JPH0477754B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD The present invention relates to a 7-formylamino-cephem compound that is useful as a therapeutic agent for bacterial infections and as a raw material compound for synthesizing various cefem compounds, and a method for producing the same. Prior Art Cephalosporin C homologues produced by fungi and cephamycin C homologues produced by actinomycetes are
It is known that it is converted into the corresponding 7-amino-cephem compound or 77-methoxy-7-amino-cepheme compound, respectively, by D-amino acid oxidase and cephalosporin acylase of microorganisms. However, 7-formylamino-
There is no known method for producing 7-formylamino-7-amino-cephem compounds from cefem compounds using microorganisms. Problems to be Solved by the Invention If 7-formylamino-deacetylcephalosporin C can be converted into a 7-formylamino-7-amino-cephalosporin compound, various compounds can be produced using the target compound as a raw material compound. This means that a 7-formylamino-cephem compound can be produced. Means for Solving the Problems The present inventors have discovered that 7-formylamino-deacetylcephalosporin C is a 7-formylamino-7
After conducting various studies on methods for converting 7-formylamino-deacetylcephalosporin C into -amino-cephalosporin compounds using microorganisms, we found that 7-formylamino-deacetylcephalosporin C was converted into 7-formylamino-7-
The compound obtained here is a new compound, and furthermore, this compound can be converted to a 7-formylamino-7-amino-cephem compound using Pseudomonas bacteria. It was discovered that the compound thus obtained was a new compound, and as a result of further research based on these findings, the present invention was completed. The present invention provides (1) general formula [Wherein, R is hydrogen or HOOC-( _CH2 ) ₃ -CO
- indicates a group. ] or its salt,
(2) Equation The HOOC-(CH ₂ ) ₃ -CO-NH- group at the 7-position of the compound or its salt is replaced with NH ₂ -
A formula characterized by contacting a culture of a microorganism belonging to the genus Pseudomonas or a processed product thereof having the ability to convert into A method for producing a compound represented by or a salt thereof, and formula (3) In the compound represented by or its salt, the 7th position of the compound is

[Formula] A formula characterized by contacting a culture of a microorganism belonging to the genus Trigonopsis or a treated product thereof, which has the ability to convert a group into a HOOC-(CH ₂ ) ₃ -CO-NH- group. This is a method for producing a compound represented by or a salt thereof. The microorganisms used in the step of converting the compound () of the present invention into compound (-2) include:
Any microorganism that belongs to the genus Trigonobsis and has the ability to convert compound () to compound (-2) can be used. Examples of the microorganism include Trigonopsis variabilis, and specific examples thereof include Trigonobsis variabilis IFO 0670 and IFO 0755 strains. The microorganisms with the above IFO numbers have been deposited with the Institute for Fermentation (IFO), and are included in the Institute For Fermentation, Osaka List of Cultures, 1984, 7th edition published by the Institute.
Cultures 1984 seventh edition). The IFO deposit dates are shown below.

[Table] The mycological properties of Trigonobsis variabilis are listed in Yeasts; A Taxonomic Study, 1970, No. 2.
Edition, pp. 1353-1357, editor J. Lodder.
Same as that published by North Holland Prb Corporation. The compound (-2) of the present invention is converted into the compound (-1)
The microorganism used in the process of converting the compound (-2) belongs to the genus Pseudomonas.
Any microorganism can be used as long as it has the ability to convert into compound (-1). A specific example of the microorganism is Pseudomonas sp. UK-2221 strain isolated from the soil of Fukuchiyama City, Hyogo Prefecture. The mycological properties of the UK-2221 strain are as follows. (a) Morphology When observed after culturing on a broth agar slant for 5 days at 24°C, the cells were rod-shaped with a diameter of 0.5 to 1.0 μm and a length of 0.8 to 2.0 μm, and motility by flagella was observed. The flagellum is extremely simple. Does not form spores. In addition, Gram staining is negative and does not show acid-fast properties. (b) Growth status on various media, cultured at 24°C and observed over 1 to 14 days. Broth agar plate culture: Colonies are colorless, round,
The surface is convex and circular, and the periphery is glossy. Does not produce diffusible dyes. Broth agar slant culture: Medium growth, shiny filamentous growth, colorless, and does not produce diffusible pigment. Meat juice liquid culture: Grows weakly in a turbid manner, and precipitates are also observed, but no bacterial membrane is formed. Meat juice gelatin puncture culture: Shows weak growth. Does not liquefy. Litmus milk: The reducing ability of litmus is not recognized. No peptonization activity was observed either. (c) Physiological properties Nitrate reduction: - Denitrification reaction: - MR (methyl red) test: - VP (Vouges-Proskauer) test:
- Production of indole: - Production of hydrogen sulfide (lead acetate paper): - Hydrolysis of starch: - Utilization of citric acid (Kozel, Christensen and Simmons media): + (- in Simmons) Utilization of inorganic nitrogen sources Potassium nitrate :+ Ammonium sulfate:+ Pigment production (King A, B and Mannito yeast extract agar media): Diffusible pigment production was not observed. King A medium: 10g glycerin, 20g peptone
g, magnesium chloride 1.4g, ammonium sulfate 10g, agar 15g, distilled water 1000ml, PH7.2 King B medium: glycerin 10g, peptone 20
g, potassium monohydrogen phosphate 1.5g, magnesium sulfate 1.5g, agar 15g, PH7.2 Urease: + Oxidase: + (weak) Catalase: + Growth range PH: Grows between PH4.3 and 7.0, but the optimum pH is is 5.0
~6.0. Temperature: Grows at 14-32℃, but the optimum temperature is 18℃
~26℃ Attitude towards oxygen: absolutely aerobic. O-F (oxidative-fermentative) test (Hugh Leifson method): Non-degradable. Production of acids and gases from sugars:

[Table] DNA GC (guanine + cytosine) content:
UK-2221 strain with mycological properties of 68.3±1.5% or more,
Bergey's Manual of Determinative Bacteriology, 8th Edition and International Journal of Systematic Bacteriology, Vol. 30, pp. 225-420, 1980)
and the magazine's validation list.
