JPS58134093A - Novel antibiotic substance oa-6129e and preparation thereof - Google Patents

Abstract

NEW MATERIAL:An antibiotic substance OA-6129E of the formula (R<1> is H or benzyl; R<2> and R<3> are H or together form isopropylidene) having the following properties: Specific rotatory power: [alpha]<24>D=12.6 deg. (C=1.0, CHCl3). USE:A synthetic intermediate for carbapenem antibiotic substances which are dehydrodipeptidase inhibitors or a precursor for the antibiotic substances in the living body. PROCESS:A microorganism, e.g. Streptomyces sp. OA-6129 (FERM-P No.11), capable of producing the antibiotic substance OA-6129E, and belonging to the genus Streptomyces is cultivated at 4-9pH and 20-40 deg.C.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to novel antibiotics and derivatives thereof, and further focuses on novel antibiotics and derivatives thereof, which have the following formula (wherein 7?1 represents a hydrogen atom or a substituted or unsubstituted benzyl group, R' and R8 each represents a hydrogen atom or -.

7-oxo-1-azabicyclo [3゜g, o
) Antibiotics having a hept-2-ene-2-carmenic acid skeleton (hereinafter referred to as carbapenem antibiotics) are generally
Highly antibacterial and l-lactamer 111111.

7-oquinol-l-azobiccyclor:3.2.0)hept-2-ene-2-cal Ganic acid derivatives have been produced [for example, Chenamycin (Journal Otsu Antibiotics, Vol. 3 (111)
? I year), 1-1! Page entry: Chenamycins (No. 17)
Annual Interscience Conference on Anti-Vital Agents and Chemotherapy,
Abstract No. 80 and No. 81 (1977)), N-Acetylchenamycin (West German Patent!!6521581)
(No. 19-7), Ori/4 Diphosphoric Acids (Journal/
No. 211 Antibiotics, Volume 32 (1179)
, pp. 287-304), ps-s (Journal Otsu.
Antibiotics, 3! Volume (1979), 262
~286 negative), PS-6 and ps-'r (journal
Odoantibiotics, Volume 33 (1980), 1
128-11B'r pages), JK17927A,, Substance and 1179!7A, Substance (JP-A-53-103401, JP-A-53-109997), KA-66
43-A91) Quality (% open/close publication No. 55-139380)
and various carbapenem antibiotics (Japanese Unexamined Patent Application Publication No. 56-5478) obtained by total synthesis.

The above Shita KA-664a-Ap@ substance is an antibiotic represented by the following formula, and also includes 417927A and A1'1
92'lA, the substance is an antibiotic shown by the following formula H -, but recently, through examination of the culture conditions of the producing bacteria of these substances, Al79LI shown by the following formula is a substance structurally similar to the substance. ? It was discovered and proposed that Substance D (2,3-dihydro form) could be produced (Japanese Patent Application Laid-Open No. 1983-1989).
-24129).

On the other hand, the present inventors previously prepared an antibiotic 0111129B (%Zengsho 55-1) represented by the following formula.
44507), and the chemotactic antibiotics 0A-61291 and 0A-8111*C replacing 6- of the antibiotic.
I proposed each heading in the regulations (Tokugansho!! 8-'
1358! No. 9 and patent application Sho% 5s-1yossn4).

The present inventors found that among the 0A-11211 group antibiotics, the KA-6643-A substance and the AI against the 179QIA substance? Considering that there is a possibility that a substance corresponding to the il 27D substance exists, the above OA-fi 1
After studying the culture conditions for the bacteria producing Group 1 antibiotics, a substance represented by the above formula (1-a) was discovered, and the present invention was completed.

The above formula (1-a), which is one of the compounds provided by the present invention,
) has weaker antibacterial activity than the 0A-612f1 group antibiotics, but it is effective against highly lactam-sensitive bioassay bacteria such as Comamonas teris B-996 (C
otrwtinaaa tarriganaB-99
ft) on an assay plate containing horse serum, the present inventors decided to name this compound "Antibiotic OA-6129A'J".

