JPS6034556B2 - Antibiotic C-15003 - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel antibiotic C-15003, a method for producing the same, and a method for producing derivatives from the antibiotic. The present inventors collected various types of soil samples, searched for the antibiotics produced by microorganisms isolated from them, and found that certain microorganisms produce novel antibiotics. We have learned that the microorganism belongs to the genus Nocardia, that the antibiotic can be accumulated in the culture by culturing the microorganism in a suitable nutrient tower, and that we can obtain a donor from the resulting antibiotic. As a result of this knowledge and further research, we have completed the present invention. That is, the present invention
General formula (1) [wherein R represents or]. ] and ■Antibiotic C-15003-producing bacteria belonging to the genus Nocardia are cultured on rented land, and antibiotic C-15003 is produced and accumulated in the culture, which is then collected. This is a method for producing antibiotic C-15003. In the present invention, "antibiotic C-15003J" refers to a generic term for the three compounds represented by the above general formula (1), a mixture of two or three, or a single compound of each.
In the general formula (1), the R-CO-CH compound is called "antibiotic C-1500-3" or simply c-1.
500 Snake-3", R is -CO-CH2-CH2CH3
The compound is referred to as "antibiotic C-1500-3" or simply {C-1500-3', where R is -CO-CH2-
The compound of CHKUS Hanji is called “Antibiotic C-1500-4”
” or simply “C-1500 crime-4” and R is the compound [general formula (0)] [antibiotic C-1500 is -C
J or simply "C-1500 is -C", respectively. Examples of bacteria that produce the antibiotic C-15003 of the present invention include actinomycetes NnC-15, which the present inventors isolated from soil during the search for antibiotic-producing bacteria.
Examples include the 003 stock. The mycological properties of NoC-15003 strain were determined by the methods of Shearing and Gottlieb [International. Journal of Systematic Bacteriology {lntematioMI JomM1 ofS$t
matic mother cteriolo plow), Volume 16, 313
Pages 340 to 340, 1966 King], 2 is o,
The results observed over 21 days are as follows. 1 Morphological characteristics The basal hyphae elongate and branch well both on agar and liquid media. Most of them are 0.8 to 1.2 mm in diameter, and sometimes they are divided into birch-like or branched short hyphae.
It grows well on various classification media, and aerial fungi grow on the substrate mycelia, but fascicles (50-200 mm x 200 x 1
000 m) and often grows on them. Most of the shapes of aerial hyphae are curved or straight;
In rare cases, a loose spiral can be seen. When a mature culture is examined under a microscope, there are few cases where the spores are thought to be chained, and when a bacterial suspension taken from the culture surface is examined under a microscope, it is found that the spores are oblong (0.8 to 1.2 Mountain m×
4.8 to 6.8 m) and oval (0.8 to 1.2
Many segmented spore-like particles (1.0 to 2.0 rm) were observed, and their surfaces were smooth when observed using an electron microscope. 2. Bacterial composition This bacterial strain was added to the ISP port. 28℃ in the underground modified tower of No. 1, 8.66
The cells were cultured by shaking for ~90 hours, and the bacterial cells were collected and washed with rice bran. Bee the above bacteria. Becker et al.'s method (Applied Microbiology)
gy), 12 萱, 421 pages, 196 Sanno] and M.