When compared with the species listed in the list), the UK-2221 strain is
It is a Gram-negative bacillus, does not form spores, is motile by monopolar hairs, is absolutely aerobic, is catalase positive, has a non-degradable O-F test, and has DNA GC (guanine + It is thought to belong to the genus Pseudomonas, as the cytosine content is 68.3 ± 1.5%.
This bacterium was designated as Pseudomonas sp. UK-2221. The above Pseudomonas sp. UK-2221 strain was deposited with the Fermentation Research Institute (IFO) on August 31, 1984 under accession number IFO14366, and this microorganism was On September 7, it was deposited with the Microbial Research Institute (FRI), Agency of Industrial Science and Technology, Ministry of International Trade and Industry, under the accession number FERM P-7836, and the deposit was changed to a deposit under the Budapest Treaty, and the deposit was transferred under the accession number FERM BP-637. It is stored at the same research institute (FRI). Trigonobsis bacteria used in the method of the present invention,
Bacteria of the genus Pseudomonas are generally susceptible to changes in their properties, and can be easily mutated by artificial mutagenesis methods using, for example, ultraviolet rays, X-rays, or chemicals (e.g., nitrosoguanidine, ethyl methanesulfonic acid). Any mutant strain that has the conversion ability of the method of the present invention can be used in the method of the present invention. The "culture" used in the method of the present invention refers to one obtained by culturing the microorganisms used in the method of the present invention. The medium used for culturing the above-mentioned microorganisms may be either liquid or solid as long as it contains a nutrient source that can be used by the microorganism, but it is preferable to use a liquid medium when a large amount is to be obtained. The medium contains a carbon source that can be assimilated by the microorganism,
Digestible nitrogen sources, inorganic substances, micronutrients, etc. are appropriately blended. Examples of carbon sources include glucose, maltose, lactose, blackstrap molasses, oils and fats (e.g. soybean oil, olive oil, etc.), organic acids (e.g. citric acid,
Succinic acid, gluconic acid, etc.) that can be assimilated by bacteria are used as appropriate. Examples of nitrogen sources include organic and inorganic nitrogen compounds such as soy flour, cotton flour, corn staple liquor, dried yeast, yeast extract, meat extract, peptone, urea, ammonium sulfate, ammonium nitrate, ammonium chloride, and ammonium phosphate. compounds are available. In addition, as the inorganic salt, for example, inorganic salts necessary for culturing microorganisms, such as sodium chloride, potassium chloride, calcium carbonate, magnesium sulfate, monopotassium phosphate, and disodium phosphate, are used alone or in appropriate combinations. . In addition, the medium used for culturing Trigonopsis bacteria contains D-
For the purpose of inducing the activity of amino acid oxidase, D-
D-type amino acids such as alanine and D-methionine or D-α-aminoadipic acid may be added to the medium. The culturing method may be static culture, shaking culture, or aeration/agitation culture. For large-scale treatment, it is desirable to use so-called deep aeration agitation culture. It goes without saying that the conditions for culturing Trigonobcis bacteria vary depending on the condition and composition of the medium, the type of strain, the means of cultivation, etc., but they are usually around 20°C.
At a temperature of -45°C, more preferably about 24-37°C, the pH of the medium is adjusted to about 6-9, more preferably about 6-8. Culture time is approximately 24 to 144
time, more preferably about 24 to 120 hours. It goes without saying that the conditions for culturing Pseudomonas bacteria vary depending on the state and composition of the medium, the type of strain, the means of cultivation, etc., but they are usually around 20°C.
At a temperature of -45°C, more preferably about 24-37°C, the pH of the medium is adjusted to about 6-9, more preferably about 6-8. Culture time is approximately 24~168
It's time. The "processed product" used in the present invention refers to the culture obtained as described above, subjected to physicochemical treatments such as filtration, centrifugation, ultrasonication, French press treatment, osmotic shock method, freezing, thawing, alumina polishing, etc. Break,
It refers to bacterial cells obtained by lytic enzyme treatment, surfactant or organic solvent treatment, or crushed bacterial cells containing enzymes involved in the reaction of the method of the present invention. Furthermore, the enzyme obtained by purification by a known method or the bacterial cells or the enzyme immobilized by a known method can also be used. The concentration of compound () in the reaction solution in the reaction carried out by bringing the compound () into contact with the culture of the above-mentioned microorganism or its treated product is approximately 0.5 to 20 mg/ml,
More preferably, it is about 5 to 10 mg/ml. The amount of microorganisms is about 0.1 to 1 g/ml, more preferably about 0.1 to 0.3 g/ml in terms of wet bacterial weight. When using a treated product, the concentration of the treated product is calculated from the weight of the wet bacterial cells obtained. The pH of the reaction solution is approximately 6 to 10.
