JPS6344581A - Substance yl-0358m-a and production thereof - Google Patents

Abstract

NEW MATERIAL:A substance YL-0358M-A expressed by formula I. USE:Useful as a medicine, particularly an antitumor agent and antimicrobial agent capable of exhibiting antimicrobial action on various bacteria, particularly Gram-positive bacteria as well as cytotoxic action on various tumorous cells. PREPARATION:A strain, e.g. Saccharopolyspora glyceopurpurea YL-0358M (FERM-P No. 8862), having the ability to produce the substance YL-0358M-A and belonging to the genus Saccharopolyspora is used and cultivated to collect the aimed substance expressed by formula I from the resultant culture fluid. A culture medium containing a carbon source, e.g. D-xylose, etc., nitrogen source, e.g. meat extract, etc., metal salt, e.g. Na, etc., is used as a culture medium used for cultivation and the cultivation is preferably carried out at about 28 deg.C and a pH of the culture medium within the range of 6-8. The cultivation period is normally about 1-5 days.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a substance YL-0358M-A useful as a medicine, particularly an antitumor agent and an antibacterial agent, and a method for producing the substance by fermentation.

(Problems to be solved by the invention, means for solving them) The substance YL-0358M-A according to the present invention is a novel compound specified by the following chemical structural formula and π physical and chemical properties.

Chemical structural formula: % Formula %: (1) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum in methanol is shown in FIG.

(2) Infrared absorption spectrum: Figure 2 shows an infrared plate spectrum obtained by the potassium bromide tablet method.

(31 Hydrogen nuclear magnetic resonance spectrum; 500 MHz IH nuclear magnetic resonance spectrum in deuterated chloroform is shown in FIG. 3.

(4) Carbon-13-Nuclear Magnetic Resonance Spectrum: The 125 MHz 13c nuclear magnetic resonance spectrum in deuterated chloroform is shown in FIG.

(5) Mass spectrometry: 390 (MH
) and 412 (M+ Na”).

(6) Molecular weight = 389 (7) Molecular formula: C22H31N05 (8) Specific rotation: [α]^+88° (c' = 1. methanol) (
9) Melting point: 93°C αQ Appearance: White crystal + Il+ Distinction between basic, neutral, and acidic: Basic substance f
i2 Solubility: methanol, ethanol, acetone.

Soluble in ethyl acetate and chloroform 03 Rf value of thin layer chromatography: Silica gel 60 F254 (manufactured by Merck & Co., Ltd.) was used. Detection is [JV
2541m6 Table 1 aa X-ray diffraction Crystals suitable for X-ray analysis were obtained from the acetonitrile solution.

Crystallographic data are shown below. C22H31NO5
・2 H2O, Mr = -425,52, monoclinic system, space group C2, a = 22.70f11. b = 9.51
8(61,'c = 10.786(6)X, β=90
．． 51 (51', z = 4. dc = 1.21
CnKa (λ = 1.5418
4A) 1982, including 40 reflections with one intensity of zero, using a 4-axis automatic diffractometer AFc5R manufactured by Rigaku.
(2θ<125°) reflection intensities were measured.

The structure was solved using the direct method and the Fourier synthesis method, and the refinement was performed using the diagonal least squares method. Attribution of each atom was made based on the height of the peak in the electron density distribution map, temperature, etc. Hydrogen atoms were found in the electron density distribution map obtained by the difference Fourier method and included in the refinement. The final reliability factor is 4.0 for the 1830 reflections with IFOI > 3σ.
It was 1%.

Table 2 shows the coordinates of non-hydrogen atoms in YL-0358M-A.

Table 2 The value of B in the table is the equivalent temperature factor calculated from the anisotropic temperature factor; B=8 x” (ut + ut +
03), /3. Here 'l1u21u3 is the principal component ring of the mean square deviation matrix U. The value inside is the anisotropy, (Σ(B8 K2Ui )2/3)
Defined by Furthermore, the values in parentheses are standard deviations.

(Effects of the Invention) The substance YL-0358M-A exhibits an antibacterial effect against various bacterial strains and gram-positive bacteria, and also has a cytotoxic effect by compressing various tumor cells. These effects are shown below along with measurement methods.

(11 Antibacterial effect experimental method Add methanol to YL-0358M-A substance, add O
Make a solution of OOμg/ml. This solution was impregnated into a thin paper disk (manufactured by Toyo Seisaku Shin) with a diameter of 8 mm for antibacterial activity measurement, and after removing the excess liquid, Penoku-disk assay was performed on each coral test bacteria. The test bacteria are 3
Growth inhibition zone diameter (mm) after culturing at 7°C for 16 hours
) is measured.

