JPH0436276A - Antibiotic ma-638-2-b, its production and antitumor agent comprising antibiotic ma-638-2-b as active ingredient - Google Patents

Abstract

NEW MATERIAL:An antibiotic MA-638-2-B shown by the formula. Properties: neutral white powder. Specific rotatory power: [alpha] -322.7 deg. (C=0.022g/ml CH3OH); Molecular formula: C13H14O5: molecular weight: m/z 250. Elemental analysis: C 62.35%, H 5.59%, O 32.06%. Melting point: 128-130 deg.C. Solubility: readily soluble in ethyl acetate, acetone or methanol and slightly soluble in water. Color reaction: positive in iodine gas and KMnO4 reagent. USE:An antitumor agent showing antibacterial activity against Gram-positive and Gram-negative bacteria and having low toxicity. PREPARATION:A fungus belonging to the genus Fusarium, capable of producing antibiotic MA-638-2-B (FERM-P 11,480) is cultured under an aerobic condition in a nutritive medium, and antibiotic MA-638-2-B is obtained by extraction from the filtrate, followed by purification.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel antibiotic, a method for producing the same, and an antitumor agent containing the same as an active ingredient.

[Conventional technology]

Various antibiotics have been discovered and developed over the years, but
BACKGROUND OF THE INVENTION A problem with antibiotics is that continued use can cause bacteria to develop resistance to the antibiotics used. Also, cancer (
With continued use of antibiotics, tumors such as cancer can develop resistance to the antibiotics being used.

[Problem to be solved by the invention]

Therefore, the emergence of new antibiotics is always desired.

In particular, the emergence of new antibiotics with antitumor activity and less toxicity is desired.

Therefore, the present invention aims to provide a novel antibiotic with antitumor activity and less toxicity.

[Means to solve the problem]

The inventors have discovered that Fusarium MA
-638-2 produces a novel antibiotic MA-6382-B, which has antitumor activity and antibacterial activity against Durum-positive bacteria and Durum-negative bacteria, and is less toxic, This led to the completion of the present invention.

The above Fusarium MA-638-2 is a microorganism discovered by the present inventors in a soil sample collected in Takatsuki City, Osaka Prefecture, and has been deposited with the Microbial Industrial Laboratory, Agency of Industrial Science and Technology, Ministry of International Trade and Industry. The number is 1148
It is No. 0.

The Fusarium MA-638-2 strain that produces the antibiotic MA-638-2-B of the present invention has the following mycological properties.

(1) Quantitative culture of Fusarium MA-638 is usually carried out at 30°C.
The morphological characteristics of the two strains are that they form aerial hyphae and spores.

(2) The growth condition on the standing wheat germ extract agar medium or starch (starch) yeast extract medium is good, with white aerial hyphae forming in a felt shape and purple aerial hyphae growing in a felt shape. . Produces soluble red pigment.

(3) Note 111 侃1! First, we investigated the physiological properties of Fusarium MA, -638-2 strain.
Table 2 shows the assimilability of carbon sources.

Table 1 +: Utilized + 10: Used considerably The mycological properties of Fusarium MA-638-2 strain are as described above, and natural and genetic engineering methods for this Fusarium MA-638-2 strain Artificial mutant strains including 1jMA-638-2-B can also be produced, and these can also be used as producing strains of antibiotic 1jMA-638-2B.

In the present invention, in producing antibiotic MA-638-2B, the culture of antibiotic MA-638-2B producing bacteria is carried out by
- It is carried out according to the culture method for mold (mold).

This will be explained in detail as follows.

The antibiotic MA-638-2-B of the present invention is produced by using antibiotic MA-638-2B-producing bacteria belonging to the genus Fusarium, such as Fusarium MA-638-2 strain, as a carbon source and nitrogen that can be utilized under aerobic conditions. produced by growing in a nutrient medium containing sources. As a specific means, it is preferable to use shaking culture, deep culture, or aeration agitation culture in a liquid medium. It is preferable to inoculate the suspension or culture solution into a medium and perform aeration and agitation culture.