, more preferably about 7.5 to 8.5.
The reaction temperature is about 15-40℃, more preferably about 15-40℃
It is 37℃. The reaction time is about 4 to 48 hours, more preferably about 8 to 16 hours. The reaction may be carried out under static conditions or under conditions of shaking, aeration or stirring, preferably shaking, aeration or stirring. In order to obtain the target compound (-2) in good yield, the objective can be achieved by adding a catalase inhibitor to the reaction solution. Examples of catalase inhibitors include inorganic azides such as sodium azide, ascorbic acid, 3-amino-1,
2,3-triazole and the like are used. The progress and completion of the reaction is confirmed by checking the disappearance of antibacterial properties against E. coli, thin layer chromatography, or high performance liquid chromatography (HPLC). After the reaction is complete, the reaction solution is centrifuged and
The bacterial cells and the like are removed, and the supernatant is separated and purified to obtain the target compound (-2). Of course, the method of the present invention may be carried out by adding the compound () to the medium when culturing the microorganism in the medium. In this case, the amount of compound () added is about 1 to 20 mg/ml, more preferably about 2 to 10 mg/ml. In addition, culture temperature,
The pH of the medium and the culture time are the same as those in the culture described above. In the reaction step, the compound ()

[Formula] The group is once converted to the HOOC-CO-(CH ₂ ) ₃ -CO-NH- group, but the group continues to be HOOC-(CH ₂ ) ₃ -CO-
It is converted into an NH- group to produce compound (-2). The concentration of compound (-2) in the reaction solution in the reaction carried out by bringing compound (-2) into contact with the culture of the above-mentioned microorganism or its treated product is about 2 to 20
mg/ml, more preferably about 4 to 10 mg/ml. The amount of microorganisms is approximately 0.1 to 1 g/wet weight of microorganisms.
ml, more preferably about 0.1 to 0.3 g/ml.
When using a treated product, the concentration of the treated product is calculated from the weight of the wet bacterial cells obtained. The pH of the reaction solution is
It is adjusted to about 6 to 10, more preferably about 6.5 to 7.5. The reaction temperature is about 15-40°C, more preferably about 15-37°C. Reaction time is approximately 4-66 hours,
More preferably about 16 to 54 hours. The reaction may be carried out under static conditions or under conditions of shaking, aeration or stirring, preferably shaking, aeration or stirring. The reaction involves adding compound (-2) to the medium,
The microorganism used in the method of the present invention may be cultured in the medium. The compound in this case (
The amount of -2) added is about 2 to 20 mg/ml, more preferably about 4 to 10 mg/ml. In addition, the culture temperature, pH of the medium, and culture time are the same as those in the above-mentioned culture. To isolate compound (-2) or compound (-1) from the reaction solution, appropriate combinations of means generally used for isolating water-soluble acidic substances are used. That is, adsorption chromatography using various carriers, ion exchange chromatography using ion exchangers, molecular sieve chromatography, reverse phase liquid chromatography, vacuum concentration, freeze drying, and other means may be used alone or in combination in any order. used repeatedly or repeatedly. As the carrier, activated carbon, adsorbent resin, anion exchange resin (anion type), powdered cellulose, etc. are used, or carriers having properties such as molecular sieves are also used. Elution solvents vary depending on the type of carrier, but include water-soluble organic solvents, aqueous solutions such as acetone, methanol, ethanol, propanol, butanol, isopropanol, and isobutanol, or aqueous solutions of acids, alkalis, buffers, or inorganic or organic salts. etc. are used. To explain the example in more detail, after the reaction is completed, the reaction solution is adjusted to pH around 6 to 7, and since the substance is an acidic substance, the reaction solution is mixed with anion exchange resin [Amberlite IRA-68, 402, 410]. (manufactured by Rohm & Haas, USA), Dowex-1 (manufactured by Dow & Chemical, USA), Diaion SA-21C (manufactured by Mitsubishi Chemical Corporation), etc.
