JPH0699444B2 - Antibiotic TAN-547 and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel antibiotic TAN-547, salts thereof and a method for producing the same.

The present inventors have isolated a large number of microorganisms from soil for the purpose of searching for new antibiotics, and when separating and searching for the antibiotics produced by the microorganisms, certain microorganisms produce novel antibiotics, Knowing that the microorganism is a new strain belonging to the genus Resobacter, and that the antibiotic showing antibacterial activity can be accumulated in the medium by culturing the microorganism in an appropriate medium, etc., the antibiotic was isolated. , It was confirmed from the physicochemical and biological properties that the antibiotic is a new antibiotic, and it was named antibiotic TAN-547. Of the components of antibiotic TAN-547, the three components obtained in large quantities were named antibiotics TAN-547A, B, C.

Furthermore, the three components other than the above were named antibiotics TAN-547D, E, F.

The present inventors have completed the present invention as a result of further research based on these findings.

The present invention provides (1) the following formula [Wherein R ¹ represents formylamino, R ² represents alanyl-alanyl, or R ¹ represents hydrogen, and R ² represents hydrogen, alanyl, or alanyl-alanyl], a cephem antibiotic TAN -547 or salts thereof, and (2) belonging to the genus Resobacter, the following formula [Wherein R ¹ represents formylamino, R ² represents alanyl-alanyl, or R ¹ represents hydrogen and R ² represents hydrogen, alanyl or alanyl-alanyl]
A method for producing the antibiotic TAN-547 or a salt thereof, which comprises culturing a microorganism having an ability to produce TAN-547 in a medium, allowing the antibiotic to be produced and accumulated in the culture, and collecting the antibiotic.

Antibiotics TAN-547C, D, E and F have the following formula [In the formula, when R ¹ is formylamino, R ² represents alanyl-alanyl, and when R ¹ is hydrogen, R ² represents hydrogen, alanyl, or alanyl-alanyl]; And alanyl-alanyl has the formula Formylamino has the formula Are each represented by. R ¹ and R ² in the antibiotics TAN-547C to F components are as shown below.

In the present specification, the antibiotics TAN-547C, D, E and F are collectively referred to as "the antibiotic TAN-547" or as the name of a mixture of two or three of them or as the name of a single compound respectively. Sometimes "TAN-547" is simply used.

In addition, in the present specification, a microorganism having the ability to produce one or more of the antibiotics TAN-547C, D, E and F may be referred to as "antibiotic TAN-547-producing bacterium".

As the antibiotic TAN-547 producing bacterium used in the present invention,
Antibiotics TAN-54 belonging to the genus Lysobacter
Any microorganism can be used as long as it has the ability to produce 7. An example thereof is the new bacterial species Lysobacter lactamgenus. A specific example thereof is the Resobacter lactamgenus YK-90 strain (hereinafter also referred to as “YK-90 strain”) collected from soil in Niimi City, Okayama Prefecture.

The mycological properties of the YK-90 strain are listed below.

(A) Morphology After culturing on a broth agar slope for 5 days at 24 ° C, the cells were rod-shaped with a diameter of 0.7-0.9 μm and a length of 1.0-3.5 μm and a length of 6.5.
It may show a long rod shape of ~ 8.0 μm. No flagella, but motility due to gliding is observed. Does not form spores. Gram stain is negative and does not show acid resistance.

(B) Growth state on various media Cultures were carried out at 24 ° C. and observed for 1 to 14 days.

Meat broth agar plate culture: The colonies are opaque, pale yellow, round, convex on the surface, and rounded on the edges. No diffusible dye is formed.

Meat broth agar slope culture: shows medium spread-like growth,
Opaque, pale yellow.

Meat juice liquid culture: Grows in a thin cloudy state and forms a pellicle. There is no precipitation.

Meat broth gelatin stab culture: Grows well on the surface and is also observed in the middle and deep areas. It liquefies into layers and bags and has a strong liquefaction activity.

Litmus milk: Litmus has a weak reducing ability. The peptone-forming activity is strong, but no coagulation is observed.

(C) Physiological properties Nitrate reduction: + Denitrification reaction:-MR (methyl red) test:-VP (Fouges-Proscauer) test:-Indole formation:-Hydrogen sulfide formation (TSI agar and lead acetate paper) :-TSI agar: meat extract 4g, peptone 15g, lactose 10g, sucrose 10g, glucose 1g, sodium chloride 5g, sodium thiosulfate 0.08g,
Sodium sulfite 0.4 g, ferrous sulfate 0.2 g, phenol red 0.02 g, agar 15 g, distilled water 1000 ml, pH 7.4 Starch hydrolysis: − Utilization of citric acid (Cosel, Christensen and Simmons media): + Utilization of nitrogen source I) Potassium nitrate: -II) Ammonium sulphate: + III) Sodium glutamate: + Pigment formation (King A, B and Mannit yeast extract agar medium): No diffusible pigment formation is observed.

