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    • 专利摘要:
    Novel antibiotics C-19393S2 and H2, which are produced by cultivating a microorganism belonging to the genus Streptomyces, are useful as a bactericide or disinfectant, and particularly, Antibiotic C-19393S2 produces a synergistic effect with penicillin and/or cephalosporin antibiotic agents.
    公开号:SU1075984A3
    申请号:SU802878699
    申请日:1980-02-01
    公开日:1984-02-23
    发明作者:Имада Акира；Харада Сецуо；Асаи Мицуко
    申请人:Такеда Кемикал Индастриз,Л.Т.Д. (Фирма)；
    IPC主号:
    专利说明:

    This invention relates to the microbiological industry and relates to the production of a new antibiotic. The aim of the invention is to obtain a new antibiotic of the general formula xI-y KHCOCHj, where is the antibiotic C-19393 S and / or R-H is the antibiotic C-19393 N Shz-y. -cC / g-0 o - NHCOCHj CC is an antibiotic C-19393 S where, R-SO, H and / or R-I is an antibiotic C-19393 H which is that Streptomyces C-19393 strain (IFo 13,886) cultured in a nutrient medium containing carbon and nitrogen sources, and the target product is isolated from the liquid culture medium as a mixture and / or individual antibiotics. To obtain the antibiotic C-19393 S and / or Hj of the invention, microorganisms are used that produce the antibiotic C-19393 S.j and / or Hj., Belonging to the Streptomyces sp. A typical example of such a microorganism is the strain Streptomyces sp C-19393, which is obtained by sowing a suspension of soil samples collected in Sweden in sterile water for a culture medium consisting of 1% soluble starch, 0.05% urea, 0.05% polypeptone (Daigo Nutritive Chem.Ltd) 0.05% yeast extract (Dif with uSA), 0.02% potassium phosphate, 0.02% potassium chloride, 0.01% magnesium sulfate, 5 mg / ml bleomycin and 2% agar, pH 7.0 and repeated purification of the microorganism from the colony obtained during cultivation for 2 weeks; Strain C-19393 species of Streptomyces is the following microbiological characteristics. Morphological characteristics. Aerial mycelium, having a width of about 1, is pulled from a well-branched vegetative mycelium and spins off from the main axis monopodially to form side chains. Straight or slightly curved spore chains are observed at the end of the side chain. Each dispute has a cylindrical shape (0.35-0.55-0.7-1.4) and has a smooth surface. No special organs, such as spherical sporangia, sclerotia, and also mobile 2 | no dispute is observed. Cultural characteristics. The cultural characteristics of the strain in different environments are presented in table.1. The characteristics shown were observed after cultivation at 28 ° C for 2 weeks. Table 1
    Nitrate sucrose
    Agar, Moderate
    Glucose asparasunter- -. gynic agar
    Glycerin, aspar ginovy agar
    Moderate
    Starch Agar
    Colorless No
    White
    -
     I I
     I
    Ocher
    Nutrient Agar
    Tyrizine
    agar
     I I
    Yeast Extract Maltose Extract Agar
    Oats no-flour
    "I j agar
    Strain C-19393 grows abundantly on medium containing 2% oatmeal, 2% tomato paste, 0.2% BovriE and 2% agar, having a pH of 7.0, and abundant formation of a greyish yellow mycelium occurs. Soluble pigment does not form.
    Physiological characteristics.
    Temperature range of growth: the lower limit is below the upper limit of 32-35 C; The optimum temperature is 26.5-30 ° C.
    Gelatin thins, starch hydrolyzes, peptonizes skim milk, but does not coagulate. Melanoid pigments tyrosine-agar, and peptone-yeast extract-iron-agar does not form.
    The following carbon sources are well absorbed by glycerin, D-xenose, L-arabinose, D-glucose, D-galactose, D-fructose, maltose, rhamnose starch. Poorly absorbed inositol, cellulose. Does not assimilate D-mannitol, sharose, raffinose.
    The strain Streptorayces C-19393 deposited in the Institute of fermentation in Fermentation Reseach last Agency of Ind.Science and TechnoCogy PERM-P. Osaka numbered iFo 13886 and on the American Tour Cuture CoXection numbered AMCC 31486.