It is passed through a Cl ^- or ACO ^- type carrier to adsorb biological substances. The effective substance is eluted using saline or a buffer solution for the elution of the adsorbed substance. To desalt the eluate, make the eluate neutral or slightly acidic, perform activated carbon chromatography (manufactured by Takeda Pharmaceutical Co., Ltd.), and elute with hydrous alcohol. The eluate containing the active substance is then concentrated under reduced pressure at low temperature, and the concentrated solution is passed through a Cl ^- type resin such as DEAE or QAE Sephadex (Pharmacia, Sweden) to adsorb the antibiotic. The adsorbed antibiotic is eluted and fractionated with dilute saline. Effective fractions are detected by high performance liquid chromatography, collected and subjected to activated carbon chromatography for desalting. Concentrate the eluate and lyophilize the concentrate. Add acetone to the dry powder and collect the resulting precipitate. In addition, the above compound () and compound ()
may be formed into a salt by conventional means, and when used as a medicine such as a therapeutic agent for bacterial infections, the salt may be a pharmacologically acceptable salt such as a lithium salt, a sodium salt, Examples include barium salts, calcium salts, magnesium salts, etc. When used as synthetic intermediates, in addition to the above salts, for example, ammonium salts, methylamine salts, diethylamine salts, trimethylamine salts, tetrabutylammonium salts, pyridine salts, etc. can be mentioned. The salt of the compound thus obtained can also be converted into other salts using means known per se. For example, the obtained salt is dissolved in water, and the pH is adjusted to about 2 to 3 while cooling with dilute hydrochloric acid. The aqueous solution is immediately adjusted to a pH of about 7 to 8, containing a dilute alkaline aqueous solution such as sodium hydroxide, calcium hydroxide, methylamine, tetrabutylammonium hydroxide, etc., and the aqueous solution is chromatographed on activated carbon. After applying it to a graphite and washing the inorganic or organic salts with water, the antibiotic salts are eluted. The physicochemical properties of the disodium salt of compound (-2) obtained in Example 1 described below are as follows. (1) Appearance: White powder (2) Molecular weight: The molecular ion peak determined by SIMS method is observed at m/z432 (M+H) ⁺ . (3) Elemental analysis value: (40℃, 8 hours drying under reduced pressure) Actual value Calculated value C 38.06 38.19 H 3.81 3.66 N 9.97 9.54 O 30.88 S 7.68 7.28 Na 10.44 (4) Molecular formula: C ₁₄ H ₁₅ N ₃ O ₈ SNa ₂ 0.5H ₂ O (5) Ultraviolet absorption spectrum (in water): Maximum value:
259nm (E1% 1cm=224) (6) Infrared absorption spectrum (KBr): Main wave numbers (cm ^-1 ), Figure 1 3400, 3230, 3010, 1775, 1700, 1690, 1610,
1580, 1410, 1305, 1240, 1150, 1065, 1045,
1000, 850, 800, 710, 515 (7) Nuclear magnetic resonance (PMR) spectrum: 100MHz,
In heavy water, δppm J(H ₂ ) 1.75−2.15 2H, m 2.15−2.55 4H, m 3.38 1H, d, J=18 3.70 1H, d, J=18 4.27 2H, s, 5.39 1H, s, 8.21 1H, s, (8) High performance liquid chromatography: Model 6000A/660/440 manufactured by Waters (USA) Column: YMC-Pack A-312 (manufactured by Yamamura Kagaku Kenkyusho) Mobile phase: 0.01M phosphate buffer (PH6.3) ), 2ml/
min detection: 254 nm Rt=3.1 min. The physicochemical properties of compound (-1) obtained in Example 2 described below are as follows. (1) Appearance: White powder (2) Molecular weight: The molecular ion peak determined by SIMS method is observed at m/z 296 (M+Na) ⁺ . (3) Molecular formula {C ₉ H ₁₁ N ₃ O ₅ S (273) (4) Ultraviolet absorption spectrum (in water): Maximum value;
259nm (E1% 1cm=365) (5) Circular dichroism spectrum (in water): [θ] ^H ₂ ^O ₂₂₉ −
33300±5000 and [θ] ^H ₂ ^O ₂₆₀ +32400±5000 (6) Infrared absorption spectrum: main wave number (cm ^-1 ),
Figure 2 3400, 2980, 1760, 1680, 1600, 1510, 1410,
1380, 1290, 1240, 1180, 1140, 1070, 1040,
1000, 860, 790, 700, 500 (7) Nuclear magnetic resonance (PMR) spectrum: 400MH ₂ ,
In heavy water, δppm J (H ₂ ), 3.43 1H, d, J = 17.6 3.65 1H, d, J = 17.6 4.21 1H, d, J = 12.9 4.25 1H, d, J = 12.9 5.19 1H, s 8.17 1H, s (8) High performance liquid chromatography: manufactured by Hitachi, Ltd.
Model638-50 Column: YMC-PaCk A-312 Mobile phase: 0.01M phosphate buffer (PH6.3), 2ml/
min Detection: 254nm Rt=3.0min The compound (), which is the raw material for the method of the present invention, can be used as an antibiotic by culturing Rhizobacter bacteria, for example.
TAN-5474 (or sometimes simply referred to as TAN-547A) is produced and accumulated, and collected.
This can then be obtained by subjecting it to a hydrolysis reaction. Antibiotic TAN- belonging to the above Rhizobacter genus
Examples of 547A-producing bacteria include Rhizobacter lactamgenus YK-90 strain, which belongs to a new bacterial species collected from soil. The Rhizobacter lactamgenus YK-90 strain was acquired by the Fermentation Research Institute (IFO) on September 14, 1983.