King A medium: Glycerin 10g, peptone 20g, magnesium chloride 1.4g, ammonium sulfate 10g, agar 15g, distilled water 1000m
l, pH7.2 King B medium: Glycerin 10g, Peptone 20g, Potassium monohydrogen phosphate 1.5g, Magnesium sulfate 1.5g, Agar 15g, pH7.2 Urease:-Oxidase: + Catalase: ± (false positive) Growth range I) pH: It grows at pH 5.4 to 7.6, but the optimum pH is 5.6 to 6.6.

II) Temperature: It grows at 10 to 30 ℃, but the optimum temperature is 15 to 27 ℃.

 Attitude toward oxygen: aerobic or facultative anaerobic.

OF (oxidative-far-mental) test [Hugh-Leifson method]: non-decomposition type.

Acid and gas production from sugar: GC (guanine-cytosine) content of DNA: 75.8 ± 1.5% Microcyst forming ability:-Carboxymethylcellulose degrading ability: + Colloidal chitin degrading ability: + YK-90 strain having the above-mentioned mycological characteristics
Bergey's Mannual of Determinative Bacteriol
ogy) 8th Edition and International Journal
Of systematic bacteriology (Internat
ional Journal of Systematic Bacteriology) Volume 30 22
When compared with the species described on pages 5 to 420 (1980), Vol. 32, pages 146 to 149 (1982), the YK-90 strain shows motility due to yellow gliding without flagella. It is a negative bacillus, is aerobic, has a high GC content of DNA, and is not capable of forming microcysts, so it is appropriate to belong to the genus Resobacter. Therefore, the YK-90 strain was compared with known species of the genus Resobacter. Known species of the genus Resobacter include International Journal of Systematic Bacteriology Vol. 28, 367-39.
Only 4 and 1 subtypes listed on page 3 (1978) are known. Then, when the characteristics of these 5 species (46 strains are described in the above-mentioned document) and the YK-90 strain were compared, the following properties of the YK-90 strain, namely 1) oxidative-far-mental The test was non-degradable, 2) no production of acid and gas from sucrose and lactose was observed, 3) it had the ability to reduce nitrate, 4) no formation of soluble pigment was observed, and 5) starch No strain has been found that exhibits all of these properties in that it has no hydrolytic ability and 6) there is no utilization of nitrate.

Therefore, the YK-90 strain was recognized as a strain belonging to a new bacterial species,
The new strain is designated as Lysobacter lactamgenus (Lysobacter
lactamgenus).

The above Resobacter lactamgenus YK-90 strain is
Contract number IFO to the Institute of Fermentation (IFO) on September 14, 2014
It was deposited as 14288, and this microorganism was found in September 1983.
Ministry of International Trade and Industry, Institute of Industrial Technology, Institute of Microbial Technology (FR)
I) was deposited as deposit number FERM P-7247, and the deposit was converted into a deposit under the Budabest Treaty, and the deposit number
It is stored in the same laboratory (FRI) as FERM BP-575.

The properties of the Rhizobacter bacterium used in the present invention are generally liable to change, for example, ultraviolet rays, X-rays, chemicals (eg, nitrosoguanidine, ethylmethanesulfonic acid).
Although it can be easily mutated by an artificial mutagenesis method using, etc., any mutant strain is a target of the present invention.
Any product capable of producing TAN-547 can be used in the present invention.

When TAN-547-producing bacteria are cultured, carbon sources include glucose, maltose, lactose, molasses, fats and oils (eg, soybean oil, olive oil, etc.), organic acids (eg, citric acid, succinic acid, gluconic acid). Etc.) that can be assimilated by the bacterium are appropriately used. As the nitrogen source, for example, soybean flour, cotton seed flour, corn steep liquor, dried yeast,
Organic or inorganic nitrogen compounds such as yeast extract, meat extract, peptone, urea, ammonium sulfate, ammonium nitrate, ammonium chloride and ammonium phosphate can be used. As the inorganic salt, for example, inorganic salts necessary for culturing ordinary bacteria such as sodium chloride, potassium chloride, calcium carbonate, magnesium sulfate, monopotassium phosphate, and disodium phosphate can be used alone or in appropriate combination. . In addition, sulfur compounds that can be assimilated by bacteria that produce the antibiotic TAN-547 belonging to the genus Resobacter, such as sulfates (eg, ammonium sulfate), thiosulfates (eg,
Inorganic sulfur compounds such as ammonium thiosulfate), sulfites (eg, ammonium sulfite), sulfur-containing amino acids (eg, cystine, cysteine, L-thiazolidine-4-carboxylic acid), hypotaurine, sulfur-containing peptides (eg, glutathione), etc. When the organic sulfur compound (1) or a mixture thereof is added to the medium, the production amount of the target product may increase.

