NO802574L - PROCEDURE FOR THE PREPARATION OF ANTIBIOTICS KA-6643 - Google Patents

Description

The present invention relates to the production of

antibiotic KA-6643.

Various strains of soil microorganisms have been isolated to investigate their ability to produce antibiotics. In the investigations leading to the present invention, it has been found that a recently isolated strain, referred to as KC-6643, is capable of producing two forms of new antibiotics with excellent antibacterial activities against both gram-positive and gram-negative bacteria, and it has been successful to isolate such antibiotics from the culture fluid.

These two forms of antibiotics produced according to the invention are structurally closely related to each other. As can be seen from their physico-chemical properties and biological properties to be described later, the antibiotic is a new substance that is different from all known antibiotics.

According to one aspect of the present invention, an antibiotic is provided which comprises at least one substance represented by the formula (I):

where R means a hydrogen atom or -SO^H.

According to another aspect of the present invention, it comprises a method for producing such an antibiotic consisting of the steps of cultivating a microorganism belonging to the genus Streptomyces and isolating the antibiotic substance from the culture liquid.

In the present application, the mixture of the two forms of the antibiotic where R is H and -SO^H respectively is designated as the substance KA-6643. The form where R in formula (i) is a hydrogen atom is further referred to as the substance KA-6643-A, while the other where R

in formula (I) is SO^ H. is designated as substance KA-6643-B.

Other objects, features and advantages of the invention will be readily apparent from the following description of certain preferred examples thereof, seen in connection with the accompanying drawings, although variations and modifications may be carried out within the scope of the invention.

In the drawings are:

Fig. 1 an ultraviolet absorption spectrum diagram of a sodium salt of the substance KA-6643-A; Fig. 2 an infrared absorption spectrum diagram of the sodium salt in fig. 1; Fig. 3 a "<*>"H nuclear magnetic resonance spectrum diagram of the sodium salt in fig. 1; Fig. 4 is an infrared absorption spectrum diagram of the methyl ester of the substance KA-6643-A; Fig. 5 an "H nuclear magnetic resonance spectrum diagram of the methyl ester from Fig. 4; Fig. 6 a c nuclear magnetic resonance spectrum diagram of the methyl ester from Fig. 4; Fig. 7 an ultraviolet absorption spectrum diagram of a disodium salt of the substance KA-6643-B; Fig. 8 an infrared absorption spectrum diagram of the disodium salt from Fig. 7; and Fig. 9 a nuclear magnetic resonance spectrum diagram of the disodium salt from Fig. 7. The organism which according to the invention produces the substance KA- 6643 has the following taxonomic characteristics. It should be noted that the morphological features in different iSP media are observed when cultured at 27°C for 21 days using standard methods and that the color is expressed for the mature culture in accordance with the classification in the ColorHarmony Manual ( Container Corporation of America), unless otherwise noted. ;I. Morphological characteristics;The strain producing the substance KA-6643 (hereafter referred to as "Strain KC-6643") forms an airy mycelium in distinct equal media that is individually branched. The sporophore is produced on the airy mycelium whereby straight spore chains are provided. Each spore is 0.4 to 0.5 µm x 0.9 to 1.0 µm in size, has an oval to cylindrical shape, and forms chains of 20 or more spores whose surfaces are smooth. The spore chains are often fused so that they form a membrane-like structure, and the division of the substrate mycelium is observed in some media used in taxonomic studies. II. Cultivation characteristics in different media; 1) Sucrose nitrate agar; Growth: Good, flat spreading, colorless - brownish yellow (2fb) Aerial mycelium: Moderate, powdery, white (a) ; Soluble pigment: None; 2) Glucose-asparagine- agar;Growth: Moderate, flat and buttery, cream (1 1/2 approx ) - pastel yellow (2hb);Aerial mycelium: None;Soluble pigment: None;3) Glycerol-asparagine agar;Growth: Good, warty, wrinkled, brownish yellow (2fg) - bamboo (2gc) Aerial mycelium: Good, powdery, pearly (3ba) ;Soluble pigment: None;4) Starch-inorganic salt agar;Growth: Good, wrinkled, buttery, bamboo (2gc) - honey yellow ;(2ic) ; Aerial mycelium: Good, powdery, white (a) - pearl (3ba); Soluble pigment: None; 5) Tyrosine agar; Growth: Good, significantly warty, wrinkled, gelatinous cinnamon; Aerial mycelium: Good, powdery, pearl (3ba); Soluble pigment: None; 6) Nutrient agar; Growth: Medium, slightly warty, wrinkled, gelatinous colorless-light yellow (1 1/2 ea); Aerial mycelium: None; Soluble pigment: None; 7) Yeast extract/malt extract agar; Growth : Abundant, wrinkled, smeared ktig - gelatinous mustard (2/e) Aerial mycelium: Good, powdery, white (a) - pearl (3ba) ;Soluble pigment: None;8)Oatmeal agar ;Growth: Moderate, flat growth, cream (1 1/2 ca ) -butter yellow ;(1 1/2 ga);Aerial mycelium: Moderate, powdery, white (a) Soluble pigment: None ;9) Peptone-g jaer-iron-agar;Growth: Good, considerably warty, wrinkled, buttery, pale yellow ;(1 1/2 ea);Aerial mycelium: None;Soluble pigment: None;III. Physiological properties; 1) Growth temperature range: 3 - 33°C; 2) Optimum growth temperature range: 27'- 33°C; 3) Liquefaction of gelatin: positive; 4) Hydrolysis of starch: Positive; 5) Coagulation of skimmed milk : Negative; Peptisation of skimmed milk: Positive; 6) Formation of melanoid pigment: Negative; IV. Utilization of carbon sources; Utilization of carbon sources in Pridham and Gottlieb agar medium was examined with the following results: Utilized carbon sources: L-arabinose, D-xylose, D -glucose, D-furactose, inositol and L-rhamnose Unutilized carbon sources: Sucrose, raffinose and D-mannitol V. The present strain contains LL-diaminopimelic acid in the cell walls. ;In consideration of the characteristics stated above, the strain KC-6643 is assumed to belong to the genus Streptomyces, but shows characteristic properties such as e.g. fragmentation of the substrate mycelium, fusion of the spore chains and the like which is slightly different from the typical mycelia of the genus Streptomyces. It is, however, well known that the strains classified as belonging to the genus Streptomyces often adopt specific structures such as e.g. fragmentation of mold in the medium and abnormality in the spore chains, and therefore it is considered correct to classify the strain KC-6643 as belonging to the genus Streptomyces. ;When strains of a nature in which the spore chains are straight, the surface of the spore is smooth, the aerial mycelium is white, the back of the colony shows no characteristic color, and they do not produce any soluble pigment or melanoid pigment, are sought among known strains indicated in "Bergy's Manual of Determinative Bacteriology", 8th ed., ISP by Shirling and Gottlieb, and "The Actinomycetes" by S.A. Waksman, 2 (1961), seven possible strains can be