WO1986003205A1 - Antibiotic, process for their preparation, and microorganisms producing the same - Google Patents

Abstract

An antibiotic, TAN-588, produced by bacteria belonging to genus Empedobacter or Risobacter, p-nitrobenzyl- or benzhydryl ester derivatives thereof, N-deacetyl derivatives thereof, and salts thereof have an anti-bacterial effect on Gram-positive and Gram-negative bacteria, and can be used as agents for treating bacterial diseases of mammal, fowl, etc.

Description

 Specification

 Antibiotics, their production and microorganisms

Technical field

 The present invention relates to a novel antibiotic useful as an antibacterial agent, a method for producing the same, and a microorganism that can be used for the production.

Background art

 Conventionally, sulfazecin and isosulfazecin are known as antibiotics that inhibit the cell wall synthesis produced by pacteria (Nature, vol. 289, 590-591, 1981). 26700, 26823, 26875, 26970, 26812, etc. have also been discovered (Nature, 291: 489-491, 1981). These are mainly known as antibiotics that have antibacterial properties against Gram-negative bacteria. Recently, reports such as SQ 28332, 28502, 28503 have also been reported (J. Antibiotics, 36, 1245-1251, 1252-1257, 1983).

Disclosure of the invention

 The present inventors isolated a large number of microorganisms from soil for the purpose of searching for novel antibiotics, and searched for antibiotics produced by the microorganisms.As a result, it was found that certain microorganisms produce novel antibiotics. The microorganism is a new species belonging to the genus Endobacter and the genus Rhizopactor. By culturing the microorganism in an appropriate medium, an antibiotic having antibacterial activity against Gram-positive bacteria and Gram-negative bacteria can be obtained. Knowing that it can accumulate in the culture medium, isolating this antibiotic, confirming that it is a new antibiotic from its physicochemical and biological properties, and identifying it as an antibiotic TAN-588.

 The present inventors have further found that the above-mentioned antibiotic TAN-588 has an N-acetyl group and a carboxy group, and can release the acetyl group.

OMPI As a result of further research, the present invention was completed. The present invention relates to (1) an antibiotic TAN-588, its paranitrobenzil,

Or benzhydryl esters or their N-deacetyl derivatives or

Are their salts,

 (2) the antibiotic T AN belonging to the genus Endobacter or Rhizobacta

-Incubate microorganisms capable of producing 588 in the culture medium and add antibiotics to the culture.

Special feature is to produce and accumulate the substance TAN-588 and collect it.

A method for producing the antibiotic TAN-588 or a salt thereof,

 (3) The DNA content of GNA (guanine + cytosine) in DNA exceeds 70%

Lactamgenus,

(4) A white colony is formed, and 0—F (oxidative

Rative) The test is non-decomposable and has no assimilation of inorganic nitrogen source.

Bhakta * Arbus, ·

 (5) Production of N-deacetyl derivative of antibiotic TAN-588 or a salt thereof, which is characterized by deacetylating antibiotic TAN-588 or a salt thereof.

Law, and

 (6), introducing a benzhydryl group into the antibiotic TAM-588 or a salt thereof;

The resulting compound is anti-) S, and the antibiotic TAN-588 Benzhydryl ester

Antibiotics, which are then subjected to a deacetylation reaction.

Preparation of Benzhydryl ester form of N-Decetyl form of TAN-588

It is a construction method.

 In the present specification, the antibiotic TAN-588 may be simply referred to as "TAN-588".

 The antibiotic TAN-588 producing bacteria used in the method of the present invention include:

 Genus Empedobacter or Lysobacter

If it belongs to the genus and has the ability to produce the antibiotic TAN-588

O PI

WIPO, _v Any microorganism may be used. Examples of the production bacteria belonging to the genus Endobacter include, for example, a new bacterium, Endobacter lactamgenus.

(Empedobacter lactamgenus). As a specific example, the present inventors have collected Endobacta ♦ lactamgenus YK-258 (hereinafter also referred to as “YK-258”) collected from soil in Masuda, Shimane, Japan. Is raised.

 The mycological properties of the Υ-258 strain are as follows.

 (a) Form

After observation at 24 ^e C for 5 days on the gravy agar slope, the cells were 0.4-

It has a long shape of 0.6 ^ m, length of 2.0-3.Q〃ra, and may have a filament shape of 12-30111. There are no flagella and no motility. It does not form spores and has a negative Gram stain, indicating no acid resistance.

 (b) Growth on various media

 The cells were cultured at 24 ° C and observed for 1 to U days.

 (1) Gravy agar plate culture: The colony is translucent, light yellow, round, and has a head-shaped, perimeter; No diffusible dye is formed.

 ② Gravy agar slant culture: Shows good spread-like growth and shows a pale yellow or amber color.

 ③ Liquid broth culture: Grows in a mixed state, precipitates, and forms a pellicle.

 (4) Juice gelatin stab culture: Grows mainly in the upper part and liquefies like a crater. Liquefaction activity is comparatively weak.

 ⑤ Litmus. Milk: No activity of reducing, peptizing or coagulating litmus.

 (c) Physiological properties

 ① Reduction of nitrate:-

② Denitrification reaction:-

O PI ③ MR (Methyle ': / de) test: 1

 ④ VP (Forges. Pros power) test: —

 Generation of Pin Doll: — ·

 水 素 Production of hydrogen sulfide (TS I agar and lead acetate paper):-

⑦Hydrolysis of starch: —

 ⑧Utilization of citric acid (Coselle, Christensen and Zines media):

 利用 Utilization of inorganic nitrogen source-i) Potassium nitrate: —

 ii) Aluminum sulfate:

 (4) Pigment formation (King A, B and mannitol yeast agar medium): No diffusible pigment formation is observed.

 ⑪ ゥ Rare one: one '-'

 Poxidase: One

 ⑲ Catalase: One

 @ Growing S

 i) pH: Grows at pH 5.4-5, but the optimal pH is 5.8-6.6.

 Medium: Glucose 0.1%, Discharge extract 0.01%, ammonium sulfate 0.1%, salt 0.1%, magnesium sulfate (7-hydrate) 0.05%, phosphate buffer Q.1M (separate sterilization).

 i i) Temperature: Growing at 20-32 ° C, the optimal temperature is 24-3 It;

 Medium: broth liquid medium.

 ⑮ Attitude toward oxygen: aerobic

 ⑮ 0—F (oxidative-fermentative) test [Hugh-Leifson method]: Non-decomposed type.

© Production and utilization of acid and gas from sugar: Acid gas availability

 (Putton water) (Peptone water) (Davis medium)

L-arabinose

 D—Kinrose

 D-Darcos

 D — Mannose

 D-fructose

 D—Galactoose

 Malt sugar

 Sucrose

 Lactose

 Trendy Rose

 D—Sorbit

 D — Mannite

 Ino sit

 Glycerin

 Starch

G GC (guanine-cytosine) content of DNA: 74.4% ± 1.5% (Tm method) 分解 能 Polysaccharide resolution:

 Carboquin methyl cellulose: +

 Colloidal chitin:

 Sodium alginate: one

 Susceptibility to pactinomycin: resistance

The YK-258 strain having the above mycological properties was purified using Purges' Manual Deter- minateif * P'Bergey's annLaL of Determinative Bacteriology) Eighth Edition and International ♦ Journals, Jobs, Systematics,

(International Journal of Systematic B acteriology) Younger brother, Vol. 30, pp. 225-420 (1980), and compared with the species described in Vol. It is aerobic, has no ability to generate acid and gas from sugar, and has a high GC content in DXA. Therefore, it is appropriate to belong to the genus Flapobacterium. However, it has been pointed out from the viewpoint of bacterial taxonomy that the species of the genus Flavobacterium described so far are heterogeneous, and in recent years the definition of the genus Flavobacterium has been revised. Was performed in the International National Journal of Systematics 'Bacteriology', Vol. 29, pp. 416-426 (1979). References and Manuals ♦ Review According to the book “Annual Review of Microbiology”, Vol. 37, pp. 233-252 (1983), strain YK-258 is from the genus Flavobacterium. However, it is more appropriate to claim that it belongs to the genus Endopacta. However, there is no description of a species whose GC content exceeds 70%. _C, designated & Rakuta Mugenusu (.E mpedobacter lactamgeniis) - where the YK- 258 strain, found as a strain belonging to斩菌species, E down Bae a該新species Dobakuta

Lactamgenus YK- 258 strains from February 20th, 1984 (February 20th, 1984), Fermentation Research Institute (IF 0, 2-chome, 17-3 Honcho-cho, Yodogawa-ku, Osaka, Osaka, Japan) No. 85) as IF 0 14322. In addition, the microorganism was transferred to the Research Institute of Microorganisms and Technology (FRI, 1-3-1 Higashi, Yatabe-cho, Tsukuba-gun, Ibaraki, Japan) on March 26, 1984, from the Ministry of International Trade and Industry of Japan. Deposited under accession number FE RM P-1 7558. Examples of the bacterium producing the TAN-588 antibiotic belonging to the genus Rhizobacta used in the method of the present invention include, for example, a new strain of lysobacter albus. (Lysobacter albus). As a specific example, the present inventors may abbreviate Rhizobacta albus sp. Nov. YK-422 strain (hereinafter referred to as “YK-422 strain”) collected from soil in Kanzaki-gun, Shiga Prefecture, Japan. ).