The nutrient source of the medium is not particularly limited, and carbon sources, nitrogen sources, and others commonly used for culturing microorganisms can be contained in the medium and used as nutrient sources. As a medium component, for example, glucose, galactose, sucrose, maltose, lactose, raffinose, inositol, mannitol, molasses, glycerin, dextrin, starch, soybean oil, cottonseed oil, etc. are used as a carbon source, and glucose and starch are particularly used. preferable. Examples of nitrogen sources include soybean flour, peanut flour, cottonseed oil, fishmeal, cornstarch liquor, peptone,
Meat extract, yeast, yeast extract, malt, rice bran, bran,
Asparagine, sodium nitrate, ammonium nitrate, ammonium sulfate, etc. are used, and soybean flour and peptone are particularly preferred.Inorganic salts include common salt, phosphate, calcium carbonate, calcium chloride, trace metal salts (for example, manganese, zinc, copper, molybdenum, iron, boron, cobalt,
V:L salts, phosphates, hydrochlorides, nitrates, etc. of selenium, vanadium, iodine, etc.) are added as appropriate. For liquid culture, silicone oil, vegetable oil, animal oil, mineral oil, liquid paraffin, surfactant, etc. are appropriately used as antifoaming agents.

The pH of the medium is preferably neutral to slightly acidic, particularly around pH 5.5, and the culture temperature is preferably 25 to 33°C, especially around 30°C.

Antibiotic MA638-2 produced during the course of culture
Changes in the titer of -B over time can be determined using a paper disk assay method using Bacillus subtilis as the test bacterium [for example, using a 6-layer filter paper Th1nC thin type manufactured by Toyo Roshi Co., Ltd. or Advantesoku Toyo Co., Ltd.]. can be measured. Usually 72
Antibiotic WMA-638-2-B with ~216 hours of culture
(hereinafter simply referred to as rMA-638-2-BJ) reaches its highest level.

After culturing, MA-638-2-B is produced by removing bacterial cells and other solid parts by filtration using diatomaceous earth as a filter aid or by centrifugation, and extracting and purifying the filtrate or supernatant. can get.

In addition, by utilizing the physicochemical properties of MA-638-2-B, for example, MA-638-2-B can be prepared using an adsorbent.
2-B can be collected.

As the occlusal agent, for example, activated carbon or adsorption resin Amberlite XAD-2, XAD4, XAD-
7 etc. (all manufactured by Rohm and Haas) or Diaion HPIO1HP20, HP20AG, HP
50 (both manufactured by Mitsubishi Chemical Industries, Ltd.) are used.

When using such an adsorbent, MA-6382-B is prepared by passing a liquid containing MA-638-2-B through a layer of the above-mentioned adsorbent to remove impurities contained in the liquid containing MA-638-2B. or MA-638-2
- After adsorbing B, methanol water, acetone water, n-
Obtained by elution using butanol water or the like.

In addition, MA-638-2-B can be obtained by collecting neutral fat-soluble substances from culture fluid using an organic solvent such as chloroform, ethyl acetate, or n-phthanol, which is immiscible with water. It is also possible to extract B from the culture filtrate or aqueous solution. Furthermore, antibiotic MA
By washing the organic solvent layer containing -6382-B with dilute alkaline water, dilute acidic water, etc., it is also possible to remove mixed acidic properties or basic substances.

In purifying the MA-638-2-B obtained in this manner, for example, partition column chromatography using cellulose such as Avicel (manufactured by Asahi Kasei Corporation) or Sephadex LH-20 (manufactured by Pharmasoa), Adsorption column chromatography using a carrier such as silica gel, alumina, or florisil, reverse phase column chromatography using a reverse phase carrier, or MA-6
An extraction method that utilizes the difference in the distribution ratio of 38-2-B and mixed impurities to the solvent, or a countercurrent distribution method, etc. is adopted as a purification method, and these purification methods are used alone or in combination as appropriate, and if necessary, it is repeated. By using it as M
A-638-2-B can be purified.

Furthermore, like general fat-soluble antibiotics, MA-638-2-B exists in the bacterial cell portion of the culture solution depending on the culture conditions. In this case, extract using a hydrophilic organic solvent such as alcohol or acetone, remove the solvent from the extract, and
After making it into an aqueous solution, it can be extracted and purified in the same manner as from the culture filtrate.

MA-638-2-B thus obtained has the following physicochemical and biological properties.

(1) Properties of the substance Neutral white powder (2) Specific rotation [α] ”,'-322,7°C= 0.022g/〆 CH,0H (3) Molecular formula C1s H+ 405 (4) Molecular weight
m/z 250 (5) Ultraviolet absorption spectrum in methyl alcohol (concentration of MA-638-2B 70.
The ultraviolet absorption spectrum measured at
As shown in the figure, 214.5 nm (t = 8300 f
/mol cm).