Results: Table 3 A: Mueller-Hinton agar medium (manufactured by Eiken) B Nihard infusion agar medium (manufactured by Eiken) (2) In vitro cytotoxicity method using tumor cells: L 121 adjusted to I X 105 cells/mZ
01) (establishedcell 1ine
) r P3882) (Primary culture
each cell suspension (cell 5us
Pension) Add 4 μt of YL-0358M-A substance of each concentration dissolved in methanol to 1 ml, and
After culturing for 3 days in a CO2 inc'bator at °C, viable cells were counted by Trivan blue staining.

Cytotoxic activity (IC50 value) was determined by calculating the cell growth inhibition rate at each concentration using the number of cells without addition of the drug as a control.

It was calculated by plotting it on a graph.

Medium used 1) RPMI-1640+10% Neonatal serum 2)
RPMI-1640+ 5% bovine serum + 5μM
2-Hydroxyethyl disulfide (manufacturing method) YL-0358M-A Ministry of Finance cultivated YL-0358M-A substance-producing bacteria belonging to the genus Satucharopolyspora.

It can be produced by collecting YL-0358M-A substance from a culture.

An example of YL-0358M-A substance-producing bacteria used in this production method is Satsucalopolyspora griseopurpurea (5acch), which was isolated from the soil of Hatoma Island, Okinawa Prefecture.
allopolyspora griseopurpur
ea #=哄) YL-0358M strain (Feikoken Bacterial Collection No. 8
862). The mycological properties of this strain are described below.

l. Morphology This strain forms relatively long, branched basal hyphae on various agar media, and one basal spore is usually observed at the end of the short sporophyte that branches from the basal hyphae. . The basal hyphae exhibit a gray-purple or purplish-gray color on tyrosine agar, yeast malt agar, and starch inorganic salt agar.

As the culture progresses, the basal hyphae rupture into rod-like shapes.

Aerial hyphae formed on glycerin/asparagine agar medium, tyrosine agar medium, etc. are white (powder-like), and spore chains are observed at the tips.

Under an electron microscope, the spores are approximately oval in shape with a diameter of 04 μm −0.
,6pm In addition, the spore surface is covered with a sheath K that has a hair-like structure.

2. Properties on various agar media Properties on various agar media are as shown below. Unless otherwise specified, the samples were cultured at 28°C for 21 days and observed according to conventional methods. Color descriptions were based on color standards (Japan Color Research Institute).

Table 4 (Note) G: Growth and bacterial flora color on the colony surface A: Epiphytion of aerial mycelium and its hue R: Hue on the back side S: Soluble pigment 3, physiological properties Table 5 (Note) The growth temperature is at each temperature ( 5, 10, 15, 20, 25
, 28, 30° 33, 37. 40. 45. 50°C), and as a result of observation from 7 to 21 days, the effect on milk was 3.
Observation results from 3 to 21 days at 7°C. Otherwise, unless otherwise specified, the results are shown after 2 weeks at 28°C.

4. Assimilation of carbon sources (Pridham-Godliep agar medium, 28°C culture) Table 6 5. Analysis LEC of cell walls and whole cell hydrolyzed extracts, etc.
The method of HVALIER et al. (LECHVALIER, MP
.

et al; PP277 283 in DIET
Z, Aet al ed,.

DAP) was detected. In addition, it was confirmed that the sugar components contained arabinose and galactose, but no mycolic acid was detected.

To summarize the above properties, the basal hyphae of YL-0358Ma exhibit a grayish-purple to purplish-gray color on tyrosine agar medium, etc., and as the culture progresses, they rupture into rod-like shapes, and short hyphae branch from the basal hyphae. A single basal spore is usually observed at the end of the sporophore. The white powdery aerial hyphae form ten or more spore chains at the tip, and the spore surface is covered with a sheath with a hair-like structure. Glycerin/asparagine agar medium, starch/
Inorganic salt agar medium.

Produces a brown soluble pigment on yeast malt agar media, etc. Analysis of the cell wall detected meso-diaminopimelic acid (meso-=DAP) as a characteristic amino acid, and arabinose and galactose as sugar components.

From these results, the YL-'0358 M strain was found to be 5accharopolyspora.
) genus (J, Laceys.

Journal of General Microbiology.

