KR810001056B1 - Process for preparing antibiotic c-15003 - Google Patents

Abstract

Antibiotics C-15003 P-3 (I; R = COCHME2), C-15003 P-3' (II; R = COPr) and C-15003 P-4 (III; R = COCH2CHMe2) are produced ferm. with Nocardia C-15003. Thus, a preculture of Nocardia was inoculated into a pH 7 medium contg. dextrin 5, corn steep liquor 3, polypeptone 0.1, and CaCO3 0.5 % and inoculated at 28oC for 90 hr. The titer at this time was 2.5μg C-15003 ml. C-15003 was purified by extn. into EtoAc, pptn. from petroleum ether, and chromatog. on silica gel. C-15003 had fungicidal and leukemia-inhibiting properties.

Description

Preparation of Antibiotic C-15003

The present invention relates to the preparation of novel antibiotic C-15003.

The present inventors collect samples of various kinds of soils, search for the antibiotics produced against the microorganisms separated therefrom, and produce a new antibiotic substance of any kind of microorganisms. By cultivating this microorganism in the genus, and by culturing this microorganism in a hostile nutrient medium, it was found that this antibiotic could be accumulated in the culture line, and furthermore, it was found that the derivative was obtained from the obtained antibiotic. The invention was completed. That is, the present invention is characterized by culturing antibiotic C-15003 producing bacteria belonging to the genus Nokaldia represented by the following general formula (I) in a medium, producing and accumulating antibiotic C-15003 in the culture, and collecting the same. Preparation of antibiotic C-15003.

In the present invention, the term "antibiotic C-15003" refers to a generic term, two or three mixtures, or a single compound of three compounds represented by the general formula (I).

의 화합물을 ＂항생물질 C-15003 P-3＂ 혹은 ＂C-15003 P-3＂라고 하고, R이 -CO-CH₂-CH₂CH₃의 화합물을 ＂항생물질 C-15003 P-3'＂ 혹은 단지 ＂C-15003 P-3'＂라 하고, R이

의 화합물을 ＂항생물질 C-15003 P-4＂ 혹은 단지 ＂C-15003 P-4＂라고, 각각 칭하는 것으로 한다.Moreover, in general formula (I), R is

The compound of is called “antibiotic C-15003 P-3” or “C-15003 P-3”, and R is a compound of -CO-CH ₂ -CH ₂ CH ₃ . ＂Or just＂ C-15003 P-3 '＂and R is

The compound of is referred to as "antibiotic C-15003 P-4" or only "C-15003 P-4", respectively.

Examples of the bacterium producing the antibiotic C-15003 of the present invention include the actinomycetes No. C-15003 strain etc. are mentioned.

No. Examination of C-15003 strains according to methods of shearing and hightrib (Inter National Journal of Systematic Bacteriology, Vol. 16, pp. 313-340, 1966). The result of observing over 28 days at 28 degreeC is as follows.

1) Morphological characteristics

Parasitic hyphae are well elongated and branched in both agar and liquid media.

The major part of the direct connection is 0.8-1.2 micrometers, and sometimes it divides into a bacilli or a branched short mycelium.

It grows well on various sorting mediums, and the mycelia grows in parasitic fungi, but often forms a supernatant (50-200 μm × 200-1000 μm), and often grows on them.

Most of the shape of the mycelia is curved or linear, and sometimes a gentle spiral shape can be seen.

When spontaneous culture was observed, spores were not considered to be in a chain state. As a result, microscopic suspensions were collected from their culture surfaces, and long ovals (0.8-1.2 μm × 4.8-6.8 μm) and ellipsoids (0.8) were observed. Segmented spore-shaped thing of (-1.2 * 1.0-2.0micrometer) was observed a lot, and the surface was smooth by the observation by the electron microscope.

2) Cell composition

The strain was shaken and cultured at 28 ° C. for 66 to 90 hours in a modified medium of I.S.P No. 1, and the cells were collected and washed.

The cells were treated by non-backers [Applied Microbiology, Vol. 12, p. 421, 1964], and by M. Virechbarrier [Journal, Observatory, and Clinical]. ㆍ As a result of diaminopimelic acid and sugar composition in cell cells according to the Journal of Laboratory and Clinical Medicine Vol. 71, p. 934, 1968, the former is a meso-torm, the latter The presence of droplets corresponding to galactose and arabineoche was confirmed.

3) Determination of the classification medium

This strain grows relatively well on various media, and the parasitic mycelia are colorless to pale yellow at the beginning of culture, and then pale brown or yellowish brown.