Also, heavy metals such as ferrous sulfate and copper sulfate, vitamins
Vitamins such as B ₁ and biotin are also added as needed. Further, a defoaming agent such as silicone oil or polyalkylene glycol ether or a surfactant may be added to the medium. Other organic or inorganic substances that help the growth of bacteria and accelerate the production of TAN-547 may be appropriately added.

The culturing method may be the same as a general antibiotic production method, and may be solid culture or liquid culture. In the case of liquid culture, any of static culture, stirring culture, shaking culture, aeration culture, etc. may be carried out, but aeration stirring culture is particularly preferable. The culture temperature is preferably about 10 ° C to 30 ° C, and the pH of the medium is about 4 to 8 for about 8 hours to 168 hours, preferably 24 hours to 144 hours.

In order to collect the target antibiotic TAN-547 from the culture, a separation means usually used for collecting the metabolite produced by the microorganism from the culture of the microorganism is appropriately used. For example, the antibiotic TAN-547 has the property of being a water-soluble basic substance (a basic substance as a whole molecule although it also contains a weak acidic group as a functional group), and is mainly contained in the culture solution. The microbial cells are removed by adding a super-auxiliary agent to the culture medium or by centrifugation or centrifugation. A means for bringing the obtained culture solution into contact with an appropriate carrier to adsorb the active ingredient in the solution, then desorbing the active substance with an appropriate solvent, and collecting and collecting the active ingredient is advantageously used. As the carrier for chromatography, use the difference in the adsorptivity of compounds such as activated carbon, silica gel, powdered cellulose, adsorbent resin, or the difference in the functional groups of compounds such as cation exchange resin, cation exchange cellulose, cation exchange sephadex. Those that utilize or utilize the difference in molecular weight of compounds such as cephadex are advantageously used. Combinations of the target compounds to be eluted from these carriers differ depending on the type and properties of the carrier. For example, aqueous solution of water-soluble organic solvent, that is, water-containing acetone, water-containing alcohols, acid, alkali, buffer A liquid or an aqueous solution containing an inorganic or organic salt is appropriately combined and used. In addition, the crude substances containing antibiotics obtained by these chromatographies were subjected to preparative high-performance liquid chromatography,
Purified products are also obtained.

More specifically, as a carrier, a cation exchange resin such as Dowex-50W (manufactured by Dow Chemical Co.,
US), Amberlite IR-120 (Rohm and Haas Company, USA), Diaion SK 116 (Mitsubishi Chemical Industries Co., Ltd.), etc., the antibacterial substance in the liquid is adsorbed and contains salts or acids. It is eluted with an aqueous solution or buffer solution of. Alternatively, a cation exchange molecular sieving resin such as CM-Sephadex (Farmasia Huaine.
Antibiotics can be adsorbed on a carrier such as Chemicals Co., Ltd. (Sweden) and eluted with a salt or acid-containing aqueous solution or buffer. To remove salts and coloring substances from these eluates, activated carbon for chromatography (Takeda Pharmaceutical Co., Ltd.) or adsorbent resin such as Diaion HP-20 (Mitsubishi Kasei Co., Ltd.), Amberlite XAD -II (Rohm and Haas Company, USA) is advantageously used. The fractionated elution fraction is pulverized through steps such as concentration and freeze-drying. When the powder thus obtained has poor purity, high performance liquid chromatography is advantageously used for further purification. Examples of carriers used include TS
K gel (manufactured by Toyo Soda Co., Ltd.), YMC gel (manufactured by Yamamura Chemical Research Institute) and the like can be mentioned, and a mixed solution of methanol or acetonitrile and an aqueous solution containing an inorganic salt or a buffer solution is used as the moving layer. TAN-547 is isolated as a salt of mineral acids such as hydrochloric acid, sulfuric acid, phosphoric acid or organic acids such as formic acid, acetic acid and oxalic acid.

The physicochemical properties of TAN-547 / hydrochloride obtained in Examples 1 and 2 described below are as follows.

(I) TAN-547C dihydrochloride 1) Appearance: White powder 2) Specific optical rotation: ▲ [α] ²⁵ _D ▼ + 25.1 ° ± 15 ° (c = 0.4
9, water) 3) Measured molecular weight: SIMS method, (M + H) ⁺ 830 4) Estimated molecular formula: C ₃₂ H ₅₁ N ₁₁ O ₁₃ S ・ 2HCl ・ (3H ₂ O) 5) Elemental analysis value (%) : Measured value ^{* 1} Calculated value ^{* 2} C, 39.61 ± 2.0 C, 40.17 H, 6.54 ± 1.0 H, 6.22 N, 15.92 ± 1.5 N, 16.10 O, 26.75 S, 3.41 ± 1.0 S, 3.35 Cl, 6.41 ± 1.5 Cl , 7.41 * 1; The sample was dried on phosphorus pentoxide at room temperature for 15 hours under reduced pressure. * 2: Calculated as including 3 mol of attached water.

6) UV spectrum: See FIG.