    The Shp1m Streptomyces C-19393 (iFo 13886) is cultivated in a nutrient medium containing sources of carbon and nitrogen. Glucose, starchC, glycerol decastrin, sucrose, jelly from millet, malyassa can be used as carbon sources. An example of a nitrogen source is meat extract, dried yeast, yeast extract, soy flour, corn extract, wheat buds, cotton blossom, ammonium sulfate, ammonium nitrate. If necessary, inorganic continuation can be used. Table 1
    Elephant
    White bone
    - colorless
    Grayish -
     1 yellow
    Colorless
    White
    0
    cic salts such as calcium carbonate, sodium chloride, potassium chloride, phosphoric acid salt, as well as organic and inorganic substances that contribute
    5 growth of the microorganism and can be added to the nutrient medium in appropriate quantities.
    In addition, heavy metal salts can be added to the medium, as
    0 ferrous sulfate, copper sulfate, as well as vitamins B, biotin. .
    Antifoam and surface active agents, such as ethylene masses, polyalkylene glycol ether, can be added to the medium.
    Cultivation is carried out in solid or liquid medium under aeration conditions. Cultivation is carried out at 15-32 s, pH 4-8 for 8-168 h, preferably 24-144 h.
    0
    The antibiotic C-19393 S and H are produced extracellularly and in the fermentation broth, the resulting culture is separated from the microbial cells and the upper liquid layer by centrifugation or filtration, and then the antibiotic is separated from the upper liquid layer. However, an antibiotic can also be obtained directly from fermentation -; -,
    0 foot broth.
    Isolation of C-19393 82 and Hj can be carried out according to the procedure commonly used to isolate the metabolites produced by microorganisms. For example, since C-19393 S2 and H2 is a water-soluble acidic compound, produced mainly outside the cell, a technique involving removing microbial cells by filtration or centrifugation and separating, purifying and collecting the active substance from the filtrate is usually used to isolate C -19393 S and N. Thus, different means using the difference in solubility and degree of solubility in different solvents, the difference in the nature of the precipitation and the deposition rate and the difference in adsorption capacity, and the same ion exchange chromatography,. molecular sieve chromatography under reduced pressure and freeze-dry; and the like. can be used on their own or in a suitable combination, in any order and with any number of repetitions. Examples of suitable adsorbents are activated carbon, adsorption mixtures, anion exchange resin (anion type), powdered cellulose, silica gel, and the like. or carriers possessing the properties of molecular sieves. Examples of eluting solvents that can be used are aqueous solutions of water-soluble solvents, such as acetone, methanol, ethanol, propanol, butanol, isopropanol, isobutanol and the like, or aqueous solutions of acids or alkalis, or buffer or aqueous solutions of inorganic or organic salts, although the solvents used may vary depending on the type of carrier. The fermentation broth obtained after the completion of the cultivation, is filtered using filter media to remove microbial cells. The resulting filtrate is passed through a column of activated carbon under neutral or slightly acidic conditions and the adsorbed antibiotic C-19393 S is eluted with water or a hydrophilic solvent system. Most of the antibacterial activity is found in the aqueous eluate. Since the antibiotic C-19393 S is naturally acidic, such anion exchange resins of type C or AcO, such as Amberdite 1PA-400, 402 and 410, can be used for further purification. From the resulting antibiotic eluate, the active substance is further eluted with an aqueous solution of sodium chloride or with a buffer solution. The desalting of the eluate is achieved by weak acidification of the eluate and chromatographic treatment of the eluate with activated carbon, followed by elution with an aqueous solution of alcohol, etc. The eluate containing the active compound is concentrated under reduced pressure at low temperature and ethanol or methanol is added to the concentrate. The resulting precipitate is removed by filtration and the resulting aqueous-alcoholic solution is again concentrated under reduced pressure. Acetone or a similar compound is added to the concentrated precipitate and the precipitate is separated by filtration. The resulting powder can be subjected to additional chromatographic treatment using column chromatography with a combination of DEAE or QAE Sephadex phases in the SS-form and an adsorbent — XAD resin or a highly porous Diaion HP-20 type resin. The aqueous solution of the powder obtained above was adsorbed onto DEAE Sephadex A-25 (cCP-form) and, after washing with water, the column was eluted with a 0.4 M aqueous solution of sodium chloride. The pH of the eluate is set to 5, and it is column chromatographed using activated carbon. Elution from an activated carbon column was carried out using aqueous isobutanol, however, the best result is obtained by elution under neutral or slightly alkaline conditions, which are created by adding dilute aqueous ammonia solution and the like. Then, the resulting compound is subjected to column chromatography using XAD-H or HP-20. The adsorbed active substance is fractionally eluted with water. The active fractions were collected and concentrated, and the concentrate was subjected to chromatography on a QAE-Sephadex A-25 column (CE-form), after which it was eluted with a 0.02 M aqueous solution of sodium chloride. The eluate obtained is desalted by chromatography on activated carbon in the same manner as described above. The eluate is concentrated and the concentrate is subjected to column chromatography using an XAD-11 phase, followed by elution and fractionation with water. It was found that the fractions obtained give a single peak on the liquid chromatogram, as will be shown below. The active fractions are combined and concentrated to dryness under reduced pressure, at low temperature and acetone or a similar compound is added to the residue to form C-19393 S2. The fermentation broth, obtained after the cultivation is completed, is iltrated with filter aid to remove the microbial plumes. The resulting eltrate was passed through a column of activated carbon under neutal or weakly alkaline conditions.
