JPS5859996A - Novel aureolic acid antibiotic substance and its preparation - Google Patents

Abstract

NEW MATERIAL:A compond of the formula (R' is H or methyl; R<2> is acetyl or isobutyryl). USE:An antimicrobial and antitumor agent against Gram-positive bacteria. PROCESS:A microorganism, belonging to the genus Streptomyces, and having the ability to produce an aureolic acid antibiotic substance, e.g. Streptomyces aburaviensis PA-39856 (FERM-P No.6129), is cultivated in a culture medium preferably at 5.5-8.5pH and 25-32 deg.C for 2-6 days.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a novel aureolic acid (Aureolic acid).
cid) antibiotic, its production method, and a new strain of Streptomyces arabiensis that produces the antibiotic.

The novel aureolic acid antibiotic provided by the present invention is
It is obtained by separating and collecting from a culture obtained by culturing the antibiotic-producing strain of the present invention belonging to the genus Streptomyces in a medium, and has the following structural formula.

(In the formula, R/ represents hydrogen or methyl, and RJ represents acetyl or isobutyryl, respectively.) The antibiotics of the present invention include known aureolic acid antibiotics, Chromomycin A2 and Olivomycin. in) A,
It is produced in the culture medium together with B. However, this antibiotic has single element analysis values, melting point, specific rotation, infrared absorption spectrum, and H-FT nuclear magnetic resonance spectrum.

Constituent Fatty Acid 1 The corrected values in thin-layer chromatography using various solvent systems and analytical retention times in high-performance liquid chromatography are known for chromomycin and SynA, which are known aureolic acid antibiotics.

~, ~, AP (Special Public Showa θ-/ll!;9/, Special Public Show 33-71'l-2,'Special Public Show 3!;-72397
, JP-A No. 3; 2-102202, etc.), Olivomain (
A.

B, C, D (British patent 1/327g and others), Mithramycin A,, B, C
(U.S. Patent No. 3.906.093, etc.), dielbesidin (
Gel be -1-cidine) (U.S. Patent 3
．． j j /: Otsu/others), BP-76,97g (
French Patent No. 11I33, 3θ3, etc.), Variamycin (Special Publication No. 11
t9-'I θ711 and others), and tidrimycin (
It is a new nooleolic acid antibiotic that is significantly different from Citrimycin (Japanese Patent Application Laid-Open No. 5-3393, et al.). 'H-F'T nucleus [The major structural features of the antibiotic of the present invention obtained from analysis results by resonance spectroscopy and gas chromatography are that it has 7 or 2 acetic acids as esters in the molecule, and It has only one methoxyl group. The following Table 1 shows a structural comparison with the main known aureolic acid antibiotics. (The numbers indicate the number of substituents) (The following is a blank space) The novel aureolic acid antibiotic of the present invention is Streptomyces arabiensis PA-39136 (Streptomyces arabiensis PA-39136)
eptomyees aburaviensis PA
-39za 56). This strain is /971
It is an actinomycete that was isolated from a soil sample in June, 2013.

From the results of taxonomic examination, Streptomyces aburabiensis (Streptomyces aburabiensis)
nais) (ATCC231rls9). This strain has been deposited with the Institute of Microbial Technology, Yatabe-cho, Tsukuba-gun, Ibaraki Prefecture, since May 2, 1981, as Microtechnology Research Institute Bacteria Collection No. 6/29. The mycological properties are shown below.

(a) Morphological properties (21'C on Pennet's agar medium)
/ 1 day culture) Sporangia, flagellated spores, and sclerotia were all observed, and no division due to fragmentation was observed in the basal hyphae. One strain showed good growth on this medium,
In addition, formation of aerial mycelia and settlement of spores are also good. Spore-bearing hyphae grow on aerial hyphae, branch simply, and have a straight shape.

The surface structure of the spores observed by electron microscopy is smooth.

(Left below) (c) Growth temperature (Culture for 2 weeks on Benne, Moon, agar medium T.

