KR790001354B1 - A process for producing the antibiotic complex - Google Patents

Abstract

A broad-spectrum antibacterial agent against aminoglycosides resistant bacteria, antibiotic Bu-2183 complex, was prepd. by culturing pseudomonas D946-B83 (ATCC 31086) in a ordinary liquid medium contg. digestable nitrogen and carbon sources aerobically at 4-37≰C, pH 4.5-11 and separating into its active components, antibiotic Bu-2183A (I; R = COCH2CH3), BU-2183 A2 (R = CO(CH2)2CH3), Bu-2183B (R = COCH3), and Bu-2183D (R = H).

Description

Method for producing antibiotic combination

1 is an infrared absorption spectrum of Bu-2183A free base when pelleted in potassium embrittlement,

Figure 2 shows the internal standard material measured by JEOL 60MHz NMR spectrometer (type TNM-C-60HL) of Bu-2183A in which hydrochloride was dissolved in heavy water using 2,2-dimethyl-2-cysilapentane-5-sulfonate. Quantum magnetic resonance spectrum,

3 is an infrared absorption spectrum of Bu-2183A ₂ free base when pelleted in potassium embrittlement,

4 is a proton of Bu-2183A ₂ in which hydrochloride is dissolved in heavy water using 2,2-dimethyl-2-silapentane-5-sulfonate as an internal standard measured by a JEOL 60 MHz spectrometer (type TNM-C-60HL). Magnetic resonance spectrum,

5 is an infrared absorption spectrum of Bu-2183B free base when pelletized from potassium embrittlement,

6 is a proton of Bu-2183A in which hydrochloride is dissolved in heavy water using 2,2-dimethyl-2-silapentane-5-sulfonate as an internal standard measured by a JEOL 60 MHz NMR spectrometer (type TNM-C-60HL). Magnetic resonance spectrum,

7 is an infrared absorption spectrum of Bu-2183D free base when pelleted in potassium embrittlement,

8 shows Bu-2183D dissolved in heavy water using hydrochloric acid in 2,2-dimethyl-2-silapentane-5-sulfonate as an internal standard measured by a JEOL 60MHx NMR spectrometer (type TNM-C-60HL). Quantum magnetic resonance spectrum.

Various antibiotics are known in the art, including some aminoglycosite antibiotics, such as kanamycin, kentamycin, streptomycin, neomycin, toppramycin and paromomycin. However, there is a need for a new broad-spectrum antibiotic with antimicrobial effects against aminoglycoside resistant bacteria, such as Pseudomonas aeruginosa.

A new strain of Pseudomonas aeruginosa, designated as strain D946-B83 in the Bristol-Anti-Storage Registered strain, is a low-affinity soil bacterium isolated from soil samples in India. This strain has been stored in US Supplemental Registered Strains (A. T. C. C.) and added as a permanent storage strain as A. T. C. C 30086.

The new amino glycoside complex of the present invention has at least five components, and three of Bu-2183A, A ₂ and B are active in vivo and are inactive in two of Bu-2183C and D. It turns out.

Antibiotic complex Bu-2183 and the antibiotic components active in the three types of the above-mentioned range of antibacterial activity, and most of the aminoglycosite resistant strains including Pseudomonas aeruginosa are suppressed. The antibiotics Bu-2183, Bu-2183A, Bu-2183A ₂ , and Bu-2183B are valuable as antibacterial agents, as nutrients in feeds and as therapeutics for poultry or animals, including humans. Gram is useful for treating infectious diseases caused by sex bacteria and Gram-negative bacteria, particularly aminoglycosite resistant bacteria. Bu-2183D, one of the new bioinactive ingredients in fermentation seed complex, is a bioactive ingredient useful as an intermediate in semi-synthetic preparation (N-Asurence) of Bu-2183A, A ₂ and B.

[microbe]

The bacterium producing B-2183 antibiotics, defined as the P. aeruginosa D946-B83 strain and A. T. C. C 31086 strains, is aerobic, amalogenic and Gram-negative bacteria. The bacteria are rod-shaped and have multiple flagella on one end. The strain D946-B83 is a low temperature affinity bacterium capable of ending at 4 ° C. and thus cannot be grown at 41 ° C. When cultured in glutamate-containing medium and skim milk solution, fluorescent pigments are generated and H.Iizuka & Konagata, An Aflempt at Grouping of the Genus Pseudomons J. Gen Appl. Mic. 5 Vol. 9: 73- 82, 1963) do not form a pigment. It does not produce citrine oxidase. Morphological, cultural and physiological characteristics of this strain D946-B83 are described below.

