JPS6172789A - 7-formylamino-cephem compound and its production - Google Patents

Abstract

NEW MATERIAL:A compound of formula I [R is H, HOOC-(CH2)3-CO-] and its salt. USE:Remedy for bacterial infectious diseases and synthetic intermediate of cephem compounds. PREPARATION:A compound of formula II or its salt is brought into contact with a culture mixture of a microorganism in Pseudomonas which is capable of converting HOOC-(CH2)3-CO-NH- group into NH2- group such as Pseudomonas sp. Uk-2221 strain (FERm BP-637) or its treated product, preferably at a pH of 6.5-7.5 at 15-54hr.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention provides 7-formylamino-cephem which is useful as a therapeutic agent for bacterial infections and also useful as a raw material compound for synthesizing various cephem compounds. Prior Art Cephamycin C homologues produced by fungi and cephamycin C homologs produced by actinomycetes are
It is known that D-amino acid oxidase and cephalosporin acylase of microorganisms convert it into the corresponding 7-amitoce7em compound or 7-medoxy7-aminocepheme compound, respectively. However, the production of 7-formylamino-cephem compounds by microorganisms from novel cephem compounds produced
There is no known Ig production method for formylami-two-amino-cephem compounds.

Problem to be solved by the invention 7-Formylamitodiacetylcephatetrasporin (4
If it can be converted to a -7-hoymiμ amino-7-amino-cephem compound, it will be near 1. Using the target compound as a raw material compound, it is possible to produce rJ 7-formylamino-cephem compounds.

Means for Solving the Problems The present inventors have proposed various methods for converting 7-formylamituzoacetylcephalosvorin C into a 7-homimino-7-amino-cephem compound using microorganisms. As a result of investigation, it was found that 7-formylamino-deacetymu-cephalonuporin C can be converted into a 7-phymylamino-7-gutally ρ-amino-cephem compound by Trigonopunus bacteria, and the compound obtained here is a new compound. In addition, this compound was further analyzed using Pseudomonas spp.
The present invention was completed based on the knowledge that the compound can be converted into a formylamitofuaminocephem compound and that the compound thus obtained is a new compound.As a result of further research based on these findings, the present invention was completed.

The present invention is based on the general formula (IJ [wherein R is Mizuki or HOOC-(CH2)3-Co
- indicates a group. ] or its salt.

(2) HCIOC-(cH2)3-Co-NU-Is at the 7-position of the compound or a salt thereof is added to [2
- Pseudomonas (Pseudomonas) with the ability to convert into
do-monas), a method for producing a compound represented by the formula or a mass thereof, which comprises contacting a culture of a hoe organism belonging to the species Do-monas or a treated product thereof, and (3
) to the compound represented by the formula or its salt, the compound ( C
H2) A method for producing a compound represented by the formula or a salt thereof, which comprises contacting a culture of a microorganism belonging to the genus Trigonopsis or a treated product thereof, which has the ability to convert into a 3-Co-NH- group. be.

The microorganisms used in the step of converting compound (II) to compound (I-2) of the present invention include acute microorganisms that belong to the genus Trigonopsis and have the ability to convert compound (I[) into compound (I-2). If so, either can be used. The microorganism includes, for example, Trigonopsia parabilis (Trigonopsia varia).
bilia), and its body side is, for example, Atribonobsis palabilis O.
'O0671, Engineering F'0 0755 strains are mentioned.

The microorganisms numbered F'0 above have been deposited with the Institute for Fermentation Research (IF'O) and published in the Institute's List of Cultures 11984, 7th edition (In5t
Itute F'or Fermentation,
0sakaList of Cultures l 9
g4 aevent) 1 edition). The deposit date of IF'O is shown below.

The mycological properties of Atribonobsis palabilis were investigated in the Yeasts Taxonomy Study (Yeasts;
A Taxonomic 5tudy) 1970 2nd edition, pp. 1353-1357 1 Editor J, Lad
der.

North Holland Pub Corpora
It is the same as that described in the publication of tion.

The microorganism used in the step of converting compound (I-2) to compound (I-1) of the present invention belongs to the genus Pseudomonas and has the ability to convert compound (I-2) to compound (I-1). Any microorganism can be used as long as it has the following. A specific example of such a microorganism is Pseudomonas spp., which was isolated from Dobe in Fukuchiyama City, Hyogo Prefecture.
SP (Paeudomonaa ap) UK
-2221 strain is mentioned.

The chemical properties of the UK-2221 strain are as follows.

(a) Morphology Observation after culturing on a broth agar slant at 24°C for 5 days revealed that the cells were 0.5-1.0 μm in diameter and 0.8-2.0 μm in length.
It is rod-shaped and motile due to flagella. The flagellum is monociliated. Does not form spores. In addition, Gram staining is negative and does not show acid-fast properties.

(b) Growth status on each culture medium The cells were cultured at 24°C and observed for 1 to 14 days.

■Meat juice agar plate culture: Colonies are colorless and round, with a convex circular surface and a glossy periphery. Does not produce diffusible dyes.

■ Broth agar slant culture: medium growth, shiny filamentous growth, colorless, no formation of diffusible pigments.

■ Broth 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.

■Lidomas Mi〃ri: The reducing ability of Lidomas is not recognized. No peptonization activity was observed either.

