PL127697B1 - Process for preparing novel antibiotic - Google Patents

Description

The subject of the invention is a method for producing a new antibiotic. The method according to the invention produces an antibiotic with the general formula shown in the figure, in which R stands for a hydrogen atom or residues with the formula -SOaH. Various strains of microorganisms occurring in the soil have been isolated. to test their ability to produce antibiotics. During the research work leading to the development of this invention, it was found that the newly isolated strain as KC-6643 has the ability to produce two forms of a new antibiotic with the general formula shown in the drawing, which has excellent antibacterial properties against both gram-positive and gram-positive bacteria. and gram-negative, and such antibiotics were successfully isolated from the culture broth. These two forms of the antibiotic produced according to the invention are very similar in structure. As is clear from their physico-chemical and biological properties presented below, the antibiotic with the formula shown in the drawing is a new substance different from other known antibiotics. According to the invention, a method for producing an antibiotic with the formula shown in the drawing, optionally in the form of a salt or ester consists in cultivating the microorganisms Sfcreptomyces sp KC-6643 (ATCC 31493 and FERM-P/4467) 10 15 20 25 30 and then isolating the antibiotic substance from the culture broth. Further part of the description, the mixture of two forms of antibiotic with the formula shown in the drawing, in which R stands for hydrogen atom and the group with the formula -SO3H, respectively, is designated as substance KA-6643. The form in which R in the given formula represents a hydrogen atom is hereinafter designated as substance KA-6643-A, while the other form in which R in the given formula is a group of formula -SO3H is designated as substance KA-6643-B. The method of producing the antibiotic according to the invention is presented with reference to examples and drawings, in which Fig. 1 shows a record of the ultraviolet absorption spectrum of the sodium salt of the substance KA-6643-A, Fig. 2 - a record of the infrared absorption spectrum of the sodium salt. substance KA-6643-A, Fig. 3 - recording of the 1H nuclear magnetic resonance spectrum for the sodium salt of substance KA-6643-A, Fig. 4 - recording of the infrared absorption spectrum of the methyl ester of substance KA-6643-A, fig. 5 - recording of the *H nuclear magnetic resonance spectrum *H of the methyl ester of the substance KA-6643-A, Fig. 6 - recording of the WC nuclear magnetic resonance spectrum of the methyl ester of the substance KA-6643-A, Fig. 7 - recording of the spectrum ultraviolet absorption of the disodium salt of the substance KA-6643-A, Fig. 8 - recording of the infrared absorption spectrum for the disodium salt of the substance KA-6643-B, Fig. 9 - recording of the nuclear magnetic resonance spectrum *H of the disodium salt of the substance KA-6643-B. The microorganism producing the antibiotic substance K-6643 according to the invention has the following taxonomic properties. It should be noted that the morphological features in the various ISP media are presented only as the result of observation of a culture carried out for 21 days at 27°C by standard methods, with the color of the mature culture determined in accordance with the current color classification according to the Color Harmony Manual (Container). Corporation of America), except where otherwise noted.I. Morphological features. The strain producing the KC-6643 substance, hereinafter referred to as the KC-6643 strain, allows the formation of single-branched surface mycelium in various environments. The ovary capsule is formed on the mycelium, which produces straight chains of spores. Each spore is 0.4-0.5 nm x 0.9-1.0 nm in size, oval or cylindrical in shape and the spores are connected to form a chain of 20 or more spores, which has a smooth surface. The spore chains are often held together in a rigid structure and mycelial separation is observed in certain environments used for taxonomic research. II. Culture characteristics in different environments: (1) Sucrose-nitrate agar Growth: good, flat spreading, colorless - eyelid (2fb) Surface mycelium: moderate, powdery, white (a) Soluble pigment - none (2) Agar Glucose-aspartic * Growth: moderate flat and buttery, creamy (1- - ca) pastel yellow (2 hb) Surface mycelium - none Soluble pigment - none (3) Glycerol-aspartate agar Growth: good, covered with warts, wrinkled combined, buff-yellow (2fg) bamboo color (2gc) Surface mycelium: good, powdery, pearly (3ba) Soluble pigment - none (4) Starch and inorganic salt agar Growth: good, rough butyric bamboo color (2gc) and golden honey (2ic) Surface mycelium: good, powdery, white Soluble pigment - none (5) Tyrosine agar Growth: good, densely covered with warts, rough, gelatinous, cinnamon (31e) Surface mycelium: good, powdery, pearly ( 3ba) Soluble Pigment - None (6) Nutrient Agar Growth: Medium, Slightly Nodulated, Rough Gelatinous,- Colorless Light Yellow 1" (l-.ea) Surface Mycelium: None Soluble Pigment - None (7) Yeast Extract and Extract Agar malt Growth: lush, rough, buttery-gelatinous, mustard {21 e) Surface mycelium: good, powdery, white (a) pearly (3ba) Soluble pigment - none (8) Oatmeal agar Growth: moderate, flat spread , creamy. (1- ca) butter-yellow (1-ga) 2 2 Surface mycelium: moderate, powdery, white (a) Soluble pigment - none (9) Iron, yeast-peptone agar Growth: good, very densely covered with warts kami, rough buttery, light yellow (1— ea) Surface mycelium - none Soluble pigment - none III. Physiological features (1) Proofing temperature range: 3-35°C (2) Optimal growth temperature range 27-33°C (3) Conversion of gelatin into liquid state: positive (4) Starch hydrolysis: positive (5) Milk coagulation skimmed milk - negative Peptonization of skimmed milk: positive (6) Formation of melanoid pigment: negative. IV. The use of carbon carriers. Studies on the use of carbon carriers in the Pridham and Gottlieb agar environment were carried out with the following results: Carbon carriers used: L-arabinose, D-xyl, D-glucose, D-fructose, inositol and L-rhamnose. Unused carbon carriers: sucrose, raffinose and D-mannitol.V. The present strain contains LL-diaminopimelic acid in the cell wall. Taking into account the established features presented above, the KC-6643 strain can be considered to belong to the genus Sterptomyces, but it has characteristic features such as fragmentation of mycelium and fusion of spore chains, which are slightly different from the typical features of mycelium of the genus Streptomyces. This does not It is less commonly known that strains classified as belonging to the genus Streptomyces often have specific features such as, for example, fragmentation of mycelium in the environment or abnormalities in the structure of spore chains, and it has therefore been considered appropriate to classify strain KC-6643 as belonging to the genus Streptomyces. myces. When looking for strains that form straight spore chains, have a smooth spore surface, have a white surface mycelium, show no distinctive color on the other side of the colony, and do not produce any soluble pigment or melanoid pigment among the strains of the mentioned publications: "Bergy's Manual of Determinant Bacteriology", 8th edition, ISP of Shirling and Gottlieb and in the publication "The Actinomycetes" S,A. Waksmana, 2 (1961) it can be indicated that they may belong to one of three possible strains including: Streptomyces albovinaceus (ISP 5136), Streptomyces aureocir- curatu s ( ISP 5386), Streptomyces baarnensis (ISP 5232), Streptomyces chrysomallus (ISP 5128), Streptomyces candidas (ISP 5,141), Streptomyces qouqeroti (ISP 5324), amd Streptomyces griseoloalbus (ISP 5468). The Streptomyces albovinaceous strain (ISP 5136) differs from the KC-6643 strain in that it shows poor growth in sucrose-nitrate agar and other environments at a temperature of 5°C, has a poor ability to convert gelatin into liquid form and in addition, it has a low capacity to utilize carbon carriers (that is, it uses D-mannitol and not inositol. Streptomyces aureocirculatus (ISP 5386) differs from the KC-6643 strain in that it has a poor ability to form surface mycelium and grows at a rapid rate temperature of 37°C, but weakly at 5°C, it has a poor ability to hydrolyze starch, as well as different degrees of ability to use carbon carriers (i.e. it uses D-mannitol and not L-rhamnose). Streptomyces candidas (ISP) 5141) differs from strain KC-6643 in that it shows poor growth in sucrose-nitrate agar and almost does not grow at 5°C, and also differs in its ability to utilize carbon carriers (i.e., it uses D -mannitol, not inositol). Streptomyces gougeroti (ISP 5324) differs from strain KC-6643 in its poor growth in sucrose-nitrate agar, poor formation of its surface mycelium and low ability to convert gelatin into liquid form, peptize fat milk and use carbon carriers (that is, it uses D-manitol, not inositol or L-rhamnose). Streptomyces griseoloalbus (ISP 5468) differs from the KC-6643 strain in that it grows at 37°C but does not develop at 5°C and also has the ability to convert gelatin into liquid form and use carbon carriers. (that is, it uses D-mannitol) as well as a slightly yellowish color of the surface mycelium. Streptomyces buarnensis (ISP 5232) is a relatively similar strain to the KC-6643 strain in terms of its culture characteristics, but differs in that it exhibits poor growth in temperature of 5°C and produces a brown pigment soluble in gelatin, and also has a poor ability to use carbon carriers (that is, it uses iD-mannitpl, not insitol). Streptomyces Chrysemallus (ISP 51.28) is very similar in culture characteristics to strain KC-6643, but does not grow at 5°C or in a skimmed milk environment. It also differs from strain KC-6643 in that it produces a yellow soluble pigment in several media and a different ability to use carbon carriers (that is, it uses D-mannitol, not inositol). 