[Morphological features]

The D946-B83 strain is limber, does not form apoptosis and is characterized by Gram-negative bacilli. Bacterial cells are straight and sometimes bent along the long axis, with multiple flagella on one end. As a stock of bacteria, Pori-β-hydroxy botrate does not contain much (R. Y, Stanier, NJ Palleron: 8M Doudoroff! The Aerobic Pseudomonads: A taxonomic J. Gen. Microbiol 43: 159-271, 1966) . It does not form shells or capillaries that enclose cells.

[Characteristics of culture]

Orthodontic agar and yeast extracts on agar medium:

The growth is very good, and after a day, the colony reaches a diameter of 0.5-1.5 mm. The surface of the colony is soft with the skin. It is opaque with a white creamy color and later pale yellow. It is slightly sticky and does not form pigments that can spread on the medium.

Normal liquid medium and yeast extract liquid medium:

It grows well and grows a lot. At first it becomes cloudy and later precipitates out and sometimes surface grows.

Yeast Extract Agar Plan: Grows only on the surface, not under anaerobic conditions.

Chemically defined inorganic salt media:

Normal growth when glucose or lactose is added as a carbon source.

Requirements for growth factors: none.

Growth temperature: Growth is inhibited at 4 ℃.

Grows well at 10-32 ° C. Slightly grown at 37 ° C. No growth at 41 ° C.

Effect of medium pH: Not growing totally at pH 4.0. Growth is inhibited at pH 4.5 and pH 10.5-11.0, grows well at pH 5.5-9.5.

Effect of salt: Not growing in 12% salt. Growth is suppressed in 6-9%, but usually grows in 5% or less.

The morphological and culture characteristics described above are similar to those of the Pseudomonas aeruginosa tube.

[Physiological characteristics]

Strain D946-B83 produces fluorescent pigments that luminesce in certain media but do not produce other bacteria. That is, Pseudomonas fluorescence produces a large amount of fluorescence among known Pseudomonas aeruginosa (Table 1).

Physiological and biochemical properties of strain D946-B83 are shown in Table 2. The carbohydrate utilization of the strain and the availability of other carbon sources are shown in Table 3 by comparing two known species of Pseudomonas aeruginosa with Pseudomonas fluorescence and Pseudomonas erugino.

[Morphological characteristics]

Strain D946-B83 seems to belong to the Pseudomonas aeruginosa in view of the morphological, culture and physiological properties described above. According to the morphological theory reported by Stern and his collaborators (RY Stanier, NJ Palleron and M. Doudoroff: The Aerobic Pseudomonads: A Taxonomic Study. J. Gen. Micrbiol 43: 159-271, 1966), strain D -946-B8 is closer to Pneumomonas fluorescence, except for its egg-yolk negative response, inositol insolubility and negative oxidation products. Of the 13 forms of aerobic Pseudomonas aeruginosa tested by Steen, only Xdomonas maltophilia reported negative oxidation. However, this strain is very different from D946-B83 because it has very little fluorescent dye, requires methionine, does not grow at 40 ° C at all, there is no arginine dehydrolase, and its dilution is different. As such, strain D946-B83 is considered to belong to a new species in Pseudomonas aeruginosa.

[Production of antibiotics]

Antibiotic complex Bu-2183 is a substance produced by culturing a novel Pseudomonas aeruginosa strain D-946-B 83, A. T. C. C 31086, in aerobic state, usually in a deep liquid medium. The bacterium grows in a common medium containing assimilable carbon sources such as assimilable carbohydrates. Preferred carbon sources are glucose, fructose, mannose, glycerol. Normal medium is also an anabolic nitrogen source, ie fish meal, soy flour, peptone and the like. Nutritive inorganic salts are also contained in the culture medium, for example sodium potassium, ammonium, calcium, phosphate, sulfate, chloride, emulsified, nitrate, carbonate, and the like.

