JPS58128385A - Novel antibiotic oa-6129 a, b1 and b2, and their isopropylidene derivative - Google Patents

Abstract

NEW MATERIAL:An antibiotic OA-6129 shown by the formula (R<1> is H or OH; R<2> and R<3> are H or isopropylidene; R<4> is H, substituted or unsubstituted benzyl) and its salt when R<4> is H. USE:An antibacterial agent. Having strong antibacterial activity and beta-lactamase inhibitory action, enhancing synergistically the antibacterial activity of penicillin- based or cephalosporin-based antibacterial agent against bacteria capable of producing beta-lactamase. PROCESS:A bacterium[e.g., Streptomyces sp. OA-6129(FERM-P 11)]belonging to the genus Streptomyces, capable of producing antibiotic OA-6129, is cultivated usually under aerobic conditions in a nutritive medium preferably by liquid culture. Each antibiotic OA-6129 accumulated in the culture is water-soluble, and exists mainly outside of a mold, so that the desired substance is recovered by filtration, extraction, etc. from a filtrate from which the mold is removed through various kinds of purifying processes after the cultivation is over.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to novel antibiotics and their derivatives.
and R3 is a hydrogen atom or a combination of 7 improviride groups/
The present invention relates to antibiotics represented by Cl (-C), and 1t4 represents a hydrogen atom or a substituted to Cl or unsubstituted benzyl group, and novel derivatives thereof.

7-oxo-1-azabicyclo [3゜2.0
] Antibiotics having a 7')-2-ene-2-carboxylic acid skeleton generally have high antibacterial activity and β-lactamase inhibitory activity, and have traditionally been treated using two fermentation methods, semi-synthetic methods, and total synthetic methods. various 7-oxo-1-azabicyclo[3.2
0] Hept-2-ene-2-carboxylic acid derivatives have been produced [for example, Chenamycin (Journal on Antibiotics, Vol. 32 (1979)).

1-12), Epichenamycins (17th Interscience Conference on Microbial Science and Chemotherapy, Abstract No. 8)
0 and 81 (1977)), N-acetylchenamycin (West German Patent No. 2652681 (1977)),
), olivanic acids (Journal on Antibiotics, Vol. 32 (1979), 287-304
page).

PS-5 (Journal on Antibiotics,
32 (1979), pp. 262-286).

PS-6 (Public Patent Publication No. 54-59'295).

PS-7 (Public Patent Publication It'd No. 54-92983), etc.].

The compound of the formula (1) that is feared by obscurity is:

7-oxo-1-azabicyclo[3.2.0]hept-
A novel product not described in conventional literature that has a structural feature of having a panthiinyl group at the 3-position of the 2-ene-2-carboxylic acid skeleton and an ethyl group or 1-hydroxyethyl group at the 6-position. Antibiotics.

In addition, it is a novel categorical ketal derivative characterized in that the hydroxyl group of the pantoyl group is impubilidened. This antibiotic is referred to as "Antibiotic 0A-612".
9j, and more specifically, 1.9j of the above formula (1).
l:t2. l'(, 3 and 1t4 represent hydrogen atoms [Antibiotic 0A-6129Aj.

1 represents a hydroxyl group, 1 (2, It3 and 1t4
Among antibiotics in which is a hydrogen atom, those with a 5,6-cis configuration are referred to as [Antibiotic 0A-6129B,j, and those with the same (,5,6-) lance configuration as [Antibiotics@ It is abbreviated as 0A-6129B,,j.

(In the formula, R1 represents a hydrogen atom or a hydroxyl group) Antibiotic 0A-6129 is unique in that it is stable compared to previously proposed antibiotics with the same basic skeleton, and The compound and its salt have two strong antibacterial properties and I-
In addition to having lactamase inhibitory activity, it also has the ability to synergistically enhance the antibacterial activity of anti-cleavage substances such as penicillins and cephalosporins that f'll' against β-lactamase producing bacteria. It is useful as a drug.

Further, a derivative represented by the formula 2 (wherein 1 has the above meaning and 1 represents a substituted or unsubstituted benzyl group) is 2
Compared to the antibiotic of formula (1-a), it has improved solubility in various organic solvents, so it can be easily applied to various organic synthesis reactions, especially for compounds where llj' represents a hydroxyl group. It is useful as an intermediate for the development of useful derivatives since only the hydroxyl group of the pantoyl group can be selectively protected.

In the substituted benzyl group used herein, the atom group on the top of benzene includes 1, for example, a lower alkyl group such as methyl or ethyl; a lower alkoxy group such as methoxy or ethoxy; a halogen atom such as chlorine or fluorine; or a nitro group. can be mentioned. Such substituted benzyl groups include, for example, p
-nitrobenzyl group, p-bromobenzyl group, p-methylbenzyl group, 2,4-nitrobenzyl group, p-methoxybenzyl group and the like. In the above formula (1), the [substituted or unsubstituted benzyl group] represented by R' is preferably a benzyl group or a p-nitrobenzyl group.

2 of the compound of formula (I) when Jt' represents a hydrogen atom
The carboxyl group at the -position can also be in the form of a salt; examples of such salts include one example, the sodium salt.

Alkali metal salts such as potassium salts and lithium salts; alkaline earth metal salts such as calcium salts and magnesium salts; other metal salts such as aluminum salts; ammonium salts;
Includes salts with primary, secondary or tertiary amines such as monoethylamine, dimethylamine, trimethylamine, monoethanolamine, jetanolamine; salts with organic bases such as benzathine salts, prohydroline salts, etc. ,
Among these, pharmaceutically acceptable salts are preferred, especially.

Alkali metal salts such as sodium salts and potassium salts are preferred.

According to the invention, IL2. The antibiotic 0A-6129 of the formula (1-a) in which 1% 3 and the hydrogen atom are obtained by culturing the antibiotic 0A-6129-producing microorganism in a nutrient medium and from the culture β - Antibiotic 0A-6 described in - having lactamase inhibitory activity
It can be produced by a method consisting of collecting 1,29.

The antibiotic 0A-6129 production box used in the present invention may be any type of antibiotic belonging to any genus as long as it has the ability to produce antibiotic 0A-6129 having the above-mentioned physical and chemical properties and biological properties. can be used and selected from a wide range of microorganisms.

Therefore, a person skilled in the art can search for a bad strain suitable for the purpose of the present invention as follows.
Bacteria producing antibiotic OA-6129 used in the present invention can be easily obtained.

That is, a bioassay agar plate containing β-lactam-sensitive bacteria as the test bacteria, and various types of β-lactam-sensitive bacteria were prepared on this plate.
The cultured tear fluid of the soil breakage isolate was assayed using a bioassay agar plate supplemented with lactamase.

We search for soil isolates that give cultured tears that give an 1811 inhibition circle on the former agar plate, and that give a smaller inhibition circle on some of the latter agar plates than that of the 011 one.

Next, the active ingredient in the culture solution of the soil-isolated bacteria is adsorbed on activated carbon, and the eluate condensate is developed using paper chromatography and thin layer chromatography, and bioautographing is performed using β-lactam-sensitive bacteria as the test bacteria. If antibiotic 0A-6129 is detected, it can be said that the bacterium is an antibiotic 0A-6129 producing bacterium that can be used in the method of the present invention.

This search method will be further explained using a specific example as follows.

As the bioassay agar plate 10 for β-lactam sensitivity box, the Comamonas assay plate described below was used, and the Comamonas assay plate to which β-lactamase produced by Bacillus cereus 569 was added, and Citrobacter freundii 1. Two Komamo assay plates and M assay plates to which β-lactamase produced by -9 was added were prepared. On the other hand, the culture solution of soil-isolated bacteria was made into pulp disks with eight fin diameters, placed on each assay plate, and cultured at 35°C for 20 hours.

The inhibition circle is given by the Comamonas assay plate, and the inhibition circle on the Comamonas assay plate standard plate or the Comamonas assay plate M assay plate is smaller than the inhibition circle on the Comamonas assay plate.

/ Select soil isolates fed with cultured tear fluid.

Next, 2% (W/V
) amount of special Shirasagi activated carbon (manufactured by Shikinichi Yakuhin Kogyo Co., Ltd.) was added and stirred for 15 minutes.