(C) Physiological properties ■ Nitrate reduction knee ■ Denitrification reaction knee ■ MR (methyl red) test knee ■ VP (Voges-Grosskaue/L/) 7-Nut2 - ■ Indole formation knee ■ Hydrogen sulfide formation (acetic acid (Lead paper) Knee ■ Starch hydrolysis Knee ■ Utilization of citric acid (Kozel, Christensen, and Simmons media): + (- in Simmons) ■ Utilization of inorganic crab source D) Potassium nitrate: + ■) Ammonium sulfate: + [ [Strain] Production of pigment (King A, B and Mannitol 3 mother extract agar media): Production of diffusible pigment was not observed.

King A medium: Glycerin 10f, Begton 20g, m
Magnesium chloride 14g, ammonium sulfate 10g, agar 15g, distilled water 1000 edition C2 pH 7.2 King B medium: Glycerin 1 (1, peptone 20g, potassium phosphate-hydrogen LEM, magnesium sulfate L59.
15 g of agar, pH 7.2 ■Urease: + ■Ogisidase: + (weak) ◎Catalase: + ■Growth range□! I) PH: PH4,3-7.
II) Temperature: Grows at 14-32°C, but the optimum pEI value is 18
~26C ■Attitude toward oxygen: absolutely aerobic.

@0-F (oxidative fermentative) test [Hugh-Leifson]
Method]: Non-decomposable type.

◎Generation of acid and gas from sugar: L-arabinose l-i,
-D-Mannose i --10 l ・D-Fufuctose +-i-+ D-Gafuctose i -1-Wheat bud sugar 1 - ” --Stain Sugar i--- Lactose i -: +: Positive, soil : False positive, - bicyclic @ DNA GC (guanine + 7 tons) content: 6 & 3 soil 1.5% UK-2221 strain having the following mycological properties was treated with Percy's Manual μ Op Determinative. Battererioisie 8th edition and International Journal of Stematic Bacteriology 3rd edition
Volume 0, pp. 225-420 (1980) and the same magazine's Validation 1st.
), strain UK-2221 is a Guhum-negative bacillus, does not form spores, moves by monopolar hairs, is obligate aerobic, and is positive for catalase.

0-F' test is non-degradable, I) GC of HA
(Guanine + 7 tons) content is 6 & 3 ± 1.5%, so Pseudomonas (paeudo-mona)
Pseudomonas s) is considered to belong to the genus Pseudomonas y,
・x sp (Paeudomonas sp) U
I decided to call it K-2221.

The Pseudomonas sp. UK-2221 strain mentioned above was deposited with the Fermentation Research Institute (UFO) on August 31, 1984 under the accession number IF'014366, and the zero-defeat organism was It has been deposited with the Research Institute of Microbiology (rRI), Agency of Industrial Science and Technology, Ministry of International Trade and Industry on September 7, 1984, under accession number F'ERM P-7836.

The properties of the Trigonopsis and Pseudomonas FA bacteria used in the method of the present invention are generally susceptible to change.
Although it can be easily mutated by artificial mutation means such as ethylmethane (phonic acid), any mutant strain that has the conversion ability of the method of the present invention should be used in the method of the present invention. I can do it.

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 strain.
When obtaining large amounts of material, it is preferable to use a liquid medium.

The medium contains a carbon source that can be assimilated by the microorganisms.

Digestible nitrogen sources, inorganic substances, nutrients, etc. are appropriately added. Examples of carbon sources include those that can be assimilated by prisoners, such as glucose, maltose, lactose, blackstrap molasses, oils and fats (e.g., soybean oil, olive oil, etc.), organic acid FA (e.g., citric acid, succinic acid, gluconic acid, etc.) is used as appropriate. Nitrogen sources include, for example, soybean flour, cottonseed flour, corn staple liquor, dried yeast, B mother extract, meat extract, peptone, urea, ammonium sulfate, and ammonium nitrate.

Organic nitrogen compounds and inorganic nitrogen compounds such as ammonium chloride and ammonium phosphate can be used. Examples of inorganic salts include sodium chloride.

Potassium chloride, calcium carbonate, magnesium sulfate, potassium phosphate, disodium phosphate, etc.□1
Inorganic salts necessary for the cultivation of normal microorganisms are used alone or in appropriate combinations. In addition, D-type amino acids such as D-awonine and D-methionine or D-α-aminoadipic acid are added to the medium used for culturing Trigonopsis bacteria in order to induce the activity of D-amino acid oxidase. You may.

Cultivation may be carried out by static culture, shaking culture, or aerated agitation culture. For the treatment of gluttony, so-called deep aeration agitation culture is preferable.

The conditions for culturing Trigonopsis bacteria include the state and composition of the medium,
Although it naturally varies depending on the type of strain, culture method, etc., the temperature is usually 20°C to 45°C, more preferably about 24°C to 37°C, and the pE of the medium is about 6 to 45°C. 9, more preferably about 6-8. The culture time is about 24 to 144 hours, more preferably about 2 hours.
It is between 4 and 120#.

It goes without saying that the conditions for culturing Pseudomonas waste cocoons vary depending on the condition-1 composition of the medium, the type of strain, the means of cultivation, etc., but they are usually at a temperature of 20°C to 45°C, more preferably at a temperature of about 20°C to 45°C. At a temperature of 24-37°C, the pE of the medium is adjusted to about 6-9, more preferably about 6-8.

