JPH0328435B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel antibiotic and its derivatives, more specifically, the following formula: (In the formula, R ¹ represents a hydrogen atom or a substituted or unsubstituted benzyl group, and R ² and R ³ each represent a hydrogen atom or together represent an isopropylidene group.

The present invention relates to a compound represented by the following formula, a salt of the compound when R ¹ , R ² and R ³ represent a hydrogen atom, and a method for producing the same. The following formula Antibiotics having a 7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid skeleton (hereinafter referred to as carbapenem antibiotics) generally have high antibacterial activity and β-lactamase inhibitory activity. Conventionally, various 7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid derivatives have been produced by fermentation, semi-synthesis, and total synthesis methods. [For example, thienamycin (Journal of Antibiotics, Vol. 32 (1979), pages 1-12), epithienamycins (17th Interscience Conference on Antimicrobial Diseases and Chemotherapy, Abstracts No. 80 and 81 (1977)), N-acetylthienamycin (West German Patent No. 2652681 (1977)), Olibanic acids (Journal of Antibiotics, Vol. 32 (1979), 287-304), PS-5 (Journal of Antibiotics, Vol. 32 (1979), pp. 262-286), PS-6 and PS-7 (Journal of Antibiotics, Vol. 33 (1979), pp. 262-286), 1980), pp. 1128-1137), No. 17927A ₁ substance and No. 17927A ₂ substance (JP-A-53-103401, JP-A-53-109997), KA-6643-A substance (PT. JP-A No. 55-139380) and various carbapenem antibiotics by total synthesis method (JP-A No. 56-5478)
Publication No.), etc.]. The above KA-6643-A substance has the following formula No.17927A ₁ and No.17927A ₂ substances are each represented by the following formula. However, recently, through examination of the culture conditions of the bacteria that produce these substances, it has been found that the following substances have been found to be structurally similar to these substances: No.17927D substance (2,3-dihydro form) shown by
It was discovered and proposed that the production of
55-24129). On the other hand, the present inventors previously calculated the following formula Antibiotic OA-6129-B (patent application 1982-
144507), as well as the 6-position of the antibiotic.

