JPS6334157B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel antibiotic, more specifically, the following formula: The present invention relates to a compound represented by the above, salts and esters thereof, and a method for producing the compound. 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. 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 Agents and Chemotherapy), , Abstract No. 80 and No.
81 (1977)), N-acetylthienamycin (West German Patent No. 2652681 (1977)), olivanic acids (Journal of Antibiotics, Vol. 32 (1979), pp. 287-304), PS -5 (Journal of Antibiotics, Vol. 32 (1979), pp. 262-286), PS-6 and PS-7 (Journal of Antibiotics, Vol. 33 (1980), pp. 1128-1137) Page), No. 17927A, Substances and
No. 17927A ₂ substances (Japanese Patent Application Laid-Open No. 53-103401 and JP-A No. 53-109997), etc.). No.17927A ₁ substance listed at the end above and No.
17927A _{The two} substances have the following formulas: 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 We newly discovered and proposed the antibiotic OA-6129B shown by (Japanese Patent Application No. 144507/1982). The present inventors have determined that the above-mentioned No. 17927A,
Similar to the relationship of No.17927D substance, the above antibiotic OA
Considering that there is a possibility that a 2,3-dihydro form also exists for -6129B, the antibiotic OA-6129B
As a result of examining the culture conditions of the producing bacteria, a substance represented by the above formula was discovered, and the present invention was completed. The compound represented by the above formula is an antibiotic OA-
Although its antibacterial activity is weaker than that of 6129B, it is highly susceptible to β-lactams such as Comamonas terrigena B-996.
The present inventors have classified this compound as an "antibiotic."
We decided to name it "OA-6129D". This antibiotic OA-6129D can be converted to antibiotic OA-6129B with high antibacterial potency by dehydrogenating under appropriate conditions without eliminating the pantetheinyl group at the 3-position. for example,
Antibiotic OA-6129B-producing bacteria or processed products thereof,
By acting on the antibiotic OA-6129D with a cell disruption solution or a portion thereof or a completely purified enzyme solution, it can be converted to the antibiotic OA-6129B, and the hydroxyl group in the side chain of the antibiotic OA-6129D is required. After protection by a method known per se,
For example, as described in JP-A-54-76593, the pantotheinyl group in the 3-position side chain of the antibiotic OA-6129D is preliminarily removed by a chemical or biological method by dehydrogenation, and the following formula is obtained. 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. Since the compound represented by the above formula 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 sodium salts, potassium salts,
Alkali metal salts such as lithium salts; alkaline earth metal salts such as calcium salts and magnesium salts;
Other metal salts such as aluminum salts; ammonium salts; salts with primary, secondary or tertiary amines such as monoethylamine, dimethylamine, trimethylamine, monoethanolamine, diethanolamine; organic salts such as benzathine salts, procaine salts Salts with bases are included, and among these, pharmaceutically acceptable salts are preferred, and alkali metal salts such as sodium salts and potassium salts are particularly preferred. Examples of the ester of the compound of the formula include benzyl ester, p-nitrobenzyl ester, p-methoxybenzyl, p-promobenzyl ester, benzhydryl ester, trityl ester, phenacyl ester, phthalidyl ester, phthalimidomethyl ester. , pivaloyloxymethyl ester, methoxymethyl ester, methylthiomethyl ester, 2,2,2-trichloroethyl ester and the like. According to the invention, the antibiotic OA-
6129D is produced by culturing the antibiotic OA-6129D-producing microorganism in a nutrient medium and extracting the antibiotic from the culture.
It can be produced by a method consisting of collecting OA-6129D. The antibiotic OA-6129D-producing bacteria used in the present invention is an antibiotic OA-6129D-producing bacterium having the above-mentioned chemical structure.
As long as it has the ability to produce 6129D,
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 bacteria producing the antibiotic OA-6129D used in the present invention. Typical bacteria producing antibiotic OA-6129D include antibiotic OA-6129D, which belongs to the genus Streptomyces.
6129D-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 diameter, 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. Bakas.
and EJBackus), Journal of Abiotic 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 and dissolved on medium growth of yellow ash [2 dc] to light gray yellow brown [3 ge]. No sex pigments are observed. (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 [2fe] growing well. 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], bright brown-gray [3fe] to Bright grayish-reddish brown [5fe] aerial mycelium grows on it, and the 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. 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 prosthetic 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 tone 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 initial stage of culture, and as the culture continues, it becomes yellowish brown.