Collect the precipitate by centrifugation, wash with distilled water of the same volume as the culture solution using this precipitate, and centrifuge again to collect the precipitate. To this precipitate was added 50% (V/V) acetone water in an amount equivalent to half the volume of the culture P solution used above, and after stirring at room temperature for 30 minutes, the mixture was centrifuged to obtain a clear solution. Using a rotary evaporator, this 2U liquid was heated to 30-35%
Concentrate at °C.

A 20 times larger amount of basal fluid was obtained than the culture r solution used above.

Pour this concentrated liquid into 80% of
0% acetonitrile/Tris/EDTA [120 ml of acetonitrile, 30 ml of pi-I 7.5'4M Tris(hydroxymethyl)aminomethane-hydrochloric acid buffer.
After 16 hours of paper chromatography using a solvent with pI (consisting of 1 ml of an aqueous solution of Ko M ethylenediaminetetraacetate sodium IJ um salt of 7,5),
Bioautography is performed using Comamonas terrigena B-996 as a test bacterium.

Then, soil isolates showing an inhibition zone at the same migration distance () (, / value) as one antibiotic in one building of antibiotic 10A-6129 are selected as candidates for production of antibiotic 0A-61.29.

Regarding the candidate bacteria selected in this way.

What about paper chromatographies? 1 chromatography to confirm the productivity of antibiotic 0A-6129.

Accordingly, those skilled in the art can easily search for antibiotic 0A-6129-producing bacteria suitable for the purpose of the present invention.

Typical antibiotic 0A-6129 production boxes searched as above include antibiotic 0A-6129 producing bacteria belonging to the genus Streptomyces, and a suitable example is Fukuoka City, Fukuoka Prefecture. The present inventors discovered 0A-612 in actinomycetes isolated from soil collected near Oko Shrine.
There are nine numbered bacterial strains.

The national characteristics of this 0A-6129 strain are as follows 13- It is.

1) Morphology Under the microscope, well-branched basal hyphae are found to be straight to curved (S
The aerial mycelium of traight to flexible is elongated, and two whorled branches are not observed. 10 mature spore chains
It consists of more than one elliptical to cylindrical spores, and no spore receptacles are observed. The size of the spores is 0.6-1. OX 0
．． The surface of the spore is smooth, measuring 7 to 2.5 microns.

Flagellated spores are not observed.

2) Growth status in various media Unless otherwise specified, cultivation was carried out at 28° to 30°C. In addition, the description of the color tone is H, D.

Tresner and B., J., Back.
s), Journal of Applied Microbiology (Journal of Applied Microbiology)
d Microbiology) Volume 11, No. 4, (19
1963) according to the method on pages 335 to 338, the code shown in [ ] [C1-1, M code (code)) H=+Intainer Corporation of America Color No. 1-Mony Manual No. 1/ ( Container
Corporation of America Co
1or I-Armony Manual) was used.

(1) Sucrose/nitrate agar medium: Yellow fire (2
Aerial mycelia of yellow fire [2 dC] to grayish yellow pink [5 dc] are grown on the moderately severed neck of dc) to bright brown color C3ge), and no soluble pigments are observed.

(2) Glucose-asparagine agar medium: Aerial mycelium of light gray Cd) is grown on the good growth of pale yellow [2db] to bright olive brown [2ge]. In addition, this aerial mycelium becomes pink (5 dc) later. No soluble pigments are observed.

(3) Glycerin-asparagine agar medium (IS
l) Medium-5): Good growth of mild yellow pink (4gc) to light brown [4ie], and light gray (d) to light gray reddish brown [5fe]
: ] Aerial mycelia are attached. No soluble pigments are observed.

(4) Starch inorganic salt agar medium (ISP medium-4): Good growth of light yellow [2 db] to gray [2 fe]] Aerial mycelia of light gray (d) are attached. No soluble dyes are produced.

(5) Tyrosine agar medium (ISP medium-7): Fee (3
ec) to light brown (4ie) growth with light gray (d
)~Grows aerial mycelia of bright tea [3fe]. The medium will have a very slight brown tinge.

(6) Nutrient agar medium: Good growth of light yellow (2db) or bright yellow [2fb] to light olive brown (2ge).

It grows bright gray-reddish-brown (5fe) aerial mycelia. No soluble pigments are observed.

(7) Yeast extract/malt extract agar medium (I
SI) Medium-2): Gentle yellow-pink C4g c) to bright brown (4ie)
On top of the good growth, aerial mycelia of pink (5 dc) or slightly later bright gray (d) are attached. No soluble pigments are observed.

(8) Oatmeal agar medium (ISP medium-3):
Price (3ec) to bright orange-yellow (3e a).

Or good growth of bright tea (3ge).

Aerial mycelium of light brown color (3fe) to light gray-reddish brown (5fe) grows, and the medium around the fungal colony has a slightly brown color.

(9) Malate lime agar medium: medium growth of dark to yellow [2dC] with light ash (
d) ~ Light grayish-reddish brown (5fe) aerial mycelia are grown, and no soluble pigment is observed. A common area of calcium salt can be seen around the Shokenai village.

=17- (10) Glucose peptone gelatin medium (20
℃ culture) Pale yellow (2 dl) to brown with good growth and white (
b) - Aerial mycelia of pink (5c I)) are attached. When cultured for a long period of time (about 3 weeks or more), a brown soluble pigment was produced.

3) Physiological properties (1) Growth temperature range Yeast extract/malt extract agar medium (ISP medium-2
) using 10°, 20°, 25°, 30°.

As a result of experiments at temperatures of 34°, 37°, 40°, 45°, and 50°C, almost no growth occurred at 37°C. It does not grow at all above 40°C. Growth was observed at all other temperatures. The optimum growth temperature range appears to be 20-30°C.

(2) Liquefaction of gelatin: Liquefaction.

(3) Starch hydrolysis m: decomposes.

18- (4) Coagulation and peptonization of skimmed milk: Coagulation chopper 7 [but it is pegtonized.

(5) Production of melanin-like pigment: No production of melanin-like pigment was observed in peptone yeast iron agar medium (ISP medium-6) and tryptone yeast extract prosthetic medium (ISP medium-11). Although it shows a very slight brown color on tyrosine agar medium, there is only a trace of melanin production.

4) Assimilation of each carbon source (using Glitonium Gotopu base medium) (1) L-arabinose + (2) D-xylose + (3) D-glucose (4), l) - Fructose + (5) Sucrose elaborate (6)
Ynonthole - (7) L-Rhamnose + (8) Raffinose - (9) L)-Mannitol ++ is assimilated.

- is not assimilated.

According to the scientific characteristics of ``2'', the 0A-6129 strain is Stre.
It is a strain belonging to the genus ptomyces, and the shape of the aerial hyphae is shown in Sections I, F (Section Rec
if-1exibiles), and the spore surface was smooth. The color of aerial mycelia is light gray (d) in most media, such as oatmeal agar, glycerin/aparagine agar, starch/inorganic salt agar, etc.

It is a strain of Gray 5eries. However, in case of Nucrose nitrate agar, yeast extract/malt extract agar, and glucose/asparagine agar medium, depending on the %1 at the time of culture, a reddish color (5 dc) may be exhibited.

However, the color of the basic medium chain threads is amber to yellow in any medium at the initial stage of culture, and as the culture continues, the color changes to yellowish brown to brown or brown. Melanin pigment was not found in peptone yeast extract iron agar medium and tryptone yeast extract broth, and other water bath pigments were not produced in many media. But tyrosine agar.

A slight brown pigment was observed in the glucose-peptone-gelatin medium and oatmeal agar medium.

The present inventors identified this bacterium as Streptomyces sp. OA.
-6129 (8 treptomyces sp,
OA-6129) 2 The Ministry of International Trade and Industry, Agency of Industrial Science and Technology, Institute of Microbial Technology
It has been internationally deposited as P-11).

The antibiotic 0A-6129 of the present invention is the antibiotic 0A-6
The 129-producing bacterium, j91J, is produced by inoculating spores or hyphae of the Streptomyces sp.

As the nutrient source, those commonly used as nutrient sources for actinomycetes, such as assimilable nutrient sources such as carbohydrates, oxygen sources, and inorganic salts, can be used. Examples are glucose, glycerin, maltose, sucrose, and molasses.

Carbohydrates such as dextrin and starch, and soybean oil.

