JPS59204199A - Substance f6 for promoting antibacterial property and its preparation - Google Patents

Abstract

NEW MATERIAL:The antibacterial property promoting subsance F6 of formula and its salt. USE:It promotes the antibacterial activity of beta-lactam antibiotic substance. PREPARATION:A bacterial strain belonging to Pseudomonas genus and capable of producing the compound of formula [e.g. Pseudomonas acidophila G-6302 (FERM-P No.4344), Pseudomonas mesoacidophila SB-72310 (FERM-P No.4653), etc.] is cultured in a medium, and the compound of formula is collected from the cultured product.

Description

Detailed Description of the Invention The present inventors isolated a large number of microorganisms from soil for the purpose of searching for new antibiotics, and when searching for the antibiotics they produced, certain microorganisms were found to be novel antibacterial agents. that the microorganism belongs to the genus Pseudomonas, and that by culturing the microorganism in an appropriate medium, a substance that enhances the antibacterial action of β-lactam antibiotics against Durham-negative bacteria is accumulated in the medium. After learning about scales, we isolated the scales, and based on its physicochemical and biological properties, we recognized that the antibacterial enhancer was a new active substance, and called it antibacterial enhancer F6. It was to be.

This antibacterial enhancer F6 is a compound represented by the following formula (I).

That is, the present invention provides (1) a compound represented by formula (I) and a salt thereof, and (2) a bacterium producing the compound of formula (I) belonging to the genus Pseudomonas, cultured in a medium, and a culture containing the compound represented by formula (I). ) is a method for producing r# of the compound, which is characterized by producing and accumulating the compound, and collecting the compound.

In the present application note, the compound represented by formula CI) may be simply abbreviated as F6 in the following description.

In the present invention, the compound (E'6) of formula (1) can be produced by a microorganism belonging to the genus Pseudomonas, and the microorganism may be any bacterium that has the ability to produce the compound of formula (I). For example, Pseudomonas acidophila (P.
seudomonasacidophila ) G −
6302 strain and Pseudomonas mesoacidophila (
Pseudomonas mesoacidophila
) SB-72310 strain is mentioned.

The above oa-6302 strain and 5E-72310 strain were obtained from the Institute of Microbial Technology, Agency of Industrial Science and Technology (FBI).

1-1-3, Tsuka, Yatabe-cho, Tsukuba-gun, Ibaraki Prefecture, Japan).

Fermentation Research Institute (IFO, No. 5, Jusanhonmachi 2-17es, Yodogawa-ku, Osaka, Osaka, Japan) and The American Type Culture Collection (ATCC, T
he American Type Cu1tu
reCollection+ l 2301 Par
klawn Drive.

Rockville, Maryland 208
52 USA) as follows.

Among the above microorganisms, the mycological properties of strain G-6302 are disclosed in Japanese Patent Application Laid-Open No. 163501/1983 and US Patent No. 4.2.
No. 29,436 (patent date, West) M October 2, 1980
1), and the mycological properties of the 5B-72310 strain are described in Japanese Patent Application Laid-Open No. 55-49394 and U.S. Patent No. 4.225.
．． No. 586 (patent date: September 30, 1980).

The properties of the Pseudomonas bacteria used in the present invention are generally susceptible to change, for example, when exposed to ultraviolet light.

Any mutant strain that can be easily mutated by artificial mutation means using X-rays, chemicals, etc., and has the ability to produce the compound of formula (I) that is the object of the present invention, It can be used in the present invention.

When culturing Honkan, carbon sources include evaglucose, sucrose, and maltose.

Those that can be assimilated by bacteria are used as appropriate, such as blackstrap molasses, glycerol, oils and fats (eg, soybean oil, olive oil, etc.), organic acids (eg, citric acid, succinic acid, gluconic acid, etc.).

Examples of nitrogen sources include soybean flour, cottonseed flour, non-nutip liquor, dried yeast, yeast extract, meat extract,
peptone, urea.