It becomes grayish-yellow or brown in color. Melanin pigment was not observed in peptone/yeast extract/iron agar medium or tryptone/yeast extract/pross, 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 identified this bacterium as Streptomyces sp. OA-6129, and it has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Microbiological Research Institute No. 5714, and This bacterium was transferred to the International Deposit under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedures on May 1, 1980, as FIKEN Article No. 11. The antibiotic OA-6129D of the present invention is an antibiotic OA-6129D.
−6129D-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-6129D-producing bacteria can be used as a nutritional source.
Any nutrient source that produces 6129D 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-6129D 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 cultivation of antibiotic OA-6129D-producing bacteria in such a nutrient medium can be carried out according to the method commonly used in the production of antibiotics using general actinomycetes. It is usually preferred to culture under aerobic conditions, usually with stirring and/or
Alternatively, it can be carried out with ventilation. In addition, as a culture method, static culture, shaking culture, and submerged culture with aeration and agitation can all be used.
Submerged culture is advantageous. The culture temperature that can be used is such that the growth of antibiotic OA-6129D-producing bacteria is not substantially inhibited.
There is no particular restriction as long as it can produce 6129D, 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-6129D 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-6129D 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. Thus, the antibiotic OA accumulated in the culture
Since -6129D is water-soluble and mainly exists outside the bacterial cells, it is advantageous to use filtration, centrifugation,
The bacterial cells are removed by a separation method known per se, such as extraction, and recovered from the liquid, supernatant liquid, extract liquid, 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 etc. at low pH and transfer of the solvent layer to high pH water layer; activated carbon, Amberlite XAD (manufactured by Rohm and Haas),
Adsorption with Diaion HP-20 (manufactured by Mitsubishi Kasei Corporation) and elution with methanol water, acetone water, etc.; Dowex 1×2 (manufactured by Dow Chemical Corporation),
QAE-Sephadex A-25 (manufactured by Pharmacia), DEAL-Cellulose Watmann DE-32
(manufactured by Watmann), DEAL-Sephadex A-
Adsorption and elution with ion exchange resins such as 25 (manufactured by Pharmacia); gel filtration with Cephadex G-10 (manufactured by Pharmacia), Bio-Gel P-2 (manufactured by Bio-Rad); cellulose, Avicel SF ( Column chromatography (manufactured by American Viscose Co., Ltd.); forced precipitation by adding a solvent such as acetone; freeze-drying; these methods are used alone or in appropriate combinations, and in some cases repeatedly. . Thus, antibiotic OA with the above-mentioned properties
−6129D is obtained. Antibiotic OA- produced by the above fermentation method
6129D, i.e. a compound of the above formula, generally has 5
The hydrogen atoms bonded to the - and 6-position carbon atoms each have a trans or cis configuration, and the above formula includes 5,6-trans form, 5,6-cis form, or a mixture thereof. Both are 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 bandoyl 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 S is an asymmetric carbon atom, the antibiotic OA-6129D has S
It can exist in either the - form or the R- form, or in the racemic form. The present antibiotic OA-6129D 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 a derivative, or it can be 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-6129D into its salt or ester form can be carried out according to methods known per se. For example, a salt of the antibiotic OA-6129D can be prepared by treating free antibiotic OA-6129D 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 earth bases such as calcium hydroxide and magnesium hydroxide; Metal hydroxides; monoethylamine, dimethylamine, trimethylamine,
Examples include primary, secondary, or tertiary organic amines such as monoethanolamine, diethanolamine, benzathine, and brocaine. Furthermore, esterification of antibiotic OA-6129D can be carried out in the same manner as for esterification of antibiotic PS-5, etc., for example, by the method described in JP-A-54-84589. Specifically, antibiotic OA−
6129D 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-6129D provided by the present invention
or its salts or esters are useful as synthetic intermediates for various carbapenem antibiotics (see Reference Examples 1 to 5 below), and the antibiotics can be used as dehydrodipeptidase inhibitors or as carbapenem antibiotics. It is thought that it is useful as a drug. Next, the present invention will be further explained with reference to Examples. Example 1: 6-(1-hydroxyethyl)-3-pantetheinyl-7-oxo-1-azabicyclo[3.2.0]
