties or its salts: (1) appearance: white powder; (2) molecular formula: C10H17N3O4; (3) ultraviolet absorption spectra: giving terminal absorption (in the form of its aqueous solution), etc. USE:An therapeutic agent for fungal infectious diseases. PREPARATION:Microorganisms classified as Streptomyces sp. such as Streptomyces Chartreusis A-344 (FERM BP-2691) are put to culture, normally with deep aeration culture technique, under a pH of ca.7 for the medium at pref. 24-30 deg.C.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a novel compound TAN-1171 and salts thereof useful as therapeutic agents for fungal infections, methods for their production, and related microorganisms.

Background of the Invention In recent years, deep-seated mycoses have increased due to a decline in in-vivo immune function due to frequent use of cancer chemotherapeutic agents, immunotherapeutic agents, and adrenocortical hormone agents, as well as a downturn caused by administration of antibacterial agents. It is becoming a big problem in the medical field. For deep or superficial mycosis, chemotherapy with polyene antibiotics, flucytosine, imidazole antifungal agents, etc. is used; conventional antifungal agents have strong side effects and are resistant. Fungal infections are a major problem in the field of chemotherapy, as they are often unsatisfactory due to the easy appearance of bacteria.

Problems to be Solved by the Invention Medications for treating fungal infections are still extremely unsatisfactory, and there is a need for new antifungal agents that are clinically effective and have fewer side effects.

Means for Solving the Problems The present inventors have isolated microorganisms from a large number of soil samples and conducted exploratory research to obtain new antifungal agents from their metabolites. As a result, they discovered that certain actinomycetes produce antibiotics with antifungal activity in culture.
Isolate this substance purely from the culture and confirm from its physicochemical properties that it is the antibiotic or a new substance,
This was decided to be called TAN-1171.

The present inventors have completed the present invention as a result of further research based on these findings.

Namely, (1) the present invention provides antibiotic TAN-1171 and its salts, (2) Streptomyces
Antibiotic TAN-], which is characterized by culturing a microorganism belonging to the genus ces) in a medium and having the ability to produce the antibiotic TAN-1171, producing and accumulating the antibiotic in the culture, and collecting the microorganism. ,], 7] or a method for producing a salt thereof, and (3) Streptomyces chartreuis/
Regarding A-34.4.

In addition, in the specification, [antibiotic TAN117N is sometimes simply referred to as rTAN-], 17] or J.

Any microorganism may be used as a producing microorganism for the antibiotic TAN-1171 as long as it has the ability to produce the antibiotic TAN-1, I71. Streptomyces
Chart Ruins A344 (Streptomyc
es Chartreusis A-3, 44)
The most effective example of this is the strain named or its related strains. Hereinafter, this bacterium may be abbreviated as A344.

The morphological characteristics and culture findings on the classification medium are as follows, for example.

In Honkan, aerial hyphae are formed under the normal classification name, and they show simple branching, and spore-forming hyphae have a spiral shape. More than 10 spores are chained together, the surface is thorn-like, and the size is 14-1.8 μm x 0.8-1.2
It is μm. No formation of sporangia, i+ trichospores, or sclerotia was observed on a normal classification medium.

The growth condition on Honkan's classification medium is as clear. Unless otherwise specified, these are culture findings observed at 28°C for 21 days. In addition, the information in parentheses indicates color, harmony,
Manual 4th edition (Kuntina Corporain Yon)
of America (1958).

The physiological properties of A.-344 bacteria are as follows.

(1) Growth temperature range: Grows at 132-385℃, but 3
It shows better growth at 5-35.8°C.