[Formula] The group is CH ₃ CH ₂ − and

[Formula] The antibiotics OA-6129A and OA-6129C were newly discovered and proposed respectively (Japanese Patent Application No. 135829/1982 and
−170864). The present inventors also found that among the OA-6129 group antibiotics,
Considering that there is a possibility that there is a substance that corresponds to the KA-6643-A substance or the No. 17927D substance as opposed to the No. 17927A substance, and as a result of examining the culture conditions of the OA-6129 group antibiotic-producing bacteria, A substance represented by the above formula (-a) was discovered, and the present invention was completed. The compound represented by the formula (-a), which is one of the compounds provided by the present invention, has a weaker antibacterial activity than the OA-6129 group antibiotics, but is effective against β-lactam-highly susceptible biotrophs, such as Comamonas terrigena. B-996 (Comamonas terrigena B-996)
The present inventors decided to name this compound "Antibiotic OA-6129E" because it showed weak antibacterial activity on an assay board containing horse serum. This antibiotic OA-6129E is a novel OA-6129E with high antibacterial potency that corresponds to the antibiotic by dehydrogenation under appropriate conditions known per se without eliminating the pantetheinyl group at the 3-position. 6129
There is a high possibility that it can be converted into a group antibiotic. In addition, after protecting the hydroxyl group in the side chain of antibiotic OA-6129E as necessary, dehydrogenation is performed by a method known per se, for example, as described in JP-A-54-76593, to produce antibiotic OA-6129E. The pantotheinyl group in the 3-position side chain of is removed by a chemical or biological method in advance to form the following formula. After producing the compound represented by and protecting or modifying the amino group in the side chain at the 3-position as desired,
It is also possible to dehydrogenate and induce carbapenem antibiotics in the same manner as above. In particular, compounds other than formula (-a) provided by the present invention, such as carboxylic acid esters derived from the OA-6129E and cyclic ketals of the esters, are more lipophilic than the antibiotic OA-6129E itself. Because of the improved properties, it is easier to handle in lipophilic organic solvents, and is expected to be used as an intermediate for semi-synthetic carbapenem antibiotics. Since the compound represented by the above formula (-a) provided by the present invention has a carboxyl group at the 2-position, it can also take the form of a salt or an ester. Examples of such salts include alkali metal salts such as sodium, potassium and lithium salts; alkaline earth metal salts such as calcium and magnesium salts; other metal salts such as aluminum salts; ammonium salts, monoethylamine, dimethyl amine, trimethylamine, monoethanolamine,
Includes salts with primary, secondary or tertiary amines such as diethanolamine; salts with organic bases such as benzathine salts and procaine salts, among which pharmaceutically acceptable salts are preferred, particularly sodium salts. Alkali metal salts such as , potassium salts are preferred. Further, examples of the ester of the compound of formula (-a) include benzyl ester, p-nitrobenzyl ester, p-methoxybenzyl, p-bromobenzyl ester, benzhydryl ester,
Trityl ester, phenacyl ester, phthalidyl ester, phthalimidomethyl ester, pivaloyloxymethyl ester, methoxymethyl ester, methylthiomethyl ester, 2,2,
Examples include substituted or unsubstituted benzyl esters such as 2-trichloroethyl ester. Furthermore, the antibiotic OA- provided by the present invention
Yet another derivative of 6129E is one in which the α- and γ-positions of the pantoyl group are simultaneously etherified.
formula (In the formula, R ¹ has the above-mentioned meaning) An isopropylidene derivative represented by the following is mentioned as a representative compound, and as an equivalent of the compound,
Other cyclic ketals or cyclic acetals can be mentioned. According to the invention, the antibiotic OA of the formula ()
-6129E and/or their derivatives can be obtained by first culturing the antibiotic OA-6129E-producing microorganism in a nutrient medium, collecting the antibiotic OA-6129E from the culture, and optionally injecting the antibiotic itself. It can be produced by a method consisting of esterification by a known method and further cyclic acetalization or cyclic ketalization. The antibiotic OA-6129E-producing bacteria used in the present invention is an antibiotic OA-6129E-producing bacterium that has the above-mentioned chemical structure.
As long as it has the ability to produce 6129E,
Bacteria belonging to any genus can be used and can be selected from a wide range of microorganisms. A search for a strain suitable for the purpose of the present invention can be carried out by a method known per se, and by this means, those skilled in the art can easily obtain the antibiotic OA-6129E-producing bacteria used in the present invention. Typical bacteria producing antibiotic OA-6129E include antibiotic OA-6129E, which belongs to the genus Streptomyces.