This antibiotic 0A-81291 can be obtained by dehydrogenating it under suitable conditions known per se without eliminating the nonantetinyl group at the 3-position. There is a possibility that it can be converted into 0A-61211 group antibiotics.

Furthermore, after the hydroxyl groups in the side chains of the antibiotic OA-612*E are coated with a film, the antibiotic is dehydrogenated by a method known per se, for example, as described in JP-A-54-76593. The /4 toteinyl group in the side chain at the 3-position of substance OA-6129E was previously removed by a chemical or biological method to produce a compound represented by the following formula, and the amino group in the side chain at the 53-position was removed. After preservation or modification as desired, it is also possible to dehydrogenate in the same manner as above to induce carbapenem antibiotics.

In particular, compounds other than formula (l-α) provided by the present invention,
For example, the carlinic acid ester derived from the 0A-6129B and the cyclic ketalized product of the ester have improved fat solubility compared to the antibiotic 0A-6129E itself, and therefore cannot be handled in lipophilic organic solvents. It is expected that it will be used as a semi-synthetic intermediate for carbapenem antibiotics, and as a compound with even greater usefulness.

Since the compound represented by the above formula (1-g) provided by the present invention has a xyl 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;・・・
).

Monium salt; monoethyl aine, dimethyl aine t7゜1:
:.

Includes salts with primary, secondary or tertiary amines such as trimethylamine, monoethanolamine and jetanolamine; salts with organic bases such as benzathine salts and glocaine salts, among which are pharmaceutically acceptable salts. Preferred are salts that can be used, particularly alkali metal salts such as sodium salts and potassium salts.

Further, as an ester of the compound of formula (1-a), t'i,
nt, benzyl ester, p-nitrobenzyl ester, p-methoxypencil, p-bromopencyl ester, benzhydryl ester, trityl ester, phenacyl ester, phthalisol ester, phthalimidomethyl ester, piparoyloxymethyl ester, methoxymethyl ester, methylthiomethyl ester, 2
．． Examples include substituted or unsubstituted benzo esters such as 2.2-trichloroethyl ester.

11 Further, another derivative of the antibiotic OA-6129H further used in the present invention includes a- and r-
A typical compound is an isoglopylidene derivative represented by the formula (菖-h) (wherein R1 has the meaning described above) in which the - position is simultaneously etherified. Ketals or cyclic acetals can be mentioned.

According to the present invention, the antibiotic 0A-612 of the formula (1)
9E and/or their derivatives are first treated with the antibiotic 0A.
-411! Cultivating IE-producing microorganisms in a nutrient medium, collecting antibiotic 0A-6129AI' from the culture,
Furthermore, if necessary, the antibiotic can be produced by a method comprising esterifying the antibiotic by a method known per se, and further converting it into a cyclic acetal or a cyclic ketal.

The antibiotic 0A-6129B producing bacteria used in the present invention are:
Antibiotic 0A-6129A with the chemical structure described above;'
Microorganisms belonging to any genus can be used, as long as they have the ability to produce microorganisms, 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 strain producing the antibiotic 0A-6129E used in the present invention.

Typical bacteria producing antibiotic 0A-6129E include:
Antibiotic OA-6129E belonging to the genus Stregutomyces
Production bacteria are included, and a suitable example thereof is an actinomycete isolated from nine soils collected near Oko Shrine in Fukuoka City, Fukuoka Prefecture, and the strain numbered by the present inventors as 0A-6129@ strain. can be mentioned.

The mycological properties of this 0A-6129@ strain are as follows.

1) Morphology Under the microscope, well-branched basal hyphae are found to be straight to curved (S
traight-flemwtsua), the aerial branches are elongated, and whorled branches are not observed. The mature spore chain is 1
It consists of zero or more spores in the shape of an oval to cylinder, and no sporangia are observed. Spore size is a6~LOXα7~2
At about 5 ocrons, the surface of the spores is smooth. Flagellated spores are not observed.