 The bacteriological properties of the YK-422 strain are as follows.

 (a) Form

 After 2 days of culture in broth liquid culture, the cells are 0.4 to 0.7 μιη in diameter, 2.0 to 4.4 μα in length, and may show filaments of 20 to 30 μm. No flagella, no motility. It does not form spores or microcytosis and is negative for Gram stain and does not show acid resistance.

 (b) Growth on various media

 The cells were cultured at 24 ° C and observed for 1 to 14 days. -

(1) Gravy agar plate culture: The colony is translucent white, round, convex and convex on the surface, and the entire periphery is mucoyal. No diffusible dye is formed.

② Gravy agar slant culture: Shows good growth of itofushi and shows white color.

 (3) Broth liquid culture: grows in a lightly mixed state, forms a small amount of precipitate, and forms a weak bacterial ring.

 ④ Nuts gelatin stab culture: grows on top and liquefies in layers-liquefaction activity is relatively strong.

 ⑤Litmus · Milk: Reduction of litmus and peptone formation are observed, but no coagulation is observed.

 (4) Dried yeast plate: A lysis circle is formed around the colony, and it shows mobility due to gliding.

 (c) Physiological properties

 ① Reduction of nitrate:-

② Denitrification reaction:- ③ MR (methyl red) test: 1

 ④ V P (Forges Proscowell) Test: ―

 Generation of Pindol: —

 水 素 Production of hydrogen sulfide (TS I agar and lead acetate paper):-

加 水 Hydrolysis of starch: 1

 ⑧Utilization of citrate (Christensen and Simmons medium): + 利用 Utilization of inorganic nitrogen source

 i) Potassium nitrate: —

 Π) Ammonium sulfate: —

 ⑩Dye formation (King Α, Β and Mannit yeast extract agar media)

: No diffusible dye was formed.

 ⑪ ゥ Rease: One

 Poxidase: Ten '

 ⑱ Catalase: +

 ⑭Range of growth

 i) pH: Grows at pH 4.6 to 8.2, but the optimal pH is 6.3 to 7.9.

 Medium: glucose 0.1%, yeast extract 0. (U%, ammonium sulfate G .1%, salt 0.1%, magnesium sulfate (7-hydrate) 0.05%, pH is soda or sulfuric acid Adjust with.

 i i) Temperature: Growing at 14 ~ 32 ° C, the optimal temperature is 21 ~ 28 ° (.

 Medium: broth liquid medium.

 ⑮ Attitude toward oxygen: aerobic

 ⑱ 0—F (oxidative-fluent) test: Hugh * Leifson method]: Non-decomposition type.

® Generation and Utilization of Acid and Gas from Sugar Acid gas availability

 (Peptone water) (Peptide-bis medium)

L-arabinose

D—Xylose + D—Dalco

D—Manno

D—Fructose

D—Galactooth

Malt sugar

Sucrose + lactose

Trenoleose 4-

D—Sorbit

D—Mannit '4-Boar' /

Glycerin

Starch

G DMA GC (guanine + cytosine) content: 70.2% ± 1.5% (Tm method) ⑬Multiple resolution:

 Carboxymethylcellulose: +

 Colloidal chitin: +

 Sodium alginate:

The YK-422 strain having the above mycological properties was obtained from Vergy's Manual of Determinative. Bacteria's Manual of Determinative Bacteriology, 8th Edition and International II. • Journal “Ob” Systematic Bacteriology, Volume 30, 225-420 (1980), Volume 32, pp. 146-149 (1982) When compared with the species listed in the validation list, it is a gram-negative bacillus that shows mucoidal growth, and some filamentous bacteria are also observed, showing motility due to gliding and having the resolution of colloidal chitin and dried yeast. However, since it does not form micromixes and has a high GC content in DNA, it is appropriate to belong to the genus Rhizobacta. However, the bacterial species of the genus Rhizobacta described so far (International Journal of the Systemic. Bacteriology, Vol. 28, pp. 367-393 (1978)) The tests differed in that the test was non-degradable, did not have any distinct color in the colonies, and had no assimilation of inorganic nitrogen sources. So YK—

The strain 422 was identified as belonging to a new strain, and the new strain was named Lysobacter albns sp. Nov. YK-422.

 The above-mentioned Resobacta 'Arbus sp. Nov. ΥΚ-422 was assigned to IF0 on October δ, 1984 (1984) as accession number IF 0 14384, and to FRI on November 14, 1984 (1984). Deposited under accession number FERM II-7938.

 The properties of the bacteria of the genus Endobacter and the genus Rhizobacter used in the present invention are generally liable to change. It can be easily mutated by artificial mutation means, and any mutant having the ability to produce TAN-588, which is the object of the present invention, can be used in the present invention.

In culturing TAN-588 producing bacteria, carbon sources include, for example, glucose, fructose, maltose, solvable starch, and dextrin. Ingredients that can be assimilated by bacteria such as oils, fats (eg, soybean oil, olive oil, etc.), and organic acids (eg, citric acid, succinic acid, dalconic acid, etc.) are used as appropriate. As the nitrogen source, for example, organic nitrogen compounds such as soybean flour, sweeping powder, corn gluten, meal, dry yeast, yeast extract, meat extract, peptone, and urea can be used. As the inorganic salt, for example, inorganic salts necessary for culturing normal bacteria, such as sodium chloride, chloride chloride, calcium carbonate, magnesium sulfate, lithium phosphate, sodium phosphate, etc., are used alone. Alternatively, they are used in combination as appropriate. - In addition, heavy metals, such as ferrous, copper sulfate, Vita Mi B _1, are added as necessary such as Vita Mi emissions such as Piochin. Furthermore, an antifoaming agent such as silicone oil / polyalkylene glycol ether or a surfactant may be added to the medium. In addition, organic and inorganic substances that support the growth of bacteria and promote the production of TAN-588 may be appropriately added. '' The cultivation method may be the same as that of 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, and aeration culture may be performed, but aeration and agitation culture is particularly preferred. The culture temperature is preferably about 15 ° (: to 32 ° C., and the pH of the medium is about 5 to 8 for about 8 to 168 hours, preferably for 24 to 144 hours.

 In order to collect the desired antibiotic TAN-588 from the culture, a separation method usually used to collect a metabolite produced by a microorganism from a culture of the microorganism is appropriately used. For example, the antibiotic TAN-588 exhibits the properties of a water-soluble acidic substance and is mainly contained in the culture filtrate.Therefore, first, a filtration aid is added to the culture, and the cells are removed by filtration or centrifugation to obtain The obtained culture filtrate is brought into contact with an appropriate carrier to adsorb the active ingredient in the filtrate, and then the active substance is desorbed with an appropriate solvent, and a means for separating and collecting is advantageously used.

O PI Used. Use the difference in the adsorptivity of compounds such as activated carbon, silica gel, powdered cellulose, and adsorbent resin as the carrier for chromatography, or use the difference in the functional groups of compounds such as anion-exchange resin and anion-exchange cellulose, or It is advantageous to use the difference in molecular weight of compounds such as sieve carriers. 'In order to elute the target compound from these carriers, combinations vary depending on the type and properties of the carrier.For example, aqueous solutions of water-soluble organic solvents, that is, aqueous acetone, aqueous alcohols, etc., or acids, alkalis, etc. A buffer, an aqueous solution containing an inorganic or organic salt, or the like is used in appropriate combination. In addition, the crude substance of the present antibiotic obtained by these chromatography can be subjected to preparative high performance liquid chromatography for further purification. Concentrate the eluate, which has been desalted by activated carbon chromatography, and use the ion-paired extraction method from the concentrate, i.e., recover the antibacterial substance using an organic solvent containing quaternary alkyl ammonium halide. Can also.

 More specifically, an anion exchange resin such as DEX-1 (manufactured by Dow & Chemical Co., USA) as a carrier, Amberlite

 Filtration using IRA-68, 400, 402, 4i0 (manufactured by K. And Haas, USA), Diaion SA—2iA and C (manufactured by Mitsubishi Chemical Industries, Ltd., Japan) This antibiotic is adsorbed and eluted with an aqueous solution or buffer containing salts or acids. Also, anion exchange cellulose such as

D E-32 (Pittman, UK), D E A E-cellulose (Brown

Or anion exchange molecular sieve resin such as D E

A E — or Q A E — Sefadex (Falmasha, Sweden

(Den), etc. to absorb the antibiotics into water containing salt or acid.