Culture time is approximately 24-168 hours.

The "processed product" used in the present invention refers to the culture obtained above that undergoes physicochemical treatment such as filtration, centrifugation, 9M sonication, French press treatment, osmotic shock method, freezing,
It refers to bacterial cells obtained by the melting method, alumina grinding, lytic enzyme treatment, 51iL surfactant or organic solvent treatment, or a crushed bacterial cell material containing enzymes involved in the reaction of the method of the present invention. Furthermore, the enzyme obtained by M production using a known method, or the bacterial cells or the enzyme immobilized using a known method can also be used.

Compounds (
It) tDa degree is about 0.5-20'! v/at.

More preferably about 5-101RL! / River t. The amount of microorganisms is about 0.1 to 1 q/pet, more preferably about 0.1 to 0.39/pet, 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, it is adjusted to about 75 to 8.5. The reaction temperature is about 15-40°C, more preferably about 15-37°C.
Dechiru. 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, and it is particularly preferable to carry out the reaction under conditions of shaking, aeration, or stirring.

In order to obtain the target compound (I-2) in good yield, the objective can be achieved by adding a catalase inhibitor to the reaction solution. Examples of catalase inhibitors used include inorganic azides such as sodium azide, ascorbic acid, and 3-amino-1,2°3-triacyl. 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 completed, the reaction solution is centrifuged to remove bacterial cells, and the supernatant is separated and purified to obtain the target compound (I
-2) is obtained.

Of course, when culturing the microorganism in a medium, chemical compounds (I
The method of the invention may be carried out by adding I) to the culture medium. In this case, the amount of compound (It) added is about 1 to 20 M'J/ljt, more preferably about 2 to 10 M'J/ljt.
Cape/wt. In addition, the culture temperature, pH of the medium, and culture time are the same as those for the culture described above.

In the reaction step, ":,>CTi of compound (1)
(CH2) 3-Go-NU- group is once HOOC-
Co-(cH2)3-co-NH- group, which is subsequently converted into HOOC-(c[2)3-co-NH-
- group to produce the compound (nee 2).

The concentration of compound (I-2) in the reaction solution in the reaction in which compound Cl-2) is brought into contact with the culture of the microorganism or the treated product thereof is about 2 to 20 gl, more preferably about 4 to 10 gl. Cape/GL.

The amount of microorganisms is about 0.1 to 1 liter/liter in terms of wet bacterial weight.

1 more preferably about 0.1-0.
3g/1ttl. 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 about 6 to 10, more preferably about 6.5.
Adjusted to ~7.5. The reaction temperature is about 15-40°C, more preferably about 15-37°C. The reaction time is about 4
~66 hours, more preferably about 16-54 hours.

The reaction may be carried out under static conditions or under conditions of shaking, 1 aeration, or stirring, and particularly preferably shaking, 9 aeration, or stirring.

The reaction may be carried out by adding the compound (Ni-2) to a medium and culturing the microorganism used in the method of the present invention in the medium. In this case, the amount of compound (I-2) added is: About 2-20q/11, more preferably about 4-10M9
/vtl. In addition, the culture temperature, pH of the medium, and culture time are the same as those in the culture described above.

Compound (nee 2) or compound (nee 1) from the reaction solution
To isolate the substance, 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, 9-molecular sieve chromatography, reverse phase liquid chromatography, vacuum concentration, and freeze drying can be used alone or in any order. Used in combination or repeatedly.

As a carrier, activity, adsorption resin, anion exchange resin (
Anionic type), powdered cellulose, etc. are used, or carriers with 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/1/, ethanol, Demavanop, Getano-μ, isopropano-μ, and Imbutano-P, or acids, alkalis, buffers, or An aqueous solution of an inorganic or organic salt is used.

To describe the example in more detail, after the reaction is completed, the reaction solution't-pHl is adjusted to about 6 to 7, and since the substance is an acidic substance, the reaction solution is mixed with anion exchange resin [Amberlite RA-68, 402゜410 (ROHM &
Manufactured by Haas, ffim).

Antibiotics are adsorbed by passing through C1i or ACO-type carriers such as DOWEX-1 (manufactured by Dow & Chemical Co., USA) and Diaion 5A-21C (manufactured by Mitsubishi Kasei Kocha Co., Ltd.). The effective substance is eluted using saline or a saline solution as the eluate for the adsorbed substance. To desalt the eluate, make the eluate neutral or slightly acidic and use activated carbon (
(manufactured by Takeda Pharmaceutical Company Limited) chromatography.
Elutes with hydrous alcohol, etc.

Next, the eluate containing the active substance is concentrated under reduced pressure at low temperature, and the concentrated solution is passed through a C1'-type resin of DIAI or QAE Sephadex (manufactured by Pharmacia, Nueden),
Absorbs antibiotics. The adsorbed antibiotic is eluted and fractionated with fine salt water. 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-mentioned compound (It) and compound (I) may be formed into salts by conventional means, and the salts include:
Thin G [When used as a medicine such as a treatment agent for dyed surfaces,
Pharmacologically acceptable salts such as lithium salts, sodium salts, barium salts.

In addition to the above salts, when used as a synthetic intermediate, for example, ammonium salt, methylamine salt, diethylamine salt, trimethylamine salt, etc. Amine salt, tetrabutylammonium salt,
Examples include pyridine salt.

