WO1986005185A1 - Antibiotique, procede de preparation et microorganisme - Google Patents

Antibiotique, procede de preparation et microorganisme Download PDF

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Publication number
WO1986005185A1
WO1986005185A1 PCT/JP1985/000095 JP8500095W WO8605185A1 WO 1986005185 A1 WO1986005185 A1 WO 1986005185A1 JP 8500095 W JP8500095 W JP 8500095W WO 8605185 A1 WO8605185 A1 WO 8605185A1
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WIPO (PCT)
Prior art keywords
tan
antibiotic
deacetyl
salt
culture
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PCT/JP1985/000095
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English (en)
Japanese (ja)
Inventor
Hideo Ono
Yukimasa Nozaki
Setsuo Harada
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Takeda Chemical Industries, Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Takeda Chemical Industries, Ltd. filed Critical Takeda Chemical Industries, Ltd.
Priority to PCT/JP1985/000095 priority Critical patent/WO1986005185A1/fr
Priority to EP85301948A priority patent/EP0157544A3/fr
Priority to US06/714,084 priority patent/US4656288A/en
Priority to ES541441A priority patent/ES8702428A1/es
Priority to DK135785A priority patent/DK135785A/da
Priority to CA000477912A priority patent/CA1238594A/fr
Priority to HU851207A priority patent/HU194310B/hu
Priority to CN85101679A priority patent/CN1010103B/zh
Priority to ES555584A priority patent/ES8707251A1/es
Publication of WO1986005185A1 publication Critical patent/WO1986005185A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the present invention relates to a novel antibiotic useful as an antibacterial agent, a method for producing the same, and a microorganism that can be used for the production.
  • the present inventors isolated a large number of microorganisms from soil for the purpose of searching for new antibiotics, and searched for antibiotics produced by the microorganisms.As a result, it was found that certain microorganisms produce new antibiotics.
  • the microorganism is a new species belonging to the genus Endobacter and the genus Rhizobacter, and by culturing the microorganism in an appropriate medium, an antibiotic exhibiting antibacterial activity against Gram-positive bacteria and Gram-negative bacteria can be obtained. Knowing that it can accumulate in the medium, etc., isolate this antibiotic, confirm from its physicochemical and biological properties that the antibiotic is a novel antibiotic, I decided to call it 588.
  • the present inventors have also found that the above-mentioned antibiotic TAN-588 has an N-acetyl group and a carboxy group in the molecule, and that the acetyl group is eliminated. I found that I could do it.
  • the bacteria of the genus Endopactor and the lysobacterium are capable of producing and accumulating the N-deacetyl body of the above-mentioned antibiotic TAN-588.
  • a mixed culture of Endobacter bacteria or Rhizobacter bacteria having the ability to produce 588 and Z or its N-deacetyl compound, and a mixed culture of AN-588 N-deacetyl compound with antibiotics It was found that a larger amount could be produced and accumulated than without.
  • the present invention relates to (1) an antibiotic TAN-588, a paranitrobenzyl or benzhydryl ester thereof, or an N-deacetyl derivative thereof or a salt thereof;
  • Rhizopater albus which forms a white colony and whose 0-F (oxidative-fantative) test is non-degradable and has no assimilation of inorganic nitrogen sources
  • This is a method for producing an N-deacetyl derivative of the antibiotic AN-588 or a salt thereof, which is characterized by collecting the same.
  • antibiotic TAN-588 may be simply referred to as “TAN-588”.
  • the antibiotic AN-588 used in the method of the present invention and the bacterium that produces no or its ⁇ '-deacetyl derivative belong to the genus Empedobacter or Lysobacter, and the antibiotic TA-588. And / or any microorganism having an ability to produce its N-deacetyl derivative.
  • a production bacterium belonging to the genus Endobacter there is, for example, a new bacterium species Emped bacterium lactamgenus.
  • YK-258 strain Lacta mugenus strain collected from the soil of Masuda ⁇ , Shimane Prefecture, Japan by the present inventors. can give.
  • the mycological properties of the Y-258 strain are as follows.
  • the cells were cultured at 24 ° C and observed for 1 to U days.
  • Broth agar flat culture The colony is translucent, light yellow, circular, head-shaped, and rim-shaped. No diffusible dye is formed.
  • Liquid broth culture Grows in a mixed state, precipitates, and forms a pellicle.
  • Juice gelatin stab culture Grows mainly in the upper part and liquefies like a crater. Liquefaction activity is relatively weak.
  • ⁇ Grows at ⁇ 5.4 to 8.5, but the optimal ⁇ is 5.8 to 6.6.
  • -Medium 0.1% Darcose, 0.01% yeast extract, 0.1% vanadium sulfate, 0.1% salt, 0.05% magnesium sulfate (7-hydrate), phosphate buffer Q.1M (separate sterilization).
  • the YK-258 strain having the above mycological properties was prepared by using the Verges Manual, Minuteif, and Bergey's Mann et al. Determinative B acteriology) Eighth Edition and International Journal 'OB' Systematic Bacteriology
  • the YK-258 strain is more likely to be used than the genus Flavobacterium. It is more appropriate to attribute it to the genus Endopactor. However, the GC content of the DNA exceeds 70%. Therefore, the YK-258 strain was identified as belonging to the ⁇ strain, and the new strain was named as Ninbedaku Yuichi * Empedobacter iactamgens.
  • Antibiotic T AN belonging to the genus Lysobacter used in the method of the present invention
  • bacteria producing 588 and / or its N-deacetyl form examples include Lysobacter albus, a new strain.
  • Lysobacter albus a new strain.
  • the present inventors may abbreviate Rhizobacta. Arbus sp. Nov. YK-422 strain (hereinafter referred to as "YK-422 strain") collected from soil in Kanzaki-gun, Shiga Prefecture, Japan. ).