However, only IO"C was cultured until 3 weeks later) IO"
C Almost no growth at 21°C, good formation of aerial mycelia at 37°C.
Good growth is observed, but formation of aerial hyphae is not observed, and no growth occurs above t'c (d) Physiological properties (cultured at 2°C/4' days) Melanoid pigment producing ability Negative tyrosinase Reaction Negative Coagulation of milk Negative Peptonization of milk Positive Liquefaction ability of gelatin Positive Ice-melting ability of starch Positive (e) Availability of carbon source Carbon source with good growth D- Carbon source with no glucose growth L-arabinose, D-xylose, D-fructose, sucrose, inocyto-J.

From the properties of L-rhamnose, raffinose, and D-mannitol, it is clear that this strain belongs to the genus Streptonus.

The Actinomycetes by Waxman
inomicetss) Volume 2 (/9 o°/year), shearing and Gottlieb glue, S, P, (Int
ernationalStreptomyces Pr
project) report [International Journal
Of Systematic Bacteriology (In't
ernational Journal orSyst
matic Bacteriology) Volume 1I 6
9 pages.

279 pages (/96 J'), Volume 79, page 39 (79
22, p. 26j (7972)] and Bergey's Manual of Determinants to Bacteriology.
f Determinative Bacteriol
When searching for closely related species of strain PA-39136 in the literature on new species of actinomycetes, Streptomyces arabinose (St.
reptomyces aburabiensis)
(ATCC23L!r Otsu 9) C International Journal of Systematic Bacteriology Volume IV, Copy Page (796 years), Bird's Manual of Determinative Bacteriology G Edition, 76 /page (/97'1) and Journal of Antibiotics (Journal o
fAntibiotics) Volume 1O, 2θ! Page</9>
! ;7 years)] is listed as the most closely related species. This strain is Streptoices arabiensis (Str.
As a result of a simultaneous comparison experiment with a standard strain of (Eptomycas aburaviensig), differences were observed in milk coagulation, but other properties were in very good agreement. Based on the above findings, this strain was selected.

New strain Streptomyces arabiensis PA-3
It was named this time as 9za56.

In the present invention, Streptomyces arabiensis PA-3 water jr31. Not only natural and artificial mutant strains of the strain, but also all strains belonging to the genus Streptonus that produce the novel aureolic acid antibiotic of the present invention can be used and are included in the present invention.

Next, a method for producing the novel aureolic acid antibiotic provided by the present invention will be described. The present invention is carried out by culturing the above-mentioned strain producing the antibiotic of the present invention in a nutrient medium under aerobic conditions, and separating and collecting the antibiotic of the present invention from the culture.

In principle, cultivation may be carried out according to general microbial culture methods, but deep culture using a liquid medium is advantageous. As a general rule, the medium is a carbon source and a nitrogen source.

Including inorganic salts. Also, vitamins as needed,
Precursor substances etc. may also be added. Examples of carbon sources include glucose, starch, and dextrin.

Sucrose, glycerin, molasses, organic acids, etc. are used alone or in mixtures. Examples of nitrogen sources include soybean flour and cornstiny liquor.

Meat extract, yeast extract, cottonseed flour, peptone, casein,
Tomato paste, wheat germ W, ammonium sulfate, ammonium nitrate, etc. can be used alone or as a mixture. Examples of inorganic salts include 'calcium carbonate, sodium chloride, and potassium chloride.

Examples include magnesium sulfate and potassium phosphate, which are added to the culture medium as needed. The pH of the medium is approximately 53~
is preferred. The culture temperature is preferably about 2θ to 0°C, particularly preferably 2j to 32°C.

The culture time depends largely on the scale of fermentation, but it takes 2 to 6 days for mass production. If severe foaming occurs during culturing, add vegetable oil, for example, before or during culturing.
Antifoaming agents such as lard and polypropylene glycol may be added as appropriate.