Carbon sources such as peanut oil, fats and oils such as lard; peptone, meat extract, soybean flour, cottonseed flour, dried yeast, corn steep liquor, yeast extract, skim milk, casein, sodium nitrate, ammonium nitrate, ammonium sulfate, etc. Desirable element source; inorganic salts such as dipotassium phosphate monochloride, calcium carbonate, and magnesium sulfate can be used, and if necessary, trace metals such as cobalt and manganese can be added. Other nutritional sources include antibiotic 0A-61.
Any nutrient source can be used as long as the antibiotic 0A-6129 is produced using 29-producing bacteria, and any known culture material for actinomycetes can be used. Further, in order to suppress foaming during heat sterilization and culturing, an antifoaming agent such as silicone or vegetable oil may be added.

The blending ratio of the nutritional sources as described in item 1 is not particularly limited and can be varied over a wide range.

A person skilled in the art can easily determine the optimal composition and blending ratio of the nutrient source for the antibiotic 0A-6129-producing bacteria to be used by simple small-scale experiments.

The nutrient medium can be sterilized prior to cultivation, and before or after this sterilization, the p i-I of the medium is in the range of 4 to 9,
In particular, it is advantageous to adjust the pl (in the range of 6 to 8).

In principle, the antibiotic oA-6129-producing bacteria can be cultured in such a nutrient medium according to a method commonly used in the production of antibiotics using actinomycetes. It is usually preferable to culture under aerobic conditions, and the culture can usually be carried out with stirring and/or with aeration. Also.

As a culture method, both static culture and submerged culture with shaking culture and aeration and agitation can be used.

Submerged culture is advantageous.

The vine culture temperature used is not particularly limited as long as the growth of antibiotic 0A-6129 producing bacteria is not substantially inhibited and antibiotic 0A-6129 can be produced, and it depends on the production strain used. Generally temperatures in the range 20-40°G, preferably 25-35°C are suitable, although this can vary.

In addition, in order to perform culture appropriately, as necessary.

During cultivation, the p I-I of the culture was increased from 4 to 9. In particular, it can be adjusted to a range of 6 to 8.

In the case of large-scale mass culture, it is best to perform seed culture as appropriate, inoculate this into a nutrient medium, and perform liquid culture.

Cultivation can normally be continued until sufficient accumulation of antibiotic 0A-6129 occurs. The culture time varies depending on the composition of the medium, culture temperature, production strain used, etc., but it is usually 30~
The range is 90 hours.

As for the culture conditions to be used, those skilled in the art can easily determine the optimal conditions through simple experiments, depending on the characteristics of the production strain used.

What is the amount of antibiotic 0A-6129 accumulated during culture?

Each antibiotic 0A-6129A, 0A-6129B.

0A-6129132 can be quantified by the bioassay method and bioautography described later, and thereby the optimum accumulation amount can be easily determined.

Thus, each antibiotic 0A-6i accumulated in the culture.
29 is water-soluble and exists outside the bacterial body as a king, so
After culturing, the bacterial cells are removed by a separation method known per se, such as strain filtration, centrifugation, extraction, etc., and recovered from the liquid, supernatant, extract, etc.

Recovery can be carried out by various methods known per se,
In particular, methods often used for recovering carboxylic acid type antibiotics are advantageously applied. For example, solvent extraction with ethyl acetate, n-butanol, etc. at low pH and transfer of the solvent layer to a high pi-I water layer; activated carbon, Amberlite XAD (manufactured by Rohm and Haas), Ri( Adsorption using Yaion 1-IP-20 (manufactured by Mitsubishi Kasei Corporation), etc.
methanol water.

Elution with acetone water, etc.: DOWEX 1×2 (manufactured by Dow Chemical Company), QAH-Sephadex A-25 (manufactured by Pharmasha Company), DEAE-Cellulose Whatman DE
-32 (Whatman Company.

DEAE - Adsorption and elution with ion exchange resin such as Sephadex A-25 (manufactured by Pharmasha); 9G - Sephadex G-10 (manufactured by Pharmasha);
Kel P-2 (manufactured by Bio-Rad) 9 etc. if Yolkel
"jM; Cellulose, Abyssel SJi" (American
Column chromatography (manufactured by Viscose Co., Ltd.); forced precipitation by adding a solvent such as acetone; and freeze-drying.

The behavior of antibiotic 0A-6129 during the recovery and purification process can be quantitatively measured by the bioassay method and bioautography described below.

Thus, antibiotic 0A-612 with the properties described above
9 is obtained.

Antibiotic 0A-6129 produced by the above fermentation method, that is, the compound of formula (1-a) in which al, R2 and 3 of formula (1) represent hydrogen atoms, generally Hydrogen atoms bonded to carbon atoms have a trans configuration with each other. Also.

In the compound of formula (1) where R1 represents a hydroxyl group, the hydrogen atoms bonded to the 5- and 6-position carbon atoms generally have a trans or cis configuration. In addition, 3
The bunchyinyl group bonded to the carbon atom at -position has one asymmetric carbon atom.

It may exist in either the D-form or the L-form, or in racemic form.

The present antibiotics 0A-6129A, B, and B2 are generally more stable in their salt forms than in their free forms, so they can be used for the pharmaceutical purposes described below or as intermediates for further conversion into derivatives. When used as a salt or subjected to the above-mentioned purification process, it is preferable to treat it in the form of a salt.

Conversion of antibiotics 0A-6129A, B, and B2 into their salt forms can be carried out by treating the antibiotics with inorganic or organic bases according to known methods. Examples of inorganic or organic bases that can be used in this salt-forming reaction include alkali metal hydroxides such as sulfur hydroxide, potassium hydroxide, and lithium hydroxide; calcium hydroxide, and magnesium hydroxide. Hydroxides of alkaline earth metals; primary such as monoethylamine, dimethylamine, trimethylamine, monoethanolamine, jetanolamine, benzathine, protylene.

Examples include secondary or tertiary organic amines.

- Antibiotic 0A-6129A obtained as described above,
B and B2 can also be changed to (substituted or unsubstituted benzyl) esters. The esterification can be carried out according to methods known per se.

For example, antibiotic 0A-6129A, B, or B
2 or a salt thereof substituted or unsubstituted benzyl halide (e.g. benzyl chloride, benzyl 29-bromide, p-nitrobenzyl chloride, p-nitrobenzyl bromide, p-methoxybenzylfuromite, 2,4-dinitrobenzyl chloride, It can be converted into an ester by reacting with p-bromobenzyl bromide (p-bromobenzyl bromide). This esterification reaction is generally preferably carried out in an inert liquid medium, and examples of the inert liquid medium that can be used include halogenated hydrocarbons such as chloroform and methylene chloride; dimethylformamide and hexamethylphosphoramide. amides such as dimethyl sulfoxide; ethers such as tetrahydrofuran and dioxane; ethyl acetate, n-butyl acetate,
and ketones such as acetone and methyl ethylgtone. Each of these liquid media may be used alone, or two or more types may be mixed as necessary.

30- The reaction temperature is not critical, the type of halide used,
It can be changed to J-Mu range depending on the classification of I-body media, etc.
The temperature can be arbitrarily selected within the temperature range at which the antibiotics 0A-6129A, B, and B2 do not decompose significantly, but generally the temperature is 60°C or lower, preferably in the range of 0 to 40°C, and more preferably A range from 5°C to room temperature is advantageous.

In addition, in the above reaction, trimethylamine, triethylamine, pyridine may be used as necessary.

A reaction accelerator such as dicyclohexylcarbodiimide may be added.

Under such conditions, the reaction generally takes place within 1 to 24 hours.

Usually it can be completed in 3 to 12 hours.

In addition, antibiotics (JA
-6129A, B, or 13□ does not necessarily need to be isolated, and the antibiotic 0A-612
9. A culture of the producing bacteria or a culture solution after separating the bacterial cells from the culture can be used.

Alternatively, crude antibiotic 0A-6129A at least partially purified according to the isolation and purification method described above.

B1 or B2 can also be used. Such partially purified products include 2, for example, an activated carbon adsorption elution concentrate of the culture solution;
Diaion 1 (P-20 (manufactured by Mitsubishi Chemical Industries, Ltd.) adsorption and elution concentrate of the culture solution; the concentrate was adsorbed on QAJ!J-Sephadex (manufactured by Pharmasha) and phosphate buffer with a gradient salt concentration was added. Concentrates eluted with water and desalted with activated carbon; butanol extract concentrates at pH 3.5 at low temperatures, and the like.