(6) Infrared absorption spectrum The infrared absorption spectrum measured by the KBrBr method is the second
As shown in the figure.

(7) Nuclear magnetic resonance (NMR) spectrum CDC+, measured in liquid i using tetramethylsilane as a reference substance.
The 'H-nuclear magnetic resonance vector is shown in FIG. 3, and the '3c-nuclear magnetic resonance vector is shown in FIG. 4. The chemical shift values in FIG. 4 are as follows.

+3cm ~ North Spectrum δ (ppm) 18.604 29.450 29.733 30.017 30, 300 30.5B3 30, 867 31, 150 41.510 62; 717 69, 975 73, 078 87.904 127, 363 12B, 226 132, 543 149, 865 159, 564 172, 542 197, 742 206, 902 207, 091 (8) Elemental analysis Carbon 62.35% Hydrogen 5.59% Oxygen 32.06% (9) Melting point 128~ 130°C Oω Solubility Easy to ethyl acetate, acetone, methanol 'R1 Slightly soluble in water tm Color reaction Positive for ioso gas, positive for KMnO, reagent 02) Thin layer chromatography adsorbent Silica gel developing solvent system Benzene: Acetone-2 :lRf=0.
63 Side antibacterial spectrum Bacillus subtilis Ba5illus 5ubt as a Durham-positive bacterium
ilis and Escherichia coli as a Durham-negative bacterium.
richia coli and MA- against them.
The antibacterial spectrum of 638-2-B was measured by the agardilution method. The results are shown in Table 3.

Table (b) Antitumor Effect The therapeutic effect of MA-638-2-B on P388 leukemia in mice was tested by the following method. The test method is as shown below, and the results are as shown in Table 4.

Six mice were used for each dose, and the antitumor activity in Table 4 is based on the average survival days (C
) to the median survival days (T) of the treatment group was expressed as a percentage. In addition, the number of survivors in Table 4 is expressed in such a manner that the number of mice used is the denominator and the number of surviving mice is the numerator, so that the number of survivors in the mice used can be easily understood.

P388 subcultured in DBA/2 mice was intraperitoneally cultured in six BDF + mice (female, 5 weeks old, Nippon Talea).
24 hours after inoculating XIO' leukemia cells,
MA-638-2-B was administered intraperitoneally once a day for 5 consecutive days, a total of 5 times.

Table 4 Antitumor activity (T/C) is generally considered to have antitumor activity if it is 125 to 130, but as shown in Table 4, MA-638-2-B has antitumor activity. (T/C) exceeds the above value, and it is recognized that it has antitumor activity.

In addition, as some of the test mice were observed to survive for 30 days or more, we hope to further develop MA638-2-B's antitumor activity by conducting further research on the purification method and usage method. You can expect that it will be possible.

0ω acutely toxic BDF, LD when administered intraperitoneally to mice (♀, 5 weeks old, Nippon Talea). was 200a/kg, and it can be judged that the toxicity is low.

By analyzing the above data (specific optical rotation value, molecular formula, molecular weight, elemental analysis value, etc.), confirmation of the presence of double bonds such as carbonyl groups by ultraviolet absorption spectrum, analogy of functional groups by infrared absorption spectrum, nuclear magnetic resonance The planar structure of MA-638-2-B was determined to be as follows. Also, as a result of comparing this with known substances, M
A-638-2-B was determined to be a new substance.

Next, an antitumor agent containing MA-638-2-B as an active ingredient will be described.

The antitumor agent containing MA-638-2-B as an active ingredient can be administered either orally or parenterally.

When administered orally, it can be used in the form of soft or hard capsules, tablets, granules, fine granules, powders, enteric coated agents, or suspensions. For parenteral administration, injections, drips,
It can be used as a halved product, an emulsion, or a suspension, and it can also be used in a dosage form that maintains mucosal or transdermal absorption as an ointment, cream, liquid, poultice, or tape.

Cleavage of the antitumor agent containing MA-638-2-B as an active ingredient can be carried out as appropriate using medically acceptable surfactants, excipients, lubricants, adjuvants, etc. It is possible to obtain cleavage of solids, semi-solids, liquids, etc. Additionally, stabilizers, colorants, fragrances, and the like may be used. The amount of MA-638-2-B in the preparation may be appropriately determined depending on the target tumor, usage method, etc.