2) In various media! Unless otherwise specified, culturing was carried out at 5'28° to 30°C. In addition, H. D. Tresner and Eshu ΔKas (H, D, Trea-saran)
d E, J, Baeksa), Zoyana/l/
II Osci azolide microbiozoi (Jour
nalof Applied Mierobiolog
y) Volume 11, No. 4, (1963) 335-338
According to the method on the page, enter the code [CHM code (c
ode)) is the color of Container Corporation of America.
3-A71/ (ContainerCorporat
ion of kngrica's Co1or Harm
onyManual) f was used.

(1) Sucrose/nitrate agar medium: plate (2
dc) - Medium growth of light gray-yellow-brown (3gg), aerial mycelium of 2 dc to gray-yellow-pink [5 dc] was attached and grown, and no soluble pigments were observed. □1::・0. .

(2) Glucose-asparagine agar medium: Bright gray (d) aerial mycelia are grown on the good growth of pale yellow (2d6) to bright olive brown (C2cte). This aerial mycelium later becomes a grayish-yellow hyphae (5 da). No soluble pigments are observed.

(3) Glycerin-asparagine agar medium (ISF
Medium-S): On top of good growth of mild yellowish/red C4ye) to light brown [41-], light gray ((() to bright gray reddish brown [5/
#] Aerial mycelia are attached. No soluble pigments are observed.

(4) Starch inorganic salt agar medium (ISF medium-4): light yellow C2db] to gray [j! Bright gray [d] aerial mycelium grows on the good growth of /#). No soluble color lA mineral formation.

(5) Tyrosine agar medium (ISF medium-7): gray yellow (8
gg) ~ Light brown (4(s) growth, light gray [d
]~Grows bright straw ash (3/#) aerial mycelia. The medium will have a very slight brown tinge.

(6) Nutrient agar medium: On top of good growth of pale yellow (2d6) or bright yellow [2fb) to bright olive brown C2ya], bright gray reddish brown C
afe] aerial mycelium. No soluble pigments are observed.

()) Yeast extract/malt extract agar medium (Is
P medium-2): On the good growth of gentle yellow pink C4ye] to bright brown (4i-), aerial mycelia of gray yellow pink [54c] or slightly later bright gray [d] are attached.Dissolution No sex pigments are observed.

(8) Oatmeal agar medium<rsp@ji-3):
On top of good growth of gray-yellow [3#C] to bright orange-yellow [3#α] or bright gray-yellow-brown [3gg], bright straw-gray [:3/#) to bright gray-reddish brown [5/#] Aerial mycelium grows on the bacteria, and the culture medium around the fungal colony takes on a slightly brown color.

(9) Malic acid lime agar medium: Aerial mycelium of light gray [d] to light gray reddish brown (5/#) grows and dissolves on medium growth of dark to plate (P!da). No sex pigments are observed. A dissolution zone of calcium salts can be seen around the growing bacterial colonies.

(III Glucose-Pegtone-Rlatin Medium (2)
0C culture) Pale yellow (2d&) ~ white with good growth of brown (6) ~
Grows on gray-yellow-pink (5c&) aerial mycelia. Long-term culture (
(approximately 3 weeks or more), a dry soluble pigment is produced.

3) Physiological properties (1) Growth temperature range Yeast extract/malt extract agar medium (ISF medium-2
) using 10”,!O”,! 5°, 3G”, 34°,
As a result of experiments at temperatures of 37°, 4G", 4!i@, and 50C, there is almost no growth at 3C. At 40C or higher, no growth occurs at all. The temperature range seems to be 20 to 30C.

(2) Liquefaction of r-latin: Liquefy.

(3) Starch hydrolysis: Decompose.

(4) Coagulation and pegtonization of skim milk: Although it does not coagulate,
Turn into pegton.

(5) Production of melanin-like pigments on yeast iron agar medium (ISP medium-5)
) and tryptone yeast extract broth medium (IS
No production of melanin-like pigment was observed in P 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 various anthrax sources (Gridham Gotley! using agar medium) (1) L-arabinose + (2) D-xylose + (3) D-glucose + (4) D-7 lactose + (5) Shake Close Suspicious (6)
Inositol - (7) L-Rhamnose + (8) Raffinose - (9) L-Mannitol + + is assimilated, - is not assimilated.