32 strains are known that have the ability to produce a product similar to the metabolite of strain KC-6643. These strains belong to 11 species including Streptomyces cattleya, Streptomyces flavoqgiseus, Streptomyces ilivaseus, Streptomyces flavovirence, Streptomyces flaves, Streptomyces fla- 6 * voviridis, Streptomyces algenteolus, Streptomyces shioyaensis, Streptomyces lipmanii, Streptomyces cremeus and Streptomyces gedanensis. However, none of these known strains have the characteristics that the surface mycelium they produce is white in color and that they use inositol and not D-mannitol. Based on the results of this analysis, it can be concluded that the KC-6643 strain is a new strain belonging to the genus Streptomyces and was designated Streptomyces sp. KC-6643. The strain was deposited at the Fermentation Research Institute of the Agency of Industrial Science Technology based in No. 1—3, Higashi 1-chome, Ystabe- is -machi, Tsukubagun, Ibaragi-ken, Japan, on April 7, 1978 and received approval No. FERN-P-4467. The strain was also deposited in the American Type Culture CoUectiori based in No. 12301 Parklawn Drive, Rockville, Maryland,. 20 United States of America on March 12, 1979 and Obtained Approval No. ATCC 31498. Cultures of this strain can be carried out by the usual methods used for cultures of acti-: nomycetes. A variety of materials can be used as carbon carriers. Starches, glycerol, maltose, dextrin, fructose, molasses and the like are preferably used singly or in mixtures. Hydrocarbons, organic acids, vegetable oils and the like may also be used. Soybean flour, yeast extracts, dry yeast, peptone, meat extracts, corn extracts, Casamine acid, soluble distillates, ammonium chloride, ammonium sulfate, urea, ammonium nitrate and the like are used as nitrogen carriers. These carriers can also be used individually or in mixtures. If necessary, inorganic salts such as sodium chloride, potassium phosphate, sodium phosphate, magnesium sulfate, calcium chloride, calcium carbonate, calcium hydroxide, cobalt chloride, zinc sulfate, iron chloride, iron sulfate are also added. and the like, as well as trace amounts of heavy metals. In addition, optionally, organic and inorganic substances are also added, respectively, which enhance the development of the mycelium and possibly facilitate the production of KC-6643. When cultivating with aeration, it is preferable to add substances that prevent foam formation, such as fatty oil, silicone oil or paraffin. 50 Although the discussed strain can be cultivated in a solid environment, cultivation in a liquid environment, especially culture in the case of disturbance, is considered more advantageous for use in the production of ordinary antibiotics. 55 Such culture is carried out under aerobic conditions at a temperature ranging from 3° to 35°C, and preferably at 27° to 33°C. Antibiotic KC-6643 can be produced in both shaken and bulk culture. 80 during such cultivation for 2 to 10 days, the active substance is accumulated in the breeding broth. Cultivation is stopped when the maximum amount of product in the culture broth is reached. Various known methods can be used to isolate the secretion of the KC-6643 antibiotic substance from the culture broth and to purify the isolated substance, such as: is usually used for these purposes. For example, the culture broth is first centrifuged or filtered to remove the mycelium and the filtrate is subjected to various combinations of further treatments. The filtrate is passed through an alkaline anion exchange resin to absorb the desired antibiotic. io formed on the resin; and could then be isolated by washing with a saline solution or a water-methanol salt solution. Examples of such ion exchange resins are strongly basic anion exchange resins, such as DOWEX 1X2 manufactured by The Dow Chemical Co., Diaion PA-318, 316, 306S and 312 manufactured by Mithubishi Chem. Ind. Ltd. Amberlite IRA-400, 401 and 410 manufactured by Rohm and Haas Co. and the like, and weakly basic anion exchange resins such as Amberlite 20 IRA-68. Alternatively, the filtrate is absorbed on an absorbent such as activated carbon and the desired product is obtained by washing with aqueous methanol, aqueous acetone or the like. 25 Then the active fraction containing the antibiotic substance KA-6643 is passed through a strong alkaline anion exchange resin such as, for example, Dowex 1X|2 to absorb the antibiotic substance KA-6643, and the desired substance is eluted gradually by gradually changing the salt concentration in the buffer solution phosphate. In this washing operation, the substance KA-6643-A is washed in an initial step with subsequent washing of the substance KA-6643-B, thereby separating the two substances from each other. Alternatively, the active fraction is passed through a column packed with a moderately polar Iud, non-polar, hydrophobic polymer with a cross-linked structure such as, for example, Amberlite XAD-2 or Diaion HP-20 to separate the KA-6643-A substance and the substance tions of KA-6643-B using the differences in absorption capacity and between them. The substances 45 KA-6643-A and KA-6643-B' obtained in the above manner are subjected to gel filtration using a gel such as Biogel P-2 produced by BIO.RAD.Lab. or Sephadex G-10 from Pharmacia Fine Chemicals or reversed phase chromatography using a Bondapack Cis column from Waters Associates, Inc. thus obtaining high purity of the substance KA-6643-A and the substance KA-6643-B. If necessary, both the substances KA-6643-A 55 and KA-6643-B are converted in a known manner into alkali metal salts, salts alkaline earth metals, primary, secondary and tertiary amine salts and quaternary ammonium salts. The substance KA-6643 produced by the method of the invention has the ability to have antibacterial activity against both gram-positive and gram-negative bacteria. . Moreover, this substance has an excellent antibacterial