It can be eluted with a solution or a buffer. Activated carbon for chromatography (Takeda) to remove salts, coloring substances, etc. in these eluates

^WIP . ,

, Mi, '

' Pharmaceutical Co., Ltd., Japan) or adsorbent resin such as Diaion HP-20 (Mitsubishi Kasei Kogyo Co., Ltd., Japan), Amberlite XAD- 辽 (Rohm & Haas Co., USA) are advantageous Used for The fractionated elution fraction is made into a powder through processes such as concentration and freeze-drying. When the purity of the powder thus obtained is poor, high-performance liquid chromatography is advantageously used for further purification. Examples of the carrier to be used include TSK gel (manufactured by Toyo Soda Co., Ltd., Japan) and YMC gel (manufactured by Yamamura Chemical Research Laboratories, Japan). The moving layer is made of an acidic material such as methanol or acetonitrile. A mixed solution with an aqueous solution or a buffer solution is used. The quaternary alkyl monoxide used in the above-mentioned ion-pair oil extraction method is, for example, tri-n-butyl methyl ammonium chloride, tetra η-ventil ammonium chloride. And η'-tetradecyl methylbenzylammonium sigma-ride, etc. As the organic solvent, methylene chloride, chloroform, dichloroethane and the like are usually used.

 In the purification process, TAN-588 is present in the form of salts bound to the salts used in the buffer and to the ions in the buffer, such as sodium, calcium, lithium, calcium, ammonium and the like. In this case, the corresponding salt is isolated as the corresponding salt when subjected to activated carbon chromatography at the same pH, and the eluate is adjusted to a pH of about 5 to 2, preferably pH about 4.5 to 3, and the activated carbon chromatography is performed. To give a free form.

TA-588 thus obtained has two peaks on reversed-phase high performance liquid chromatography. If these peaks are referred to as Α and Β, the following phenomena are observed. When the peaks of A and そ れ ぞ れ are respectively collected by preparative high-performance liquid chromatography, a fairly single A and B can be obtained. These are left at room temperature in the pH 3, 5, and 7 buffer solution. Any pH After about an hour, A becomes B, B becomes A, and A: B changes to a 1: 1 equilibrium mixture. Therefore, it seems that TAN-588 itself cannot be separated into Α and Β by the currently known separation technology. However, if TAN-588 is a ρ-nitropentyl benzyl ester or a benzhydryl, it can be divided into A-type compounds and B-type compounds, respectively.

 As a method for deacetylating the antibiotic TAN-588 or a salt thereof, a known deacetylation reaction is employed.

 As an example, for example, first, a benzyl or benzhydryl group is introduced into TAN-588 to form an ester form, and then the acetyl group of TAN-588 is eliminated. It is performed by removing the Nichrill group.

 In order to introduce the above-mentioned para-nitro benzyl group, a compound capable of introducing a para-nitro σ-benzyl group is reacted with TAN-588 or a salt thereof. Examples of the compound into which the paranitrobenzyl group can be introduced include, for example, paranitrobenzyl sigma-ide, benzyl chloride at the paranitole.

The use of the compound capable of introducing a paranitrobenzyl group is about 1 to 5 equivalents, preferably about 1 to 2 equivalents. The anti-Jt & is preferably performed in a solvent, and examples of the solvent include dimethylformamide (DMF), dimethylacetamide (DMAA), and tetrahydrofuran (THF). In this reaction, for example, triethylamine (Et ₃ N), pyridine and the like are used in an amount of about 0.1 to about 10 to promote the reaction. 1.5 equivalents, preferably about 0.1 to 0.2 equivalents, may be added.

The reaction temperature is about 0 to 40 ° C., more preferably about 20 ° (: to 30 ° C.), and the reaction time is about 0.5 to 8 hours, more preferably about 1 to 4 hours. Preferably. To introduce the above benzhydryl group, a compound capable of introducing a benzhydryl group is reacted with TAN-588 or a salt thereof. Examples of the compound into which the benzhydryl group can be introduced include, for example, diphenyldiazomethane, diphenylmethylbutamide and the like. The amount of the compound capable of introducing a benzhydryl group is about 1 to 6 equivalents, preferably about 2 to 4 equivalents. The reaction is preferably carried out in a solvent, such as THF, dioxane, ethyl acetate, dichloromethane or the like. In the reaction, in order to accelerate the reaction, a small amount of, for example, dilute hydrochloric acid, dilute sulfuric acid, dilute phosphoric acid, or the like is added, for example, about 0.01 to 1.0, and equivalents of PH are added to about 1 to 3, preferably It is preferable to adjust the value to approximately i.5 to around 2.5. The reaction temperature is about -10 ° C to + 50 ° C, more preferably 0 ° C to 30 ° C, and the reaction time is about 30 minutes to about 8 hours, and more preferably about 1 to 3 hours. The reaction is preferably carried out with stirring.

 The ester obtained above can be collected using a commonly used separation and purification means. As the means, for example, the target substance is extracted into an organic layer with, for example, dichloromethane, chloroform, or the like, the extract is concentrated, and the concentrate is added to ether or hexane to convert the ester to a crystalline powder. Start out. This ester is isolated into two components by the silica gel method, but may be used as a mixture when proceeding to the next reaction.

 Further, the para-pentoxy benzyl ester or benzhydryl of TAN-588 obtained above is subjected to a deacetylation reaction.

 Examples of the deacetylation reaction include an imino ether method, a solvolysis method, and a hydrolysis method using an enzyme.

When the iminoether method is used, for example, the raw material compound is reacted with phosphorus pentachloride, phosgene, phosphorus trichloride, oxychlorine, or the like. The above reaction reagent is preferably used in an amount of about 1 to 5 equivalents, more preferably about 1.5 to 3 equivalents. New It is convenient to carry out the reaction in the presence of a solvent such as methylene chloride, dichloroethane, chloroform, carbon tetrachloride, and trichloroethane. In order to promote the reaction, for example, pyridin, Ν, Ν-dimethylanilin, triethylamine, aniline, toluidine and the like may be used in excess, for example, about 3 to 20 equivalents, more preferably about 5 to 10 equivalents.

 The deacetylation reaction is carried out at a reaction temperature of about 130 ° C to 0 ° C, more preferably at 115 ° C to -5 ° C, and a reaction time of about 15 minutes to 8 hours, more preferably about 30 minutes to 2 hours. Good to do. The reaction is conveniently carried out with stirring.

 An excess of methanol is added to the reaction solution in order to convert imino chloride formed as an intermediate into an imino ether, and is added at about -30 to 0, preferably about 115 to 15 ° C. Stir for 15 minutes to 2 hours, preferably for about 30 minutes to 1 hour, and add about 10 ° (to about 40 ° C, preferably about 20 to 30 ° C for about 30 minutes to 2 hours) to complete the reaction. Stirring Further, dilute hydrochloric acid is added to the reaction solution to break C-N bond Reaction temperature is about 10 ° C to 40 ° C, preferably about 20 ° C to 30 ° C, and reaction time is about 15 minutes to 15 minutes. At 2:00, it is about 30 minutes to 1 hour.To use the solvolysis method, for example, dissolve the starting compound in methanol, ethanol or a mixed solution of them with water, and add about 20 ° C to reflux temperature, preferably at about 50 ° C to reflux temperature, for about 0.5 to 30 hours, preferably for about 2 to 8 hours. Let

 The reaction solution thus obtained is neutralized and the product is extracted with water-miscible L, an organic solvent such as methylene chloride, getyl ether, ethyl ethylate, and the extract is concentrated. Thus, a deacetyl-form paranitrobenzyl or benzhydryl form of TAN-588 is obtained.

Next, in order to remove the ester group as a final step, for example, an acid hydrolysis method, a catalytic reduction method, or the like is used. When the acid hydrolysis method is used, the reaction is carried out using, for example, about 3 to 20 equivalents of an acid, such as trifluoroacetic acid, formic acid, or hydrochloric acid, based on the starting compound. It is also preferable to add about 1 to 5 equivalents, preferably about 2 to 4 equivalents, of anisol. In this reaction, as a solvent, for example, methylene chloride, chloroform, THF, ethyl acetate or the like is used.

 The reaction temperature is about −30 ° C. to 0 ° C., more preferably about −20 ° C. ^ −— 10 ° C., and the reaction time is about 0.5 to 8 hours, more preferably about 1 to 4 hours. .

 When the catalytic reduction method is used, for example, palladium, platinum, their oxides and the like are used as a catalyst and reacted under a stream of hydrogen.

 The reaction temperature is about 0 to 50 ° C, more preferably about 10 ° (: to 40 ° C), and the reaction time is about 0.1 to 6 hours, more preferably about 0.2 to 2 hours.

 The free carboxylic acid thus formed is separated and collected by removing impurities in the reaction mixture by filtration or chromatography, for example, using activated carbon, an adsorbent resin, etc., and then concentrating and freeze-drying. , Can be purified.

 In each of the above steps, when the obtained compound is a mixture of isomers, for example, column chromatography, for example, silica gel, Sephadex LH-20 (Pharmacia, Sweden) as a carrier, Diaion HP-20, etc. Chromatography using preparative method, or recrystallization method. Preparative reversed-layer chromatography [Examples of carrier: YMC gel, TSK gel, Examples of mobile phase: buffer or buffer containing methanol or acetonitrile] ] Can be separated into each isomer component.

 The physicochemical properties of the TAN-588 sodium salt (mixture of A and B) obtained in Example 1 described below are shown below.