Based on the above mycological properties, the OA-6119 strain is Strap.
It is a strain belonging to the genus tomyaaa, and the shape of the aerial mycelium is similar to section RF (5aattoKoRaettf-j
vs (6 (Jgs)), and the spore cost is smooth and the color tone of aerial mycelia is similar to that of most media, i.e., oatmeal agar, glycerin agar/#line agar, and starch/inorganic salt agar. Light gray [d], gray type (Gr
ay sgrjgg) strain. However, depending on the culture period, Shuku U-su/Nitrate Agar, Yeast Extract/Mat Extract Agar, and U-Dulcose/Asuno Kaiugin Agar media produce grayish-yellow-pink (sdC) to reddish (Rad)
aadayi##) may occur. 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, the color changes to yellowish brown to grayish yellowish brown or brown. Melanin pigment was not observed in peptone yeast extract iron agar medium and tryptone yeast extract prosthesis, 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 rlatin medium, and oatmeal agar medium.

The inventors hereby present the book 1i1 - Stregutomyces sp.
A-6129 (!3treptoyces ap,
0A-6129), and has been internationally deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, as FAIKEN Article No. 11.

Antibiotic of the present invention OA-6129A'ti, antibiotic 0
It is produced by inoculating spores or m filaments of A-61!9A' producing bacteria, for example, the above-mentioned Streft 1 Ses ap, oA-site, into a nutrient-containing medium and growing aerobically.

As the nutrient source, those normally used as nutrient sources for actinomycetes, such as assimilable nutrient sources such as carbohydrates, nutrient 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; pegtone, meat extract, soy flour,
Nitrogen sources such as cottonseed flour, dried yeast, corn steep liquor, yeast extract, skim milk, casein, sodium nitrate, ammonium nitrate, ammonium sulfate; dipotassium phosphate,
Inorganic salts such as 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, as a nutrient source, if antibiotic 0A-6129Et- is produced using antibiotic 0A-61211A'-producing bacteria,
Any nutrient source can be used, and any known culture material for actinomycetes can be used. Further, in order to suppress foaming during heat sterilization and culturing, antifoaming agents such as silicone and 411 oil can also 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 those skilled in the art will be able to determine the optimal nutrient source composition and blending ratio for the antibiotic 0A-s12eE-producing bacteria to be used. If so, it can be easily determined by a simple random voice experiment.

The nutrient medium can also be sterilized prior to nutrient feeding, and the pH of the medium may be adjusted to a pH range of 4 to 9, especially at
It is advantageous to adjust H to a range of 6 to 8.

The cultivation of antibiotic 0A-8129E-producing bacteria in such a nutrient medium is, in principle, carried out in accordance with the method commonly used in the production of antibiotics using general actinomycetes, usually under aerobic conditions. It is preferable to culture under
This can usually be carried out with stirring and/or with ventilation. 9.Culture methods include static culture, shaking culture,
Although any submerged culture with aeration and agitation can be used, submerged culture is preferred.

The culture temperature that can be used is that the growth of antibiotic 0A-6119E production is not substantially inhibited.
'a- There is no particular restriction as long as it can be produced, and it can be changed depending on the production strain used, but generally 20 to 40C1 is preferred! Temperatures within the range of 5-3[:' are preferred.

In addition, 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, as necessary during the culture.

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 generally be continued until sufficient antibiotic OA-sxteE has accumulated. The culture time varies depending on the composition of the medium, culture temperature, production strain used, etc., but is usually 30 minutes.
~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-sxteE accumulated in the culture? - Lactam highly sensitive bioassay Cocoons, such as Comamonas terrirus B-91161, can be quantified by bioassay and bioautography, thereby making it easy to determine the optimum accumulation amount.