The antibiotic of the present invention is produced and accumulated in the culture obtained in this way, and is contained in both the furnace solution and the bacterial cell extract. The substances produced at the same time are determined from the analysis results of ultraviolet absorption spectra, infrared absorption spectra, 'H-FT nuclear magnetic resonance spectra, thin layer d-mography in various solvent systems of the constituent fatty acids, and high performance liquid chromatography. The main ones are chromomycin, chromomycin, olibomycin A, and olibomycin B, which are known aureolic acid antibiotics.

After the culture is completed, the antibiotic of the present invention is separated from the culture.

For collection, use any conventional method for separating and collecting fermentation products as appropriate. For example, filtration, centrifugation, solvent extraction, adsorption/desorption using an active adsorbent, chromatography, partition chromatography using a reversed-phase packing material, countercurrent partition extraction, etc. may be appropriately combined.

The antibiotic of the present invention purified by the above method is a yellow crystalline powder whose properties are as shown in the table below. However, in Table 1, compounds 1 to (■) are compounds having the groups shown below as R/ and R2 in the formula (■).

Compound I: R'=hydrogen, R1=isobutyryl〃■:R1
: Methyl, R2=Isobutyrylttffl: R'=Hydrogen, R-Facetyl -zz: R'=Methyl, R2=Cicetyl (the following is a blank space) The antibiotic of the present invention having the above properties has antitumor activity and Duram positive Has antibacterial activity against bacteria.

/) Antitumor activity The antitumor activity of the antibiotic of the present invention against P311 leukemia in mice is shown.

[Activity measurement method]

BDF, mice are inoculated with P3rg leukemia cells at /θ6, and the next day, the test substance is administered intraperitoneally in the range of 0-'IOW/klj (treatment group). The test substance non-administration group is combined with the control group, and the number of survival days (days) in both groups is determined.

[Comparison standard]

Based on the survival rate (ILS) and chemotherapy index (CI) shown in the following formula.

(Average survival days of control group) LSmaz CI: □ ILSJ (7% = (In the formula, IL830% is the amount of test substance (Q / kq) required to make ILS 3θ%, IL S m
ax is the amount of test substance (#/&g) at which ILS is maximum. )
The higher the CI, the wider the safety margin, and it can be said that the drug is highly safe to administer.

〔result〕

The following table shows the antitumor activity of the objective antibiotic of the present invention. Compound 1 in the table. ■, [, ■ are compounds with the same meanings as above.

As is clear from the following table, all of the nine antibiotics of the present invention have antitumor activity, and up to a certain dose, an increase in ILS is observed depending on the amount of the test substance. Compound 1. IL of up to 6θ% for II and up to 76% for compound ■
It showed S. In addition, according to the study results by CI, compounds ■,
■ showed particularly high safety.

(ILS (%)) (CI) Probiotic activity The antibiotic of the present invention has activity against Durham-positive bacteria. The antibacterial spectrum is shown below.

However, compound 1. I[,1,lV is a compound with the same meaning as above. Moreover, the -' small inhibitory concentration is a value determined by the agar dilution method, and the number of inoculated bacteria at that time was 704.

(Hereinafter in the margin) The antibiotic of the present invention having antitumor cancer activity and antibacterial activity as described above is useful as an antitumor agent or a therapeutic agent for infectious diseases caused by Durham-positive bacteria.

Administered orally or parenterally to humans or animals. For example, pharmaceutically acceptable excipients (lactose, sucrose, starch, calcium carbonate, gelatin, sodium benzoate, etc.), fillers (starch, kaolin, lactose,
bentonite, talc, etc.), lubricants (stearic acid,
Granules are made by adding pharmaceutical ingredients such as boric acid, talc, etc., and disintegrants (starch, carboxymethyl cellulose, etc.).

Oral administration as tablets, capsules, powders, etc.

Alternatively, it can be dissolved in ethanol, glycerin, propylene glycol, etc. and administered parenterally as an injection. Although the antibiotic of the present invention differs depending on the therapeutic purpose, the 7-day dose for adults when used for tumor treatment is approximately θ/q-
It is 29.

Next, production examples of the antibiotic of the present invention will be shown, but the following examples are not intended to limit the present invention in any way.