The salt of the compound thus obtained can also be converted into other salts using means known per se. For example, dissolve the obtained salt in water and cool it with dilute hydrochloric acid until the phi is about 2.
Adjust to ~3. The pH of the aqueous solution is immediately adjusted to about 7 to 8 with a dilute alkaline aqueous solution containing, for example, sodium hydroxide, calscum hydroxide, methyμ amine, tetragutylammonium hydroxide, etc., to produce the desired salt, and the aqueous solution is then heated with activated carbon. chromatography of
After washing the inorganic or organic salts with water, the antibiotic salts are eluted.

The physicochemical properties of the disodium salt of compound (I-2) obtained in Example 1 described below are as follows.

(1) Appearance: White powder (27 molecular weight rsIMs method molecular ion peak is m
/z, 432 (M-1-H).

(3) Elemental analysis value: (40tE, 8 hours drying under reduced pressure)
Actual value Calculated value C38,063B, 19 H3,813,66 N 9.97 9.540
30.88S 7.68
7.28Na 10
．． 44(4) Molecular formula: C14H15N30BSN
&2 ・0.5Ei 20(5) Ultraviolet absorption Subek)
A/ (Underwater): Maximum @I: 259 nm (EI 24) (6) Infrared absorption Subek) /L' (KB
r): Main wave number (α-1), Fig. 1 3400, 3230.3010.1775.1700.
1690°1610, 1580.1410.1305.
1240.1150°1065.1045.1000.
850. 800. 710° (7) Nuclear magnetic resonance (PMR) spectrum/L': 100MHz.

In heavy water, δppm ff (Hz) 1.75-2.15 2H,m 2.15-2.55 4H,m 3.38 1H,d, J-18 3.70 111! , d, J-184,2
72H, a.

5.39 1H* s* &21 1H, a.

(8) '￥IMH Body Bear Matography: Waters Inc. (USA) tJ Model 6000A/66
0/440 column: YMC-Pack A-3
12 (Yamamura Kagaku Kenkyushin) Transfer, 11:o, 01M phosphate buffer (PH6,3):
2r//min Detection: 254nm Rt -3, 1win.

The physicochemical properties of the compound (I-1) obtained in Example 2 described below are as follows.

(1) Appearance: White powder (2) Molecular weight: The molecular ion peak measured by SIMS method is m
A 296 (M4-NIL) was observed.

(3) Molecular formula: C9E11□tr3o5s (2
73) (4) Ultraviolet absorption subek)/L' (in water): maximum fit; 259 nm (L: i theory = -365) (5
) Circular dichroism spectrum lv (underwater): [θ] Yoni 333
00±5000 and (θ)ii: +32400 5
000 (6) External absorption spectrum //: Main wave number (Cm
-1).

Figure 2 3400.2980, 1760.1680. 1600
．． 1510°1410, 13g0. 1290. 1
240. 1180. 1140°1070.1040
．． 1000. 860. 790. 700° (
7) Nuclear magnetism co-I! 1% (PMR) Nuvector: 400M
δppm J (FI2) in H2+fI water.

' 3.43 1H, d, J-17-63, 65
1L d, J-17,6 4.21 1H, d, , T-12,94,2
5111, d, J-129 5,191H, a Model 638-50 Column: YMC-Pack A-312 Mobile phase: 0.
01M phosphate buffer (pH 6,3), 2F//win
.

Detection: 254nm Rtm3. Qmin Compound (II) which is a raw material for the method of the present invention is, for example, Rhizopacta=! Antibiotic TAN-5 was produced by culturing i4 bacteria.
tcTAN-547A (also sometimes referred to simply as tcTAN-547A) can be obtained by accumulating t-products, collecting them, and then subjecting them to a hydrolysis reaction.

Antibiotic TAN-547A belonging to the above Rhizobacter genus
An example of a produced ulva is Rhizobacter lactamgenus YK-90 strain, which belongs to a new bacterial species and was collected from soil. The Rhizobacter phtatumgenus YK-
The 90 stocks were given accession number IF'0 14 to the Fermentation Research Institute (F'O) on September 14, 1983.
It has been deposited as No. 288. In addition, the original protozoan was born in the Showa 5
On September 19, 1983, it was submitted to the Microbial Research Institute (F'RI), Agency of Industrial Science and Technology, Ministry of International Trade and Industry, with accession number F'ER.
MP-7247, the deposit was converted to a deposit under the Budapest Treaty, and has accession number F'lRM.
It is stored at the same research institute (F'R Engineering) as BP-575.

The antibiotic TAN-547A is shown in the following four packaging formats.

Compound (II) is, for example, the antibiotic T obtained above.
It can be produced by subjecting AN-547A to a hydrolysis reaction. The hydrolysis reaction was performed using TAN-547A
This is done by treating it 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, and treated.

Examples of the aqueous solution include buffers commonly used in chemical reactions, such as phosphates, borates, citrates, carbonates, hydrochlorides, and vinegar.
, sodium hydroxide, glycine, perroner p, borax,
Examples include aqueous solutions whose pH has been adjusted with ammonium salts, aminomethylpropane di-μ, and the like.

=1 The hydrolysis reaction of B is carried out at a temperature of about 0°C to 80°C, preferably about 20°C to 40°C, for about 1 hour to 72°C.
The reaction is carried out for a period of time, preferably about 4 hours to 40 hours.

