JPH10201494A - Production of dehydrorabelomycin and its use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain dehydroravelomycin having a cell proliferation-inhibiting action and useful as a chemotherapeutic agent against malignant tumors by culturing a dehydrorabelomycin-producing microorganism. SOLUTION: This method for producing dehydrorabelomycin comprises culturing a dehydrorabelomycin-producing microorganism belonging to the genus Streptomyces, such as Streptomyces s.p. NA 30664 strain (FERM P-15907), to produce and accumulate the dehydroravelomycin of the formula, and subsequently collecting the dehydroravelomycin from the culture solution. The NA 30664 strain is cultured in a culture medium containing water-soluble nutritive sources suitable for the usual culture of actinomyces in an aerobic condition at 20-37 deg.C.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing dehydrolaberomycin and its use. The compounds of the present invention, that is, dehydrolaberomycin and laveromycin have cytostatic effects and are expected as bioactive substances used as chemotherapeutic agents against malignant tumors.

[0002]

Conventionally, adriamycin, bleomycin, cisplatin, etoposide and the like have been known as chemotherapeutic agents for malignant tumors. In addition, it is disclosed that laveromycin having an antibacterial activity can be obtained by culturing Streptomyces olymbacium ATCC 21549, and that dehydrolaberomycin is produced by chemically dehydrating laveromycin. (JP-A-47-1344, USP
-3721684).

[0003]

SUMMARY OF THE INVENTION In view of problems such as side effects, drug resistance, and efficacy against solid cancers, the invention of new compounds suitable for these uses is awaited.

[0004]

The present inventors have conducted various searches for metabolites of microorganisms and found that one strain belonging to the genus Streptomyces produces a compound having a strong growth inhibitory effect on animal cells. This was identified as dehydrolaberomycin. The present invention has been completed based on the above findings. That is, the present invention provides the following formula, wherein a dehydrolaberomycin-producing bacterium belonging to the genus Streptomyces is cultured to produce and accumulate dehydrolaberomycin, and dehydrolaberomycin is collected from the culture solution.

[0005]

Embedded image And a process for producing dehydrolaberomycin represented by

[0006] The present invention further provides dehydroveromycin,
The present invention relates to an antitumor agent containing laveromycin or a pharmacologically acceptable salt thereof as an active ingredient.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION As a representative of the bacteria producing dehydrolaberomycin, NA strain 30664 (Seikoken No. 15907, FERM P-15907; hereinafter referred to as NA)
30664 strains). This strain is mutated by a conventional method such as irradiation, and among the mutant strains,
Better productivity strains can also be obtained. Below NA
The bacteriological properties of the 30664 strain are shown. 1. Morphological properties

[0008] 1. Morphological properties Observation after 2 weeks at 27 ° C. shows that the aerial hyphae are simply branched, the tip is helical, and no ring-shaped branches are formed. Neither sporangia nor zoospores are observed. Spore surface is smooth, spores are cylindrical, size 0.6 ~
0.8 × 0.9 to 1.2 μm. In addition, spores are formed in a chain of 10 or more.

[0009] 2. Physiological properties 1 Moderate growth range: 24 to 37 ° C. 2 Nitrate reduction: Negative 3 Starch hydrolysis: Positive 4 Coagulation of skim milk powder: Negative 5 Peptonization of skim milk powder: Positive 6 Generation of melanin-like pigment: Negative 3. Utilization of carbon source D-glucose: + L-arabinose: + D-xylose: + D-fructose: + sucrose:-L-rhamnose: + raffinose:-inositol: + D-mannitol: +

[0010] 4. Diaminopimelic acid in cell wall Diaminopimelic acid in cell wall is in LL form. In summary, this strain is a cell wall LL- diaminopimelic acid, the type of menaquinone primarily MK-9 (H _8),
It is MK-9 (H _6). According to the method of the International Streptomyces genus project (abbreviated as ISP), the spore-forming mycelium forms Section Spi.
spores, the spore surface is smooth and the color of the mature hypha is red (Red color-series),
It does not produce melanin-like pigments and the medium has a yellow tint. In addition, the color of the underlying mycelium exhibits a light yellow to light yellow brown color. As a carbon source, L-arabinose, D-xylose, D-glucose, D-fructose, inositol, L-rhamnose, D-mannitol are used, and sucrose and raffinose are not used.