  • the mycological properties of 422 are as follows.
  • the cells should be 0.4 to 0.7 m in length, 2.0 to 4.4 m in length, and 20 to 30 m in filament length. There is also. No flagella, no motility. It does not form spores or microcysts and has a negative Gram stain, indicating no acid resistance.
  • Broth liquid culture grows in a lightly mixed state, forms a small amount of precipitate, and forms a weak bacterial ring.
  • Dried yeast plate A lysis circle is formed around the colony, and it shows mobility due to gliding.
  • ⁇ ' Adjusted with caustic soda or sulfuric acid.
  • the YK-422 strain having the above-mentioned mycological properties was obtained by using the Burges' Manual. National 'Journal of the Systematic Pakterio Sigma, Vol. 30, pp. 225-420 (1980), Vol. 32, U6-149 (1982), and the validity of the literature.
  • ⁇ When compared with the species in the list, it is a gram-negative bacillus that shows mucoidal growth, some filamentous bacteria are also observed, showing motility by grinding, and the ability to degrade colloidal chitin and dry yeast It does not form micromixes and has a high GC content in DNA, so it is appropriate to belong to the genus Rhizobacta.
  • Rhizopactor International 'Journal', 'Ob', 'Systematic', Pacteriology, Volume 28, pp. 367-393. They differed in that they were degraded, did not have any distinct color in the colonies, and did not have the assimilation of inorganic nitrogen sources. Therefore, YK-422 bead was identified as a strain belonging to a new strain, and the new strain was named Rhizobacta'arbus (Lysobacter alb sp.) Sp. Nov. Y-422.
  • the bacteria belonging to the genus Acinetobacter used in the mixed culture used in the method of the present invention belong to the genus Acinetobacter (.ki netob acter), and have the ability to produce the antibiotics TA-588 and Z or the N-deacetyl derivative thereof. Any microorganism may be used as long as it has the ability to produce and accumulate a significant amount of the N-deacetyl derivative of the antibiotic TA-588 by co-cultivation with Escherichia coli or Rhizobacta.
  • the term "produce and accumulate a significant amount” as used above means that the microorganism of the genus Annetobacter is used. However, the target compound is produced and accumulated in a larger amount than the amount of the N-deacetyl product of the antibiotic TAN-588 when the Endobacter species or the Rhizobacter species are used alone. Means
  • microorganism belonging to the genus Acinetobacter examples include, for example, Acinetobacter sp.
  • Acinetobacter sp examples include, for example, Acinetobacter sp.
  • the present inventors may refer to a strain of yeast YSP-504 (hereinafter referred to as “YK-504”) isolated from fresh water collected from Yodogawa-ku, Osaka-shi, Osaka, Japan. )).
  • the mycological properties of the YK-504 strain are as follows.
  • the cells were found to be short rod-shaped or spherical with a diameter of 0 to 1.5 ⁇ 111 and a length of 1.1 to 2.1111. There are many. No flagella. No motility. It does not form spores and has a negative Gram stain, indicating no acid resistance.
  • the cells were cultured for 2 to 4 days and observed for 4 to 14 days.
  • Juice agar plate culture Colonies are small and dot-like, the surface is convex, and the periphery is all square. No diffusible dye is formed.
  • Liquid broth culture Grows turbid, precipitates, and does not form a pellicle.
  • 5Litmus ⁇ Milk Reduction of litmus, peptone formation and coagulation ( ⁇ , no negative activity is observed.
  • Carboxymethyl cellulose one
  • Colloidal chitin —
  • the above-mentioned facilitator Patricia sp. 504 strain was deposited with I F0 on January 31, 1985 as accession number I F 0 14420.
  • the microorganism has been deposited with the FRI on February 12, 1985 as a deposit under the Budapest Treaty, under the accession number F ERM II-709.
  • the properties of the bacteria of the genus Endobacter or Rhizobacter used in the present invention are generally liable to change.
  • ultraviolet rays, X-rays, and chemicals eg, nitrosoguanidine, ethyl methanesulfonic acid
  • Any mutant which can be easily mutated by means of artificial mutation and which has the ability to produce TA 588-588 and / or its N-deacetyl derivative, which is the subject of the present invention, can be used in the present invention.
  • the bacteria of the genus Acinetobacter used in the present invention generally change their properties easily, and are easily mutated by, for example, artificial mutation using ultraviolet rays, X-rays, chemicals (eg, nitrosoguanidine, ethyl methanesulfonic acid) and the like. Any mutant strain can be mixed with a microorganism belonging to the genus Endobactor or Lysobacter, which is capable of producing the antibiotic TAN-588 and / or its N-deacetyl derivative.
  • carbon sources include, for example, darcose, fructose, maltoses, and solvable starch, dextrin, oils and fats (eg, , Soybean oil, olive oil, etc.), and organic acids (eg, citric acid, succinic acid, dalconic acid, etc.) that can be used by bacteria can be used as appropriate.
  • organic nitrogen compounds such as soybean flour, cottonseed flour, corn gluten ⁇ meal, dried yeast, yeast extract, meat extract, and peptone.urea can be used.
  • the inorganic salts include, for example, sodium chloride, potassium chloride, calcium carbonate, magnesium sulfate, monolithium phosphate, and sodium phosphate required for culturing normal bacteria, alone or in combination. used.
  • Heavy metals such as ferric sulfate and copper sulfate, and vitamins such as vitamin B-biotin are also added as necessary.
  • an antifoaming agent such as silicone oil / polyalkylene dali coal ether or a surfactant may be added to the medium.
  • Other organic and inorganic substances that promote the growth of bacteria and promote the production of TAN-58 ⁇ and / or its deacetyl form may be added as appropriate.