The method of bi-lechvale (Journal of Laboratory and Clinical Medicine)
As a result of examining the diaminopimelic acid and sugar composition in the bacterial cells according to ``Dicine'' Vol. 71, p. 984, 1999], it was found that the former was meso-form, and the latter had spots corresponding to galactose and arabinose. Ta. 3. Characteristics of the classification tower This strain grows relatively well on various media, and its basal hyphae are colorless to pale yellow at the initial stage of cultivation, and exhibit pale yellow-brown or clump-brown color. It also produces a yellow to yellow-brown soluble pigment in various sorting media. Aerial mycelium is powdery, generally of medium growth, and white to yellow or tan in color. The properties of this strain on various classification media are shown in Table 1. Table 1 No. C-15003 Various properties on the vermilion classification medium

[I] Growth on sucrose/nitrate agar medium (G): Abundant, yellow (3ia) * or pale yellowish brown 4
31c), Eastoid formation Aerial hyphae (AM): Poor, white Soluble pigment (SP): None or yellowish brown 'o' Glycerol/nitrate agar medium G: Moderate, pale yellow (Next a) * Eastoid formation Formation AM: Moderate, white SP: None Glucose/asparagine agar tower ground G: Moderate, light yellow (3pa) *None bright yellow (book a)
) *AM: poor, white SP: light yellow (Sa) * 0 Glycerol/asparagine agar medium G: moderate,
Pale yellow (Sediment a) * Floccule formation AM: poor, white P: none ■ Starch agar medium G: moderate, pale yellow (pine a) * or pale yellowish brown (cloth a)
* East formation AM: Abundant, pale yellow (next a) * Sp: None N Nutrient agar medium G: Moderate, pale yellow (SA) * or yellow (ZP) *
East body formation AM: Poor, white SP: None [Calcium malate agar medium G: Moderate. Pale yellow (Sa)* or pale yellowish brown (Ki-a)* East formation AN: Medium, white or pale yellow (Sa)*SP: None Yeast extract/malt extract agar Nagisaji G: Medium , pale yellowish brown (3ic) * to light brown (3ia) * East formation AM: medium, white to pale yellow (next a) *SP: no skin Oatmilk agar toji G: medium, pale yellow (Xa) )
*or yellow (23) * East body formation AM: poor, white or pale yellow SP: none (nu) Beptone/yeast extract/iron agar medium G: moderate, yellow (Z3) *AM: none SP: yellow (Name) * (Le) Tyrosine agar medium G: medium, pale yellow (Xa) *no yellow (bead a) *fascicle formation AM: moderate, white to pale yellow (next a) *S
P: Tan (3ie)* *: Color Harmony Manual, 4th edition, (Container Corporation of America, 19
Physiological properties The physiological properties of this strain are shown in Table 2. That is, the growth temperature range is 1〆○ to 38°C, and the temperature range in which aerial mycelium is well established on an agar medium (ISPM.2) is 20pm to 35q0. Table 2 M. C-
Physiological properties of 15003 Vermilion Growth temperature range: 120-38
q0 Aerial mycelial growth temperature 20oo~35q0 Gelatin liquefaction:
Positive Starch hydrolysis: Positive Nitrate reduction: Positive Milk/veptonization: Positive Milk/coagulation: Negative Casein resolution: Positive Melanin-like pigment formation (Beptone/yeast extract iron agar medium): Negative, (Tyrosine agar medium): Enteric Tyrosine degradation: Positive xanthine degradation Bear: Negative Hypoxanthine degradation ability: Negative Lysozyme resistance: Positive Salt tolerance: 2% 5 Utilization of various carbon sources Priedham and Gottlieb method [Journal of Bacteriology. The usability of various carbon sources was examined using the medium described in J. (Joumalof Mother Actiolo Stage) 5 Biwa, p. 107, 1948] and a basal medium to which 0.1% yeast extract (Bacto) was added. The results are shown in Table 3. Table 3 No. C-15003 Vermillion carbon source Usable carbon source Growth *
D-xylose 10-day D-arabinose ++ D-glucose 1-day ○-galactose ++ D-fructose
State L-Rhamnose ++ D-Mannose Kawano Seuucrose Yokohama Lactose Maltose
Master + Trehalose
Toka Raffinose ±Melipiose ++ i-inositol D-sorbitol D-mannitol M glycerol −± soluble starch
+ 10 vs. control − − *: Basal medium supplemented with 0.1% yeast extract Note; River: Abundant growth juice: Relatively good growth 10: Growth observed ±: Slight growth −: No growth 6 Other miscellaneous Properties Collect bacterial cells using the method shown in 2) above, and mix them with J.
Marmer et al.'s method [Journal of Molecular
Biology (JoumalofMolecularB
iolo period), p. 208, 1961].
When prepared and tested for the GC content of DNA, it was approximately 71 mol%.
Met. Gram staining of the vegetative hyphae of this strain was positive. The ship mentioned above. Various properties of C-1500 strain
The Actinomycetes (meActi) by Waxman
nomycetes), Volume 2, The Williams & Wilkins Company, 1961, R.I. BergeyS Ma
nual of Detenni Misaki tiVe mother cter
iolo plow), 8th edition, 1974 King and other literature. This strain is Nocardia (Nocar).
Although it is thought to belong to group m of the genus Dia), no species with the above-mentioned properties was found among the known strains, and it was identified as a new bacterial species. This strain No. C-15003 group, FERM-P No. to the Institute of Microbial Technology, Agency of Industrial Science and Technology. 3
IFO-1372 to Fermentation Research Institute as 992
6, respectively. T As mentioned above, a ship. C-15003% is a new species of the genus Manocardia, but as a general property of microorganisms, it can mutate naturally or by mutagens. For example, there are many mutant strains obtained by irradiating single spores with X-rays, gamma rays, ultraviolet rays, etc., culturing 0 on media containing various drugs, mutating them by other means, or naturally occurring strains. Even if it is a mutant strain, etc., it is not enough to make it a substantially different species when compared with the mycological properties described above or the mycological properties shown below, and furthermore, it is not sufficient to classify it as a different species.