 1) Appearance: white powder

,, WIPO ~ " twenty three

 Specific rotation: [α] 19.0 ° ± 10 ° (c = 0.5, in water)

 D

) Elements whose constituent elements consist of C, H, N, 0 and Na:% pentoxide

 Material dried at 40 ° C on a Lin for 6 hours

Actual value Calculated value ' ^:

 C, 38.5 Sat 2.0 C, 39.61

 H, 4.5 ± 0 H, 3.99

 N, 9.1 ± 1.5, 9.24

 0, 39.58

 Na, 6.9 ± 1.5 Na, 7.58

 Calculated as including 0.5 mole of attached water)

) Moisture content: 3.0 ± 1.5% (hot-air method)

) The molecular ion peaks by the SIMS method are as follows:

 ヽ ヽ ヽ

m / z '611 (2M + Na) ", 317 (M + Na), 29

+ Η)

) 'Molecular formula:

) Ultraviolet absorption (UV) spectrum (in water): Fig. 1 λ216nm (E = 130, shoulder)

 max lcm

) Infrared absorption (IR) spectrum (KBr method):

 The main absorption (wave number) of the absorption spectrum (Fig. 2) due to the bromide power failure is as follows.

3450, 1780, 1730, 1660, 1550, 1385, 1320, 1290, 1260, 1200, 1120, 1040, 980, 910, 810, 770, 690, 600, 540cm one ¹

) ¹³ C—nuclear magnetic resonance (N'MR) spectrum (100 MHz, in heavy water):

 A signal is observed.

 182.02 (s), 177.30 (s), 173.79 (s), 173.30 (s), 173.25 (s),

One OMPI

, IPO 172.58 (s), 96.97 (s), 96.92 (s), 74.27 (t), 72.63 (t),

 55.57 (d), 55.34 (d), 31.92 (t), 31.08 (t), 30.98 (t), 24.58

 (.ppm (however, s; singlet, d; doublet, t; triplet,

 q; quartet)

) Circular dichroism (CD) spectrum (underwater):

 A negative Cottton effect is shown at 232 ± 3 nm.

 Solubility:.

 Soluble; water, dimethyl sulfoxide.

 Poorly soluble; ethyl acetate, kuguchiguchi form, getyl ether.

) Color reaction:

 Positive; ninhydrin reaction

 Negative: Greig's Leeback, Sakaguchi, Ehrlich, Baton, De

 Lagen-Dolph reaction

) Amino acid analysis: known to be hydrolyzed in 6 N hydrochloric acid for 20 hours at 105 ° C

 Serine is detected as amino acid.

) Stability: Stable at pH 5 in aqueous solution, slightly unstable at pH 3 and 7,

 Unstable at pH 9.

) Thin layer chromatography (TLC) (Cellulose f, Tokyo Chemical Industry Co., Ltd.)

 Made by company, Japan):

 Solvent system Rf value Acetonitrile: water (4: 1) 0 33

Butanol: acid fraud: water (1: 1: 1) 0 77

Acetonitrile: 3% ammonium sulfate (4: 1) 0 28) Distinguishing between acidic, neutral and basic: neutral substances

) High Performance Liquid Chromatography (HP LC) (Carrier: YMC A-312,

(O PI Manufactured by Yamamura Chemical Laboratory, Japan, mobile phase: 4%, methanol / 0.01M phosphate buffer (PH6.3), 2 mlZmin):

 Rt = 4.3 and 4.8 (min)

 The physicochemical properties of the paranitrobenzyl ester of TAN-588 (mixture of A and B) obtained in Example 4 described below are shown below.

 1) Appearance: white powder

2) Specific rotation: [] — + 16.3 ° ± 5. (C = 0.485, CHC13 in ₃ )

3) Molecular weight: 407 (by SMS method)

 4) Element analysis value:

 Calculated: C, 50.13; H.4.21; N.10.32; 0, 35.35

 Found: C, 50.26; Η, 4.32; Ν, ΙΟ.31

5) Molecular _{formula: C 17 Η 17 Ν 3,} ο 3 '.

6) 'UV spectrum: λ ^{Μθ0 Η} (Ε%) = 262 ± 2 (281 ± 20),

 max lcm

 214 ± 2 (278 ± 20, shoulder)

 7) IR spectrum: KBr method, Fig. 3

3400, 3080, 2960, 1805, 1760, 1680, 1610, 1520, 1450, 1380, 1350, 1270, 1180, 1105, 1050, 1015, 970, 905, 850, 740, 690, 600, 540 cm- ¹

8) NMR spectrum: 90Mgz, CD Cl ₃ in S ppm J (Hz)

2.05 (3H, s), 2.3-3.3 (4H, m), 4.10 (lH, m), 4.5-5.l (2H, m), 5.35 (2H, s), 6.2o (lH, d _> like) _> 7.55 (2H, dd.like) ₎ 8.27 (2H, d, like)

9) T L C:

 Carrier: silica gel (Merck, West Germany)

 Developing solvent: chloroform: methanol (19: 1)

OMPI Rf value, 0.25 and 0.32

10) Acidity. Neutral and basic: 'Neutral substance

 The physicochemical properties of TAN-588A paranit σ benzyl ester obtained in Example 4 described below are shown below.

 1) Appearance: white powder

 Q

2) Specific rotation: [α] + 97.3 ° ± 15 ° (C = 0.48, in CHC 1 ₃₎

3) Molecular weight: 407 (by SMS method)

 4) Elemental Xin value:

 Calculated: C, 50.13; H, 4.21; .10.32; 0, 35.35

 Found: C, 50.20; H, 4.22;, .13

δ) Molecular formula: C ₁₇ H ₁₇ N ₃ 〇 ₉

6) UV spectrum: ^{46 0 H} E ^i% on λ) = 262 ± 2 nm (280 ± 30),

 max lcm

 214 earth 2 nm (276 ± 30, shoulder)

7) ¹³ C-NMR _{spectrum: (100MHz, CDC 1 3)} ,

 173.70 (s), 171.o3 (s), 170.72 (s), 165.09 (s), 148.06 (s), 141.38 (s), 128.86 (d), 123.91 (d), 9i.82 (s), 71.60 (t), 67.29 (t), 53.00 (d) / 29.09 (t), 27.49 (t), 22.64 (q). ppm. 8) IR spectrum: KBr method Fig. 4

3400, 3080, 2950, 1805, 1775, 1760, 1680, 1610, 1530, 1450, 1380, 1350, 1300, 1275, 1190, 1105, 1060, 1020, 980, 910, 850, 740, 700, 600, 540 cm " ¹

 9) TLC:

 Carrier: silica gel (Merck, West Germany)

Developing solvent, quenched form: methanol (19: 1) Rf value '0.25

10) Distinguishing between acidic, neutral and basic: neutral substances

 The physicochemical properties of TAN-588B para-nitrate benzyl ester obtained in Example 4 described below are shown below. 1) Appearance: white powder

 20

2) Specific rotation: [α] 64.5. ± 15 ° (C = 0.50, in CHC 1 ₃₎

 D

 3) Molecular weight: 407 (by SMS method)

4) Element value:

 Calculated: C, 50.13; H.4.21; N.10.32; 0, 35.35

 Found: C, 50.10; H.4.21; Ν, 10.15

5) Molecular _{formula: C 17 H 17 N 3 0} 3

6) UV spectrum: λ ^{Μθ0 η} (Ε%) = 262 soil 2 Μ (282 ± 30),

 -max icm *.

 214 ± 2 nm (280 ± 30, shoulder)

7) ^13C— NMR spectrum: (100MHz, CD Cl ₃ ),

 173.59 (s), 170.86 (s), 170.61 (s), 165.06 (s), i48.12 (s),

 1.24Cs), 128.96 (d), 123.96 (d), 91.69 (s), 74.60 (t), 67.39 (t), 51.94 (d), 29.11 (t), 27.38 (t), 22.67 (q) .ppm .

 8) IR spectrum: KBr method, Fig. 5

3400, 3090. 2950, 1805, 1760, 1680, 1610, 1530, 1450, 1380, 1355, 1270, 1180, 1105, 1055, 1015, 965, 910, 855, 740, 695, 600, 540 cm " ¹

9) 1 ¹し 〇: Choha 1-588 octaparanitone Same conditions as for benzyl ester

 Rf value, 0.32

O PI 10) Distinguishing between acidic, neutral and basic: neutral substances

 The physicochemical properties of TAN-588 benzhydryl ester (mixture of A; and B types) obtained in Example 5 described below are shown below.