  • the cultivation method may be the same as a general antibiotic production method, and may be solid culture or liquid culture.
  • any of static culture, stirring culture, shaking culture, and aeration culture may be performed, but aeration and agitation culture is particularly preferred.
  • the culture temperature is preferably about 15 ° (: to about 32 ° C, and the pH of the medium is about 5 to 8 for about 8 to 168 hours, preferably about 24 to 144 hours.
  • the mixed culture method for the production of the N-deacetyl form of the antibiotic TAN-588 should be performed in accordance with the culture method for the production of the antibiotic TAN-588 and / or its N-deacetyl form as described above. With power.
  • a TLC-bioautography method using pseudomonas.eruginosa C-U1 as a test bacterium is used.
  • the mixed culture is carried out using the microorganism of the genus Annetobacter of the present invention
  • the antibiotic TAN which is the target substance
  • the mixed culture method of the present invention is advantageous in industrial production.
  • antibiotic TAN-588 and Z or its N-deacetyl derivative are appropriate.
  • the antibiotic TAN-588 and its N-deacetyl derivative are water-soluble and are mainly contained in the culture filtrate. Therefore, a filtration aid is first added to the culture, followed by filtration or eccentric separation.
  • a means for removing the cells, bringing the resulting culture filtrate into contact with a suitable carrier to adsorb the active ingredient in the filtrate, desorbing the active substance with a suitable solvent, and separating and collecting the fraction is advantageously used.
  • a chromatographic carrier use the difference in the adsorptivity of compounds such as activated carbon, silica gel, powder cell mouth, and adsorbent resin, or use the difference in the functional groups of compounds such as ion-exchange resin and ion-exchange cellulose. It is advantageous to use the difference in molecular weight of compounds such as molecular sieves L and sex carriers. In order to elute the target compound from these carriers, combinations vary depending on the type and properties of the carriers.
  • aqueous solutions of water-soluble organic solvents that is, aqueous acetates and aqueous alcohols, or acids and alkalis
  • a buffer an aqueous solution containing an inorganic or organic salt, or the like is used in appropriate combination.
  • high-performance liquid mouth chromatography for preparative separation of the crude substance of this antibiotic obtained by these chromatographys (HP LC) for further purification.
  • the eluate desalted by activated carbon chromatography can be concentrated, and the antibiotic can be recovered from the concentrate using an ion-pair extraction method, that is, a quaternary alkylammonium it can.
  • TAN-588 is an acidic substance
  • an anion exchange resin such as Dowexu-1 (manufactured by Dow & Chemical Co., USA) and Amberlite IRA-68, 400, 402, 410 are used as carriers. (Roam & Haas Co., USA), Diaion SA-21A and C (Mitsubishi Kasei Kogyo Co., Ltd., Japan), etc., absorb the antibiotics in the filtrate and contain salts or acids. Is eluted with an aqueous solution or buffer solution.
  • Anion exchange cell openings such as DE-32 (Wattman, UK), DEAE-cellulose (Brown, Germany), etc., or anion-exchange molecular sieve resins such as DEAE- or QAE-Sephadex
  • the antibiotic can be adsorbed to a carrier such as [Falmasha, Sweden] and eluted with an aqueous solution or buffer containing salts or acids. To remove salts, coloring substances, etc.
  • Activated carbon for chromatography manufactured by Takeda Pharmaceutical Co., Ltd., Japan
  • an adsorbent resin such as Diaion HP-2.0 or S-207 (Mitsubishi Kasei Kogyo Co., Ltd., Japan), Amberlite XAD- ⁇ (Mouth And Haas Co., USA), etc.
  • the fractionated elution fractions are made into powder through processes such as concentration and freeze-drying. When the purity of the powder thus obtained is poor, high-performance liquid chromatography is advantageously used for further purification.
  • the carrier used examples include TS ⁇ ⁇ gel (manufactured by Toyo Soda Co., Ltd., Japan) and YMC gel (manufactured by Yamamura Chemical Research Laboratories, Japan).
  • the moving layer is formed of methanol or acetonitrile and an acidic aqueous solution. Alternatively, a mixed solution with a buffer solution or the like is used.
  • Ions mentioned above • Quaternary alkyl ammonium used in the paired extraction method ⁇
  • For halides for example, tri-n-butylmethylammonium chloride, tetra-n-pentylammonium chloride, ⁇ -tetradecyldimethylbenzyl There are ammonium chloride and the like.
  • the organic solvent methylene chloride, chloroform and dichloroethane are usually used.
  • ⁇ -deacetyl form of TAN-588 is an amphoteric substance and has a stronger basic property. Therefore, as a carrier, a cation exchange resin such as Dowex 50W (manufactured by Dow And Chemical Co., USA), Amberley G. Using IR-120B (manufactured by ANDOM Haas Co., USA), Diaion SK-U0 (manufactured by Mitsubishi Kasei Kogyo Co., Ltd., Japan), etc., the antibiotic in the filtrate is adsorbed, It is eluted with an aqueous solution or buffer containing salts or acids or alkalis.
  • a cation exchange resin such as Dowex 50W (manufactured by Dow And Chemical Co., USA), Amberley G. Using IR-120B (manufactured by ANDOM Haas Co., USA), Diaion SK-U0 (manufactured by Mitsubishi Kasei Kogyo Co., Ltd., Japan), etc.
  • the antibiotic can be adsorbed on a carrier such as a cation exchange molecular sieve resin such as CVI-Sephadex (Pharmacia, Sweden) and eluted with a salt-containing aqueous solution or buffer.
  • a carrier such as a cation exchange molecular sieve resin such as CVI-Sephadex (Pharmacia, Sweden)
  • activated carbon for chromatography or an adsorbent resin such as Dia SP-207 or HP-20 is advantageously used.