Thus, antibiotic 0A-612 accumulated in the culture.
Since 9A' 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 V-filtration, centrifugation, or extraction. liquid,
It is recovered from the supernatant liquid, extract liquid, etc.

Collection can be carried out by methods known per se,
In particular, methods often used for recovery of calcium acid type antibiotics are advantageously applied. For example, solvent extraction and dissolution with ethyl acetate, 5-butanol, etc. at low pH;
・Transfer from soot layer to high pH water layer □・: Activated carbon, Anno-Lite XAD (Roam・
Adsorption using Diaion HP-20° (manufactured by Mitsubishi Kasei), elution with methanol water and acetone suigo; DOWEX 1×2 (manufactured by Dow Kezcal), QAE-Sepha Dex A-25 (manufactured by Pharmacia), DEAE-Cellulose Whatman DE-'
! Adsorption and elution with ion exchange resins such as 12 (Whatman), DEAE-Sephadex A-25 (Pharmacia); Sephadex G-10, (Pharmacia), Bio-Rul P-2 ( (manufactured by Bio-Rad), R-filtration by Go; column chromatography of cellulose, Avicel SF (manufactured by American Viscose), etc.; forced precipitation by adding a solvent such as acetone; freeze-drying, etc. Alternatively, they may be used in appropriate combinations, and in some cases, iterated as follows. ,,゛・ Thus, the antibiotic OA-sig having the above-mentioned properties
sE is obtained.

Antibiotic 0A-6129E produced by the above fermentation method
That is, in the compound of the formula (1-a), the hydrogen atoms bonded to the 5- and 6-position carbon atoms each have a trans or cis configuration, and the compound of the formula (summer-α) 5. ! -) lance isomer, 5.6-cis isomer, or a mixture thereof. The attachment of the secondary hydroxyl group of the 9th, 2-position and 3-position carbon atoms, the /ltoyl group in the 3-position side chain is the root carbon atom and the 1-hydroxyethyl side chain at the 6-position. Each of the carbon atoms to which the hydroxyl group is bonded is an asymmetric carbon atom, so nuclear antibiotic 0A-6129
1'#i It can exist in either the S-type or the R-type, or in the race ζ type.

The present antibiotic 0A-61*taE is generally more stable in the form of Shiomatasha ester than in the free form.
When used as an intermediate for conversion into a derivative, or when subjected to the purification process described above, it is preferable to treat it in the form of a salt or ester.

The conversion of the antibiotic (jA-6129A') into its salt or ester form can be carried out according to methods known per se. For example, the salt of the antibiotic OA-6129B can be converted from the free antibiotic 0A-6129E into an inorganic or organic Inorganic or organic bases that can be used in this salt-forming reaction include alkali metal hydroxides such as sodium hydroxide, potassium hydroxide, and lithium hydroxide. ; alkaline earth metal hydroxides such as calcium hydroxide, magnesium hydroxide; primary, secondary or secondary such as monoethylamine, zomethylamine, trimethylamine, monoethanolamine, jetanolamine, benzathine, glocaine; Examples include tertiary organic amines.

Moreover, the esterification of antibiotic 0A-6129A' was carried out in the same manner as the esterification of antibiotic PS-shiro.

For example, the method described in JP-A-54-84589 can be used. Specifically, antibiotic 0A-612
9E: Salts with organic bases or alkali metals or alkali metal salts of dimethylformamide, tetrahydrofuran, dioxane, hexamethylphosphoria, acetonitrile, acetone, etc. at about -50C.
% to 50C, preferably at a temperature of about -1OC to 30C.

It can be easily formed by stirring and reacting with 1.0 to 10 equivalents of RX (in the formula, X represents a halodane atom and R represents an ester residue) for several minutes to several hours. - Is the antibiotic OA-6129E obtained as described above substituted or unsubstituted? Sol ester.

, 1:1. ．． 1. .

a- of the notoyl group bonded to the 3-position carbon atom
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 quality 0A-6129E can be converted into their cyclic ketal or cyclic acetal derivatives by reacting with ketone or aldehyde derivatives. This reaction is.