mentioned including Streptomyces albovinaceus (ISP 5136), Streptomyces aureocirculatus (ISP 5386), Streptomyces baarnensis (ISP 5232), Streptomyces chrysomallus (ISP 5128), Streptomyces candidas. (ISP 5141), Streptomyces gougeroti (ISP 5324) and Streptomyces griseoloalbus (ISP 5468). ;Streptomyces albovinaceous (ISP 5136) differs from strain KC-6643 in that it grows poorly in sucrose-nitrate agar medium and grows poorly at 5°C in various media, and has poor ability to liquefy gelatin and to utilize carbon sources (i.e. it utilizes D-mannitol and not inositol). Streptomyces aureocirculatus (ISP 5386) differs from strain KC-6643 in that it forms poor aerial mycelium and grows at 37°C but poorly at 5°C, and that it hydrolyses starch weakly and exhibits different degrees of ability to utilize carbon sources (i.e. it utilizes D-mannitol and not L-rhamnose). Streptomyces candidas (ISP 5141) differs from strain KC-6643 in that it grows poorly in a sucrose-nitrate agar medium and barely grows at 5°C, and also utilizes carbon sources poorly (i.e., utilizes D-mannitol but not inositol). Streptomyces gougeroti (ISP 5324) differs from strain KC-6643 in that it grows poorly in a sucrose-nitrate agar medium and forms poor aerial mycelium, is poor in liquefying gelatin and in peptonizing skimmed milk and utilization of carbon sources (i.e., it utilizes L -mannitol but neither inositol nor L-rhamnose). Streptomyces griseoloalbus (ISP 5468) differs from strain KC-6643 in that it grows at 37°C but not at 5°C, and also in liquefaction of gelatin, utilization of carbon sources; (i.e., it utilizes D-marinitol) and weakly yellowish color of the aerial mycelium. Streptomyces baarnensis (ISP 5232) is relatively similar in cultivation nature compared to strain KC-6643 but differs in that it grows poorly at 5°C and produces a brown, soluble pigment in a gelatin medium, and also has a poor ability to utilize carbon sources (i.e. it utilizes D-mannitol but not inositol).. Streptomyces chrysomallus (ISP 5128) has growth characteristics very similar to those of strain KC-6643, but does not grow at 5°C or in a skimmed milk medium. furthermore, it differs from strain KC-6643 in that it produces a yellow, soluble pigment in many media and has the ability to utilize carbon sources (i.e. it utilizes D-mannitol and not inositol). As a strain that can produce a product similar to a metabolite of strain KC-6643, 32 strains belonging to 11 species are known including Streptomyces cattleya, Streptomyces flavogriseus, streptomyces olivaseus, Streptomyces flavovirence, Streptomyces flaves, Streptomyces flavoviridis, Streptomyces algenteolus, Streptomyces shioyaensis, Streptomyces lipmanii, Streptomyces cremeus and Streptomyces gedanensis. However, none of these known strains have such a nature that the aerial mycelium is white in color and that inositol and not D-mannitol is utilized. ;From the above results, it is concluded that the strain KC-6643 is a new strain belonging to the genus Streptomyces and it is designated as Streptomyces sp.KC-6643. This strain has been deposited in the Fermentation Research institute of the Agency of Industrial Science&Technology located at No. 1-3, Higashi 1-chomé, Yatabe-machi, Tsukuba-gun, Ibaragi-ken, Japan, on April 7, 1978, and has been assigned receipt no. .FERM-P 4467. The strain has also been deposited in the American Type Culture Collection located at Parklawn Drive 12301, Rockville, Maryland, the United States of America, on March 12, 1979, and has been assigned receipt no. ATCC 31493. In carrying out the method according to the invention, not only the strain KC-6643 itself but also its spontaneous and artificial mutants can be used. The cultivation of the present strain can be carried out using any usual method for the cultivation of actinomycetes. A number of sources can be used as a carbon source in the media. Preferably, starch, glycerol, maltose, dextrin, fructose, molasses and the like are used alone or in combination. Hydrocarbons, organic acids, vegetable oils and the like can also be used. Nitrogen sources include soybean meal, yeast extracts, dry yeast, peptone, meat extracts, corn-molding liquids, casamino acid, soluble substances from the distillery, ammonium chloride, ammonium sulphate, urea, sodium nitrate and the like. These sources can be used alone or in combination. If necessary, inorganic salts can be added, e.g. sodium chloride, potassium phosphate, sodium phosphate, magnesium sulphate, calcium chloride, calcium carbonate, calcium hydroxide, cobalt chloride, zinc sulphate, iron chloride, iron sulphate and the like, and traces of heavy metals. furthermore, organic and inorganic substances can be added to help the growth of the mycelium of the substance KA-6643 and possibly facilitate its production. When an aerated cultivation method is used, it is further preferred to add an anti-moulding agent such as e.g. a fatty oil, silicone oil or paraffin. Although the cultivation of the strain in a solid medium is possible, cultivation in a liquid is preferred, especially a submerged cultivation as in the case where antibiotics are produced. Such cultivation is carried out under