Anything that has the property of producing P-3, P,3 and/or P-4 can be used in the method of the present invention. For example, No. By subjecting C-15003 vermilion to various mutation treatments, almost no soluble pigment is produced;
Base hyphae are colorless, yellow-green, reddish-brown to light red, hyphae easily split into short, camphor-like or branched hyphae, and many aerial hyphae are white or aerial. Mutant strains with almost no adhesion have been obtained. The medium used for culturing the bacteria that primarily produces antibiotic C-15003 may be liquid or solid as long as it contains nutrients that can be used by the strain, but it is more appropriate to use a liquid medium when processing large quantities. It is. The medium contains M. Carbon sources that can be assimilated by C-15003 beads, nitrogen sources that can be digested, inorganic substances, collected nutrients, etc. are appropriately blended. Carbon sources include, for example, glucose, lactose, sucrose, maltose, dextrin, starch, glycerin, mannitol, sorbitol, and other fats and oils (eg, soybean oil, lard oil, chicken oil, etc.), and nitrogen sources, such as meat extract. , yeast extract, dried yeast, soybean flour, corn stave liquor, veptone, cottonseed flour, hot molasses, urea, ammonium salts (eg, ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium acetate, etc.), and others are used. In addition, salts containing sodium, potassium, ursium, magnesium, etc., iron, manganese, zinc,
Metal salts such as cobalt and nickel, salts such as phosphoric acid and boric acid, and salts of organic acids such as acetic acid and propion are used as appropriate. Other amino acids (e.g., glutamic acid, aspartic acid, alanine, glycine, lysine, metinine, proline, etc.), peptides, tribeptide, etc.), vitamins (e.g., B, &, nicotinic acid, B,2, VC, E, etc.) ), nucleic acids (eg, purine, pyridine, derivatives thereof, etc.), etc. may be included. Of course, inorganic or organic acids, alkalis, buffers, etc. may be added for the purpose of adjusting the pH of the medium, or appropriate amounts of oils and fats, surfactants, etc. may be added for the purpose of defoaming. The culturing method may be static culture, shaking koji culture, aerated strain culture, or the like. Needless to say, for large-scale treatment, it is desirable to use so-called deep aeration fly culture. It goes without saying that the culture conditions vary depending on the state and composition of the medium, the type of strain, the culture method, etc., but they are usually 2000~35o0.
It is best to select the initial pH to be around neutral at a temperature of . In particular, the temperature during the middle stage of culture is 23°C to 30°C, and the initial pH is
A condition of 6.5 to 7.5 is desirable. The culture period also varies depending on the conditions described above, but it is preferable to culture until the desired antibiotic concentration is maximized. The time required for this is usually about 2 to 6 days in the case of shake culture or aerated oak culture using a liquid medium. The titers of these antibiotics were measured using Tetrahymena pyriformis W strain (Tetraph m).
ona, p.ifo skin leW) as test microorganisms in a test medium [tryptose-beptone (manufactured by Difco) for 20 hours, yeast extract (manufactured by Difco) for 1 night, glucose for 2 hours, distilled water 1000 and 1 molar phosphate buffer. A liquid dilution assay method in which the growth is measured by turbidity after culturing at 28 qo for 4 to 4 hours using a solution with a pH of 7.010, and a thin layer chromatography method (hereinafter abbreviated as TLC) described later.