    and the adsorbed antibiotic C-1939 Hj is eluted with a hydrophilic solvent system. Since the antibiotic Cr-19393 H is acidic in nature, anio-exchange, resins in the form of C1 or CH COO, such as Amberit IRA-400, 402, 410, Duex-1, Diaion A-21A and G. The active substance, additionally eluted with an aqueous solution of chlorate n atri or buffer solution. The desalting of the eluate is carried out by its neutralization or weak acidification and subsequent chromatography through activated
    coal, followed by elution with an aqueous solution of alcohol, etc. The eluate containing the active compound is concentrated under reduced pressure at low temperature and methanol and ethanol are added to the concentrate. The resulting precipitate is removed by filtration and the resulting aqueous alcohol solution is again concentrated under reduced pressure. For further purification of the resulting concentrate, column chromatography can be successfully used using a combination of DEAE or QAE Sephadex in the C-form, as well as an adsorption resin XAD or Diaion PH-20, i.e. the previously obtained concentrate is passed through Diaion HP-2O and fractionally eluted with water. The active fractions are concentrated and the concentrate is passed through DEAE Sephadex. A-25 / se-form. After washing with a 0.02 M aqueous solution of sodium chloride, the elution was carried out with a 0.05 M aqueous solution of sodium chloride. Then, the resulting eluate is passed through Diaion HP-20, which is treated with an aqueous solution of sodium chloride and the elution is carried out with an aqueous solution of sodium chloride containing methanol. Both of the eluate salines are chromatographed using activated carbon, in the same manner as described above. The resulting eluate is concentrated and the concentrate is subjected to column chromatography using Diaion HP-20 (50-100 mesh) and fracioino is eluted with water. The active fractions are drained and 4 are concentrated and the concentrate is subjected to chromatography on a column using a Sephadex A-25 iCt-QAE phase) which is treated with a 0.02 M solution of sodium chloride -. Fractionation is carried out using a 0.04 M aqueous solution of sodium chloride and the active fractions are desalted.
    in the same way as described above using chromatography through activated carbon. The eluate is concentrated and the concentrate is subjected to chromatography on a column using Avice E (crystalline cellulose) - and the column is eluted and fractionated with 90% aqueous propanol solution. The eluate obtained is concentrated and the concentrate is subjected to chromatography on a column with XAD-11 (100-200 mesh), I after which elution and incorporation with water is carried out. The active fractions are concentrated and the concentrate is subjected to preparative liquid chromatography, after which elution and fractionation is carried out using meta-containing phosphate buffer. Fractions ,. giving one peak to xpOMaTorpaivivie, combine and concentrate and concentrate the concentrate by column chromatography using HP-20 (100-200 mesh), after which elution is carried out with water to remove the buffer. Eluted active fractions are concentrated under reduced pressure at low temperature and the concentrate is freeze-dried to obtain C-19393 Ey as a white powder.
    The compounds of the invention may form a metal salt and an ammonium salt. Examples of metal salts include sodium salt, potassium salt, lithium salt, and the like.
    The C-19393 S dinatrine salt obtained in Example 1 has the following physical and chemical properties: appearance — white powder; specific force: (, 5, in water),
    Elemental analysis,% (the sample was dried over phosphorus pentoxide with during bh): C 36.29f1.0; H 3.72 tO, 5; N, 5; Na 9.70-t il, 0; Q 31.09tl, 0; S 13.1311.0.
    The amount of oxygen is calculated. on balance, i.e. subtracting the contents of the other components.