Deposited with accession number IFO 14288. This microorganism was deposited with the Microbial Research Institute (FRI), Agency of Industrial Science and Technology, Ministry of International Trade and Industry on September 19, 1983, under the accession number FERMP-7247, and the deposit was made under the Budapest Treaty. It has been transferred and is now stored at the same research institute (FRI) under the accession number FERM BP-575. The above antibiotic TAN-547A is represented by the following structural formula. Compound () can be produced, for example, by subjecting the antibiotic TAN-547A obtained above to a hydrolysis reaction. The hydrolysis reaction is
This is done by treating TAN-547A under alkaline conditions. For alkalinity, the raw material compound TAN-547A is added to an aqueous solution whose pH is adjusted to about 7 to 11, preferably about 9 to 9.7. Examples of the aqueous solution include buffers commonly used in chemical reactions, such as phosphate, borate, citrate, carbonate, hydrochloride, acetate, sodium hydroxide, glycine, etc. , veronal, borax, ammonium salts, aminomethylpropanediol, and the like, such as aqueous solutions adjusted to the above pH. The above hydrolysis reaction is carried out at a temperature of about 0°C to 80°C.
C., preferably about 20.degree. C. to 40.degree. C., for about 1 hour to 72 hours, preferably about 4 hours to 40 hours. In order to isolate the compound () from the reaction solution, appropriate combinations of means generally used for isolating water-soluble acidic substances are used. That is, adsorption chromatography using various carriers, ion exchange chromatography using ion exchangers, molecular sieve chromatography, reverse phase liquid chromatography, vacuum concentration, freeze drying, and other means may be used alone or in combination in any order. used repeatedly or repeatedly. The compound (-2) obtained in this way is
Compounds useful as therapeutic agents for bacterial infections (-
It is useful as a synthetic intermediate for producing 1). Next, the biological properties of compound (-1) will be described. The antibacterial spectrum of the sodium salt of compound (-1) against various microorganisms is shown in Table 1. As is clear from this table, compound (-1) exhibits antibacterial properties against Gram-negative bacteria.

[Table] In addition, the sodium salt of compound (-1) is stable against various β-lactames. Table 2 shows the stability of compound (-1) and DCP against β-lactamase.

[Table] Also, 1g/1g of compound (-1) sodium salt
Compound (-1) is considered to have low toxicity since no deaths were observed even when the amount of the compound (-1) was subcutaneously administered to mice. As is clear from the above data, the compound (-1) obtained by the present invention exhibits antibacterial activity against Gram-negative bacteria and can be said to be a low-toxicity antibiotic. Therefore, compound (-1) or a salt thereof is suitable for mammals (e.g., mice, cows, horses, dogs,
It can be used to treat bacterial infections in humans), poultry (e.g., chickens, acyl), etc. In order to use compound (-1) as a therapeutic agent for bacterial infections, for example, compound (-1)
is administered parenterally as an injection to the above-mentioned mammal subcutaneously or intramuscularly at a dose of about 5 to 200 mg/Kg/day, preferably about 20 to 100 mg/Kg/eye. In addition, as an oral preparation, compound (-1) is prepared as a capsule, and the amount of compound (-1) is about 20 to 400 mg/
Kg/day, preferably about 40 to 200 mg/Kg/day. Moreover, compound (-1) or its salt can be used as a disinfectant. For example, as a solution prepared by dissolving compound (-1) in distilled water at a concentration of 0.05 to 0.4 W/V%, or as an ointment containing about 1 to 100 mg, preferably about 4 to 50 mg, of compound (-1) per gram. By applying it to the hands, feet, eyes, ears, etc. of the above-mentioned mammals, it can be used to sterilize and disinfect these parts. Compound (-1) is also useful as a synthetic intermediate for the synthesis of semisynthetic cefem compounds useful as therapeutic agents for bacterial infections. As is clear from a comparison of the stability of cephalosporin C and deacetyl cephalosporin C in aqueous solutions, cephalosporin derivatives generally have a
If the position is acetylated, it becomes unstable. Compound (-1) has -CH ₂ OH at the 3-position, and the acetylated corresponding compound (3-position -
It exhibits much more stable properties in aqueous solution than CH ₂ OCOCH ₃ ). This shows that when carrying out a synthetic reaction, compound (-1) is easy to handle and can therefore be advantageously used as a synthetic intermediate. EXAMPLES Next, the present invention will be described in more detail with reference to reference examples and examples. Note that the percentage of the medium indicates weight/volume %. Reference example 1 Rhizobacter lactamgenus YK-90 (IFO 14288, FERM BP) grown on a nutrient agar slope
-575) strain with 2% glucose, soluble
0.5% precipitated calcium carbonate in an aqueous solution (PH7.0) containing 3% starch, 1% raw soybean flour, 1% corn steep liquor, 0.5% polypeptone (manufactured by Daigo Nutritional Chemical Co., Ltd.), and 0.3% salt. The mixture was inoculated into three 200 ml Erlenmeyer flasks containing 40 ml of a medium supplemented with the following, cultured with shaking at 24°C for 48 hours, and the resulting culture was used as a seed culture. Next, dextrin 3%, raw soybean flour 1.5%, corn gluten meal 1.5%, polypeptone 0.2
%, sodium thiosulfate (PH6.5) containing 0.5% precipitated calcium carbonate was added to each 200 ml Erlenmeyer flask with 40 Kl each.