  • the fractionated elution fraction is powdered through steps such as concentration and freeze-drying. If the powder obtained in this way has a low density, high-performance liquid chromatography is advantageously used for coarse purification.
  • the carrier, moving bed, etc. are the same as in the case of the refinement of TAN-5 ⁇ .
  • TAN-588 is present in the form of salts used as salts, cations in a buffer solution, such as sodium, calcium, lithium, calcium, and amphibious ions. In this case, it is isolated as the corresponding salt by applying it to activated carbon at the same pH and applying it to ⁇ -matography. The eluate is adjusted to a pH of about 4.5 to 3, preferably about 4.5 to 3, and chromatographed on activated carbon to obtain a free product.
  • the N-deacetyl form of TAN-588 can be obtained as a zwitterion compound by activated carbon chromatography at near neutral pH.
  • the N-deacetyl form of TAN-588 can form a salt with a strong acid because it has a stronger basic property.
  • the strong acid include hydrochloric acid, phosphoric acid, sulfuric acid, and trifluoroacetic acid.
  • the TAN-588 thus obtained has two peaks on reversed-phase high performance liquid chromatography. If these peaks are referred to as A and B, the following phenomenon is observed. When the peaks of A and B are respectively collected by preparative high-performance liquid chromatography, a fairly single A and B are obtained.
  • a paranitrobenzyl or benzhydryl group is introduced into TAN-588 to form an ester form.
  • the acetyl group of 8 is also eliminated, and this is carried out by eliminating the para-nitrobenzil or Bench drillyl group, if necessary.
  • a compound capable of introducing a para-nitrobenzyl group is reacted with TAN-588 or a salt thereof.
  • the compound into which the paranitrobenzyl group can be introduced include, for example, paranitrobenzylbutamide and paranitrobenzyl chloride.
  • the amount of the compound capable of introducing a paranitrobenzyl group is about 1 to 5 equivalents, preferably about 1 to 2 equivalents.
  • the reaction is preferably performed in a solvent.
  • the solvent include dimethylformamide (DMF), dimethylacetamide (DMAA), and tetrahydrofuran (THF).
  • THF tetrahydrofuran
  • Et 3 N triethylamine
  • pyridine pyridine and the like may be added in an amount of about 0.1 to 0.5 equivalent, preferably about 0.1 to 0.2 equivalent to promote the reaction.
  • the reaction temperature is about O ° C to 40 ° C, more preferably about 20 ° (: to 30 ° C, and the reaction time is about 0.5 to 8 hours, more preferably about 1 to 4 hours. It is preferred to carry out with stirring.
  • a compound capable of introducing a benzhydryl group is reacted with TAN-588 or a salt thereof.
  • the compound into which the benzhydryl group can be introduced include, for example, diphenyldiazomethane, diphenylmethylbromide and the like.
  • the amount of the compound capable of introducing a benzhydryl group is about 1 to 6 equivalents, preferably about 2 to 4 equivalents.
  • the reaction is preferably carried out in a solvent, and examples of the solvent include THF, dioxane, ethyl acetate, digrometan and the like.
  • reaction To promote the reaction, a small amount of, for example, about 0.01 to 0.0 equivalents of dilute hydrochloric acid, dilute sulfuric acid, or dilute phosphoric acid is added to add PH to about 1 to 3, preferably about It is preferable to adjust to around 1.5 to 2.5.
  • the reaction temperature is about 110 ° C. to T 50 ° C., more preferably 0 to 30 ° C., and the reaction time is about 30 minutes to about 8 hours, more preferably about 1 to 3 hours.
  • the reaction is preferably performed with stirring.
  • the ester obtained above can be collected using a commonly used separation and purification means.
  • the target substance is, for example, dichlorme
  • the extract is concentrated into an organic layer with tan or black form, and the extract is concentrated. When the concentrate is added to ether or hexane, the ester is precipitated as a crystalline powder.
  • This ester compound is isolated into two components by the silylation gel method, but may be used as a mixture when proceeding to the next reaction.
  • Examples of the deacetylation reaction include an iminoether method, a solvolysis method, and a hydrolysis method using an enzyme.
  • the raw material compounds are also reacted with phosphorus pentachloride, phosgene, phosphorus trichloride, and oxychloride.
  • the reaction reagent is preferably used in an amount of about 1 to 5 equivalents, more preferably about 1.5 to 3 equivalents.
  • the reaction is conveniently carried out in the presence of a solvent such as, for example, methylene chloride, dichloroethane, chloroform, carbon tetrachloride, trichloromethane.
  • pyridine, ⁇ , ⁇ -dimethylylaniline, triethylamine, aniline, toluidine and the like may be used in excess, for example, about 3 to 20 equivalents, and more preferably about 5 to 10 equivalents.
  • the deacetylation reaction is carried out at a reaction temperature of about 130 ° C to 0 ° C, more preferably at 11 ° C to -5, and a reaction time of about 15 minutes to 8 hours, more preferably about 30 minutes to 2 hours. Is good.
  • the reaction is conveniently carried out with stirring.
  • reaction solution An excess of methanol is added to the reaction solution in order to convert imino chloride formed as an intermediate into imino ether, and the resulting mixture is added at about 130 ° (: 0 ° C, preferably about 15 ° C to 15 ° C).
  • the mixture is stirred for 2 hours, and then dilute hydrochloric acid is added to the reaction solution to cleave the C—N bond.
  • the reaction temperature is about i Ot; 4040 ° C., preferably about 20 to 30 ° C.
  • the reaction time is about 15 minutes to 2 hours, preferably about 30 minutes to 1 hour.