    Molecular weight (calculated on the basis of the content of 2 Na atoms / molecule) is 528-429 molecular formula, С Н Н Н Наа2Од32. UV spectrum: spectrum taken in water and maximum wavelengths were as follows: D 240 f 12 nm / E.2 296120 / and 285 ± 2 nm / E2 245420)
     IR spectrum; spec tr was measured with; samples in tablets KBG and had main peaks (wave numbers) were: 3450, 3220, 3000, -1770, 1700, 1630, 1515, 1395,
    65 1240-1260, 1100, 1050, 1010, 980, 950, 915, 900j 860, 815, 795, 760 715, 670, 625, 590, 530. (cm). Thin-layer chromatography CeCeueose F. Solvent system. The value of R a) propane: water (4: 1) 0.3310.1 b) butanol: acetic acid: water (2: 2: 1) 0.54 ± 0.1 s) propanol: ethanol-water (5: 2 : 3) 0.62 tO, l Solubility: insoluble in chloroform, ethyl acetate, acetone; partially soluble in ethanol, wutanol, pyridine; soluble in methanol, dimethyl sulfoxide, acetic acid; easily soluble in water. Color reactions: positive Ehrlich reaction with potassium permanganate; negative — reactions with ningi-drin, Greig-Libak, Dragendorff, with ferric chloride, and the Sakagushi reaction. The sodium salt of C-19393 Hj obtained in Example 2, shown below, has the following physical and chemical properties. Appearance - white powder. Elemental analysis (determined for a sample of dried over phosphorus pentoxide at 40 ° C for 6 hours): C 45.34 ± 1.0; H 4.98 ± 0.5; N 7.48 ± 0.5; Na 6.15 tl, 0; 6 8.51ll, 0. Molecular weight (calculated on the basis of the content of one Na atom per molecule) 426-322. Molecular formula of NaSOg. Specific rotation: fo (3. ° 134 ° (with 0.156, in water). UV spectrum: the measured spectrum has the following maximum wavelengths: Amax.NO / E 242+ ± 2 nm / 395 ± 20) and 28912 nm ( 314f20) IR spectrum: the spectrum measured in a tablet of KBG has the following major pIH (wave numbers): 3400, 298.0, 2940, 1770, 1630, 1530 1390, 1265, 1215, 1130, 1090, 1065 1040, 1000, 920, 840, 820, 790, 7 620, 540, 450 / cm. Circular dichroism spectrum (in uDOZ: cat's positive effect at 234 nm and cotton's negative effect at 206, 258, 292 mm. Thin-layer chromatography (using cellulose). Solvent system. R a value) propanol: water (4: 1) 0.45 tO, l b) butanol: acetic acid: water (2: 1: 1) 0.67, tO, l c) butanol: pyridine: acetic acid: water (15: 3: 2: 12), upper layer 0.5510.1 Color reactions: Ehrlich positive reactions and with potassium permanganate; negative - with ninhydrin, Greig-Libak, Dragendorf with ferric chloride and Sakagushi reaction. Solubility: insoluble in chloroform, ethyl acetate; moderately soluble in acetone, ethanol, and butanol; soluble in methanol and water. Antibiotics C-19393 S, and Nl are antibiotics of the lactal type 5. The antimicrobial spectrum of the sodium salt of the antibiotic C-19393 82 is presented in table 2. Table 2 Escherichia Cooper Staphydococcus aureus 209P12,5 Sarcina t utea IFo 323212,5
    Continuation of table 2
    subtilis
    12.5
    i
    cereus
    100
    Wednesday - broth agar.
    The antibiotic C-19393 S also has a strong beta-lactamaena inhibitory activity and, therefore, it significantly increases the sensitivity of various bacteria that are resistant to the action of pinicillin and / or cephalosporin, to the action of these agents.
    The antibiotic has activity against gram-positive game of negative bacteria and therefore it can be used in the treatment of bacterial infections in mammals, such as mice, rats, dogs, humans.
    For use of C-19393 82 in the treatment of infection by a microorganism of the E.coti species, the antibiotic is dissolved in physiological saline in order to prepare an injection for injection, which can be used parenterally, for example, subcutaneously or intramuscularly at a dose of 2-200 mg / kg / day, preferably 5-50 mg / kg / day. In the case of oral administration, antibiotic C-19393 S is mixed with lactose and encapsulated in order to obtain a capsular preparation that can be used with a dose of 10-500 mg / kg / day, preferably 20-200 mg / kg / day.
    In addition, the antibiotic C-19393 obtained according to the invention can be used as a disinfectant drug. For example, a liquid preparation that can be obtained by dissolving the antibiotic C-19393 S in distilled water with a concentration of 0.11, 0 w / v% or an ointment containing 2-50 mg, preferably 5-20 mg . C-19393 S2 per g of white petrolatum or lanolin can be used as a base as a bactericide or disinfectant ± wah for treating upper and lower limbs, eyes, ears, etc. in the above animals.