In addition, yellow to yellow brown soluble pigments are produced in various sorting media.

The aerial mycelia are powdery, usually medium, and show a white to yellow or pale yellow brown color.

The composition and state of the strain on the various sorting media are as shown in the first table.

Table 1 No. C-15003 Determination on Classification Media

(A) Sucrose and hepatitis agar medium

Growth (G): Rich, yellow (3ia) * to pale yellow brown (3lc) *, supernatant formation

Mycelia (AM): poor, white

Soluble pigment (SP): absent or light yellowish brown

(B) Glycerol ㆍ Nitrate agar medium

G: Medium, pale yellow (2ca)

AM: Medium, white

SP: None

(C) glucose and asparagine agar medium

G: Medium, Pale yellow (2pa) ※, upset body formation ※

AM: poor, white

SP: Light yellow (2pa) ※

(D) Glycerol and asparagine agar medium

G: Medium, pale yellow (2ca)

AM: poor, white

SP: None

(E) starch agar medium

G: Medium, pale yellow (2ca) to pale yellow brown (2ea)

AM: Rich, pale yellow (2ca) ※

SP: None

(F) nutritional agar medium

G: Medium, pale yellow (2ca) *, to yellow (2ga) *, upset formation

AM: poor, white

SP: None

Calcium tungrate calcium agar medium

G: medium, light yellow (2ca) *, to light yellow brown (2ea) *, supernatant formation

AM: Medium, white to pale yellow (2ca) *

SP: None

(H) Yeast extract, malt extract agar medium

G: medium, light yellow brown (3lc) *, to light brown (3la) *, supernatant formation

AM: Medium, white to pale yellow (2ca) *

SP: None

Oatmeal agar badge

G: Medium, pale yellow (2ca) *, to yellow (2ga) *, upset formation

AM: poor, white to pale yellow

SP: None

(Tea) peptone, yeast extract, iron agar medium

G: Medium, yellow (2ga) *

AM: None

SP: Yellow (2ga) ※

(K) Tyrosine agar medium

G: Medium, pale yellow (2ca) *, to yellow (3ea) *, supernatant formation

AM: Medium, white to pale yellow (2ca) *

SP: yellow brown (3ie) ※

※ Color name symbol by Color Harmony Manual, 4th edition (Container, Corporation of America, 1958)

4) Physiological Properties

Physiological properties of this strain are shown in Table 2.

That is, the growth temperature range is 12 ° C to 38 ° C, and the temperature range where the mycelia are well formed on the agar medium (ISP No. 2) is 20 ° C to 35 ° C.

Table 2 No. Physiological Growth of C-15003

Growth temperature range: 12 ℃ ~ 38 ℃ Nitric acid salt

Mycelial Growth Temperature: 20 ℃ ~ 35 ℃ Milk / Peptonation: Positive

Gelatin liquefaction: positive milk, coagulation: negative

Starch hydrolysis: positive casein resolution: positive

Melanin-like pigmentation (peptone, yeast extract, iron agar medium): negative,

(Tyrosine agar medium): Positive Hippoxanthin Resolution: Negative

Tyrosine resolution: positive Lysozyme resistance: positive

Xanthine resolution: negative salt resistance: 2%

5) Availability of various carbon sources

The medium described in the method of Freedam and Gotrib (Journal of Bactoriology, Vol. 56, p. 107, page 1948), and a basal medium containing 0.1% of yeast extract (Bacto) added thereto. It was used to test the availability of various carbon sources, and the results are shown in Table 3.

Table 3 No. Carbon Source Availability of C-15003

※ Basic medium added with yeast extract 0.1%

: 풍부한 발육 ±: 약간 발육함week

: Rich development ±: Slightly developed

: 비교적 양호한 발육 - : 발육하지 않음

: Relatively good development-: not developing

+: Admits development

6) Other Properties

The cells were collected by the method described in the above 2), and DNA was prepared according to the method of Jay and Maa Maah (Journal of Molecular Biology 3, p. 208, 1961). When the GC content of the DNA was examined, it was about 71 mol%.

The mycelia of the strain were gram stained and were positive.

No. described above. C-15003 The Quality of the Lord by S. Waxman, The Actinomycetes, Vol. 2, The Williams and Wilkins, Camp., 1961. I searched according to Nan and N. E. Gibbons, Burgess, Manual, Of, DataMinitive, Bacteriog, 8th edition, 1974, and other literature.