Production of sodium heptane-2-carboxylate (antibiotic OA-6129D) by fermentation method (A) Put 100 ml of the seed culture medium (S-1) with the following composition into a 500 ml Erlenmeyer flask, and add 120 ml of the seed medium (S-1) with the following composition by the usual method. Sterilize for 15 minutes at °C. On the other hand, Streptomyces sp. OA-6129 (Streptmyces sp.
sp. The culture was carried out with shaking at a diameter of 7 cm).
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, 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 0.5 72 4.2 90 12.5 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) Fishmeal 1.0 〃 Meat 3.0 〃 CaCO ₃ 0.3 〃 K ₂ HPO ₄ 0.2 〃 MgSO ₄ 0.2 〃 PH 7.2 with NaOH Dissolved in 0.01M phosphate buffer with pH5.5 separately, adjusted to
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 basket centrifuge to obtain 900 culture fluids. 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 13 eluates, from fraction 6 to fraction 18, 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 3.0% sodium chloride. Fractionate the eluate one by one to obtain a total of 15 fractions, from fraction 7 to fraction 21.
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, the active fractions from fraction 52 to fraction 59 were 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.
Transferred to a packed column (8 x 100 cm), developed with the above buffer solution, and determined the activity by bioassay to 7.4.
Collected. This active fraction was adsorbed onto a QAE Sephadex A-25 (manufactured by Pharmacia) packed column (8 x 100 cm) that had been equilibrated with the above buffer solution, and the pH of the eluate was carefully adjusted with 10% NaOH.
After adjusting to 8.4, add 0.01M phosphate buffer (PH
8.4) to a Diaion HP-20 packed column (4 x 100 cm) that had been equilibrated in advance, and the concentration was 0.
Elute with acetone water increasing linearly from to 30% (total 6.0). One section was 15 ml, the eluate was fractionated, and the antibiotic OA-6129B and D sections from section 45 to section 64 were collected to obtain 300 ml. This was freeze-dried to obtain 2.2 g of pale yellow powder. The obtained 2.2 g of powder of OA-6129B and D categories was dissolved in a small amount of water, and Sephadex G-
10 (manufactured by Pharmacia) packed column (1.5 x 80
cm), developed with the above buffer, and collected 30 ml of active fraction by bioassay. This active fraction is equilibrated in advance with the above buffer and adsorbed onto a QAE Sephadex A-25 (manufactured by Pharmacia) packed column (2.5 x 40 cm). 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%. The eluate was fractionated into 15 ml portions, and a total of approximately 250 ml of active fractions, fractions 20 to 36, were collected by bioassay. 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 69 from 25 was antibiotic OA−
6129B and D categories, and a total of 100 ml of fractions 70 to 79 was antibiotic OA-6129A category. By freeze-drying each section, OA−
6129B and D sodium salt (mixture) is 410
mg, 54 mg of sodium salt of OA-6129A was obtained. Example 2: 6-(1-hydroxyethyl)-3-pantetheinyl-7-oxo-1-azabicyclo[3.2.0]
Heptane-2-carboxylic acid (antibiotic OA-
Production of p-nitrobenzyl ester of 6129D) The antibiotic OA-6129B obtained in Example 1 and
Pale yellow powder consisting of sodium salt of OA-6129D
Dissolve 600 mg in 30 ml of dimethylformamide, add 2.0 ml of triethylamine under ice cooling, and while stirring, dissolve p dissolved in a small amount of dimethylformamide.
- 2.7 g of nitrobenzyl bromide were added. After reacting at the same temperature for 5 minutes, the reaction was continued at room temperature for 3 hours. Pour the reaction solution into 100 ml of methylene chloride and add 30 ml of 0.1 M phosphate buffer (PH6.8) saturated with sodium chloride.
Washed twice with ml. Furthermore, 60 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 filled with benzene/acetone (1/1)
It was adsorbed onto 60 g of silica gel, and developed sequentially with benzene/acetone mixed solvent (1/1), benzene/acetone mixed solvent (1/3), and acetone. When the fractions eluted with benzene/acetone mixed solvent (1/3) and acetone are collected and concentrated, benzene/acetone
An antibiotic that exhibits UV absorption at an Rf value of 0.52 on silica gel TLC developed with an acetone mixed solvent (1:4), and also exhibits antibacterial activity on an assay plate containing horse serum.
p-nitrobenzyl ester of OA-6129D is 240
mg obtained. In addition, when the fractions eluted with acetone are collected and concentrated, the Rf value is determined by TLC on silica gel developed with a mixed solvent of benzene/acetone (1:4).
250 mg of p-nitrobenzyl ester of antibiotic OA-6129B having a value of 0.23 was obtained. Antibiotic OA-6129D obtained as above
The physical properties of p-nitrobenzyl ester were as follows. Optical rotation [α] ²⁴ _D 11.7° (C=1.0, CH ₂ Cl ₂ ) IR spectrum ν ^CHCl3 _nax cm ^-1 : 1750 (β-lactam, ester,
1660 (amide) UV spectrum λ ^CH2Cl2 _nax nm (ε): 268 (5500) NMR spectrum (CDCl ₃ ) δ: 0.90 (3H, s,