It was measured by The novel antibiotic C-
1500*-3, P-3' and/or P-4 are produced and accumulated, and these are contained in both the furnace liquid and the bacterial cell extract. These substances were also searched by TLC method. That is, the culture is separated into microbial cells and sap by filtration or centrifugation, and the sap is directly extracted with the same amount of ethyl acetate as the sap. The same amount of 70% acetone water as the furnace solution was added to the bacterial cells, and the mixture was incubated for 1 hour at 20:00 and then filtered. Acetone in the furnace solution is distilled off, and the resulting water and furnace solution are extracted with ethyl acetate. A 10M concentrated solution of each extract was placed on a silica gel glass plate (West German Merck & Co., Kiesergen hexavalent 0.25
Thin layer chromatography (20 x 20) as a carrier
TLC) (solvent: chloroform/methanol = 9
:1), measured from the intensity of the absorption image detected by irradiating 2,537 people with ultraviolet rays. C-15 produced in culture
In order to obtain 00 Kabuto-3, P-3 and/or F-4, since this group of substances is neutral fat-soluble, separation and purification methods commonly used to collect such microbial metabolites are required. will be used as appropriate. For example, there are methods that utilize differences in solubility with impurities, methods that utilize differences in adsorption affinity of various adsorbents such as activated carbon, macroporous nonionic resins, silica gel, and alumina, and methods that use ion exchange resins to remove impurities. may be used alone, in combination, or repeatedly. As mentioned above, C-1500-3, P-3', and P-4 are contained in both the culture furnace solution and the bacterial cells, so when using these adsorbents, they can be used directly in the culture solution or by solvent extraction. Afterwards, the bacterial cells are extracted with a solvent and adsorbed onto an adsorbent for separation and purification. When extracting with a solvent,

[11] Either method can be used, such as solvent extraction from the whole culture without separating the bacterial cells, or {21 solvent extraction from each of the bacterial cells and culture solution separated by filtration or centrifugation. When extracting the furnace liquid and the bacterial cells separately, it is advantageous to carry out the following method. Suitable solvents for extraction from furnace liquid include organic solvents that are immiscible with water, such as fatty acid esters such as ethyl acetate and amyl acetate, alcohols such as butanol, halogenated hydrocarbons such as chloroform, and methylisobutyl ketone. Ketones are used. Extraction is carried out at around neutrality, preferably from a culture solution adjusted to pH 7 using ethyl acetate. After washing the extract with water, it was concentrated under reduced pressure, and a non-disruptive solvent such as petroleum ether or n-hexane was added to obtain the crude substance 1 containing the active ingredients.
Collect. This includes antibiotic C-150 on TLC.
Since many spots other than 03 are observed, the following purification steps are used in stages. In other words, various types of adsorption chromatography are effective as commonly used purification methods, and commonly used carriers such as silica gel, alumina, and macroporous nonionic adsorption resins can be used as adsorbents, but crude Silica gel is most effectively used for purification of substance 1, starting with a non-polar solvent such as petroleum alcohol or n-hexane, and adding a polar solvent such as ethyl acetate, acetone, ethanol, or methanol. Antibiotic C-15003 is thereby dissolved. One example is silica gel (West German Merck
0.05 to 0.2 side) as a carrier, column chromatography was performed while increasing the mixing ratio of n-hexane and ethyl acetate sequentially, and the eluate was examined by TLC.
The fractions containing 3 are collected, concentrated under reduced pressure, and petroleum ether or n-hexane is added to obtain crude material 0. Since this still contains other impurities, the next purification is performed. For example, purification is performed using a second silica gel column using a different solvent system. In this case, developing solvents include halogen-containing hydrocarbons such as dichloromethane and chloroform, as well as polar solvents such as alcohols such as ethanol and methanol, and ketones such as acetone and methyl ethyl ketone. Substance C-15003
Separate and collect. The combination of solvents for the first and second silica gel columns may be reversed, and other commonly used organic organic solvents may be combined as appropriate. When using macroporous adsorption resin as a means of purifying crude substance 0, to elute antibiotic C-15003,
A mixture of lower alcohols, lower ketones, or esters and water is used. Examples of lower alcohols include methanol, ethanol, propanol, and butanol; examples of lower ketones include acetone and methyl ethyl ketone; and examples of esters include ethyl acetate. An example is to dissolve crude substance 0 in 60% methanol water,
It is passed through a Diaion HP-10 (Mitsubishi Kasei) column for adsorption, washed with 70% methanol water, and then eluted with 90% methanol water to elute the target antibiotic C-15003. In either method, the obtained antibiotic C-
The fraction of 15003 was concentrated under reduced pressure, and 5 to 8
When double the amount of ethyl acetate is added and left to stand, antibiotic C-150
03 crystals are precipitated. This crystal contains C-1500 spots-3, P-3 and P-
4, which are then further separated using the adsorbent described above. That is, using silica gel or macroporous nonionic adsorption resin, it is possible to separate and elute with the above-mentioned solvents. For example, when using silica gel, n-hexane, ethyl acetate, or chloroform/methanol can be used. Developed with a solvent of the system, C
-1500 is eluted in the order of -4, P-3, and P-3, so after detection by TLC, C-1500 is eluted in the order of -4, P-3, and P-3.