Organic metal nitrides and inorganic nitrogen compounds such as ammonium sulfate, ammonium nitrate, ammonium chloride, and ammonium phosphate can be used. In addition, as inorganic salts, for example, inorganic salts necessary for the cultivation of normal bacteria such as thorium chloride, potassium chloride, calcium suboxide, magnesium sulfate, potassium phosphate, and disodium phosphate can be used alone or in appropriate combinations. be done. Furthermore, sulfur compounds that can be assimilated, such as inorganic sulfur compounds such as sodium sulfate and sodium thiosulfate, cystine, and cysteine.

It has been found that addition of organic sulfur compounds such as methionine increases the amount of target product produced. Particularly preferred are cysteine and sodium thiosulfate.

The concentration of sulfur compounds in the medium is 0.01 to 1.01#/
V% is more preferably 0.02 to 0.5 w/v%. Addition of sulfur compounds to the culture medium increases the amount of the compound of formula (I) produced, which is extremely advantageous industrially.

In addition, precious metals such as ferrous sulfate and tin sulfate, and vitamin B
Also, vitamins such as biotin are added as necessary. Furthermore, antifoaming agents and surfactants such as silicone oil and polyalkylene glycol ether may be added to the medium. Organic or inorganic substances that aid the growth of the bacteria and promote the production of the compound of formula (I) may be added as appropriate.

The culturing method may be the same as a general antibiotic production method, and solid culture or liquid culture may be used. For liquid culture, static culture, l! fi stirring culture.

Although any method such as shaking culture, 2-aeration culture, etc. may be carried out, aeration agitation culture is particularly preferred. Also, the culture temperature is approximately 15
℃~35tC range is preferred, and the pH of the medium is about 4~8
The culture is carried out for approximately 8 hours to 168 hours, preferably 24 hours to 144 hours.

Since the produced compound of formula (I) mainly exists in the culture 77''i solution, it is necessary to separate the culture into a supernatant and bacterial cells by centrifuging or filtration, and purify the supernatant. However, it is also possible to purify directly from the culture.

The titer of this substance can be measured, for example, by the Escherichia cup method or the Vapordinuk method.

In order to collect the compound of formula (I) from the culture, any means commonly used for collecting metabolites produced by microorganisms can be appropriately used. For example, after removing all bacterial cells by centrifugation, a method is generally used in which effective substances are separated, collected, and purified from the P solution. That is, differences in solubility and solubility in appropriate solvents, differences in precipitation methods and rates from solutions, differences in various adsorption affinities, ion exchange chromatography with ion exchangers or vacuum concentration, freeze drying, crystallization, recrystallization. , drying, etc. may be used alone or in combination in any order, or repeatedly.

An example of this is as follows. In other words, after the cultivation is completed, the culture solution is used as needed for bacterial cells. After separation, it is passed through an ion exchange resin to adsorb the compound of formula (I). Since this ion exchange resin is an acidic substance, anion exchange resin [Amberlite RA-400, 40
2. Column manufactured by Rohm & Haus, USA; Dowex 1, manufactured by Dow & Chemical Co., USA; Diaion 5A-21A ÷ Hon, manufactured by Mitsubishi Kasei (Japan)] C1 type, etc. can be advantageously used. To elute the adsorbed substance, an aqueous solution such as common salt is used to elute the effective substance. Desalting of the eluate is carried out by activity column chromatography. The adsorbed compound of formula (I) is then eluted using a hydrophilic organic solvent system. The hydrophilic organic solvent system is acetone.