【formula】) 0.98(3H,s,

[Formula]) 1.34 (2H, d, J=6.5Hz, CH ₃ −CH) 1.75 to 2.20 (3H, m, C−4H ₂ , OH) 2.30 to 2.80 (5H, m, S−CH ₂ −CH ₂ -NH,
NH-CH ₂ -CH ₂ -CO, OH) 3.15-4.50 (12H, m, C-3H, C-5H, C
−6H, C−8H, NH−CH ₂ −CH ₂ −CO, S
−CH ₂ −CH ₂ −NH,

[Formula], O - CH ₂ - C, OH) 4.77 (1H, d, J = 7.0Hz, C - 2H) 5.23 (2H, s, CH _{2 -} Ar) 6.70 (1H, br, NH) 7.30 - 7.60 (3H, m, Ar・H , NH) 8.17 (2H, d, J=8.0Hz, Ar・H ) Mass spectrum (FD) m/z 611 (M+1) Reference example 1: 6-(1-hydroxyethyl) -Production of 3-isopropylidenepantetheinyl-7-oxo-1-azabicyclo[3.2.0]heptane-2-carboxylic acid p-nitrobenzyl ester Add 52 mg of p-nitrobenzyl ester of antibiotic OA-6129D to 4.0 ml of acetone, 0.5 ml of 2,2-dimethoxypropane, and 200 mg of sodium sulfate (anhydrous).
Dissolve p in a mixed solvent of p
- Add 1.5 mg of toluenesulfonic acid. After reacting for 30 minutes, 20 ul of triethylamine was added to the 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, 20 ml of 0.1 M phosphate buffer (PH8.4), 20 ml of 0.1 M phosphate buffer (PH
6.8) Washed sequentially with 20 ml. The extract layer was dehydrated with sodium sulfate (anhydrous) and then evaporated under reduced pressure. The residue was dissolved in a small amount of methylene chloride and adsorbed on a 5 g column of silica gel packed with a mixed solvent of benzene/acetone (10/1). Benzene/acetone mixed solvent (10/1), (5/1), (3/1), (1/
1) and (1/3) sequentially, and the fractions eluted with benzene/acetone mixed solvent (1/1) to (1/3) are collected and concentrated, resulting in benzene/acetone mixed solvent (1:1). silica gel developed with
31 mg of the title substance with an Rf value of 0.35 was obtained by TLC. The physical properties of this substance were as follows. Optical rotation [α] ²⁴ _D 20.3° (c=1.0, CH ₂ Cl ₂ ) IR spectrum υ ^CHCl3 _nax cm ^-1 : 1750 (β-lactam, ester)
1660 (amide) UV spectrum λ ^CH2Cl2 _nax nm (ε): 268 (7500) NMR spectrum (CDCl ₃ ) δ: 0.96 (3H, s,