The seeds were sterilized at 120°C for 20 minutes, inoculated with 1 ml of the inoculum, and cultured with shaking at 24°C and 200 revolutions/min for 72 hours. PH the culture solution (20) obtained above with 7% oxalic acid.
After adjusting to 3.5, add Hyflo Super Cell (manufactured by John's Manville Products, USA),
A solution (16) was obtained through filtration. After adjusting the pH of the liquid to 6.8, it was passed through a column filled with activated carbon (1). After washing the column with water (3), apply antibiotics.
TAN-547 was eluted with 8% isobutanol-N/200 hydrochloric acid (8). After concentrating the eluate to 1.8
Concentrate Amberlite CG-50 (H ⁺ type, 1.4)
(manufactured by Rohm and Haas, USA). After washing the column with water (4.5), the column was eluted and fractionated with N/100 hydrochloric acid (9). The effective fraction was collected and concentrated, and the concentrated solution was adjusted to pH 73, and then passed through a column filled with Diaion HP-20 (50-100 mesh, 0.5) (manufactured by Mitsubishi Chemical Industries, Ltd.). After washing the column with 0.01M phosphate buffer (PH7.3, 1.5), wash the column with 0.01M phosphate buffer (PH3.5, 5).
) and fractionated. After collecting the effective fraction and adjusting the eluate to pH 7.2, it was passed through a column packed with activated carbon (100 ml). After washing the column with water (300 ml), 8% isobutanol-N/200 hydrochloric acid (600 ml) was added. )
It was eluted. After concentrating the eluate, the concentrated solution was passed through a column packed with CM-Sephadex C25 (Na ⁺ type, 200 ml) (manufactured by Pharmacia Huain Chemical, Sweden), and 0.02 M-saline solution (6
) and fractionated. Each fraction was subjected to liquid chromatography analysis, and fractions containing TAN-547A as the main component were collected. After adjusting the TAN-547A main component fraction to pH 7.2, it was passed through a column packed with activated carbon (10 ml), washed with water (30 ml), and then washed with 8% isobutanol-N/200 hydrochloric acid (70 ml). It was eluted. The eluate was concentrated and then lyophilized to obtain a crude powder (61 mg) of TAN-547A dihydrochloride. Crude powder (61 mg) of TAN-547A dihydrochloride was subjected to preparative high performance liquid chromatography using YMC-GEL ODS I-15 (manufactured by Yamamura Kagaku Kenkyusho) as a carrier, and 0.02M phosphate buffer solution was applied. (PH3.0) and fractionated. Each fraction was subjected to liquid chromatography analysis, and portions showing a single peak were collected. After adjusting the effective category to PH7.5 with 1N NaOH, PH with 1N HCl
After readjusting to 3.0 and passing through a column packed with activated carbon (5 ml) and washing the column with water (25 ml),
Elution was performed with 8% isobutanol water (25 ml). The eluate was concentrated and lyophilized to obtain a white powder (40 mg) of TAN-547A dihydrochloride. The physicochemical properties of TAN-547A dihydrochloride obtained above are shown below. TAN-547A dihydrochloride: 1 Appearance: White powder 2 Specific rotation: [α] ²⁵ _D +71.8°±20° (c=0.50,
(in water) 3 Molecular weight measurement: SIMS method, (M+H) ⁺ 688 4 Estimated molecular formula: C ₂₆ H ₄₁ N ₉ O ₁₁ S・2HCl・
(3H ₂ O) 5 Elemental analysis value (%): Actual value ^*1 Calculated value ^*2 C, 38.29±2.0 C, 38.33 H, 6.48±1.0 H, 6.06 N, 15.11±1.5 N, 15.47 O, 27.49 S, 4.12±1.0 S, 3.94 Cl, 8.71±1.5 Cl, 8.70 *1, Sample dried under reduced pressure over phosphorus pentoxide at room temperature for 15 hours. *Calculated assuming that 2 to 3 moles of attached water are included. 6 Ultraviolet absorption (UV) spectrum: λ ^H ₂ ^O _nax 260 ± 2 nm (E1% 1 cm = 117 ± 20) 7 Circular dichroism (CD) spectrum: [θ] ^H ₂ ^O _{228 ± 2} −30900 ± 5000 and [θ] ^H ₂ ^O _260±2
+
29500±5000 (-: Kotton effect of -; +: Kotton effect of +) 8 Infrared absorption (IR) spectrum: The main wave numbers (cm ^-1 ) in potassium bromide are as follows. 3420, 3250, 3080, 3000, 1775, 1730, 1670,
1510, 1450, 1400, 1260, 1165, 1060, 980,
860,510 9 Nuclear magnetic resonance ( ¹³ C-NMR) spectrum: In heavy water, at least the following signal at 100 MHz is observed (δppm). 179.84(s), 177.42(s), 176.05(s), 173.75(s),
171.12(s), 166.40(d), 162.16(s), 159.62(s),
134.86(s), 117.40(s), 79.64(s), 72.66(d), 67.16
(t), 68.94(d), 57.34(d), 56.31(d), 52.03(d),
43.59(t), 41.23(t), 37.36(t), 32.80(t), 28.98(t),