aerobic conditions at temperatures in the range 3 to 35°C, preferably 27 to 33°C. The antibiotic KA-6643 can be produced either by shaking culture or tank culture. When such cultivation is continued for 2 to 10 days, an active substance is collected in the culture liquid. Cultivation ends at the time when the amount of the product reaches a maximum in the culture liquid. In order to isolate the substance KA-6643 from the culture fluid thus obtained and to purify the isolated substance, different methods can be used which are usually used for such purposes. The culture liquid can, for example, first be centrifuged or filtered to remove the mycelium from there, and the filtrate is subjected to any freely chosen combination of the following treatment methods. The filtrate can be passed through a basic anion exchange resin to allow the desired product to be absorbed onto the resin, and the desired product is obtained by elution with a salt solution or an aqueous methanolic solution of the salt. Examples of such ion exchange resins include strongly basic anion exchange resins, e.g. "Dowex" 1x2 (TheDow Chemical Co.), "Diaion" PA-318, ;316, 306S, 308 and 312 (MitsubishiChem. Ind.,Ltd), "Amberlite" IRA-400, 401 and 410 (Rohm&Haas Co.) and the like, and weakly basic anion exchange resins such as e.g. "Amberlite" lRA-68. Alternatively, the culture filtrate can be subjected to adsorption with an adsorbent such as e.g. activated carbon, and the desired product is obtained by elution with aqueous methanol, aqueous acetone or a similar solvent. ;The active fraction including the substance KA-6643 is then passed through a strongly basic anion exchange resin, e.g. "Dowex" 1x2, to absorb the substance KA-6643, after which the substance is stepwise eluted with a gradual change of the salt concentration in a phosphate buffer solution. In this elution operation, the substance KA-6643-A is eluted in an early step, with subsequent elution of the substance KA-6643-B, whereby the substances KA-6643-A and KA-6643-B are separated from each other. Alternatively, the active fraction can be passed through a column packed with a moderately polar or non-polar, hydrophobic, cross-linked polymer such as e.g. "Amberlite" XAD-2 or "Diaion" HP-20 to separate the substances KA-6643-A and KA-6643-B due to different adsorptivity between them. ;The thus obtained substances KA-6643-A and KA-6643-B are subjected to further gel filtration using a gel, e.g. "Biogel" P-2 (BIO.RAD Lab.) or "Sephadex" G-10 (Pharmacia FineChemicals), or subjected to a reverse phase chromatography using a column packed with "Bondapack" C,0 (Waters ;lo Associates, Inc.), whereby very pure substances KA-6643-A and KA-6643-B are obtained. If necessary, both substance KA-6643-A and KA-6643-B can be converted in any usual way into alkali metal salts, alkaline earth metal salts, primary, secondary or tertiary amine salts or quaternary ammonium salts thereof. The substance KA-6643 produced according to the invention has the following physico-chemical properties and biological properties, and exhibits antibacterial activity against gram-positive and gram-negative bacteria. Furthermore, the substance has excellent activity against bacteria that are resistant to various types of fi-lactam antibiotics, and has /3-lactamase-inhibiting activity. ;IN. Physico-chemical properties; A) The sodium salt of the substance KA- 6643- A; 1) Appearance: White powder; 2) Melting point: Gradually decomposed above 145°C; 3) Ultraviolet absorption spectrum: When measured in an aqueous solution, the spectrum is mainly the same as shown in fig. 1 with maximum absorption at 240 nm and 288 nm and minimal absorption at 265 nm. 4) Infrared absorption spectrum: When absorbed in KBr, the substance essentially has the same spectrum as shown in fig. 2. 5) Solubility in solvents: Slightly soluble in water, but essentially insoluble in acetone, chloroform, ethyl acetate and petroleum ether. 6) Nuclear magnetic resonance spectrum ''"H nuclear magnetic resonance spectrum: When measuring i, the spectrum is essentially the same as shown in Fig. 3. ;7) Thin layer chromatography; Rf value: 0.6 (plate: TLC aluminum sheet-cellulose ^ 254' 0 .2 nm (Merck&Co., Inc.); solvent: butanol-isopropyl alcohol-water (7:7:6)) ;Rf value: 0.55 (plate: TLC aluminum sheet-silica gel 60F254»0.2 mm (Merck&Co., Inc.), solvent: isopropyl-alcohol-water (7:1)) ;8) High performance liquid chromatography; Elution time: 10.5 minutes; Column: "yj-Bondapack" C^g (Waters Associates, Inc.), ;4 x 300 mm ;Solvent: Methanol-0.0lm phosphate buffer solution (1:9), pH 6.8 ;Flow rate: 1 m^</>min.;9) Molecular weight: 364 (calculated from the value obtained by mass spectrometry of the monomethyl ester). 10) Molecular formula: C-^H^^OgSNa (determined from the molecular formula of the methyl ester calculated from the results of mass spectrometry). 11) Stability: The half-life at different pH values is as follows: ; Solvent:;pH 4.0-7.0: Mcllvanine's buffer solution (sodium phosphate citric acid) ;pH 10.0: 0.lm sodium carbonate-sodium bicarbonate buffer solution ;Measurement:;Ultraviolet absorption spectroscopy;<*>J. Antibiot., 32, 295-304 (1979)