To isolate compound (I[) 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. Mano is used repeatedly.

Compound (I-2) thus obtained is useful as a synthetic intermediate for producing a compound (nee 1) useful as a therapeutic agent for cocoon infection.

Next, the biological properties of compound (I-1) will be described.

The antibacterial spectrum of the sodium salt of compound (I-1) against various microorganisms is shown in Table 1. As is clear from this table, compound (I-1) exhibits antibacterial properties against Guhum-negative bacteria.

Table 1 Escherichia coli CP13
11 Neudomonas aeruginosa C14119 Staphylococcus aureus F'DA209P
<8 Note) Medium Nutrient agar medium (pH 7,0) containing diamino pimelic acid (20q/J) Compound (I-1) O used: 1000 μg/*/ Also, sodium salt of compound (I-1) is stable to various β-tuctamases. Table 2 shows compounds (I-1) and D
The stability of CPC towards β-lactamase is shown.

(3) Amount of compound used: Compound (I-1) is ioo.

p 9/ml, other compounds 100 μ9/ml Bacterial strain used: yNeedomonas aeruginosa,
(Pseudomonas aeruginos
a) C141 medium Nidiaminopimelic acid (20 ml/
J) Nutrient agar medium containing t″ (pH 7,0) Note 1: DC: PC Nigeacetylcephalosporin C
Note 2: CMC: Cephamycin C Note 3: Bacillus cereus
Beninlinase (manufactured by Calbiochem, USA), 0.048 liter/go Note 4: Enteropacter cloacani (Enteropacter cloacani)
Bacter cloacae) origin, 0.02
5 unitZdr Also, compound (I-1) sodium salt f;r:1f/k
Compound (I-1) is considered to have low toxicity since no deaths were observed even when the amount of 1.5 g was subcutaneously administered to mice.

As is clear from the above data, the compound (I-1) obtained by the present invention exhibits antifungal activity against Gram-negative moss, and can be said to be a low-toxicity antibiotic.

Therefore, Compound (I-1) Takeso salt is used for the treatment of bacterial infections in mammals (e.g., mice, animals, horses, dogs, humans), poultry (e.g., chickens, ahi/I/), etc. be able to.

Compound (I-1) is used as a therapeutic agent for infectious diseases, for example, by administering compound (I-1) as an injection parenterally to the above-mentioned mammal subcutaneously or intramuscularly at about 5 to 200 M7kf1. days, preferably about 20 to 1
Administer 001f//kQ for 1 day. In addition, as an oral agent, the compound (Ni 1) was used as Kab7 preparation, and the compound (I-1'J
about 20 to 400 g/97 days, preferably about 40 to 200 g/9/day.

Moreover, compound (I-1) or a salt thereof can be used as a disinfectant. For example, compound (nee 1) is about 0
．． As a solution dissolved in distilled water at a concentration of 0.05 to 0.4 W/V%, or as an ointment containing about 1 to 100 η, preferably about 4 to 50 q, of compound (I-1), the above-mentioned By applying it to the hands, feet, eyes, ears, etc. of animals, it can be used to sterilize and disinfect these parts.

Furthermore, the compound (nee 1) is useful as a synthetic intermediate for the synthesis of semi-synthetic cephem compounds useful as therapeutic agents for H1 bacterial infections.

As is clear from a comparison of the stability of cephalosporin C and deacetyl cephalosporin C in aqueous solutions, cephalosporin conductors generally become unstable when the 3-position is acetylated. Compound (nee 1) has -CH2 at the 3rd position
0H, acetylated corresponding compound (3-position -
CH20COCH3) shows a much more stable property 5t in aqueous solution. This shows that when carrying out a synthetic reaction, compound (I-1) can be easily handled and thus can be advantageously used as a synthetic intermediate.

Example 4 Solitary Shrine

Reference Example 1 Zopacter octumgemes YK-90, (IF'0 14288°7'ERM) grown on nutrient agar slope
BP-575) was mixed with 2% glucose, 3% Solp μ starch, and 1% raw soybean flour.

Precipitated carbonate Calcolumn α 5% in an aqueous solution (pH 7.0) containing 1% Corn Nuteep Liquor, 0.5% Polypeptone (manufactured by Daigo Nutritional Chemical Co., Ltd.) and 0.3% salt.
3 200-capacity Erlenmeyer flasks containing 40 d of medium supplemented with
Trees are inoculated and cultured with shaking at 24°C for 48 hours, and the culture is used as a seed plot.

Next, dextrin 3%, raw soybean flour 1.5%, corn/
Gluten Me/L/L 5%, Polypeptone 0.2%,
Aqueous solution containing 0.1% sodium thiosulfate (pH 6)
, 5) K sedimentation carbonic acid column 4000 #t of medium supplemented with 0.5% 200 s #) 40 t each in Erlenmeyer flasks
The mixture was dispensed into 1 mt portions and sterilized at 120° C. for 20 minutes, then inoculated with 1 mt portion of the incubator, and cultured with shaking at 24° C. and 200 revolutions/min for 72 hours.