7) CD spectrum: 8) IR spectrum: Major wavenumber (cm ^-1 ) in potassium bromide
Is as follows. See FIG.

3350, 3250, 3070, 3000, 2950, 1780, 1735, 1665, 1530, 1450, 1400, 1300, 1250, 1160, 1060, 790, 520.

9) ¹³ C-NMR spectrum: The signal at 100 MHz in heavy water is at least found below (δ ppm).

179.79 (s), 178.04 (s), 177.47 (s), 177.38
(S), 176.12 (s), 173.47 (s), 170.02 (s), 166.32
(D), 162.07 (s), 159.52 (s), 133.11 (s), 122.70
(S), 79.57 (s), 72.86 (s), 67.10 (t), 65.88
(D), 57.28 (d), 55.87 (d), 53.07 (d), 52.35
(D), 51.62 (d), 43.52 (d), 41.52 (d), 37.27
(T), 32.73 (t), 29.25 (t), 28.48 (t), 27.36
(T), 23.41 (t), 19.45 (q), 19.42 (q), 19.25
(Q), (s; singlet, d; doublet, t; triplet, q; quartet) 10) Amino acid analysis value: (in constant boiling hydrochloric acid (5.5N-HCl), 110
Sample hydrolyzed at 15 ℃ for 15 hours. ) Alanine: 3.1 mol α-amino-adipic acid: 1.1 mol 11) TLC: (Spot film, cellulose (manufactured by Tokyo Chemical Industry Co., Ltd.). Solvent system, acetonitrile: 3% ammonium sulfate (1: 1),) Rf = 0.60 12) HPLC: (carrier, YMC pack A312 (manufactured by Yamamura Chemical Laboratory), mobile phase, 2% methanol / 0.01M phosphate buffer (pH 3.
0), 2 ml / min. ) Rt = 11.7 (min) 13) Solubility: Easy solubility: water, hydrous acetone, hydrous alcohol Poor solubility: dimethyl sulfoxide, methanol, acetone, ethyl acetate 14) Color reaction: Positive: Ninhydrin, Greig Lee Back, Sakaguchi reaction Negative: Ehrlich, Burton reaction, potassium permanganate 15) Stability: unstable in acidic and basic aqueous solution, unstable in neutral aqueous solution 16) Substance classification: amphoteric substance (dihydrochloric acid) (Salt is a neutral substance) (ii) TAN-547D ・ monohydrochloride: 1) Appearance: white powder 2) Specific rotation: ▲ [α] ²⁵ _D ▼ + 53.5 ° ± 10 ° (c = 0.5)
1, water) 3) Measured molecular weight: SIMS method, (M + H) ⁺ 645 4) Estimated molecular formula: C ₂₅ H ₄₀ N ₈ O ₁₀ S · HCl · (3H ₂ O) 5) Elemental analysis value (%) : Actual value ^{* 1} Calculated value ^{* 2} C, 40.30 ± 2.0 C, 40.84 H, 6.44 ± 1.0 H, 6.44 N, 15.34 ± 1.5 N, 15.24 O, 28.29 S, 4.39 ± 1.0 S, 4.36 Cl, 4.66 ± 1.5 Cl , 4.82 * 1, Samples were dried on phosphorus pentoxide at room temperature for 15 hours under reduced pressure. * Calculated as containing a few moles of attached water.

6) Ultraviolet absorption (UV) spectrum: See FIG.

7) Circular dichroism (CD) spectrum: (-:-Kotton effect; +: + Kotton effect) 8) Infrared absorption (IR) spectrum: The main wave numbers (cm ^-1 ) in potassium bromide are as follows. See FIG.

3400,3250,3075,2950, 1765,1735,1665,1540, 1450,1400,1350,1270, 1160,1115,1065,1030,980,750,540.

9) The magnetic resonance ( ¹³ C-NMR) spectrum: 100 MHz in heavy water
The signal at is found at least below (δppm).

179.52 (s), 177.43 (s), 176.05 (s), 173.72
(S), 171.61 (s), 167.89 (s), 159.57 (s), 134.40
(S), 118.99 (s), 72.64 (d), 67.23 (t), 61.99
(D), 60.13 (d), 57.32 (d), 56.28 (d), 51.97
(D), 43.53 (t), 41.18 (t), 37.58 (t), 32.75
(T), 28.94 (t), 28.43 (t), 27.46 (t), 23.85
(T), 19.70 (q) 10) Amino acid analytical value: in constant boiling hydrochloric acid (5.5N-HCl), 110
Sample hydrolyzed at 15 ℃ for 15 hours.

Alanine: about 1 mol α-amino-adipic acid; about 1 mol 11) Thin layer chromatography (TLC): spot film, cellulose (manufactured by Tokyo Chemical Industry Co., Ltd.).