  • the starting compound is dissolved in methanol, ethanol or a mixed solution thereof with water, and the mixture is heated at about 20 ° C. to reflux temperature, preferably at about 50 ° C. to reflux temperature.
  • the reaction is carried out for 0.5 to 30 hours, preferably for about 2 to 8 hours.
  • reaction solution thus obtained is neutralized, and the product is extracted with an organic solvent that is immiscible with water, such as methylene chloride, getyl ether, and ethyl ethyl ester, and the extract is concentrated. 588 deacetyl-form paranitrobenzyl or Bench drills are obtained.
  • an acid hydrolyzing method for example, an acid hydrolyzing method, a direct contact reduction method, or the like is used.
  • trifluoroacetic acid formic acid, hydrochloric acid or the like is used as an acid in about 3 to 20 equivalents of the raw compound for reaction. It is preferable to add about 1 to 5 equivalents, preferably about 2 to 4 equivalents, of anisol.
  • a solvent for example, methylene chloride, chloroform, THF, ethyl acetate and the like are used.
  • Anti-Temperature about 130 ° C. to 0 ° C., more preferably about 120 ° C. to 1 ° C., and reaction time; about Q. 5 to 8 hours, more preferably about 1 to 4 hours. is there.
  • the catalytic reduction method for example, palladium, platinum, their oxides and the like are used as a catalyst and reacted under a stream of hydrogen.
  • the reaction temperature is about 0 to 50 ° C, more preferably about 10 ° (: to 40 ° C), and the reaction time is about 6 hours, more preferably about 2 to 2 hours.
  • the free carboxylic acid compound thus formed is separated by filtration of impurities in the reaction solution or removal by mouth chromatography, for example, by a method using activated carbon, an adsorbent resin, etc., followed by concentration and freeze-drying. , Collection, Can be purified.
  • the main absorption (wave number) of the absorption spectrum (Fig. 2) due to the bromide power failure is as follows.
  • Soluble water, dimethyl sulfoxide.
  • Amino acid analysis known to hydrolyze in 6 N-hydrochloric acid at L05 ° C for 20 hours Serine is detected as an amino acid.
  • Stability Stable at pH 5 in aqueous solution, slightly unstable at pH 3 and 7, unstable at pH 9.
  • TLC Thin layer chromatography
  • High Performance Liquid Chromatography (HP LC) (Carrier: YMC A-312, manufactured by Yamamura Chemical Laboratory, Japan, mobile phase: 4%, methanolic 0.01M phosphate buffer (PH6.3), 2 tnl / mln ):
  • silica gel (Merck, West Germany)
  • 3400 3090, 2950, 1805, 1760, 1680, 1610, 1530, 1450, 1380, 1355, 1270, 1180, 1105, 1055, 1015, 965, 910,
  • UV absorption (UV) spectrum (in methanol)
  • 3400 3050, 2970, 1800, 1780, 1740, 1600, 1500, 1460 ; 1305, 1270, 1190, 1110, 1060, 980, 920, 880, 850, 750, 710, 650, 620, 605 cm " 1
  • the main absorptions are as follows.
  • HP LC Equipment, carrier, and speed are the same as those of the above-mentioned benzhydryl ester (mixture of type A and type B) of deacetyl.
  • the main absorption is as follows.
  • TAN-588 has an acetyl group bonded to nitrogen and a carboxyl group.
  • TAN-588 sodium salt The therapeutic effects of TAN-588 sodium salt on experimental mouse infections are shown in Table 2. Infectious orchid administration method ED soCmg / kg) Staphylococcus ⁇ aureus 308A-1 subcutaneous 25.0 In addition, TAN-588 sodium salt was administered subcutaneously to mice at 400 mg / kg, or 4,000 mg Zkg was orally administered to mice. No acute toxicity was observed.
  • the biological properties of the N-deacetyl derivative of TAN-588 (a mixture of type A and type B) are described.
  • the mixture of the A-type and B-type compounds and the A-type and B-type compounds are equivalent in biological properties.
  • Table 3 shows the antibacterial spectrum of the N-deacetyl derivative of TAN-588 against various microorganisms.
  • Test bacteria Minimum growth inhibitory concentration (Note 1) Staphylococcus aureus FDA 209P 50 Micrococcus luteus IFO 12708 6.25 Bacillus subtilis NIIU PCI 219 12.5 Bacillus cereus FM 550 Escherichia coli NIfU JC 25 Salmonella typhimurium IFO 12529 50 Citrobacter iF ⁇ Indy IFO 12681 50 Klebsiella pneumoniae IFO 3317 100 Serratia Marce 'Socense IFO 12648 25 Proteus ⁇ Mirabilis.
  • ATCC 21100 100 Proteus, Bulgaris IFO 3988 100 Proteus Morgani — IFO 3168> 100 Shu Domonas aerugino —sa IFO 3080 50 Alkaligenes, Fucalis IFO 13111 100 Acacia tobacco ICO 13006 50 (Note 1) Bacterium, antibiotics ⁇ Medium 3
  • N-deacetyl form of TAN-588 can be used in various 3-lactamases. It is stable against.
  • Table 4 shows the results of examining the stability against two species of; 8-lactamase using Escherichia coli PG 8 as a test bacterium.
  • the numerical values indicate the diameter of the blocking circle (mm).
  • Medium Nutrient agar medium (PH7.0) containing diaminopimelic acid (20 mg /).
  • PCG benzylpenicillin, CPC: cephalosporin C, CMC: cephamycin C;
  • the drug concentration is T8-588 ⁇ '-Decetyl form is 1000 , All others are 100 gz mo
  • e 1 Bacillus ⁇ Derived from B. cereus, manufactured by Calbio Chemical Co. (USA).
  • TAN-588 its paranitrene or benzhydryl-ester form, or its N-deacetyl form [hereinafter, these are collectively referred to as compound (II)].