The culture solution (zoJ) obtained above was adjusted to pH 3 with t-7% oxalic acid.
, 5, Hyflo Superverse Iv (manufactured by John's Mambi μ Products, USA) was added and filtered to obtain a furnace solution (161). After adjusting the pH of the p solution to 6.8, it was passed through a column filled with activated carbon (1j). After washing the column with water (3j), antibiotic TAN-547 was eluted with 8% isobutano/L/-47200 hydrochloric acid (8j). After concentrating the eluate to 184, convert the concentrated solution to Amberly) CG
-50< H+ type, 1.41) (ROHM & l)
The sample was passed through a column manufactured by Co., Ltd., USA). Water (4,51
) and then eluted and fractionated with N/100 hydrochloric acid (9J). The effective fraction was collected and concentrated, and the eluate was adjusted to pH 73, and then passed through a column packed with Diaion EIP-20 (50-100 mesh, 0.51) (manufactured by Mitsubishi Chemical Industries, Ltd.). 0.01M phosphate buffer (pH 7
After washing the column with 0.01M phosphate buffer (pH 3, 5, 5J), the column was eluted and fractionated with 0.01M phosphate buffer (pH 3, 5, 5J). After collecting the effective fraction and adjusting the eluate to pH 92, activated carbon (100y
7) was passed through a column packed with 8% isobutano-/I/-N/20 after washing the column with water (300 rt).
It was eluted with 0 hydrochloric acid (600txt). After concentrating the eluate,
The concentrated solution was transferred to CM-Sephadex C25 (Na+ type, 20
0rj? ) (manufactured by Faman Fine Chemical Co., Ltd.)
Sweden) was passed through a column packed with 0.02
Elution fractionation was performed using M-saline solution (6). 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 (10g4?) and water (
After washing the column with 30xi), 8% inbutanol-
It was eluted with N/200 hydrochloric acid (70 g).

After condensing the eluate, it was lyophilized and TA1'? -547A dihydrochloride crude powder (611 rg) was obtained.

Crude powder (6111 g) of TAN-547A dibulk salt was subjected to preparative high-performance liquid chromatography using YMC-GEL ODS 1-15 (Yamamura Kagaku Kenkyusho! II) as a carrier, and 0.02 M phosphorus was used. Buffer solution (pH 3,0
) and fractionated. Each fraction was subjected to liquid chromatography analysis, and portions showing a single peak were collected. After adjusting the effective range to pH 7.5 with 1N NaOH, 1N HCl
The pH was readjusted to 3, Q, the column was passed through a column packed with activated carbon (5sm?), the column was washed with water (25x), and the column was eluted with 8% isobutano-water (25x). The eluate was concentrated and lyophilized to obtain a white powder (40MQ) 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] Crab + 71.8° ± 20'C0-0
．． 50. in water) 3) Molecular weight measurement [: 3 IMS method 1 (M4-H)
”4) Estimated molecular formula: C25H, IN901IS-2H
C1・(3H2o) Ne1 5) Elemental analysis value %: Actual value Calculation “2G, 3a
29±ZOC, 3 & 33 L 6.48±LQ H, 6,061'i, 1
5. i l±1.5 N, 15.470.27.49 S, 4.12±1. OS, 3.94C1, &7
1±L5 C1,8,70*1. Samples were placed on phosphorus pentoxide for 15 hours at room temperature.

Dry under reduced pressure. Calculated assuming that 2 to 3 moles of attached water are included.

6) Ultraviolet absorption (UY) spectrum/L/:λH2°26
0±2nm (E”%-117±20)max
1F) Circular dichroism (CD) spectrum: C&)%JNoteYu+29500f5000 (-knee effect; +:+ effect) 8) Infrared absorption (IR) spectrum) /I/: Potassium bromide The main wave numbers (ex-'') are as follows.

3420, 3250. 3080. 3000°17
75, 1730. 1670. 1510°1450
, 1400. 1260. 1165°1060,
980. 860. 5109) Nuclear magnetic resonance (C
-NMR) Spect/I/double water, lQQMHz
At least 1 signal is observed below (δppm).

17'184(8), 177.42iB>, 1
76.0 Das), 173.75(a).

17L12(s), 166.4(Xd), 1i16(8
), 159.62(8).

13486(s), 117.4(Xs), 79
．． 64Cs), 72.66(d).

67, 1αt), 65.94d), 57.3
4dl, 56.31(d).

52.03(d), 43.59(tl, 41
23(t), 37.36(t).

32-80(t), 2&9yot), 28.
6o(t), 2y, 5o(t).

23.50 (t), 19.7 Hama ψ (S; singlet, d multi-doublet, t; triplet, q; quack!
10) Amino acid analysis GL: Constant boiling point hydrochloric acid (5,5N-HC
Samples hydrolyzed in I) at 110°C for 15 hours.

Alanine i 0.86 moy α-amino-adipic acid i 0.94 μm1) if
Chromatography (TLC) Nispot film, 7μ loin (manufactured by Tokyo Kasei Kogyo Co., Ltd.) 6 r8v & system, acetonitrile/I/: 3% ammonium sulfate (1:1), Rr value 0.52 12) High performance liquid chromatography (E!PLC):
Carrier, YMC bag A312 (manufactured by Yamamura Kagaku Kenkyushin), mobile phase, 2% methanol/'10.01M phosphate buffer (p
H3,0), 2g//min.