Solvent system, acetonitrile: 3% ammonium sulfate (1: 1),
Rf = 0.50 12) High Performance Liquid Chromatography (HPLC): Carrier, YMC Pack A312 (manufactured by Yamamura Chemical Laboratory), mobile phase, 5% methanol
/0.01 M phosphate buffer (pH 3.0), 2 ml / (min.) Rt = 4.2 (min) The following physical properties are the same for both D, E and F components (hydrochloride).

13) Solubility: Easy solubility: water, water-containing acetone, water-containing alcohol Poor solubility: dimethylsulfoxide, methanol, acetone, ethyl acetate 14) Color reaction: Positive: Ninhydrin, Greig Leeback, Sakaguchi reaction Negative: Ehrlich, Burton reaction, potassium permanganate 15) Stability: unstable in acidic and neutral aqueous solution, unstable in basic aqueous solution 16) Substance classification: amphoteric substance (dihydrochloride is neutral substance) (iii ) TAN-547E ・ Dihydrochloride 1) Appearance: White powder 2) Specific optical rotation: ▲ [α] ²⁵ _D ▼ + 31.1 ° ± 10 ° (c = 0.5
1, water) 3) Measured molecular weight: SIMS method, (M + H) ⁺ 716 4) Estimated molecular formula: C ₂₈ H ₄₅ N ₉ O ₁₁ S ・ 2HCl ・ (3H ₂ O) 5) Elemental analysis value (%) : Measured value ^{* 1} Calculated value ^{* 2} C, 39.86 ± 2.0 C, 39.90 H, 6.28 ± 1.0 H, 6.34 N, 14.64 ± 1.5 N, 14.96 O, 26.58 S, 3.79 ± 1.0 S, 3.80 Cl, 7.83 ± 1.5 Cl , 8.41 * 1, * 2, Same condition as D 6) UV spectrum: See Fig. 5.

7) CD spectrum: 8) IR spectrum: Major wavenumber (cm ^-1 ) in potassium bromide
Is as follows. See FIG.

3375, 3260, 3220, 3075, 2950, 1770, 1735, 1660, 1540, 1455, 1400, 1345, 1250, 1160, 1115, 1065, 1035, 980, 880, 815, 540 9) ¹³ C-NMR spectrum: heavy The signal at 100MHz in water is at least found below (δppm).

179.48 (s), 177.82 (s), 177.36 (s), 176.12
(S), 173.46 (s), 171.49 (s), 167.83 (s), 159.54
(S), 134.37 (s), 118.99 (s), 72.84 (d), 67.19
(T), 61.95 (d), 60.10 (d), 57.31 (d), 55.96
(D), 53.04 (d), 51.63 (d), 43.53 (t), 41.47
(T), 37.53 (t), 32.72 (t), 29.14 (t), 28.38
(T), 27.43 (t), 23.81 (t), 19.44 (q) (2) 10) Amino acid analysis value: (same condition as D) Alanine: about 2 mol α-amino-adipic acid: about 1 mol 11 ) TLC: (same condition as D) Rf = 0.54 12) HPLC: (same condition as D) Rt = 4.9 (min) (iv) TAN-547F / dihydrochloride: 1) Appearance: white powder 2) Specific rotation : ▲ [α] ²⁵ _D ▼ + 5.8 ° ± 3 ° (c = 0.4
9, water) 3) Measured molecular weight: SIMS method, (M + H) ⁺ 787 4) Estimated molecular formula: C ₃₁ H ₅₀ N ₁₀ O ₁₂ S ・ 2HCl ・ (3H ₂ O) 5) Elemental analysis value (%) : Actual value ^{* 1} Calculated value ^{* 2} C, 40.27 ± 2.0 C, 40.74 H, 6.52 ± 1.0 H, 6.40 N, 14.52 ± 1.5 N, 15.33 O, 26.26 S, 2.92 ± 1.0 S, 3.51 Cl, 7.12 ± 1.5 Cl , 7.76 * 1, * 2, Same condition as D 6) UV spectrum: See Fig. 7.

7) CD spectrum: 8) IR spectrum: Major wavenumber (cm ^-1 ) in potassium bromide
Is as follows. See FIG.

3360,3250,3070,3000, 2950,1770,1735,1660, 1535,1455,1395,1345, 1240,1160,1115,1065, 1030, 980, 960, 870, 510 9) ¹³ C-NMR spectrum: heavy The signal at 100MHz in water is at least found below (δppm).

179.39 (s), 177.87 (s), 177.26 (s), 176.47
(S), 176.15 (s), 173.45 (s), 170.68 (s), 168.00
(S), 159.65 (s), 133.51 (s), 121.03 (s), 72.90
(D), 67.15 (t), 62.04 (d), 60.21 (d), 56.84
(D), 55.91 (d), 53.02 (d), 52.44 (d), 51.77
(D), 43.64 (t), 41.54 (t), 37.47 (t), 32.54
(T), 29.30 (t), 28.56 (t), 27.41 (t), 23.77
(T), 19.55 (q), 19.44 (q), 19.37 (q) 10) Amino acid analysis value: (same condition as D) Alanine: about 3 mol α-amino-adipic acid: about 1 mol 11) TLC: ( Same condition as D) Rf = 0.58 12) HPLC: (Same condition as D) Rt = 6.5 (min) Next, the biological properties of TAN-547 will be described. TAN-547
The first antibacterial spectrum of (hydrochloride) against various microorganisms is
As shown in the table.