effect against bacteria resistant to various types of f-mactam antibiotics and has inhibitory activity against β-lactamase. This substance has the following physico-chemical and biological properties. I. Physico-chemical properties. A) Sodium salt of the substance KA-6643-A (1) Appearance: White powder (2) Melting point: gradual decomposition above 145°C (3) Ultraviolet absorption spectrum: When measured in in the aqueous solution, the spectrum obtained is exactly the same as shown in the attached drawing, Figure 1, with absorption maximum at 240 nm and 288 nm and absorption minimum at 265 nm. (4) Infrared absorption spectrum: When measured in KBr, the substance has essentially the same spectrum as shown in Figure 2. (5) Solubility in solvents: Readily soluble in water, but very slightly soluble. in acetone, chloroform, ethyl acetate and petroleum ether. (6) Nuclear magnetic resonance spectrum: Spectrum *!! nuclear magnetic resonance: When measured at D20, the spectrum is essentially the same as shown in Figure 3. (7) Thin layer chromatography: Rf value: 0.6/plate: aluminum sheet coated with F254 cellulose, 0 .2 mm for thin layer chromatography (Merck 2 mm 60F254 for thin layer chromatography (Merck X Associates, Inc.), 4X(300 mm) Solvent: Methanol-0.01M phosphate buffer solution (1:9) pH 6.8 Flow rate: 1 ml/min (9) Molecular weight: 364 (calculated from the value obtained as a result of monomethyl ester mass spectrometry) (10) Molecular formula: C14H17N206 SNa (determined on the basis of the molecular formula of methyl ester calculated from the results of mass spectrometry) -(11) Stability: The half-life at various pH values is as follows: PH 4.0 6.0 7.0 7.8 10.0 KC-6643-A [t-^-(h)] 33 144 187 110 2.8 MM-4550* 5.5 18 10.5 8.5 19127 697 9 10 20 Solvent: pH 4.0—7.0: Mcllvaine buffer solution (sodium phosphate—citric acid) pH 10.0 0.1M sodium carbonate—sodium acid carbonate buffer solution 5 Measurement: » Absorption spectroscopy in ultraviolet . * J. Antibiot., 32,295 - 304 (1979) B) Monomethyl ester of the substance KC-6643-A (1) Appearance: Colorless solid 10 (2) Molecular weight: 356 (mass spectrometry) X (3) Elemental analysis for C16H20N2O6S : C H N S Calculated(%) 50.55 5.66 7.86 9.00 . » Found (%) 50.38 5.48 7.59 8.60 (4) Molecular formula: CMHa^jOsS (5) Ultraviolet absorption spectrum: The spectrum in methanol has an absorption maximum at 243 nm and 300 nm and a minimum absorption at 274 nm. (6) Infrared absorption spectrum 'When measured in KBr, the substance has a spectrum exactly identical to that shown in the attached drawing, Fig. 4.' .l (7) Nuclear magnetic resonance spectrum 25 1) 1H nuclear magnetic resonance: When measured in CDCl using tetramethylsilane as an internal standard, spectra were obtained exactly the same as those shown in the attached drawing, Fig. 6. 3§ (8) Chromatography thin-layer Rf value: 0.44/plate: aluminum sheet silica gel for thin-layer chromatography 60 F^, 0.2 mm (Merck Xi Co., Inc.) solvent: methylene chloride-methanol (9:11) 35 (9) Specific rotation [a] « —96.0° (cl. CH2C12) C) Disodium salt of the substance KA-6643-. P (1) Appearance: White powder (2) Melting point: No specific temperature is indicated, the substance turns gradually from yellow to brown at temperatures above about 130°C. % (3) Elemental analysis: C H N S 32.5% 3.48ft 5.17% 11.00% 45 (4) Ultraviolet absorption spectrum: Exactly the same as shown in the accompanying figure 7 and having the maximum absorption bands at 240 nm and 285 nm and the minimum absorption band at 265 nm. M (5) Infrared absorption spectrum: The infrared absorption spectrum of the test substance in the KBr tablets is exactly the same as shown in the attached drawing, Fig. 8. (6) Nuclear magnetic resonance spectrum 55 When carrying out the measurement in D2O, the tetramethylsilane as an external standard, obtaining a nuclear magnetic resonance spectrum exactly the same as shown in the attached drawing, Fig. 9. 60 (7) Solubility in solvent: Readily and soluble in water, but definitely insoluble in organic solvents such as acetone, chloroform, ethyl acetate, petroleum ether and the like. «5 (8) Specific rotation: [aJ2* -145° (Cl, HzO) (9) Stability: The half-life at different pH values is as follows: pH 4.0 6.0 7.0 7.8 10, 0 t—-(h) 2 ' 21.8 210 170 130 7.9 The solvent and measurement are the same as above for KA-6643-A. (30) Thin layer chronomatography: The results are shown in Table 1. (11) Paper chromatography Filter paper: Toyo filter paper No. 51 manufactured by Toyo Filter Pater oC., Ltd) Solvent: Acetonitrile-water (3 : 2) ascending method Rf value : 0.9 (12) High Performance Liquid Chromatography Column; n-Bondapack Ci8 (Waters Associates, Inc.) 4X300 mm Solvent: 0.1M phosphate buffer solution, pH6.8 Flow rate: 1 ml/min Residence time: 23.5 minutes (13) High voltage paper electrophoresis Using a high voltage instrument for paper electrophoresis and migration # in a 1/30M phosphate buffer solution at pH 7.0 and 3000 V within 30 minutes, this substance moves towards the anode by 8.5 cm. Penicillin N tested under the same conditions moved by 4.4 cm. II. Biological properties (1) Antibacterial spectra Antibacterial spectra illustrating activity against various microorganisms are shown in Table 2 in comparison to the spectra of aminobenzyl penicillin (AB-Pc) and cefoxitin (CFX). As shown by