Rt=a8 (min) 13) f8 solubility: Easy solubility: water, aqueous acetone, aqueous alcohol soluble rainbow methane oxide, methano-A/, acetone, ethyl acetate 14) Wide coloration: Positive: Ninhydrin, Brave Reback, Sakaguchi reaction Negative double μL, Burton reaction, potassium permanganate 15) Stability: Unstable in acidic and basic aqueous solutions, slightly unstable in neutral aqueous solutions 16) Substance classification: Amphoteric substance (dihydrochloride is a neutral substance) Reference example 2 TAN-547A (1,11) was dissolved in 0.02M hydrogen phosphate aqueous solution (200Ml), and pH 9 was added with 2N sodium hydroxide aqueous solution. , adjusted to 4. solution p
) The mixture was stirred at room temperature for 33 hours while adding 2N sodium hydroxide every 5 hours to maintain I from 9.0 to 9.4. Add water (100yrt) to the reaction solution and adjust the pH to 7.0! After II, QAE Sephadex-25 (C1 type 2,
100g/) (manufactured by Pharmacia, Sweden) was passed through a t-packed column, and 0.02M phosphate buffer (p
Elution fractionation was carried out using H7,0). The eluted fractions were subjected to high performance liquid chromatography analysis, and fractions showing a single peak were collected and adjusted to pli 7,0. Pour the collected solution on activated carbon (5
0 det), and water (150 liters)
) and then eluted with 8% inbutanol (300pt). The eluate was concentrated and then lyophilized to obtain a white powder (253q) of the sodium salt of compound (I[).

The physicochemical properties of the sodium salt of compound (I[) obtained above are as follows.

(1) Shape: White powder (2) Specific rotation: [α] Sweet 146.5° ± 30° (
cxO, 51, water) (3) Molecular weight: 438 (by SIMS method) (4) Elemental analysis value B4J; C15H1gN40BSNC15H1
As expected value Calculated value C39,65±ZO39,48 H464±0.5 4.64 N IZ20±10 12.28 0 3 no-rg
-87,28±L0 7.02 ffa 5.2±LO5,04 (5) Ultraviolet absorption spectrum ~ λ (Ej, 4) 258±2 nm (209±50) (
6) ILt (KB): Main peak 3430.3240, 3020, 1770.16
80°1610.1520,1410,1375,13
00°1220.1145,1070.1040.10
00°800, 710. 540es”””(7)
13c nuclear magnetic resonance, X = hek)! V (100MH
z, in heavy water): δ 179.65(a), 17
7.22 (ml, 17L4 Hama 8).

, 1 166.30 ((L), 162.01(a),
132.76 (s), 12Z50 (s).

79.47(8), 66.03(d),
63.75(t), 57.33(d).

37.28(t), 3L71Xt), 2EL2
2(t), 23.4(Xt)92m (s: singlet, d: dou
blet, t: triplet t-represented respectively) (8) High performance liquid chromatography (manufactured by Waters, USA): Support: YMC-PaK A-312 (manufactured by Yamamura Kagaku Kenkyushin) Transfer tl19i1: 2% methanol-0, 01M phosphate buffer (pH 3,0), Rt-43 minutes (9) Positive color reaction: Negative reaction with ninhydrin: Erich, Greig Liebach, Sakaguchi reaction (transfer) Solubility Easily soluble in water.

Soluble in methanol.

Elutes with diethyl ether ρ and acetic acid ethyl μ.

Example 1 Trigonodes V. palabilis 11'0 0755t-
It was inoculated into a seed medium (40 gJ dispensed into a 200 ml fufusco) having the following composition. Composition of seed medium = (E
j) Glucose 209, KH2P0゜49,
MgSO44112019, CaCl2 0.59.

H3BO4α1 'i -(NH4)6MO7024・
4H2040q, MgSO4412040av-ZnS
O44H2040"i', CuSO4・5H20459
, F'eSO4'7H2025Mf.

Thiamine hydrochloride 4 parts, biotin 20 pg, Dh-α-awonine 4 g (pHao). Cultured with shaking at 28°C for 2 days,
The Igt was added to the main medium (composition is the same as the seed medium,
However, DL-methionine 2 is substituted for DL-α-afnin.
59/j) and cultured in a shaking tray at 28°C for 3 days. Bacteria were collected using a high-speed refrigerated centrifuge, and a washed body was obtained by washing with distilled water. Washed bacterial cells (15 g of wet bacterial cells) t-140y/
10mM in α1M pyrophosphate buffer (plI&0)
Compound (II) 440M! ! 4 x lf) dissolved in distilled water and added with 200y/volume triangular
The reaction was allowed to proceed at 8° C. for 16 hours with shaking. The reaction solution was sterilized using a high speed refrigerated centrifuge to obtain 91 zt of supernatant synovial fluid.

Add 1fC water (500we) to the supernatant and adjust the pH to 7.0K.
After F adjustment, QAK Sephadex A-25 (C1-type).

200y/) t-packed column t-passed, 0.0
Elution fractionation was carried out using 5M phosphate buffer (pH 7.0). The effective fractions were collected and passed through a column packed with activated carbon (200 Mt) and washed with water (400 tl) followed by 8% isobutano-μ water (1000 ff/).

8% isobutano-/'-N/100 ammonia water (40
It eluted at 0y/). Eluate? After passing through a column filled with qAm Sephadex A-25 (C1-type, 20 (M/)), 0.03MIJ acid buffer (p
elution fractionation was carried out using uy, 0). Each fraction was subjected to liquid chromatography analysis, and fractions showing a single peak were collected and passed through nine columns packed with activated carbon (300y/) tl-. Wash the column with water (900++t) followed by 8% isobutano-water (900rt).