Bu-2183 composite products can grow sufficiently, for example, at 10-32 ° C., but the most preferred temperature is 28-30 ° C. ambient temperature. Usually the product is obtained optimally for 3-5 days. The optimum pH of the medium is about 5.5-9.5 but most preferably the pH of the medium is adjusted to about 7.0. In the case of tank fermentation, it is preferable to inoculate a juicy culture by slope or soil culture or to inoculate a lyophilized culture solution of a strain to generate a vegetable inoculation in a liquid medium. In this way, the active inoculation is obtained and then aseptically transferred to the fermentation tank medium.

The preferred method of preparing the complex of Bu-2183 antibiotics is as follows. Using a known agar slope of Pseudomonas aeruginosa 946-B 83, the inoculation medium containing 3% glucose, 2% fish meal, 0.5% soy flour, 0.2% peptone and 0.6% calcium carbonate was adjusted to pH7.0 before sterilization and Inoculate. The medium was incubated with a rotary stirrer (250 rpm) for 48 hours at 28 ° C., and 2 ml of the culture medium was 2% glycerol, 2% linseed powder, 1% peanut powder, 2% fish meal, 0.3% (NH ₄ ) ₂ SO in a 500 ml Ellenmeyer flask. ₄ ) and 100 ml of fermentation broth containing 0.5% calcium carbonate. The antibiotic product peaks after 3-5 days with stirring at 28 ° C. Antimicrobial activity in fermentation broth is measured by paper disc-agar infiltration identification using Bacillus subtilis PCI 219 as a test strain. Strain D966-883 produces 1,500-2,000 mcg / ml antibiotic complex by a stirred flask fermentation method.

After the optimal medium has been obtained (as measured by the identification method described above), the medium is adjusted to pH approximately 2 to separate the basic water soluble antibiotic complex from the mycelium and dissolved in an aqueous fermentation medium. The medium is filtered, preferably filtered through a filter aid and neutralized with filtrate containing antibiotics to adjust to about pH 7. The neutralized filtrate is passed through a cation exchange resin, suitably ammonium type IRC-50 ampelite. The resin is washed with water and diluted with ammonia water (N / 50) and the antibiotic complex is eluted from the resin with a suitable eluent, ie 1 / 2N, NH ₄ OH. The eluates are combined, concentrated under reduced pressure and evaporated or lyophilized to yield crude BU-2183 antibiotic complex. The crude solid thus obtained is loaded onto TLC and contains at least three active ingredients of BU-2183A, A ₂ and B and at least two inactive ingredients of BU-2183C and D. The BU-2183 complex can be separated into its components BU-2183A, A ₂ , B, C and D using a cation exchange resin, preferably ammonite Ambelite CG-50. The complex is dissolved in water, passed through the resin, washed with water, diluted with ammonium hydroxide (1 / 40N) and eluted with a suitable eluent. Ammonium monoxide (1 / 20N) separates BU-2183A and B. Subsequent elution of the column with richer ammonium hydroxide (1 / 10N) elutes BU-2183C and D, which are ninhydrin positive but bioinert. To obtain pure BU-2183A ₂ , further BU-2183A fractions were subjected to column chromatography to separate BU-2183A ₂ and BU-2183A. To completely separate and purify each component, the above-described chromatographic separation process may be repeated. As shown in Table 4, the two TLC systems (S-117 and S-122) are suitable for separating the four components of BU-2183A, B, C and D. During chromatographic purification of BU-2183A as described in detail in the following examples, it was found that the bioactive amino glycoside component was indicated here as BU-2183A ₂ . BU-2183A ₂ is different from BU-2183A by TLC-based S-117 and S-122 as shown in Table 5 below.

BU-2183 Characteristic Data of Antimicrobial Components

BU-2183A

The antimicrobial substrate BU-2183A is a white amorphous base, soluble in water, slightly soluble in methanol and ethanol, in particular in n-butanol, acetone and other organic solvents.

Antimicrobial agent BU-2183A forms salts with acids, and pharmaceutically acceptable acid addition salts are also included within the scope of the present invention. Examples of pharmaceutically acceptable acid addition salts are organic and inorganic non-toxic salts, in particular hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, stearic acid, propionic acid, tartaric acid, malic acid, benzoic acid, succinic acid and the like.

