JPH01168660A - Yl-0710m compound and production thereof - Google Patents

Abstract

NEW MATERIAL:The YL-0710M compound of formula having the following properties. Appearance, pale-yellow amorphous solid; nature, basic; solubility, soluble in methanol, ethanol, acetone, ethyl acetate, butyl acetate, benzene, toluene and chloroform, scarcely soluble in water and almost insoluble in hexane; molecular weight, 573[M+1] and 595[M+Na] (by FAB-Mass); molecular formula, C29H40N4O8. USE:An antibacterial agent and antitumor agent. It exhibits excellent action especially against Gram-positive bacteria and strong cytotoxic action against various tumor cells. PREPARATION:The objective compound of formula can be produced by culturing YL-0710M strain (FERM P-9728) belonging to Streptomyces genus and capable of producing YL-0710M compound under aerobic condition and subjecting the cultured product to extraction, separation and purification.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a novel YL-0710M compound having antibacterial and cytotoxic effects and a method for producing the same.

(Problems to be solved by the invention, means for solving them) The compound of the present invention has the following planar chemical structural formula:
This is the L-0710M compound.

Further, in the production method of the present invention, YL-0710M compound-producing bacteria belonging to the genus Streptomyces are cultured.

This is a method for producing the above YL-0710M compound, which comprises collecting the YL-0710M compound from a culture.

(Effects of the Invention) The YL-0710M compound exhibits antibacterial activity against various bacterial strains and Durham-positive bacteria, and also has a strong cytotoxic effect against various tumor cells, so it can be used as an antibacterial agent and an antitumor agent. Useful.

(1) Antibacterial effect Table 1 shows the minimum inhibitory concentration (MIC) of the YL-0710M compound against various bacteria.

(2) In vitro cytotoxicity test method using tumor cells: L121 adjusted to 1 x 10' cells/mt
4 μt of YL-0710M compound at each concentration dissolved in ethanol was added to 1 ml of each cell suspension of 0'' and P2S5''.

After culturing for 3 days in a carbon dioxide incubator at 37°C.

Surviving cells were counted by Trivan blue staining method. Cytotoxic activity was determined by calculating the cell proliferation inhibition rate at each concentration using the number of cells without the addition of the drug as a control, and plotting the results on a graph.

Medium used: It RPMI-1640+ 10% newborn calf serum mW RPMI-1640+ 5% beef tallow serum +5
μM2-hydroxyethyl disulfide Table 2 (Production method) The YL-0710M compound can be produced by culturing a YL-0710M compound-producing bacterium belonging to the genus Streptomyces and collecting the YL-0710M compound from the culture. .

An example of YL-0710M compound-producing bacteria used in this production method is Streptomyces nisp.
yces sp,) YL-0710M strain (Feikoken Bibori No. 9728). The mycological properties of this strain are described below.

(1) Morphology This strain grows on various synthetic and organic media, particularly starch/inorganic salt agar, yeast malt agar, and peptone/yeast/iron agar. Aerial mycelium grows well on starch/inorganic salt agar medium and oatmeal agar medium, and its color is grayish-white to gray. According to light microscopy, aerial hyphae are simply branched.

It shows a spiral shape with an open tip (0pen 5piral).

The shape is section spirales (S). By observation with an electron microscope, a chain of about 50 spores was observed, the size of the spores was 0.5 to 0.7 x O, 8 to 1.0 μm, and the surface structure was thorn-like. Whorled branches, sporangia, and sclerotia formation are not observed.

(2) Properties on various agar media Properties on various agar media are as shown below. Unless otherwise specified, the cells were cultured at 28° C. for 21 days and observed according to conventional methods. The description of color tone was based on the color standard (Japan Color Research Institute).

Table 3 (3) Physiological properties Table 4 (Note) The growth temperature is at each temperature (5, 10, 15, 20, 25
,28,30,33°37.40,45.50°C),
Observation results from days 7 to 21.