the data presented, the substance KA-6643 has intense antibacterial activity against both gram-negative and gram-positive bacteria, including bacteria resistant to various antibiotics such as penicillins and cephalosporins. (2) ^Inhibitor action - β-lactamase (i) Antibacterial action - increased action against bacteria resistant to the action of 0-lactam compounds Penassy scale (12 g of which was dissolved in 500 ml of distilled water, Kyoei Pharm. Co. ., Ltd) were inoculated with Escherdchia coli EC-1 (PC ase) and Citrobacter 24 m and producing 0-lactamase, and 10 ml of this inoculated agent were transferred to a Petri dish with a diameter of 9 cm and solidified to obtain a test medium. agar medium, 8 mm diameter discs were introduced on which 30 g of an aqueous solution of antibiotics were placed, and then cultures were carried out at a temperature of 37°C for 11 127 697 12 Table 1 | Plate I TLC aluminum sheet kieserite gel 60 F254 0.2 mm (Merck Co., Inc.) 1 1 TLC aluminum cellulose sheet F254 0.1 mm (Merck Co., Inc.) Solvent Butanol-ethanol-water (top layer 4:1:5) Isopropanol-water (7 : 3) Butanol—acetic acid—water (12:3:5) Butanol—isopropanol—water (7:7:6) Rf value 0.13 0.85 0.16 0.46 Tested bacterium Staphylococcus aureus 209P JC- 1 Staphylococcus epidermidis 1228 Bacillus subtilis 6633 Escherichia coli NIHJ JC-2 Klebsiella pneumoniae 602 Serratia marcascans NHL Enterobacter cloacae 977 Enterobacter aerogenes 972 Hafnia alvei 978 Pseudomonas aeruginosa A-3 Pseudomonas aeruginosa 1 0490 Pseudomonas putida 5121 Proteus vulgaris 874 Proteus morganii 3168 Proteus rettgeri 13501 Proteus inconstans Kmll5 Escherichia coli EC-1 (PC ase I) EC-59 (PC ase II) EC-68 (PC ase IV) Citrobacter 1 (PC ase) 24 (CS ase) Klebsiella 25 (PC ase) 32 (CS ase) Table 2 Minimum inhibitor concentration (mcg/1) KA-6643-A (sodium salt) 0.39 0.78 0.2 0.05 0.2 0.2 0.78 0.1 0.2 6.25 6, 25 1.56 0.39 0.39 1.56 0.78 0.2 0.05 0.05 0.78 1.56 0.78 3.13 KA-6643-B (disodium salt) 6.25 6 .25 6.25 1.56 6.25 6.25 6.25 3.13 6.25 50 25 100 12.5 12.5 50 12.5 3.13 1.56 3.13 6.25 12.5 6 .25 12.5 AB-Pc 0.05 0.78 0.025 6.25 12.5 50 100 6.25 50 100 100 100 100 100 0.2 100 100 100 100 100 100 100 100 CFI 1.56 1.56 1.56 6.25 0.78 102.5 100 100 6.25 100 100 100 6.25 6.25 1.56 6.25 3.13 6.25 6.25 100 100 6.25 100 period 18 hours and the diameters of inhibition around each disc were successively measured. The results are shown in Table 3. (ii) β-lactamase inhibitor activity When used as enzyme (3-lactamase amido- penicillin hydrolase BC 3.5.2.6. {Tokyo Chem.Syn. Co., Ltd) obtained from bacteria belonging to the genus Bacillus. The I50 value for the KA-6643tA substance was 0.5 ng/ml and for the KA-6643-B substance 12.8 ng/ml. The I50 value was measured according to the method of C. Reading and M. Cole (Antimior. Agents i Chemoth .11, 852-857(1977)). Accordingly, it can be expected that the substances KA-6643-A and KA-6643-B will show a strong synergistic effect when combined with other antibiotics (3-lactams. Table 3 50 55 60 Antibiotic composition (B-Pc * (1 mg/m2) KA-6643-A (10 y/m?) KA-6643-B (10 y/m2) AB-Pc (1 mm/m2) + 1 KA-6643-A (10 y/m2) m2) AB-Pc 91 mg/m2) + KA-6643-B (H) yAn2) Inhibited zone diameter (mm) Environment containing E.coli EC-1 — 18.0 — 20.0 23.0 Environment containing Citrobacter 24 — 16.0 — » 20.0 14.0 | 65 * AB-Pc: Aminobenzyl penicillin127 697 13 14 Taking into account the numerous features noted above, it was decided that the substances of the invention are β-lactam antibiotics. There is a known substance MM 4550 (MC696-SY2-A), which has a similar spectrum absorption in the ultraviolet * to the spectrum to the substances produced by the method according to the invention. The known antibiotic differs from the substances KA-6643-A and KA-6643-B in the following properties. (1) Paper electrophoresis. The compared substances were subjected to paper electrophoresis at a voltage of 300 V for 25 minutes. Solvent: 0.1 B phosphate buffer solution. ' Solvent: 0.1 phosphate buffer solution (pH 8.0) KA-6643-A: moves towards the anode by a distance of 5 cm.MM 4550. Moves towards the anode by 10.z cm (2) High liquid chromatography efficiency. Column: - j3-Bondapack Ci8 (Water Associated, Inc.) 4X30 cm Solvent: 0.05 M phosphate buffer solution (pH 7.0) Flow rate: 1 ml/min Residence time: KA-6643 -A 1 hour KA-6643-B 30 minutes MM45J50-B 17 minutes From these results it was concluded that the substance KA/6643 is a new compound different from the known J3-lactam antibiotics. If the substance KA-6643 prepared according to the invention has in carboxyl group in the molecule can be used in the form of a free acid, but it is preferably used in the form of a salt. Suitable salts are, for example, alkali metal salts or alkaline earth metal salts, i.e. salts of sodium, potassium and calcium, ammonium salts, and sodium salts. such as ammonium salt of dimethylamine, trimethylamine and others like that. In addition, the substance may be used in the form of lower alkyl esters such as methyl ester, ethyl ester and the like, as well as higher fatty acid esters. The process of the invention is explained in detail in the examples below, in which parts and percentages are given by weight, if any. not otherwise specified. Example I: (a) 301 liquid medium was prepared consisting of 3% starch, 1.5% soy flour, 0.28% monobasic potassium phosphate, 0.18% dibasic phosphate dibasic hydrate sodium, 0.0005% cobalt chloride hexahydrate, 0.06% magnesium sulfate heptahydrate and 0.001% iron sulfate, and the pH of which was adjusted to 6.0. 