Antibacterial compounds and process for production thereof

Abstract

The antibacterial compound S551-II (Reductiomycin) is produced by culturing a microorganism belonging to the genus Streptomyces.S551-II-A is prepared by sublimation of the compound S551-II.

Description

SUMMARY OF THE INVENTION

The present invention relates to new antibacterial compounds S551-II (Reductiomycin) and S551-II-A. The compound S551-II is produced by culturing a microorganism belonging to the genus Streptomyces in a nutrient medium. The compound S551-II-A is produced by sublimating the compound S551-II.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the infrared absorption spectrum of the compound S551-II.

FIG. 2 shows the infrared absorption spectrum of the compound S551-II-A.

FIG. 3 shows the nuclear magnetic resonance spectrum of the compound S551-II.

DETAILED EXPLANATION OF THE INVENTION

The present invention relates to new antibacterial compounds represented by the following general formula: ##STR1## (the compound having the following formula ##STR2## is referred to as Compound S551-II hereinafter) or ##STR3## (the compound having the following formula ##STR4## is referred to as S551-II-A hereinafter.).

The compound S551-II is also designated as Reductiomycin.

The antibacterial compound S551-II is produced by culturing a microorganism belonging to the genus Streptomyces and being capable of producing the antibacterial compound in a nutrient medium. The antibacterial compound is accumulated in the culture medium and is isolated therefrom.

The antibacterial compound S551-II-A is produced by sublimation of the compound S551-II.

The present compound S551-II has the following physicochemical properties.

Appearance: A yellow fine crystal.

Melting point: 215.degree. C. (decompose).

[.alpha.].sub.D.sup.23.degree. : +281.degree. (c=0.30, acetone).

Molecular weight: 293 (by the mass spectrometry).

Elementary analysis: C: 57.36%, H: 5.25%, N: 4.73%. Molecular formula: C.sub.14 H.sub.15 O.sub.6 N.

Ultraviolet absorption spectrum (the maximum value, .epsilon. value): 273 nm (28655) (in methanol); 288 nm (21600) (in 0.1 N HCl-methanol).

Color reactions:

Ehrlich's reaction (in HCl): positive Ferric chloride reaction: positive Nitroprusside reaction (in alkaline): positive 2,4-Dinitrophenylhydrazine reaction: positive Pine-shaving reaction: negative Ninhydrin reaction: negative Schiff's reaction: negative Sakaguchi's reaction: negative

Infrared absorption spectrum by the paste method is illustrated in FIG. 1.

PMR spectrum measured in CDCl.sub.3 by using TMS as an internal standard is illustrated in FIG. 3.

Based on the foregoing data, the compound S551-II is considered to have the structural formula shown earlier.

The compound S551-II-A has the following physicochemical properties.

Appearance: A yellowish crystal

Melting point: 215.degree. C. (sublimate)

[.alpha.].sub.D.sup.23.degree. : 0.degree. (c=0.30, dimethylsulfoxide)

Molecular weight: 233 (by the mass spectrometry). Elementary analysis: C: 60.98%, H: 4.59%, N: 6.06%. Molecular formula: C.sub.12 H.sub.11 O.sub.4 N.

Ultraviolet absorption spectrum (the maximum value, .epsilon. value): 260 nm (52900) (in methanol and 0.1 N-NaOH.)

Color reactions:

Ehrlich's reaction (in HCl): positive Ferric chloride reaction: positive 2,4-dinitrophenylhydrazine reaction: positive Ninhydrin reaction: negative Schiff's reaction: negative Sakaguchi's reaction: negative Nitroprusside reaction: negative

Solubilities: The compound is soluble in acetone, dimethylsulfoxide and alkaline solution, slightly soluble in methanol and insoluble in bezene, chloroform ethylether and hexane.

Infrared absorption spectrum of S551-II-A by the paste method is illustrated in FIG. 2.

Based on the foreging data, the compound S551-II-A is considered to have the structural formula shown earlier.

Now, the process for producing S551-II is described below.

The new antibacterial compound S551-II is produced by culturing a microorganism belonging to the genus Streptomyces and being capable of producing the compound. A suitable microorganism belongs to Streptomyces griseorubiginosus. Its typical strain is Streptomyces griseorubiginosus KY 11448 (FERM-P 3836) (NRRL 11,268).

The strain has the following properties.

I. MORPHOLOGY

The spore forming mycelium shows flexous simple branching. A chain of ten or more spores is formed.

The surface of the spore is smooth and the diameter of its minor axis and major axis are 1.0.mu. and 2.5.mu., respectively. The sporophore is formed on the aerial mycelium.