=+78.5°(C, 1, 0, 물)이다.The BU-2183A component reacts positively with ninhydrin and anthrone reagents, but negatively with Tolens, Pelling and Sarcas Gucci reagents. The specific light intensity of BU-2183A base is

= + 78.5 ° (C, 1, 0, water).

A sample of BU-2183A precipitated in ethanol and analyzed was C ₁₅ H ₃₁ N ₃ O ₉ C ₂ H ₅ OH, H ₂ O.

Elemental Analysis Calculation: C, 44.24: H, 8.52: N, 9.11: O, 38.13

Experimental value: 44.25: 8.08: 9.11: 38.56 (different)

BU-2183A and di-N-acetate are colorless needles and have a melting point of 149-150 °. Molecular weight was measured by Osmodetri and found to be 481. Elemental analysis was C ₁₉ H ₃₅ N ₃ O ₁₁ .H ₂ O.

Calculated Value: C, 45.68: H, 7.47: N, 8.41: O, 38.44

Experimental value: 45.73: 7.49: 8.19: 38.59 (different)

BU-2183A is a weak base and there are titratable groups with pKa values of 6.90 and 9.40 in water. The approximate molecular weight calculated from the titration data is 398. The BU-2183A component is absorbed only at the ultraviolet end. When used as a pelletizing agent in potassium embrittlement, as shown in FIG. ¹ , the characteristic absorption bands were substantially observed in the following wave number (cm ⁻¹ ) of the infrared spectrum: 1,635 and 1,570 (amide) and 1,080 and 1,020 (hydroxyl group). ) The nuclear magnetic resonance spectrum of BU-2183A hydrochloride when dissolved in heavy water at the concentration of about 8% is shown in Figure 2.The spectrum shows that the propionyl group at .12 (3H, t, J = 7.5 Hz) and 2.29 (2H, q, J = 7.5 Hz).

The structure was determined to be as follows.

BU-2183A ₂

Like the BU-2183A described above, the antibiotic BU-2183A is a white amorphous base, soluble in water, slightly soluble in methanol and ethanol and practically insoluble in n-butanol acetone and other organic solvents. Acids form salts and pharmaceutically acceptable acid addition salts of BU-2183 bases are within the scope of this invention. BU-2183A ₂ has a positive reaction with ninhydrin and anthrone, and negative reactions with Tolens, Pelling and Sarcasagucci.

=+79.1°(C, 0.43, H₂O)이다.BU-2183A ₂ base and non-photon

= + 79.1 ° (C, 0.43, H ₂ O).

Sample analysis results of BU-2183A ₂ isolated as carbonate are as follows.

Elemental analysis: C ₁₆ H ₃₃ N ₃ OP · 1 / 2H ₂ CO ₃

Calculated Value: C, 44.79: H, 7.75: N, 9.50: O, 37.96

Experience: 44.35 7.83 9.21 38.61 (different)

Component BU-2183A ₂ is absorbed at the ultraviolet end. Infrared spectrum as shown in FIG. 3 is shown when pelletizing potassium potassium embrittlement. The nuclear magnetic resonance spectrum of BU-2183A ₂ hydrochloride when about 6% was dissolved in D ₂ O at the concentration is shown in FIG. BU-2183A ₂ is different from BU-2183A and BU-2183B, B, which is the presence of n-butyl groups in the nuclear magnetic resonance spectrum, 0.92 (3H, t), 1.63 (2H, 6th) and 2.30 (2H, t) ppm And components A and B have propionyl or acetyl groups.

The structure of BU-2183A ₂ was measured as follows.

BU-2183B

Antibiotic BU-2183B is similar in appearance and solubility to BU-2183A and BU-2183A ₂ , ie white amorphous base, soluble in water, slightly soluble in methanol and ethanol, practically insoluble in n-butanol, acetone and other organic solvents to be.

Antibiotic BU-2183B can form salts with acids and pharmaceutically acceptable acid addition salts of antibiotics fall within the scope of the present invention.

Component BU-2183B reacts positively with ninhydrin and anthrone reagents but negatively with Tolens, Falling and Sarcas Gucci reagents.

=85°(C₂, 1.0물)이다.The specific light intensity of BU-2183B base is

= 85 ° (C ₂ , 1.0 water).

Elemental analysis of the component BU-2183B sample when the precipitation was made with ethanol is C ₁₄ H ₂₉ N ₃ O ₉ · C ₂ H ₅ OH · H ₂ O.

