JPS61268685A - Goo69c substance - Google Patents

Abstract

NEW MATERIAL:The GOO69C substance of formula and its salt. It has the following physical and chemical properties. Appearance, pale brown powder; molecular weight, 287 (by fast atomic-bombardment mass-spectrometry); solubility, easily soluble in water and hardly soluble in ether and chloroform; etc. USE:Carcinostatic agent and antibacterial agent. PREPARATION:The titled substance can be produced e.g. by culturing Streptomyces brunneogriseus Furumai et Okuda subsp. bannaensis GOO69 strain (FERM-BP 706) capable of producing GOO69C substance usually in a liquid medium under aeration and agitation preferably at 25-40 deg.C usually for 30-100hr.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a GO069C substance represented by the formula and a salt thereof.

The GO069G substance and its salts of the present invention have anticancer and antibacterial effects and are useful as medicines.

The GO069G substance and its salt of the present invention are new substances that have not been described in any literature.

An object of the present invention is to provide a new substance having anticancer and antifungal effects.

1i Stage Wataru j The GO069G substance of the present invention is represented by the above formula (I) and has the following physical and chemical properties.

(1) Molecular weight: 287 (first atomic bombardment mass spectrometry method).

(2) Solubility in solvents: easily soluble in water, slightly soluble in ether and chloroform.

(3) Infrared absorption spectrum, ν (cm-')
:Ilax 3350.1770.1670.163011390.
The 1350.1135.1055° chart is shown in Figure 1.

(4) Proton NMR spectrum, δ (ppm),
In 020: 1°58 (3H, d%J-7H2), 2.89 (1
H, d, J-17H2), 3, 40 (1H, dd,
J-17H2, 2,5H7), 3.86 (IH, dd, J-11,5H2.

6.7H7) 4.48 (1H, dlJ-2, 5H2), 5.47
(1H, d, J-2, 5H7”).

(5) Ultraviolet absorption spectrum, in H2O: 220-3
No characteristic absorption is shown at 50 nl.

The chart is shown in Figure 2.

GOO69C substance belongs to a new group of antibiotics, its chemical name is 3-(alanyl-3-serino)-7-oxo-4-
Oxa-1-azabicyclo[3°2.0]hebutane or 2-(alanyl-3-serino)flavam.

The GO069G substance of the present invention can be produced by culturing GOO69C substance-producing bacteria in a medium, and separating and collecting the GO069G substance from the resulting culture.

As the GOO69C substance-producing bacteria, any bacteria belonging to the genus Streptomyces and capable of producing the GOO69G substance can be used. For example, Streptomyces pruneogriseus furumaieto is a strain newly isolated by the present inventors from the soil of Xishuangbanna, Yunnan Province, People's Republic of China, and was found to belong to Streptomyces pruneogriseus as described below. Octa subspecies vannaensis GO069 strain (Strel) tomyces br
unneogriseusFurusai et
0kuda 5ubsp, bannaensis
GOO69) can be used advantageously.

This GO069G substance-producing bacterium has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology under accession number 706.

The mycological properties of the GO069 strain are as follows.

(a) Morphology When strain GO069 is observed under a microscope, aerial hyphae with a spiral formation extend from well-branched basal hyphae, and no whorled branches are observed. A mature spore chain is a chain of 10 or more spores, and the size of the spores is 0.96-1.4 x 0.8-0.96.
The surface of the spore is smooth, on the order of microns.

(b) Growth status on various media (1) Glucose-asparagine agar medium (cultured at 28°C) White to pale pink-white aerial mycelia are grown on the growing light yellow tea, and the solubility of the light yellow tea is slightly Produces pigment.

(2) Glycerin-asparagine agar medium (ISP medium -5, cultured at 28°C) Gray-white aerial mycelium grows on the pale yellow growth, producing a slightly pale yellow soluble pigment.

(3) Starch/inorganic salt agar medium (ISP medium-4, 2
(cultivated at 8°C) Bright brownish aerial mycelium grows on the growing light olive, and no production of soluble pigments is observed.

(4) Yeast/malt agar medium (ISP medium-2, 28
℃ culture) Aerial mycelium of brown ash grows on the dark brown growth, producing a slight brown soluble pigment.

(5) Tyrosine agar medium (cultured at 28°C) Aerial mycelium with a bright yellow color grows on the dark brown growth, producing a dark brown soluble pigment.

(6) Nutrient agar medium (cultured at 28°C) Growth is brown, aerial mycelia do not attach, and brown soluble pigment is produced.

