JPS63211275A - Novel antibiotic 55-b - Google Patents

Abstract

NEW MATERIAL:Novel antibiotic 55-B shown by the formula. USE:An antibiotic having antitumor action. PREPARATION:Myrothecium verrucaria strain (FERM P-8880) is cultivated and 55-B is collected from the culture mixture to give 55-B. 55-B has the following chemical and physical properties. Melting point: 173-175 deg.C. Specific rotatory power [alpha]D<20> (C=0.35, methanol). Solubility: soluble in chloroform, ethyl acetate, acetone and methanol, slightly soluble in benzene and insoluble in water and n-hexane. Color reaction: positive in 2,4-diphenylhydrazine reagent and negative in FeCl3, indophenol reagent.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel antibiotic 55-B and a method for producing the same.

The present inventors succeeded in isolating a novel antibiotic from the culture solution of Myrothecium bercariae strain 55, and named this substance 55-B.

55-B has the following physical and chemical properties.

1) Properties of substance Melting point 173-175°C 2) Specific optical rotation [α) 20 = -13.7°C (C = 0.35. MeOH
) 3) Molecular formula CzoH220e 4) Molecular weight 358 5) Ultraviolet absorption spectrum The ultraviolet absorption spectrum measured in methanol is the first
As shown in the figure, 246rv (ε-8370)
shows maximum absorption.

6) Infrared absorption spectrum The infrared absorption spectrum measured by the KBrBr method is as shown in FIG.

7) Nuclear Magnetic Resonance Spectrum Measurement was carried out in a CDα3 solution using tetramethylsilane as a reference substance.

The main peaks of the If(-N?fR spectrum are shown in Table 3, and the main peaks of the I'C-NMR spectrum are shown in Table 4.

Table 3 IH-NMR δ 0.039 4.842 0, 124 4.971 0, 214 5.035 0, 659 5.084 0, 7 1 0 5.4470.742
5.565 0,793 5.638 1,3 44 5.757 1.425 7.014 1,557 7.027 1,628 7.227 1.686 1,76 2 1.789 2,296 2,45 5 2, 5 40 2.553 2, 69 9 2, 7 0 9 3, 1 4 1 3, 1 48 3, 2 65 4, 77 6 Table 4 Large-NMR δ 7.7' CHzl 6, 3
CH 8, 8 C11224, 9C 27, 2CH3 28, 5CH2 28, 6CHs 42, I C 42, 4G 45, 6 CH 52, 4CH2 74, 6C0 98, 50-C-0 115, 8CH2 1・34.7 C 140,3CH 146,3C1 181,20-C-0 192, OC=0 208.6 C-0 8) Solubility Soluble in chloroform, ethyl acetate, acetone, methanol.

Slightly soluble in benzene, insoluble in n-hexane 9) Color reaction 2. Positive for 4-dinitrophenylhydrazine reagent Peα
3. Negative for indophenol reagent 10) Thin layer chromatography adsorbent Silica gel developing solvent system Rf Benzene: Acetone: Methanol - 85: 10: 5 0.53 Benzene: Ethyl acetate = 75: 25 0.
35 hexane: acetone = 70: 30
0.35 chloroform:acetone-90: 10
By analyzing data of 0.30 or more, the planar structure of 55-B was determined as shown below.

Jf1 55-B is derived from Myrothecium bercariae 5 according to the present invention.
It is produced by culturing 55-B (Keikoken Bacteria No. 8880) and collecting 55-B from the culture.

The Myrothecium bella strain 55 that produces the novel antibiotic 55-B of the present invention has the following mycological properties.

Cultivation is usually carried out at 30°C. Myrothecium belcalia 5
The morphological characteristics of the five strains are as follows: Hyphae are colorless, smooth, thin-walled, and rarely branched.

Conidia are lemon-shaped and collect as black sticky masses when cultured on slants.

Conidia cluster together in the form of line-colored droplets.

The conidiophores are closely attached to each other. Its terminal end gives rise to three 2° branches.

When cultured on slants, the mycelium becomes woolly. Light brown with a whitish tinge,
Yellow conidiating structures are scattered or clustered in black conidial fawns with white margins.

The culture in the present invention is carried out in accordance with the culture method for general fungi, and is preferably carried out by shaking culture or aerated agitation culture in a liquid medium.The medium components include, for example, glucose, galactose, sucrose as a carbon source,
Maltose, lactose, raffinose, inositol, mannitol.

Carbohydrate, glycerin, dextrin, starch, soybean oil.

Cottonseed oil and the like are particularly preferred, as glucose and nitrogen sources, soybean flour, peanut flour, cottonseed flour, Farmamine, fishmeal,
Corn steep liquor, peptone.

Meat extract, yeast, toast extract, asparagine,
Sodium nitrate, ammonium nitrate, ammonium sulfate, etc., and inorganic salts such as common salt, phosphate, calcium carbonate, calcium chloride, trace metal salts, etc. are added as appropriate. For liquid culture, use silicone oil, vegetable oil,
A surfactant or the like is appropriately used as an antifoaming agent.

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

Changes over time in the titer of 55-B produced over the course of culture were measured using paper discs (manufactured by Toyo Kagaku Sangyo ■, diameter FJ m) with Staphylococcus aureus as the test bacterium.
Th1ck) assay method.

The production amount of 55-B usually reaches its maximum value after 72 to 216 hours of culture.

55-B can be obtained by removing bacterial cells and other solid parts after the completion of culture by a filtration operation using diatomaceous earth or the like as a filtration aid or by centrifugation, and then extracting and purifying from the filtration or supernatant. .