It is thought that this strain belongs to group III of the genus Nocerdia, but among the known strains, no species having the above properties are found, and it was recognized as a new species.

This strain No. The C-15003 shares have been deposited with the application acceptance number 3992 to the Japan Institute of Industrial Technology, Microorganism Industrial Technology Research Institute, and IFO-13726 to the Fermentation Research Institute of Japan Foundation.

As described above, the No. The C-15003 strain is a new species of the genus Nocaldia, but as a general property of the microorganism it can cause mutations either naturally or by mutagenic agents.

For example, even if it was a lot of mutant strains obtained by mutating by irradiation of X-rays, gamma rays, ultraviolet rays, and the like, separation of single spores, culture on a medium containing various drugs, and other means, or mutant strains obtained naturally, In comparison with one bacteriologic state or as shown below, the C-15003 strains P-3, P-3 'and / or P-4 are substantially different. What has the property to produce can be used for the method of this invention.

For example, C-15003 strains are variously mutated to produce little soluble pigment, parasitic mycelium colorless, yellowish green, reddish brown to light red, mycelium as a bacilli or branched short mycelium. Mutant strains that are easy to be divided and have many mycelial and rarely grow white or mycelia are obtained.

The medium used for the cultivation of the antibiotic C-15003 producing bacterium may be liquid or solid as long as it contains a nutrient source available to the strain, but it is more appropriate to use a liquid medium when processing a large amount. The medium contains a carbon source capable of equalizing the C-15003 strain, a nitrogen source capable of being burned, an inorganic substance, a micronutrient, and the like.

As a carbon source, for example, glucose, lactose, sucrose, maltose, dextrin, starch, glycerol, mannitol, sorbitol, etc., fats and oils (e.g., soybean oil, lard oil, chicken oil, etc.), etc. Yeast extract, dry yeast, soy flour, corn steep liquor, peptone, cottonseed flour, molasses, urea, ammonium salts (e.g. ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium acetate, etc.).

Salts containing sodium, potassium, calcium, magnesium and the like, metal salts such as iron, manganese, zinc, cobalt and nickel, salts such as phosphoric acid and boric acid, and salts of organic acids such as acetic acid and propionic acid are suitably used.

Other amino acids (e.g. glutamic acid, aspartic acid, aranine, glycine, lysine, methionine, proline, etc.), peptides (e.g. dipeptides, tripeptides, etc.), vitamins (e.g., B ₁ , B ₂ , nicotinic acid, B ₁₂ , Vc, E and the like), hexanes (eg, purine, pyrimidine and derivatives thereof) and the like.

Of course, an inorganic or organic acid, an alkali, a buffer, etc. may be added for the purpose of adjusting pH of a medium, or an appropriate amount of fats and oils and surface cyclic agents may be added for the purpose of defoaming.

As a means of culturing, a means such as stationary culture, vibration culture or aeration stirring culture may be used.

It goes without saying that it is preferable to carry out so-called deep-air agitation culturing for a large amount of processing.

Naturally, the conditions of the culture are not constant depending on the condition of the medium, the composition, the type of the strain, the means of the cultivation, and the like, but these are usually selected at a temperature of 20 ° C to 35 ° C near the neutral.

Especially, the temperature of culture medium is 23 degreeC-30 degreeC, and the conditions of a starting pH of 6.5-7.5 are preferable.

The incubation period is not constant according to the above conditions, but it is better to incubate until the desired concentration of antibiotic is maximized.

The time required is about 2 to 6 days in the case of vibration culture using a liquid medium or aeration stirring culture.

The titer of these antibiotics was measured using tetrapimena pyriformis W strain (tetraphymena pyriformisw) as a test microorganism (20 g of assay medium [tripros peptone (made by Diffco)], 1 g of yeast extract (made by Diffco), 2 g of glucose, Incubated at 28 ° C. for 44 hours to 48 hours using 1000 ml of distilled water and 1 mole phosphate buffer pH 7.0, 10 ml], and liquid gray assay to measure the growth in turbidity, and thin layer chromatography (hereinafter referred to as TLC). Was measured. New antibiotics C-15003 P-3, P-3 'and / or P-4 are produced and accumulated in the culture thus cultured, and these are contained in any of the filtrate and the cell extract.

In addition, these substances were searched by the TLC method.

That is, the culture is divided into cells and filtrate by filtration or centrifugation, and the filtrate is extracted with the same amount of ethyl acetate as the filtrate as it is.