100 ml samples of the environment were introduced into 500 ml conical flasks and sterilized, then the mycelium of the KC-6643 strain was inoculated and shock culture was carried out at 27°C for 4 days at 220 rpm. (b) After the culture was completed, Celite (Johns Manville Co., Inc.) was added as a filter aid and the mycelium was removed by filtration. 25 liters of the filtrate were passed through a column (6 x 30 cm) packed with a strong basic anion exchange resin. Diaion PA316 (type Cl", Mitsubishi Chem. Ind., Ltd.). The column was washed with water and eluted with a 0.01 M phosphate buffer solution at pH 7.0 containing 0.5 M sodium chloride to collect the active substance fraction. The obtained fraction of the active substance was passed through a column (4X30 cm) filled with activated carbon and, after washing with water, it was then treated with a 50% by volume aqueous solution of acetone to collect the active substance fraction. The collected fraction was in turn passed through a column (4XJ40cm) filled with ¬ on alkaline anion exchange resin - Amberlite 15 IRA-68 (Cl~ type) and again the column was washed with water and then eluted with a 0.01 M phosphate buffer solution at pH 7.0 containing 0.2 M sodium chloride to collect the fractions active substance. In turn, these collected active substance fractions were passed through a column (3X30 cm) filled with activated carbon, then the column was washed with water and eluted with a 50% aqueous acetone solution to collect the active substance fraction, 25 (c) The fraction obtained in this way was passed through a column (4X30 cm) filled with strong alkaline Dowex 1X2 anion exchange resin (grain size 0.152-0.076 mm, Cl- type), the column was washed with water and eluted using the phenomenon of concentration and velocity gradient. flow of 3 ml/min, between a 0.01 M phosphate buffer solution at pH 7.0 containing 0.1 M sodium salt and a solution containing 0.4 M sodium chloride, dividing the fractions into two further fractions, one of which is 35 one fraction is washed initially and the other fraction is washed later, and the fractions separated in this way are collected separately. (d) Initially, the active substance fractions collected in the amount of 11 were washed and concentrated to approximately 100 ml under reduced pressure at a temperature below 30°C. The obtained concentrate was passed through a column filled with Diaion HP-20, which had previously been washed with a 20% salt solution, and then the column was eluted with distilled water at a flow rate of 4 ml/min. The active substance fractions were collected and concentrated to 2 ml below 30°C, and then subjected to cooling and drying to obtain 80 mg of KA-6643-A powder. (e) The second fraction of the active substance washed out was subjected to a similar treatment as in step (d), resulting in 240 mg of KA-6643-B powder. Example II: (a) Composition containing 2% starch, 1.5% soy flour, 0.28% monobasic potassium phosphate, 0.18% dibasic disodium phosphate dodecahydrate, 0.0005% cobalt chloride hexahydrate, 0.05% magnesium sulfate heptahydrate and 0.001% sulfate the iron was adjusted to pH 6.0 and sterilized, after which the mycelium of KG-6643, 60 was inoculated, and then a preliminary culture was carried out for a period of 48 hours at a temperature of 27°C to obtain the first inoculation environment. In each of two tanks with a volume of 200 l, 1001 of the composition specified above, but with the addition of 1% cottonseed oil, were placed. Cultures of the first inoculation in the amount of 500 ml were inoculated into the environment in each tank and further cultivation was carried out at a temperature of 30°C using the aeration mixing method, at 300 revolutions per minute and an air stream speed of 50 rpm for a period 4 days. (b) After completion of the culture, 10% by volume of Dicalite Perlite 4109 (Dicalite Orient Co., Ltd) was added to the culture broth in an amount of 170 liters, serving as an auxiliary filtration agent, and the mycelium was removed by filtration. The conductivity obtained in an amount of 150 1 of the filtrate was adjusted to 0.15 S/m and passed through a column <21%X4i5cm) filled with a strongly alkaline anion exchange medium Diaion PA316 (type Cl - Mitsubishi Chem. Ind., Ltd) at a flow rate of 20© l/min. After washing with a 0.01 M phosphate buffer solution at pH 7.0, the resins were eluted with a 2% (v/v) mixture of methanol and a 0.01 M phosphate buffer solution at pH 7.0 2M against sodium chloride at a flow rate of 150 ml/min, collecting traction of the active substance. The collected fraction of the active substance was passed through a column (16X10 cm) filled with a Diaion HP-20 preparation, which was treated with a 10% w/v sodium chloride solution at a flow rate of 400 ml/min and after washing with 10% w/v sodium chloride solution was eluted with deionized water at a flow rate of 200 ml/min, collecting the fractions of the active substance. (c) The active fraction thus collected was passed through a column (6JX, 70 cm) filled with the strongly basic Amberlite IRA-458 type Cl" ion exchange resin from Rohm and Haas Co.) at a flow rate of 50 ml/min and the column was eluted with 0, 01 M phosphate buffer solution at pH 7.0 containing 0.15 M sodium chloride. The remaining fraction of the clear substance was eluted with 0.01 M phosphate buffer solution at pH 7.0 containing 2M sodium chloride. (d The initially eluted fraction obtained in step (c) was subjected to the same treatment as in step 700 mg of powdered KA-6643-A. (e) The second eluted fraction of the active substance was passed through the column (6X ,? 