【formula】) 1.02(3H,s,

[Formula]) 1.36 (3H, d, J = 7.0Hz, CH ₃ - CH) 1.42 (3H, s,

【formula】) 1.46(3H,s,

[Formula]) 1.60 ~ 2.20 (2H, m, C-4H ₂ ) 2.42 (2H, t, J = 6.5Hz, NH-CH ₂ -CH ₂
−CO) 2.50 to 2.70 (2H, m, S−CH ₂ −CH ₂ −NH) 2.85 (1H, d, J=5.0Hz, OH) 3.15 to 3.85 (8H, m, S−CH ₂ −CH ₂ − N.H.
NH- _CH2 - _CH2 -CO, C-3H, C-6H,
O-CH ₂ -C) 4.04 (1H, s,

[Formula]) 3.90~4.20 (2H, m, C-5H, C-8H) 4.77 (1H, d, J=7.5Hz, C-2H) 5.27 (2H, s, CH ₂ -Ar) 6.42 (1H, br, NH) 6.97 (1H, br, NH) 7.50 (2H, d, J=8.5Hz, Ar・H ) 8.18 (2H, d, J=8.5Hz, Ar・H ) Nass spectrum (FD) m/z :651 (M+1) Reference Example 2 Production of 6-(1-acetoxyethyl)-3-isopropylidenepantetheinyl-7-oxo-1-azabicyclo[3.2.0]heptane-2-carboxylic acid p-nitrobenzyl ester 25 mg of the compound produced in Reference Example 1 was dissolved in 1.0 ml of pyridine, and acetic anhydride was added with stirring under ice cooling.
Added 0.32ml. 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, stir for 10 minutes, then add 50 ml of methylene chloride.
Pour into 20ml of 0.1M phosphate buffer (PH6.8), 0.1M
It was washed successively with 40 ml of phosphate buffer (PH8.4) and then with 20 ml of 0.1M phosphate buffer (PH6.8). The extract was dehydrated with sodium sulfate (anhydrous) and then evaporated under reduced pressure.
The residue was dissolved in a small amount of methylene chloride and adsorbed on a 5 g column of silica gel packed with benzene, and benzene and a benzene/acetone mixed solvent (10/1), (5/1), (3/1), (2 /1),
(1/1) and (1/5) were developed sequentially. The fractions eluted with a benzene/acetone mixed solvent (1/1) were collected and concentrated, and then analyzed using silica gel TLC developed with a benzene/acetone mixed solvent (1/1).
18 mg of the title substance exhibiting UV absorption at an Rf value of 0.61 was obtained.
The physical properties of this substance were as follows. Optical rotation [α] ²⁴ _D 12.5° (c=1.0, CH ₂ Cl ₂ ) IR spectrum ν ^CHCl3 _nax cm ^-1 : 1768 (β-lactam) 1740 (ester) 1665 (amide) UV spectrum λ ^CH2Cl2 _nax nm (ε ): 268 (10100) NMR spectrum (CDCl ₃ ) δ: 0.97 (3H, s,