Volume 88, p. 75*1975)
a) Actual comparison with KCCA-0170 strain, and also for reference.
+sis) Compared with the KC-6606 strain in the literature (
T, Deushi et a+ Journal of Massacres
Antibiotics, vol. 32, p. 173, 197
8 years). The results are shown in Table 71 below.

As shown in the table, the KYL-0358M strain is KCCA-0
Although there are many similarities with strain 170 and KC-6606, the growth temperature range is low to low and it exhibits a lye purple color. The color tone of growth is significantly different from both strains. In addition, there are differences from known bacterial species in physiological properties, such as weakly positive production of melanin-like pigments and positive coagulation of milk. The above matters YL
The -0358M strain is considered to be a new species of the genus Satucharopolispora because its characteristics are different from known bacterial species belonging to the genus Satucharopolispora. Therefore, based on the characteristic growth color, Satucharopolyspora griseopurpurea
Varnish pea knob (S accharopolyspo)
ragriaeopurpurea 5uzuKt,
NACAI, and 5AITOHsp, nov)
YL-0358M as the type strain.
The strain has been entrusted to the Institute of Microbial Technology, Agency of Industrial Science and Technology under accession number 8862.

(Culture method) The microorganisms used in the production method of the present invention are obtained by isolation from natural soil, but they can be easily obtained by restoring the freeze-dried product of the strain deposited with the Microbial Technology Research Institute. This microorganism can be easily mutated either artificially or naturally.
Strain L-0358M is an actinomycete isolated from nature, 8.
It also includes strains that have been artificially mutated using ultraviolet rays, X-rays, chemical agents, etc., as well as natural mutant strains thereof. Cultivation of the YL-0358M-A substance-producing bacteria is carried out according to the cultivation method of general microorganisms, but the deep culture method using a liquid medium is usually advantageous. As a medium used for culture.

Any medium may be used as long as it contains a nutrient source used by the producing bacteria. In other words, a synthetic medium, a semi-synthetic medium, and a natural medium are used, and the composition of the medium is, for example, D as a carbon source.
-xylose, glucose, D-fructose, L-rhamnose, mannitol, glycerin, dextrin,
Starch, vegetable oil, etc. are used as nitrogen sources, and meat extract and peptone are used as nitrogen sources.

Gluten meal, cottonseed meal, soybean flour, peanut flour.

Fishmeal, cornsteep liquor, dried yeast, yeast extract,
Ammonium sulfate, ammonium nitrate.

Urea and other organic or inorganic nitrogen sources are used. Also, as metal salts NA, K, CIL, Zn, Fe
Sulfates, nitrates, chlorides, carbonates, phosphates, etc. are added as necessary. Furthermore, if necessary, antibiotic production accelerators or antifoaming agents such as methionine, cysteine, cystine, methyl oleate, lard oil, silicone oil, and surfactants are used.

As for the culture conditions, it is generally advantageous to culture under aerobic conditions, and the culture temperature is preferably in the range of about 15 to 37°C.
Preferably, the temperature is around 28°C, and the pH of the medium is around 5-10. Good results are obtained by keeping it preferably in the range of about 6-8.

The culture period varies depending on the composition of the medium, temperature, etc.
Generally, it takes about 1 to 5 days, and the target substance is selectively accumulated at the end of the culture.

In order to isolate and collect the object of the present invention from a culture, a conventional method for isolating antibiotics from a microbial culture is applied. Since the YM-0358M-A substance is contained in the culture solution, after removing the bacterial cells by centrifugation or filtration,
The active substance is extracted from the filtrate. In other words, the general method utilizes differences in solubility and solubility in appropriate solvents, differences in precipitability from solutions and precipitation rates9, differences in adsorption affinity for various adsorbents, and differences in distribution between two liquid phases. 1. Separation, collection, and purification by means used in the production of antibiotics.

This method can be used alone as needed, and can be applied in any combination or repetition in any order.
The present invention will be explained in more detail with reference to Examples.

Example: 2.0% white dextrin, 0.5% glucose, 0.5% polypeptone, 0.5% yeast extract, 05% corn steeple bone liquor, 0.52% rain heart infusion and carbonic acid. A medium containing 0.2% calcium (pH 8.0) was prepared, and 60 ml of this was dispensed into 500 ml Erlenmeyer flasks, sterilized at 120°C for 20 minutes, and then grown on YS agar medium. The mycelia of Sancaloborispora griseopurpurea were scraped and inoculated, and cultured with shaking at 28°C for 48 hours to prepare a seed culture. Next, a medium (p
81 f(7,0) were prepared, and 60 ml of each was dispensed into 500 ml Erlenmeyer flasks, which were sterilized at 120° C. for 20 minutes and inoculated with a seed culture solution at a rate of 3.0%. 2
When the shaking culture is continued for 72 hours at 8°C, the antibacterial activity against Bacillus subtilis ATCC6633 strain reaches its maximum.