Single or mixed solvents of lower ketones such as methyl ethyl gutone and methisobutyl ketone, lower alcohols such as methano-A', ethano-/L/, ingropanol, propano-/L/, butanol, etc. A mixed solution of and water is used. Next, after concentrating the eluate containing the effective substance, acetone or the like is added to collect the precipitate, which is then washed with acetone, ether, etc. and dried to obtain a light brown powder. To further purify the obtained powder, Q
AE Sephadex (Sweden) 7μ Masia)
Column chromatography can be advantageously used. That is, QAE Sephadex A-25 was phosphate buffered,
After washing with (pH 6,0), a solution of the previously obtained powder in water is passed through and adsorbed, and eluted with M/25 phosphate buffer.

After adjusting the pH of the F6-containing fraction to 6 or less, it is passed through the activated carbon column again, washed with water, and eluted with 8 V/V% isobutanol water. Otherwise, the effective fraction is concentrated under reduced pressure and then freeze-dried to obtain F6 powder.

F6 forms metal barrels or ammonium salts. Examples of metal salts include sodium salts, potassium salts, and lithium salts.

Physicochemical properties of F6 The physicochemical properties of the F6 monosodium salt obtained in Example 1 are as follows.

1) Melting point = 221-229 W (decomposed) 2) Color and shape of substance = colorless powder 3) Elemental analysis: 4 (dried under reduced pressure over phosphorus pentoxide for 1.6 hours).

Actual measurement as C, 7H28N30□3SNa”H20
α (%) Calculated value (support)] c 36.4
0 36.76H5,785,44 N 7.70 7.56s
5.76 5.77Na 4
, 0 4.144) Ultraviolet absorption spectrum: Only terminal absorption (no specific absorption above 210 nm) 5) Optical external absorption spectrum)/shi: Absorption spectrum by potassium bromide tablet) V is shown in Figure 1. show.

N-CH3000H) γ) Nuclear magnetic resonance spectrum/1/(Do A100MH2)
All −CH3 signals are observed in aZ and +a.

8) Solubility in solvents: Petroleum ether, hexane, diethyl ether.

Insoluble in benzene, ethyl acetate and chloroform. Slightly soluble in ethanol, pyridine, and acetone. methanol,
Soluble in dimethyl sulfoxide. Easily soluble in water.

9) Color reaction: Positive for Daley-Goorie-Partsk reaction and potassium permanganate reagent. Negative for ninhydrin reagent, Sakaguchi reaction, and Molish reagent.

10) Stability: Aqueous solutions with pH 3 to pH 9 are stable when heated at 60°C for 10 minutes.

11) Distinction between basic, neutral, and acidic: Acidic substances 12) Amino acid analysis = Hydrolyzed with 61J hydrochloric acid +10tl for 18 hours and analyzed by automatic amino acid analysis, β-alanine,
Glucosamine and 4-hydroxy-5-hydroxymethylproline were detected.

The physicochemical properties of F6 as shown above, its decomposition reactions, various compounds obtained by decomposition, and the spectrum data of F6 itself (IEISPMRlCM
, R, mass spectrum, etc.), F6 was identified as a compound represented by formula (1).

Next, the biological properties of the compound represented by formula (I) will be described. This compound exhibits the effect of enhancing the antibacterial properties of β-lactam antibiotics, and the following is a complete example of the data.

Table 1: Antibacterial enhancement effect of cefmetuxime and mecillinum by F6 obtained in Example 1 (Test bacteria: Escherichia coli IFO+2734) Table 1 * A gravy agar medium was impregnated with the drugs as described, and an 8-diameter paper was Tested using Dinuku.

※※25μ4 sample solution on a paper disk with a diameter of 8 ms
was soaked in and placed on agar.

The compound of the present invention represented by formula (I) exhibited significant antibacterial activity in synergy with cefmetuxime in mice infected with Escherichia coli. Additionally, compounds of formula (I) can be used in combination with these antibiotics to treat mammals (e.g., mice, rats, dogs, humans) and poultry (e.g., chicken, ahi/I).
/) can be used for the treatment of infectious diseases caused by Kokiwa bacterium.