(2) Seratin liquefaction positive (3) Starch hydrolysis/positive (4) Skimmed milk coagulation/peptonization 2 negative (5) Production of melanin-like pigment° positive (peptone/yeast/iron agar medium and tryptone/yeast liquid) Medium) (6) Nitrate reduction: Negative (Interna/Jonal Streptomyces Project N008 medium) (7) Utilization of carbon source (Bridham Cottrieb agar medium) Commonly used carbon sources Inositol D-Mannitol D-Glucose, D-fructose, raffinose, moderately utilized carbon source L-arabinose, D-kinlose, unutilized carbon source rhamnose, sucrose LL diaminopimelic acid was detected in the hydrochloric acid hydrolyzate of A-344 bacterial cells. . This suggests that Honkan belongs to the genus Streptomyces. As a result of attempting to compare the 1344 bacterium with existing bacterial species based on its morphological characteristics, culture findings, and physiological properties, this bacterium was identified as Streptomyces chartolysis, and Streptomyces chartolysis A344 (Streptomyces Cha
rtreusis A-344). Streptomyces chartreinus used in the present invention
A-344 has been transferred to the Fermentation Research Institute (IFO) since December 5, 1999, with accession number IFO1,4,976.
In addition, from December 18, 1999, the Ministry of International Trade and Industry, Agency of Industrial Science and Technology, Microbial Research Institute (FRI') received the accession number FERM.
Each has been deposited as BP2691.

As a general characteristic of Streptomyces, it is highly variable and is not Streptomyces. Therefore, as mentioned above, the properties of Honkan are not constant, and various mutant strains can be easily obtained. However, even with these mutant strains, the antibiotic TAN-117]
It can be used in the method of the present invention as long as it does not lose its ability to produce. Of course, these mutations may be derived from natural causes or may be artificially induced using various mutagenic agents (eg, ultraviolet rays, X-rays, radiation, nitrosoguanidine, etc.).

During cultivation in the method of the present invention, a medium containing a carbon source that can be assimilated by microorganisms, a nitrogen source that can be digested, and inorganic salts is generally used.

In addition, the culture medium may contain micronutrients, growth-promoting substances, etc. as necessary.
Trace amounts of active substances such as precursors may also be added. In general, glucose and sucrose are carbon sources that can be assimilated by microorganisms.
There are things like molasses, starch, and glycerin. Digestible nitrogen sources include meat extract, soybean flour, cornstarch liquor, peptone, casein, and cottonseed meal.
and inorganic nitrogen compounds such as ammonium nitrate compounds, all of which can be used effectively. Culture may be carried out by surface culture method, or usually by deep aeration culture method. When using the deep aeration culture method, the nature of the medium should be around neutrality, and the temperature during culture should be between 20 and 20℃.
It is best to maintain the temperature around 36°C, preferably between 24 and 30°C. However, the culture conditions such as the culture composition, liquid nature of the medium, culture temperature, and number of agitation should be adjusted and selected as appropriate to obtain favorable results depending on the type of bacterial strain used and external conditions. Needless to say.

To collect TAN-1171 of the present invention from a culture solution,
Considering that the substance is a water-soluble amphoteric substance, as shown in the Examples below, separation and purification means commonly used to collect such microbial metabolites are appropriately used. For example, a method utilizing the difference in solubility with impurities, chromatography, etc. may be used alone or in combination.

The carriers used in chroma I/graphy include conventional inorganic and organic carriers such as activated carbon, high porous resins, ion exchange resins, alumina, cellulose, ion exchange cellulose, Sephatex, etc. used.

In particular, since the compound TAN-1171 is a water-soluble amphoteric substance, it can be effectively used in carriers having ion exchange ability, such as ion exchange resins and ion exchange Sephadex. As the ion exchange resin, cation exchange resin 1 such as Amberlite 1R120, IRC-50, CG-5
0 (manufactured by Rohm and Haas, USA), TOWEX 50W (manufactured by Tau Chemical, USA), and anion exchange resins such as Amberlite IRA-4,02.
1RA68 (manufactured by Rohm and Haas, USA)
For example, CM-Sephadex C-25 (manufactured by Pharmacia, Sweden) is used as the ion exchange Sephadex.