  • compound (II) Or their salts have antibacterial activity against Gram-positive and Gram-negative bacteria and have low toxicity. Therefore, the compound (I) of the present invention or a salt thereof is useful for the treatment of bacterial infectious diseases in mammals [eg, rats, mice, dogs, cats, livestock (such as horses). Can be used.
  • the compound (I) of the present invention or a salt thereof as a therapeutic agent for bacterial infections
  • they can be administered orally as tablets, capsules, etc., together with pharmacologically acceptable carriers, excipients, diluents, etc., and parenterally as injections.
  • diluents and the like in the case of injections include physiological saline.
  • An example of a carrier in the case of a capsule is lactose.
  • the dose is about 5 to 50 mgZkgZ days, preferably about 10 mgZkgZ days for compound (I) in the case of an oral preparation, and about 2.5 to 25 mgZkgZ days, preferably about 5 to 5 in the case of a parenteral preparation. 20 mg kg Z days.
  • the compound (I) or a salt thereof obtained by the present invention can be used as a bactericide or disinfectant.
  • a compound (I) a solution prepared by dissolving in distilled water at a concentration of 0.01 to 0.1 WZV%, or vaseline or lanolin as a base, 0.2 to 20 mg, preferably 1 to 1 g of compound (I) per 1 g As an ointment containing 10 mg, it can be used for disinfection and disinfection of human and animal hands, feet, eyes and ears.
  • the compound (I) obtained by the method of the present invention is also a very promising compound as a synthetic intermediate for a new drug.
  • compound (I) a novel antibiotic.
  • FIG. 1 shows the UV absorption spectrum of TAN-588 (an equilibrium mixture of A and B).
  • FIGS. 2, 3, 4, 5, 6, 7, 7, 8, 9, 1Q, 11 and 12 show TAN-588 ( Equilibrium mixture of A and B), p-nitrobenzyl ester of TA-588 (mixture of A and B), TAN-588 para-n-benzyl ester (type A), TAN-588 para-nitro-benzyl ester (A) B), T AN—588 Benzhydryl ester
  • the content of the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.
  • the percentage in the medium indicates the weight Z volume% unless otherwise specified.
  • This culture solution was inoculated into a 50 J2 tank containing 30 J2 of the above medium supplemented with 0.05% of an antifoaming agent Actol (manufactured by Takeda Pharmaceutical Co., Ltd.), and aerated at 24 ° C.
  • the cells were cultured for 48 hours under the conditions of 50 J2 / min, 200 rpm and Z minutes.
  • This culture solution 6 J2 was converted to an aqueous solution (PH6.5) containing dextrin 3% raw soy flour 1.5%, corn gluten meal 1.5%, polypeptide 0.2%, sodium thiosulfate 0.1_%.
  • the fermenter was inoculated into a 200 J2 fermenter containing a medium supplemented with 0.05% Factol, and cultured at 17 ° C for 66 hours under the conditions of aeration of 200 J2 / min and 150 rpm. After adjusting the culture solution (230J2) from the two
  • Lactamgenus YK-258 strain (IFO 14322, FERBP-699) grown on nutrient agar with glucose 2%, solubil
  • This culture solution 602 was converted into an aqueous solution (PH6.5) containing 3% dextrin, 1.5% raw soy flour, 1.5% corn gluten ⁇ meal, 1.5% polypeptone, and sodium sodium thiosulfate.
  • the tank was inoculated into a tank having a capacity of 2000 J2 containing 1200 J2 of medium supplemented with 0.05%, and cultured at 17 ° C for 90 hours under the conditions of aeration of 2000 £ / min and 120 rotations.
  • the culture solution thus obtained was filtered using Hyflo Super Cell (manufactured by Johns Manville, USA).
  • the filtrate (1150 ⁇ ) was subjected to column chromatography on Amberlite IRA-402 (C1 type) 40 ⁇ .
  • Antibiotics were eluted with 2% saline (200 J2), and the eluate was subjected to column chromatography on activated carbon (20 J2).
  • the 8% iso-butanol eluate (81J2) was subjected to column chromatography on Amberlite IR-68 (C1 type),
  • the eluate (54 ⁇ ) was again subjected to column chromatography on activated carbon (10J2), and the antibiotic was eluted with 8% aqueous isobutanol.
  • the eluate (80) is concentrated under reduced pressure, and the concentrated solution (5 £) is reduced to ⁇ 4.5, followed by 2% tree ⁇ 1-year-old octylmethylammonium chloride ⁇ nomethylene chloride solution (2.5J2 X 2).
  • the extract was treated with a 1.6% sodium iodide solution to transfer the antibiotic to the aqueous layer, and the transferred solution was concentrated.
  • the concentrate (1.5J2) was desalted by chromatography on activated carbon (0.5J2), and the eluate was concentrated.
  • the concentrated solution was subjected to column chromatography with QA II-Sephade's Sox (C1 type) 200 mJ2.
  • the fraction was eluted with 0.03M saline to obtain an active fraction (1.3J2).
  • the active fraction was desalted by chromatography on activated carbon (500 mJ2), and the eluate was concentrated and lyophilized to obtain a white powder of TAN-588 (3.56 g).
  • One-sided extraction Since approximately 50% of the antibiotic remained in the wastewater layer, the aqueous layer (5 was subjected to column chromatography with QAE-Sephadex (Type C1) 1J2.
  • the antibiotic was treated with Q.03M and 0.05M NaCl.
  • the column was eluted with water, and the eluate was subjected to activated carbon (column chromatography on column 2).
  • the salt-removed solution (22) was again added to a 2% tri-n-octylmethylammonium chloride.
  • the extract was treated with a sodium iodide solution and subjected to a step of desalting activated carbon to obtain a white powder of TAN-588 sodium salt (3.18 g).