To use a compound of formula (1), for example, in the treatment of E. coli infections, 0.5 to 1 oy of a compound of formula (I) can be mixed with 0.5 to 2.0 g of cefmetuxime in 1 oz of physiological saline.
It is dissolved in 0 to 100 ml, mixed with an intravenous drip solution, and administered to adults two to three times a day.

Furthermore, the compound of formula (I) is an extremely promising compound as a synthetic intermediate for new pharmaceuticals.

LD5 when the compound of formula (I) is administered intravenously to mice
° was >If/kg.

Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto.

Percentages are weight/volume % unless otherwise specified.
shows.

Example 1 Pseudomonas mesoacidophila SB-72310 (rERM-P 4653
; IFO1aaa + 1ATcc-31433), gluco-7-1%, polypeptone (manufactured by Rokupo Nutritional Chemical Co., Ltd.) 0.5%, meat extract 0.5%, salt 0.5% (pH 7).
.

Next, glycero/v 3%, Gumu cornu 0%, polypeptone 0.5%, meat extract 0.5%, NaC 10.5%
, medium 120e consisting of 0.1% cysteine was mixed with 20O4
The pH of the liquid was adjusted to 7.0 with 30% sodium hydroxide solution, and the mixture was heated at 120℃ for 2 hours.
After steam sterilization for 0 minutes, the seed culture was inoculated. Next, the temperature was 28°C, the aeration was 1201/min, and the stirring speed was 180.
The cells were cultured for 78 hours under r, p, m conditions. This culture was grown using Amberlite E R-68 (CI) (ROHM, USA).
It was passed through a column packed with 30e (manufactured by Andhaas).

After washing with 90 g of water, elution was carried out with 240 e of 2% saline. After collecting the effective fraction and adjusting the pH to 4.0, the activated carbon column (15
6), washed with 45e of water, and eluted with 7 v/v% isogetanol water. After concentrating the effective fraction under reduced pressure to 100 liters, Mechnol-acetone (100st/-1
00ml) was added and the precipitated precipitate was dried under reduced pressure to obtain 25f of crude material.

The crude substance 24f obtained here was dissolved in 500 ml of water, and then mixed with Q A E 5ephadex A-25 (Sweden), /l/Masia, buffered with M/25 phosphate buffer (pH 6,0). Column 1000g/manufactured by
Ic was passed through and adsorbed. Next, elution was performed using the same buffer. Collect the effective fraction and apply it to activated carbon column 400
d was passed. Wash the same column with 1200ml of water,
Elution was performed with 8v/v% inbutanol water, and the loading fractions were collected and adjusted to p and H5.5, concentrated under reduced pressure, and then freeze-dried to obtain a powder of 810q. Dissolve this powder in 20wt water and repeat Q A E -5ep.
hadex A-25 (C1) column (100*/)
was passed through and adsorbed. M/251 phosphate buffer (p
Elution was performed with H6.0), and the effective fraction was concentrated and desalted using an activity column (50 ml). f3 V
/V% Inbutanol eluate was condensed to powder 4
214 (/ was obtained. This powder was dissolved in +ov/v% methanol water and adsorbed on 5ephadex LM-20 (manufactured by Pharmacia Co., Ltd.), and the same solution was dissolved in itl/;
It was developed with thread and elution was performed. The F6-containing fractions were collected, concentrated, and lyophilized to yield 37 μm of white powder. The optical external absorption spectrum is shown in FIG.

5rJ) JtJNm c 36.40. H5,78
, N 7.70°S 5.76, Na 4.0 (%
).

[Brief explanation of the drawing]

FIG. 1 shows the infrared absorption of F6 monosodium salt obtained in Example 1.

Claims (2)

[Claims] (1) Formula %formula% A compound represented by and its salt. (2) A method for producing the compound of the above formula, which comprises culturing a bacterium that produces the compound of the formula belonging to the genus Pseudomonas in a medium, producing and accumulating the compound of the above formula in the culture, and collecting the compound. .