Generally, it is preferable to use the reaction reagent as a solvent,
Examples of reaction reagents that can be used include acetone, 2.
2-dimethoxygulon I, formaldehyde, 2.2
-V methoxyethane, acetaldehyde, benzaldehyde, glopionaldehyde, acetophenol, diphenylketone, cyclohexanone, sopenzolketone, 1
．． 1, diphenylacetone, etc., and these reaction reagents may be used individually, or two or more types may be mixed and used as required.

In addition, if necessary, antibiotics OA-61 for reaction reagents may be added.
An inert solvent can also be mixed in consideration of the solubility of the 29E ester.

The reaction temperature is not critical and can be varied widely depending on the liquid medium used, seed field, etc.
The temperature can be arbitrarily selected within the temperature range in which the '19E ester does not decompose significantly, but generally the temperature is 60C or less,
Preferably in the range of -40 to 40p, more preferably O
A range from C to room temperature is advantageous.

In addition, in the above reaction, if necessary, anhydrous p-1
Add reaction accelerators such as luenesulfonic acid% lontolufluoride etherate and zinc chloride.

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

The cyclic ketal of the ester thus obtained or #i, J
The Il-type acetal derivative can be isolated from the reaction mixture according to separation and purification methods known per se. For example, after the completion of the reaction, firstly, in order to inactivate the excess reaction accelerator, etc., it is treated with amines, and then the treated solution is made of a non-polar material that is substantially immiscible with water, such as ethyl acetate, benzene, or black diform. Pour into organic solvent. ml from the mixture
Wash with an aqueous medium to remove water-soluble impurities such as living organisms.

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, according to a method known per se.

A desired cyclic ketal or cyclic acetal derivative is obtained by appropriately combining chromatography using silica glu, Biobeads (manufactured by Bioland), Sephadex LH-1G (manufactured by Pharmacia), etc. as carriers, and using it repeatedly as necessary. can be isolated.

The antibiotic OA-8129E or its salt or ester provided by the present invention is useful as a synthetic intermediate for various carbapenem antibiotics, and #antibiotic is dehydrodipe! As a titase inhibitor or Cal/
In vivo precursors of penem antibiotics (grodrugs)
It is considered useful as a.

Next, the present invention will be further explained with reference to Examples.

Example 1 Production (a seed culture medium (S-1) with the following composition of 1O〇- was placed in a 4s00d Erlenmeyer flask, and 1
Sterilized at 20C for 15 minutes. Meanwhile, Streptozu Seth
SP 0A-6129 (Strgpttnycga
ap, OA-6129) strain (Feikoken Joyori 1st
After the spores of No. 1) were sufficiently attached, the platinum loops were inoculated into the seed medium and cultured with shaking at 28C for 48 hours using a rotary shaker (200 rpm, amplitude Ill).

This seed mother culture solution 11 was mixed into a production medium (GAf-
1) Inoculate a 200 (liter) fermenter containing 1,000 I,
Aerated agitation culture was carried out for 90 hours under the conditions of stirring at 28C, 150 rpm, and aeration at 50017 sin. As an antifoaming agent, silicone K M-78.1. (manufactured by Shin-Etsu Chemical Co., Ltd.) t-0.07% was used. Sample the culture solution over time and measure the antibacterial activity of the centrifuged supernatant. The measurement results at each time were as shown in the table below.

48 to 8 72 翫6 90 11L3 me) 15%<W/V)
Yeast extract a5 〃4 Teto starch zO# Ca CO, α2 〃 pH (before sterilization) 7.

Glycerin &O%<W/V) Fishmeal
LO〃 Meat &O〃Caco,
α3 〃、HPO40,2
%(W/V) MfSo, 0.2
#Adjust the pH to 7.2 with NaOH, separately dissolve in pH'A, s (LOIAf phosphate buffer, and autoclave to 1 to 9/clIIG, !s) and sterilize the nine vitamin B□to, o o o s% (W/
V) Addition.