 1) Appearance: colorless crystal

 2) Melting point: 153-155 ° C (decomposition point)

 twenty three

3) Specific ^{rotation: [α] 9.2 ° ϋ5 0} (C = 0.52, in CHC 1 ₃₎

 D

 4) Molecular weight: m "z 438 (M +) (E I-MS method)

 5) Element analysis value:

 Calculated: C, 63.01; H.5.06; .6.39; 0, 25.54

 Found: C, 62.83: H, 5.32;, 6.28

6) Molecular formula: C ₂₃ H ₂₂ N ₂ 0 ₇

 7) UV spectrum: In methanol, λ max 220 ± 2 nm (50, shoulder) and

250 -260nm (E 28 ± 10, shoulder)

 lcm

 8) IR spectrum: KBr method, Fig. 6

3380, 3080, 3050, 2960, 1800, 1780, 1750, 1705, 1690, 1600, 1590, 1540, 1500, 1460, 1380, 1310, 1280, 1190, 1110, 1060, 980, 920, 880, 750, 710, 700, 650, 630, 610, 570, 550, 470 cm " ¹

9) - NMR spectrum: 90 MHz, in CD Cl _3,

 δ ppra J (Hz),

 1.97 (3H, s), 2.1-3.o (4H, m), 3.8-.2 (1H, m), 4.-5.1 (2H, m), 6.1-6.4 (iH, br), 6.97 (lH , s), 7.3 -7. (10H, m)

(m: miiltiplet, br: broad, H: roton) 10) TLC: Same conditions as those for Type A described below

 Rf values, 0.58 and 0.65

 11) Distinguishing between acidic, neutral and basic: neutral substances

 The physicochemical properties of TAN-588 Benzhydryl ester (A type) and TAN-588 Benzhydryl ester (B type) obtained in Example 5 described below are shown below.

Type A compound

 1) Appearance: colorless crystal

 2) Melting point: 97 ~ 135 ° C (slow foaming, decomposition)

3) Specific ^{rotation: ^] 1 + 44.2 ° ±} 10 ° (C = 0.505, in CHC 1 ₃₎

 D

 4) Molecular weight: molecular ion peak by EI-MS method

 m / z 438 (M)

 5) Element analysis value: '

 Calculated: C, 63.01; H.5.06; N.6.39; 0, 25.54

 Found: C, 62.62; H.5.06; .6.32

6) Molecular formula: C ₂₃ H ₂₂ N ₂ 0 ₇

 7) Ultraviolet absorption (UV) spectrum: (in methanol)

 1

 λ 220 person 2 nm (E = 290 soil 50, shoulder)

 max lcm

 1%

 And 250—260 (E = 30 ± 10, shoulder)

 lcm

 8) IR spectrum: KBr method, Fig. 7

 .3380, 3080, 3050, 1800, 1780, 1760, 1685, 1540, 1500, 1450, 1380, 1310, 1280, 1190, 1110, 1050, 980, 920,

880, 750, 710, 650, 610, 550 cm ¹

. 9) ^l H- NMR _{spectrum: iOOMHz, CDC 1 3, d} e - DMS O blend

,,, WIPO. \

- In liquid, <5 PPm J (Hz)

 1.98 (3H, s), 2.2-3.4 (4Η, πι), .10 (1H, dd, J = 8, 10), 4.4-5.0 (2H, m), 6.93 (lH, s), 7.3-7.5 ( 10H, m), 8.27 (1H, d, J = 7)

 0) Thin layer chromatography (TLC):

 Carrier, silica gel (Merck, West Germany),

 Solvent, ethyl acetate

 Right, 0.58

 1) Distinguishing between acidic, neutral and basic: neutral substances

Type B compound

 1) Appearance: colorless crystal

 2) Melting point: 157-160 ° C (decomposition point)

3) Specific ^{rotation:. [Α] 21 - 28.8} ° ± 10 ° (C = 0.5, in CH C1 ₃₎

 D

 4) Molecular weight: m / z) (E I-iMS method)

δ) Element analysis value:

 Calculated: C, 63.01 H.5.06; N, 6.39; 0, 25.54

 Obtained: C, 63.U H.5.13;, 6.30

6) Molecular formula: C ₂₃ H ₂₂ N 0

 7) J V spectrum: Medium

 1 o /

 λ 220-2 nm (E = 300 = 50, shoulder)

 max lctn

 1%

 And 250-260nm (E 26 ± 10, shoulder)

 lcm

 8) IR spectrum: KBr method, Fig. 8

3400, 3080, 3050, 1815, 1780, 1735, 1705, 1540, 1460, 1380, 1290, 1265, 1190, 1060, 980, 920, 880, 760, 715, 610, 550 cm one ¹ 9) — NMR spectrum: 100MHz, in CD Cl ₃

 δ ppm J (Hz),

 1.98 (3H, s), 2.2-3.4 (4H, m), 4.03 (1H, dd, J = 8, 10), 4.6-5.2 (2H, m), 6.32 (lH, d, J = S), 6.96 (lH, s), 7.2 -7.5 (10H, m)

 10) T L C: (Same conditions as for type A)

 Rf value, 0.65

 11) Distinguishing between acidic, neutral and basic: neutral substances

 The physicochemical properties of the benzhydryl ester (mixture of type A and type B), which is a decyl form of TAN-588, obtained in Example 6 are shown below.

1) Appearance: white powder

 twenty five

2) Specific _{rotation: [a] D - 15.2 ±} 5 ° (C = 0.5, in CHC 1 ₃₎

3) Molecular weight: m / z 396 (M +) (E I-MS method) ·

 4) Element analysis value: '

 Calculated: C, 63.63; Η, δ.09; .7.07; 0, 24.22

 Found: C, 63.63; Η.5.05; Ν, 7.02

δ) Molecular formula: C ₂₁ H ₂ . N ₂ O _e

6) U \ Skdoll: Medium

1 ⁰ ί

 λ 220 nm (E = 336 ± 50, shoulder)

 max 1cm

 1 o

 250 -260nm (E, = 32 ± 10, shoulder)

 lcm

 7) IR spectrum: KBr method, Fig. 9

3400, 3050, 2970, 1800, 1780, 1740, 1600, 1500, 1460, 1305, 1270, 1190, 1110, 1060, 980, 920, 880, 850, 750, 710, 650, 620, 605 cm " ¹

8) ¹ H—NMR spectrum: 90 MHz, in CD Cl ₃ δ ppm J (Hz),

 2.2-3.5 (4H, m), 3.7 -4.0 (2H, m), 4.4 -4.6 (iH, m), 6.97 (lH, s), 7.2-7.4 (10H, m)

9) High performance liquid mouth chromatography (HPLC):

Equipment, Model

Waters, USA) Carrier, YMC-Pack A-312 (Yamamura Chemical Research Pound, Japan) Mobile phase, 65% methanol / 0.01M phosphate buffer (PH6.3) Flow rate, 2 ! 111 [^ 11,1 ぃ .5.3 び 5.61 ^ 1

 10) Color reaction:

 Positive, ninhydrin

 Negative, ferric chloride

 11) Distinguishing between acidic, neutral and basic: basic substances

 The physicochemical properties of the deacetyl derivative of T A —588 (mixture of type A and type B) obtained in Example 7 are shown below.

 1) Appearance: white powder

2) Specific rotation: [α] "— 11 ° 2 5 ° (C = 0.1, in water)

3) Molecular weight: ηιΖζ · 231 (Μ-H) + (FD-MS method)

 4) Element analysis

 Measured value Calculated value

 C, 40.42 C, 40.17

 Η, 4.36 H, 4.64

 , 11.65 N.11.71

 O, 43.48

 (<< · Calculated as including 0.5 mol of attached water)

5) Molecular _{formula: C e H ^ NsOs ^ .SHsO} ) ) UV spectrum: Underwater

 1%

 λ 221 ± 2 nm (E, = 154 ± 20)

 max lcm

) IR spectrum: KBr method, Fig. 10

 The main absorptions are as follows.

3450, 3220, 2960, 2900, 1800, 1760, 1740, 1670, 1580, 1420, 1390, 1370, 1310, 1250, 1200, 1120, 1050, 1030, 980, 950, 920, 810, 770, 720, 690, 610, 540 cm- ¹⁾ iH- NMR spectrum:. 400 MHz in D ₂ 0, the following signal is certified

 I'm sorry. <5 pptn J (Hz

 2.52 (lH, m), 2.72 (lH.m), 2.91 (lH.m), 3.08 (ΙΗ, ιη), 4.35 (lH, m), 4.56 (ΙΗ, ι) 4.80 (lH, m)

) Circular dichroism (CD) spectrum: underwater

 -233 soil shows a negative cotton effect at 3 nm.

) Solubility

 Soluble: water

 /

 Poorly soluble: dimethylsulfoxide, ethyl ethyl sulphate, getyl ether) HPLC: Equipment, carrier, flow rate are the same as those for the above-mentioned deacetyl-form benzhydryl ester (mixture of type A and type B).

 Mobile phase, 0.01M phosphate buffer (PH6.3)

 R t: 3.1 and 3.3min

) Color reaction

 Positive: ninhydrin, host

 Negative: ferric chloride

) Distinguishing between acidic, neutral and basic: amphoteric

Physics of crystals of the deacetyl form of TAN-588 (form A) obtained in Example 8.

Ο ΡΙ IPO The chemical properties are shown below.