The cells were added with 70% acetone water in the same amount as the filtrate and stirred at 20 ° C for 1 hour and filtered.

The filtrate obtained by distilling acetone from the filtrate is extracted with ethyl acetate.

100 times the concentration of each extract was added to thin layer chromatography (TLC) using a silica gel glass plate (West Germany Melco Co., Kieselgel 60F ₂₅₄ 0.25 mm, 20 × 20) as a carrier (solvent: chloroform ethanol = 9: 1). ) It is measured from the intensity of the absorbed image detected by irradiation with 2537 Å ultraviolet rays.

To collect C-15003 P-3, P-3 'and / or P-4 produced in the culture, since the material bacteria are neutral fat-soluble, the method of the separated purification commonly used to collect these microbial metabolites. This enemy is used.

For example, the means for utilizing the difference in solubility with impurities, the means for utilizing the difference in adsorption affinity of various adsorbents such as cyclocarbon, macroporous non-ionic resin, silica gel alumina, and the means for removing impurities by ion exchange resin can be used. Each is used individually, in combination, or repeatedly. As mentioned above, since C-15003 P-3, P-3 'and P-4 are contained in both the culture filtrate and the cells, when these adsorbents are used, the culture filtrate is directly or after solvent extraction. After solvent extraction, the mixture is adsorbed on the adsorbent and purified.

In case of extraction by solvent, it can be used by (1) solvent extraction from whole culture which does not separate cells, or (2) solvent extraction from each cell and culture filtrate separated by filtration or centrifugation. Can be.

In the case of extracting the filtrate and the cells individually, it is advantageous to carry out by the following method.

Suitable solvents for extraction from the filtrate include organic solvents which are not mixed with water, for example fatty acid esters such as ethyl acetate, amyleacetate, alcohols such as butanol, halogenated hydrocarbons such as chloroform, and methyl isobutyl ketone. Ketones, such as these, are used.

Extraction is performed near neutral, Preferably, it is performed using ethyl acetate from the culture filtrate adjusted to pH 7%.

The extract is washed with water, concentrated under reduced pressure, and a crude substance I containing the active ingredient is collected by adding a nonpolar solvent such as petroleum ether or n-hexane.

In this case, the following purification process is used step by step because it is detected in a number of droplets other than C-15003 on the TLC.

That is, various adsorption chromatography is effective as a stagnation method commonly used, and as the adsorbent, carriers generally used, for example, silica gel, alumina, macroporous, nonionic adsorption resins, etc. may be used. Silica gel is most effectively used for purification in I, starting with a nonpolar solvent such as petroleum ether n-hexane, and adding a polar solvent such as ethyl acetate, acetone, ethanol, and methanol to add antibiotic C-. Elution of 15003 is performed.

As an example, silica gel (West Germany Melk 0.05-0.2 mm) is used as a carrier, column chromatography is performed while increasing the mixing ratio of n-hexane and ethyl acetate sequentially, and the eluate is irradiated with TLC to contain C-15003. The resulting mixture was concentrated under reduced pressure, and petroleum ether or n-hexane was added to obtain crude substance II.

Since it still contains another impurity in it, the following purification is performed.

For example, it refine | purifies by the 2nd silica gel column which changed the solvent system.

In this case, the developing solvent starts developing in halogenated hydrocarbons such as dichloromethane and chloroform, and adds a polar solvent such as alcohols such as ethanol and methanol, ketones such as acetone and methyl ethyl ketone, and then antibiotics C-15003. Separate the sample.

The combination of the solvent systems of the first and second silica gel columns may be reversed before and after, and other commonly used organic solvents are suitably combined. As a means for purifying crude material II, to elute antibiotic C-15003 when using macroporous adsorbent resins, lower alcohols or mixtures of lower ketones, ester oils and water are used.

As lower alcohols, for example, methanol acetate, ethanol, propanol, butanol, etc. As lower ketones, acetone, methyl ethyl ketone, ester, ethyl acetate etc. can be used, for example.

To explain one example, crude substance II was dissolved in 60% methanol water, passed through a DIION HO-10 (Mitsubishi Chemical Co., Ltd.) column, adsorbed, washed with 70% methanol water and eluted with 90% methanolic water. C-15003 elutes.

In either method, a portion of the obtained antibiotic C-15003 is concentrated under reduced pressure, and 5-8 times of ethyl acetate is added to the dried product, and the crystals of antibiotic C-15003 precipitate.