【formula】) 1.02(3H,s,

[Formula]) 1.30 (3H, d, J = 6.5Hz, CH ₃ - CH) 1.40 (3H, s,

【formula】) 1.44(3H,s,

[Formula]) 2.05 (3H, s, CH ₃ CO) 1.80-2.28 (2H, m, C-4H ₂ ) 2.40 (2H, t, J = 6.5Hz, NH-CH ₂ -CH ₂
-CO) 2.55 to 2.90 (2H, m, S-CH ₂ -CH ₂ -NH) 3.10 to 3.80 (8H, m, S-CH ₂ -CH ₂ -NH,
NH- _CH2 - _CH2 -CO, C-3H, C-6H,
O-CH ₂ -C) 4.01 (1H, 3,

[Formula]) 4.00~4.20 (1H, m, C-5H) 4.72 (1H, d, J=7.0Hz, C-2H) 4.98~5.30 (1H, m, C-8H) 5.23 (2H, s, CH ₂ −Ar) 6.34 (1H, br, NH) 6.93 (1H, br, NH) 7.48 (2H, d, J=9.0Hz, Ar・H ) 8.17 (2H, d, J=9.0Hz, Ar・H ) Mass spectrum FDm/z: 693 (M+1), IBm/z: 692 (M) Reference example 3: 6-(1-acetoxyethyl)-3-chloro-3
-isopropylidenepantetheinyl-7-oxo-1-azabicyclo[3.2.0]heptane-2
-Carboxylic acid p-nitrobenzyl ester S-
Manufacture of oxides 6-(1-acetoxyethyl)-3-isopropylidenepantetheinyl-7-oxo-1-azabicyclo[3.2.0]heptane-2-carboxylic acid p-
Dissolve 31 mg of nitrobenzyl ester in 1 ml of benzene and 1 ml of methylene chloride, cool to 0°C, and dissolve pyridine.
16μ and 24 mg of iodobenzene dichloride were added and reacted at the same temperature for 2 hours. The reaction solution was applied to a 10 ml column of silica gel, eluted with a mixed solvent of benzene and acetone (1:1), and the fraction having UV absorption at an Rf value of 0.44 was dried under reduced pressure by TLC on silica gel developed with the same solvent, yielding the title compound. 15mg of was obtained. The physical properties of this substance were as follows. IR spectrum υ ^CHCl3 _nax cm ^-1 : 1782 (β-lactam) 1740 (ester) 1665 (amide) UV spectrum λ ^CH2Cl2 _nax nm (ε): 267 (10700) NMR spectrum (CDCl ₃ ) δ: 0.94 (3H, s ，

【formula】) 1.01(3H,s,

[Formula]) 1.24 (3H, d, J = 7.0Hz, CH ₃ - CH) 1.38 (3H, s,

【formula】) 1.41(3H,s,

[Formula]) 2.04 (3H, s, CH ₃ CO) 2.10 ~ 2.50 (3H, m, C-4H, NH-CH ₂ -
CH2 _- CO) 2.70-3.90 (10H, m, C-4H, C-6H,
NH−CH ₂ −CH ₂ −CO, S−CH ₂ −CH ₂ −
NH, O-CH ₂ -C) 4.00 (1H, s,

[Formula]) 4.08~4.45 (1H, m, C-5H) 4.97 (1H, s, C-2H) 5.18 (2H, s, CH ₂ -Ar) 5.28~5.60 (1H, m, C-8H) 6.48 (1H, br, NH) 6.90 (1H, br, NH) 7.49 (2H, d, J=9.0Hz, Ar・H ) 8.15 (2H, d, J=9.0Hz, Ar・H ) Mass spectrum (FD) m/z: 743 (M+1), 707 (M-Cl) Reference example 4: 6-(1-acetoxyethyl)-3-isopropylidenepantetheinyl-7-oxo-1-azabicyclo[3.2.0]hept- Production of 2-ene-2-carboxylic acid p-nitrobenzyl ester S-oxide 6-(1-acetoxyethyl)-3-chloro-3
-Isopropylidenepantetheinyl-7-oxo-1-azabicyclo[3.2.0]heptane-2-carboxylic acid p-nitrobenzyl ester S-oxide (7.9 mg) was dissolved in benzene (1.5 ml), and triethylamine (4μ) was added thereto for 30 minutes at room temperature. After reaction for 5 minutes, silica gel
ml column, eluted with a benzene:acetone mixed solvent (1:3), and measured the Rf value using silica gel TLC developed with a benzene:acetone mixed solvent (1:1).
The fraction having a UV absorption of 0.11 was dried under reduced pressure to obtain 5.7 mg of the title compound. The physical properties of this substance were as follows. IR spectrum υ ^CHCl3 _nax cm ^-1 : 1785 (β-lactam) 1735, 1710 (ester) 1660 (amide) UV spectrum λ ^CH2Cl2 _nax nm (ε): 310 (7200) 269 (12600) NMR spectrum (CDCl ₃ ) δ :0.93(3H,s,