Fermentation liquid 95 obtained by (above) after 72 hours of incubation
Togecopperlite I & 34 (5%<W/V) amount to 01 (
(manufactured by Toko) and Wright Co., Ltd.] was added, and the bacterial cells were separated using a filter press to obtain a filtrate of 9001.

This was adsorbed onto a Diaion HP-20 (manufactured by Mitsubishi Kasei Corporation) packed column (30x300α), and distilled water
After washing with water, elution was performed with 30% (V/V) acetone water.

One division is counted as 1, and the eluate is fractionated to collect a total of 14j of eluate from fraction 0 fraction 11 to fraction 1!4. After washing with distilled water Ion, add αO1M9 phosphate buffer (p# &4
) was eluted.

The eluate was fractionated by lj, and a total of 151 eluates from fraction 6 to both fractions 20 were collected.

This eluate was applied to a Diaion HP-20 packed column (IO
This was eluted with a total of 401 ml of acetone water whose concentration increased linearly from O to 30%.

One section was 200 mj, and the eluate was fractionated. By bioassaying each fraction, fraction 6S to fraction 6
! An activity level of up to 1600- was obtained. Of this circular division, 400- each time ao lAf:', 1IIQ
1. Equilibrate with acid buffer (pH 4) and fill with 9/# Iol, P-2 (a# made by Iorad) packed beak column (8X10
0cm+), developed with the above buffer solution, and collected 7.0 It of active fraction by bioassay. QAE Sephadex A-2 in which this active fraction was equilibrated with the above buffer solution in advance.
5 (manufactured by Pharmacia) packed column (8X100cR)
This eluate was carefully purified with 10% NaOH.
After adjusting to Ht-&4, aOIMIJ acid buffer (
It was adsorbed onto a Diaion HP-20 packed column (4x100m) pre-equilibrated at pH 4), and the concentration ranged from O to 3.
Acetone water increasing linearly to 0% (total 6.0)
It was eluted. One category is designated as 15117, and the eluate is fractionated to obtain antibiotics 0A-61 from category 48 to category 67.
Collect 29E, , D and 8 sections and get 30011t.9.

Freeze-dry this 1 Repeat, 11! A brown powder of f was obtained.

,□111, Obtained 1! ! The powders of QA-6129Et, D and 8 sections of f were all dissolved in a small amount of water and pre-equilibrated with o, oxA (phosphate buffer (pH 4)) Sephadex G-10.
(Manufactured by Pharmacia) Packed column (tsx80csz)
The active fraction 3ON1 was collected by bioassay. This active fraction is adsorbed onto a QAE Sephadex A-25 (Pharmacia 11) packed column (!5 x 40 cs) that has been equilibrated with the above buffer solution. After washing with 180% of the above buffer, elution was carried out with the above buffer containing salt increasing linearly from 0% to 20%.

The eluate was fractionated by 15- and the total WSOwl was determined from fractions 1!2 to 38 as active fractions by bioassay.
Collected.

After freeze-drying, this eluate was dissolved in a small amount of water, adsorbed onto a Diaion HP-2oAG (manufactured by Mitsubishi Kasei Corporation) packed column (L5X11 Gm), washed with about 200 wLI of distilled water, and then 0% It was eluted with acetone water increasing linearly from to 6% (total L21). The eluate was fractionated into two fractions, and active fractions were obtained by bioassay. One part of the active fraction = total of fractions 1 from °i 45011
j was antibiotic OA-s1gsBt, D and 8 categories.

By freeze-drying each section, OA-61s9B,
, D and E sodium salt (mixture) coarse powder is L8f
Obtained.

Implement ill! Pale yellow coarse powder consisting of the sodium salts of the antibiotics DA-6129E, DA-6129E, D and 0A-6129B obtained in Example 1. 1.3 fl-dimethylformatrs.