The therapeutic effects of TAN-547C on mouse infections are shown in Table 2.

Table 3 shows the stability of TAN-547C against β-lactames. Compared to the control, TAN-547C (dihydrochloride) is extremely β
-Stable against lactamase.

In Table 3 above, Escherichia coli TN71
Stability of β-lactamase produced by 3 and β-lactamase produced by Klebsiella oxytoca TN1719 was represented by the stability of PCG against them.
In addition, β-lactamase produced by Serratia marcescens TN81 and β produced by Proteus vulgaris GN 4413
-Stability to lactamase was expressed with the stability of CER to them as 100.

The antibiotic TAN-547C dihydrochloride was considered to be of low toxicity because no death occurred even after subcutaneous administration of 1 g / kg to the mice.

As evidenced by these data, the antibiotic TAN-547
Is an antibiotic that exhibits antibacterial properties against Gram-positive and Gram-negative bacteria and has low toxicity to mammals.
In addition, TAN-547C is stable against β-lactamase producing strains. Therefore, TAN-547 is a mammal (eg mouse,
It can be used to treat bacterial infections in rats, rabbits, dogs, and humans).

To use TAN-547 as a therapeutic agent for bacterial infections, for example, TAN-547 is parenterally administered subcutaneously or intramuscularly to the above mammal in an amount of about 1 to 50 mg / kg / day, preferably about 5 To 20 mg / kg / day. As an oral agent, the antibiotic TAN-547 is used as a capsule, and TAN-547 is about 1 to 100 mg / kg / day, preferably about 5 to 50 mg / day.
Administer kg / day.

In addition, TAN-547 can be used as a germicide.
For example, as a liquid agent in which TAN-547 is dissolved in distilled water at a concentration of about 0.01 to 0.1 W / V%, or an ointment containing about 0.2 to 20 mg, preferably about 1 to 10 mg of TAN-547 per 1 g of the above mammals. By applying to the hands, feet, eyes, ears, etc., it can be used for sterilization and disinfection of these parts.

The antibiotic TAN-547 is also a very promising compound as a synthetic intermediate for new drugs.

Based on the properties described above, TAN-547 is considered to be a CFM antibiotic. However, the molecular formula is C ₃₂ H ₅₁ N ₁₁ O ₁₃ S,
Since no CEF antibiotics are known, C ₂₅ H ₄₀ N ₈ O ₁₀ S, C ₂₈ H ₄₅ N ₉ O ₁₁ S or C ₃₁ H ₅₀ N ₁₀ O ₁₂ S, TAN-547
Is considered to be a novel compound.

Next, the present invention will be described more specifically with reference to examples.
The percentages are weight / unless otherwise specified.
Shows% by volume.

Example 1 A strain of Rhizotactor lactamgenus YK-90 (IFO 14288, FERM BP-575) grown on a nutrient agar slope was treated with glucose 2%, soluble starch 3%, raw soybean flour 1%, corn stee. Three 200 ml Erlenmeyer flasks containing 40 ml of a medium containing 0.5% of precipitated calcium carbonate in an aqueous solution (pH 7.0) containing 1% liquor, 0.5% polypeptone (manufactured by Daigo Nutrition Chemical Co., Ltd.) and 0.3% sodium chloride. Inoculate into 2
Incubate with shaking at 4 ° C for 48 hours and use this culture as the inoculum.

Next, a medium containing 0.5% precipitated calcium carbonate in an aqueous solution (pH 6.5) containing 3% dextrin, 1.5% raw soybean flour, 1.5% corn gluten meal, 0.2% polypeptone, and 0.1% sodium thiosulfate. 4000 ml of each was dispensed into a 200 ml Erlenmeyer flask by 40 ml, and 1 ml of inoculum was inoculated into the sterilized one at 120 ° C. for 20 minutes, and cultured at 24 ° C. for 200 hours with shaking at 200 rpm.