    Antibiotic S-19393 Sj has beta-lactamase inhibitory activity and therefore, significantly increases the sensitivity of penicillin or. cephalosporin - resistant bacteria to ampicillin and
    cefotiam because of its ability to produce beta-lactamase. Accordingly, C-19393 S can be used to treat infection in mammals (e.g.
    in mice, rats, dogs, and humans), as well as in birds (for example, chickens, ducks), especially bacterial infections, caused by the action of beta-lactates — antibiotically resistant
    0 bacteria in combination with penicillin or cephalosporin.
    In the case when the antibiotic C-19393 S is used in combination with other beta-lacquer-type agents for the treatment of infectious diseases caused, for example, beta-lacto antibiotic - stable E.coEi, equal amounts of C-19393 S and ampicillin are dissolved .
    Q in physiological solution to obtain an injection solution that can be used parenterally, for example, subcutaneously or intramuscularly, with a dose of 0.1-20 mg / kg / day
    5 preferably 0.5-5 mg / kg / day. C-15393 S. can also be administered orally with a dose of 1–200 mg / kg / day, preferably 5–100 mg / kg per day, in the form of capsules, each of which contains an equal amount of C-19393 S and cephalexin.
    If the antibiotic C-19393 S is used as a disinfectant, a liquid preparation, for example, an aqueous solution containing an5 tibiotic C-19393 S with a concentration of 0.1-10% w / v and benzylpenicillin with a concentration of 0.1-1.0 wt / vol.%, or an ointment containing 5-20 mg of C-19393 S and 5-20 mg of benzylpenicylin per 1 g of white petrolatum or lanolin, as a base, can be used as a bactericide or disinfectant to treat the upper and lower limbs, eyes, ears, etc. in the above animals.
    The antibiotic C-19393 S. is also a very useful intermediate Q compound for the synthesis of new types of pharmaceutical preparations. The antibiotic is stable in aqueous solution at neutral pH. .
    Physiological properties
    five
    C-19393 H is described below.
    The antimicrobial spectrum of sodium salt C-19393 E AGAINST various microorganisms is presented in
    Table 3 and, as a consequence of the results presented in Table 3, the antibiotic C-19393 H exhibits antibacterial activity against gram-positive and gram-negative bacteria. The anti-bacterial spectrum of the action of the sodium salt of the antibiotic C-19393 H is presented in Table 3. TABL Minima Microorganism per inn of the mg / ml center of Escherlchia cof i. NIHJ Oh, §8 Sa $ mon & ea typhimus utea IFO 3232 .0,31 Bac ± Uus subtle is IFO 3S130,31 Bade (Jus cereus IFO 3460 10 Medium - broth agar. As follows from Table 3, the antibio C-19393 Hjj, obtained in accordance with the invention, has a mycotic activity against gram-positive and gram-negative bacteria and, thus, can be used to treat bacterial infections in mammals, such as mice, rats, dogs, humans, etc., as well as in birds, for example, in chickens, ducks, etc. For using C-19393 N as a treatment agent, for example, infectious E., C-19393 N. is dissolved in physiological saline in order to obtain an injection solution that can applied parengerally, for example subcutaneously or intramuscularly, with a dose of 0.1-50 mg / kg / day, preferably 0.5-20 mg / kg / day. In the case of oral administration, the antibiotic C-19393 2 is mixed with lactose and encapsulated in order to obtain a capsular preparation that can be used with a dose of 1-100 mg / kg / day7, preferably 5-50 mg / kg / day. The antibiotic C-19393 H2 obtained according to the invention may. used as a disinfectant4 For example, a liquid preparation obtained with C-19393vH2 solvent in distilled water with a concentration of 0.010, 1% w / v or an ointment containing 0.2-20 mg, preferably 1-10 mg of C-19393 H per 1 g of white petrolatum or lanolin, as a base, can be used as a bactericide or disinfectant for upper and lower limbs; eyes, ears, etc. in the above animals. The antibiotic C-19393 H may be useful as an intermediate agent for the synthesis of new types of pharmaceutical preparations. The antibiotic of the invention is stable in an aqueous solution at a neutral pH. Example. The culture of the strain Streptomyces C-19393 (1 FO 13886, ATS C 31486) is grown in 200 ml of culture medium, placed in a 1 l Erlenmey flask with the formation of spores. Then, the resulting spores are suspended in sterile water at a concentration of 1.210 live cells / ml. The spore suspension is diluted with sterile water to a volume 10 times larger than the initial volume and 1 ml of