To give a more specific example of a method for collecting TAN-1171 from a culture solution, first, culture soil clear or culture solution obtained by separation by centrifugation or filtration is treated with ion exchange resin Amberlite 1R120 (ammonium After washing with water, the active substance is eluted with dilute ammonia water. After concentrating the eluate to remove ammonia,
Furthermore, it is passed through an Amberlite IRC-50 (type) column. After washing the column with water, it is eluted with dilute ammonia water, the active fraction is collected and concentrated, and CM-Sephatex C-
25 (Na type) column chromatography,
Expand with water. The active fractions are collected, concentrated, and lyophilized to obtain a crude powder.

The obtained crude powder is dissolved in water and further adsorbed on a column of CM-Sephatex C-25 (H type), developed with diluted sodium chloride water, and the active fractions are collected and concentrated. After desalting the concentrate using a desalting device, it is freeze-dried to obtain an intermediate purified powder.

Dissolve this in water and reuse CM-Sephadex (H type)
After column chromatography, it was developed with diluted sodium chloride water, HPLC showed a single peak, both fractions were collected and concentrated, desalted using a desalting device, and lyophilized.
A purified powder of '1-1471 (educt) is obtained.

After desalting both fractions shown by a single peak in "-", the pH was adjusted to 3 with dilute hydrochloric acid, passed through a column of Biogel P-2 (Bio-Rad, USA), developed with water, and the fraction showing a single peak was extracted. By collecting, concentrating, and lyophilizing, a white powder of TAN-1171 hydrochloride can be obtained.

Moreover, if it is difficult to obtain purified powder by such column chromatography (if the content is low and there are many impurities), high performance liquid chromatography can also be used.

The physicochemical properties of the TAN-1171 educt thus obtained are as follows.

■) Shape: White powder 2) Specific rotation [α] 26-9!1.4° (cm 0.2
8.0.21 Hydrochloric acid) 3) Mass spectrometry value (Sl-MS method):
m/z244 (M1H) 4) Molecular formula: C1oH17N3o4 5) Elemental analysis value - Calculated value (as C,.H17N304/H2O) - C,45,97: H17,33,N,
16.08 Actual value: C, 46, 26, H17, 54;
N, 16.036) Ultraviolet absorption spectrum: terminal absorption (in aqueous solution) 7) Infrared absorption spectrum (cm' by KBr tablet method) 34] 0 3050 1665. +590.1390
．． 1265.1110 (see Figure 1) 8) 1H nuclear magnetic resonance spectrum (300MHz, in heavy water) 5.90 (1H, dq, J=5.9, 2.2), 5.
56 (1H, dQ, J = 5.82.2), 4.45 (
ill, d, J=4.7), 4.11(II), Q,
J=7.1), 3.62 (1H, dt, J=7.8,
3.2), 3.23 (III, m), 2.63 (11
1, ddQ, J=17.9, 8.0, 2.4), 2.
23(1H,brd,J-17,9), 1.55(
3H, d, J = 7.1) 9)”C nuclear magnetic resonance spectrum (75MHz, chemical shift in heavy water, δppm)
: 179.7(s), 175.4(8), 135.
5((+), 130.5(d)65.9(d),
59.8(d), 54.9(d), -52,2(
d) 39.0 (t), 20.3 (q) 10) Solubility Easy to dissolve, water soluble Dimethyl sulfokind 4 Difficult m: Methanol 11) Thin layer chromatography (support, silica gel glass plate 60 F, 5. , 0.25 mm, manufactured by Merck & Co., West Germany) Developing solvent Rfn-butanol-acetic acid-water (2': 1: 1) 0.32 acetoni I/Ryroux water (2: I) 0.1712) High performance liquid chromatography carrier LiChrospher ] OORP-18(e
) 5μm (Merck)/lX125mm Solvent system 3% acetonitrile 10.02M! J acid buffer (pH 5,0) - 0,005M sodium octanesulfonate flow rate: 1. Od/min retention time (1,): 6N min + 3) positive color reaction, ninhydrin, Ehrlino Hibberton Greig
Rieber, talin molybdic acid reagent negative; Itame, I. Lagentorff reagent 14) Acidic, neutral, basic 1 ampholytic Also, since TAN-1171 is an amphoteric substance, it can be diluted with hydrochloric acid, Sulfuric acid, phosphoric acid, monoformic acid.