Antibiotic 0A-6129A thus obtained.

The ester of B1 or J3□ can be separated from the two reaction mixtures or purified by various methods known per se in the antibiotic field. For example, after the completion of the two reactions, first the two reaction solutions are poured into an aqueous medium to remove water-bathable impurities such as by-products. At that time p l-
In order to keep I approximately neutral, it is desirable to use a neutral buffer as the aqueous medium. This mixture is then diluted with, for example, ethyl acetate.

Antibiotic 0A-6129A, treated with a non-polar organic solvent that is substantially immiscible with water, such as benzene or chloroform;
The ester of B or B2 is extracted into the organic solvent layer. During this extraction, salts such as common salt, ammonium sulfate, etc. can be added to enhance the extraction efficiency due to the salting out effect.

The solvent layer is dried with dehydrated sodium sulfate, and then filtrated using a method known per se, such as Bio-Beads 5-X3 (manufactured by Bio-Rad) or Sephadex LH-20 (manufactured by Pharmacia). ; silica gel, alumina,
The ester can be isolated by using an appropriate combination of adsorption chromatography using Florisil (manufactured by Florizin) or the like as a carrier and repeating it as necessary.

33- Antibiotics 0A-6129A, B obtained as above
, or a substituted or unsubstituted benzyl ester of B2,
The hydroxyl groups at the α- and γ-positions of the pantoyl group bonded to the 3-position carbon atom can be simultaneously converted into an etherified cyclic ketal or cyclic acetal derivative. The ketal or acetalization can be carried out by methods known per se.

For example, antibiotics 0A-6129A, B, or B2 can be converted into their cyclic ketal or cyclic acetal derivatives by reacting with their ester ketone or aldehyde derivatives. This reaction is generally preferably carried out using a reaction reagent as a solvent, and examples of reaction reagents that can be used include:

For example, acetone, 2,2-dimethoxypropane.

Formaldehyde, 2,2-dimethoxyethane.

Examples include acetaldehyde, benzaldehyde, propionaldehyde, acetophenone, diphenyl ketone, cyclohexanone, dibenzyl ketone, 1,1 diphenylacetone, etc. These reaction reagents may be used alone or in combination as necessary. It is also possible to use a mixture of two or more.

In addition, if necessary, antibiotics 0A-61 for the reaction reagent may be added.
Considering the solubility of the ester of 29A, B, or B2, an inert solvent can also be mixed.

The reaction temperature is not critical and can be varied widely depending on the type of liquid medium used, etc.
The temperature can be arbitrarily selected within a temperature range in which the ester of No. 29 does not decompose significantly, but the temperature is generally 60°C or lower, preferably in the range of -40 to 40°C, and more preferably in the range of 0°C to
Room temperature ranges are advantageous.

In addition, in the above reaction, if necessary, anhydrous p-
A reaction accelerator such as toluene sulfonic acid, boron tolufluoride etherate, zinc chloride, etc. is added.

Under such conditions, the reaction can be completed within about 30 minutes to 12 hours, usually within 2 to 5 hours.

The cyclic cutal or cyclic acetal derivatives of the esters thus obtained can be separated by methods known per se.

It can be isolated from the reaction mixture according to purification methods. For example, after the completion of two reactions, first, in order to inactivate excess reaction accelerator, etc., treatment is performed with amines.

The treatment solution is poured into a nonpolar organic solvent that is substantially immiscible with water, such as ethyl acetate, benzene, or chloroform. In order to remove water-soluble impurities such as by-products from the mixed liquid, it is washed with an aqueous medium.

At that time, it is desirable to use a neutral buffer as the aqueous medium. Next, the organic solvent layer is concentrated to dryness, and then, for example, 2 is added according to a method known per se.

Silica gel, Biopeace (manufactured by BioRad).

A desired cyclic ketal or cyclic acetal derivative can be isolated by appropriately combining chromatography using Sephadex LIE (-20 (manufactured by Pharmacia) or the like as a carrier and repeating it as necessary).

Antibiotics 0A-6!29A, B provided by the present invention
, or B2 or its salts have a wide range of antibacterial activity and exhibit very strong antibacterial activity against Durum-positive bacteria belonging to various microorganisms such as Staphylococcus, Sarcina, and Bacillus, and furthermore, It also shows very strong antibacterial activity against Durham-negative bacteria belonging to the genus Comamonas, etc.

Also, uninvented antibiotics 0A-6129A and B.

and B2 is 2 e.g. Escherichia sp., Klebsiella sp.
It exhibits fairly strong antibacterial activity against Durham-negative bacteria belonging to the genus Groteus.

Particularly one antibiotic of the present invention 0A-6129A, B.

and B2 have fairly strong antibacterial activity against Durum-negative bacteria belonging to the genera Citrobacter, Proteus, Enterobacter, Klebsiella, Serratia, etc., which are resistant to antibiotics with β-lactams. It is distinctive in that it shows

The antibacterial spectra of the antibiotics 0A-6129A, B, and B2 of the present invention are demonstrated by measuring the minimum inhibitory concentration on various pathogenic test teeth as described below.

The above antibacterial activity was measured as follows. That is, the Japanese Society of Chemotherapy Standard Method
of Chemotherapy: The r
evised me day tod of determi
nation of MICvalueChemo
therapy 22. 1126-1128. 1
The test was carried out using the agar medium dilution method based on 974).

Antibiotics were diluted with IVI150 PBS pi-(7.3).

Prepare a 2-fold dilution series and add 1 ml of this solution to an agar medium (
Heart Infusion Agar Difc
(manufactured by Company O) was mixed in a Petri dish with a diameter of 9crrL to form a flat plate.

For the inoculum, one platinum loop of 5tock culture was inoculated into trypto soy broth medium (manufactured by Eiken Kagaku Co., Ltd.), and cultured for 18 hours at 37°C. Dilute to ml.

Plates were inoculated using a microplanter. The flat plate is 37
Culture was carried out at 18°C for 18 hours, and the lowest concentration of antibiotic that completely inhibited bacterial growth was defined as the MIC.

Furthermore, the antibiotic 0A-6129A provided by the present invention
, B, and B2 ester impropylidene derivatives have increased solubility in fat-soluble organic solvents due to the protection of the hydroxyl group of the bunchyinyl group bonded to the 3-position carbon atom. Furthermore, it is useful as a reaction intermediate for producing useful derivatives.

Next, the present invention will be further explained by examples. In addition, the qualitative and quantitative analysis of the antibacterial active substance used in the following examples was performed by the method described in -F.

(1) Bioassay method Comamonas terrigen cultured overnight on Nutriendo agar.
a) B-996 (DWM cells suspended in New!J endo broth and derived from the cells)
A seed mother liquid having a nm absorbance of 0.04 is prepared. A 1% seed mother liquor was inoculated into an agar medium made of 0.8% Kyokuto Powder Bouillon (Kyokuto Pharmaceutical Industries Co., Ltd.) and 1% Bacto Agar (Difco), and 9 cf of each 7 me
Dispense it into an n-diameter veterinary dish and assimilate it to prepare a Comamonas assay plate.

(2) Bioautography In the above-mentioned bioassay method, instead of using a 9 cm diameter Hetori dish, a dish measuring 32 Cm vertically by 24 α horizontally is used, and 100 ml of agar medium inoculated with the test node is dispensed and solidified to prepare a large assay plate.

After developing the test solution, Paper Chromato P paper was pasted on the agar surface of the large assay plate prepared above, removed after 15 minutes, and the large assay plate was incubated at 35°C for 20 hours.

The Rf value of the paper chromatogram can be calculated from the position of the inhibition zone (qualitatively), and semi-quantitative can be determined from the size of the inhibition zone. When using a thin layer chromatography plate, apply it to the surface of the agar using a thin paper so that the component side touches the plate, remove it after 15 minutes, and perform qualitative and semi-quantitative analysis using the same procedure as above.

Example 1 Production of antibiotics 0A-6129A, B1 and B2 by fermentation method (A) A 500 ml Erlenmeyer flask (VCI OOml) was charged with a seed culture medium (S-1) having the following composition, and heated to 120°C by a conventional method. sterilized for 15 minutes.