the diluted suspension is used to inoculate 40 ml of the seed medium in a 200 ml Erlenmeyer flask. The inoculated broth medium is then cultured in a rotation knot for 2 days. The resulting culture was used to inoculate 500 ml of the inoculum medium placed in a 2 liter Sakagushi flask and the inoculated inoculum was cultured in a spray at for. 2 days. The seed culture thus obtained is transferred to a 50 liter stainless steel fermenter containing 30 liters of the seed medium containing 15 ml of Aktokol and cultured for 3 days, with aeration at a rate of 30 l / min and at a stirring speed of 280 rpm. min The culture broth is transferred to a 2 m fermenter containing 1.2 basic culture medium and cultivated at 30 ° C for 5 days at a speed of aeration of 840 ml / min and a stirring speed of 180 rpm. The seeded medium used above is supplemented with 20 g of glucose, 30 g of soluble starch, 10 g of raw soy flour, 10 g of corn extract, 5 g of polypeptone, 3 g of sodium chloride and 5 g of precipitated calcium carbonate per 1 l of medium, the pH of which is set to 7.0 before sterilization, and the main culture media used above is supplemented with Zo g of glucose, 30 g of soluble starch, 15 g of defatted soy flour, 15 g of cotton seed flour, 0.25 g of potassium hydrogen phosphate, 0.6 g potassium hydrogen phosphate, 0.02 g cobalt chloride and 0.5 g Aktocol per liter Reda, wherein the pH is adjusted to 7.0 before sterlizatsiey. All media used above are sterilized with
     within 20 min.
    i
    The fermentation broth thus obtained is filtered using Hyf2o-Superce to obtain 1230 liters of filtrate, the pH of which is then set to 6.3 and passed through a column packed with 100 liters of activated carbon. Then the antibiotic C-19393 82 and H are eluted from the column with 300 l of water and 700 l of 7% aqueous solution of pa isobutanol, respectively. The containing C-19393 82 is passed through a column with Dowex 1-2 (CE-form, 2 l) and the column was washed with b l vgdy and eluted with 32 l of 5% aqueous solution of sodium chloride. The eluate is adjusted to pH 5 and passed through a column with 4 liters of activated carbon. After washing 12 liters of water with antibiotic, 7 liters of 8% aqueous isobutanol and 12 liters of a mixture of 8% isobutanol — N / 20 are eluted. aqueous ammonia and the resulting eluate is concentrated to a volume of 150 ml under reduced pressure. 1350 ml of methanol was added to the concentrate and the precipitate as a result was removed by filtration. The filtrate is concentrated to a volume of 200 ml and passed through a column with a phase of 300 ml of DEAE-Sephadex; A 25 (C-form). The column was successively washed with 0.1 M and 0.2 M aqueous solutions of sodium chloride (900 ml each) and then the antibiotic was eluted with 1500 ml of 0.4 M aqueous solution of sodium chloride. The pH of the eluate is set to 5 and passed through a column of 500 ml of activated carbon. After washing with 1.5 l of water, the column was eluted with 2.5 l of isobutanol mixture N / 20 aqueous ammonia (8:92) and the eluate was concentrated to dryness and acetone was added to the residue to form 2.4 g of light yellow powder. After dissolving the powder in a small amount of water, the resulting solution is passed through a column packed with 1.2 l of Am-. berlit XAD-11 (100–200 mesh), and eluted (by fractions) with water. The fractions with antibiological activity are collected and concentrated and the concentrate is passed through a column with 200 MP CAA-Sephadex A 25 (SG: form). After washing with 600 ml of a 0.1 M aqueous solution of sodium chloride, the column was eluted with 1.2 L of a 0.2 M aqueous solution of sodium chloride. The eluate is adjusted to pH 5 and passed through a column of 600 MP of activated carbon. After washing the column with 1.8 l of water, the column was eluted with 3 l of a mixture of 8% isobutanol — N / 20 aqueous ammonia. The eluate is concentrated to dryness and acetone is added to the residue to obtain 1.07 g of powder. 620 mg of the powder thus obtained is dissolved in a small amount of water and the resulting solution is passed through a column packed with 360 ml of Amberlite XAD-11 (100200 mesh). The column is then eluted and fractionated with water, and each of the fractions detecting antibiological activity is analyzed by liquid chromatography as described above, giving one peak, combined and concentrated to dryness, and acetone is added to the concentrate to obtain 136 mg of the antibiotic C-19393 82 (disodium salt ) in the form of a white powder.