4, P-3', and P-3 fractions can be concentrated under reduced pressure and ethyl acetate can be added to obtain respective crystals. When using a macroporous nonionic adsorption resin, a diagonal elution method that changes the mixing ratio of alcohols, ketones, or esters with water, such as 60% methanol water containing 5% salt and 95% methanol water, is recommended. When using the oblique elution method using C-1500, C-1500 was eluted in the order of -3, P-3, and P-4, and each fraction was detected by TLC and concentrated under reduced pressure.
It is isolated and collected as crystals from ethyl acetate. These crystals contain ethyl acetate as a crystal solvent, so 70o
The physicochemical properties determined after drying over phosphorus pentoxide under reduced pressure for 8 hours are as follows (Table 4). As mentioned above in Table 4, comparisons were made with known antibiotic groups based on the molecular formula, antimicrobial activity and antitumor-stimulating properties described below, but no group such as the present antibiotic C-15003 was observed. However, when we searched for substances that show similar ultraviolet absorption among plant ingredients and other natural organic compounds, we found the maytanacin group, and in particular, the maytancine group, which contains two nitrogen atoms from its molecular formula, was identified. Presumed. Maytanacin is obtained as a plant component, and its structure is shown in Figure 1 and published in the Journal of the American Chemical Society.
rican Chemical Society), Vol. G7, p. 5297 (1975). The mass vector data for maytanacin is shown below. M 11 {a] Maytanasin 545 {a) = KO 10th NC0, M+-(a+b), 485-CH3 Maytanasin 485 47
0Meitanashin 485-CI450 C-1500 Group-3, P-3, P-4 also m/e
Since 48F470450 is observed, it can be easily inferred that the basic skeleton is the same as metanacin, but the acyl group at the 3-position is different, and the antibiotic C-150
It can be seen that 03 is a new compound. When C-15003F-3, P-3, and P-4 were subjected to alkaline decomposition and the resulting carboxylic acids were examined using gas chromatography, it was found that isobutyric acid and C-1500 were derived from C-1500 Snake-3. Normabutyric acid, C-15 from 3'
Isococyanic acid was obtained from 00-4. From the above data, C-1500 group-3, P-3, P-4
The estimated structure of is shown in FIG. Figure 1 Biological Activity A Antimicrobial Activity Trypticase Soy agar medium (manufactured by BBL) was used as a test medium, and the ability to inhibit the growth of the following microorganisms was tested by the paper disk method. That is, on the following microorganism-containing plate culture, 0.02 ml of a solution of 300 ml of C-1500 stain-3, P-3', and P-4 was added to a paper disk (Toyo Seisakusho, thin type,
The growth inhibiting ability was tested by adding it to a pig (diameter: 8 pigs).
As a result, it showed no activity against the following microorganisms.
Escherichia coli Proteus pulgaris, Proteus mirapilis, Pseudomonas aeruginosa, Staphylocotcus aureus, Pacillus. subtilis, Bacillus cereus, Klebsiella pneumoniae, Serratia marcescens, Mycobacterium avium, while assay medium [disodium phosphate 3.5
evening, phosphate-potassium 0.5 evening, yeast extract (Difco)
5 evenings, glucose 10 evenings, agar 15 evenings, distilled water loo0
Using an agar plate (pH 7.0), prepare Talaromyces avellaneus (Talaromycesave).
When the growth ability was examined using C.
-1500 group-3 and P-3'', 30 muta/similar,
C-1500 Group-4 showed growth inhibiting power at 1.50 mountains/ground. In addition, Tetraphymena pyriformis (Tetraphymena pyriformjs) W
The strain was used as the test microorganism, and the assay site [tryptose/beptone (Tifco) 20 days, yeast extract 1 day, glucose 2 hours]
Evening, distilled water 1000ml, 1M phosphate buffer pH 7.0,
10'] and cultured for 0,4 hours to 48 hours, and the ability of the antibiotic to inhibit the growth of the microorganism was examined by liquid dilution assay. As a result, C-1500 is -3 and P-3' is 10 muta/'; C-1500 is -4 is 0.50 muta/'
was found to inhibit the growth of the microorganism. B. C-1500*-3, P-3' and P- against anti-tumor active Koshito P plaque 8 (1 x 1 P cells/mouse, mouse, peritoneal transplant)
The therapeutic effect of No. 4 (continuous intraperitoneal administration for 9 days) was investigated. As a result, the survival rate of mice by these substances was symmetrical, and an anti-tumor effect of 200% was observed at 9/k9/sunset of 0.0062. In an acute toxicity test using C-1500 mice as test materials, C-1500
When P-3, P-3 and P-4 were injected intraperitoneally, all of the antibiotics had a LD,oo value of 0.6250M/k9.