Among these crystals, C-15003 P-3, P-3 'and P-4 are contained, which are then separated again using the above adsorbent.

That is, using silica gel or macroporous nonionic adsorptive resin, it is possible to separate and elute with the above-mentioned solvent, respectively. For example, when using silica gel, it is developed with a solvent of n-hexane, ethyl acetate or chloroform methanol. , Elute in order of C-15003 P-4, P-3 ', P-3, and after detection by TLC, the C-15003 P-4, P-3', P-3 fractions are concentrated under reduced pressure, Ethyl acetate can be added to obtain respective crystals.

In addition, when using a macroporous nonionic adsorptive resin, a gradient elution method for changing the mixing ratio of alcohols, ketones, esters and water, for example, 60% methanol and 95% methanol, containing 5% salt When the gradient elution method used is used, it is eluted in the order of C-15003 P-3, P-3 'P-4, and each fraction is concentrated under reduced pressure after detection by TLC, respectively, and isolated as a crystal from ethyl acetate.

Since these crystals contain ethyl acetate as a crystal solvent, the physicochemical properties measured after drying for 8 hours on rinse pentaoxide at 70 ° C. under reduced pressure are as follows. Table 4

TABLE 4

Compared with the known antibiotic group in the molecular formula, antimicrobial ring and anti-tumor activity described later, as described above, the group such as this antibiotic C-15003 was not identified.

However, when a substance which exhibits the same ultraviolet absorption is searched in the group of plant components and other natural organic compounds, a maytanacin group is mentioned, and it is estimated that it is a maytanacin group which contains two nitrogens especially in molecular formula.

Maytanasin is obtained as a plant component, the structure of which is shown in (I ') and in Journal of American Chemical Society, Vol. 97, pp. 5294, (1975). Reported.

In addition, the data of the mass spectrum of maytanacin are expressed as follows.

As for C-15003 P-3, P-3 ', and P-4, m / e 485, 470, and 450 are also identified, and the basic skeleton is the same as maytanasin, but it is easily estimated that the third acyl group is different. , Antibiotic C-15003-new compound.

C-15003 P-3, P-3 ', and P-4 are attached to alkali decomposition, and the resulting carboxylic acid is irradiated with gas chromatography. In C-15003 P-3, isobutyl acid and C-15003 P-3 Normal butyric acid was obtained from ', and isovaleric acid was obtained from C-15003 P-4. In the above data, the estimation structures of C-15003 P-3, P-3 ', and P-4 are shown in (I').

Biocompatibility

A) antimicrobial shouts

Trypticase Soy agar medium (BBl) was used as a test medium, and the development inhibition function for the microorganism shown next was examined by the paper / disk method.

That is, 0.02 ml of a solution of 300 µm / ml of C-15003 P-3, P-3 'and P-4 was impregnated into a paper / disc (Toyo Corporation, thin diameter 8 mm) on the following microbial plate containing medium. The growth hypothesis was examined by

As a result, it showed no activity against the following microorganisms. Escherichia coli, proteus, bulgari, proteus, miraviris, sudmonas, aeruginosa, Staphylococcus aureus, Bacillus subtilis, batillus, cerius, pneumonia, ceratimercesense, mycobacterium avium,

On the other hand, tararomices was prepared using agar plate of assay medium 2 [3.5 g of sodium phosphate, 0.5 g of first potassium phosphate, 5 g of yeast extract (dipco), 10 g of glucose, 15 g of agar, 1000 ml of distilled water, pH7.0]. ㆍ To examine the growth ability of Taromyses avellaneus as a test bacterium, C-15003 P-3 and P-3 'were 3 µg / ml and C-15003 P-4 was 1.5 µg / ml. Indicates.

Further, as a test microorganism, tetrapimena pyriphormis W strain was used as a test medium [20 g of tripeptopeptone (dipco), 1 g of yeast extract, 2 g of glucose, 1000 ml of distilled water, 10 ml of 1 molar phosphate buffer solution having pH 7.0] Was cultured at 28 ° C. for 44 to 48 hours, and the microbial oil repellent ability of this antibiotic was examined by liquid dilution assay.

As a result, it was confirmed that C-15003 P-3 and P-3 'inhibited the growth of this microorganism at 1 µg / ml, or C-15003 P-4 at 0.5 µg / ml.

B) antitumor activity

To investigate the therapeutic effect of C-15003 P-3 P-3 'and C-15003 P-4 (9 days continuous intraperitoneal administration) on tumor cell P 338 (1 × 10 ⁶ cells / mouse, mouse, intraperitoneal transplantation) It was.