8% isobutano-tv-N/100 ammonia water (50
0zl). The eluate was concentrated and lyophilized to obtain the disodium salt of compound (I-2) (360 kg).

Example 2 Pseudomonas sp. UK-2221 (IF'0
14366, F'ERM P-7836) with 1% peptone, 0.5% meat extract, 0.1% yeast extract, 1V
filV acid 0.05%, Mail 0.5%, pH 1
200 g of a medium (0,0) was dispensed into an IJ five-tricornered flask, inoculated in an amount of 1 platinum loop, and incubated at 30°C.

The cells were cultured with shaking for 7 days. Bacteria were collected by centrifugation and suspended in 0.1M potassium phosphate buffer (pH 7.0) to give a wet bacterial mass of 500 Da/Nl. That 20g4! Compound (I-2) obtained in Example 1 was added to 1511f/xl
The mixture was mixed with 60 txl of αIM'J potassium phosphate buffer containing the same concentration, and allowed to stand at 37°C for 48 hours to react. After centrifuging the reaction solution to remove the reagent, the pH of the supernatant was adjusted to 12.
and subjected to column chromatography for active R (5d). Wash the column with water (10gJ
) and then eluted with water (10 gl) and 8% isobutymu alcohol/I/ (40 gl). The eluate was concentrated under reduced pressure, and then the concentrated solution (1el) was purified with qAE-Sephadex A.
-25 (C1 type) 5- was subjected to column chromatography. After washing the column with water (25st/), further α0
It was washed with 2M saline (25 ml) and fractionated and eluted in 5 el portions with 0.05M saline. E! It was analyzed by PLC, and fractionated liquids (20 liters of bonito flakes) showing only a single peak of the target product were collected, the pH was corrected to 6.9, and the mixture was subjected to column chromatography on activated carbon (5we). After washing the column with water (25gt), add 8% isobutylene
AμCO water (25srl) and 8% isobutylene acetate
Elution was performed with Co#-N/100 ammonia water (25 grams). The eluate was concentrated under reduced pressure and then freeze-dried to obtain the educt &5q of compound (I-1) as a white powder.

Effects of the Invention Compound (I) of the present invention can be used as a raw material compound for synthesizing cephem compounds useful as therapeutic agents for bacterial infections.

In addition, the compound (I-1) in which R is hydrogen in the compound (I) of the present invention is a stable compound, and thus is easy to handle when used as a raw material compound.

[Brief explanation of drawings]

Figures 1 and 2 show the compound (I) obtained in Example 1.
-2) and the infrared absorption spectra of compound (I-1) obtained in Example 2 are shown. Procedural Amendment (Salmon) August 2, 1985 1. Indication of the Case 1989 Patent Application No. 195985 2. Title of the Invention 7. Formylaminorcephem Compound and Process for Producing the Same 3. Person Making the Amendment Related Patent Applicant Address 2-27 Doshomachi, Higashi-ku, Osaka Name (2)
93) Takeda Pharmaceutical Co., Ltd. Representative Tadashi Kura
Iku 4 Division 4, Agent Address: 2-17-85-6 Jusohonmachi, Yodoyo-ku, Osaka City,
Contents of the amendment (1) "Deposited.
It is kept at the same research institute (FR■). ” is corrected. (2) r FERM P-78 on page 47, line 3 of the same book
Correct 36J to rFERM BP-637J. Notification of change in accession number Akihiro on August 2, 2011 1. Indication of the case 1982 Patent Application No. 195985 2. Title of the invention 7. -Formylamino-cephem compound and its manufacturing method 3. Case of the person who filed the procedure. Relationship Patent Applicant Address 2-27 Doshomachi, Higashi-ku, Osaka Name Name
(293) Takeda Pharmaceutical Co., Ltd. Representative: Kurabayashi Ikushibu 4, Agent address: 2-17-85-5 Jusohonmachi, Yodoyo-ku, Osaka City,
Name of former depository institution: Institute of Microbiology, Agency of Industrial Science and Technology 68 Former depository number: Microbiological Research Institute No. 7836 (FEBM P-7836)
7. Name of the new depositary institution: Institute of Microbial Technology, Agency of Industrial Science and Technology 8 New deposit number: FERM BP-637 (FERM BP-637) 9
, List of attached documents (1) One or more documents certifying the new accession number

Claims (3)

[Claims] (1) General formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [In the formula, R is hydrogen or HOOC-(CH_2)_3-
Indicates a CO- group. ] or its salt. (2) Add HOOC-(CH_2)_3-CO-NH- group at the 7th position of the compound to NH_
A method for producing a compound represented by the formula ▲There are mathematical formulas, chemical formulas, tables, etc.▼ or a salt thereof, which is characterized by contacting a culture of a microorganism belonging to the genus Pseudomonas that has the ability to convert into a 2-group, or a processed product thereof. . (3) In the compound or its salt represented by the formula ▲There are mathematical formulas, chemical formulas, tables, etc.▼, the group at the 7th position of the compound is HOOC- (CH_2)_3-CO- A method for producing a compound represented by the formula ▲There are mathematical formulas, chemical formulas, tables, etc.▼ or a salt thereof, which is characterized by contacting a culture of a microorganism belonging to the genus Trigonopsis that has the ability to convert into an NH- group, or a processed product thereof. .