Example (A) Fermentation process (a) Seed culture Seed medium Soluble starch 05% Glucose 05% Polypeptone θj% Beef extract θ! % Yeast extract 023% Sodium chloride 023% Deionized water (pH 70, before sterilization) A 21-volume Erlenmeyer flask containing 100 ml of seed medium was inoculated with a seed culture slant of Streptomyces arabiens PA-39tS, and the seed culture slant was inoculated every minute. 21'C, 11 with O rotation
Perform shaking culture for 1 hour.

(b) Main culture - Fermentation medium Soybean flour 15% Corn steep liquor 05% Gluco, Su 2θ% Glycerin
05% Sodium chloride 03% Calcium carbonate 03% Water (pH 70, before sterilization) Add 100 txl of culture solution obtained from seed culture to fermentation medium 2
01 was inoculated into a 30g jar containing 0.01 airflow per minute and an internal pressure of 03 kg/clt2. Culture at 2°C for 3 days with stirring at θθrpm.

(B) Separation step The culture solution obtained in the main culture was adjusted to pH 3 to 10g with 10% hydrochloric acid, subjected to Sharpless centrifugation, and the supernatant liquid (1)
J and wet bacterial cells (2) g, 2 kfl were obtained. Sodium chloride is added to the supernatant liquid (1) to a concentration of 7θ%, and stirring and extraction with ethyl acetate is performed twice to obtain ethyl acetate layer 33; This is washed with deionized water at pH 3 to Hiroshi, then washed with a small amount of deionized water at around neutrality, and concentrated. The concentrate is dissolved in a small amount of ethyl acetate, cyclohexane is added, and the resulting precipitate is collected and dried to form a coarse powder (
3) Get / & 3 fl. Further, methanol is added to the wet bacterial cells (2), stirred, and then filtered to obtain a methanol solution/jl. After concentrating this, adjust the concentration to 53=q with 10% hydrochloric acid, perform extraction with ethyl acetate twice, wash with deionized water at pH 3~Hiro, wash with a small amount of deionized water again at around neutrality, and concentrate. do. Dissolve the concentrate in ethyl acetate, add cyclohexane, collect the resulting precipitate, and dry it to obtain a coarse powder (
4) Obtain i + 9. The coarse powders (3) and (4) obtained in this step are chromomycin A, a known aureolic acid-based antimicrobial substance other than the antibiotic of the present invention.

Also includes chromomycin, olibomycin A, and olibomycin B.

0 Purification Step I The coarse powder (3) obtained in the above step (B) and (
The mixture of 4) is purified by column chromatography using licage gel as follows.

1 toy of iron-free silica gel to which 10% water has been added (Silica Gel 60. Manufactured by Merck & Co., West Germany)
Coarse powder Z was packed in a column and dissolved in a small amount of developing solvent.
θf is absorbed. The developing solvent used is a mixture of [chloroform:methanol:water=-2jθ:/j:/] with 0.7% acetic acid added. Thin layer chromatography of this eluate in the same solvent system yields several active fractions. A for each active category in the order of elution.
, B, C, D, E, F, G, and H groups. The active fractions were collected and concentrated, dissolved in ethyl acetate, and cyclohexane was added. The resulting precipitate was collected and dried, resulting in 6 groups of 0.
6θf, 0 group 0! ;3f.

A mixture of group F/Ot y-y, group A 12zaf, group 1 and group ozry is obtained as a yellow to orange powder, and trace amounts of groups A, B, E and H are also obtained. By further repeating the above purification process, finally C group 6f, 0 group 2. Of
, F Senbutsu 79. Group G obtains 3f.

ρ) Purification step ■ Dissolve the F group 'A7'l obtained in the above step C) in small methanol and add Lichroprep RP-/10-20θη per 7 times.
1 (manufactured by Merck & Co., West Germany) column, GCH-2θ.