, and the LDo value was 0.313 females/k9. As mentioned above, the present antibiotic C-15003 has a strong ability to inhibit the growth of filamentous fungi and protozoa, and is therefore useful as a preventive agent or an antiprotozoal agent. Furthermore, since antibiotic C-15003 exhibits a life-prolonging effect on mammals (eg, mice) with canker sores, it is expected to be useful as an antitumor agent. This antibiotic C-
To use 15003 as a preventive and antiprotozoal agent,
It can be advantageously used, for example, in examining the bacterial ecology of soil, activated sludge, or animal body fluids. In other words, when separating useful bacteria from soil, or when examining the effects of bacteria other than protozoa or ruts in the operation and analysis of the activated sludge method used for water replenishment treatment, signs of survival in the sample may be detected. Alternatively, it is possible to selectively develop bacterial ecology without allowing protozoa to develop. Specifically, a test sample is added to a liquid or solid medium, and 0.1 of a 1% methanol-containing aqueous solution containing 10 to 100 pieces of this antibiotic per 1 inch of the medium is added. , to culture. Above C-15
Group 00-3, P-3, and P-4 are new compounds, all of which have the same skeleton, and are also used as intermediate raw materials for more useful pharmaceutical compounds. That is, due to the deacylation reaction, C-100, which has a hydroxyl group at the 3-position, becomes -0 [maytansinol (manasi
nol)] can be obtained. In this case, since the acyl group is located at the beta-position of the carbonyl position, the commonly used reductive aqueous reaction can be advantageously used. That is, using a complex metal hydride compound [e.g., lithium aluminum hydride (LiAIH4]) at low temperatures (e.g., -20 to 0°C), other functional groups, such as carbonyl groups, epoxy groups, carbon-carbon double bonds, etc. No influence, 3rd place C-
Meitane seal can be obtained by hydrolyzing the ester bond. The physicochemical properties of maytansinol obtained here are in good agreement with those described by Kupchan et al. [see Journal of the American Chemical Society, Vol. 97, pp. 5294-5295 (King, 1973). ]. Example 1 Nocardia No. cultured on yeast extract malt extract slanted agar tower
．． C-15003 (m013720FERM-P ground.
3992, 2% glucose, 3% soluble starch, 1% raw soybean flour, 1% cornstarch liquor, 0.5% polybeptone, NaCI in a 200' Erlenmeyer flask.
O. 40 containing 3%, CaC030.5%, pH 7.0
The seeds were inoculated into a seed medium and cultured on a rotating shaker for 4 hours.
A seed culture solution was obtained. 0.0 of the obtained seed culture solution. 200 [in the Erlenmeyer flask, 5% dextrin, 3% corn staple liquor]
, polybeptone 0.1%, CaC030.5%, PH7
．． It was transplanted to the main pagoda of 4 tubes including 0 and cultured on a rotary shaking koji machine for 280 hours for 90 hours. This culture solution was assayed by broth dilution method using Tetrahymena pyriformis W as the test bacterium and Antibiotic C-1500 Group-3 as a standard product, and showed a production titer of 25 r/ml. Example 2 10' of the seed culture solution obtained in Example 1 was added to the seed medium 5
00 hearts were transplanted into two customer Sakaguchi flasks, 2800,
Culture was carried out on a reciprocating shaker for 4 hours. 500ml of this culture was transferred to a 50ml stainless steel tank containing 30ml of seed stock, 2800ml, and vented to 3.
0 so/min, Azusa 280 revolutions/min (1/phantom T), internal pressure lk
A seed culture solution was obtained by culturing for 48 hours under the condition of 9 evenings/day. The obtained seed culture solution was transferred to a 200-ml stainless steel plate containing 100 ml of the same main medium as shown in Example 1 with a transplantation rate of 10%.
Transplanted to a steel tank, 2800, ventilation 100 m/min, fly Azusa 200 revolutions/min (1'2 DT), internal pressure lk9 m/min.
The cells were cultured for 90 hours under uniform conditions. The obtained culture solution showed a production titer of 25 Muta/di using the same assay method as in Example 1.