0cm) filled with the Diaion HP-20 preparation, was previously treated with a 20% w/v aqueous sodium chloride solution and then the column was eluted with deionized water at a flow rate of 20 ml/min, collecting the active substance fraction. method, the fractions of the active substance were diluted with deionized water to obtain an electrical conductivity of approximately 0.07 S/m and passed through a column (3X30 cm) filled with a weakly basic anion exchange resin DEAE-Sephadex A-25 (from Pharmacia Fine Chemicale) with a pH adjusted to 7, 0 using a 0.01 M phosphate buffer solution, and then the column was washed with water and eluted with a phosphate buffer solution at pH 7.0 containing 0.15 M sodium chloride at a flow rate of 35 ml/min to collect the substance fraction active. The collected eluate was concentrated under reduced pressure at a temperature below 30°C and then passed through a column (3.5 X an aqueous solution of hydrated sodium chloride and the column was eluted with deionized water at a flow rate of 20 ml/min to collect the active substance fraction. The fractions of the active substance were concentrated under reduced pressure to 2 ml and dried by freezing to obtain 190 mg of powdered substance KA-6643-B. The powder thus obtained was dissolved in 1 ml of deionized water and passed through a column (2X30 cm) filled with Diaion. HP-20, 15 and then the column was eluted with deionized water at a flow rate of 1 ml/min to collect the active substance fraction. The active substance fraction collected in this way was concentrated under reduced pressure and passed through a Sefadex G-10 column, and then the diagram was developed with deionized water at a flow rate of 0.2 ml/min. The active fraction was collected, concentrated under reduced pressure to approximately 2 ml and then freeze-dried to obtain 80 mg of powdered product. (f) §0 mg of the powder obtained in this way was dissolved in 0.1 ml of 0.1 M phosphate buffer solution with pH 6.8 and passed through a column (0.8 x 120 cm) filled with Bondapack 30 Cis (Polasil B/Waters Associatts, Inc.) for high-performance liquid chromatography treated with a 0.01 M phosphate buffer solution, and then the column was eluted with the same buffer solution at a flow rate of 6 ml/min, collecting the substance fractions. active. The active fraction was passed through a column (0.9X5 cm) filled with activated carbon. Ko-. the column was washed with deionized water and eluted with a 50% w/v aqueous solution of acetone, collecting the active substance fractions. The active fraction was concentrated under reduced pressure to approximately 1 ml and freeze-dried to obtain 35 mg of purified KA-6643-B powder. Example III. 120 mg of the powder obtained in Example I (d) or Example II (d) was dissolved in 1 ml of deionized water and then passed through a column (0.8 x 60 cm) packed with Bondapack Ci8 (Polasil B/Waters Asso¬ ciates, Inc.) for high-performance liquid chromatography and the column was eluted with a 0.01 M phosphate buffer solution at pH 6.8 containing 2% methanol at a flow rate of 6 mVmin, collecting the active substance fractions. method, the fractions of the active substance were concentrated to 5 ml under reduced pressure and passed through a column (2X15 cm) filled with Diaion HP-20 preparation, which was washed with sodium chloride solution and then eluted with deionized water at a flow rate of 1 ml/min 60 collecting fractions of the active substance. These fractions were concentrated to 1 ml and then passed through a column (3X150 cm) packed with Sephadex G-10 resin and the column eluted with deionized water at a flow rate of 1 ml/min. The obtained fraction of the active substance was concentrated to 2 ml under reduced pressure and freeze-dried to obtain 4 mg of the purified product of the sodium salt KA-6643-A. Example IV. Methyl ester of the antibiotic KA-6643-A. 0.06 ml of methyl iodide was added to a solution of the antibiotic KA-6643-A (5 mg) in dry dimethylformamide (1 ml). The solution was stirred at room temperature for 3 hours. The reaction mixture was evaporated to dryness under reduced pressure. The residue was chromatographed on two HF254 silica gel plates previously washed (E. Merk, 2GXt20XiO.05 cm) using a mixture of methylene chloride and methanol in the ratio 9:1. The Rf 0.34 zone corresponding to the title compound was collected and eluted with the same solvent system to obtain the title compound in pure form. x 235 (-1.427 KBr): ultraviolet absorption spectrum UV* (methanol) nuclear magnetic resonance spectrum 1HNMIKCDCI3): "CNMR(CDCl3)5: mass spectrum (m) of 357 (MH+): Elemental analysis for calculated found CD (cO. l), methanol (0): 143—144°C -96.0°C (cl, CHaCl2) 1780, 1715, 1620, 1500, 1435, 1370, 1330, 0260 cm"1 244 (e 17,000), 299 ( n,O0Wnm 1.29, 1.49 (each 3H, s. C(CH3)CH8) 2.11 (3H, s, COCH;,) 2.98 (1H, dd, J = 15.0, 23, OHz, H-4) 3.55 (1H, d, J = 5.7Hz, H-6) 3.87 (3H, s, OCH3) 4.25 <2H, m, H-4, H-5) 6.7 (1H, d, J = 13.6Hz, SO-CH=CH) 7.56 (1H, dd, J = 10.8, 13.6Hz, CHaCH-NH). . 