The culture solution (20 l) obtained above was adjusted to pH 3.5 with 7% oxalic acid, and Hyflo Supercell (manufactured by Jyon's Malvin Product Co., USA) was added, and the solution (16 l) was obtained. . The solution was adjusted to pH 6.8 and then passed through a column packed with activated carbon (1). After washing the column with water (3 l), the antibiotic TAN-547 was eluted with 8% isobutanol-N / 200 hydrochloric acid (8 l). After concentrating the eluate to 1.8 liters, the concentrate was passed through a column of Amberlite CG-50 (H ⁺ , 1.4 liter) (Rohm and Haas Company, USA). After washing the column with water (4.5 l), elution fractionation was performed with N / 100 hydrochloric acid (9 l). After collecting effective fractions and concentrating them and adjusting the concentrate to pH 7.3, Diaion H
It was passed through a column packed with P-20 (50-100 mesh, 0.5 l) (manufactured by Mitsubishi Kasei Co., Ltd.). The column was washed with 0.01 M phosphate buffer (pH 7.3, 1.5 l) and then eluted with 0.01 M phosphate buffer (pH 3.5, 5 l). After collecting the effective fractions and adjusting the eluate to pH 7.2, pass through a column packed with activated carbon (100 ml) and wash the column with water (300 ml), then 8%
Elution with isobutanol-N / 200 hydrochloric acid (600 ml). After concentrating the eluate, concentrate the CM-Sephadex C25 (Na ⁺ type, 2
(00 ml) (Falmatia Huaine Chemical Co., Sweden) was passed through the column, and elution fractionation was carried out with 0.02 M saline solution (6 l). Each fraction was analyzed by liquid chromatography, and the fractions containing TAN-547A, B, and C as the main components were collected.

After adjusting the main component fraction of TAN-547A to pH 7.2, activated carbon (10 ml)
After passing through a column packed with water, the column was washed with water (30 ml) and then eluted with 8% isobutanol N / 200 hydrochloric acid (70 ml). The eluate was concentrated and freeze-dried to obtain a crude powder of TAN-547A dihydrochloride (61 mg).

The same operation was performed for the main component fractions of TAN-547B and C.
A crude powder of 7B dihydrochloride (144 mg) and a crude powder of TAN-547C dihydrochloride (226 mg) were obtained.

YMC-GE using TAN-547A dihydrochloride crude powder (61 mg) as a carrier
It was subjected to preparative high performance liquid chromatography using L ODS I-15 (manufactured by Yamamura Chemical Laboratory), and 0.02M phosphate buffer solution (p
H3.0) for elution fractionation. Each fraction was subjected to liquid chromatography analysis, and a portion showing a single peak was collected. The effective fraction was adjusted to pH 7.5 with 1N NaOH, readjusted to pH 3.0 with 1N HCl, passed through a column packed with activated carbon (5 ml), washed with water (25 ml), and then washed with 8% It was eluted with butanol water (25 ml). After concentrating the eluate, freeze-dry it and TA
A white powder of N-547A dihydrochloride (40 mg) was obtained.

Similarly, the crude powder of TAN-547B, C dihydrochloride was subjected to preparative high performance liquid chromatography to obtain a white powder of TAN-547B dihydrochloride (96 mg) and a white powder of TAN-547C dihydrochloride (112 mg). )was gotten.

Example 2 Resobacter lactamgenus YK-90 (IFO 14288, FERM BP-575) grown on a nutrient agar slope was added to glucose 2.
%, Soluble starch 3%, raw soybean powder 1%, corn stay liquor 1%, polypeptone 0.5%, salt 0.3% in an aqueous solution (pH 7.0) containing calcium carbonate 0.5%
Two 2 liter Sakaguchi flasks containing 500 ml of the medium to which was added were inoculated and cultured at 24 ° C. for 48 hours with reciprocal shaking. The whole amount of this culture solution was inoculated into a tank having a capacity of 200 liters containing 120 liters of a medium containing 0.05% of the antifoaming agent ACTCOL (manufactured by Takeda Pharmaceutical Co., Ltd.) in the above-mentioned medium, and an aeration rate of 120 liters / minute at 150 rpm at 24 ° C It was cultured under the condition of minute for 48 hours. 3% dextrin, 1.5% raw soybean flour, 1.5 corn gluten meal
%, Polypeptone 0.2%, sodium thiosulfate 0.1% in an aqueous solution (pH 6.5) containing 0.5% of precipitated calcium carbonate,
600 liters containing 4000 l of culture medium supplemented with 0.05% Actol
Inoculate to a tank of 0l, aeration rate of 4000l / min at 24 ℃, 120rpm /
It was cultured for 66 hours under the condition of minute.

The culture broth (3900 l) obtained above was adjusted to pH 6.1 with 2N hydrochloric acid, Hyflo Supercell was added, and the mixture was washed with water to obtain a liquid (4370 l). The solution was adjusted to pH 7.0 and then passed through a column packed with Dowex-50W (Na ⁺ type, 50-100 mesh, 120 liters). The column was washed with water (360l) and then eluted with 2M saline (1800l). The eluate was passed through a column of activated carbon (60 l), washed with water (180 l), and then eluted with 8% isobutanol-N / 200 hydrochloric acid (420 l). Concentrate the eluate to 40 liters, adjust the concentrate to pH 7.3, and then use Diaion HP-20 (40
It was passed through a column packed with l). The column was washed with 0.01 M phosphate buffer (pH 7.3, 80 l) and then eluted with 0.01 M phosphate buffer (pH 3.5, 400 l).