Example 3 Culture solution 95 obtained in Example 2 was added with Hyflo Super Cell 2K9 (Johnsmanville Products, USA) and stirred well. The mixture was filtered using a pressurized filter to obtain 85 kg of furnace liquid and 32 kg of wet bacterial cells. Add 30 ml of ethyl acetate to 85 ml of furnace liquid to extract Nawa Azusa, and repeat this operation twice. Combine the ethyl acetate layers, wash twice with 30ml of water, and add 50ml of anhydrous sodium sulfate.
After drying for 30 minutes, the mixture was concentrated under reduced pressure to 200 degrees centigrade, petroleum ether was added, and the precipitate was collected in a furnace (53 days). To the obtained crude substance 1, 100% of ethyl acetate was added and stirred, the insoluble matter was removed in a furnace, and the furnace solution was mixed with silica gel (Merck & Co., West Germany).
05 to 0.2 side) After stirring for 10 minutes, ethyl acetate was distilled off under reduced pressure and placed on the upper end of a silica gel column (400 mm) prepared in advance. (3:1) 500 i, n
-Hexane/ethyl acetate (1:1) 500%, n-hexane/ethyl acetate (1:3) 500%, ethyl acetate 500%, ethyl acetate/methanol (50:1) for 1 night. Fractionate the eluate to 100M. 1' of each flank was concentrated to dryness, 0.1' of ethyl acetate was added, and 2. 5 Spot on the skin, add developing solvent, ethyl acetate,
Develop about 17c with methanol (19:1). After development, the absorption image was examined under ultraviolet light (2537A) and Rfo. 6
~Furaction with absorption around 0.6 part■23~2
Collect up to 8' and concentrate under reduced pressure to about 20'. Petroleum ether 150 is added to the concentrate to obtain a crude substance of 015. Example 4 To 32 kg of the bacterial cells obtained in Example 3, 50 g of 70% acetosome water was added, and after 3 hours of toyo extraction, the mixture was heated in a pressurized filter. Repeat the extraction with 50 mL of 70% acetone water, combine the filtrate obtained in the same manner, and remove the acetone by concentrating under reduced pressure. The obtained aqueous liquid and Diaion HP-10
(Mitsubishi Kasei) 5. Pour into the column, wash with 20. water and 50% methanol water, and then ship with 90% methanol water. The eluate was concentrated under reduced pressure for 3 days, then 3 portions of water and 3 portions of ethyl acetate were added and mixed, and this operation was repeated twice. The ethyl acetate layers were combined, washed with water, added anhydrous sodium sulfate, dried, concentrated under reduced pressure to 200M, added petroleum ether, and filtered out the precipitate (No. 28). The obtained crude material was purified using a silica gel column in the same manner as in Example 3 to obtain 08.0% of the crude material. Example 5 1.5 μl of the crude material obtained in Example 3 was dissolved in 10 μl of ethyl acetate, and silica gel (Merck & Co., West Germany, 0.05-0.0
．． 2) After adding 4 parts and stirring well, ethyl acetate was distilled off under reduced pressure. Place it on the upper end of a silica gel column 300 prepared in advance, wash it with 500 g of chloroform, add 500 g of chloroform/methanol (50:1), 500 g of chloroform/methanol (20:1), 500 g of chloroform/methanol ( 10:1) elutes at 500'. The eluate was fractionated into 25% fractions, 0.5% of each fraction was concentrated under reduced pressure, 0.05% of ethyl acetate was added, and this was used as a sample for silica gel thin layer chromatography (developing solvent Chloroform/methanol 9:1). Rf
o. Fraction Yuzu showing an absorption image at 2537A between 50 and 0.60. 3940 was concentrated to dryness under reduced pressure, and ethyl acetate was added to it and left to stand, giving 150 crystals of antibiotic C-15003.
You get a fence. Antibiotic C-1500 obtained above 150 parts 9 parts is dissolved on 15 parts methanol, and 300 parts salt and 15 parts water are added and dissolved. Diaion HP-10 (Mitsubishi Kasei) 200 fence with a diameter of 1
．． 8. Adjust by pouring 600ml of 50% methanol water containing 5% salt into the skin column. After pouring the previously prepared sample solution, add 30% methanol water containing 5% salt to 60% methanol water.
0' and 60% methanol water containing 5% salt 1.
Perform oblique elution between 5 hours and 95% methanol water for 1.5 hours. After elution, the solution was fractionated into 15 fractions and each fraction was covered with a thin layer of silica gel. Detection is done by subjecting it to matography. C-15003P-3 with fraction 145-153
, C-15003P with fraction 167-18000
-3 and P-4, fractions 185-19 P-C and C-1
5 - -4 is shipped. Collect each and concentrate to 50% water.