28.60(t), 27.50(t), 23.50(t), 19.75(q) (s: singlet, d: doublet, t: triplet, q: quartet) 10 Amino acid analysis value: Constant boiling point hydrochloric acid (5.5N-HCl)
Samples hydrolyzed at 110°C for 15 hours. Alanine: 0.86 mol α-amino-adipic acid: 0.94 mol 11 Thin layer chromatography (TLC): Spot film, cellulose (manufactured by Tokyo Kasei Kogyo Co., Ltd.). Solvent system, acetonitrile: 3% ammonium sulfate (1:1), Rf = 0.52 12 High performance liquid chromatography (HPLC): Support, YMC back A312 (manufactured by Yamamura Chemical Research Institute), mobile phase, 2% methanol/0.01M phosphorus Acid buffer (PH3.0), 2ml/min. Rt=5.8 (min) 13 Solubility: Easily soluble: water, aqueous acetone, aqueous alcohol Poorly soluble: dimethyl sulfoxide, methanol, acetone, ethyl acetate14 Color reaction: Positive: ninhydrin, Greig-Liebak,
Sakaguchi reaction Negative: Ehrlich, Burton reaction, potassium permanganate15 Stability: Unstable in acidic and basic aqueous solutions, very unstable in neutral aqueous solutions16 Substance classification: Amphoteric substances (dihydrochloride is neutral Material) Reference Example 2 TAN-547A (1.0 g) was dissolved in a 0.02M aqueous disodium hydrogen phosphate solution (200 ml), and the pH was adjusted to 9.4 with a 2N aqueous sodium hydroxide solution. pH of solution
Add 2N sodium hydroxide to maintain the temperature from 9.0 to 9.4.
Stirred at room temperature for 33 hours with hourly additions. After adding water (100ml) to the reaction solution and adjusting the pH to 7.0,
The mixture was passed through a column packed with QAE Sephadex A-25 (Cl ^- form, 100 ml) (Pharmacia, Sweden), and eluted and fractionated with 0.02 M phosphate buffer (PH7.0). The eluted fractions were subjected to high performance liquid chromatography analysis, and fractions showing a single peak were collected and adjusted to pH 7.0. Pour the collected solution on activated carbon (50
ml) and water (150 ml).
After washing with water, elution was performed with 8% isobutanol (300 ml). The eluate was concentrated and then lyophilized to obtain a white powder (253 mg) of the sodium salt of compound (2). The physicochemical properties of the sodium salt of compound () obtained above are as follows. (1) Shape: White powder (2) Specific rotation: [α] ²⁵ _D +146.5°±30° (c=0.51
,
water) (3) Molecular weight: 438 (by SIMS method) (4) Elemental analysis value (%); C ₁₅ H ₁₉ N ₄ O ₈ SNa・H ₂ O
Actual value Calculated value C 39.65±20 39.48 H 4.64±0.5 4.64 N 12.20±1.0 12.28 O 31.55 S 7.28±1.0 7.02 Na 5.2±1.0 5.04 (5) Ultraviolet absorption spectrum λH ₂ Omax (E1% 1cm) 258± 2nm (209±50) (6) Infrared absorption spectrum (KBr method): Main peaks 3430, 3240, 3020, 1770, 1680, 1610, 1520,
1410, 1375, 1300, 1220, 1145, 1070, 1040,
1000, 800, 710, 540cm ^-1 (7) ¹³ C nuclear magnetic resonance spectrum (100MHz, in heavy water): δ 179.65(s), 177.22(s), 171.43(s),
166.3(d), 162.01(s), 132.76(s), 122.50(s), 79.47
(s), 66.03(d), 63.75(t), 57.33(d), 37.28(t),
32.70(t), 28.22(t), 23.40(t)ppm (s: singlet, d: doublet, t:
triplet) (8) High performance liquid chromatography (manufactured by Waters, USA): Support: YMC-PaX A-312 (manufactured by Yamamura Kagaku Kenkyusho) Mobile phase: 2% methanol-0.01M phosphate buffer (PH3.0), Rt=2.3 minutes (9) Positive color reaction: Ninhydrin reaction Negative: Ehrlich, Greig Liebak,
Sakaguchi reaction (10) Solubility Easily soluble in water. Soluble in methanol. Slightly soluble in diethyl ether and ethyl acetate. Example 1 Trigonobsis variabilis IFO 0755 was inoculated into a seed medium (40 ml dispensed into a 200 ml flask) having the following composition. Composition of seed medium:
(In 1) Glucose 20g, KH ₂ PO ₄ 4g,
_MgSO4・_7H2O 1g, _CaCl2 0.5g, _{H3BO4 0.1}
g, (NH ₄ ) ₆ Mo ₇ O ₂₄・4H ₂ O 40 mg, MnSO ₄・
4H ₂ O 40mg, ZnSO ₄・7H ₂ O 40mg, CuSO ₄・
5H ₂ O 45mg, FeSO ₄・7H ₂ O 25mg, thiamine hydrochloride 4mg, biotin 20μg, DL-α-alanine 4g
(PH6.0). Cultured with shaking at 28℃ for 2 days, and 1 ml of the culture was
Main medium (composition is the same as the seed medium, but
DL-methionine 25 instead of DL-α-alanine
g/) and cultured with shaking at 28°C for 3 days. Bacteria were collected using a high-speed refrigerated centrifuge, and washed cells were obtained by washing with distilled water. Washed bacteria (15g of wet bacteria)
into 40ml of 0.1M pyrophosphate buffer (PH8.0),