[0011] Based on the above properties, edited by Earl Buffanand and NE Gibbons, Bergey's Manual of Detergent Bacteriology (Bergey's Manual of Defect Bacteriology)
erminative Bacteriology), 8th edition, 1974, and as a result of the search,
The A30664 strain was found to belong to the genus Streptomyces. Therefore, this strain was transformed into Streptomyces sp.
No. 4. The strain has been deposited with the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology as FERM P-15907.

The production of dehydrolaberomycin according to the present invention is carried out by fermentation with the above-mentioned strain NA30664. The cultivation of this strain is carried out at 20-37 ° C, preferably 20-30
It is performed under aerobic conditions in a medium containing a water-soluble nutrient suitable for cultivation of normal actinomycetes in a temperature range of ° C.

The medium to be used for the purpose of the main culture may contain nutrient sources that can be used by actinomycetes. The medium composition includes sugars such as glucose, fructose and sucrose; carbon sources such as starch and glycerin; Nitrogen sources such as organic nitrogen such as soybean flour and dried yeast or inorganic nitrogen such as ammonium sulfate and ammonium chloride can be used alone or in combination.

Further, sodium chloride, calcium carbonate,
Ferrous sulfate, magnesium sulfate, zinc sulfate, various vitamins, etc. can also be used for the purpose of promoting and regulating growth.If necessary, silicone, vegetable oil or synthetic antifoaming agent can be added to the medium to prevent foaming. It is possible. NA
Various methods can be used to preserve the 30664 strain, such as freeze-drying and subculturing to an agar slant medium.

When producing dehydrolaberomycin,
One platinum loop spore is scraped from the agar slant medium, transplanted into a shake flask, and cultured at about 25 to about 30 ° C. for 2 to 5 days to obtain a seed. Seed culture can be repeated according to the scale of the main culture to obtain a large amount of inoculum. During the main culture, a certain amount of the inoculum thus obtained was inoculated into a fermentation vessel, and about 2
Stir with aeration at 0 to 35 ° C, preferably about 25 to about 30 ° C, for 3 to 6 days.

The obtained culture solution is separated into cells and culture filtrate by means such as centrifugation or filtration, and the cells are extracted with a water-miscible solvent such as acetone or methanol. The organic solvent is removed from the eluate under reduced pressure. In this water suspension,
A target compound is extracted into an organic solvent layer by adding a solvent such as ethyl acetate or chloroform. If necessary, washing the organic solvent layer with a solution such as saturated saline is effective in removing water-soluble impurities. The extract is dehydrated with anhydrous sodium sulfate or the like, and then the solvent is distilled off, whereby an extract containing dehydrolaberomycin can be obtained. Further, in order to purify dehydrolaberomycin, ordinary means for purifying fat-soluble low-molecular substances can be applied. For example, silica gel chromatography using hexane-ethyl acetate as a developing solvent is performed, and the obtained active fraction is concentrated to dryness to isolate green crystalline dehydrolaberomycin.

The dehydrolaberomycin thus obtained has the following physicochemical properties. (1) Appearance: green crystal (2) Molecular weight: FAB-MS m / z 321 (M + H) ⁺ (3) Molecular formula: C ₁₉ H ₁₂ O ₅ (4) Solubility in solvent: soluble in dimethyl sulfoxide, chloroform, hexane Insoluble in water. (5) Color reaction: Positive for phosphomolybdic acid-sulfuric acid, iodine vapor reaction. (6) Rf value by thin-layer chromatography on silica gel: developing solvent of hexane: ethyl acetate (4: 1) shows 0.43. (7) ¹ H-NMR spectrum: FIG. 1 shows a spectrum measured in deuterated chloroform. (8) ¹³ C-NMR spectrum: FIG. 2 shows a spectrum measured in deuterated chloroform.

On the other hand, laveromycin can be produced and obtained using Streptomyces Olymbacius ATCC 21549 in accordance with the description in JP-A-47-1344 or US Pat. No. 3,721,684.

[0019]

[Action] Dehydrolaberomycin derived from cervical cancer HeL
It was examined antiproliferative activity against a-S ₃ cells. HeL
a-S ₃ cells were cultured at a ratio of 1.5 × 10 ⁴ cells / well to 96
The cells were seeded on a well test plate, cultured at 37 ° C. in a 5% CO ₂ incubator for 24 hours, and then the present compound was added to the culture at various concentrations. 72 hours later, MTT
The growth inhibition rate against HeLa-S ₃ cells of the present compounds was determined by the law. Growth inhibition rate (GI)% = (1-X / C) × 100 C: control absorbance, X: sample absorbance The results are shown in Table 1.