2! ; Using Oxmx2 (manufactured by Umetani Seiki Co., Ltd.),
θ:so] and subjected to high performance liquid chromatography using a mixed solution ('PHR2j). 210 by ultraviolet absorption detector
When the absorption at nm was examined, Compound I, the antibiotic of the present invention, had a retention capacity of about /W Ox1. Compound (1) elutes at about 2 ml, so each fraction is collected, concentrated, and extracted with ethyl acetate. The resulting ethyl acetate layer was
After washing thoroughly with 0% saline and distilled water, it was concentrated, dissolved in a small amount of ethyl acetate, and the precipitate formed by adding cyclohexane was collected and dried. Compound (13 g) and compound (17 g) were purified as pure compounds. Obtained as a standard specimen. Similarly, group G [Acetonitrile: 30 Tartaric acid - Sodium tartrate = Gu! ;:! When subjected to high performance liquid chromatography using a mixed solution (pH 3, 2! ; ') of compound 1 (17
fl ) and compound IV (129) are obtained as pure standards. Furthermore, when Groups C and D were purified in the same manner, both groups were separated into their respective co-components.

From group C in descending order of retention capacity are the known aureolic acid antibiotics olibomycin A and chromomaino A;
From group D, olibomycin B and romomain A
I got 3 points.

[Brief explanation of the drawing]

Figures 1, 2, 3, and 2 are the ultraviolet absorption spectra of compounds t, n, m, and 2 measured in ethanol and ethanol, respectively, and Figures S and 6 show them. FIG. 7 and FIG. 1 show compound 1. The infrared absorption spectra of potassium bromide tablets of I[, 1 and ■,
Figure 9, Figure 1θ, Figure 1/Figure 72 shows compounds i, n, m and ■, respectively, in deuterated chloroform at room temperature.
'H-FT measured at 100 h using MS as internal standard
A nuclear magnetic resonance spectrum is shown. Patent applicant: Shionogi & Co., Ltd. Figure 1 Figure 2 Figure 3 Figure 9 - Figure 1θ (ppm19876543210 Figure 1/Figure 72 IψmI@IB 7'6 5
4 3 2 1 0 Procedural amendment (
spontaneous) 1. Display of the case 1982 Patent Application No. 159307 2 Title of the invention New aureolic acid antibiotic and its manufacturing method 3 Person making the amendment Relationship to the case Patent applicant 4 Address of agent Sagisu, Fukushima-ku, Osaka City 5-12-4, 553 Shiosugi Pharmaceutical Co., Ltd. Patent Department (Telephone: 06-458-5861) Column for claims and detailed description of the invention of the specification to be amended. Contents of B's amendment (1) The scope of claims in the specification is corrected as shown in the attached sheet. (2) Correct the structural formula on the last line of page 3 of the specification to the following structural formula. Z List of Attached Documents Attachment 1 or more copies (Attachment) 2. Claims (1) An aureolic acid antibiotic having the following structural formula. (In the formula, Hl represents hydrogen or methyl, and RJ represents acetyl or isobutyryl, respectively.) (2) An aureolic acid antibiotic-producing bacterium belonging to the genus Streptomyces is cultured in a medium, and the following structural formula is derived from the resulting culture. 1. A method for producing a novel aureolic acid antibiotic, comprising collecting at least one aureolic acid antibiotic having the following. (In the formula, Hl represents hydrogen or methyl, R2 represents acetyl or isobutyryl, respectively6) (3) Streptomyces arabiensis PA-39 j6° (or more)

Claims (1)

[Claims] (1) An aureolic acid antibiotic having the following structural formula. (In the formula, Bl represents hydrogen or methyl, and HJ represents acetyl or isobutyryl, respectively.) (2) An aureolic acid antibiotic-producing bacterium belonging to the genus Streptomyces was cultured in a medium, and the following structural formula was obtained from the resulting culture. 1. A method for producing a novel onileolic acid antibiotic, which comprises collecting at least one type of aureolic acid antibiotic having the following properties. (In the formula, a/ represents hydrogen or methyl, and - represents acetyl or isobutyryl, respectively.) (3), Streptomyces arabiens PA-39t36゜