JPS6366155A - Novel antibiotic jietacins and manufacture - Google Patents

Abstract

(57) [Abstract] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a new antibiotic Genocin class useful as an antiparasitic agent or a pharmaceutically acceptable salt thereof, and a method for producing the same.

[Conventional technology]

Known antiparasitic agents include, for example, antibiotics such as Ebarme, cutin, paromomycin, and synthetic agents such as levamisole and nicrosamide.

These are used as medicines and veterinary drugs to treat nematode infections and tapeworm infections, and their effectiveness has been recognized.

[Problem that the invention seeks to solve]

However, all known antiparasitic agents have problems such as toxicity, side effects, and resistance, and antiparasitic agents with less toxicity are desired.

In such a situation, it is a medical necessity for humans and animals to provide substances with antiparasitic activity.

[Means for solving problems]

Therefore, the present inventors, in order to solve the above problems,
With the aim of searching for new physiologically active substances, we isolated bacterial strains from various soils and continued research on their products.
It has been found that a substance having parasite movement inhibiting activity is produced in a culture of the KP-197 strain.

Next, a substance having parasite movement inhibiting activity was separated and purified from the culture, and as a result, two types of substances were isolated.As a result of examining the physical and chemical properties of the substances, it was found that they had the chemical structure as described below. It turns out. Since no other substance with such a chemical structure has been found, these substances are collectively called the antibiotic dietacin (Jielacin), and the substance in which R is an isopropyl group is called dietacin A, and the substance in which R is an isobutyl group is called dietacin A. We decided to call this substance Dietacin B. The present invention was completed based on this knowledge.

That is, the present invention provides the formula (wherein R represents an isopropyl or isobutyl group)
The substance dietacin represented by or a pharmaceutically acceptable salt thereof and a microorganism belonging to the genus Streptomyces and having the ability to produce the substance dietacin (1) are cultured in a culture medium, and the substance dietacin [■] is added to the culture medium. The present invention provides a method for producing the substance dietacin (1) or a pharmaceutically acceptable salt thereof, which is characterized by accumulating and collecting dietacin (hereinafter sometimes simply referred to as dietacins).

The physical properties of Dietacin A and B are as follows.

(r) Antiparasitic antibiotic yr Dietacin A (1) White powder (2) Elemental analysis C+ s Hx 4 N z 02 Calculated value: C69,57; H11,04; N 9.
02χ experimental value: C70,40; H11,09i N
7.68χ(3) Molecular weight and molecular formula Dietacin A mass spectrum at m/z 311 (M
+H) Ten molecular ion peaks were observed, and the high-resolution mass spectrum showed m/z 293 (M-OH)
Since the + ion peak was observed, the molecular formula was changed to C
+eH24Nz Ox. Therefore, the molecular weight of dietacin A is 310.

(4) Ultraviolet absorption spectrum shows a characteristic absorption maximum at 228 nm as shown in Figure 1, and 2
It shows absorption at the shoulder of 50 nm.

(5) Infrared absorption spectrum The infrared absorption spectrum measured by the KBrBr method is as shown in FIG. 2, and the wave numbers of the main absorption maximums are as follows.

2950.2870.1705.1475.1415.
1380.1270.1090,960 (cm-') (6) Nuclear magnetic resonance spectrum 'H measured in deuterated chloroform using a Parian X L-400, 400MH, NMR spectrometer
The NMR spectrum is shown in FIG.

(7) Color reaction Anisaldehyde-Sulfuric acid Positive Potassium permanganate Positive Sulfuric acid Positive Ninhydrin Positive Dragendorff Positive (8) Solubility in solvents Soluble in acetone, ethyl acetate, chloroform, n-hexane, dimethyl sulfoxide , insoluble in water and methanol, (9) The structure in which R is an isopropyl group in chemical structural formula (1) was determined by comparing the above physicochemical properties with the physicochemical properties of known similar compounds. It was determined to be 14-methyl-1-vinylazooxydipentadecan-8-one.

(II) Antiparasitic antibiotic Dietacin B (1) Colorless oil (2) Molecular weight and molecular formula In high-resolution mass spectra of Dietacin B,
By observing an ion peak of (M-OH) in soil 307, the molecular formula was determined to be C+ qHsbNzot.

Therefore, the molecular weight of dietacin B is 324.

(3) Ultraviolet absorption spectrum Same as Dietacin A (4) Infrared absorption spectrum Same as Dietacin A (5) Color reaction Same as Dietacin A (6) Solubility in solvent Same as Dietacin A (7) Chemical structure Dietacin A is a similar substance with a different number of carbon atoms in the alkyl side chain in the structure.