Suspend in a solution containing 10mM sodium azide, add 440mg of compound () dissolved in 4ml of distilled water, and react in a 200ml Erlenmeyer flask with shaking at 28°C for 16 hours. I let it happen. The reaction solution was sterilized using a high-speed refrigerated centrifuge, and the supernatant liquid was
Got ml. After adding water (500ml) to the supernatant and adjusting the pH to 7.0,
The mixture was passed through a column packed with QAE Sephadex A-25 (Cl ^- form, 200 ml) and eluted and fractionated with 0.05 M phosphate buffer (PH7.0). The effective fraction was collected and passed through a column packed with activated carbon (200 ml), and after washing the column with water (400 ml), 8% isophtanol water (1000 ml), 8% isobutanol-N/100 ammonia water (400 ml) It was eluted. After concentrating the eluate, QAE Sephadex A-25 (Cl ^- form, 200
ml) and was eluted and fractionated with 0.03M phosphate buffer (PH7.0). Each fraction was subjected to liquid chromatography analysis, and fractions showing a single peak were collected and passed through a column packed with activated carbon (300). After washing the column with water (900 ml), it was eluted with 8% isophtanol water (900 ml) and 8% isobutanol-N/100 ammonia water (500 ml). After concentrating the eluate, the compound (
-2) disodium salt (360 mg) was obtained. Example 2 Pseudomonas sp. UK-2221 (IFO
14366, FERM BP-637) with peptone 1%, meat extract 0.5%, yeast extract 0.1%, glutaric acid 0.05
%, NaCl 0.5%, PH10.0 200ml
One platinum loopful of the mixture was inoculated into an Erlenmeyer flask and cultured with shaking at 30°C for 7 days. Collect bacteria by centrifugation and add 0.1M so that the wet bacterial mass is 500mg/ml.
Suspended in potassium phosphate buffer ((PH7.0)).
20ml of the compound (-2) obtained in Example 1
The mixture was mixed with 60 ml of 0.1 M potassium phosphate buffer containing 15 mg/ml of the mixture, and allowed to stand at 37°C for 48 hours to react. After centrifuging the reaction solution and removing bacterial cells,
The pH of the supernatant was corrected to 7.2, and the supernatant was subjected to column chromatography using activated carbon (5 ml). After washing the column with water (10 ml), it was eluted with water (10 ml) and 8% isobutyl alcohol (40 ml). After the eluate was concentrated under reduced pressure, the concentrated solution (1 ml) was subjected to column chromatography using 5 ml of QAE-Sephadex A-25 (Cl type). After washing the column with water (25 ml), further washing with 0.02M saline (25 ml),
Fractional elution was carried out in 5 ml portions with 0.05M saline. Analyze each fraction by HPLC, collect the fractions (20 ml) showing only a single peak of the target product, and adjust the pH to 6.9.
The mixture was corrected and subjected to column chromatography on activated carbon (5 ml). After washing the column with water (25 ml), it was eluted with 8% isobutyl alcohol water (25 ml) and 8% isobutyl alcohol-N/100 ammonia water (25 ml). Compound-1) was obtained by concentrating the eluate under reduced pressure and then freeze-drying it.
8.5 mg of the educt was obtained as a white powder. Effects of the Invention The compound () of the present invention can be used as a raw material compound for synthesizing a cefem compound useful as a therapeutic agent for bacterial infections. In addition, the compound (-1) in which R is hydrogen in the compound () of the present invention is a stable compound, so it is easy to handle when used as a raw material compound.

[Brief explanation of drawings]

1 and 2 show infrared absorption spectra of compound (-2) obtained in Example 1 and compound (-1) obtained in Example 2, respectively.

Claims (1)

[Claims] 1. General formula [Wherein, R is hydrogen or HOOC-( _CH2 ) ₃ -CO
- indicates a group. ] or its salt. 2 formulas The HOOC-(CH ₂ ) ₃ -CO-NH- group at the 7-position of the compound or its salt is replaced with NH ₂ -
A formula characterized by contacting a culture of a microorganism belonging to the genus Pseudomonas or a processed product thereof that has the ability to convert into A method for producing a compound represented by or a salt thereof. 3 formulas A culture of a microorganism belonging to the genus Trigonobsis that has the ability to convert the [Formula] group at the 7-position of the compound into a HOOC-(CH ₂ ) ₃ -CO-NH- group, or a treatment thereof, into a compound represented by or a salt thereof. An expression characterized by bringing objects into contact A method for producing a compound represented by or a salt thereof.