On the other hand, Streptomyces sp. OA-6129 (S
treptomyces sp, OA-6129
) The spores of the bacterial strain were sufficiently attached, and the platinum loops were inoculated into the seed medium and cultured with shaking at 28° C. for 48 hours in a rotary shaker (2 oorpm, amplitude: 7 crn). 200 ml of this seed culture solution was added to the seed culture medium (S
E-4) Inoculate into a 307 jar 7-armenter containing 15i, stir at 288C, 400 rpm and 7.
Aerated agitation culture was performed for 90 hours under aeration conditions of 511/min. As an antifoaming agent, 0.07% of silicone KM-751 (manufactured by Shin-Etsu Chemical Co., Ltd.) was used.

CB) The seed mother culture solution 21 after 24-hour cultivation obtained in (A) above was mixed with 100% of the production medium (GM-1) having the following composition.
Inoculate a 2001 volume fermentation tank containing A and heat at 28°C.
Aerated agitation culture was performed for 90 hours under the conditions of stirring at 200 rpm and aeration for 50117 min.

0.07% silicone KM-75 (mentioned above) as an antifoaming agent
used.

The culture solution was sampled over time, and the antibacterial activity of the centrifuged supernatant was measured.

The measurement results at each time were as shown in the table below.

Culture time (hours) Antibacterial titer (μφ 4.8
2.672
11.590
Composition of 24.0 seed medium (S-1): Meat 1.5% (W/V) Yeast extract 0.5 Potato starch 2.0 CaC030.2 pH (before sterilization) 7.0 Seed medium ( Composition of SE-4): Beef extract 0.3% (W/V) Tryptone 0.5 Glucose 0.1 Soluble starch 2.4 Yeast extract 05 CaCO30,
4 Meat
0. 5p I-I (before sterilization) 7.5 Composition of production medium (GM-1): Glycerin 8.0% (W/V) fishmeal
1.0% (W/■) Meat 3. O Cact30. Adjust the pH to 7.2 with 2HPO, 0.2 Mg5O, 0,2' tt NaOH, and 0.0 to 5. 00005% (W/V) of vitamin B1□ dissolved in OIMIJ acid buffer and sterilized in an autoclave at 1 kg/dG for 5 minutes was added.

(C) 5% (W/V) amount of Dobukopari) A3 in the fermented liquid 1001 after 90 hours of culture obtained in (B) above.
4 (manufactured by Takko Perlite Co., Ltd.) was added, and the bacterial cells were separated using a basket centrifuge.

A culture P solution of 90A' was obtained.

This is Diaion FLP-20 (manufactured by Mitsubishi Kasei Corporation)
Adsorb onto a packed column (IOXlookTL).

-46= After washing with distilled water 51, it was eluted with 30% (V/V) acetone water.

The eluate was fractionated, with each section designated as 1 no. Bioassay active fraction 8 to fraction 15-]: A total of 81 eluates were collected, and this was adsorbed onto a Diaion PA, 306S (manufactured by Mitsubishi Kasei Corporation) packed column (8 x 60 cm).
After washing with distilled water 11, it was eluted with 30% saline. 500
The eluate was fractionated by 1 ml, and a total of 54 eluates from bioassay active fraction 7 and 16 fractions were collected. This eluate contained antibiotics 0A-6129A, B1 and B2. Add 300 g of common salt to 6.07 g of the bath solution, and add it to the Diaion I-1, P-20 packed column (6X
After washing with 500ml of distilled water, acetone water with a concentration increasing linearly from 0% to 40% total 4. It was eluted with Ol.

1 division is 17rnl! The eluate was fractionated.

Approximately 1 from bioassay active fraction 20 to fraction 130
．． Antibiotics 0A-6129B are included in the bath water of 8.
B2 and some antibiotic 0A-6129A.

The approximately 700 TLl eluate from bioassay active fraction 131 to fraction 170 contained antibiotic 0A-6129A.

The above two sections were each freeze-dried to obtain a brown powder.

[Purification of antibiotic 0A-6129A] A brown powder obtained by freeze-drying the eluate containing antibiotic 0A-6129A was dissolved in a small amount of distilled water to obtain Biogel P-2 (manufactured by Bioland). It was introduced into a packed column (8×100 cm), developed with distilled water, and an active fraction of 1.01 was collected by bioassay. This active fraction was adsorbed onto a QAE-Sephadex A-25 (Pharmacia 21) packed column (4X 40 cra) equilibrated with 0.01M phosphate buffer (pH 8,4) and washed with the above buffer 200111. Afterwards, elution was performed with saline increasing linearly in concentration from 0% to 4% (total 3.07). The eluate was fractionated into 15 ml portions, and bioassay was performed to obtain fractions 51 to 70 (two total fractions of 300 mL).
An active fraction of rnl was obtained.

This fraction was lyophilized to give a tan powder.

Dissolve this powder in a small amount of distilled water, add 5g of salt,
It was adsorbed onto a Diaion i (P-20A G (manufactured by Mitsubishi Kasei Corporation) packed column (2X50cIn)).

After washing with 50 ml of 55 saline solution, it was washed with 100 Inl of distilled water. Elute with acetone water whose concentration increases linearly from 0% to 30% (total of 1.0.7%).
m1, and the eluate was fractionated. By bioassay, active fraction 35 to fraction 45 total 110m7!
was collected and freeze-dried to obtain 52 cm of yellowish brown powder.

52 mg of antibiotic 0A-6129A coarse powder.

It was dissolved in a small amount of distilled water, introduced into a Sephadex G-10 (manufactured by Pharmacia) packed column (2X 80 am), developed with distilled water, and the active fraction 3 was determined by bioassay.
0ml was collected. This active fraction in advance.

QAg-Sephadex A-25 (manufactured by Pharmacia) equilibrated with 0.01M phosphate buffer (pi48.4)
It was adsorbed onto a packed column (2 x 30 cm), washed with 50 ml of the buffer described above, and eluted with saline (total 800 ml) whose concentration increases linearly from 0% to 5%. , were fractionated. A total of 25Tn from active fraction 36 to fraction 40 was determined by bioassay.
I collected l.

Add 4g of salt to both of these, and
0AG (manufactured by Mitsubishi Kasei Corporation) packed column (2X 40
cm) and washed with 50 ml of distilled water.

Acetone water whose concentration increases linearly from 0% to 30% (
A total of 800 m1) is eluted, and 1 car is 5 m/! The eluate was fractionated.

A total of 65 ml of active fractions 105 to 117 were collected by bioassay.

By freeze-drying this, 21m9 of pale yellow powder was obtained.

The obtained freeze-dried sample had the following characteristics.

(1) Shape: Pale yellow powder 51- (2) Specific optical rotation: [α]. , 11.6° (C=1.0°0.01M phosphate buffer, pH 8.4) However, the value is when ε of λrnax 300 nm is 5600 in ultraviolet absorption.

(3) Molecular formula Theoretical molecular formula C2oH3oN30.8Na (M, W
, -479) (4) Ultraviolet absorption spectrum (5) Main peaks of infrared absorption spectrum (KBr) 1760 (β-lactam) 1660 (amide) 1600 (carboxylate) (6) Nuclear magnetic resonance spectrum (solvent:] )B2) (internal standard: DSS) δ (ppm) 1.00 (3H, t, J=7.5Hz, CH2-CH
8) 1.60-2.00 (2I-I, m, CH2-C
H,)2.48 (211,t, J=6.51(Z
.

N -CB2-C)-12-CO) 2.8 (1-3,65(11I-I, m, C
-4H2, C-6H.

S -Cf(2-C17I2-N, N-CB2-
CB2-CO.

C-CB2-01-I) (Paper Chromatography Toyo F Shiya 50 Developing solvent Acetonitrile:Water (8:2) Detection method Comamonas terrigena B99
Bioautography jtf value by 6 0.53 (8) Color reaction Reaction to ninhydrin: Negative Reaction to Ehrlich reagent: Positive (9) Elemental analysis, unmeasurable due to melting point hygroscopicity [Antibiotic 0A-6129B and B2 purification ] The eluate containing antibiotic OA-'6129 B1 and B2 in soil,
The brown powder obtained by freeze-drying was dissolved in a small amount of distilled water, introduced into a Biogel P-2 (manufactured by Bio-Rad) packed column (8 x 100 cWL), developed with distilled water, and the active fraction was determined by bioassay. Minute 1. I collected Ol. This active fraction was prepared in advance at 0. OIM/acid buffer (
QAg-Sephadex A equilibrated with pi(8,4)
-25 (manufactured by Pharmacia) packed column (4X 4.
Adsorb to Ocm).