6129E-producing bacteria are included, and a suitable example thereof is an actinomycete isolated from soil collected near Sumiyoshi Shrine in Fukuoka City, Fukuoka Prefecture, which the present inventors have identified as OA-
Examples include strains numbered 6129 strains. The mycological properties of this OA-6129 strain are as follows. 1. Morphology Under the microscope, the well-branched basal hyphae are straight to
Elongates curved (Straight~flexuous) aerial hyphae,
Whorled branches are not observed. A mature spore chain consists of ten or more oval to cylindrical spores, and no sporangia are observed. Spore size is 0.6-1.0×0.7
Approximately 2.5 microns in size, the spore surface is smooth.
Flagellated spores are not observed. 2 Growth conditions in various media Cultivation was carried out at 28° to 30°C unless otherwise specified.
In addition, descriptions of color tones are mainly based on HDTresner and E.G. Bakus.
and EJBackus), Journal of Applied Microbiology
Applied Microbiology) Volume 11, No. 4, (1963)
According to the method on pages 335 to 338, the code in [ ] [CHM code] is based on the Color Harmony Manual of Container Corporation of America.
America's Color Harmony Manual) was used. (1) Seuculose/nitrate agar medium: Aerial mycelia of yellow ash [2 dc] to gray yellow pink [5 dc] are grown on medium growth of yellow ash [2 dc] to light gray yellow brown [3 ge], and soluble. No pigment is visible. (2) Glucose-asparagine agar medium: Bright gray [d] aerial mycelia are attached to the good growth of light yellow [2db] to bright olive brown [2ge]. This aerial mycelium later becomes grayish-yellow-pink [5 dc]. No soluble pigments are observed. (3) Glycerin-asparagine agar medium (ISP medium-5): Good growth of gentle yellow-pink [4gc] to light brown [4ie], and light gray [d] to light gray-reddish brown [5fe]. Grows hyphae. No soluble pigments are observed. (4) Starch inorganic salt agar medium (ISP medium-4): Bright gray [d] aerial mycelium grows on light yellow [2db] to gray brown [2fe] growth. No soluble dyes are produced. (5) Tyrosine agar medium (ISP medium-7): Aerial mycelium of light gray [d] to light brown gray [3fe] grows on gray-yellow [3ec] to light brown [4ie] growth. The medium will have a very slight brown tinge. (6) Nutrient agar medium: Aerial mycelium of light gray-reddish brown [5fe] grows on a well-growing medium of light yellow [2db] or bright yellow [2fb] to light olive brown [2ge]. No soluble pigments are observed. (7) Yeast extract/malt extract agar medium (ISP
Medium-2): On the good growth of gentle yellow-pink [4gc] to bright brown [4ie], aerial mycelia of gray-yellow-pink [5dc] or slightly later bright gray [d] are attached. No soluble pigments are observed. (8) Oatmeal agar medium (ISP medium-3): On top of good growth of gray-yellow [3ec] to bright orange-yellow [3ea], or bright gray-yellow-brown [3ge], light brown-gray [3fe] to light gray Reddish-brown [5fe] aerial mycelium grows, and the culture medium around the fungal colony is slightly brown. (9) Malate lime agar medium: Aerial mycelium of light gray [d] to light gray reddish brown [5fe] grows on medium growth of dark to yellow gray [2dc], with no soluble pigments. I can't do it. A dissolution zone of calcium salts can be seen around the growing bacterial colonies. (10) Glucose-peptone-gelatin medium (20℃
Culture) White [B] to gray-yellow pink [5CB] aerial mycelia grow on the pale yellow [2dB] to brown good growth.
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: 10°, 20°, 25°, 30°, 34°, 37°, using yeast extract/malt extract agar medium (ISP medium-2).
As a result of experiments at temperatures of 40°, 45°, and 50°C, almost no growth occurred at 37°C. It does not grow at all above 40℃. Growth was observed at all other temperatures. The optimal growth temperature range seems to be 20-30°C. (2) Liquefaction of gelatin: Liquefaction. (3) Starch hydrolysis: decomposes. (4) Coagulation and peptonization of skim milk: It does not coagulate, but it converts into peptonization. (5) Production of melanin-like pigment: Peptone yeast iron agar medium (ISP medium)
5) and tryptone yeast extract broth medium (ISP medium-11), no production of melanin-like pigment was observed. Although it shows a very slight brown color on tyrosine agar medium, there is only a trace of melanin production. 4 Assimilation of various carbon sources (using Pridham-Gotlieb agar medium) (1) L-arabinose + (2) D-xylose + (3) D-glucose + (4) D-fructose + (5) Seuculose Doubtful (6 ) Inositol - (7) L-rhamnose + (8) Raffinose - (9) L-mannite ++ + is assimilated, - is not assimilated. Based on the above mycological properties, the OA-6129 strain is
It is a strain belonging to the genus Streptomyces, and the shape of the aerial hyphae is Section RF (Section RF).
Rectiflexibiles), and the spore surface was smooth. The color of the aerial mycelium is light gray [d] in most media, such as oatmeal agar, glycerin/asparagine agar, starch/inorganic salt agar, etc., and is a gray series strain.