B) Monomethyl ester of. the substance KA-6643-A

1) Appearance: Colorless solid

2) Molecular weight: 356 (mass spectrometry)

3) Elemental analysis (as cj_5H2oN2°6S^

4) Molecular formula: ci5H2o<N>2°6S

5) Ultraviolet absorption spectrum: The spectrum in methanol has maximum absorption at 243 nm and 300 nm and minimal

absorption at 2 74 nm.

6) Infrared absorption spectrum

When absorbed in KBr, the substance essentially has the spectrum shown in fig. 4.

7) Nuclear magnetic resonance spectrum

1) H nuclear magnetic resonance:

When measuring iCDCl^ using tetramethylsilane as an internal standard, the spectrum obtained is essentially the same as that shown in fig. 5.

13

2) C nuclear magnetic resonance spectrum:

When measuring in CDCl^ and using tetramethylsilane as an internal standard, the obtained spectrum is essentially the same

which is shown in fig. 6.

8) Thin layer chromatography

Rf value: 0.44 (plate: TLC aluminum sheet-silica gel 60F2g<4.>0.2 mm (Merck&Co., Inc.), solvent: methylene chloride-methanol (9:11)).

9) Specific rotation: ta]^<3>- 96.0° (cl, CH2Cl2)

C) The disodium salt of the substance KA-6643-B

1) Appearance: White powder

2) Melting point: No specific point indicated and it gradually turned yellow to brown at temperatures above approx. 130°C.

3) Elementary analysis:

4) Ultraviolet absorption spectrum: Essentially the same as shown in fig. 7 and with maximum absorption at 240 nm and 285 nm and a minimum absorption band at 265 nm. 5) Infrared absorption spectrum: The infrared absorption spectrum of the substance taken up in KBr tablets is essentially the same as shown in fig. 8.

6) Nuclear magnetic resonance spectrum:

When measuring iD20 and using tetramethylsilane as an external standard, the nuclear magnetic resonance spectrum is essentially the same as shown in fig. 9. 7) Solubility in solvents: Easily soluble in water, but essentially insoluble in organic solvents such as e.g. acetone, chloroform, ethyl acetate, petroleum ether and the like. 24 o

8) Specific rotatability: [a]D - 145 (cl, H20)

9) Stability: The half-life at different pH values is as follows:

The solvent and the measurements are the same as in the substance KA-6643-A.

10) Thin layer chromatography: The results are shown in table 1.

11)<p>apyr chromatography

Filter paper: Toyo filter paper no. 51 (Toyo Filter Paper

Co.,Ltd.) Solvent: Acetonitrile/water (3:2), ascending method Rf value: 0.9

12) Liquid chromatography with high performance

Column: '^j-Bondapack" C-^g (Waters Associates, Inc.),

4 x 300 mm

Solvent: 0.lm phosphate buffer solution, pH 6.8 Flow rate: 1 ml/min.

Retention time: 23.5 minutes

13) High voltage paper electrophoresis

When a high-voltage paper electrophoresis device is used and the substance is moved in a l/30m phosphate buffer solution (pH 7.0) at 3000 V for 30 min., it moves 8.5 cm towards the anode. Penicillin N' tested in a similar manner moved 4.4 cm towards the anode.

II. Biological properties

1) Antibacterial spectrum

The antibacterial spectra against various microorganisms are shown in Table 2 in comparison with the spectra of amino-benzyl-penicillin (AB-Pc) and cefoxitin (CFX).

As can be seen from the foregoing, the substance KA-6643 exhibits intense antibacterial activity against gram-negative and gram-positive bacteria, including those resistant to various penicillin and cephalosporin antibiotics.

2)/3-lactamase inhibitory activity

i) Antibacterial activity-improving effect against

/3-lactam-resistant bacteria

In penassay agar (of which 12 g was dissolved in 500 ml of distilled water, Kyoei pharm. Co., Ltd.) Escherichia coli EC-1 (PC-ase) and Citrobacter 24 (CS-ase) were inoculated as

var/3-lactamase-producing organisms, and 10 ml of the inoculated medium was floated on a 9 cm diameter Petri dish and fixed to provide a test medium.