The structure in which R is an isobutyl group in formula (1) was determined by comparing the above physicochemical properties with those of known similar compounds, and is I5-methyl-1-vinylazooxy. It was determined to be hexadecane-8-one.

The microorganisms that have the ability to produce this dietacin belong to the genus Streptomyces, and for example, the K1)-197 strain, which belongs to the genus Streptomyces and was isolated by the present inventors,
This is an example of the strain most effectively used in the present invention, and the mycological properties of this strain are as follows.

(1) Morphological characteristics Vegetative hyphae develop well on various agar media, and no division is observed. Aerial mycelium grows slightly on starch inorganic salt agar media and appears white. When observed under a microscope, the aerial hyphae appear linear, and chains of 20 or more spores are observed. The size of the spores is 0.7-1. OXo, 7 μm and cylindrical. The surface of the spore is smooth. No sclerotia, sporangia and zoospores are found.

(II) Properties on various media E, B, Shirling
) and the method of D. Gottlieb (International Journal on Systematic Bacteriology, vol. 16, p. 313)
The following table shows the culture properties of this producing bacterium, which were investigated by (1966). The color tone was determined as a standard color using the Color Harmony Manual, 4th edition (Container Corporation on America, Chicago, 1958), and the code is also written in parentheses along with the color chart base. The following are the results of observations in each culture medium at 27°C for 2 weeks unless otherwise specified.

Culture properties (III) Physiological properties (1) Production of melanin pigment Positive (a) Tyrosine agar Positive (b) Peptone/yeast iron agar Positive (c) Glucose/peptone/gelatin medium (21-23°C) Positive (ni) ) Tryptone yeast solution positive (2) Tyrosinase reaction positive (3) Production of hydrogen sulfide
Negative (4) Nitrate reduction
Positive (5) Liquefaction of gelatin (21-23℃) (glucose-peptone-gelatin medium) Positive (6) Hydrolysis of starch Positive (7) Coagulation of skim milk (37℃) Negative (8) Peptone in skim milk (37℃) Positive (9) Growth temperature range 1
5-38℃ (10) Utilization of carbon source (Priedum-Gotlieb agar medium) Utilize; D-glucose, I)-fructosu,
Mannose, slightly utilized; D-mannitol, L-arabinose, D-xylose, sufrose, not utilized; L-rhamnose, i-inositol, raffinose, melibiose, salicin, (11) Cellulose degradation Negative (IV)
Cell Wall Composition Diaminopimelic acid in the cell wall is in the LL form.

The mycological properties of this bacterium are summarized as follows.

Diaminopimelic acid in the cell wall is of the LL type. Aerial hyphae have a linear morphology and form long spore chains. The surface of the spore is smooth. As for the properties of the culture, vegetative mycelia exhibit a beige color tone, and aerial mycelia exhibit a white color. Melanin is produced as a soluble pigment.

From these results, this bacterial strain belongs to the genus Streptomyces, and is classified according to Pridham and Tresner's classification (Burge's Manual on Determinative Batteriology, 8th edition, pp. 748-829, 1974). It is considered to be a bacterial species belonging to the White series.

This strain is Streptomyces sp. KP.
-197 (KP-IQ?), which has been deposited with the Institute of Microbiology, Agency of Industrial Science and Technology (National Institute of Microbiology, Japan, August 8889).

In the present invention, first, belonging to the genus Streso1 Myces,
Microorganisms capable of producing the substance dietacin (1) are cultured in a suitable medium. Examples of microorganisms used for culturing include the strains listed in ``2'', but mutant strains thereof can also be used in the present invention as long as they have the ability to produce dietacins.

The microorganisms used for culturing can be appropriately selected and used depending on the purpose of producing dietacin (1), such as both the substance dietacin A and the substance dietacin B, only one of them, or a strain that is more productive for one than the other. These strains can be obtained by improving appropriate strains.

Mutant strains of the above-mentioned strains can also be used in the method of the present invention as long as they have the ability to produce dietacins. Bacterial strains belonging to the genus Streptomyces that have the ability to produce other dietacins can also be used.

As the medium, a synthetic medium or a natural medium containing carbon sources, nitrogen sources, and inorganic substances suitable for normal culture of actinomycetes, as well as other nutrients as necessary, can be used.