 1) Appearance: colorless crystal

 2) Melting point: 177-181 ° C (decomposition point) *

3) Specific rotation: [α] + 124 ° ± 20 ° (C = 0.1, underwater)

4) Molecular weight: mZz 23KM H) + (FD-MS method)

 5) Elemental Xin value:

 Actual measured value

 C, 41.57 C, 41.75

 H, 4.39 H, 4.38

 , 12.11, 12.17

 0, 41.71

 6) Molecular formula: CsH sOs

 7) U V Studio: Underwater

 1%

 A 221 ± 2 nm (E = 151 ± 20)

 max lcm

 8) IR spectrum: KBr method, Fig. 11

 The main absorption is as follows.

 3450, 3220, 2950, 2900, 1800, 1735, 1660, 1580,

 1440, 1420, 1400, 1360, 1340, 1310, 1280, 1200,

 1160, 1110, 1050, 1025, 980, 940, S20, 810,

770, 710, 690, 600, 540 cm " ¹

9) th- MR spectrum: 400 MHz, in D ₂ 0, the following signal is certified

 I'm sorry. 5 ppm J (Hz)

 2.52 (lH, m), 2.72 (lH, m), 2.91 (lH.m), 3.08 (lH.m), 4.34 (lH.m), 4.55 (lH'm), 4.78 (IH.m)

IF, 10) CD spectrum: underwater

 238 soil shows a negative Coton effect at 3 nm.

 11) Solubility:

 Soluble: water

 Poorly soluble: dimethylsulfoxide, ethyl sulphate, kokuguchiform, jietizolete

 12) HP L C: Conditions are the same as for mixture of type A and type B above

 Rt, 3.3min

 13) Distinguishing between acidic, neutral and basic: amphoteric substances

 The physicochemical properties of the powder of the deacetyl derivative (form B) of TAN-588 obtained in Example 9 are shown below.

 1) Appearance: white powder

^¾ 2) Molecular weight: m / z 23KM + H) (FD-MS method)

 3) Elemental Xin value:

 Measured value Calculated value

 C, 40.98 C, 40.17

 H, 4.88 H, 4.64

 N.12.17 N.11.71

 0, 43.48

(· ^: ·· Calculated as including 0.5 mole of attached water)

4) Molecular formula: C ₈ H _t. N ₂ 0 ₈

 δ) Shi 'V spectrum: Underwater

 1%

 (A SSi S nn E = 133 = 20)

 max lcm

 6) IR spectrum: KBr method, Fig. 12

The main absorption is as follows 3440, 2980, 1800, 1760, 1670, 1570, 1520, 1390, 1290, 1250, 1190, 1090, 1050, 990, 920, 810, 760, 720, 690 cm- ¹

7) LH-NMR spectrum: 400 MHz, in D ₂ 0, the following signal is Me certification. S ppm J (Hz)

 2.52 (lH.m), 2.72 (lH.m), 2.90 (lH, m), 3.08 (lH.m), 4.44 (lH.m), 4.68 (iH.m), 4.86 (lH, m)

 8) CD spectrum: underwater

 A negative Cotten effect is shown at 224 ± 2 nm.

 9) Solubility:

 Soluble: water

 Poorly soluble: dimethyl sulfoxide, ethyl ester, chloroform, dimethyl ether '*

 10) HP L C: Conditions are the same as for the mixture of type A and type B above

Rt, 3. lrain

 11) Acidic, neutral and basic: amphoteric

 From the physicochemical properties and the reaction steps described above, it is understood that TAN-588 has an acetyl group bonded to nitrogen and a carboxyl group.

Next, the biological properties of TAN-588 are described. The antibacterial spectrum of TAN-588 sodium salt (an equilibrium mixture of A and B) against various microorganisms is shown in Table 1. Minimum growth inhibitory concentration (Note 1)

¾ n no

 Staphylococcus ♦ ゥ Reus FDA 209P 31 3 Micrococcus Reteus IFO 12708 0.39 Bacillus subtilis NIHJ PCI 219 3,13 Chinores cereus FDA 512.5 ヱ Sher ヒ ia coli NIHJ JC 25 u0 Salmonella チIFO 12529 50 Citro Noku Kufichi Freundi IFO 12681 1 on Clef Shera Niumodoni: IFO 3317 inn Serratia Ma) Rese, '/ Thessos IFO 12648 50

 i Proteus mirabilis ATCC 21100 25 Proteus bulgaris IFO 3988 25

 Say

 Proteus Morgani IFO 3168 100 Ciudamonas Ergino IFO 3080> 100 Arca Ligenes Huecaris IFO 13111 50 Acinetpa, Kuta, Calcophysics I F0 13006 25

(Note 1) Bacterioantipiotic medium 3

(Difco Laboratories, USA); 17.5g, Bact ♦ East ♦ Extract (Difco Laboratories, USA); 5.0g, Bact Aga (Difco Laboratories, USA) 20 g, distilled water; 1000 n (without pH adjustment), and about 10 ^s colony-forming unity m as an inoculum.

The therapeutic effects of TAN-588 sodium salt on experimental mouse infections are shown in Table 2. Infectious bacteria Administration method ED ₅ o (mg / kg)

1 Staphylococcus, Sokas * Pareus 308A- 丄 subcutaneous 25.0 1 Furthermore, acute toxicity does not occur even if 400 mg g of TAN-588 sodium salt is administered subcutaneously to mice or 400 mg / kg orally to mice. I was not able to admit.

 Next, the biological properties of the deacetyl derivative of TAN-588 (a mixture of type A and type B) are described. The mixture of the A-type and B-type compounds and the A-type and B-type compounds are equivalent in biological properties.

The antibacterial spectrum of the deacetyl derivative of TAN-588 against various microorganisms is as shown in Table 3.

Table 3

 Test bacteria, growth inhibitory concentration (Note 1)

 ζ / m

 Staphyloco '/ Cas Aureus FDA 129P 50 Micrococcus * Luteus iFO 12708 6.25 Bacillus subtilis NIHJ PCI 219 12.5 Bacillus cereus FDA 550 Essieri Hia coli NIHJ JC 225 Salmonella Cifiba strodium IF0 529 Indy IF0 12681 50 Klebsiella pneumoniae IF0 3317 100 Serratia marcescens IF0 12648 25 Proteus * Mirabilis ATCC 21100 100 Proteus vulgaris IF0 3988 100 Proteus morgina.IF0 3168> 100 Arca Ligenes-Fucalis IF0 13 111 100 Fast-actor Calco acetates IF0 13006 50

(Note i) Bacterium 'Antipiotic' Medium 3

(Difco Laboratories, USA); 17.5g, Bact 'East Extract (Difco Laboratories, USA); 50g Noct Agar (Difco Laboratories, USA) ; 20 g, using distilled water lOOOm ^ (PH unadjusted) was used about ¹⁰⁶ colony Fomi ring as inoculum-Yuni' preparative Ζπι · ^.

In addition, the tansyl form of TAN-588 is suitable for various yS-lactamases. And stable. Table 4 shows the results of examining the stability against two kinds of ^ -lactamase using Escherichia coli PG 8 as a test bacterium. Table 4

The numerical values indicate the diameter of the blocking circle (mm). Medium: Nutrient agar medium (PH 7.0) containing diaminopimelic acid (20 mg ^). PCG: benzylpenicillin, CPC: cephalosporin C, CMC: cephamycin C. Drug concentration is TAN-588 deacetyl is ^ JOO gZm and all others are lOO / zg / m ^

 · 1: Derived from Bacillus, manufactured by Calbio Chemical (USA), << '2: Enteropactor • Derived from Croca, · 3: No blocking circle diameter.

 As described above, TA -588, its paranitrobenzyl- or benzhydryl monoester, or their acetyl derivatives: hereinafter, these are collectively referred to as compound (I). ] Or their salts have antibacterial activity against Gram-positive and Gram-negative bacteria and have low toxicity. Therefore, the compound (I) of the present invention or a salt thereof can be used for the treatment of bacterial infectious diseases such as mammals [eg, rats, mice, dogs, cats, livestock (such as horses), humans] and poultry. It can be used for

In order to use the compound (I) of the present invention or a salt thereof as a therapeutic agent for bacterial infection, the compound is orally administered as a tablet tablet or the like together with a pharmacologically acceptable carrier, excipient, diluent and the like. Parenterally as an injection, etc. be able to. Examples of diluents and the like in the case of injections include physiological saline. Examples of carriers in the case of capsules include lactose. In the case of oral preparations, the compound

(I) is about 5 to 50 mg / kg / day, preferably about 10 to 25 mg / kg / day, and in the case of parenteral preparation, about 2.5 to 25 mg / kgZ day, preferably about 5 to 201 ^ 1 ^ / Day.

 Further, the compound (I) or a salt thereof obtained by the present invention can be used as a bactericide or a disinfectant. For example, as a compound (I), a solution prepared by dissolving in distilled water at a concentration of 0.01 to 0.1 W / V%, or dicellin or lanolin as a base, and 0.2 to 20 mg, preferably 1 As an ointment containing up to 10 mg, it can be used for sterilization and disinfection of human and animal hands, feet, eyes and ears.

 The compound (I) obtained by the method of the present invention is also a very promising compound as an intermediate for a new drug.

 From the physicochemical properties and biological properties described above, compound (I) is a new antibiotic.

 BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 shows the UV absorption spectrum of TAN-588 (an equilibrium mixture of A and B). FIGS. 2, 3, 4, 5, 6, 7, 7, 8, 9, 10, 11 and 12 show TAN-588 ( Equilibrium mixture of A and B), p-nitrobenzyl ester of TAN-588 (mixture of A and B), p-nitrobenzyl ester of TA-588 (type A), p-nitrobenzyl ester of TA-588 (type B), TAN-588 588 Benzhydryl ester (mixture of A and B), TAN—588 Benzhydryl ester (A type), Τ AN—588 Benzhydryl ester (B type), TAN—588 decetyl benzhydryl ester (A and B) Of TAN-588

PO Benzhydryl ester of tyl form (A type), Benzhydryl ester of TAN-588 deacetyl group (Form B), deacetyl of TAN-588 (mixture of A and B), Decetyl form of TAN-588 (Type A) Infrared absorption spectrum of the decyl form of TAN-588 (B-type).

BEST MODE FOR CARRYING OUT THE INVENTION

 Next, the content of the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto. The percentages in the medium are weight / volume% unless otherwise specified.

Example 1

 Strain of Endopactor Lactamgenus YK-258 (IF 0 14322, F ERM P-7558) grown on nutrient agar slopes, 2% glucose, 3% soluble starch, 1% raw soy flour , Polypton (manufactured by Daiichi Kagaku Co., Ltd., Japan) was inoculated into a two-volume flask containing a 500n medium containing 0.5% sedimentable calcium carbonate in an aqueous solution (pH 7.0) containing 0.5% and 0.3% salt. The cells were cultured with shaking for 24 hours at 24 hours. The entire volume of the culture was added to the above medium with 0.05% of the antifoaming agent Actcole (manufactured by Takeda Pharmaceutical Co., Ltd., Japan), inoculated into a 50-volume tank containing 30 ^ medium, and aerated at 24 ° C. The cells were cultured for 48 hours under the conditions of an amount of 50 / minute and 200 rotations of Z minutes. This culture solution 6 ^ was converted to an aqueous solution (pH 6.5) containing dextrin 3% raw soy flour 1.5%, corn gluten meal i.5%, polycarbonate 0.2% and sodium thiosulfate 0.1%. The seeds were inoculated into a 200-meter fermenter containing a medium supplemented with 0.05% of factor-coal and cultured at 17 ° C for 66 hours under the conditions of aeration of 200 / min and 150 rpm.

After adjusting the pH of the culture solution (230) from the two furnaces to 230, filtration was performed using 9 kg of a hyphen-opening / super-cell (Jyons Manville, USA). After adjusting the filtrate (200) to pH 6, amberlite IRA-402 (C1 type, 10, ROHM 'And' Haas Co., USA) column chromatography. The antibiotic was eluted with 2% saline, and the eluate (53 was adjusted to pH 6 and then subjected to activated carbon chromatography (5, Takeda Pharmaceutical Co., Ltd., Japan). The antibiotic was 8% isobutanol. The eluate (U) was concentrated under reduced pressure to 5. The concentrated solution was adjusted to pH 6 and 2% tri-n-octylmethylammonium chloride methylene chloride solution (2.5 · ^ X2 The extract was treated with 1.6% aqueous sodium iodide (2.5) to dissolve the antibiotics in the aqueous layer.The aqueous layer was concentrated, and the concentrated solution was subjected to activated carbon chromatography (500 m). ) And eluted with 8% isobutanol.The eluate was concentrated and freeze-dried to obtain 1.41 g of a coarse powder.The coarse powder (1.4 g) was dissolved in water (lOOn), and the solution was dissolved. QAE-Sephadex A-25 (C1 type, manufactured by Fuarmasha, Sweden) 200m column chromatography The fraction was collected, adjusted to 600m, pH 5.1, desalted with activated carbon chromatograph, concentrated, eluted, lyophilized, and powdered. This powder was dissolved in water and subjected to preparative high performance liquid chromatography using YMC—Pack S H-343 (Yamamura Chemical Laboratory, Japan) as a carrier. The fraction containing the antibiotic was collected, desalted by charcoal chromatography, the eluate was concentrated, freeze-dried, and the TAN-588 sodium salt was eluted. A white powder (lUmg) was obtained.

Example 2

Lactamgenus YK-258 strain (IFO 14322, F ERM P-7558) grown on nutrient agar was grown at 2% glucose, 3% soluble soy starch, 1% raw soy flour, 0.5% polyptone, Two 2-volume flasks containing 500i medium supplemented with 0.5% precipitated calcium carbonate in an aqueous solution (PH7.0) containing 0.3% salt were inoculated into two reciprocal shake flasks and cultured at 24 ° C for 48 hours with reciprocal shaking. Add the entire amount of this culture to the above medium and add 0.05% of antifoaming agent The cells were inoculated into 200 tanks containing 120 and cultured at 24 ° C for 48 hours under the conditions of aeration of 200 / min and 150 rotations of Z minutes. 60% of this culture solution was added to an aqueous solution (pH 6.5) containing dextrin 3%, raw soy flour] _. 5%, corn / dartene meal 1.5%, polypeptone 0.2%, and sodium thiosulfate 0.1%. The cells were inoculated into a 2000-volume tank containing 1200 medium supplemented with 0.05% tocol, and cultured at i7 ° C for 90 hours under the conditions of aeration of 2000 / min and 120 rpm.

 The culture solution thus obtained was filtered using a Hyf-mouth ♦ Super Cell (manufactured by Jyon's Manville, USA). The filtrate (1150th sample was subjected to column chromatography on Amberlite IRA-402 (C1 type) 40 · ^. The antibiotic was eluted with 2% saline (200), and the eluate was washed with activated carbon (20%). 8% iso-butanol eluate (81_ ^) was subjected to column chromatography on Amber Light IRA-68 (Ci type) 10 and eluted with 1% saline. (54) was again subjected to column chromatography on activated carbon (10), and the antibiotic was eluted with 8% aqueous isobutanol.The eluate (80) was concentrated under reduced pressure, and the concentrated solution (5) was concentrated to PH4. After the correction to 5, the oil was leached with a 2% tri-n-one-year-old butyl methyl acetate 'chloride / methylene chloride solution (2.5 ^ X 2) The oil effluent was treated with a 丄 .6% sodium iodide solution The antibiotics were transferred to the aqueous layer, and the tillage solution was concentrated. The salt solution was subjected to desalting by chromatography and the eluate was concentrated The concentrated solution was subjected to QAE-Sephadex (C1 type) 200m_ecD column chromatography, eluted with 0.03M saline, and the active fraction (1.3) The active fraction was desalted by chromatography on activated carbon (500 m), the eluate was concentrated and lyophilized to give a white powder of TAN-588 (3.56 g). The aqueous layer (5) was subjected to column chromatography on Q AE-Sephaade's Sox (Type C1) 1 since approximately 50% of the antibiotic remained in the antimicrobial agent at 0.03M and saline. And elute the eluate with activated carbon (2 ^)

, "R It was subjected to column chromatography. The salt-removing solution (2 was re-oiled (112) with 2% tri-η-butyl acetyl methyl ♦ chloride chloride solution). The extract was treated with a sodium iodide solution and activated carbon was removed. After a desalting step, a white powder of TAN-588 sodium salt (3.1g) was obtained.

Example 3

 Nov. YK-422 strain (IF 0 14384, F ERM P-7938) grown on nutrient agar with 2% glucose, 3% starch, 3% starch, 1% raw soy flour, polypeptone ( A medium containing 3.5% (uncorrected PH) was inoculated into a 2 · volume flask containing 500 m, and cultured with reciprocal shaking at 48 ° C for 48 hours. The medium was inoculated into a 200-volume tank containing medium 120 supplemented with 0.05%, and cultured for 48 hours at 28 ° C under the conditions of aeration of 12 G / min and 180 rotations of Z. This culture broth was dextrinized for 120 hours. Inoculate a 6000 tank containing a medium 4000 containing 3%, 3% raw soy flour, and 0.2% polyliptone, and 0.05% factorcol in an aqueous solution (uncorrected PH), 22 ° C. The cells were cultured for 66 hours under the conditions of aeration at 4000 / min and 12 rotations / min.

The culture solution thus obtained was filtered using Hyflo 'Supercell (manufactured by Johns Manville, USA). The filtrate (4360) was subjected to column chromatography on Amberlite IRA-402 (C1 type) 400 · ^. Antibiotics were eluted with 2% saline (2000) and the eluate was subjected to column chromatography on activated carbon (160). The 8% isobutanol eluate (640 ^ :) was subjected to column chromatography using Amberlite IRA-68 (C1 type) 40, and eluted with 1% saline. The eluate (200) was again subjected to column chromatography on activated carbon (80 ^), and the antibiotic was eluted with 8% aqueous isobutanol. The eluate (400) is concentrated under reduced pressure, the concentrate is lyophilized, and the lyophilized product is lyophilized. Ton was added to obtain a sodium salt of TAN-588 (620 g) as a precipitate. This

Powder contains 57% TAN-588 sodium salt as a result of HPLC analysis.