55-B can be collected by utilizing its physicochemical properties, for example, using an adsorbent. Examples of adsorbents include activated carbon, adsorption resins such as Amberlite XAD-2, XAD-4, and XAD-7 (manufactured by Rohm and Haas), or Diaion HPIO and HP2.
0. IP20AG, HP50, etc. (manufactured by Mitsubishi Chemical Industries, Ltd.) are used.

55-B can be obtained by passing a liquid containing 55B through a layer of adsorbent as described above to adsorb and remove impurities contained in the liquid containing 55-B, or by adsorbing 55-B and then adding methanol water, It can be obtained by elution using acetone water, n-butanol water, etc.

Neutral fat-soluble substances can also be extracted from the culture medium or from the aqueous solution using organic solvents that are immiscible with water, such as chloroform, ethyl acetate, n-butanol, alone or in combination. It is possible. Furthermore, it is also possible to remove mixed acidic or basic substances by washing the organic solvent layer containing 55-B with dilute alkaline water, dilute acidic water, or the like.

In order to purify the 55-B obtained in this way, partition column chromatography using cellulose such as Avicel (manufactured by Asahi Kasei Corporation) or Sephadex LH-20 (manufactured by Pharmacia), silica gel, alumina, etc. Adsorption column chromatography using supports such as Florisil.

Effective methods include reversed-phase column chromatography using a reversed-phase carrier, an extraction method that utilizes the difference in the distribution ratio of 55-B and mixed impurities to the solvent, or a countercurrent distribution method.

55-B can be purified by repeatedly using the above purification means alone or in appropriate combinations.

Alternatively, 55-B, like general lipid-soluble antibiotics, exists in the bacterial cell portion of the culture solution depending on the culture conditions. In this case, alcohol.

It can be extracted using a hydrophilic organic solvent such as acetone, the solvent is removed from the extract, and then the extract is made into an aqueous solution, followed by extraction and purification in the same manner as in the culture.

55-B has antibacterial activity against certain types of bacteria, and has been shown to have an antitumor effect, with a life-prolonging effect on Ehrlich's ascites cancer using mice.

1) Antibacterial Spectrum The minimum inhibitory concentration of 55-B against general Durham-positive and Durham-negative bacteria was measured by the agardilution method. The results are shown in Table 7.

Table 7 Aspergillus niger IFO44161
100Pen1cilliu chrysogenum
IFO489750Pusarium oxyspo
run+ IFO5880100Sa1005ach
aro cerevisiae 5QCand
ida albicans 50Stap
hylococcus aureus IFO3060
100Bacillus 5ubtilis IFO1
2210100M1crococcus roseus
IFO376850Escherichia col
t K-121FO3301> 200Pseudom
onas putida IFO3738> 200S
erratia marcescens IFO126
48> 20OAlkaligenes faecal
is IFO12669> 200Arthrobac
ter globiformis IFO121401
100 Corynebacterium xerosis
IFQ 12684 100 Bacillus br
evis IFO33311002) Antitumor Effect The therapeutic effect of 55-B on Ehrlich carcinoma in mice was tested by the following method. The results are shown in Table 8.

In addition, the antitumor activity in the table is the average survival days of the untreated group (C)
The ratio of mean survival days (T) of the treatment group to that of the treatment group is shown as a percentage.

Test method 2X'IO'' tumor cells were injected into ICR mice (♀.

After 24 hours, 55-B was intraperitoneally administered once a day for a total of 7 times.

Table 8 0.023 328 5/6 0.015 230 4/6 0.008 230 4/6 The present invention will be explained in more detail with reference to Examples below.

Example 55-B 55 strains of Myrothecium bercariae were added to medium 5E shown in medium composition-1.Glucose 30g Asparagine 2g KH2PO41g MgSO4・7H201, g Naci! ' 0.5g CaC1z ・2HzO0,5g 4
゜Yeast extract 0.5g Fe(j!2・6H202r@ZnSO4・2H203rrxr Distilled water 1000ml) Inoculated and cultured with shaking at 30°C for 6 days.After the culture was completed, the culture solution was centrifuged and the resulting oral information 51 to 31 Add ethyl acetate and extract three times.

On the other hand, methyl alcohol 3I for bacterial cells! Add and stir, then sip. After concentrating the methyl alcohol of this extract under reduced pressure, the residue contains 3I of water! was added, and the fish was extracted three times with 311 ethyl acetate. This was combined with the extract from the oral presentation mentioned above, the solvent was concentrated under reduced pressure, and the resulting oily residue was mixed with benzene 10-
was added to remove impurities, and then subjected to silica gel column chromatography. The above benzene solution was passed through a 1 cm x 75 cm O column of silica gel (manufactured by Wako Pure Chemical Industries, Ltd.) filled with benzene in advance, and the benzene:acetone-95;
5 was eluted, and both fractions containing 55-B were collected and concentrated under reduced pressure to obtain 55-B65nw.

[Brief explanation of the drawing]

FIG. 1 shows the ultraviolet absorption spectrum of the novel antibiotic 55-B. FIG. 2 shows the infrared absorption spectrum of the novel antibiotic 55-B. Procedural amendment June 24, 1988 1, Indication of case, 1988 Patent Application No. 046273 2, Title of invention 4, Spontaneity by agent 6, Number of inventions increased by amendment None 7, Invention subject to amendment As shown in Detailed Explanation Column 8, Contents of Amendment Attachment, Contents of Amendment 1, Structural formula on page 6 of the specification is amended as follows. 2. On page 7, line 16, "toast extract" is corrected to "yeast extract."

Claims (2)

[Claims] (1) Novel antibiotic 55-B. (2) A method for producing novel antibiotic 55-B, which comprises culturing Myrothecium bercariae 55 strain and collecting novel antibiotic 55-B from the culture.