    P. im e p 2. The eluate of the antibiotic C-19393 H2, obtained in example 1, is passed through a column with Doinks 1-2 (cC-form, 12 l) and the column is washed with 6 l of water and eluted with 180 l of 5% solution of aqueous sodium chloride. The eluate obtained is passed through a column packed with 25 liters of activated carbon. After
    washing with 75 l of water the desired antibiotic is eluted with 175 isobutanol with water (7:93) and the eluate is concentrated to a volume of 2-3 l under reduced pressure. 15 L of methanol was added to the concentrate and the precipitate thus obtained was removed by filtration. The filtrate was concentrated to a volume of 2 liters and passed through a column of 5 liters of Diaion HP-20 (50 mesh). Then the column is eluted and fractionated with 5 l of water and 10 l of methanol: water (1: 9). The active fractions are combined and concentrated and the concentrate is passed through a column of 3 liters of DEAE-Sephadex.
    A-25 (CE-form). The column is then washed with 9 L of a 0.02 M aqueous solution of sodium chloride and the antibiotic is eluted and 12 L of a 0.05 M solution of sodium chloride are fractionated. The active fractions are passed through a column packed with 2 liters of Diaion HP-20 (50 mesh), which is treated with 4 liters of an aqueous solution of sodium chloride, the active substance is eluted and fractionated with 10 liters of methanol: 5% aqueous solution of sodium chloride (5:95 ) and 10 liters of a mixture of methanol: 5% aqueous solution of sodium chloride (1: 9). The active fractions were passed through a column of 500 ml of activated carbon and, after washing with 1.5 liters of water, eluted with 2.5 liters of 7% aqueous isobutanol. The eluate is concentrated and the concentrate is passed through a column with 1 l of Diaion HP-20 (50-110 mesh) and zlyuyut and fractionated with water. The fractions with antibiological activity are combined and concentrated and the concentrate is passed through a column equipped with 200 ml of QAE-Svfadex A-25 phase (CE-form), which is pretreated with .400 ml of a 0.02 M aqueous solution of sodium chloride. Then, the clone is sequentially washed with 0.02 M, 0.03 M, and 0.04 M aqueous solutions of sodium chloride (1 l each). The active fractions were passed through a column of 200 ml of activated carbon and, after washing with 0.6 l of water, eluted with 1 l of 7% aqueous butanol. The eluate is concentrated and propanol is added to the concentrate in order to obtain 90% aqueous propanol, which is then subjected to chromatography on a stick with Avicel, which is treated with 90% aqueous solution of propanol. The column is then eluted with 90% aqueous propanol. The active fractions are concentrated and the concentrate is subjected to chromatography on a caustic with 350 ml of Amberlite XAD-11 (100-200 mesh) and the column is eluted
    water The active fractions are concentrated and the concentrate is subjected to high pressure preparative liquid chromatography using PP-18 as a carrier and eluted with 10% methanol in 0.02 M phosphate buffer (pH 6.3). The active fractions are passed through a column with 40 ml of Diaion HP-20 (100200 mesh) and fractionally eluted with water.
    O Every faction that detects an. timicrobial activity, subjected to liquid chromatographic analysis described above. Fractions giving one peak are combined and
    5 is freeze dried to obtain 12 mg of C-19393 2 as a white powder.
    Froze 1150 l of broth culture filtrate is obtained according to the method similar to that described in example 1 and its pH is set to 50 g, and 100 l of activated carbon is passed through the column. After washing with 300 l of water,
    5 column, elute with 350 l of a mixture of a solution of isobutanol with 0.02 N sodium hydroxide (8:92).
    The eluate obtained is passed through a column packed with 10 liters of Diaion SA-21A (eG-form) and, after washing with 30 liters of water, the column is eluted with 100 liters of a 5% aqueous solution of sodium chloride. Then, the eluate was passed through a column with 20 liters of Diaion HP-20 (50 mesh), which was treated with 40 liters of a 5% aqueous solution of sodium chloride and the column was eluted with 20 liters of 5% aqueous solution of NaCE. Then antibiotic
    C-19393 S and H elute with 40 L of a C column and 60 L of a mixture of methanol: 5% aqueous solution of NaCg (5:95). The resulting eluate, containing the anti-biotic C-19393 S2, is adjusted to a pH of 5 and passed through a column packed with 2 liters of activated carbon. After washing with 6 L of water, the column was eluted with 10 L of a mixture of 8% isobutanol N / 20 aqueous ammonia and the resulting eluate was concentrated. The concentrate is passed through a column, filled with 200 ml of AE-Sephadex A-25 (Ct-form), and after washing with 1 l of a 0.02 M aqueous solution of chloride
    A 5 column sodium was eluted with 1 L of a 0.4 M aqueous solution of sodium chloride. After desalting the eluite by chromatography using activated carbon, it is subjected to chromatography through Amberlite XAD-11 and treated in the same manner as in Example 1 to obtain 100 mg of the disodium salt with the antibiotic C-19393 as a white powder.