In this agar medium, pulp disks with a diameter of 8 cm were placed into which 30 ug of aqueous solutions of antibiotics were introduced, respectively, followed by cultivation at 37 o C for 18 hours and then measurement of the diameter of an inhibition zone around each disk. The results are listed in Table 3.

ii) β-lactamase inhibitory activity

When penicillin-amido-hydrolase EC3.5.2.6 (TokyoChem. Syn. Co., Ltd.) obtained from bacteria belonging to the genus Bacillus is used as the /3-lactamase enzyme, the i5Q value for

the substance KA-6643-A 0.5 ng/ml and for the substance KA-6643-B

12.8 ng/ml.

The I 5 - 0 value was measured according to the method of C. Reading and M. Cole (Antimicr. Agents and Chemoth. 11, 852-857 (1977)).

It can therefore be expected that the substances KA-6643-A and KA-6643-B will show pronounced synergistic effects in combination with other/3-lactam antibiotics.

In light of the various properties indicated above, it has been determined that the substances produced according to the invention are /3-lactam-based antibiotics. While it is a known substance, i.e. MM 4550 (MC696-SY2-A), which has a similar ultraviolet spectrum to the present substances, this known antibiotic differs from the substances KA-6643-A and KA-6643-B in the following points.

1) paper electrophoresis

Subjected to paper electrophoresis at 3000 V for 25 min. Solvent: 0.lm phosphate buffer solution (pH 8.0) KA-6643-A: Moved 5 cm towards anode side

• MM 4550: Moved 10.2 cm towards the anode side

2) high performance liquid chromatography

Column: "u-Bondapack" C18(waters Associates, Inc.)

4 x 30 cm

Solvent: 0.05m phosphate buffer solution (pH 7.0) Flow rate: 1 ml/min

Retention time: KA-6643-A 1 hour

KA-6643-B 30 min.

MM 4550 17 min.

From the above results, it has been found that the substance KA-6643 is a new compound which is different from the known β-lactam antibiotic.

Since the substance KA-6643. produced according to the invention has a carboxyl group in the molecule, it can be used in the form of a free acid, but it is preferably used in the form of salts. Suitable salts are, for example, salts of alkali or alkaline earth metals such as e.g. sodium, potassium, calcium and the like; and ammonium salts such as ammonium, trimethylamine, dimethylamine and the like. Furthermore, the substance can be used in the form of lower alkyl esters such as methyl ester, ethyl ester and the like, or higher fatty acid esters.

The invention shall be illustrated by means of the following examples where all parts and percentages are given by weight unless otherwise stated.

Example 1

i) 30 1 of a liquid medium containing 3% starch, 1.5% soybean meal, 0.28% monobasic potassium phosphate, 0.18% dibasic sodium phosphate dodecahydrate, 0.0005% cobalt chloride hexahydrate,

0.05% magnesium sulfate heptahydrate and 0.001% ferrous sulfate adjusted to pH 6.0 was provided, and 100 mL aliquots of the medium were each introduced into a 500 mL Erlenmeyer flask and sterilized. Mycelium of strain KC-6643 was inoculated into the medium and cultured with shaking at 27°C for 4 days at 220 rpm.

ii) After completion of cultivation, "Celite" (Johns-Manville Co.., Inc.) was added as a filter aid to the culture liquid, and the mycelium was removed by filtration. The filtrate (25 L) was passed through a column (6 x 30 cm) of a strongly basic anion exchange resin, "Diaion" PA316 (Cl type, Mitsubishi Chem. Ind., Ltd.), followed by washing with water and elution with a 0.0 µm phosphate buffer solution of pH 7.0 containing 0.5 M sodium chloride, to collect one active faction. The resulting active fraction was passed through a column (4 x 30 cm) of activated carbon and, after washing with water, then treated with 50 v/v% aqueous acetone to collect an active fraction. The collected active fraction was passed through a column (4. x 40 cm) of a basic anion exchange resin, "Amberlite" IRA-68 (Cl type), followed by washing with water and then elution with a 0.0 µm phosphate buffer f is solution with pH 7.0 containing 0.2m sodium chloride, for

to gather an active faction. The active fraction thus obtained was passed through a column (3 x 30 cm) of activated carbon, which was washed with water and eluted with 50 v/v% aqueous acetone to collect an active fraction.