Acid addition salts can be formed with inorganic or organic acids such as acetic acid, tartaric acid, citric acid, etc., and physiologically acceptable salts can be formed with thorium salts, potassium salts, calcium salts, triethylamine salts, etc.

For example, the physicochemical properties of the hydrochloride obtained by the method of Example 2 are as follows.

1) Shape: white powder 2) Specific rotation [a No 92.7° (c = 0.4
7.0.2N Hydrochloric acid) 3) Mass spectrometry value (SI-MS method): m/z2444) Molecular formula: C,, H17N3'O
,-HC(!5) elemental analysis value: (C10H17N30
．． -11Cρ・I], O) Calculated value・C, 40, 34; H, 6177; N,
14.11; CQ 11.91 Actual value C, 40, 53; H, 6, 65; N,
14.32Cρ, 12. ．． 2g 6) Ultraviolet absorption spectrum Terminal absorption (in aqueous solution) 7)
Infrared absorption spectrum (by KBr tablet method: cm') 3400 2930 1670, 154 13g5
．． 1280. 1210°11, +0.1000.
795. 720 (see Figure 2) 8)'] (Nuclear magnetic resonance spectrum (300 MHz, in heavy water) 5,93 (1H, dQ, J = 5.9.2.1),
5.61 (1H, dQ, J = 5.9°2.1),
4.48 (111, d, J=5.4), 4.12 (I
ll, Q, J=7.1) 3.97(Ill, dL, J=
8.2, 3.3), 3.56 (Ill, m), 2.
82 (1H, ddq, J=18.5.8.3.2,31
2.53 (III, br d, J18.4), 1.5
5 (3H, d, J = 7.1) 9) 13c nuclear magnetic resonance spectrum (75 MHz, chemical shift in heavy water, δ
ppm) 178.7 (S), 173.5 (S), 13L7 (
d), 130.4(d).

65.8(d), 59.6(d), 53.0(
d), 52.0(d).

37.2 (t), 19.4 (Q) 10) Solubility: easily soluble in water, slightly soluble in dimethyl sulfoxide, methanol 11) Thin layer chromatography (support, silica gel glass plate 60 F 254.0.25 mm, West Germany, (manufactured by Merck & Co.) Developing solvent ]]1n-butanol-acetic acid-water 2:1:1)0.32acetonitrile-water (2:1) 0.1712)For high performance liquid chromatography carrier, 1Chrospher l 00 RP-1
8(e) 5 μm, 4 X l 25 mm (Merck) I medium system: 3% acetonitrile 10.02M phosphate buffer (p
H5,0) -0,005M Sodium Octane Sulfonate Flow Rate: 1. Od/min Retention time (tR): 6.1 min 13) Color reaction positive, ninhydrin, Ehrlich, Barton Gray-Gooley Hassock, phosphomolybutenic acid reagent 7 8 Negative, Sakaro, Drakendorff reagent 14) Acidic, medium Amphoteric compound TAN-1171 generates alanine and an unknown amino acid by acid hydrolysis, and in combination with other physicochemical properties, it was judged to be a novel dipeptide.

Next, the biological properties of TAN-1171 will be described.

Filter paper discs (manufactured by Toyo Seisaku Shin, diameter 8 mm) immersed in a 1000 μg/filtered aqueous solution of antibiotic TAN-1171 were plated on edible agar plates for various fungi, and after culturing at a predetermined concentration for a predetermined time, the filter paper discs were Table 1 shows the diameter of the growth inhibition circle formed around. In the table, "o" indicates that no inhibition circle was observed.