Pass the eluate through a column of activated carbon (10 l) and water (30 l)
After washing with water, it was eluted with 8% isobutanol-N / 200 hydrochloric acid (70 l). The eluate was concentrated to 2 liters, the concentrate was adjusted to pH 7.3, and then passed through a column packed with Diaion HP-20 (50-100 mesh, 4 liters). 0.01M phosphate buffer (pH7.3,12
After washing with l), it was eluted and fractionated with 0.01 M phosphate buffer (pH 3.5, 40 l). Each fraction was subjected to analysis by liquid chromatography and separated into two groups, a fraction containing TAN-547A, B, C as a main component and a fraction containing TAN-547D, E, F as a main component. TAN-547D, E, F main component fractions were collected and passed through a column of activated carbon (300 ml),
After washing with water (900 ml), it was eluted with 8% isobutanol-N / 200 hydrochloric acid (2100 ml). After concentrating the eluate, CM-
It was passed through a column packed with Sephadex C25 (Na ⁺ type, 300 ml) and eluted and fractionated with 0.02 M saline (12 l). Each fraction was submitted to liquid chromatographic analysis, and TAN-547D, E, F
Fractions containing each as the main component were collected.

The TAN-547F main component fractions were collected, passed through a column of activated carbon (50 ml), washed with water (150 ml), and then eluted with 8% isobutanol-N / 200 hydrochloric acid (350 ml). After concentrating the eluate,
Lyophilization gave a crude powder of TAN-547F (1.0 g). TA
The same operation was performed for the main component fractions of N-547D and E.
Coarse powder (0.3g) and TAN-547E coarse powder (0.6g) were obtained.

TAN-547F coarse powder (1.0 g) as a carrier, YMC-GEL ODS
It was subjected to preparative high performance liquid chromatography using 30/60 (manufactured by Yamamura Chemical Laboratory), and eluted and fractionated with 2% methanol-0.02M phosphate buffer (pH 3.0). Each fraction was subjected to analysis by liquid chromatography, and the fraction containing TAN-547F as a main component was collected, adjusted to pH 7.1, and then passed through a column of activated carbon (20 ml). After washing the column with water (60 ml), the column was eluted with 8% isobutanol-N / 200 hydrochloric acid (140 ml), and the eluate was concentrated. The concentrate was used as a carrier and subjected to preparative high performance liquid chromatography using TSK-GEL, LS-410 (manufactured by Toyo Soda Co., Ltd.), and 1% methanol-0.01 M phosphate buffer (pH 3.
Elution fractionation was carried out in 0). Each fraction was subjected to liquid chromatography analysis, and fractions showing a single peak were collected. The effective fraction was adjusted to pH 7.0 with 1N NaOH, readjusted to pH 3.0 with 1N HCl, passed through a column packed with activated carbon (10 ml), washed with water (50 ml), and then washed with 8% isopropanol. It was eluted with butanol water (70 ml). After concentrating the eluate, freeze-dry it and TA
A white powder of N-547F dihydrochloride (69 mg) was obtained. TAN-54
Similarly, the crude powder of 7D and E was subjected to preparative high performance liquid chromatography to obtain white powder of TAN-547D monohydrochloride (30 mg), TAN.
A white powder of -547E dihydrochloride (63 mg) was obtained.

[Brief description of drawings]

Figures 1, 3, 5, and 7 show the antibiotic TAN-
The UV absorption spectra of 547C ・ dihydrochloride, D ・ monohydrochloride, E ・ dihydrochloride and F ・ dihydrochloride are shown respectively,
Figures 4, 6 and 8 show the antibiotic TAN-547C.
The infrared absorption spectra of dihydrochloride, D. monohydrochloride, E. dihydrochloride, and F. dihydrochloride are shown respectively.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI technical display location C12P 35/00 Z 8412-4B 35/08 8412-4B // A61K 31/545 ADZ 9454-4C 37 / 02 8314-4C (C12P 21/02 C12R 1:01) (C12P 35/00 C12R 1:01) (C12P 35/08 C12R 1:01)

Claims (2)

[Claims] 1. The following formula [Wherein R ¹ represents formylamino, R ² represents alanyl-alanyl, or R ¹ represents hydrogen, and R ² represents hydrogen, alanyl, or alanyl-alanyl], a cephem antibiotic TAN -547 or salt thereof. 2. The following formula belonging to the genus Resobacter [Wherein R ¹ represents formylamino, R ² represents alanyl-alanyl, or R ¹ represents hydrogen and R ² represents hydrogen, alanyl or alanyl-alanyl]
A method for producing the antibiotic TAN-547 or a salt thereof, which comprises culturing a microorganism having the ability to produce TAN-547 in a medium, allowing the antibiotic to be produced and accumulated in the culture, and collecting the antibiotic.