The effect on milk was observed at 37°C for 3 to 21 days. 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 5 (Note) +; Growing; Growth questionable -; Not growing (5) Analysis of diaminopimelic acid (DAP) LEC
The method of HVALIER et al. (LECHVALIEI:R,
MP, et al; PP 277-238 in
DIETZ, A et al ed, Actin
omyceteTaXOnOmy+ SIM 5pec
ial publication No. 6+ 198
Results of analysis of acid hydrolysates of nine bacterial strains according to 0).

LL-diaminopimelic acid was detected.

To summarize the above properties, the YL-0710M strain is.

Aerial hyphae formed on various agar media are simply branched, the shape is section spirales (S), and chains of about 50 spores are observed. As for the hue, the growth is pale yellow-brown to yellow-brown, the aerial mycelia are gray-white to gray, and no production of soluble pigments or melanin-like pigments is observed. Furthermore, LL-diaminopimelic acid was detected by analysis of the acid hydrolyzed product of the bacterial cells.

Based on the above properties, the two strains are Streptomyces (Streptomyces).
Known bacterial strains similar to the nine strains, which are considered to belong to the genus Ptomyces), were
Op Detaminedip Bacteriology (Berge
y's Manual of Determinative
e Bacteriology) 8th edition, 197
International Journal of Systematic Bacteriology (International Journal of Systematic Bacteriology)
ional Journal of Systemat
ic Bacteriology) Volume 18, No. 2,
pp. 69-189, No. 4, pp. 279-392 (1968
), Volume 19, No. 4, pp. 391-512 (1969
) Vol. 22, No. 4, pp. 265-394 (1972
) Searched from J. As a result, Streptomyces olivaceus (S, oli
vaceus) and Streftomyces ioyanensis (S, 5ioyanensis). Both strains are similar to this strain in terms of properties such as the shape of aerial mycelia, color tone of colonies, and the presence or absence of pigment production on the surface structure of spores, but they are similar to this strain in terms of their ability to assimilate carbon sources (Table 6) and on various media. The color tone of the aerial mycelium (Table 7) differs slightly between 9 and 9.
It does not match this strain. Based on the above results, this strain was determined to be a new species belonging to the genus Streptomyces, and Strepto-myces sp.
) YL-0710M strain. This strain is.

Microbiology Research Institute No. 972 at the Institute of Microbial Technology, Agency of Industrial Science and Technology
It has been deposited as No. 8.

Table 6 Comparison of YL-0710M with similar bacterial species (carbon source assimilation ability) Table 7 Comparison of YL-0710M with similar bacterial species (color tone of aerial hyphae) The bacterial strains used in the present invention are other actinomycetes As seen in 1, it is easy to cause mutations either artificially or naturally. It also includes bacterial strains that have been mutated and natural mutant strains thereof.

The microorganisms belonging to the new bacterial species of the present invention were 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. . In the present invention, the YL-0710M compound-producing bacteria belonging to the genus Sprebutomyces is cultured according to the culture method of general microorganisms, but the deep culture method using a liquid medium is usually advantageous. The medium used for culturing may be any medium as long as it contains a nutrient source used by the producing bacteria. That is, a synthetic medium, a semi-synthetic medium or a natural medium is used, and the composition of the medium is, for example, carbon sources such as D-xylose, glucose, D-fracdose, L-rhamnose, mannitol, glycerin,
Dextrin, starch, vegetable oil, etc.

Nitrogen sources include meat extract, peptone, 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, Mg, Ca, Zn
, Fe and other sulfates, nitrates, chlorides, carbonates, phosphates, etc. are added as necessary. More if necessary.

Antibiotic production accelerators or antifoaming agents such as methionine, cysteine, cystine, methyl oleate, lard oil, silicone oil, and surfactants are used as appropriate.

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 18 to 35°C.
Preferably, the temperature is around 30°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., but is generally about 1 to 5 days, and the target substance is selectively accumulated at the end of the culture.