54- After washing with 200 ml of the above buffer, elution was performed with saline increasing linearly from 0% to 4% a degree (total 3.07). The eluate was fractionated into 15 ml portions and bioassay was performed from fraction 51 to fraction 70.

A total of 3001 nl of active fraction was obtained.

Both portions were freeze-dried to obtain a yellowish brown powder.

Dissolve this powder in a small amount of distilled water, add 5g of salt,
Diaion I-f P-20A G (Mitsubishi Kasei (
Co., Ltd.] packed column (2X 50 Crn).

After washing with 50 ml of 5% saline, it was washed with 100 ml of distilled water. The eluate was eluted with acetone water whose concentration increased linearly from 0% to 30% (total 1.07), each volume was 10 ml, and the eluate was fractionated. Two fractions were obtained by bioassay: active fraction 15 to fraction 24, totaling 100 ml, and active fraction 26 to fraction 35, totaling 100 ml. The former activity category included antibiotic 0A-6129B, and the latter activity category included anti-55-biotic 0A-612913□. By freeze-drying the outside of both areas, OA, -6129B, +
7) 84.0 m of yellowish brown powder9. Antibiotic 0A-612
470 ml of a tan powder of 9B2 was obtained.

[Purification of antibiotic 0A-6129B] Antibiotic 0A-
6129B, coarse powder 840 ~.

Dissolved in a small amount of distilled water, introduced into a Sephadex G-10 (manufactured by Pharmacia) packed column (2 x 80 cm), developed with distilled water, and determined by bioassay that the active fraction was 35 m
Collected 1. This active fraction in advance.

Q equilibrated with 001M phosphate buffer (pH 8.4,)
It was adsorbed onto an AE-Sephadex A-25 (manufactured by Pharmacia) packed column (2 x 30 cm), and the buffer solution 5 described in "1"
After washing with 0ml, elution was performed with saline whose concentration increased linearly from 0% to 5% (total 800ml), and the eluate was fractionated into 5ml portions. A total of 25 ml of fractions 39 to 43 having an ultraviolet maximum absorption at 300 nm was collected.

Add 4g of salt to both portions and use Diaion HP-20.
AG (manufactured by Mitsubishi Kasei Corporation) packed column (2X40ffi
) and develop with distilled water.

The eluate was fractionated into 5 ml portions.

A total of 100 fractions, from fractions 51 to 70, were collected by examining the ultraviolet maximum absorption at 300 nm in the same manner as above. By freeze-drying this, 21m9 of pale yellow powder was obtained.

21 m9 of this pale yellow powder was dissolved in a small amount of distilled water.

Activated carbon [manufactured by Wako Tsumugi Kogyo Co., Ltd.] packed column (1,5
After washing with 20 ml of distilled water, elution was performed with Inglovir alcohol water (20,011 liters in total) whose concentration increases linearly from 0% to 50%, and the eluate was fractionated into 2 ml portions.

A total of 30 ml of both fractions 18 to 32 having maximum ultraviolet absorption at 300 nm was collected and freeze-dried to obtain pale yellow powder B my.

The obtained freeze-dried sample showed the following characteristics.

(1) Shape: Pale yellow powder (2) Specific rotation: (α) 24.2° (c-
(3) Molecular formula C2oH3oN3
08SNa (M, W, 4.95) (4) Ultraviolet absorption spectrum (5) Infrared absorption spectrum 1650 (amide) 1590 (carboxylate) (6) Nuclear magnetic resonance spectrum (solvent: D20. Internal 5
8-2.47 (2I-I, t, J=6.5l-1
z, NH-Ci(2-CI-12-CO) 2.7 5 ~ 3.7 0 (l l If,
m, C-4H2.

C-6I-I, 5-CH2-CH2-NH.

NH-CH2-CI-f2-C0.

3.93 (L H,s, HO-δl-l-C0)(
7) Paper chromatography Toyosawa paper 5
0 Developing solvent Acetonitrile 10.1. M tris(hydroxymethyl)aminomethane-hydrochloric acid buffer (pH 7,5) 1 0.1M ethylenediamine (pH 7,5) = l 20
:30:1 Detection method Comamonas terigena
Bioautography R/value by B996 0.17 (8) High pressure f paper electrophoresis p i (8,6 veronal buffer, Toyo f paper, washing 51
Electrophoresis was carried out at 1500V for 30 minutes.

R, m (li 0567 (l, m value is based on the mobility of PS-5 sodium salt as 1.0) (9) High performance liquid chromatography packing material; Microbondapak C18 column; 7.8
mm (inner diameter) x 30 (Nippon Waters Limited) Mobile phase: 0.01M ammonium phosphate buffer containing 3% acetonitrile (pH 7,5) Flow rate i 1.5 ml/min Detection method;
Ultraviolet wavelength: 301 nm Under the above conditions, the retention time is 13.9 minutes.

[Purification of antibiotic 0A-6129B2] Antibiotic 0A-
6129B2 crude powder of 470~ was dissolved in a small amount of distilled water, introduced into a Sephadex G-10 (Pharmacia Co., Ltd. I) packed column (2X 80 on), and developed with distilled water.
3Qml of active fraction was collected by bioassay. This active fraction in advance.

It was adsorbed on a QAE-Sephadex A-25 (manufactured by Pharmacia) packed column (2X 30 Crn) equilibrated with 0.011V phosphate buffer (pI-I8.4), and after washing with 50 ml of the above buffer, the concentration Elution was performed with saline (total 800 mg) in which the concentration increased linearly from 0% to 5%, and the eluate was fractionated into 5 rnl portions. A total of 3 fractions from fraction 35 to fraction 41 having maximum ultraviolet absorption at 300 nm
Collected 5ml.

Add 4g of salt to both of these and use Diaion IIP-2.
0AG (manufactured by Mitsubishi Kasei Corporation) packed column (2X 40
cm) and washed with 50 ml of distilled water.

Elute with acetone water (total 800 ml) whose limit increases linearly from 0% to 20%, and 5 ra for 1 car.
The eluate was fractionated.

As above, a total of 70 rnl of fractions 112 to 125 was collected by examining the ultraviolet maximum absorption at 300 nm.

By freeze-drying this, pale yellow powder 23■ was obtained.

The obtained freeze-dried sample showed the following characteristics.

(1) Shape: pale yellow powder (2) Specific rotation: [α] 14.7° (C=l, Q
.

0.01M phosphate buffer, pH 8,4) (3) Molecular formula
C2o1(36N, 088Na(M,W.

495) 62- (4) Ultraviolet absorption spectrum (5) Main peaks of infrared absorption spectrum (KBr) 1660 (amide) 1600 (carboxylate) (6) Nuclear magnetic resonance spectrum (solvent: D, , O, internal 2 , 45 (2H,t, J=6.5H,,Nl-l
-CH2-C1(2-CO) 2.75-3.60 (111-I, m, C-41
-1,.

C-61 (,8-CII2-CI-12-NH.

63- (7) Paper chromatography Toyo F Shiya 50 Developing solvent Acetonitrile/0.1 M Tris (
Hydroxymethyl) aminomethane-hydrochloric acid buffer (pH 7.5) 10.1M ethylenediamine aqueous solution 1 (pII 7.5) = 120730/1 Detection method Comamonas terigena
Bioautograin Rf value by B 996: 0.17 (8) High-pressure one-paper electrophoresis Electrophoresis was performed at 1500 V for 30 minutes using Veronal buffer of pH 8.6 and Toyo F Shiya 51.

The Rm value of 0.67 and the CRm value are based on the mobility of PS-5 sodium salt being 1.0.

(9) High performance liquid chromatography packing material; Microbondapak C18 column; 7.
8 mm (inner diameter) Detection method: Ultraviolet wavelength 301 nm Under the above conditions, retention time is 22.5 minutes.

Example 2 Preparation of benzyl ester of antibiotic 0A-6129A 44.6 d of antibiotic 0A-6129A sodium salt was mixed with 3.6 d of dimethylformamide. Dissolve in Q ml and cool with water.
0.25 ml of triethylamine was added, and while stirring, 18 ml of benzyl bromide Q was added. 3 at the same temperature
After reacting for 0 minutes, the reaction was continued for 3 hours at room temperature.