I was The powder (5 g) thus obtained was dissolved in water and subjected to QAE-Sephadex (C1 type) 200 m column chromatography. The active fraction (1.2) was eluted and fractionated with 0.03 M saline. The active fraction was desalted by activated carbon (500) close-up chromatography, and the eluate was concentrated, lyophilized, and tanned.

One 588 white powder (2.50 g) was obtained.

 This purified powder of TAN-588 has Rf value of TLC, Rt value of HPLC,

 It is identical to the TAN-588 sodium salt obtained in Example 1 above by IR, UV, CD and NMR spectrum and antibacterial spectrum.

Example 4

 TAN-588 sodium salt (400 mg) was dissolved in DMF (4 m, triethylamine 00), and benzylbutaneamide (800 mg) was added.

The mixture was stirred at room temperature for 3 hours. 'Reaction liquid 0.01M phosphate buffer (PH8.3, 50m) was added, and oil was removed twice with ethyl acetate (50n). The extract was washed with water, and the resulting oil was concentrated to a powder with ethyl acetate-petroleum benzene * 507 mg). TAN—588—A—paranitrobenzyl ester and TAX—588—B—paranitole benzyl A mixture of esters was obtained. The resulting powder was subjected to cephadex using ethyl acetate: methanol = 19: 1 as a mobile phase, and subjected to H-20 chromatographic chromatography to obtain TAN-588-A-paranitrobensiles.

Ter (105 mg), TAN-588-B-paranitrobenzyl ester (67 mg) and a mixture of both (280 mg) were obtained.

Example 5

 Benzophenone hydrazone 58.8g, 1,1,3,3-tetramethylguani

Gin 42m ^ 150mg CH ₂ C 1 ₂ 500m. After cooling the mixture to 0 ° (to ~ 15 ° C), add 74 g of m-chloroperbenzoic acid (70% purity) and add 40 g at 0 ° C.

OMPI WIPO, Stirred for minutes. After washing the reaction solution with water, it was dehydrated with sodium sulfate, and the solvent was distilled off to obtain diphenyldiazomethane.

31 g of TA-588 was suspended in THF, and the whole amount of diphenyldiazomethane obtained above was dissolved in 150 m of THF and added. After cooling the mixture to 0 ° C, 2N HC1, 60m was added dropwise, and the mixture was stirred at room temperature for 1 hour. 2 NHC 1 lom ^ and the mixture was stirred for another hour, and the CH ₂ C1 ₂ 3 added. The obtained solution was washed with water, concentrated, and ether was added to the residue to obtain 28 g of TAN-588 benzhydryl ester (mixture of A-type and B-type) white crystalline powder.

 The above mixture (8 g) was applied to a silica gel (180 m column chromatography) and eluted with a solvent system of chloroform: methanol (97: 3) .The compound of type B eluted first and the compound of type A The fractions were concentrated to give TAN-588 benzhydryl ester Form A (433 mg), Form B (40 Og) and a mixture of Form A and Form B (476 mg). Obtained as colorless crystals.

Example 6

26 g (59 mmol) of TAN-588 benzhydryl ester (mixture of Form A and Form B) were suspended in CH ₂ Cl ₂ and cooled to 20 ° C. To the mixture were added 49 m of pyridine and 37.6 g of phosphorus pentachloride, and the mixture was stirred at 110 to -15 ° C for 50 minutes. After lowering the temperature to 30 ° C., 180 m of MeOH was added, and the mixture was stirred at —5 ° to 115 ^a C for 30 minutes, and further stirred at room temperature for 1 hour. After adding 300m of 1N HC1 and stirring at room temperature for 45 minutes, 50% sodium phosphate 100mM 2NaOH (ca 500i) was added to the reaction solution to adjust the pH of the aqueous layer to 8.0. CH ₂ divided C 1 _2-layer and an aqueous layer, the aqueous layer was further extracted with _{CH 2 C 1 2 (600m ^} ), the combined CH ₂ C1 ₂ layer, after concentration, E residue - by adding ether T AN — 17.9 g of a powder of benzhydryl ester (mixture of type A and type B) of the decetyl form of 588 was obtained.

Example 7

O PI

WIPO 396 mg of benzhydryl ester (mixture of Form A and Form B) of the decyl form of TAN'—588 was suspended in 10 m of CH ₂ C 1 ₂ and cooled to −20 ° C. Add Disodium 434 and Trifluoro oxalic acid 924—20 ° ~ -10. The mixture was stirred at C for 40 minutes. The CH ₂ C 1 ₂ 280m was added to the reaction solution, 0.1MH ₃ P 0 ₊ - extracted with a ₂ HP 0 ₊ solution (pH 7.3) (420m). The extract was adjusted to pH 5.5, concentrated, passed through a column filled with Diaion HP-20 (50-100 mesh, 100 m), washed with water, and eluted and fractionated with 40% MeOH water. Fractions showing antibacterial properties were collected, concentrated, and freeze-dried to obtain 143 mg of a white powder of a deacetyl derivative of TAN-588 (mixture of type A and type B).

Example 8

 When 100 mg of a white powder of a decyl form of TAN-588 (mixture of Form A and Form B) was dissolved in water and allowed to stand at 7 ° C overnight, colorless crystals precipitated. The precipitated crystals were collected by filtration to obtain 40 mg of a deacetyl derivative (form A) of TAN-588.

Embodiment 9.

 657 mg of TAN-588 benzhydryl ester (form B) was subjected to the same reaction and treatment as in Example 2 to obtain 200 mg of benzhydryl ester (form B) of a deacetyl derivative of TAN-588. 180 mg of the compound was dissolved in 18 m of THF: water (1: 1), 90 rag of L0% palladium carbon was added, and the mixture was stirred in a hydrogen stream. After the reaction solution was filtered, the filtrate was concentrated, the aqueous layer was washed with getyl ether, and the aqueous layer was concentrated and freeze-dried to obtain 77 mg of a TAN-588 powder of a deacetyl group (form B). .

Industrial applicability

 Antibiotic AN-588. Its paranitrobenzyl- or benzhydryl ester form, or their N-decetyl form or their salts have antibacterial activity against Gram-positive and negative bacteria, and can be used in mammals, poultry, etc.

OMPI

Can be used as a therapeutic agent for bacterial infections

OMPI

WIPO

Claims

The scope of the claims

1. Antibiotics having the following physicochemical properties as sodium salts at the carboquine group: TAN—588;

(1) Appearance: white powder

 (2) Elemental analysis value (%) whose constituent elements are C, H, N, 0 and, 40 ° on phosphorus pentachloride (:, sample dried for 6 hours:

 Actual value calculated value C, 38.5 ± 2.0 C, 39.61

 H, 4.5 ± 1.0 H, 3.99

 , 9.1 ± 1.5 N, 9.24

 〇, 39.58

 Na, 7.58

(· ^;; Calculated as including 0.5 mole of attached water) ·

(3) Molecular formula: C _{1 Q} H ぃ> 1 ₂ 0 ₇丄 ヽ⁷ 3

 (4) The molecular ion peaks by the SIMS method are as follows

 m / z 611 (2 M Na), 317 (+ a)

 twenty three

 (δ) Specific rotation: [α] 19.0. ± 10 ° (C = 0.

5, underwater)

 D

 (6) Ultraviolet absorption spectrum (underwater): Shoulder near 216.

 (7) Infrared absorption spectrum: Main absorption by bromide-repellent tablets.

 3450, 1780, 1730, 1660, 1550, 1385, 1320, 1290, 1260, L200, 1120, 1040, 980, 910, 810, 770, 690, 600, 540cm

 (8) Solubility: Soluble in water and dimethyl sulfoxide. Poorly soluble in shauchi acid ethyl, kuchiguchi holm, and getyl ether.

(9) Color reaction: Positive for ninhydrin reaction. Greig's Reback, Mouth, Ehrlich, Barton, Dragendorf negative.

 (10) Distinguishing between acidic, neutral and basic: neutral substance (free substance is acidic substance),

Its paranite benzoyl or benzhydryl esters, or their N-deacetyl derivatives or their salts.

 2. Cultivate a microorganism that belongs to the genus D. pneumoniae or Rhizobacterium and has the ability to produce the antibiotic TAN-588 in a culture medium to produce, accumulate, and collect the antibiotic TAN-588 in the culture. A method for producing TAN-588 or a salt thereof, which is an antibiotic.

 3. Lactamgenus, which has more than 70% of GC (guanine + cytosine) content in DNA.

 4. Rhizobacta * arbus that forms white colonies and is non-degradable in the 0-F (oxidative-fermentative) test and has no assimilation of inorganic nitrogen sources.

5. A method for producing a deacetyl form of an antibiotic TAN-588 or a salt thereof, which comprises deacetylating the antibiotic TAN-588 or a salt thereof.

 6, a compound capable of introducing a benzhydryl group to the antibiotic TAN-588 or a salt thereof is reacted to form a benzhydryl ester of the antibiotic TAN-588, and then subjected to a de-T-cetylation reaction. A method for producing the decetyl-form benzhydryl ester form of TAN-588.

O PI