Calculated Value: C, 42.95: H, 8.34: N, 9.36: O, 39.35

Experimental value: 42.69: 7.78: 8.86: 40.67 (different)

Di-N-acetate of BU-2183B is a colorless prism and has a melting point of 159-162 °. No. Sumo meth Rie the molecular weight is measured by a 484 _{C 18 H 33 N 3 O 11} · H 2 O to elemental analysis

Calculated Value: C, 44.53: H, 7.27: N, 8.66: O, 39.54

Experimental value: 44.96: 7.44: 8.52: 39.08 (different)

BU-2183B is a weak base and has a titrable group with pKa values of 7.15 and 9.35 in water. The approximate molecular weight calculated from the titration data is 409.

Component BU-2183B is absorbed only at the ultraviolet end. As shown in FIG. 5 when using potassium pellets as a pellet agent, the infrared spectrum showed characteristic absorption bands at the following wave number (cm ⁻¹ ): 1,635 and 1,570 (amide) and 1,080 and 1,020 (hydroxyl group) 10 When dissolved in heavy water at the% concentration, the NMR spectrum of BU-2183B hydrochloride is as shown in FIG. 6. As shown in the NMR spectrum, BU-2183B is different from BU-2183A and A ₂ , where A and A ₂ each have an acetyl group at δ2.02 ppm (3H, S,) instead of propionyl or n-butyryl groups. .

The structure of the BU-2183B component is as follows.

Characteristic data of BU-2183D intermediate

The bioinert component BU-2183D is a white amorphous, soluble in water, slightly soluble in methanol and ethanol, insoluble in n-butanol, acetone and other organic solvents.

Compound BU-2183D can form salts with acids and such acid addition salts with bases are within the scope of the present invention.

BU-2183D reacts positively with ninhydrin and antron reagents but negatively with Tolens, Elling and Sarcas Gucci reagents.

=72.5°(C, 1.0물)이다.The specific light intensity of BU-2183D base is

= 72.5 ° (C, 1.0 water).

The sample of BU-2183B is isolated with carbonate, and the results of elemental analysis are as follows.

Elemental Analysis C ₁₂ H ₂₇ N ₃ O ₈ H ₂ CO ₃ .

Calculated Value: C, 38.70: H, 7.25: N, 10.42: O, 43.63

Experimental value: 38.86: 6.93: 10.14: 44.07 (different)

BU-2183D is a weak base and there are three titrable groups with pKa values of 6.92 (2 equivalents) and 9.68 in water.

The tri-N-acetate of BU-2183D has a melting point of 159-162 ° of the colorless prism. This salt was determined to be the same as the di-N-acetate of BU-2183B.

Component BU-2183D is absorbed only at the ultraviolet end. When pelleted in potassium embrittlement, the same infrared spectrum as in the seventh degree is shown. In the spectrum, BU-2183A, A ₂ and B, which are bioactive, do not show the amide carbonyl band present. When dissolved in heavy water at a concentration of 10%, the NMR spectrum of BU-2183D hydrochloride is equal to the eighth degree. The structure of BU-2183D is as follows.

Structural Determination of Components

Mild acid hydrolysis of BU-2183A and B ₆ (1N hydrochloric acid / methanol, 80 ° C., 3 hours) yields the same desacyl compound, C ₁₂ H ₂₇ N ₃ O ₈ , which is a chromatograph of the crude BU-2183 complex. Same as BU-2183D obtained by graphitic separation. Total N-acylation of BU-2183D yields tri-N-acetate, C ₁₈ N ₃₃ N ₃ O ₁₁ , which turned out to be the-N-acetate of BU-2183B.

Acid hydrolysis (saturated and reflux temperature at 24 hours) in methanolic hydrochloric acid yields aglycone and amino sugars with BU-2183D. Aglycones are isolated as hydrochloride crystals and have a melting point of 263-264 ° C. and a C ₆ H ₁₆ N ₂ O ₄ · H ₂ SO ₄ .