  • 2J2 medium containing 500 mJ2 of 422 strain (IF 0 14384, FE RM BP-698) containing 2% glucose, 3% soluble starch, 1% raw soy flour, and 0.5% polypeptone (uncorrected PH) , And cultured with reciprocal shaking at 24 ° C for 48 hours.
  • the entire amount of this culture solution was inoculated into a 200 J2 tank containing 120 J2 of the above medium supplemented with 0.05% of the antifoaming agent Actcol, and aerated at 120 ° C / 180 rpm at 28 ° C under an aeration rate of 120 £ / min. Cultured for 48 hours.
  • This culture solution 1202 was added to an aqueous solution containing 3% dextrin, 3% raw soy flour, and 0.2% polybeptone (uncorrected PH), containing 4000% of culture medium and containing 4000 pounds. And incubated at 22 ° C for 66 hours under the conditions of aeration of 4000 J2 / min and 120 revolutions of Z minutes.
  • the culture solution thus obtained was filtered using Hyflo ⁇ Supercell (manufactured by Jones Manville, USA).
  • the filtrate (4360J2) was subjected to column chromatography using Amberlite IRA-402 (C1 type) 400J2.
  • Antibiotics were eluted with 2% saline (2000 ⁇ ), and the eluate was subjected to column chromatography on activated carbon (160J2).
  • the 8% isobutanol eluate (640J2) was applied to Amberlite IRA-68 (C1 type) column chromatography. Eluted with 1% saline.
  • the eluate (200 J2) was again subjected to column chromatography on activated carbon (80 J2), and the antibiotic was eluted with 8% isobutanol in water.
  • the eluate (400 fi :) was concentrated under reduced pressure.
  • the concentrate was freeze-dried, and acetone was added to the freeze-dried product to obtain a sodium salt of TAN-588 (620 g) as a precipitate.
  • This powder was found to contain 57% TAN-588 sodium salt by HP LC analysis.
  • the powder (5 g) thus obtained was dissolved in water and subjected to QAE-Sephadex (C1 type) 200 mJe column chromatography.
  • the fraction was eluted with 0.03M saline to obtain an active fraction (1.2J2).
  • the active fraction was desalted by means of activated carbon (500J2 :) chromatography, and the eluate was concentrated and lyophilized to obtain a white powder of TAN-588 (2.50 g
  • the purified TAN-588 powder was obtained from the TAN obtained in Example 1 by the Rf value of TLC, the Rt value of HPLC, IR, UV, CD and NMR spectrum and antibacterial spectrum. — Same as 588 sodium salt.
  • TAN-588 sodium salt 400 mg was dissolved in DMF (4 m), triethylamine (100 parts, paranitrobenzyl bromide (800 mg)) was added, and the mixture was stirred at room temperature for 3 hours.
  • An acid buffer pH6.3.50m was added to the mixture, and ethyl acetate was extracted twice (50m. The extract was washed with water and concentrated. The oily substance obtained was powdered with ethyl acetate-petroleum benzine (507mg).
  • Example ⁇ Benzophenonhydrazone 58.8 g, 1,1,3,3-tetramethylguanidine 42rd, iodine 50 mg was dissolved in CH 2 C 1 2 500 m. After cooling the mixture to 0 ° (: — — 5 ° C, 74 g of m-chloroperbenzoic acid (70% purity) was added, and the mixture was stirred at 0 ° C for 40 minutes. The reaction solution was washed with water and dried over sodium sulfate. Then, the solvent was distilled off to obtain diphenyldiazomethane.
  • the cells were cultured under the conditions for 48 hours. This culture solution 6 ⁇ was added to an aqueous solution ( ⁇ 6.5) containing dextrin 3%, raw soy flour 1.5%, corn gluten meal 1.5%, polybuton 0.2%, sodium thiosulfate 0.1%.
  • the seeds were inoculated in a tank with a capacity of 2002 containing 120 J2 of medium supplemented with 0.05% per well and cultured at 17 C for 24 hours under the conditions of aeration of 20 QJ2 and 150 rpm.
  • the obtained medium contains N-deacetate of TAN-588 (mixture of A-type and B-type).
  • LC-Bio using C-141 as a test bacterium It was confirmed by the autograph method.
  • the total amount of this culture solution was inoculated into a tank having a capacity of 50 J2 containing 30 J2 of the above medium to which 0.05% of the antifoaming agent Actcol was added, and the aeration rate at 24 ° C was 50 J2 / min.
  • the culture was performed for 48 hours under the conditions of rotation.
  • This culture solution 6 J2 was mixed with dextrin 3%, raw soy flour 1.5%, corn and Dalten 'meal 1.5%, An aqueous solution (PH6.5) containing 0.2% of polypeptone and 0.1% of sodium thiosulfate was inoculated into a 20Q £ tank containing a medium 120J2 containing 0.05% of actcol, and aerated at 200 ° C at 17 ° C. Cultivated for 24 hours under conditions of £ min and 150 min Z.
  • the obtained medium contains the N-deacetyl derivative of TAN-588 (mixture of A-type and B-type). —Confirmed by one method of bioautography.
  • a strain of acinopactor 'SP YK-504 (1 F 0 14420, FE RM BP- 709) grown on a nutrient agar slope was prepared using glucose 2%, soluble starch 3%, raw soy flour 1%, poly ⁇ Inoculate a 2 J2 volumetric flask containing 500 ml of a medium containing 0.5% sedimentable calcium carbonate in an aqueous solution (PH 7.0) containing 0.5% ptone and 0.3% salt, and incubate at 24 ° C. The cells were cultivated with reciprocal shaking for hours.