II. CULTURE CHARACTERISTICS

______________________________________ Color of the substrate mycelium The Aerial The reverse SolubleMedium Growth Mycelium surface side pigment______________________________________Sucrose- Poor Poor White Whitenitrate Flat White(a) (a) (a) NoneagarGlucose- Moderate Moderate Cream Lightasparagine Raised Light (11/2a) Yellow Noneagar Ivory (11/2ea) (2ca)Glycerin- Moderate Moderate Cream Lightasparagine Flat Light (11/2a) Yellow Noneagar Ivory (11/2ea) (2ca)Starch Good Good Cream Light Butteragar Raised Cream (11/2a) Yellow Yellow (11/2a) (11/2 ea) (11/2ea)Tyrosine Good Good Dull Mustard Antiqueagar Raised Cream Gold Brown Gold (11/2a) (2ng) (2ni) (11/2ne)Nutrient Good Bright Bright Amberagar Raised None Gold Gold (3pc) (2nc) (2nc)Yeast Good Good Golden Golden Mustardextract- Raised Putty Brown Brown Goldmalt (11/2ec) (3pg) (3pg) (2ne)extractagarOatmeal Good Dull Dull Cloveagar Raised None Gold Gold Brown (2ng) (2ng) (3ni)______________________________________

The color indications are given according to the classifications in the Color Harmony Manual (Container Corporation of America).

III. PHYSIOLOGICAL PROPERTIES

Growth temperature: 25.degree. to 38.degree. C. Liquefaction of gelatin: negative Hydrolysis of starch: positive Coagulation and peptonization of skim milk: negative Liquefaction of skim milk: positive Formation of melanoid pigments: positive

IV. UTILIZATION OF CARBON SOURCES______________________________________Carbon source Utilization______________________________________D-Arabinose -D-Xylose -D-Glucose 2+D-Fructose 2+Sucrose -Inositol -L-Rhamnose -Raffinose +D-Mannitol 2+______________________________________

On the basis of the above observations and the description of E. Kuster, International Journal of Systematic Bacteriology 22 (3), 139 (1972), the strain is identified as Streptomyces griseorubiginosus.

As the fermentation medium employed in the present process, any synthetic or natural medium can be employed, so long as it contains a proper carbon source, a nitrogen source, inorganic materials and other necessary nutrients for the growth of the microorganism.

As the carbon source, various carbohydrates such as glucose, fructose, sucrose, galactose, xylose, sorbitol, mannitol, glycerol, starch, starch hydrolyzate liquor, molasses, blackstrap molasses, etc., various hydrocarbon such as ethane, propane, butane, n-paraffine, kerosene, etc., various organic acid such as acetic acid, fumaric acid, succinic acid, lactic acid, pyruvic acid and alcohols such as methanol, ethanol, etc. may be used.

As the nitrogen source, aqueous ammonia, various inorganic and organic ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium acetate, etc.; urea, acid amides, amines, amino acids, defatted cotton seed, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, fish meal or its digested product, defatted soybean or its digested product, soybean protein hydrolyzate; various microbial cells or its digested product, etc. may be used.

As inorganic materials, dipotassium monohydrogen phosphate, monopotassium dihydrogen phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium carbonate, magnesium phosphate, etc. may be used.

If other nutrients are necessary for the growth of the microorganisms used in the present invention, they must, of course, be present in the medium. They are sometimes supplied to the medium together with natural materials exemplified as a nitrogen source.

Culturing is carried out under aerobic conditions such as with shaking or aeration-agitation. Suitable culturing temperature is usually 23.degree. to 38.degree. C. It is desirable to keep the pH of the medium at 3 to 8, preferably around neutrality throughout culturing.

Culturing is usually carried out for 1 to 7 days to accumulate the compound S551-II in the culture liquor.

After completion of the culturing, microbial cells and precipitates are removed from the culture liquor by filtration or centrifugation and the compound S551-II may be recovered and isolated from the resultant solution by combination of ion exchange treatment, column chromatography using silica gel, etc.

The compound S551-II-A is prepared as follows. The compound S551-II is heated at a temperature of 215.degree. or more and is sublimated accompanying with deacetic acid reaction to form compound S551-II-A. The product crystallized on the glass plate was collected and washed with methanol to obtain a pure S551-II-A as crystals.

The antibacterial activity of the compound S551-II against various microorganisms by disc method (pH: no adjustment) is shown in the following Table 1.