(1) Oatmeal agar medium (ISP medium -3, 28℃
Cultivation) Aerial mycelia of the yellowing method grow on the pale yellow growth, and the soluble pigment becomes slightly yellowish.

(8) Sucrose/nitric acid agar medium (cultured at 28°C) A slight grayish-white aerial mycelium grows on the colorless growth, and no soluble pigment is observed.

(9) Cracilinico 7NO11 synthetic agar medium (cultured at 28°C) The growth is yellowish brown, aerial mycelia do not attach, and the soluble pigment is yellowish brown.

(10) Gauss N011 synthetic agar medium (cultured at 28° C.) Aerial mycelium of the yellowing method grows on the growing yellow tea, producing soluble pigment to the extent that it slightly stains the yellow tea.

(C) Physiological properties (1) Growth temperature range It grows at any temperature between 20 and 37°C.

(2) Gelatin liquefaction (28℃ culture), cellulose degradability, skim milk coagulation and peptonization (37℃ culture) were negative, starch hydrolysis, nitrate reduction, hydrogen sulfide production, melanin-like pigment Production (tryptone yeast broth, ISP medium-1; peptone yeast iron agar,
ISP medium-6 = tyrosine agar, ISP medium-7) is positive.

(3) Utilization of carbon sources (Pridham-Gotlieb agar medium, cultured at 28°C) D-xylose, inositol, D-mannitol, D
- It grows well using fructose, L-rhamnose, sucrose, raffinose, and D-glucose, but does not use cellulose or L-arabinose.

(4) Analysis of cell wall constituents L-diaminopimelic acid is detected in the whole bacterial cell hydrolyzate.

Based on these properties, known bacterial species were selected from Nonomura's Journal or Fermentation Chikunoroshii.
of Fermentation Technology
ogV) 52.

78-92 (1974), Purge Aids Manual or Deterministic Native Bacteriology, 8th Edition, (Bergey's Manual
of Determinative Bacteri
8th edition) 1974, and Handbook of C1
assignment of streptomyc
etes), 1975, GOO6
Among the 9 strains, the most green species is Streptomyces pruneogriseus furumai etoocta (Strept.
omyces brunneogriseusFur
usai et 0kuda) (Journal of Antibiotics)
antibiotics).

2U, 85-90 (1968)).

Comparing strain GOO69 with Streptomyces pruneogriseus flumaieto octa, the latter exhibits milk coagulation, peptonization, gelatin liquefaction, and utilizes L-arabinose but not inositol. Although there are some differences, the two are in good agreement. From these results, G
The OO69 strain is considered to be a subspecies of Streptomyces prunneogriseus, and Streptomyces prunneogriseus furumai et octa subsp.
iseus Ftlrulai etOkuda
5ubsp, bannaensis).

Next, a case will be described in which the GO069G substance of the present invention is produced by culturing, for example, the above-mentioned GO069G substance-producing bacteria belonging to the genus Streptomyces in a medium.

The culture method basically follows the culture method φ for general microorganisms, but the stirring aeration culture method using a liquid medium is usually advantageous.

The medium used for culture may be any medium containing a nutrient source that can be used by the GO069G substance-producing bacteria belonging to the genus Streptomyces.

That is, a synthetic medium, a semi-synthetic medium, or a natural medium can be used. The composition of the medium includes carbon sources such as glucose, mannose, glycerin, sucrose, molasses, starch, and liquefied starch, and nitrogen sources such as meat extract, casamino acids, peptone, gluten meal, cottonseed oil, and soybean. Flour, corn steep liquor, dried yeast, ammonium phosphate, ammonium sulfate,
Urea etc. are used. Inorganic salts such as metal phosphates, carbonates, and chlorides, such as disodium hydrogen phosphate, potassium dihydrogen phosphate, calcium carbonate, and magnesium chloride, may also be added as necessary. In addition, if foaming is significant during culturing, antifoaming agents such as vegetable oils such as soybean oil and linseed oil, higher alcohols such as octadecanol, tetradecanol, and heptadecanol, and various silicon compounds may be added as appropriate. good.