  • This culture solution was inoculated into a tank with a volume of 5 O J2 containing 30 J2 containing the above-mentioned medium supplemented with 0.05% of antifoaming agent, and the aeration volume at 24 ° C was 50 J2 min.
  • the culture was performed for 48 hours under the conditions of 200 rpm and Z minutes.
  • Hyflo supercell was added to the culture solution, and the mixture was filtered to obtain a filtrate (100 L).
  • the filtrate was adjusted to ⁇ 5, subjected to column chromatography using Amberlite I18-402 ( ⁇ 1-type, 10 J2), washed with water and eluted with 2% saline (509.). .
  • the eluate contained TAN-588 at a concentration of 32 g / ml.
  • the passing solution (100J2) was subjected to column chromatography on Dowex 50 WX 2 CH + , 102) to adsorb the N-deacetyl compound of TAN-588.
  • Example 13 Lysopattor grown on nutrient agar slope '' Arbus sp. Nov. Y
  • K-422 (IF 0 14384, FE RM BP-698) strain was converted to an aqueous solution containing 2% glucose, 3% solvable starch, 1% raw soybean, 1% polypeptone, 0.3% salt and 0.3% salt (PH7.0)
  • the mixture was inoculated into a 2J2 volume flask containing 500 ml of a medium to which 0.5% of sedimentable calcium carbonate was added, and cultured with reciprocal shaking at 24 for 48 hours.
  • the entire amount of this culture was inoculated into a 50 J2 tank containing 30 £ of medium containing the above-mentioned medium supplemented with 0.05% of the antifoaming agent Actcol.
  • the cells were cultured for 48 hours under the same conditions.
  • a strain of Acinetobacter sp. YK-504 (1 FO 14420, FERM BP- 709) grown on a nutrient agar slope was prepared using glucose 2%, Soluble starch 3%, raw soy flour 1%, polypeptone 0.5%. Inoculate a 2J2 volumetric flask containing 50 Qml of a medium containing 0.5% of sedimentary carbon dioxide in an aqueous solution (pH 7.0) containing 3% salt, and shake back and forth at 2'4 ° C for 48 hours. Cultured.
  • aqueous solution containing 3% of dextrin 3%, raw soybean meal 5%, corn ⁇ ⁇ gluten 'meal 1.5%, polypeptone 0.2%, sodium thiosulfate 0.1% PH6.5 was inoculated into a 200-pound tank containing a medium 120 ⁇ supplemented with 0.05% of actcol, and mixed at 24 ° C for 24 hours under the conditions of aeration of 20 minutes and 150 rpm. After culturing, the temperature was changed to 20 ° C., and mixed culture was further performed for 42 hours.
  • the obtained medium contains the N-deacetyl form of TAN-588 (mixture of A-type and B-type), indicating that TLC using Pseudomonas aeruginosa C-1 as a test bacterium. It was confirmed by one method of bioautography.
  • the antibiotic TAN-588, its para-nitrobenzene or benzhydryl ester form, or their N-deacetyl form or salts thereof have antibacterial activity against Gram-positive and negative bacteria, It can be used as a therapeutic agent for bacterial infections such as poultry.

Abstract

Le TAN-588, antibiotique produit par des bactéries appartenant au genre Empédobacter ou Rhizobacter, le p-nitrobenzyle ou son ester de benzhydryle, et son dérivé de N-désacétyle ou ses sels présentent un effet antibactérien contre les bactéries gram-positives et gram-négatives, et peuvent être utilisés en tant qu'agents de traitement de maladies d'origine bactérienne chez les mammifères et les volatiles.
PCT/JP1985/000095 1984-03-29 1985-02-28 Antibiotique, procede de preparation et microorganisme WO1986005185A1 (fr)

Priority Applications (9)

Application Number Priority Date Filing Date Title
PCT/JP1985/000095 WO1986005185A1 (fr) 1985-02-28 1985-02-28 Antibiotique, procede de preparation et microorganisme
EP85301948A EP0157544A3 (fr) 1984-03-29 1985-03-20 Antibiotiques, leur préparation et leur emploi
US06/714,084 US4656288A (en) 1984-03-29 1985-03-20 Antibiotics, their production and use
ES541441A ES8702428A1 (es) 1984-03-29 1985-03-21 Un metodo para producir antibiotico tan-588 y-o n-desacetil-antibiotico tan-588
DK135785A DK135785A (da) 1984-03-29 1985-03-26 Fremgangsmaade til fremstilling af et antibiotisk stof
CA000477912A CA1238594A (fr) 1984-03-29 1985-03-29 Antibiotiques tan-558, production et application
HU851207A HU194310B (en) 1984-03-29 1985-03-29 Process for preparing alkali metal, alkali earth metal, ammonium salt and n-deacetylated derivative of novel tan-588 antibiotic
CN85101679A CN1010103B (zh) 1984-11-29 1985-04-01 抗菌素生产和应用
ES555584A ES8707251A1 (es) 1984-03-29 1986-06-02 Un metodo para producir n-desacetil-antibiotico tan-588

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/JP1985/000095 WO1986005185A1 (fr) 1985-02-28 1985-02-28 Antibiotique, procede de preparation et microorganisme

Publications (1)

Publication Number Publication Date
WO1986005185A1 true WO1986005185A1 (fr) 1986-09-12

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PCT/JP1985/000095 WO1986005185A1 (fr) 1984-03-29 1985-02-28 Antibiotique, procede de preparation et microorganisme

Country Status (1)

Country Link
WO (1) WO1986005185A1 (fr)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Shadan Hojin Yukugakkai Pharmacia Review Henshu Iinkai "Pharmacia Review No.7, Kosei Busshitsu" (1981-10-15) Shadan Hojin Nippon Yakugakkai P.2-5 *

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