Calculated Value: C, 25.90: H, 6.52: N, 10.07: S, 11.52

Experimental Value: 26.10: 6.47: 9.93: 11.29

The 220 MHz NMR spectrum of its N, O-hexaacetate, together with mass spectra data obtained with N-acetyl-O-TMS, N-acetyl-diisopropylidene and hexa-N, O-acetyl derivatives of iglycone, shows aglycone It is likely to be the 1, 4-diamino-2, 3, 5, 6-tetraol structure of the moiety.

The di-N-acetate of iglycone is colorless needles, has a melting point of 114-115 ° and an elemental analysis of C ₁₀ H ₂₀ H ₂ O ₆ .

Calculated Value: C, 45.45: H, 7.63: N, 10.60

Experimental value: 45.37: 7.97: 10.57

Since the D-glucose-type arrangement is most functional in aglycone, 1, 4-diamino-1, 4-dideoxy-D-sorbitol is produced from 4-amino-4-deoxy-D-gluose, The product was found to be aglycone by TLC NMR analysis. The amino-sugar portion obtained after the aforementioned acid methanolysis of component B is purified by Ambelite CG-50 chromatography and separated into alpha and beta forms of methylglycosides. The N-acylated material of alpha-methylglycoside is a colorless prism, melting point 185-186 °.

Elemental Analysis: C ₉ H ₁₇ NO ₆

Calculated Value: C, 45.95: H, 7.28: N, 5.95

Experimental Value: 45.93: 7.43: 5.88

The mass spectrum of the N-acetyl derivative shows m / l 204 (M-31) peaks due to the loss of lycoside methoxy groups in the molecule. As such, the free amino sugar is C ₆ H ₁₃ NO ₅ .

The alpha and beta forms of methylglycosides are methyl 4-amino-4-deoxy-alpha- and beta-D-glucopyranosides, respectively, by infrared and hex resonance spectra of their tetra-N, O-acetyl derivatives. It turned out to be. A clear class of infrared spectra of this sugar isolated from 4-trialose (J. Antibiotics, 27, 145, 1974) is consistent with the infrared spectra of experimental samples.

Hydrolysis of Bu-2183D (reflux, 3 hours) in 6N hydrochloric acid yields the same aglycone and amino sugars isolated from hydrolysis of Bu-2183B. The crude Bu-2183 complex is hydrolysed Bu-2183C obtained by chromatographic separation in methanol containing 1N hydrochloric acid for 3 hours at reflux temperature. The aglycone moiety is the same as that of the other components of Bu-2183 and the sugar moiety is the same as methyl D-glucoside by thin layer chromatography, nuclear magnetic resonance and gas chromatography. Thus the molecular sieve of Bu-2183C is C ₁₂ H ₂₆ N ₂ O ₉ .

Based on the above data, the structure of the Bu-2183 component is as follows.

[Antibacterial activity]

In in vitro experiments, complex Bu-2183 and each antibacterial component Bu-2183 A, A ₂ and B have a wide range of antimicrobial activity.

Antimicrobial complexes and active ingredients are particularly useful for inhibiting most aminoglycoside resistant strains. Component Bu-2183A ₂ is more active than Bu-2183B but less active than Bu-2183A.

Minimum inhibitory concentrations of Bu-2183A and B were usually measured for several bacteria by 2-fold agar dilution on agar plates. Inoculation-transcription by Steers and his collaborators was used. The inoculation amount is adjusted so that overnight culture of test bacteria is diluted 10 ⁴ in cardiac extraction medium (Difco). The results are shown in Table 6, compared to cannamycin as a reference antibiotic.

The potent activity of Bu-2183A and B has moderate or weaker MIC values, while component A is 2-4 times stronger than B. However, they have broad antimicrobial activity against Gram-negative and Gram-positive bacteria, including several amino glycoside-resistant and Pseudomonas aeruginosa species.

Table 7 shows the minimum inhibitory concentrations of Bu-2183A and A ₂ against several pathogens.

Samples of Bu-2183 complex with approximately 30-40% purity were E. coli A20365 at 12.5 mg / ml concentration, Klebsiella pneumoniae D-11 at 12.5 mcg / ml concentration, and Pseudomonas ergi at 25 mcg / ml concentration. Remove labor and management A9930.

[Effect of Medium pH at Minimum Low Concentration]

Medium pH effects at minimum inhibitory concentrations of Bu-2183A were usually observed by 2-fold agar dilution at pH 6.0, 7.0, 8.0 and 9.0 using agar media. The result is that as shown in Table 8, the activity of Bu-2183A increases at alkaline pH and decreases at acidic pH.