The reaction solution was poured into 1QQmA' of ethyl acetate, washed with 20 ml of 0.01M phosphate buffer (pH 8,4) saturated with 1 sodium chloride, and the aqueous layer was further extracted with 100 ml of methylene chloride. The extracts were combined, dried over sodium sulfate (anhydrous), and then evaporated under reduced pressure. Dissolve the residue in a small amount of benzene,
Adsorb onto a Biobead 5X-3 column, elute with benzene, and collect the fraction showing UV absorption at an Rf value of 0.39 using silica gel TI, C66- developed with a mixed solvent of benzene/acetone (1/l).

It was dried under reduced pressure.

Dissolve this residue in a small amount of methylene chloride.

Benzene/acetone (2/1) was adsorbed onto a silica gel 12!90 column packed with benzene/acetone (2/1).
2/1), (1/1) and (1/3) mixed solvent and acetone, and the fractions eluted with acetone were collected and dried under reduced pressure to obtain 21.4 mg of the title compound.

This benzyl ester of antibiotic 0A-6129A exhibited the following physicochemical properties.

(1) Specific rotation [α] 31.5° ((?=1.0. C1 (2C
12) (2) Ultraviolet absorption spectrum HCI λ 22 nm (ε) = 318 (7400) a X a7- (3) Main peak of infrared absorption spectrum 1700
(ester) 1665 (amide) (4) Nuclear magnetic resonance spectrum (internal standard: TMS
) (a) When CD2C12 is used as a solvent, 2.39 (2H, t, J=6.5l-Iz, N-
Cl-1, -CI-12-CO).

2.85-3.67 (12H, m, C-41-I2
．． C-6H.

5-CH2-CH2-N, N-Cl-I2-CI(2
-Co, C-CI-(2-OH, OH or NLI),
3.93 (2I-I, m, C-5LI.

HO-(!EH-Co), 4.17 (II-I
, br, NH or OH).

5.17 (I H, d, J=1.3.0f(z,
CHH-A r ).

5.32 (I I-1, d, J=130I-Iz,
CHH=Ar).

6.73 (LH, br, N, H), 7.35
(5H, s, ArH) (b) 1.02 when CD2C12+D20 is used as a solvent (3H, t, J=7.5l
-Iz, CH2-C horse) 1.5 5~2.0 0
(2H,m, CH2-Cl-I3)2,39 (2
H, t, J=6.5H:z, N-CH2-CH,-
CO)280~3.67 (11H,m, C-4I(
2, C-6H.

S-C't(2-CI-I2-N, N-C1(2-C
FL2-CO.

C-Ck-12-Oi-I) 3.93 (IH, dt, J=3.0Hz, J=9
0f-Iz, C-5H)3,93 (LH,s, 1
(O-6-co)5,13 (11-1,d, J=1
3, OL(z, C, f(H-Ar)5, 28 (1
1(,d, J=13.0I(z, CI-(1(-A
r ) 7, 35 (5H, s, ArH) 329°Hydrolysis of this ester (6N hydrochloric acid, 115°G.

(16 hours) Cysteamine and β-alanine were confirmed in the product.

From the above physicochemical properties, the structure of antibiotic 0A-6129A is 70-, and it is thought to have a 5.6-ton and 9-ounce configuration.

Example 3 Preparation of p-nitrobenzyl ester of antibiotic 0A-6129A Antibiotic 0AI-6129A63.5 ~ was dissolved in dimethylformamide g, □ me, and 0.2 m, 1 of triethylamine was added under water cooling and stirring. Add 1.5 ml of a dimethylformamide solution containing 285 m9 of p-nitrobenzyl bromide, and react at the same temperature for 30 minutes.
1, and then allowed to react at room temperature for 3 hours.

IQQmA the reaction solution! Pour into ethyl acetate and salt to saturate 0
．． After washing with 20ml of 01M phosphate buffer (pH 8,4), the aqueous layer was further extracted again with 100ml of methylene chloride. The extracts were combined, dried over sodium sulfate (anhydrous), and then evaporated under reduced pressure.

Dissolve this residue in a small amount of methylene chloride.

Benzene/acetone (1/1) was adsorbed onto a 12 g column of silica gel packed with benzene/acetone (1/1).
1), (1/3) mixed solvent and acetone. Benzene/acetone (1/1
) with silica gel T L CVC developed with a mixed solvent.
R,/value 0.3] The fractions showing CUV absorption were collected and dried under reduced pressure to obtain 36.3 mg of the title compound.

The p-nitrobenzyl ester of antibiotic 0A-6129A exhibited the following physicochemical properties:
12) (2) Ultraviolet absorption spectrum λC'-""' nm (g): 319 (8400).

aX 270 (10500) (3) Optical external absorption spectrum HCI y 2 2 crn”: 1770 (β-lactam) aX 1700 (ester) 1665 (amide) (4) Nuclear magnetic resonance spectrum (solvent: CD2C1□;
Internal standard: TIViS) 1.04 ('3PI, t, J=7.5Hz,
CH2-Cl-l3)1,5~2.2 (3H,m,
C112-CI(3,0l-I)2.40 (2H,
t, J=6.5Hz, N-Cl-l2-CI-(2
-CO) 2,8~3.7 (12H,m, C-41 knee (2,
C-61-1゜5-cH,, -CH2-N, N-cH
2-cx-12-co.

C-C)i2-Of-I, 0I-I or Nl-1)3
．． 94 (2I(, m, C-5H, ■■0-Uran I-I
-Co)4.17 (LH, br, NH or 0I-
I) 5, 19 (IH, d, J=14Hz, Cdan・I-, I-Ar) 5.45 (LH, d, J=L
4Hz, CH4(-Ar)6.74 (IH, b
r, N1-1) 7, 63 (2H, d, J = 9
1-1z, Arl-1) 8.18 (2H, d, J
=9Hz, ArH, ) Example 4 Inglopyridenation method of antibiotic OA-6129A-p-nitrobenzyl ester 74- Antibiotic 0A-6129A-1)-Nitrobenzyl ester 11 Qmg was dissolved in 10 ml of acetone.

2. Add 2-dimethoxypropane o, srnl and 20 m9 of sodium sulfate anhydride, and add 40 m9 of pt-toluenesulfonic anhydride while stirring at room temperature. was added and reacted at the same temperature for 3 hours.

Add 1 ml of triethylamine Q to the reaction solution dropwise.

After stirring for 5 minutes, pour into 50 ml of ethyl acetate, o, 1M
After washing with 20 ml of phosphate buffer (pH=8.4).

For Sarah: Same M concentration W (pH=6.8) 20rnl
I washed it thoroughly.

The extract was dehydrated over anhydrous sodium sulfate and then evaporated under reduced pressure.

The residue was dissolved in a small amount of methylene chloride and adsorbed on a column of 5 g of silica gel packed with benzene/acetone (5/1).
1), (1/1), (2/1), and (115) were sequentially developed.

When the fraction eluted with benzene/acetone (1/1) was collected and concentrated, the It f value was 0.56 on silica gel TLC developed with a mixed solvent of benzene/acetone (1/1).
720 mg of the title compound exhibiting UV absorption was obtained.

The physicochemical properties of this compound are shown below.