However, depending on the culture period, sucrose/nitrate agar, yeast extract/malt extract agar, and glucose/asparagine agar media may exhibit a grayish-yellow-pink [5 dc] red series. In addition, the color of the basal hyphae is light yellow to grayish-yellow in any medium at the early stage of culture, and as the culture continues, it becomes yellowish-brown to yellowish-yellow.
It becomes grayish-yellow or brown in color. Melanin pigment was not observed in peptone/yeast extract/iron agar medium or tryptone/yeast extract/broth, and other water-soluble pigments were not produced in many media. However, a slight brown pigment was observed in the tyrosine agar medium, glucose peptone gelatin medium, and oatmeal agar medium. The present inventors have designated this bacterium as Streptomyces sp. OA-6129, and it has been internationally deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, as FAIKEN Article No. 11. The antibiotic OA-6129E of the present invention is an antibiotic OA-6129E.
−6129E-producing bacteria, such as the above Streptomyces
It is produced by inoculating sp. OA-6129 spores or hyphae into a nutrient-containing medium and growing aerobically. As the nutrient source, those normally used as a nutrient source for actinomycetes, such as assimilable nutrient sources such as carbohydrates, nitrogen sources, and inorganic salts, can be used. For example, carbohydrates such as glucose, glycerin, maltose, sucrose, molasses, dextrin, and starch;
Carbon sources such as oils and fats such as soybean oil, peanut oil, and lard; peptone, meat extract, soybean flour, cottonseed flour, dried yeast, corn steep liquor, yeast extract, skim milk, casein, sodium nitrate, ammonium nitrate, Nitrogen sources such as ammonium sulfate; inorganic salts such as dipotassium phosphate, common salt, calcium carbonate, and magnesium sulfate can be used, and if necessary, trace metals such as cobalt and manganese can be added. In addition, antibiotic OA-6129E-producing bacteria can be used as a nutritional source.
Any nutrient source that produces 6129E can be used, 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 nutrient sources as described above is not particularly restricted and can be varied over a wide range, and the optimal nutrient source composition and blending ratio for the antibiotic OA-6129E-producing bacteria to be used is determined by the A person skilled in the art can easily determine this through simple small-scale experiments. The nutrient medium can also be sterilized prior to culturing, and it is advantageous to adjust the pH of the medium to a range of 4 to 9, particularly 6 to 8, before or after this sterilization. In principle, the antibiotic OA-6129E-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 preferred to culture under aerobic conditions, usually with stirring and/or
Alternatively, it can be carried out with ventilation. Further, as a culture method, static culture, shaking culture, and submerged culture with aeration and agitation are advantageous. The culture temperature that can be used is such that the growth of antibiotic OA-6129E-producing bacteria is not substantially inhibited.
There is no particular restriction as long as it can produce 6129E, and it can be changed depending on the production strain used, but generally the temperature is 20 to 40°C, preferably 25°C.
Temperatures in the range ˜35° C. are preferred. Moreover, in order to carry out the culture suitably, the pH of the culture can be adjusted to a range of 4 to 9, particularly 6 to 8, during the culture, if necessary. In the case of large-scale mass cultivation, it is advantageous to perform a seed culture as appropriate, inoculate it into a nutrient medium, and perform liquid culture. Cultivation can normally be continued until sufficient accumulation of antibiotic OA-6129E occurs. The culture time varies depending on the composition of the medium, culture temperature, production strain used, etc., but is usually in the range of 30 to 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. The amount of antibiotic OA-6129E accumulated in the culture is β
- It can be quantified by a bioassay method and bioautography using a highly lactam-sensitive bioassay bacterium, such as Comamonas terrigena B-996, and thereby the optimum accumulation amount can be easily determined. The antibiotic OA− thus accumulated in the culture.
Since 6129E is water-soluble and mainly exists outside the bacterial cells, it is advantageous to remove the bacterial cells after culturing by a separation method known per se, such as filtration, centrifugation, or extraction, and extract the resulting liquid from the supernatant. It is recovered from clear liquid, extract, etc. Recovery can be carried out by various methods known per se, and in particular methods often used for recovery of 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 high pH water layer; activated carbon, Amberlite XAD (manufactured by Rohm and Haas), Diaion HP-20, (manufactured by Mitsubishi Kasei Corporation), elution with methanol water, acetone water, etc.; Dowex 1x2 (manufactured by Dow Chemical Company), QAE-Sephadex A-