As a result, the anti-tumor effect of 200% of the mouse life-span by 0.00625 mg / kg / day administration was confirmed compared with the control by these substances.

C) poisonous

In acute toxicity studies with mice, when C-15003 P-3 P-3 'and C-15003 P-4 were injected intraperitoneally, both antibiotics had a LD100 of 0.625 mg / kg and an LD50 of 0.313. was more than mg / kg.

As described above, the antibiotic C-15003 is useful as an antifungal agent or an antiprotozoal agent because of its strong growth-lowering ability against filamentous fungi and protozoa.

In addition, antibiotic C-15003 is expected to be useful as an antitumor agent because of its life-saving effect on mammals (eg mice) with tumors.

In order to use the antibiotic C-15003 as an antifungal agent and an antiprotozoal agent, it can be advantageously used, for example, when examining bacterial conditions such as soil, activated sludge or animal body fluids.

That is, when separating useful bacteria from the soil, or when testing the action of bacteria other than protozoa or fungi in the operation and analysis of the activated sludge method used for wastewater treatment, the fungus or protozoa does not develop in the sample. Can selectively develop bacterial state.

Specifically, the test sample is added to a liquid or a solid medium, and 0.1 ml of a 1% methanol-containing aqueous solution of 10 to 100 µg / ml of the antibiotic is added and cultured per 1 ml of the medium.

C-15003 P-3, P-3 ', and C-15003 P-4 are novel compounds, all of which have the same backbone, and are also used as intermediates for useful pharmaceutical compounds.

That is, by deacylation reaction, C-15003 P-0 (maytansinol) whose 3rd position is a hydroxyl group can be obtained.

위에 있기 때문에, 통상 사용되는 환원적가수분해 반응이 유리하게 이용된다.In this case, the position of the acyl group on carbonyl

Since it is in the stomach, the reductive hydrolysis reaction usually used is advantageously used.

That is, at low temperatures (eg, -20 to 0 ° C), complex metal hydrogen compounds (eg, lithium hydride (LiAlH ₄ )) are used to form other functional groups such as carbonyl groups, epoxy groups, carbon-carbon double bonds, and the like. Maytansinol can be obtained by hydrolyzing the 0-ester bond of 3rd position, without affecting.

The physical properties of maytansinol obtained here are in good agreement with the description of Kvpchans. [See Journal, Orb, American, Chemical, and Sight, Vol. 97, pp. 5294–9295 (1975).]

Example 1

Yeast Extract-Water Malt Yeast Extract Nocaldia No. In C-15003 (IFO 13726, Japan Microbiological Research Institute Application No. 3992), in a 200 ml three-neck flask, 2% glucose, 3% soluble starch, 1% soybean flour, 1% corn steep liquor, poly 40 ml of seed medium containing 0.5% peptone, 0.3% NaCl, 0.5% CaCo ₃ , and pH 7.0 were inoculated and incubated at 28 ° C. on a rotary vibrator for 48 hours to obtain a seed culture solution.

0.5 ml of the obtained seed culture solution was transplanted into a 40 ml main medium containing 5% dextrin, 3% corn steep liquor, 0.1% polypeptone, 0.5% CaCo ₃ and pH 7.0 in a 200 ml triangular flask, Incubated on a rotary vibrator for 90 hours.

This culture broth showed 25 μg / ml live titer when tetrapimena filippolis W was used as an assay and antibiotic C-1003 P-3 was used as a standard.

Example 2

들이 사카구찌((坂口)플라스코에 이식하고, 28℃로, 48시간 왕복진동기 위에서 배양하였다.10 ml of the seed culture solution obtained in Example 1, containing 2,500 ml of seed medium

Was transplanted to Sakaguchi Plasco, and incubated at 28 ° C. on a reciprocating vibrator for 48 hours.

를 함유한 50

들이 스테인레스스틸 탱크에 이식하고, 28℃, 통기 30

/분, 교반 200회전/분(1/2DT) 내압 1kg/㎠의 조건으로 48시간 배양해서 종배양액을 얻었다.Species medium of 500 ml of this culture 30

50 containing

Transplanted into a stainless steel tank, 28 ° C., aeration 30

A culture medium was obtained by culturing for 48 hours under conditions of 1 kg / cm 2 / min and stirring at 200 revolutions / minute (1 / 2DT).