The carbon source and nitrogen source used in the culture medium may be any type that can be used by the strain used. That is, various carbohydrates such as glucose, glycerol, fructose, maltose, mannitol, xylose, galactose, lipose, starch or its hydrolyzate can be used as the carbon source. The concentration is usually preferably 0.1 to 5% based on the medium. In addition, various organic acids such as gluconic acid, pyruvic acid, lactic acid, and acetic acid, various amino acids such as glycine, glutamic acid, and alanine, alcohols such as methanol and ethanol, various non-aromatic hydrocarbons such as normal paraffin, and plants. Alternatively, various animal fats and oils can also be used.

Examples of nitrogen sources include ammonium salts of various inorganic or organic acids such as ammonia, ammonium chloride, ammonium phosphate, ammonium sulfate, and ammonium nitrate, urea, peptone, NZ-amine, meat extract, yeast extract, dried yeast, and corn stew liquor. , casein hydrolyzate, fitschmeal or its digested product, soybean flour or its digested product, defatted soybean or its digested product,
Nitrogen-containing organic substances such as hydrolysates and various amino acids such as glycine, glutamic acid, and alanine can be used.

Examples of inorganic substances used include various phosphates, magnesium sulfate, common salt, and trace amounts of heavy metal salts.

Furthermore, when using a mutant strain that exhibits auxotrophy, it is of course necessary to add substances to the medium that satisfy its nutritional requirements, but this kind of nutrients must be added especially when using a medium containing natural substances. may not be necessary.

Cultivation is usually carried out under aerobic conditions such as shaking or aerated agitation culture. Practically speaking, deep aeration agitation culture is preferred. The pH of the medium is, for example, 5.0 to 8.0,
Near neutrality is preferred. The culture temperature is, for example, 20 to 40°C, but usually, for example, 26 to 32°C (preferably 27°C).
(nearly ℃). In the case of liquid culture, the culture time is usually 3~
Culture is carried out for 6 days, and the culture is terminated when the amount of accumulated genocins in the culture reaches the maximum. Culture conditions such as medium composition, liquid properties of the medium, culture temperature, stirring speed, and aeration amount are appropriately adjusted and selected depending on the type of bacterial strain used, external conditions, and the like. When foaming occurs in liquid culture, an antifoaming agent such as silicone fat, vegetable oil, or surfactant is appropriately used.

The present dietacins accumulated in the culture obtained in this way are usually produced in the culture filtrate.

To collect Genocins from culture filtrates, the means normally used for collecting metabolites from microbial cultures may be used singly, in combination in any order, or repeatedly. For example, filtration, centrifugation, dialysis,
? Means such as JI reduction, drying, freezing, adsorption, desorption, methods utilizing differences in solubility in various solvents (eg, precipitation, crystallization, recrystallization, dissolution, countercurrent distribution, etc.), chromatography, etc. are used.

Genosins are mainly chemically accumulated in the culture filtrate, so the present compound can be separated and collected from the culture filtrate after removing the bacterial cells from the culture solution.

In order to collect the main biological substance genosin from the culture solution, the culture filtrate is extracted with a non-hydrophilic organic solvent such as ethyl acetate, or the culture filtrate is extracted with activated carbon, alumina, porous synthetic polymer resin, or ion exchange resin. etc., elute with an elution solvent such as ethyl acetate, concentrate the resulting extract or eluate under reduced pressure, and then add an organic solvent such as hexane for extraction.

The obtained crude substance can be further purified by a known method commonly used for purifying fat-soluble substances, for example, column chromatography using a carrier such as silica gel or alumina.

Pharmaceutically acceptable salts of the present compound include, for example, hydrochloride, sulfate, nitrate, phosphate, carbonate, tartrate, citrate, and succinate, which can be prepared by conventional methods. can. Since this type of manufacturing method is well known, its explanation will be omitted.

〔Effect of the invention〕

Next, the antiparasitic activity and toxicity of the dietacins will be described.

Bursa nematode parasitic on pine trees
phelenchus 11gn1 colus) using the method of Kiku et al.
logical Chemistry 45 +249 (
It was determined based on locomotor activity according to (1981)).

As a result, the lethality (%) of Dietacin A against nematodes was 100% at 250ng/mj+, and 125ng/m
j! 99%, 63ng/mj! It was 86%. The lethality rate (%) of Dietacin B against nematodes is 250ng/
100% at mA', 93% at 125ng/ml, 63n
The concentration was 80% in g/ml. The activity of avermectin B1, a known antiparasitic agent, against nematodes was measured using the same method as above, and the lethality rate (%) against avermectin B1m0 nematodes was 2.5 μg.
/ml 100%, 1.25/j g/m 1
The concentration was 64%.