In order to collect the target YL-0710M compound from the culture, extraction, separation, and purification means commonly used from the culture of metabolites produced by microorganisms are appropriately used. The YL-0710M compound in the culture can be obtained from the culture as it is, or by centrifugation or by adding a translucent agent to the culture and filtration, and adding a water-immiscible organic solvent such as ethyl acetate to the culture solution. Add and extract. Further, the desired YL-0710M compound can be extracted by bringing the culture solution into contact with a suitable carrier to adsorb the desired active ingredient in the P solution, and then eluting with a suitable solvent. More specifically, 9 porous adsorption resins such as Diaion HP-20 or CHP-20P.

Or, for example, DOWEX 50. Amber Light IRC
YL-0 by contacting with an ion exchange resin such as 50
Adsorb the 710M compound. Next, in the case of the adsorption resin, methanol and ethanol.

A mixed solvent of water and an organic solvent such as acetone or acetonitrile, and in the case of the ion exchange resin, hydrochloric acid.

YL-0710M by elution with an aqueous solution such as sulfuric acid.
Compound-containing fractions can be obtained.

When extracting with an organic solvent such as ethyl acetate or butyl acetate, add these organic solvents to the culture liquid, stir and extract, and then extract the obtained extract with acidic water. By repeating the operation of neutralizing the compound and then extracting it again with the organic solvent, it is possible to obtain both components of the YL-0710M compound in higher proportions. Next, the YL-0710M compound-containing fraction obtained using each of the above-mentioned operating methods is subjected to a conventional adsorption treatment 2, such as activated carbon.

Separation can be performed by conventional methods such as column chromatography, thin layer chromatography, etc. using alumina, silica gel, cellulose, etc. as a carrier. The YL-0710M compound thus produced in the culture can be separated as the free base form, ie, the YL-0710M compound itself. The free base of the YL-0710M compound can also be reacted with an acid, such as an inorganic acid such as hydrochloric acid, sulfuric acid, or phosphoric acid, or an organic acid such as formic acid, acetic acid, citric acid, tartaric acid, or p-toluenesulfonic acid, by a conventional method. It can also be converted into salts corresponding to each acid.

(Example) Next, the present invention will be further explained with reference to Examples.

Example A medium (pH 7.0) containing 3.0% potato starch, 1.0% wheat germ, 1.0% copra meal, 0.2% feather meal, and 0.5% calcium carbonate was prepared. Dispense one 60ml volume into each 500mZ Erlenmeyer flask, and
Sprebtomyces Nis bee YL grown well on Bennett agar medium after sterilization at 20°C for 20 minutes
-0710M strain was scraped and inoculated, and incubated at 28℃ for 4 hours.
Culture with shaking for 8 hours and use as a seed culture solution. Next, 2.0% dextrin, 0.5% corn steeple, 0.5% polypeptone, and 0.5% yeast extract.

Meat extract 0.3%, plain heart infusion 0
．． 52%, calcium carbonate 0.5% (pH
8,0) was prepared, 60 ml of each was dispensed into 500 ml Erlenmeyer flasks, sterilized at 120° C. for 20 minutes, and the above seed culture solution was inoculated at a rate of 3.0%.

The cells were cultured at 28°C for 96 hours.

Radiolight≠600 was added to the culture solution thus obtained.
(manufactured by Showa Kagaku Kogyo Co., Ltd.) and stirred, then filtered 1.
8 tons of culture solution was obtained. This F solution was adjusted to pH 8.0, and ethyl acetate was added in two portions of 4 tons each, followed by stirring and extraction.

This extract was concentrated under reduced pressure to 100 nt, water at 50°C was added, and the aqueous layer was adjusted to pH 2.5 with 1N hydrochloric acid.
The target product was extracted by stirring well.