(1) Specific rotation [α] sweet 349° (c=1.0. CI-■c 1
3) (2) Ultraviolet absorption spectrum 270 (9800) (3) Nuclear magnetic resonance spectrum (solvent CI) CI,,
Internal standard TMS) 1,07 (3H,t, J=7.5Hz -CH2-
CH, ) 1.7 ~ 2.0 (2H, m, -C
I-I2-CH3)2,43 (2H,t, J=6.
5Hz, N-CH2-CH,-Co)0-) 3.97 (LH,' dt, J=3Hz, 9I
-Iz, C, -1()4,' 03 (LH,s,
Co sieve-0-)5,20 (11-I, d, J
=14]-Iz, CH-I-I-Ar)5, 4.9
(H(, d, J=14Hz, CH-1-I-Ar
)6,52 (11(,br,N1-I)6
,93 (11-1,br,NH,)7,58
('2'l=f, d, J=9Hz, Ar1-
I) 8, 15 (21-1, d, J=91-Iz,
ArI-I)-76= (4) Infrared absorption spectrum (solvent Cl-ICl5)
Wavelength (Fu-1) 1770 (β-lactam) 1660 (amide) Example 5 Preparation of p-nitrobenzyl ester of antibiotic 0A-6129B2 Addition of antibiotic 0A-6129B2) to dimethylformamide 6, □ rnl 0.2 rnl of triethylamine was added under water cooling, and while stirring, a dimethylformamide solution containing 210 m9 of p-nitrobenzyl bromide was added. After reacting at the same temperature for 5 minutes, the reaction was continued at room temperature for 3 hours. The reaction solution was poured into 100 ml of methylene chloride and washed twice with 20 mg of 0.1 M phosphate buffer (pH 6,8) saturated with 1 sodium chloride. Furthermore, the aqueous layer was re-extracted twice with methylene chloride 10078-77-me. The extracts were combined, dried over anhydrous sodium sulfate, and then evaporated under reduced pressure.

Dissolve the residue in a small amount of methylene chloride and add benzene/
It was adsorbed on a 6 g column of silica gel packed with acetone (1/1), and benzene/acetone (1/1),
The mixture was sequentially developed with a mixed solvent of /2), (1/3), and (115) and acetone.

Silica gel T eluted with acetone from benzene/acetone (115) and developed with benzene/acetone (1/4)
By LC, 85 my of the title compound showing UV absorption at f value of 0.15 was obtained.

The physicochemical properties of this compound are shown below.

(1) Specific rotation [α] 41.4° (c-: t, o, dioxane) (2) I]% spectrum KI"ts-":, 1760 (β-lactam) + nax 1695 (ester) 7Q -1640 (amide) (3) UV spectrum 271 (10500) (4) NMR spectrum (, Pyridine
-δ5) However, it is written as δ1-1-5pp'.

δ (ppm) 1,30 (6H,s, CH3-C-Cl-1,)1
, 55 (3H, d, J=7.OI■z, CI-I
L-CH)2,70 (2H,t, J=6.5Hz,
N1-I-Cf (2-C horse-Co) 2.90-4.0
5 (11H,m, C-4,1-12,C-6H
.

5-Ci-I2-CII2-NI-I, N1(-CI
-T2-CII2-6°,. -61□2-and-) "4, 10~4.50 (2f(,m, C-51-I,
C-814) 4.5 2 (IH,s, HOJ
H-CO) Example 6 Inglopyridenation method of antibiotic 0A-6129B, p-nitrobenzyl ester Antibiotic 0A-6129B2 p-nitrobenzyl ester 20~ was mixed with acetone 5.0d, 2.2-dimethoxypropane 2 ．． Oml, sodium sulfate (anhydrous) 100m9
0.51n9 of p-toluenesulfonic acid is added while stirring at room temperature.

After reacting for 30 minutes, add 6μ of triethylamine to each reaction solution.
and stirred for 5 minutes. The reaction solution was distilled off under reduced pressure, 30 ml of methylene chloride was added to the residue, and after washing with O,1+V phosphate buffer (pi(8,4) 2 Oml), the extracted layer was dehydrated with sodium sulfate (anhydrous).

It was distilled off under reduced pressure. The residue was dissolved in a small amount of methylene chloride and adsorbed onto a 2 g column of silica gel packed with benzene/acetone (2/1). Benzene/acetone (2
/1), (1/1), (1/2) in sequence, and benzene/acetone (1/1) to (1/2)
The fractions eluted with were collected and concentrated to give the title ingropylidene compound 6,5 rn9 which showed an Rf value (0,64) on silica gel TLC developed with a mixed solvent of benzene/acetone (1/4).

The physicochemical properties of this compound are shown below.

(1) Specific rotation■ [α] Sweet 55.1° (C=0.5. CH2C12
) (2) ■ ■ Spectrum 1700 (Ester) 1668 (Amide) (3) UV Spectrum 270 (11900) 82- (4) NM Spectrum (CDCI, ) 1, 37 (3
H, d, J = -7,01 (z, CH, -CH)o
,.

1.40 (3H,s, CH3-C-CI(3)P 1,43 (3H,s, CH3-C-C horse) 2.41
(2H, t, J=6.5Hz, NH-CH2-C
Horse-Co) 2.75-3.80 (111(, m,
C-4H2, C-6H.

S-CH2-Cl-12-NH, NH-CI-I2-C
H2-C0,0-CH2-C) 4,02 (II(,s, O-♂H-C04,00~
4.30 (21-I, m, C-5H, C-8
H) 5, 16 (I I-I, d, J=14.5
Hz, CHH-Ar) 5.4 4 (11-I,
d, J=1 4.5Hz, CI(I
(-Ar)6,56 (II-f, br,
NH) 83- 6.92 (IH, br, NH) 7.55 (2H, d, J=8.0Hz, Ar-1-I
)8,12 (2H,d, J=8.0f(z,
Ar ・H) [Reference example] Production of triacetyl antibiotic 0A-612982 p-nitrobenzyl ester 12 μ of antibiotic 0A-6129B2 p-nitrobenzyl ester was dissolved in 5 ml of pyridine Q, and stirred while cooling with water. While stirring, 15 m6 of acetic anhydride Q was added. After reacting at the same temperature for 5 minutes, the reaction was continued at room temperature for 3 hours. Add ice water to the reaction solution and stir for 10 minutes, then add ethyl acetate/L, 20
ml of 0.1M phosphorus buffer (pI-I6.
8) After washing with 10ml C, use the same buffer (1) H8,4) 1
oml and further washed with the same pH 6,8 buffer.

The extract was dehydrated with anhydrous sodium sulfate and then evaporated under reduced pressure. Dissolve the residue in a small amount of methylene chloride.

It was adsorbed onto a column of silica gel 2I packed with benzene/acetone (5/1), and
/1), (3/1), (1/1), (2/
1).

(115) was sequentially developed with a mixed solvent. When the fractions eluted with benzene/acetone (1/1) were collected and concentrated, silica gel TLC developed with a mixed solvent of benzene/acetone (1/3) revealed the title compound exhibiting UV absorption at an Rf value of 0.59. .9 Tn9 was obtained.

The physicochemical properties of this compound are shown below.

(1) Specific rotation Ca)%' 23.2° (c=0.5.CHCl3
) (2) ■R spectrum CI-■C13-□.

νmax crn, 1780 (β-lactam) 1735 (ester) 1672 (amide) (3) UV spectrum 270 (12000) (4) NiVI R spectrum (CDCI,) 1
．． 43 (3H, d, J=7.0I(z, CH3-
CH)2.03 (3H,s, CI(,Co)2.1
0 (3H,s, CI(3Co)2.13 (3H,
s, CH3C0)2,38 (2I-I, t,
J=6.0)Iz, NH-CH2-CI-12-Co
)2.70~3.70 (9H, m, C-4Hz,
C-6H.

86-3, 82 (ll-1,,d, J=11.5Hz,
CHH-OAc)402 (II-I, d, J
=11.5I-1z, CHH-OAc)3,97~
4.27 (11-I, m, C-5I()4,
80 (IH,s, CH-OAc)5, 10~
5.45 (IPI, m, C-8H) 5, 22
(LH, d, J=14.5Hz, CHH-Ar
)5,50 (II-I, d, J=14.5Hz
, CHH-Ar)6.29 (LH,br, N1
-I) 6.75 (IH, br, NH) 7, 63 (2H, d, J=9.0I (z, Ar
-H) 8.21 (2H, d, J=9.0Hz, A
r-H) (5) Mass spectrum (FD) m/z
: 735 (M+1) Patent applicant Sanraku Ocean Co., Ltd.

Claims (1)

[Scope of Claims] 1. A compound represented by the formula (wherein R1 represents a hydrogen atom or a hydroxyl group, and 1t2 and R3 represent a hydrogen atom or a benzyl group substituted with isopropylide or unsubstituted when combined); and A salt of the compound when tW represents a hydrogen atom. 2. The compound according to claim 1, wherein j in formula (1), 2 and W together represent an inpropylidene group, and 4 represents a substituted or unsubstituted benzyl group. 3. The compound according to claim 1 or 2, wherein the compound in which 1 mole of formula (1) represents a hydroxyl group has a 5°6-trans or 5,6-cis configuration.