When using this antitumor agent containing MA-638-2-B as an active ingredient in humans, it is desirable to use it by intravenous, intramuscular, oral, or transdermal administration.
The dose may vary as long as it is sufficient to treat the target tumor, and may vary slightly depending on the tumor symptoms, route of administration, dosage form, etc. Considering the value, in general, MA-638-2-B is 0.000 per day when administered orally.
1-200wg/kg body weight range, especially the upper limit is about 50t
g/kg body weight is preferable, and in the case of parenteral administration, MA-638-2-B is 0.0001 g/kg body weight per day.
~100■/kg body weight range, especially the upper limit is about 5a/kg
It is preferable to keep it at about 1000 mph.

〔Example〕

Next, a production example of MA-638-2-B will be shown as an example. It should be noted that the example is merely provided for the purpose of illustrating the present invention, and is not intended to limit the scope of the present invention. .

Example 1 Fusarium MA-638-2 strain was inoculated into medium 101 shown in the following medium composition-1, and cultured with shaking at 30°C for 5 days.

Soluble starch 30g soybean flour
5g KHt P Oa 1 gM g
S Oa ・7HtOIg NaCI 0.5gCa
C1t-2H,OO, 5g Yeast extract 0.5gFeCl+
・6Hz O2■ Zn5j) a ・IHz 0 3 g Distilled water 1,000 d After the culture is completed, the culture solution is centrifuged and the supernatant portion (liquid portion) obtained is 10!
61 ethyl acetate was added to the solution and extracted three times.

On the other hand, 6N methyl alcohol was added to the bacterial cell portion (solid content side portion), stirred, and then filtered. After concentrating the methyl alcohol of this filtrate under reduced pressure, 61% of water was added to the residue, and 61! , and extracted three times with ethyl acetate.

Combine the obtained extract with the extract from the supernatant portion,
The solvent was concentrated under reduced pressure, and benzene was added to the obtained oily residue to remove insoluble materials, followed by silica gel column chromatography.

That is, the above benzene solution was filled with silica gel (manufactured by Wako Pure Chemical Industries, Ltd.) 3CIX 45C1.
1 column, elute with benzene:acetone-95:5, collect both fractions containing MA-638-2-B,
MA638-2-B 2g by vacuum concentration
I got it.

The physicochemical and biological properties of MA-638-2-B obtained in Example 1 are as follows.
It was consistent with the physicochemical and biological properties of

Example 2 Fusarium MA-638-2 strain was inoculated into medium 101 shown in medium composition-2, and cultured with shaking at 30''C for 5 days.

Culture medium 24 glucose 30 g peptone
2g KHzPOa IgM g SO
a ・IHzOIg NaCl O, 5gCaCL
-2Hz OO, 5g Yeast extract 0.5gFeCl5
・6Hz O2 [ Zn5Oa ・7Hz 0 3 g
Distilled water 1,000 m After culturing, MA-638-2-8 was prepared in the same manner as in Example 1.
1.9 g was obtained.

The physicochemical and biological properties of MA-638-2-B obtained in Example 2 were also
It was consistent with the physicochemical and biological properties of

〔Effect of the invention〕

As explained above, the antibiotic MA-638-
2-B is a new substance, has antitumor activity, and has low toxicity.

[Brief explanation of the drawing]

Figure 1 shows the ultraviolet absorption spectrum of antibiotic MA-638-2-B (in methyl alcohol solution). Figure 2 shows the infrared absorption spectrum of antibiotic MA-638-2-B (KBr tablet sharpener 1). Figure 3 shows antibiotic MA-638.
-2-B'H-Nuclear Magnetic Resonance vector, and Figure 4 shows the antibiotic MA-638-2. 120-nuclear magnetic resonance spectrum of B

Claims (5)

[Claims] (1) Antibiotic MA-6 represented by the following planar structural formula
38-2-B. ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ (2) Antibiotic MA-638-2 belonging to the Fusarium genus
-Culture B-producing bacteria, and use the culture to produce antibiotic MA-6.
A method for producing the antibiotic MA-638-2-, which comprises separating and collecting MA-38-2-B. (3) Antibiotic M according to claim 2, wherein the antibiotic MA-638-2-B producing bacteria is Fusarium MA-638-2 strain.
Method for manufacturing A-638-2-B. (4) A method for producing antibiotic MA-638-2-B, wherein the antibiotic MA-638-2-B-producing bacterium is a mutant strain of Fusarium MA-638-2 strain obtained by a conventional method. (5) An antitumor agent characterized by containing antibiotic MA-638-2-B as an active ingredient.