JPS6192577A - Novel macrolide antibiotic and its production - Google Patents

Abstract

PURPOSE:To obtain the titled antibiotic useful as an additional matter for feed, remedying animals, etc., effective for infectious diseases of inspirator, by inoculating a specific mold capable of producing an antibiotic to be prepared into a medium, cultivating it aerobically, and separating the antibiotic from the culture material. CONSTITUTION:Micromonospora griseorubida A11725 strain (FERM P-705), a ray fungus capable of producing macrolide antibiotic, is inoculated into a medium consisting of dextrin, glucose, casin hydrolyzate, yeast extract, CaCO3, etc., and cultivated aerobically at 20-37 deg.C for 4-5 days, to give a culture material. The culture material is filtered off, the filtrate from which the mold and other solid substances are separated is extracted with an organic solvent such as CHCl3, etc., the organic solvent is distilled away under reduced pressure, and a crude substance containing the antibiotic to be produced is obtained. The substance is fractionated by column chromatography method, a solvent is distilled away under reduced pressure, and concentrated to dryness, to give the titled antibiotic A11725I having a structure shown by the formula, properties such as 711 molecular weight, 103-107 deg.C melting point, specific rotatory power (D-ray, at 25 deg.C, C=1, methanol), etc.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a novel macrolide antibiotic exhibiting extremely excellent antibacterial properties against Staphylococcus and Mycoglazma, and a method for producing the same.

Means to Solve the Problems The present invention utilizes Micromonospora griseorvida strain A11725 (M
ieromonogpora *griseorubi
daA11725) (Feikoken Jokyo No. 705) is cultured in a medium, and a new macrolide antibiotic (hereinafter referred to as A117251) is extracted and separated from the culture, and a method for producing the antibiotic. be.

The novel antibiotic A 117251 of the present invention is a basic nitrogen-containing compound having the following physicochemical properties.

A117251 Color property White powder Molecular formula 〇37 formula 凰No. Snow elemental analysis C: 62.34 H: 9.25 N
: 1.96 Molecular weight (from mass spectrum) 711 Melting point (or decomposition point) 103-107℃ Figure 2 IR (KBr method) Figure 1 NMR (100MHz TMS) (CDα3) Figure 3 T
LC silical CHα3:methanol (5:1) 0.48 silical CHα3:methanol = 7% aqueous ammonia (40:12
:20) Lower #1 0.72 Color reaction O Potassium permanganate aqueous solution decolorization e Ninhydrin reaction, Sakaguchi reaction, ferric chloride reaction Distinction between acids and bases Basic solubility Acidic water, methanol, acetone, acetic acid Antibacterial treatment using the agar dilution method of the antibiotic (free base) of the present invention, which is soluble in organic solvents such as ethyl and benzene, and soluble in basic water;
The spectra (indicating the minimum inhibitory concentration) are shown in the table below.

In addition to the above properties, the antibiotic A117251 of the present invention has the following properties:
A 16-membered macrolide, desosamine and 6-
It has one sugar of dioxy-2,3-di-O-methyl-hexose, has the structure expressed by , and does not have an aldehyde group in its molecule. Based on these facts, this substance is There are no known antibiotics belonging to the basic macrolide group, and it is considered a new substance.

The substance of the present invention can be used in the form of a salt with a pharmaceutically acceptable mineral acid or organic acid, for example, a salt such as a tartrate, a citrate, or a succinate.

The antibiotic of the present invention can be taken orally in the form of a tablet or powder, or can be administered intravenously.

The amount used is about 400 to 2,000 per day for an adult, and it is effectively used against respiratory infections caused by Durham-positive bacteria such as Staphylococcus.

Furthermore, the antibiotic of the present invention can be used as an antibiotic for feed addition or for animal treatment.

The novel antibiotic 79A117251 of the present invention can be obtained as follows. For example, Micromonospora griseorvida A 11725 strain (Micromonospora griseorvida A 11725 strain)
-ra IIgriseorubida A″1172
5) is aerobically cultured in a medium containing medium components commonly used for culturing microorganisms. A solid medium or a liquid medium is used as the medium, but especially for mass production, liquid medium,
Particularly suitable are aqueous media.

As a medium component, suitable carbon sources include glucose, starch, glycerin, sucrose, molasses, dextrin, and the like.

Begton, meat extract, soybean flour, casein thaw, etc. are suitable for nitrogen treatment, but cottonseed meal, corn steep liquor, nitrates, and ammonia salts can also be used.

Other inorganic substances that can be used include substances containing cations such as sodium, potassium, magnesium, calcium, cobalt, manganese, and iron, and/or substances containing anions such as chlorine, sulfuric acid, phosphorous powder, and acetic acid. Furthermore, dry yeast and yeast extract can be used as bacterial growth promoting factors. Calcium carbonate can also be added to the medium to adjust the pH of the medium.

Also silicone to suppress foaming during culturing.

Appropriate amounts of resins, animal and vegetable oils, etc. can be added to the medium.

A particularly suitable medium for carrying out the method of the present invention is a medium containing glucose, dextrin-defatted soybean flour, charcoal [s9 calcium, and cobalt chloride] as medium components.

As for the culture conditions, the well-known culture method used for producing the known antibiotic 'J-' can be used. Culture temperature is 20℃ to 37℃
℃ range, particularly temperatures between 26°C and 30°C are suitable. The number of culture days varies depending on the culture conditions, but is usually 4 to 5 days.
It is day.