The medium used was as follows.

East Nitrogen Heath (Tifco, USA)
Added 2.0% glucose and 15% agar Table 1 Antibacterial spectrum of antibiotic TAN 1171 As shown in Table 1, antibiotic TAN-11171 exhibits antibacterial activity against certain types of fungi.

Furthermore, TAN--1] 7 ] was administered at a dose of 400 mg/
No acute toxicity was observed when administered intraperitoneally to mice.

As is clear from these results, it can be said that TAN1171 is an antibiotic that exhibits antibacterial properties against fungi and is not toxic to mammals.

Therefore, TAN-+17] can be used to treat fungal infections in humans and livestock and poultry.

For this treatment, TAN-1171 can be formulated into pharmaceutical compositions in various dosage forms according to known formulation techniques.

For regular use of TAN-1171 as a treatment for, e.g., Aspergillus infections, e.g.
Dissolved in physiological saline and administered parenterally as an injection, intravenously (1), subcutaneously or intramuscularly (usually 1 to 50 mg/kg)
per day, preferably 5 to 20 mg/kg/day.

In addition, as an oral agent, the antibiotic TAN-1171 is mixed with lactose to form capsules or sugar-coated tablets manufactured by a known method.
00 mg/kg/day, preferably 5-50 mg/day
kg/day.

Moreover, TAN-1171 obtained by the present invention can be used as an external fungicide. and tifTA
N-1] 71 is usually 0. ] ~IOW/V%.

Preferably, the solution is dissolved in distilled water or the like at a concentration of 0.5 to 5 W/V%, or based on vaseline or lanolin,
Usually 02 to 100 mg of TAN-1171 per gram,
Preferably, the ointment containing 1 to 20 mg can be used for sterilizing and disinfecting the skin or mucous membranes of humans and animals.

The antibiotic TAN-1,171 is also a promising compound as a synthetic intermediate for new pharmaceuticals.

EXAMPLES Next, the present invention will be explained in more detail with examples.
The present invention is not limited by this. Percentages indicate weight/volume % unless otherwise specified.

Example 1 20% glucose, 30% soluble starch, 1.0% corn steep liquor, 10% defatted soybean flour, 05% peptone, 0.3% sodium chloride in a 3ρ volume of Sakalokorchen.
, medium 5 consisting of carbonate 05% (pH 7 adjusted)
00d was injected, sterilized, and Streptomyces chartruysis A-344 (IFO14976FERM
1) One platinum loop was inoculated from the slant culture of 2691) and cultured at 28°C for 48 hours on a reciprocating shaker at 20 cycles/min. In a 50Q capacity stainless steel tank, add Actocol (antifoaming agent, Takeda Pharmaceutical Co., Ltd., Japan) to the above medium composition.
Prepare medium 30 (added 0.005%).

500 d of sterilized and previously cultured Sakarokorhen culture solution
was inoculated into this, the aeration rate was 3012/min, and the number of stirring was 280.
A seed culture solution was obtained by performing deep culture at 28° C. for 148 hours at rpm.

20Of! Glucose 0 in stainless steel tank
5% 1 Textlin 5%, Absorbent Cotton Seed 3.5%.

Medium 12 consisting of carbonate 07% (pH 6,5)
Prepare and sterilize 0ρ by inoculating the seed culture solution 6ρ,
Airflow amount 12Of! /min, stirring number 18 Orpm and 28°
C. Culture was performed for 90 hours.

Example 2 The culture solution (100Q) obtained in Example 1 was adjusted to pH 3 and incubated with Hytulo Supercell (Johns-Manvil).
Amberly I-I R, 120 (NH, + type, I Of!: (Rohm & Haas, USA) column. After washing the column with water (30C), 0.2N-ammonia water (
50ρ).