[0020]

[Table 1] The IC ₅₀ value of the dehydrolaberomycin's growth inhibitory effect on HeLa-S ₃ cells was 0.13 μg / ml.

When the compound of the present invention is used as an antitumor agent, various methods known in the art can be applied to formulation and administration. That is, injection, oral, rectal administration and the like can be used as an administration method. The preparation may take the form of injections, powders, granules, tablets, suppositories and the like. The compounds of the present application dehydrolaberomycin and laveromycin form salts with bases, and in the present invention, alkali metal salts such as sodium and potassium, alkaline earth metal salts such as calcium,
Further, a salt with an organic base such as triethylamino may be used.

As long as the compound of the present invention is not adversely affected at the time of formulation, various auxiliaries used in pharmaceuticals, that is, carriers and other auxiliaries, such as stabilizers, preservatives, soothing agents, and emulsifiers are required. Can be used accordingly. In the preparation, the content of the compound of the present invention or a salt thereof can be varied widely depending on the preparation form and the like, and is generally 0.01 to 100% (weight), preferably 0.1 to 70%.
% (By weight), with the balance consisting of carriers and other auxiliaries commonly used for pharmaceuticals.

The dose of the compound of the present invention or a salt thereof varies depending on the symptoms and the like, but is 0.01 to 8 per adult per day.
It is about 00 mg. If continuous throwing is required, it is desirable to reduce the daily usage. Hereinafter, production methods of the compound of the present invention will be described with reference to Examples.

[0024]

【Example】

Example 1 (1) Fermentation Soybean flour 1.5%, peptone 0.5%, yeast extract 0.0
5%, glycerin 2%, glucose 0.5%, magnesium sulfate 0.005%, potassium phosphate 0.05%,
100 ml of a medium containing 0.2% calcium carbonate is dispensed into a 500 ml Erlenmeyer flask, sterilized, and then Streptomyces sp. N
One loopful of A30664 strain was inoculated and cultured on a rotary shaker at 27 ° C. for 48 hours to obtain a seed culture for main culture.
The main culture was carried out in a 30 L jar fermenter with soybean flour 1%, yeast extract 1%, glucose 3%, calcium carbonate 0.5
%, Pronal (antifoaming agent) 0.03%
L, sterilized, and inoculated with 400 ml of inoculum at 27 ° C.
The culture was aerated at 6 liters per minute for 3 days while stirring at 250 rpm. 30L Jar Fermenter 2
The culture solution obtained from the culture was filtered using a filter press to separate the filtrate from the cells.

(2) Purification 1 10 L of methanol was added to the obtained cells, stirred for 2 hours, and filtered to obtain an extract. After methanol was distilled off from this extract under reduced pressure, the resulting aqueous solution was extracted with ethyl acetate. The extract was dried over anhydrous sodium sulfate and concentrated to dryness under reduced pressure to obtain 3.8 g of an oily substance. The obtained oil was purified by silica gel column chromatography (column volume: 200 ml) using hexane-ethyl acetate (10: 1) as an eluent.
The residue was concentrated to dryness under reduced pressure to obtain 1.8 g of a crude substance. This crude substance was dissolved in a small amount of a mixed solution of chloroform-methanol and subjected to gel filtration column chromatography using Sephadex LH-20 (manufactured by Pharmacia). The collected eluate was concentrated to dryness using an evaporator, and finally crystallized in chloroform-methanol to obtain 68 mg of green crystals. The physicochemical properties of this crystal were measured. The measured values are as described above.

[0026]

As described above, since the compound of the present invention has a cell growth inhibitory action, it can be used as an antitumor agent.

[Brief description of the drawings]

FIG. 1. Dehydrolaberomycin in deuterochloroform.
¹ shows a ¹ H-NMR spectrum.

FIG. 2. Dehydrolaberomycin in deuterochloroform.
¹ shows a ¹³ C-NMR spectrum.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI (C12N 1/20 C12R 1: 465)

Claims (4)

[Claims] 1. A dehydrolaberomycin-producing bacterium belonging to the genus Streptomyces is cultured to produce and accumulate dehydrolaberomycin, and the dehydrolaberomycin is collected from the culture solution. A method for producing dehydrolaberomycin represented by the formula: 2. An antitumor agent comprising dehydrolaberomycin, laveromycin or a pharmaceutically acceptable salt thereof as an active ingredient. 3. A dehydrolaberomycin-producing bacterium belonging to the genus Streptomyces. 4. Streptomyces sp. NA 30
664 strains or mutants thereof.