iii) The fraction thus obtained was passed through a column (4 x 30 cm) of a strongly basic anion exchange resin, "Dowex" 1x2 (100 - 200 mesh, Cl -type), followed by washing with water and treatment by a concentration gradient elution with a flow rate of 3 ml/min between a solution of 0.0 ml phosphate buffer with pH 7.0 containing 0.1 ml sodium chloride and a solution containing 0.4 ml sodium chloride, whereby the fraction

split into two further fractions, one being an originally eluted fraction and the other a subsequently eluted fraction,

and collection of the two factions separately.

iv) The originally eluted active fraction which had been collected in this way (approx. 1 liter) was concentrated to approx. 100 ml under reduced pressure at below 30°C. The concentrate was passed through a column of "Diaion" HP-20 previously washed with a 20% saline solution, followed by elution with deionized water at a flow rate of 4 ml/min. An active fraction was collected and concentrated to approx. 2 ml at temperature below 30°C, followed by freeze-drying to obtain 80 mg of a crude powder of KA-6643-A.

v) The subsequently eluted active fraction was treated in the same manner as in step iv) whereby 240 mg of a crude powder of KA-6643-B was obtained.

Example 2

i) A medium with a composition of 2% starch, 1.5% soybean meal, 0.28% monobasic potassium phosphate, 0.18% dibasic sodium phosphate dodecahydrate, 0.0005% cobalt chloride hexahydrate, 0.05% magnesium sulfate heptahydrate and 0.01% ferrous sulfate and which had been adjusted to pH 6.0 and sterilized, was inoculated with mycelium of strain KC-6643, followed by precultivation at 27°C for approx. 48 hours to provide a first germ medium. In each of two tanks, each with a volume of 200 1, 100 1 of a medium with the same composition as above, but with 1% added cottonseed oil, was added. The first seed culture was inoculated into each tank in an amount of 500 ml and grown at 30°G using an aerated stirring system (300 rpm, a flow rate of 50 l/min) for 4 days.

ii) After the cultivation was completed, 10 v/v% "Dicalite<p>erlite" 4109 (Dicalite Orient Co., Ltd.) was added to the culture liquid (170 L) as a filter aid, and the mycelium was removed by filtration.

The resulting filtrate (150 L) was adjusted in conductivity to 1.5 ml/cm and passed through a column (21.5 x 45 cm) of a strongly basic anion exchange resin, "Diaion" PA316 (Cl - type, MitsubishiChem. Ind.,Ltd.) with a flow rate of 200 ml/min. After washing with a 0.0 µM phosphate buffer solution (pH 7.0), the resin was subjected to elution with a 0.0 µM phosphate buffer solution (pH 7.0) containing 2 M sodium chloride at 2 v/v % at a flow rate of 150 ml/min to gather an active faction. The active fraction thus collected was passed through a column (16 x 10 cm) of "Diaion" HP-20, which had been adjusted with a 10% w/v sodium chloride solution, at a flow rate of 400 ml/min, and, after washing with a 10% w/v solution of sodium chloride, eluted with deionized water at a flow rate of 200 ml/min to collect an active fraction.

iii) The active fraction thus collected was passed through a column (6 x 70 cm) of a strongly basic anion exchange resin, "Amberlite" IRA-458 (Cl~ type, Rohm&Haas co.) at a flow rate of 50 ml/min and eluted with a 0.0 µm phosphate buffer solution (pH 7.0) containing 0.15 µm sodium chloride. The remaining active fraction was then eluted with a 0.0 µm phosphate buffer solution (pH 7.0) containing 2 µm sodium chloride.

iv) The originally eluted fraction in step iii) was subjected to the same treatment as described in step iv) in example 1, whereby 700 mg of a crude powder of the substance KA-6643-A was obtained.

v) The subsequently eluted active fraction was passed through a column (6 x 70 cm) of "Diaion" HP-20 pretreated with a 20% w/v aqueous sodium chloride solution, and then eluted with deionized water with a flow rate of 20 ml/min to collect an active fraction. The thus collected active fraction was diluted with deionized water to obtain an electroconductivity of approx. 700^J^cm and passed through a column (3 x 30cm) of a weakly basic anion exchange resin, "DEAE-Sephadex" A-25 (Pharmacia Fine Chemicals), which had been adjusted to a pH of 7.0 using a 0, 0 µm phosphate buffer solution, followed by washing with water and elution with a phosphate buffer solution (pH 7.0) containing 0.15 M sodium chloride at a flow rate of 35 ml/min to collect an active fraction. The eluate thus collected was concentrated under reduced pressure at temperatures below 30°C and then passed through a column (3.5 x 40 cm) of "Diaion" HP-20 which had been pretreated with a 10% w/v, aqueous sodium chloride solution and eluted with deionized water at a flow rate of 20ml/min to collect an active fraction. The active fraction was concentrated under reduced pressure to approx. 2 ml and freeze-dried to obtain 190 mg of a crude powder of the substance KA-6643-B.