Adsorption and elution with ion exchange resins such as 25 (manufactured by Pharmacia), DEAE-cellulose Watmann DE-32 (manufactured by Watmann), DEAE-Sephadex A-25 (manufactured by Pharmacia); Cephadex G-10 (manufactured by Pharmacia) ), Bio Gel P-
2 (manufactured by Bio-Rad), etc.; column chromatography of cellulose, Avicel SF (manufactured by American Viscose), etc.; forced precipitation by adding a solvent such as acetone; freeze-drying, etc. They may be used alone or in appropriate combinations, and may be used repeatedly. Thus, antibiotic OA with the above-mentioned properties
−6129E is obtained. Antibiotic OA- produced by the above fermentation method
6129E, that is, the compound of the formula (-a),
Generally, the hydrogen atoms bonded to the 5- and 6-position carbon atoms each have a trans or cis configuration, and the formula (-a) has a 5,6 trans form,
Either the 5,6-cis form or a mixture thereof is included. In addition, the carbon atoms at the 2-position and 3-position, the carbon atom at the base of the secondary hydroxyl group of the pantoyl group in the side chain at the 3-position, and the hydroxyl group in the 1-hydroxyethyl side chain at the 6-position are bonded. Since each carbon atom in the antibiotic is an asymmetric carbon atom, the antibiotic
OA-6129E can exist in either the S-form or the R-form, or in racemic form. The present antibiotic OA-6129E is generally more stable in its salt or ester form than in its free form, so it can be used as an intermediate when converting into derivatives, or subjected to the purification process described above. In some cases, it is preferable to treat the compound in the form of a salt or ester. Conversion of the antibiotic OA-6129E into its salt or ester form can be carried out according to methods known per se. For example, a salt of the antibiotic OA-6129E can be prepared by treating free antibiotic OA-6129E with an inorganic or organic base. Inorganic or organic bases that can be used in this salt-forming reaction include, for example, alkali metal hydroxides such as sodium hydroxide, potassium hydroxide, and lithium hydroxide; alkaline earths such as calcium hydroxide and magnesium hydroxide; Hydroxides of similar metals; primary, secondary, or tertiary organic amines such as monoethylamine, dimethylamine, trimethylamine, monoethanolamine, diethanolamine, benzathine, and procaine. Furthermore, the esterification of the antibiotic OA-6129E can be carried out in the same manner as the esterification of the antibiotic PS-5, etc., such as the method described in JP-A-54-84589. Specifically, antibiotic OA−
6129E with an organic base or an alkali metal or alkali metal salt in an inert solvent such as dimethylformamide, tetrahydrofuran, dioxane, hexamethylphosphotriamide, acetonitrile, acetone, etc. from about -50°C to 50°C, preferably about 1.0 to 10 equivalents of RX at temperatures between -10°C and 30°C
(In the formula, X represents a halogen atom and R represents an ester residue) by stirring and reacting for several minutes to several hours. Antibiotic OA-6129E obtained as above
The substituted or unsubstituted benzyl ester of 3-
α- of the pantoyl group bonded to the carbon atom in position
and the hydroxyl group at the γ-position 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, esters of antibiotic OA-6129E can be converted into their cyclic ketal or cyclic acetal derivatives by reacting with ketone or aldehyde derivatives. This reaction is generally preferably carried out using a reaction reagent as a solvent, and examples of the reaction reagent that can be used include acetone, 2,2-dimethoxypropane, formaldehyde, 2,2-dimethoxyethane, acetaldehyde, benzaldehyde, propion Examples include aldehyde, acetophenone, diphenyl ketone, cyclohexanone, dibenzyl ketone, 1,1 diphenylacetone, etc. Each of these reaction reagents may be used alone, or two or more types may be mixed if necessary. You can also do that. Also, if necessary, antibiotics for reaction reagents may be added.
An inert solvent can also be mixed in consideration of the solubility of the ester of OA-6129E. The reaction temperature is not critical and can be varied widely depending on the type of liquid medium used, etc., and can be arbitrarily selected within the temperature range in which the ester of antibiotic OA-6129E does not decompose significantly. Generally temperatures below 60°C, preferably in the range -40 to 40°C, more preferably in the range 0°C to room temperature, are advantageous. Further, in the above reaction, a reaction accelerator such as p-toluenesulfonic anhydride, boron tolufluoride etherate, zinc chloride, etc. is added as necessary. Under these conditions, the reaction typically takes place within 30 minutes to 12 hours.