를 함유한 200

들이 스테인레스스틸 탱크에 이식하고, 28℃, 통기 100

/분, 교반 200회전/분(1/2DT) 내압 1kg/㎠의 조건으로 배양하였다.Main medium 100 similar to that obtained in Example 1 with the obtained seed culture solution at a transplantation rate of 10%

Containing 200

Transplanted into a stainless steel tank, 28 ° C, aeration 100

The culture was carried out under a condition of 1 kg / cm 2 / min, stirring 200 revolutions / minute (1 / 2DT) internal pressure.

The obtained culture had a production titer of 25 µg / ml by the same assay as in Example 1.

Example 3

에 하이프로 수퍼 셀(미국, 존스만빌 푸로덕트사) 2kg를 가해 잘 교반한다.Culture medium obtained in Example 2

2 kg of Hypro Super Cell (Jonesmanville Product, USA) is added and stirred well.

및 습균체 32kg을 얻는다.The mixture was filtered through a pressure filter and the filtrate 85

And 32 kg of wet cells.

에 아세트산에틸 30

를 가해 교반추출하고, 이 조작을 2회 반복한다.Filtrate 85

Ethyl acetate 30

Add stirring to extract, and repeat this operation twice.

씩으로 2회 수세하고 무수황산나트륨 500

을 가해서 건조후 200ml까지 감압농축하고 석유에테르를 가해, 석출되는 첨진을 여취한다.(53g)Combined ethyl acetate layer and water 30

Wash with water twice and each anhydrous sodium sulfate 500

After drying, the mixture was concentrated under reduced pressure to 200ml, petroleum ether was added and the precipitated precipitate was filtered out. (53g)

를 붓고 용출액을 100ml씩 구분한다.100 ml of ethyl acetate was added to the obtained crude substance I, and the mixture was stirred. The insoluble matter was filtered off, and 10 g of silica gel (0.05 to 0.2 mm from West Germany Merck) was added to the filtrate, followed by stirring. 500 ml of n-hexane, 500 ml of n-hexane ethyl acetate (3: 1), 500 ml of n-hexane ethyl acetate (1: 1), 500 ml of n-hexane ethyl acetate (1: 3) at the top of (400 ml) , 500 ml of ethyl acetate, ethyl acetate and methanol (50: 1) 1

Pour and separate the eluent by 100ml.

1 ml of each part was concentrated to dryness, 0.1 ml of ethyl acetate was added, and it was dripped at the position of 2.5 cm from the bottom of a silica gel glass plate (Kieselgel 60F254 0.2mm 20x20 by Merck, West Germany), a developing solvent, ethyl acetate methol (19: 1) is about 17cm to expand.

After the development, the absorbed image was irradiated under ultraviolet light (2537 Hz), and the part No. Collect 23 ~ 28 and concentrate under reduced pressure to about 20ml.

150 ml of petroleum ether is added to the concentrate to obtain 15 g of crude substance II.

Example 4

50 L of the 70% subcellular number was added to 32 kg of the cells obtained in Example 3, followed by stirring extraction for 3 hours, followed by filtration with a pressure filter.

Again, extraction was repeated with 50 L of 70% acetone water, the filtrate obtained by filtration was similarly combined, and acetone was distilled off by concentration under reduced pressure.

의 90％ 메탄놀수로 용출한다. 용출액을 3ℓ까지 감압 농축하고, 물 3ℓ, 아세트산 에틸 3ℓ을 가해 흔들어 섞고, 이 조작을 2회 반복한다.The obtained aqueous solution was poured into a column of 5 liters of Diaion HP-10 (Mitsubishi Chemical), washed with 20 liters of water and 50% methanol, and then 15

Eluted with 90% methanol. The eluate is concentrated under reduced pressure to 3 L, 3 L of water and 3 L of ethyl acetate are added, shaken, and the operation is repeated twice.

The ethyl acetate layers were combined, washed with water, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to 200 ml, and petroleum ether was added to precipitate the precipitate. (28 g)

The crude product thus obtained was purified by silica gel column in the same manner as in Example 3 to obtain 8.0 g of crude material II.

Example 5

1.5 g of crude substance II obtained in Example 3 was dissolved in 10 ml of ethyl acetate, 4 g of silica gel (0.05 to 0.2 mm from West Germany Merck) was added and stirred well, and ethyl acetate was distilled off under reduced pressure.