The crude powder thus obtained was dissolved in 1 ml of deionized water and passed through a column (2 x 30 cm) of "Diaion" HP-20 and then eluted with deionized water at a flow rate of 1 ml/min to collect a active faction. The active fraction thus collected was concentrated under reduced pressure and passed through a column of "Sephadex" G-10, followed by development with deionized water at a flow rate of 0.2 ml/min. The active fraction was collected, concentrated under reduced pressure to approx. 2 ml and then freeze-dried to obtain 80 mg of a crude powder.

vi) 80 mg of the thus obtained powder was dissolved in

0.1 ml of a 0.1 µm phosphate buffer solution (pH 6.8) and passed through a column (0.8 x 120 cm) of "Bondapack" C^g/"Polasil" b (WatersAssociates, Inc.) pretreated with a 0.1 µm phosphate buffer solution, followed by elution with the same buffer solution at a flow rate of 6 ml/min to collect an active fraction. The active fraction was passed through a column (0.9 x 5 cm) of activated carbon, washed with deionized water and eluted with 50% w/v aqueous acetone to collect an active fraction. The active fraction was concentrated under reduced pressure to approx. 1 ml and freeze-dried to achieve

35 mg of a purified powder of KA-6643-B

Example 3

120 mg of the crude powder obtained in Example 1 iv) or Example 2 iv) was dissolved in 1 ml of deionized water and then passed through a column (0.8 x 60 cm) of "Bondapack" C,Q/-"Polasil " B (Waters Associates, Inc.) for high-performance liquid chromatography and eluted with a 0.0 µm phosphate buffer solution of pH 6.8 containing 2% methanol at a flow rate of

6 ml/min to collect an active fraction.

The active fraction thus collected was concentrated to 5 ml under reduced pressure and passed through a column (2 x 15 cm) of "Diaion" HP-20, which had been washed with a saline solution followed by elution with deionized water at a flow rate of 1 ml /min to collect an active fraction. The fraction was concentrated to 1 ml and then passed through a column (3 x 150 cm) of "Sephadex" G-10 and eluted with deionized water at a flow rate of 1 ml/min. Then, the resulting active fraction was concentrated to 2 ml under reduced pressure and freeze-dried to obtain 4 mg of a purified product of a sodium salt of KA-6643-A.

Claims (10)

1. An antibiotic, characterized in that it contains at least one substance of formula (I): where R means a hydrogen atom or -SO^H , or a salt or an ester thereof. 2. Antibiotic according to claim 1, characterized in that it only comprises the substance of formula (i) where R means a hydrogen atom. 3. Antibiotic according to claim 1, characterized in that it only comprises a substance of formula (I) where R means -SO^H. 4. Antibiotic according to claim 1, characterized in that it comprises a mixture of the compounds of formula (I), where R means a hydrogen atom and -SO^ H respectively. 5. Antibiotic according to one of claims 2 to 4, characterized in that it comprises a salt or an ester of the substance or each of the substances with formula (I). 6. Antibiotic according to claim 5, characterized in that it has spectrum properties for ultraviolet absorption, infrared absorption and nuclear resonance as shown in figures 1 to 9 in the drawings. 7. Process for producing an antibiotic as claimed in claim 1, characterized in that it includes the steps of growing a microorganism belonging to the genus Streptomyces which will produce a substance or substances of formula (I) and isolating the antibiotic substance or the antibiotic substances from the culture medium. 8. Process according to claim 7, characterized in that the said microorganism used is the strain Streptomyces sp.KC -6643. 9. Process for producing an antibiotic according to claim 7, characterized in that the method is essentially as described with reference to one of the preceding examples. 10. Use of the strain designated as Streptomyces sp. 6643 and is deposited in the American Type culture collection (accession number ATCC 31493) in the method from claim 7.