Usually it can be completed in 2 to 5 hours. The cyclic ketal or cyclic acetal derivative of the ester thus obtained can be isolated from the reaction mixture according to separation and purification methods known per se. For example, after the reaction is completed, firstly, in order to inactivate the excess reaction accelerator, etc., it is treated with an amine, and then the treated solution is placed in a non-polar organic solvent that is substantially immiscible with water, such as ethyl acetate, benzene, or chloroform. Open it. 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, after concentrating the organic solvent layer to dryness, according to a method known per se, for example,
Silica gel, Biobeads (manufactured by Biorad),
A desired cyclic ketal or cyclic acetal derivative can be isolated by appropriately combining chromatography using Sephadex LH-20 (manufactured by Pharmacia) or the like as a carrier and using it repeatedly as necessary. Antibiotic OA-6129E provided by the present invention
The salts or esters are useful as synthetic intermediates for various carbapenem antibiotics, and the antibiotics can be used as dehydrodipeptidase inhibitors or as in vivo precursors (prodrugs) of carbapenem antibiotics. Potentially useful. Next, the present invention will be further explained with reference to Examples. Example 1 6-(1-hydroxy-1-methylethyl-3
-Production of sodium pantetheinyl-7-oxo-1-azabicyclo[3.2.0]heptane-2-carboxylate (antibiotic OA-6129E) by fermentation method (A) 100 ml of a seed culture medium (S-1) having the following composition was placed in a 500 ml Erlenmeyer flask and sterilized at 120° C. for 15 minutes by a conventional method. On the other hand, Streptomyces sp. OA-6129 (Streptmyces sp.
sp. 7 cm) and cultured with shaking.
This seed culture solution 1 was inoculated into a 2,000 capacity fermenter containing 1,000 volumes of production medium (GM-1) with the following composition, and stirred at 28°C, 150 rpm and 500 rpm.
Aerated agitation culture was performed for 90 hours under aerated conditions.
Silicone KM-75 as an antifoaming agent (manufactured by Shin-Etsu Chemical Co., Ltd.)
was used at 0.07%. 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 (μg/ml) 48 0.8 72 5.6 90 15.3 Composition of seed medium (S-1): Meat 1.5% (W/V) Yeast extract 0.5 〃 Potato starch 2.0 〃 CaCO ₃ 0.2 〃 PH (before sterilization) 7.0 Composition of production medium (GM-1): Glycerin 8.0% (W/V) Fish meal 1.0 〃 Meat 3.0 〃 CaCO ₃ 0.3 〃 K ₂ HPO ₄ 0.2 〃 MgSO ₄ 0.2 〃 PH with NaOH Dissolve in 0.01M phosphate buffer with pH5.5 adjusted to 7.2 and
In addition, 0.0005% (W/V) of vitamin B ₁₂ was added which had been sterilized in an autoclave at 1 kg/cm ² G for 5 minutes. (B) Fermented liquid 950 after 90 hours of culture obtained in (A) above
5% (W/V) amount of Topcoperlite No.34
[manufactured by Toko Perlite Co., Ltd.] was added, and the bacterial cells were separated using a filter press to obtain 900 culture solutions. This is Diaion HP-20 [manufactured by Mitsubishi Kasei Corporation]
Adsorb into a packed column (30 x 300 cm) and add distilled water.
After washing with 30% (V/V) acetone water, elution was performed. One division was designated as 1, and the eluate was fractionated.
A total of 14 eluates from fraction 11 to fraction 24 were collected and transferred to Diaion PA306S [Mitsubishi Kasei Corporation].
After adsorption onto a packed column (10 x 100 cm) manufactured by J.D. Co., Ltd., and washing with 10 g of distilled water, it was eluted with 0.01 M phosphate buffer (PH8.4) containing 30% sodium chloride. Fractionate the eluate one by one, from fraction 6 to fraction 20, totaling 150
The eluate was collected. This eluate was adsorbed onto a Diaion HP-20 packed column (10 x 100 cm), and the concentration was reduced to 0.
Acetone water total increasing linearly from to 30%
It eluted at 40. Each section was 200 ml, and the eluate was fractionated. By bioassaying each fraction, active fractions from fraction 55 to fraction 62 can be determined.
Obtained 1600ml. Biogel P-2 (manufactured by BioRad) was equilibrated with 0.01M phosphate buffer (PH8.4) four times in 400ml portions of this active section.
It was introduced into a packed column (8 x 100 cm), developed with the above buffer, and determined by bioassay to have an activity of 7.0.
Collected. This active fraction was adsorbed on a QAE Sephadex A-25 (manufactured by Pharmacia) packed column (8 x 100 cm) equilibrated with the above buffer solution,
The pH of this eluate was carefully adjusted to 8.4 with 10% NaOH, and then diluted with 0.01M phosphate buffer (PH8.4).
Adsorb onto a Diaion HP-20 packed column (4 x 100 cm) pre-equilibrated with
Acetone water increasing linearly up to 30% (total
6.0). One section is 15 ml, the eluate is fractionated, and the antibiotic OA-6129B ₂ , D and E sections from section 48 to section 67 are collected and 300 ml is collected.
I got it. This was freeze-dried to obtain 12 g of brown powder. The obtained 12 g of powder of OA-6129B ₂ , D and E categories was dissolved in a small amount of water and filled with Cephadex G-10 (manufactured by Pharmacia) equilibrated with 0.01M phosphate buffer (PH8.4). Column (1.5×