Any known culture method can be used, but the aerated agitation culture method in a fermentation tank is particularly suitable for mass production. In order to isolate and collect the antibiotic A117251 of the present invention from a culture, first, bacterial cells and other solid substances are removed by filtration or centrifugation, and then extracted from P by an organic solvent extraction method. is the most suitable method. As organic solvents used for extraction, chlorinated hydrocarbons such as chloroform, dichloroethylene, and trichlorethylene, fatty acid esters such as ethyl acetate, butyl acetate, and amyl acetate are used.
Any organic solvent that dissolves 1 well and is difficult to mix with water can be used.

The organic solvent extract containing A117251 is concentrated to 91/100 to 1/200 volume by distilling off the organic solvent under reduced pressure, and then this concentrated solution is diluted with hydrochloric acid, sulfuric acid, acetic acid, etc. ~3.0, separate the aqueous layer and add pn with an alkaline solution such as caustic soda, caustic potash or ammonia.
Adjust to 7.8 to 9.0 and extract again with an organic solvent.

By distilling off the solvent from this extract under reduced pressure and drying it,
A crude material containing 9 A 117251 is obtained. This crude substance is fractionated using means such as column chromatography using silica dal, countercurrent distribution method, etc., and each fraction is subjected to silica dal thin layer chromatography to confirm its components, and A117251 is purified. The containing fractions are collected, and the solvent is distilled off from them under reduced pressure, followed by drying to obtain the respective white powders.

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

Example [A] Dextrin 1%, grape s 1%, casein thawed product 0.5%, yeast extract 0.5%, calcium carbonate 0.
Contents 50 ml of medium (pu', o) containing 1%
The mixture was aliquoted into a 0-volume Erlenmeyer flask and sterilized by heating at 120°C for 20 minutes.

Micromonospora griseorvida A in 10 bottles of this culture medium.
11725 strains (MIcromOnospOra ag
One platinum loop from the slant culture of R. rige O-rubida A11725) was cultured with shaking at 30° C. for 120 hours in contact + IL. Next, this was heated and sterilized to obtain a medium 20I! having the same composition as above. 301 jar fermenter containing
Transplanted to 30°C for 72 hours at 300 r, p, m, 2 min/min.
The culture was carried out under aeration and agitation under sterile air conditions of 0.01. Next, dextrin 5%, glucose 0.5%, defatted soybean flour 3%,
Heat-sterilized medium C containing 0.2% calcium carbonate
pH 7,2) The above culture 101 was transferred to a 250 volume tank containing 200 J and 30 tl::, 120
Culture was carried out under aeration and agitation conditions at 250 r, Pm, and 100 l/min of sterile air to obtain 190 cultures.

(B) The above culture was filtered using 190 pieces of Art, and after removing bacterial cells and other solid substances, 160 pieces of p liquid was obtained. This p liquid was extracted with the same amount of ethyl acetate to obtain 160 g of an ethyl acetate solution containing the target product. This was concentrated under reduced pressure to 501, and then mixed with an aqueous hydrochloric acid solution of pH 2.5 to transfer and dissolve the target product in the aqueous layer. Furthermore, the pH of the aqueous hydrochloric acid solution was adjusted to PTL 8.5 using concentrated ammonia, extracted with 201 chloroform, and the chloroform layer was concentrated to dryness to obtain 8.5 g of a crude product.

[C) 8.5 g of the above crude product was dissolved in 50 ml of chloroform and adsorbed onto a silica dull column (3 m x 55 cm) that had been filled with chloroform in advance. Then chloroform-methanol-28thiammonia (20:1:0
．． 1) Developed with a different solvent and fractionated at 15m/d.

For each fraction, Bacillus petilis
Antibacterial activity using chloroform-methanol-7% ammonia water (40:1
The desired product was confirmed by thin layer chromatography using 2:20: lower layer as a developing solvent, and fractions containing it were collected.

The 61st fraction and the 78th fraction contained only a substance identified as A11725-1. Both fractions were condensed and dried to obtain A11725-11.2 and Q.

Effects of the Invention The novel antibiotic lτA]17251 of the present invention can be used effectively and effectively against respiratory infections caused by Durham homophytes, and can also be used as a feed additive and as an antibiotic for animal treatment.

[Brief explanation of the drawing]

Figure 1 shows the infrared absorption spectrum of the 'fr antibiotic Al1725I of the present invention, and Figure 2 shows the infrared absorption spectrum of the antibiotic A11 of the present invention.
The ultraviolet absorption spectrum diagram of 7251, Figure 3 is the nuclear magnetic co-↓ spectrum diagram of this antibiotic A117251.
Ruru.

Claims (2)

[Claims] (1) A new antibiotic A11725 with the following properties
I and its pharmaceutically acceptable salt Color property White powder Molecular formula C_3_7H_6_1NO_1_2 Molecular weight 7
11 Melting point 103-107℃ [a]^2^5_D -40.0° (C=1, methanol) Ultraviolet absorption spectrum As shown in Figure 2 Infrared absorption spectrum As shown in Figure 1 Nuclear magnetic resonance As shown in Figure 3 Color reaction as shown Decolorization with aqueous potassium permanganate solution + Ninhydrin reaction, Sakaguchi reaction, ferric chloride reaction − Distinction between acids and bases Basic solubility Soluble in acidic water, methanol, acetone, ethyl acetate, benzene; basic insoluble in water (2) Bacteria belonging to the genus Micromonospora that produces the antibiotic A11725 I are inoculated into a medium containing assimilated carbon and nitrogen sources and inorganic substances, and after culturing aerobically, the products are extracted from the culture. A method for producing a novel antibiotic A11725 I, which comprises separating and collecting it.