Radiolye #60 is added to the culture solution thus obtained.
0 (manufactured by Showa Kagaku Kogyo), stirred, and filtered to obtain 6 tons of P liquid. Add 0.1N hydroxide to this R liquid)
After adjusting the pH to 8.5 by adding a solution of 61
of ethyl acetate and stir well. After separating the ethyl acetate group.

Add 800 ml of hydrochloric acid water adjusted to pH 3.0 to this and stir well. After separating the hydrochloric acid aqueous layer, dibasic soda was added to adjust the pH to 8.5, and then 1 t of ethyl acetate was added and stirred well. The ethyl acetate layer is separated and dehydrated by adding anhydrous sodium sulfate. Next, after separating the anhydrous sodium sulfate with P, the ethyl acetate layer was concentrated under reduced pressure to obtain 460,111 g of a pale yellow substance. Obtained pale yellow substance 146
0■ in a small amount of chloroform, Wako Gel C
-200 (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) 10g was placed on a column filled with chloroform, and then aliquots of form:methanol (50:
Column chromatography using 1) as a developing solvent was performed under light-shielded conditions at ℃ Bacillus subtilis ATCC66.
Fractions showing antibacterial activity against 33 strains were collected and concentrated under reduced pressure to obtain 223 mg of a slightly yellow substance. Obtained slightly yellow substance 1
When 223 rllg is dissolved in 4mtK ethyl acetate and left at 5°C under light-shielded conditions, crystals are formed. Next, P is removed from the generated crystals and dried under reduced pressure to obtain pure YL-03.
73 mg of 58M-A substance was obtained as white crystals.

The chemical structural formula and physical and chemical properties of the substance YL-0358M-A are as described above.

[Brief explanation of the drawing]

FIG. 1 shows the ultraviolet absorption spectrum of the YL-0358M-A substance. FIG. 2 shows the infrared absorption spectrum of the YL-0358M-A material. Figure 3 shows the nuclear magnetic resonance spectrum of the YL-0358M-A substance. Figure 4 shows the +3H nuclear magnetic resonance spectrum of the YL-0358M-A substance. Figure 1 Figure 2 Figure 3 Figure 4 Procedural amendment 1, Indication of the case 1989 Patent Application No. 189114 2, Name of the invention YL-0358M-A substance and its manufacturing method 3, Person making the amendment Relationship with the case Patent applicant address 2-5-1 Nihonbashi Honmachi, Chuo-ku, Tokyo Name (467) Yamanouchi Pharmaceutical Co., Ltd. Representative Shigeo Morioka 4 Agent address 1-1-8 Azukizawa, Itabashi-ku, Tokyo Yamanouchi Pharmaceutical Co., Ltd. Patent Department 2. Lecture 4 Name (90
94) Patent attorney Seiya Fujino:. 5. Date of amendment order 7. Contents of amendment C.” (2) On page 11, line 5, "Table 4" is corrected to "Table 5." (3) Table 5J, page 13, line 2, is changed to “Table 6”, and column 12
Correct the line ``Reducing effect of nitric acid'' to ``Reducing effect of nitrate.'' (4) On page 14, line 3, ``Table 6'' is corrected to ``Table 1, Table 7''. (5) On page 16, lines 1 and 2, "It was thought of, Satucalopolispora..." was corrected to "It was thought of. Therefore, Satucalopolispora...". (6) Change “Table 7” to “Table 8” in lines 8 and 10 of page 16.
” is corrected. (7) In the 17th line, 3 lines from the bottom, "present. development" is corrected to "present development."

Claims (3)

[Claims] (1) Formula ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ YL-0358M-A substance represented by. (2) Cultivate a strain belonging to the genus Saccharopolyspora that has the ability to produce YL-0358M-A substance, and extract YL from the culture solution.
YL characterized by collecting -0358M-A substance
-0358M-A Method for producing substance. (3) The production method according to claim 2, wherein the strain belonging to the genus Saccharopolyspora is Saccharopolyspora griseobulupurea YL-0358M (Feikoken Bibori No. 8862).