[Effect of Badge]

The effect of the medium on the activity of Bu-2183A and B was measured by eight test strains. The test medium usually used agar, heart extraction agar and muller-hinton agar. Medium pH was adjusted to 8. As shown in Table 9, the maximum in vivo activity is seen when using agar as a test medium.

[In vivo Activity and Toxicity]

Bu-2183A and B were experimentally infected and administered in mice in vivo. The pathogenic bacteria used are Staphylococcus aureus smid-e.coli NIHJ and Sutomonas seruginosa A9930. Mice were infected with ₅₀ × LD ₅₀ of pathogen in a 5% suspension of gastric mucus of castrated pigs, immediately subcutaneously injected once with antibiotics, and injected twice at 0 and 3 hours after infection. Five mice were used in each group, and the animals were observed for 5 days in order to measure the median dose (PD 50).

The results are shown in Table 10. Bu-2183A and B showed in vivo activity against all three infectious bacteria.

Acute toxicity of Bu-2183A and B was measured in mice by subcutaneous and intravenous injection. Mice were observed for 15 days and the subcutaneous and intravenous doses of Bu-2183A were 2,500 mg / kg and 1,000 mg / kg, respectively. Bu-2183B was less toxic than Bu-2183A and no animals died when administered over 2,000 mg / kg intravenously or subcutaneously during the 15-day observation period.

Use of Bu-2183D

Bu-2183D is bioinert but is a valuable intermediate in the semisynthetic preparation of the bioactive components Bu-2183A, A ₂ and B. As such, the free amino group (after appropriately protecting other active functional groups) of the Bu-2183D intermediate is subjected to an acylation reaction by a known method for N-butyryl derivative Bu-2183A, N-acetyl derivative Bu-2183 or N-. The propionyl derivative Bu-2183A can be produced.

TABLE 1

TABLE 2a

TABLE 2b

1) R. Y. Stanier, N. J. Palleroni & M. Doudoroff: The Aerobic Pseudomonads: A Taxonomic Study. J. Gen. Microbiol. 43 159-271, 1966

2) M. E. Rhodes: The Characterization of Pseudomonas Fluorescens. J. Gen Microbiol. 21: 221-263, 1959

3) H. Iizuka & K. Komagata: An Attempt At Grouping of The Genus Pseudomonas. J.Gen. Appl. Micriobiol. 9: 73-82, 1963

4) H. Iizuka & K. Komagata: Taxonomy of Genus Pseudomonoas with Special Reference to their Modes of Metabolism of Carbon Compounds. J. Gen. Appl. Microbiol. 9: 93-95, 1983

5) V. B. D. Skerman: Abstracts of Microbiological Methods. Wiley-Interscience, New York, London, Sydney and Toronto, P. 364, 1969

TABLE 3a

TABLE 3b

TABLE 3c

Basal medium per liter: 40 ml of 1 M-phosphate buffer (pH 6.8); 1 g., (NH ₄ ) ₂ SO ₄ ; 20 ml of Hutner Vitamin Free Inorganic Salt Solution

TABLE 4

TABLE 5

TABLE 6a

TABLE 6b

TABLE 7a

TABLE 7b

TABLE 8

TABLE 9

TABLE 10

Ambolite IRC-50 and CG-50 are weakly acidic cation exchange resins of the carboxyl-polymethacrylic type under trade names.

Example 1

Fermentation in tank

Agar slope cultures of D946-B83 in Pseudomonas aeruginosa are inoculated in 100 ml of Seed No. 83B (3% glucose, 2% fish meal, 0.5% soy flour, 0.2% peptone and 0.6% calcium carbonate) in a 500 ml Elenmeyer flask. The flask was incubated with a rotary stirrer (250 ppm) for 3 days at 28 ° and 1 L of the seed culture medium was added to 300 L of fermentation medium No. 100F (2% glycerin, 1% parmemedia, 2% fishmeal, 2% flax seed flour, 0.3%) NH ₄ ) ₂ SO ₄ , 0.6% calcium carbonate). The tank is stirred at 30 ° at 140 rpm and through air at a rate of 200 l / min. The antimicrobial activity of the juice was measured by paper disk-agar plate method using Bacillus subtilis PCI 219 as the identified strain, and Bu-2183A was used as the identified juice material. the results are as follow:

Example 2

[extraction]

The gravy obtained is filtered through a filter at pH 2. The filtrate (19 liters) contains about 30 g of Bu-2183A, adjusted to pH 7 and stirred with 3 liters of Ambelai IRC-50 (NH ₄ type). The resin is separated and washed with 10 liters of water and 5 liters of 1 / 50N ammonium hydroxide, respectively, and stirred twice with 0.4 liters of 1 / 2N ammonium hydroxide to elute the active ingredients in vivo. The combined eluates were concentrated under reduced pressure and lyophilized to yield a white solid 18.6 (about 700 mcg / mg). This solid is dissolved in water and passed through a column of Ambolite CG-50 (ammonium type, 400 ml). The column is washed with 2.2 liters of water and 3 liters of 1 / 40N ammonia hydroxide respectively and developed with 1 / 20N ammonia hydroxide. Eluates were sorted and tested by in vivo identification and thin layer chromatography (S-117) ninhydrin in a Bacillus subtilis plate window. The appropriate fractions are driven, concentrated under reduced pressure and lyophilized to afford the following solids.

Example 3

[Manufacture of Bu-2183A]

The crude sample of Bu-2183A (2.7 g) obtained in Example 2 is dissolved in water and passed through an Ambelite CG-50 (ammonium type, 80 ml) column. The column is eluted with 1 / 20N ammonia hydroxide, and 15 ml of the eluate is collected by a fractionator. Fractions are identified by thin layer chromatography-ninhydrin and bioassay, the appropriate fractions are combined, concentrated under reduced pressure and lyophilized to give the next solid.

The pure product of Bu-2183A was analyzed by C ₁₅ H ₃₁ N ₃ O ₉ 1 / 2HCO ₃ .

Calculated Value: C, 43.45: H, 7.35: N, 9.81

Experimental Value: 43.22: 7.52: 9.49

Its infrared and nuclear magnetic resonance spectra are as shown in FIGS. 1 and 2, respectively. The summary of nuclear magnetic resonance data is as follows.

Example 4

[Manufacture of Bu-2183-B]

The crude sample of Bu-2183 (3.3 g) obtained in Example 2 is dissolved in water and placed in an column of Ambolite CG-50 (ammonium type, 130 ml). The column is eluted with 1 / 20N ammonia water and collected by distillation into 15 fractions of eluent. Fractions are tested by thin layer chromatography and biodynamics. Appropriate fractions are combined, concentrated under reduced pressure and lyophilized to afford the next solid.

The pure product of Bu-2183B was analyzed by C ₁₄ H ₂₉ N ₃ OP.½ H ₂ CO ₃ .

Calculated Value: C, 42.02: H, 7.30: N, 10.14

Experimental Value: 41.92 7.43 9.93

Its infrared and nuclear magnetic resonance spectra are in FIGS. 5 and 6. The summary of nuclear magnetic resonance data is as follows.

Example 5

[Manufacture of Bu-2183C and Bu-2183D]

The column used in Example 2 was eluted with 1.4 liters of 1 / 20N ammonia water until no further bioactive substance was eluted. Columns are developed with 1 / 10N ammonia water and the eluate is tested with thin layer chromatography ninhydrin. Collect the appropriate fractions, concentrate under reduced pressure, and freeze-dry to obtain the following solids.

Example 6

[Manufacture of Bu-2183A ₂ ]

During the purification of the components described in Example 3, new active ingredients are isolated and abbreviated as Bu-2183A ₂ . This is different from compound A according to thin layer chromatography S-117 and S-112 as follows:

The infrared and nuclear magnetic resonance spectra of Bu-2183A ₂ are shown in FIGS. 3 and 4. The summary of nuclear magnetic resonance data is as follows:

Claims (1)

Pseudomonas aeruginosa D946-B83, (ATC C 31086), was deeply cultured in an aqueous normal liquid medium containing an anabolic nitrogen source and a carbon source at pH 4.5-11.0 at a temperature of 4-37 ° C. under aerobic conditions, Method for producing an antibiotic complex Bu-2183 characterized by culturing until a substantial amount of the Bu-2183 complex produced by the production.

Images