Dissolved in d and cooled with water, triethyl azun! Lllj was added, and while stirring, a benzyl compound dissolved in a small amount of dimethylformamide and soaked in 9p-nide was added. After being allowed to react for 5 minutes at the same temperature, the reaction was continued for 3 hours at room temperature. The reaction solution was poured into 300 m methylene chloride and washed twice with 0.1 M phosphate buffer (pHas) soIIj. Furthermore, the aqueous layer 100'Id was re-extracted twice with methylene chloride 10011j. The extracts were combined, dried over sodium sulfate (anhydrous), and then evaporated under reduced pressure. Dissolve the residue in a small amount of methylene chloride and add benzene/acetate.
i・1. It was adsorbed on a 50f silica colander filled with bread (1/1), and a benzene/acetone mixed solvent (1/1)% benzene/
Developed sequentially with acetone mixed solvent (1/!I) and acetone, and collected the fractions eluted with benzene/acetone mixed solvent (1/3). This was analyzed using silica dull TLC developed with concentrated sulfate and a mixed solvent of benzene/acetone (1:3).
Antibiotic 0A-612 exhibiting UV absorption at Rf value α55
The p-nitropendyl ester of 9E is 320 Da
c6 Example 3 p-nitropendyl ester of antibiotic 0A-612'aH 3! Acetone, 10d, it! -Dimethoxyglo/tan LOmj, sodium sulfate (anhydrous)
Dissolve in 2001v of mixed solvent, and add 7.0~ of p-toluenesulfonic acid while stirring at room temperature. After reacting for 300 minutes, add 1 ml/l of triethylamine to the reaction solution.
- added and stirred for 5 minutes. The reaction solution was distilled off under reduced pressure, 100-methylene chloride was added to the residue, α1M phosphate buffer (pff&4) it0-, C11M9 phosphate buffer (
Washed sequentially with pHa 8 and 20i1. 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 onto a silica dal Sf column filled with a mixed solvent of benzene/acetone (10/1). Benzene/acetone mixed solvent (10
/1), (S/1), (3/1), (1/l)
and (1/2) sequentially, and the fractions eluted with benzene/acetone mixed solvent (1/1) to (1/2) are collected and concentrated to form benzene/acetone mixed solvent (1:1).
), 210 Da of the title substance exhibiting Rf*a,s< was obtained by TLC on silica dal.

The rostability values of this substance were as follows.

Specific optical rotation (rap l*s@(C=to, CMCI, )U
V spectrum IiCl λ ”ss(g): !88 (!!1500) always α
fossa IE spectrum νCHCl1fi″″”: 1750 (/-lactam, t+vg* stell) 111 [(amide) NMR 41) le (CDCI,, internal fi quasi-rMS
)115~! l5 (jl, 愼, to C-4) 12 g-1
70 (5H, m, NH-CM, -CH, -CO.

OH,S-C錡-CH,-NH) ZSO~185 (8H, regular, C-3H, C-6H.

&90~430 (IB, groove, C-5H) 403 (I
H, a, O-C! ! -CO)473 (IH,d
, J=7.5#g, C-2H)&2B (jIH*
a e C At) [3G (1#, 6r, NH) &93 (1#, br, NH) 7] 0 (gH, d, J=&5Hg, Ar-11)
fLl 7 (if, d, J=&5#g, Ar
-H) Mass spectrum M/Z: @@4 (M) Based on the above physical data, the title compound is presumed to have the structural formula shown below the title.

Claims (1)

[Claims] 1. Compounds represented by the formula (wherein R1 represents a hydrogen atom or a substituted or unsubstituted pencil group, and represents R1 and) and cases where Rx, Rt and Bs are hydrogen atoms A salt of the compound. The compound according to claim 1, wherein zR@, R" and Bs represent a hydrogen atom. λ R1 represents a substituted or unsubstituted pentyl group,
2. The compound according to claim 1, wherein R1 and R represent hydrogen atoms. 4 R1 is a substituted or unsubstituted pencil group,
2. A compound according to claim 1, wherein R1 and R1 together represent an isopropylidene group.翫 A microorganism belonging to the genus Stregutomyces and having the ability to produce the compound represented by the formula A method for producing the compound represented by item 2) according to claim 1, which comprises esterifying and/or glopyridenizing the compound.