【formula】) 1.00 (3H, s,

【formula】) 1.37(3H,s,

[Formula]) 1.37 (3H, d, J = 7.0Hz, CH ₃ - CH) 1.42 (3H, s,

[Formula]) 2.02 (3H, s, CH ₃ CO) 2.41 (2H, t, J = 6.5Hz, NH-CH ₂ -CH ₂
-CO) 2.80~3.90 (11H, m, C-4H ₂ , C-6H,
NH−CH ₂ −CH ₂ −CO, S−CH ₂ −CH ₂ −
NH, O-CH ₂ -C) 4.02 (1H, s,

[Formula]) 4.20~4.55 (1H, m, C-5H) 5.10~5.40 (3H, m, C-8H, CH ₂ -Ar) 6.54 (1H, br, NH) 6.93 (1H, br, NH) 7.58 (2H, d, J=8.5Hz, Ar・H ) 8.17 (2H, d, J=8.5Hz, Ar・H ) Reference example 5: 6-(1-acetoxyethyl)-3-isopropylidenepantetheinyl- Production of 7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid p-nitrobenzyl ester 6-(1-acetoxyethyl)-3-isopropylidenepantetheinyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid p-nitrobenzyl ester S-oxide 10
Dissolve mg of tributylphosphine in 2 ml of benzene.
After adding 4μ and reacting for 30 minutes at room temperature, the solvent was concentrated under reduced pressure, applied to a 6 ml silica gel column, and eluted with a benzene:acetone mixed solvent (2:1). The fraction having UV absorption at an Rf value of 0.46 by TLC was dried under reduced pressure to obtain 5.0 mg of the title compound. The physical properties of this substance are as follows. Optical rotation [α] ²⁴ _D 35.4゜ [c=0.35, CH ₂ Cl ₂ ) IR spectrum υ ^CHCl3 _nax cm ^-1 : 1780 (β-lactam) 1735, 1700 (ester) 1660 (amide) UV spectrum λ ^CH2Cl2 _nax nm (ε): 317.5 (14600) 270.5 (12700) NMR (CDCl ₃ ) δ: 0.95 (3H, s,

【formula】) 1.02(3H,s,

[Formula]) 1.37 (3H, d, J = 7.0Hz, CH ₃ - CH) 1.39 (3H, s,

【formula】) 1.44(3H,s,

[Formula]) 2.03 (3H, s, CH ₃ CO) 2.42 (2H, t, J = 6.5Hz, NH-CH ₂ -CH ₂
-CO) 2.70~3.80 (11H, m, C-4H ₂ , C-6H,
S- _CH2 - _CH2 -NH,NH- _CH2 - _CH2-
CO, O-CH ₂ -C) 4.00 (1H, s,

[Formula]) 4.10~4.45 (1H, m, C-5E) 5.00~5.40 (2H, m, C-8H, CHH-Ar) 5.41 (1H, d, J=14.0Hz, CHH-Ar) 6.50 (1H , br, NH) 6.90 (1H, br, NH) 7.57 (2H, d, J=8.0Hz, Ar・H ) 8.13 (2H, d, J=8.0Hz, Ar・H )

Claims (1)

[Claims] 1 formula Compounds represented by and their salts and esters. 2. The compound according to claim 1, wherein the salt is a sodium salt.