    EXAMPLE 4 The Yluat of the antibiotic C-19393 Hg, prepared in Example 3, is adjusted to a pH of 5 and passed through a column / packed with 2 liters of activated carbon. After washing with a lump of wax, the column was eluted with 10 L of isobutanol N / 20 aqueous ammonia (8:92) and the eluate was concentrated. The concentrate is passed through a column with 200 ml of QAE-Sephadex A-25 phase (Ci-form) and after washing with 1 l of 0.02 M aqueous solution of sodium chloride, the column is eluted with 1 l of 0.04 M aqueous solution of sodium chloride. After desalting of the eluate by Xpo 4 by trituration through activated charcoal, it is subjected to chromatography on a column with 200 ml of Diaion HP-20 (100-200 mesh) and the column is eluted and fractionated with water. The fractions giving a single peak on the liquid chromatogram are combined and concentrated, and the concentrate is freeze-dried to form 48 mg of C-19393 N sodium salt as a white powder.
    The proposed method allows to obtain a new antibiotic, which will find wide application: in medicine.
    权利要求:
    Claims (2)
    [1]
    The method of obtaining antibiotic of the General formula
    CH 3 I 4
    ch 3
    3 r
    to about
    ABOUT"
    1G x Inex 3 Soon
    where -K-ZO 3 N -antibiotic C-19393 and / or KN - antibiotic C-19393 H 2 / · '
    That is, strain 5-bruised C-19393 (ΪΒΌ 13.886) is cultivated in a nutrient medium containing sources of carbon and nitrogen, and the desired product is isolated from the liquid fraction of the culture medium as a mixture and / or individual antibiotics.
    A priority
    02.02.79 with ka C-193933 2 ;
    06.06.79 with ka C-19393H 2 .
    by featured; getting antibiotic antibiotic
    *
    1075984 A
    one
    1075984
    [2]
    2
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    同族专利:
    公开号 | 公开日
    DE3003624A1|1980-11-20|
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    PT70710A|1980-02-01|
    AU534530B2|1984-02-02|
    SE8000842L|1980-12-27|
    IT8019645D0|1980-02-01|
    FR2447922A1|1980-08-29|
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    GB2042532A|1980-09-24|
    FR2447922B1|1982-05-28|
    DK12580A|1980-08-03|
    NL8000628A|1980-08-05|
    ES8102590A1|1981-02-16|
    AU5451880A|1980-08-07|
    CH646432A5|1984-11-30|
    引用文献:
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    US4172129A|1974-03-28|1979-10-23|Beecham Group Limited|Antibiotics|
    GB1577725A|1976-06-30|1980-10-29|Beecham Group Ltd|Azetidinone derivatives|
    DE2862419D1|1977-11-12|1984-07-26|Beecham Group Plc|Derivatives of 7-oxo-1-azabicyclo-hept-2-ene-2-carboxylic acid, their preparation, pharmaceutical compositions containing them and intermediates|
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    JPS5740485A|1980-08-25|1982-03-06|Shionogi & Co Ltd|Novel antibiotic pa-39504-x3 and its preparation|
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    JPS57109786A|1980-12-26|1982-07-08|Takeda Chem Ind Ltd|Antibiotic c-19393e5 and its preparation|
    US4497742A|1981-03-04|1985-02-05|Takeda Chemical Industries, Ltd.|Isomerization of β-lactam compounds|
    US5391604A|1993-07-30|1995-02-21|Diemat, Inc.|Adhesive paste containing polymeric resin|
    WO1997041421A1|1996-04-30|1997-11-06|Metrika, Inc.|Method and device for measuring reflected optical radiation|
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    JP54011560A|JPS6125356B2|1979-02-02|1979-02-02|
    JP8114179A|JPS565496A|1979-06-26|1979-06-26|Antibiotic c-19393h2 and its preparation|
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