GO069G substance-producing bacteria usually grow at a temperature of about 20 to 40°C. Production of GO069C substance is 25-40
It is preferable to carry out the heating in a range of approximately .degree. The culture time for the main culture is usually about 30 to 100 hours, and the culture time may be further extended as the medium becomes more concentrated. During cultivation, it is preferable to blow sterile air into the medium, as is commonly done in agitated aerated cultivation. For efficient growth of microorganisms, the amount of air used for tank culture should be approximately 0.3 to 0.6 parts by volume per 1 part of the nutrient medium per minute, and stirring should be performed at approximately 200 to 400 revolutions per minute. It is better to do so. Based on the culture conditions described above, depending on the characteristics of the production strain used,
Optimum conditions can be selected and applied as appropriate.

The GO069G substance accumulated in the culture in this way is mainly contained in the culture solution, so after removing the bacterial cells from the culture by centrifugation or filtration, general antibiotics can be manufactured from the fermentation solution. It can be separated and purified by conventional means.

That is, vacuum concentration, freeze drying, solvent extraction, liquid exchange, treatment with resins such as cation exchange resins, anion exchange resins, and nonionic adsorption resins, or with adsorbents such as activated carbon, silicic acid, silica gel, and alumina. The GO069G substance can be separated, purified, and collected by using treatment, crystallization, recrystallization, or other means singly or in combination in any order, or repeatedly. still,
Confirmation of the active ingredient can be done, for example, in Saccharomyces salmon, whose growth is inhibited by the GO069G substance.
This can be carried out by a bioassay method using microorganisms such as S. omyces sake.

The salt used for the GO069C substance refers to salts commonly used in medicine, such as alkali metal salts such as sodium, potassium, and lithium, alkaline earth gold R salts such as calcium and magnesium, and organic salts such as trimethylamine and triethylamine. Amine salts, etc., and acids used include inorganic acid salts such as hydrochloric acid, nitric acid, and sulfuric acid, or formic acid,
Examples include organic acid salts such as acetic acid, oxalic acid, maleic acid, succinic acid, tartaric acid, and para-toluenesulfonic acid.

辷-mis Next, a more detailed explanation will be given with reference to examples.

Example fermentation medium (1.0% starch, 2.0% soybean meal)
%, peptone 0.5%, sodium chloride 0.4%, ammonium sulfate 0.25%, glucose 2.0%, dipotassium hydrogen phosphate 0.002%, magnesium sulfate 0.02
5%, corn staple liquor 0.25%, calcium carbonate 0.6%), pH 7.2 to 7.4, for 500 m
Dispense 100 units into 10 12-volume Erlenmeyer flasks, and
After sterilization in an autoclave at 0°C for 20 minutes, 0.5 cm2 of spores and mycelium of GO069C substance-producing bacteria (Feikokenjoyori No. 706) grown on a starch agar medium were inoculated into each flask, and sterilized at 27°C for 20 minutes. Rotary shaking culture for 2 days (22 m/min)
0 rotations, 10 CI).

Next, each i
om+ inoculation and cultured at 27°C for 3 days with rotational shaking (2 min/min).
20 rotations, 10C1) were performed.

After the culture was completed, the cells were centrifuged, and the supernatant liquid was subjected to the following separation operation.

Culture solution 5Q was mixed with MCI gel HP-20 (Mitsubishi Kasei > IQ).
Adsorb into the resin tower. After eluting the active ingredient with 0.5Q of water, the 0.5Q aqueous solution containing this active ingredient was added to Amberlite IRA-410 (manufactured by Rohm and Haas, USA).
Passed at 0.49. Amberlight I RA-41
0.59 of the aqueous solution containing the active ingredient that passed through the solution was freeze-dried to obtain 8 g of light brown powder.

Dissolve 1 g of the light brown powder in water 2-, and add C+a-OOS.
Column (15x
500 mm), the active ingredient is eluted with water, and the active eluted fraction is lyophilized.

Repeat the above operation to obtain coarse powder 801 of GO069G substance.
I got g.

The coarse powder 801III of the obtained GOO69C substance was 8-
After dissolving in water, the active ingredient was charged to Micro Bonda Pack-C+a (manufactured by Waters, 7.8X300+ei) at a rate of 50μQ each, and the active ingredient was eluted at a flow rate of 1.5-min/min using water as the mobile phase, and the retention time was 9. 0.4 to 9.9 minutes was fractionated, and the fractionated aqueous solution was freeze-dried to obtain 101 g of GO069G substance.

[Brief explanation of drawings]

Figure 1 shows the infrared absorption spectrum of the GO069C substance.
FIG. 2 shows the ultraviolet absorption spectra of the GO069G substance. (That’s all) One! “Hake J-1

Claims (1)

[Claims] (1) Formula ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ G0069C substance represented by and its salt.