This operation was repeated four times to obtain an acidic extract with a total concentration of 200 mZ. 5On+Z ethyl acetate was added to this acidic extract, and the pH of the aqueous layer was adjusted to 8.0 with 1N caustic soda.
The mixture was thoroughly stirred and re-extracted. This operation was repeated four times to obtain a total of 200 ml of an ethyl acetate extract of the YL-0710M compound. After adding Glauber's salt to this extract and thoroughly dehydrating, the Glauber's salt was removed by filtration, and the F solution was concentrated to dryness under reduced pressure.
38 ml of crude YL-0710M compound was obtained. The crude YL-0710M compound thus obtained was
cm, length 100cm of silica gel (Wakogel C-3
00 (manufactured by Wako Pure Chemical Industries, Ltd.) and developed with ethyl acetate, and the eluate was fractionated in 10 g portions using a fraction collector. The target YL-0710M compound was eluted between fraction numbers 6 and 21. Its activity is Bacillus subtilis ATCC6633
Confirm by assaying on an agar plate of the strain, and use a silica gel plate (Kieselgel 60F2 4,20
Chloroform:
The target YL-0710M compound (Rt
= 0.47) was confirmed using a UV lamp (254 nm).

Fractions with high relative purity (fraction numbers 6 to 14) were collected from this elution fraction and concentrated to dryness under reduced pressure.
4. Crude YL-0710M compound of OfDg was obtained.

In order to further purify this crude YL-0710M compound, a silica gel plate (Kieselgel 60F254°2
0 cm x 20 am (manufactured by Merck & Co.) was charged in a strip at 2 cm from the bottom, developed for 15 cm with chloroform:methanol (90:10), air-dried, and then exposed to the YL-0710M compound using a UV lamp (254 nm). (Rf = 0.47), scrape off this part, and add chloroform:methanol (90:10).
The eluate was concentrated to dryness under reduced pressure to obtain 6.0 mg of pure YL-0710M compound.

The free base of YL-0710M compound separated and purified in the above extraction 9 has the following physicochemical properties.

(1) Color and shape; pale yellow amorphous solid; (2) Distinction between acidic, neutral, and basic; basic; (3) Solubility; methanol, ethanol, acetone.

Ethyl acetate, phtyl acetate, benzene, toluene.

It dissolves in chloroform but not in water.

Almost insoluble in hexane.

(4) Ultraviolet absorption spectrum; The ultraviolet absorption spectrum of the YL-0710M compound is as shown in FIG.

(5) Molecular weight; 573 (M+1), 595 [M1N
a] (FAB-Mass) (6) Molecular formula; C29
H4ON4011 (high resolution mass spectrum) (7)
CT mass spectrum: The CI mass spectrum of the YL-0710M compound is as shown in FIG.

(8) "H-NMR spectrum; The I-H-NMR spectrum of the YL-0710M compound is as shown in FIG. 3.

(9) “-C-N MR spectrum; YL-0
The 13-C-NMR spectrum of the 710M compound is as shown in FIG.

From the above physicochemical properties, the structural formula of the YL-0710M compound is determined as follows.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the ultraviolet absorption spectrum of the YL-0710M compound. FIG. 2 shows the CI mass spectrum of the YL-0710M compound. FIG. 3 shows the IH-nuclear magnetic resonance spectrum of the YL-0710M compound. FIG. 4 &t shows the nuclear magnetic resonance spectrum of YL-0710M compound. Nagai = Figure 1: Skin length (nm)

Claims (3)

[Claims] (1) YL-0710M compound represented by the following planar structural formula. ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ (2) A method for producing a YL-0710M compound represented by the following planar structural formula, which comprises culturing a YL-0710M compound-producing bacterium belonging to the genus Streptomyces and collecting the YL-0710M compound from the culture. ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ (3) The YL-0710M compound producing bacterium is Streptomyces sp.
) YL-0710M (Feikoken Bibori No. 9728), the manufacturing method according to claim (2).