After concentrating the eluate to remove ammonia, it was passed through a column of Amberlite TR (-50 (H+ type, 5Q). After washing the column with water (1!M), 05N-ammonia water (40ρ) eluted, collected only the latter 20ρ, and
It was concentrated to /20. After concentration (1 g), the pH was adjusted to 6, and it was subjected to column chromatography on CM-Sephatex C25 (Na+ type, 3Q, Farmana, Sweden), developed with water, and fractionated at 200 d. Fraction No. 8 to 15 (after collecting and concentrating fractions 1 and 6,
Freeze-drying yielded 139 g of crude powder. Dissolve 13g of this in 150d of water and add CM-Sephadex C25.
(H+ type, passed through a 1.2f column and washed with water (36Q)
After drying, develop with 0.02M thorium chloride aqueous solution,
It was fractionated to 200d.

Fraction Nos. 33 to 45 (2,612) were collected, concentrated to 50 tIfl, and transferred to a desalting device Microasylyzer Gl (Amplex Cartridge, ACllo-
]0. After desalting using Asahi Kasei Kogyo Co., Ltd. 2 Japan), it was freeze-dried to obtain a coarse powder. 4g of l was obtained.

Dissolve 500 mg of this in 10 d of water and reuse CM-
After being subjected to column chromatography on Sephatex (H”, I0Od) and washed with water (300d), 002
The mixture was developed with M sodium chloride solution and fractionated into 10 columns. Fraction No. 2'I~50 (270〃a) was collected and concentrated, and Ushigai! When it is completely desalted using Microasylyzer G1 in a cold room at 4°C and then freeze-dried,
A white powder (1717 mg) of TAN-1171 was obtained.

After desalting the remaining half of the above concentrated solution using a microacylizer, the pH was adjusted to 3 with dilute hydrochloric acid and transferred to a column of Iogel P-2 (500d, Biorat, USA) in a low temperature room (4°C). The mixture was developed with water and fractionated to 1 Od. Fraction Nos. 40 to 44 (50d) were collected, concentrated, and lyophilized to obtain a white powder (38.3 mg) of the hydrochloride of TAN1171.

Effects of the Invention The novel antibiotic TAN-1171 of the present invention and its salts exhibit antibacterial activity against fungi, and can be administered to humans and other animals orally, parenterally, or externally to treat these fungal infections. It can be advantageously used for the treatment of.

[Brief explanation of drawings]

FIG. 1 shows the infrared absorption spectrum of TAN-11171 free form, and FIG. 2 shows the infrared absorption spectrum of TAN-1171 hydrochloride.

Claims (1)

[Scope of Claims] (1) Antibiotic TAN-1171, or a salt thereof, having the following physicochemical properties as an educt. 1) Shape: White powder 2) Molecular formula: C_1_0H_1_7N_3O_43) Ultraviolet absorption spectrum: Terminal absorption (in aqueous solution) 4) ^1H
Nuclear magnetic resonance spectrum (300 MHz, chemical shift in heavy water, δppm): 5.90 (1H, dq, J = 5.9, 2.2), 5.5
6 (1H, dq, J = 5.8, 2.2), 4.45 (1
H, d, J=4.7), 4.11 (1H, q, J=7.
1), 3.62 (1H, dt, J=7.8, 3.2),
3.23 (1H, m), 2.63 (1H, ddq, J=
17.9, 8.0, 2.4), 2.23 (1H, brd
, J=17.9), 1.55 (3H, d, J=7.1)
5)^1^3C nuclear magnetic resonance spectrum (75MHz, chemical shift in heavy water, δppm): 179.7 (s), 175.4 (s), 135.5 (d
), 130.5(d), 65.9(d), 59.8(d
), 54.9(d), 52.2(d), 39.0(t)
, 20.3(q) (2) Belongs to the genus Streptomyces and is an antibiotic TAN-1
Cultivating a microorganism capable of producing 171 in a medium,
TAN-1171, which is characterized by producing and accumulating antibiotic TAN-1171 in a culture and collecting the same.
or the method of manufacturing the salt. (3) Streptomyces chartruisis A-344
.