80 cm), developed with the above buffer solution, and collected 30 ml of active fraction by bioassay. This active fraction is adsorbed onto a QAE Sephadex A-25 (manufactured by Pharmacia) packed column (2.5 x 40 cm) that has been equilibrated with the above buffer solution. After washing with 180 ml of the above buffer, elution was carried out with the above buffer containing linearly increasing saline from 0% to 2.0%. 15ml
Fractionate the eluate and use bioassay to divide the active fraction into fractions 22 to 38, totaling approximately 250 ml.
Collected. After freeze-drying this eluate, it was dissolved in a small amount of water and used as Diaion HP-20AG (manufactured by Mitsubishi Kasei Corporation).
Adsorb onto a packed column (1.5 x 110 cm) and absorb approximately 200
After washing with 1.2 ml of distilled water, it was eluted with acetone water increasing linearly from 0% to 6% (total 1.2). The eluate was fractionated into 10 ml portions, and active fractions were obtained by bioassay. Among the active fractions, the fraction
A total of 450 ml of fraction 71 from 27 was antibiotic OA−
6129B ₂ , D and E categories. By freeze-drying each section, OA−
6129B ₂ , Sodium salts of D and E (mixture)
1.3g of coarse powder was obtained. After dissolving this powder in 3 ml of water again, use Diaion.
Adsorb it on a CHP-20 (3 x 150cm) column,
400 ml of water and water-isopropanol (400-
Elution was performed using a linear concentration gradient gradient method (400 ml). When the elution fraction shows no UV absorption and the Ridesmith color positive fraction is divided into 9 fractions and lyophilized, OA-6129E is obtained from 2 fractions.
28 mg of sodium salt was obtained. The physical properties of this OA-6129E sodium salt are as follows. UV (H ₂ O): Terminal absorption IR (KBr, cm ^-1 ): 1750 (β-lactam), 1650 (amide), 1600 (carboxylate) NMR (D ₂ O): δ0.87 (3H, s, CH ₃ ), 0.90 (3H, s, CH ₃ ),
1.31 (3H, s, CH ₃ ), 1.43 (3H, s, CH ₃ ),
1.55-2.05 (2H, m), 2.15-3.85 (13H, m),
3.95 (1H, s, O-CH-CO), 4.53 (1H, d,
7.5Hz, H-2) Example 2 6-(1-hydroxy-1-methylethyl-3
-Production of p-nitrobenzyl ester of pantetheinyl-7-oxo-1-azabicyclo[3.2.0]heptane-2-carboxylic acid (antibiotic OA-6129E) Antibiotic OA-6129B ₂ , D obtained in Example 1
Dissolve 1.3 g of pale yellow crude powder consisting of the sodium salt of OA-6129E in 50 ml of dimethylformamide, add 2.5 ml of triethylamine under ice cooling, and while stirring, dissolve 2.7 g of p-nitrobenzyl bromide dissolved in a small amount of dimethylformamide. g 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 300 ml of methylene chloride and washed twice with 50 ml of 0.1M phosphate buffer (PH6.8). Furthermore, 100 ml of the aqueous layer was re-extracted twice with 100 ml of methylene chloride. The extracts were combined, dried over sodium sulfate (anhydrous), and then evaporated under reduced pressure. The residue was dissolved in a small amount of methylene chloride and packed with benzene/acetone (1/1).
g of silica gel, developed sequentially with benzene/acetone mixed solvent (1/1), benzene/acetone mixed solvent (1/3), and acetone, and the fraction eluted with benzene/acetone mixed solvent (1/3) collected. When concentrated, silica gel developed with benzene/acetone mixed solvent (1:3)
Antibiotics that exhibit UV absorption at an Rf value of 0.55 in TLC
p-nitrobenzyl ester of OA-6129E is 320
mg obtained. Example 3 6-(1-hydroxy-1-methyl)ethyl-
3-isopropylidenepantetheinyl-7-oxo-1-azabicyclo[3.2.0]heptane-
Production of 2-carboxylic acid p-nitrobenzyl ester Add 320 mg of p-nitrobenzyl ester of antibiotic OA-6129E to 10 ml of acetone, 1.0 ml of 2,2-dimethoxypropane, and 200 mg of sodium sulfate (anhydrous).
Dissolve p in a mixed solvent of p
- Add 7.0 mg of toluenesulfonic acid. After reacting for 30 minutes, 0.1 ml of triethylamine was added to the reaction solution and stirred for 5 minutes. The reaction solution was distilled off under reduced pressure, 100 ml of methylene chloride was added to the residue, and the mixture was washed successively with 20 ml of 0.1M phosphate buffer (PH8.4) and 20ml of 0.1M phosphate buffer (PH6.8). The extract layer was dehydrated with sodium sulfate (anhydrous) and then evaporated under reduced pressure. Dissolve the residue in a small amount of methylene chloride and add benzene/
It was adsorbed onto a 5 g column of silica gel packed with an acetone mixed solvent (10/1). Benzene/acetone mixed solvent (10/1), (5/1), (3/1),
Develop sequentially with (1/1) and (1/2), and use benzene/acetone mixed solvent (1/1) to (1/2).
After collecting and concentrating the fractions eluted at up to 210 ml, the title substance with an Rf value of 0.54 was found on silica gel TLC developed with a benzene/acetone mixed solvent (1:1).
mg obtained. The physical properties of this substance were as follows. Specific optical rotation [α] ²⁴ _D 12.6° (C=1.0, CHCl ₃ ) UV spectrum λ ^CHCl3 _nax nm (ε): 268 (21500) IR spectrum ν ^CHCl3 _nax cm ^-1 : 1750 (β-lactam, ester)
1662 (amide) NMR spectrum (CDCl ₃ , internal standard TMS) δ: 0.97 (3H, s,