Table 1______________________________________Microorganism tested MIC (mcg/ml)______________________________________Aspergillus niger IAM 2026 250Aspergillus niger ATCC 6275 1,000Aspergillus oryzae NRRL 692 62.5Penicillium chrysogenum IAM 7142 125Penicillium chrysogenum O 174 62.5Mucor Spinescens IAM 6071 500Dusarium moniliforme IAM 5062 125Myrothecium verrucaria IAM 5063 125Trycophyton mentagrophytes IAM 5064 4Mrycoderma T-1 ATCC 9645 16Chactomium globosum ATCC 6025 500Microsporum gypseum IFO 5948 125Alternaria solani IFO 5924 8Cladosporium herbarum Link Fr IAM 5059 31.2Bacillus subtilis PCI 219 62.5Bacillus subtilis IAM 1026 31.2Bacillus subtilis NA 64 62.5Sarcina lutea IAM 1099 125Staphylococcus aureus FAD 209P 62.5Diplococcus pneumoniae 1000Escherichia coli IAM 1268 1000Escherichia coli ATCC 3655 500Serratia marcescens IAM 1022 >1000Proteus vulgaris HX19 IAM 1025 >1000Pseudomonas aeruginosa IAM 1156 >1000Pseudomonas fluorescens ATCC 27 >1000Salmonella enteritidis >1000Shigella sonnei E23 >100Klebsiella pneumoniae 348 >1000Candida albicans IAM 4888 >1000Saccharomyces cerevisiae IAM 4485 1000Saccharomyces rouxii M-9 1000______________________________________

The antibacterial activity of the compound S551-II-A against various microorganism by agar dilution method (pH 7.0) is shown in Table 2.

Table 2______________________________________Microorganism tested MIC (mcg/ml)______________________________________Bacillus subtilis ATCC 10707 200Staphylococcus aureus ATCC 6538P 200Klebsiella pneumoniae ATCC 10031 200Proteus vulgaris ATCC 6897 25Escherichia coli ATCC 3655 500______________________________________

As it is obvious from above description, compounds of the present invention are useful to clean and disinfect laboratory glassware and surgical instruments, and may also be used in combination with various soaps for sanitation purposes and in cleaning and sanitizing hospital rooms and areas used for the preparation of food.

Practice of certain specific embodiments of the invention is illustrated by the following representative examples.

EXAMPLE 1

Composition of fermentation medium (as same as seed medium):

______________________________________Soluble starch 2%Gluten meal 2%Defatted cotton seed 2%Dry yeast 2%CaCOhd 3 0.3%MgSO.sub.4 . 7H.sub.2 O 0.07%Silicon K966 0.1% (V/V)(antifoaming agent)______________________________________

Streptomyces griseorubiginosus KY 11448 (FERM-P No. 3836) (NRRL 11,268) is used as a seed strain.

200 ml of seed culture obtained by culturing the above seed strain in 300 ml of seed medium in a Sakaguchi-flask for 50 hrs in advance is transfered into 30 l-Jar containing 15 l of said fermentation medium. Culturing is carried out at 30.degree. C. for 72 hrs with aeration of 14 l/min. and stirring at 300 r.p.m.

After culturing, culture broth is filtrated to remove microbial cells and the filtrate is extracted with about 15 l of ethylacetate. The resulting extract is concentrated to dryness under reduced pressure. The above separated microbial cells is extracted with acetone and the resulting extract is concentrated to dryness under reduced pressure. Then, the obtained residue is ectracted with ethylacetate and concentrated to dryness under reduced pressure. The resulting two residues are collected and is subjected to chromatography using silica gel (200 ml by volume) and then washed with 500 ml of benzene. Then, elution is carried out with each of 500 ml of benzene-methanol (1% by volume), benzene-methanol (2% by volume) and benzene-methanol (3% by volume). The eluates by benzene-methanol are collected and concentrated to dryness and then resulting residue is washed with about 30 ml of ethylether-chloroform (10:1 by volume). Then, recrystallization is carried out twice from about 8 ml of chloroform and about 5 ml of acetone, whereby 30 mg of compound S551-II is obtained as yellow fine crystal.

EXAMPLE 2

Synthesis of compound S551-II-A

Compound S551-II obtained in Example 1 is put on heater and is covered with Petri dish. Then, the compound is heated at 215.degree. C. whereby compound S551-II is sublimated and the volatile component is adhered to wall of Petri dish to form crystals. The resulting crystals are collected and are washed with methanol, whereby pure compound S551-II-A is obtained.

Claims

1. Compounds represented by the following general formula: ##STR5##

2. Compound S551-II represented by the formula: ##STR6##

3. Compound S551-II-A represented by the formula: ##STR7##

4. A process for preparing compound S551-II-A which comprises sublimating compound S551-II.

5. The process according to claim 4 wherein the sublimation is carried out at a temperature of 215.degree. or more.