8.86 (1H, br-d,J = =10.8Hz, NH) 23.3, 28.1 (q, C(CH3)CH3), 29.7 (q, COCH8), 29.7(t ,C-4), 52.9 (q, OCH3), 53.9 (d, C-6), 63.7 (d, C-5), 70.6 (s, C-8), 111, 2 (d, SO-CH=CH) 131.7 (s, C-3), 134.0 (d, CH=CH= =NH), 148.2 (s, C-2), 160.2, 169.1, 175.7 (s, C=0), 340, 308, Ci5H20N2O6S: C H N 50.55 5.66 9.00 50.61 5.69 8.92 215 (-3.47^X10*) , 244 (+4,757 p-nitrobenzyl ester of the antibiotic KA-6643-A. A solution of 5 mg of the antibiotic KA-5543-A and 7 mg of p-nitrobenzyl bromide in 1 ml of dry dimethylformamide was stirred at room temperature for 3 hours. then to dryness under reduced pressure. Preparative chromatography method carried out on pre-washed HF254 silica gel plates using a mixture of methylene chloride - methanol in a ratio of 9:1 obtained 3 mg of the title compound with Rf 0.38. absorption spectrum in the sub- 1785 , 1710, 1620, 1520, red IR(KBr): 1365, 1345, 1320, 1250 cm-\ 35 40 45 50 55 60 15 ultraviolet absorption spectrum UV (methanol) nuclear magnetic resonance spectrum iHNMR(DMF- dT5: 250 (e 17,900), 305 (9,400)nn\ 1.28, 1.45 (each 3H, s, CJCHaJCHg), 2.06 (3H, s. COCH,), 4.97 (1H,, s , OH), 5.40, 5.59 (each 1H, d, J = 13.6Hz, CH2Ar) 6.34 (1H, d, J = 14.1 1 Hz, SO-CH=CH), 7, 48 (1H, dd, J = 10.5, 14.1Hz, CH=CH-NH), 7.48, 8.29 (each 2H, d, J = 8.8Hz, Arn), 10.58 (1H , br-d) J = = 10.5Hz, NH) Example VI. p-nitrobenzyl ester of the antibiotic KA-6643-B Proceeding as described in Example V, 3.5 mg of the title compound (Rf 0.32, a mixture of CH2Cl2 - methanol in the ratio of 4:1) was prepared using 5 mg of the antibiotic KA-6643- B 5 mg psnitrophoenzyl bromide. absorption spectrum in the sub- 1790, 1720, 1620, 1510, red IR(KBr): 1420, 1370, 1350, 1320, 1280-1310(br)cm-and absorption spectrum in the ultraviolet UV (methanol) spectrum of the nuclear resonator magnetic resonance iHNMR(DMF-dx)5: 251 (e 19,200), 300 (ll,400)nm, 1.58, 1.60 (each 3H, s, CCCHaJCHa), 2.06 (3H, s, COCH, ), 5.40, 5.58 (each 1H, d, J = 13.5Hz, CHjjAr), 6.37 (1H, d, J = 13.6 Hz, SO-CH=CH), 7.48 ( 1H, dd, J = 10.6, 13.6Hz,CH=CH-NH) 7.84, 8.28 (each 2H, d, J=8. 4Hz ArH), 10.61 (1H, br-d, J = = 10.6Hz, NH).127 697 19 20 Example VII. p-bromobenzyl ester of the antibiotic KA-6643-A Following the procedure given in Example V, 10 mg of the title compound (Rf 0.62) were prepared by treating 20 mg of the antibiotic KA-6643-A with 40 mg of p-bromobenzyl bromide. The obtained solid product was crystallized from an acetone-n-hexane mixture to obtain the title compound, which was then recrystallized from the same solvent to obtain well-shaped crystals in the form of needles for X-ray analysis. melting point infrared absorption spectrum IR(KBr): ultraviolet absorption spectrum UV(MeOH): 149-149.5°C 1785, 1710, 1620 cm." 228 (e 19,500), 245 (16,400), 303 (9,800)nm 10 15 Elemental analysis for: calculated found C21H23N20«SBr: C H N S 49.32 4.53 5.48 6.27 49.39 4.55 5.44 6.21 Patent claim Method of producing a new antibiotic of the formula general method shown in the drawing, in which R is a hydrogen atom or a group of the formula -SO3H, optionally in the form of a salt or ester, characterized in that Streptomyces microorganisms are grown, are KC-6643 (ATCC 31493 and FERM-P 4467), and then the antibiotic substance or substances are isolated from the culture broth and optionally converted into s51 or ester.CH, CH.OR O / ¦NO t S-CH=CHNHCOCH, COOH 0* Q 0 Q 3 60 50 40 30 20 10 FIG.1 1 I I 1 1 1 I 1 - I 1 1 ¦ 200 250 300 330 Wavelength (n m) 100, FIG.2 4000 3000 2000 1000 Wavenumber (crrr1)127 697 FIG .3 b~J -1 1 L_ 8 7 6 5 4 3 2 1 a(ppm) FIG.4 length {ali(fii)n) 5 10 - 4000 2000 1500 1000 Wave number (cm-1) 20 50 500 200 FIG.5 CHCl3 JL__v^lJl JJ JUjU LxuJ^ 987654321 6(ppm)127 697 FIG. 6 70.6 175.7 CDCl3 169.1 148.2 160.2 131.7 134.01 111.2 i^siwpw^ 52.9 63.7 535 29.7 128.1 23.3 4|iA#w in [ppm] FI6.7 100 FIG.8 N 2000 150 0 Wave number (crrT1) 220 250 300 wave length (mm ) FIG.9 _u_ju_^^UVj^ uL . 987654321 06 (ppm) ZGK 1243/1331/5 — 80 copies Price PLN 100 PL PL PL

Claims (1)

1. Patent claim 1. A method of producing a new antibiotic with the general formula shown in the drawing, in which R represents a hydrogen atom or a group of formula 2. -SO3H, optionally in the form of a salt or ester, characterized in that Streptomyces microorganisms are cultivated, are KC-6643 (ATCC 31493 and FERM-P 4467), and then the antibiotic substance or substances are isolated from the culture broth and optionally converted into s51 or ester.3. CH,4. CH.5. OR O6. / ¦N7. About t8. S-CH=CHNHCOCH,9. COOH 0*10. Q 011. Q 3 60 50 40 30 20 1012. FIG.1 1 I I 1 1 1 I 1 - I 1 1 ¦ 200 250 300 33013. • Wavelength (n m) 100.14. FIG.2 4000 3000 2000 100015. Wavenumber (crrr1)127 69716. FIG.3 b~J -1 1 L_ 8 7 6 5 4 3 2 1 a(ppm)17. FIG.4 length {ali(fii)n) 5 10 -• 4000 2000 1500 100018. Wave number (cm-1) 20 50 500 20019. FIG.520. CHCl321. JL__v^lJl JJ JUjU LxuJ^ 987654321 6(ppm)127 69722. FIG. 6 70.6 175.7 CDCl3 169.1 148.2 160.2 131.7 134.01 111.2 i^siwpw^ 52.9 63.7 535 29.7 128.123.3 4|iA#w in [ppm] FI6.7 100 FIG. 8 N 2000 1500 Wave number (crrT1) 220 250 300 wave length (mm) FIG.9 _u_ju_^^UVj^ uL . 987654321 06 (ppm) ZGK 1243/1331/5 — 80 copies Price PLN 100 PL PL PL