【formula】) 1.03(3H,s,

【formula】) 1.28(3H,s,

【formula】 1.41(3H,s,

[Formula] or

[Formula]) 1.45 (3H, s,

[Formula] and

[Formula] or

【formula】) 1.46(3H,s,

[Formula] and

[Formula] or

[Formula]) 1.65-2.15 (2H, m, C-4H ₂ ) 2.25-2.70 (5H, m, NH-CH ₂ -C H ₂ -
CO, OH, S-CH ₂ -C H ₂ -NH) 2.80-3.85 (8H, m, C-3H, C-6H,
S-C H ₂ -CH ₂ -NH, NH-C H ₂ -
CH ₂ −CO,

【formula】) 3.90~4.30 (1H, m, C-5H) 4.03(1H,s,

[Formula]) 4.73 (1H, d, J=7.5Hz, C-2H) 5.23 (1H, s, C H ₂ -Ar) 6.30 (1H, br, NH) 6.93 (1H, br, NH) 7.50 (2H , d, J=8.5Hz, Ar・H ) 8.17 (2H, d, J=8.5Hz, Ar・H ) Mass spectrum M/Z: 664 (M ⁺ ) From the above physical data, the title compound is , is presumed to have the structural formula shown below the title.

Claims (1)

[Claims] 1 formula (In the formula, R ¹ represents a hydrogen atom or a substituted or unsubstituted benzyl group, R ² and R ³ each represent a hydrogen atom, or together represent an isopropylidene group [Formula], provided that R (excluding cases where ¹ is a hydrogen atom and R ² and R ³ together represent an isopropylidene group) and salts of the compounds when R ¹ , R ² and R ³ represent a hydrogen atom . 2. The compound according to claim 1, wherein R ¹ , R ² and R ³ each represent a hydrogen atom. 3. The compound according to claim 1, wherein R ¹ represents a substituted or unsubstituted benzyl group, and R ² and R ³ represent a hydrogen atom. 4. The compound according to claim 1, wherein R ¹ represents a substituted or unsubstituted benzyl group, and R ² and R ³ together represent an isopropylidene group.