In an acute toxicity test using mice as test animals, when Jetacin A was administered intraperitoneally, even at a dose of 10 mg/kg, the mice were not lethal.

A similar administration test was conducted for avermectin BIB, a known antiparasitic agent, and as a result, administration of 1 Omg/kg caused death in mice.

These dietacins are less toxic than avermectin,
Furthermore, since it has antiparasitic activity against pine nematode at low concentrations, it is expected to exhibit excellent therapeutic effects as a therapeutic agent for parasitic infections in animals and plants.

The novel genocins of the present invention are useful as antiparasitic agents for humans and animals or plants.

〔Example〕

The present invention will be explained below with reference to Examples, but is not limited thereto.

500mj! Glucose 0.1% in Yosakaro flask,
Potato starch 2.4%, peptone 0.5%, meat extract 0.3%, yeast extract 0.5%, calcium carbonate 0.4%
A liquid medium (pH 7.0) containing 100 mA was dispensed and steam sterilized at 121°C for 15 minutes, and to this was added 1.0% glycerol, 1.0% calcium malate, 0.05% ammonium chloride, dipotassium phosphate 0.05%,
1 from a slant culture of strain KP-197 cultured at 27°C on an agar slant medium containing 0.1% yeast extract and 1.5% agar.
Platinum loopfuls were inoculated and cultured with shaking at 27°C for 3 days using a rotary shaker to obtain seeds.

2 x 301 jar fermenters with 2 x glycerol
．． 0%, cinchona flour2. θ%, NaCj 20.3%, citrulline 0.01%, cobal chloride) 0.002% liquid medium (pH 7,0) 15 p.
Steam sterilization was performed at 121°C for 30 minutes. Add to this the seed mother 6 above.
Transplant each portion and stir at a stirring speed of 25 Or, p.

Culture was carried out with aeration at 27° C. for 12 hours under aeration conditions of 15 ρ/min.

Centrifuge the culture solution using a Sharpless centrifuge (10,
00Or, p, m) The cells were separated into bacterial cells and culture supernatant.

The upper layer 30n of the obtained culture solution was diluted with 6N hydrochloric acid to p113.0.
and centrifuged in a centrifuge (10,00 (] r, p
, m) to obtain a precipitate. 151 parts of 50% acetone water was added to this precipitate, and the mixture was stirred and extracted, and the mixture was filtered to obtain an acetone extract. This extract was concentrated under reduced pressure to an aqueous solution of 61 ml. After concentration, 37 ethyl acetate! The operation of adding and shaking and extracting was performed twice. The extracted ethyl acetate extract was dried under reduced pressure to obtain an oily 1'l+ substance. Hexane 25 [) + n n was added to this concentrate and an operation of extraction was performed twice. The obtained hexane extract was concentrated under reduced pressure to obtain crude product 41K. This was charged to a silica gel column (manufactured by Merck & Co., ArL7734.160g),
Elution was performed with hexylline and ethyl acetate (30:1). Each fraction was followed by a bioassay using 7-horned nematodes, and the active fractions were collected and concentrated under reduced pressure to obtain 30 mg of crude material. Dissolve this in as little chloroform as possible and add 1/20th of the amount to a preparative reversed phase column for high performance liquid chromatography (Yamamura Kagaku Kenkyusha,
AM324 (ODS); 1ox300mm) was charged and the flow rate was 2m1/m with 60% acetonitrile water.
It was eluted at in.

Active fractions showing peaks at retention times of 18.5 minutes and 19.6 minutes were collected and dried under reduced pressure to obtain 7 J mg of Diexin A and 5 mg of Dietacin B, respectively.

[Brief explanation of drawings]

Figure 1 shows the ultraviolet absorption spectrum of the antibiotic Diexin A (measured with cyclohexane), Figure 2 shows the infrared absorption spectrum of the antibiotic (measured with KBr), and Figure 3 shows the proton nuclear magnetic resonance spectrum of the antibiotic (measured with KBr). (measured in chloroform). Figure 1

Claims (2)

[Claims] (1) Substance dietacins and their pharmaceutically acceptable salts represented by the formula ▲ Numerical formulas, chemical formulas, tables, etc. ▼ (In the formula, R represents an isopropyl or isobutyl group). (2) Belongs to the genus Streptomyces and has the formula ▲ Numerical formula, chemical formula, table, etc. ▼ (In the formula, R represents isopropyl or isobutyl group)
Cultivating a microorganism capable of producing the substance dietacin shown in a culture medium, accumulating the above compound in the culture,
A method for producing dietacins, which comprises collecting the same.