Add 100 parts of ethyl acetate to dissolve, put into a separatory funnel, stir, separate the aqueous layer, wash twice with 50 parts of water, dry the ethyl acetate layer with ramie, concentrate and leave it. Then, each crystal is precipitated. Take the crystals in a furnace and dry them. C
-1500 is -3 70m9 C-15003P・3, P-4 18 no Ta C-1500
-4 15m9 C-1500*-3 P-4 mixed crystals 1 & 9 were dissolved in 0.3' of ethyl acetate, and a silica gel glass plate (West German Merck & Co., Kieselgel 6 Misaki 2 string 0.25
Apply in a straight line at a position 2.5 arcs from the bottom edge of 3 ribs (20 x 20) and spread with ethyl acetate/methanol (19:1). After about 18 degrees of development, Rfo. 68 (P-4), Rfo. 6
The absorption images of 5(P-3) are each scraped off, extracted twice with ethyl acetate containing a small amount of water, and the extracted ethyl acetate obtained is washed with water, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and discharged. Rf value from 0.68 to C-1500 spot-4
9 out of 10, Rf value 0.68 to C-1500 spots-4 1
0 p, Rf value 0.65 force) et al. C-15500 crime-3
3/9 crystals are obtained. Example 6 One sample of the Sakaguchi flask culture solution shown in Example 2 was used.
The seeds were inoculated into a stainless steel tank and cultured for 4 hours at 28 qo, aeration at 100 rpm, and 200 rotations/min to obtain a seed culture. The obtained seed culture solution was transplanted at a transplant rate of 10% into a 2,000 ml stainless steel tank containing 1,000 liters of the main medium shown in Example 1, at 28 qo, with aeration of 1,000 m/min.
The culture was carried out for 9 hours under the conditions of 120 rotations/min (1/button T) and an internal pressure of 9 m/min. The obtained culture solution showed a production titer of 20 cells/secretion using the assay method of Example 1. Add 900 ml of acetone to the resulting culture solution and leave it for 1 hour, then add Hyflo Super Cell (manufactured by Johns Manville, USA) 20k9, stir, and filter through a pressurized filter. Add 1,700 ml of the obtained furnace liquid and 500 ml of distilled water,
Ethyl acetate 1000 sleeves P.O.
Extract using the local IELNIAK INC). The obtained ethyl acetate layer is washed with water, dried with anhydrous sodium sulfate, and concentrated under reduced pressure. When 7 liters of petroleum ale was added to the concentrated solution and the precipitate precipitated was filtered and dried, crude substance 168 was obtained, which was purified in the same manner as in Examples 3, 4, and 5 to obtain C-1500 speck-39. .5ta C-1500*-
9 of 3300 and 2.5 days of C-1500*-4 were obtained, respectively. Example 7 Fifteen legs of the antibiotic C-1500 Kyuyoshi crystal obtained in Example 5 were dissolved in one volume of tetrahydrofuran and cooled to -5°C. Add lithium aluminum hydrand 12 copy and leave for 2 hours. After adding 0.5 parts of 1% S04 water to the reaction solution, 2 parts of ethyl acetate was added for extraction. The ethyl acetate layer was washed with water, added with anhydrous sodium sulfate, dried, and concentrated under reduced pressure.
Preparative TLC using a silica gel column (solvent: ethyl acetate:methanol = 19:1) was performed in the same manner as shown in Example 5, and an absorption image around 0.25 to 0.3 was scraped off. Extract with ethyl, wash with water, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to precipitate crystals.
When the crystals are dried in a furnace, 10 pieces of C-1500-C are obtained. Melting point 174qo Elemental analysis C59.65, day 6.
58, N5.02, CI6.51, C28th 37CIN
208 calculated value C59.52, day 6.60, N4.96,
CI6.27IR1715I670・1580 (skin-1
) UV (nm) 232 (32750), 244 (sh
30850) 252 (31650) 281 (5750
) 288 (5700) The properties of this product are consistent with maytansinol.

Claims (1)

[Claims] 1. General formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [In the formula, R is represented by ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ or ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼. ] Antibiotic C-15003. 2 Antibiotic C-15003-producing bacteria belonging to the genus Nocardia were cultured in a medium, and antibiotic C-1500 was added to the culture medium.
A method for producing antibiotic C-15003, which comprises producing and accumulating C-3 and collecting the same.