It is placed on the top of 300 ml of the silica gel column prepared in advance, washed with 500 ml of chloroform, and then eluted with 500 ml of chloroform / methanol (50: 1), 500 ml of chloroform / methanol (20: 1), and 500 ml of chloroform / methanol (10: 1). The eluate is divided into 25 ml portions, 0.5 ml of each portion is concentrated under reduced pressure, 0.05 ml of ethyl acetate is added, and this is taken as a sample and pasted into thin layer chromatography. (Developing solvent: Chloroform-methanol (9: 1), Rf 0.05 ~ 0.60 at 2537 kPa) Part No. Concentration and drying of 39.40 under reduced pressure followed by addition of 2 ml of ethyl acetate yielded crystals of antibiotic C-15003.

150 mg of the antibiotic C-15003 crystal obtained above is dissolved in 15 ml of methanol, and 300 ml of sodium chloride and 15 ml of water are added thereto to dissolve.

200 ml of Diaion HP-10 (Mitsubishi Chemical Co., Ltd.) was packed in a column of 1.8 cm in diameter, and 600 ml of 50% methanol containing 5% salt was poured.

와 95％ 메타놀수 1.5ℓ와의 사이에서 경사용출을 행한다.After flowing the sample solution prepared previously, 300 ml of 60% methanol solution containing 5% salt was poured out, and 60% methanol solution containing 5% salt 1.5

And elution is carried out between and 1.5% of 95% methanol.

The eluate is separated by 15 ml and detected by attaching each part to thin layer chromatography on silica gel.

C-15003 P-3 in sections 145-153, C-15003 P-3 'and P-4 in sections 167-180, and C-15003 P-4 in sections 185-190.

Gather each of them, concentrate, add 50 ml of water and 100 ml of ethyl acetate, dissolve, place in a separatory funnel, shake, mix, separate the water layer, wash twice with 50 ml of water, and dry the ethyl acetate layer with anhydrous sodium sulfate, concentrate and leave. Each crystal is precipitated.

The crystals are filtered off and dried.

C-15003 P-3 70mg

C-15003 P-3 ', P-4 18mg

C-15003 P-4 15mg

C-15003 P-3 ', P-4 and the mixed crystal 18mg dissolved in 0.3ml of ethyl acetate, silica gel glass plates (Merck Co. West Germany selgel key 60F ₂₅₄ 0.25mm 20 × 20) of the linear position 2.5cm from the bottom of the third plate It is applied onto the bed and transferred with ethyl acetate / methanol (19: 1).

After about 18 cm of development, the absorbing phases of Rf 0.68 (P-4) and Rf 0.65 (P-3 ') were each scraped off, and separately extracted twice with ethyl acetate containing a small amount of water. After washing with water, concentrated under reduced pressure and dried with anhydrous sodium sulfate.

Crystals of C-15003 P-410 mg at Rf 0.68 and C-15003 P-3 '3 mg at Rf values 0.65 are obtained.

Example 6

1 L of the Sakaguchi flask culture medium shown in Example 2 was inoculated into a 200 L stainless steel tank containing 100 L of seed medium and incubated for 48 hours at 28 ° C, aeration 100 L / min, and stirring at 200 revolutions / min for 48 hours.

The obtained seed culture liquid was transplanted into a 2000-l stainless steel tank containing 1000 l of the main medium indicated in Example 1 at a transplantation rate of 10%, and subjected to 28 ° C, aeration 1000 l / min, stirring 120 revolutions / minute (1 / 3DT), It was incubated for 90 hours under conditions of 1 kg / cm 2 internal pressure.

The obtained culture showed a production titer of 20 µg / ml by the assay of Example 1.

900 L of acetone obtained was added to the mixture, and stirred for 1 hour. Then, 20 kg of a high pro-super cell (manufactured by Jones Manville, USA) was added, stirred, and filtered with a pressure filter.

500 L of water was added to 1700 L of the obtained filtrate, and extracted with 1000 L of ethyl acetate using Podbilniak (US, podbielniak INC). The obtained ethyl acetate layer was washed with water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. When petroleum ether was added to the concentrate and the precipitate precipitated was filtered and dried, 68 g of a crude substance was obtained, and the same method as in Examples 3, 4 and 5 below. Purification by C-15003-P-39.5g, 300 mg of C-15003 P-3 'and C-15003 P-42.5g were obtained respectively.

Claims (1)

Antibiotic C-15003 producing strain belonging to the genus Nokaldia A method for producing antibiotic C-15003, wherein C-15003 is cultured in a nutrient medium, and antibiotics C-15003 are produced and accumulated in the culture medium and collected.