JPH0365360B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention is based on the formula This relates to the G0069C substance represented by and its salt.

The G0069C substance of the present invention and its salt have anticancer and antifungal effects and are useful as medicines.

Prior Art The G0069C substance and its salt of the present invention are new substances that have not been described in any literature.

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

Disclosure of the Invention The G0069C substance of the present invention is represented by the above formula () 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 ^KBr _nax (cm ^-1 ): 3350,
1770, 1670, 1630, 1390, 1350, 1135, 1055.
The chart is shown in Figure 1.

(4) Proton NMR spectrum, δ (ppm), D ₂ O
Medium: 1.58 (3H, d, J = 7Hz), 2.89 (1H, d, J
= 17Hz), 3.40 (1H, dd, J = 17Hz, 2.5Hz),
3.86 (1H, dd, J = 11.5Hz, 6.7Hz) 4.48 (1H,
d, J = 2.5Hz), 5.47 (1H, d, J = 2.5Hz).

(5) Ultraviolet absorption spectrum in H ₂ O: No characteristic absorption between 220 and 350 nm. The chart is shown in Figure 2.

G0069C 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]heptane or 2-(alanyl-3-serino) Serino) Clabham.

The G0069C substance of the present invention can be produced by culturing G0069C substance-producing bacteria in a medium, and separating and collecting the G0069C substance from the resulting culture.

As the G0069C substance-producing bacteria, any strain belonging to the genus Streptomyces and capable of producing the G0069C substance can be used. For example, a strain of Streptomyces bruneogriseus furumai et okuda was newly isolated by the present inventors from soil in Xishuangbanna, Yunnan Province, People's Republic of China, and was found to belong to Streptomyces bruneogriseus as described below. Species Vannaensis G0069
Strain (Streptomyces brunneogriseus Furumai et al.
Okuda subsp.bannaensis G0069) can be used advantageously.

This G0069C 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 strain G0069 are as follows.

(a) Morphology Under a microscope, strain G0069 shows that 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 spore size is 0.96~
The size of the spore is approximately 1.4 x 0.80 to 0.96 microns, and the surface of the spore is smooth.

(b) Growth status on various media (1) Glucose-asparagine agar medium (28℃
Culture) White to pale pink-white aerial mycelia grow on the growing light yellow tea, producing a slight amount of soluble pigment of light yellow tea.

(2) Glucose-asparagine agar medium (ISP
Medium-5, 28°C culture) 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. Cultured at 28℃) 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,
(Cultivated at 28℃) Brown ash aerial mycelium grows on the dark brown growth, producing a slight brown soluble pigment.

(5) Tyrosine agar medium (cultured at 28°C) Bright yellowish gray aerial mycelium grows on dark brown growth, producing 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.

(7) Oatmeal agar medium (ISP medium-3, 28
℃ culture) Yellowish gray aerial mycelium grows on the pale yellow growth, and the soluble pigment has a slightly yellowish tinge.

(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) Krasilnikov No.1 synthetic agar medium (28℃
Culture) Growth is yellowish brown, aerial mycelium is not attached, and soluble pigment is yellowish brown.

(10) Gauss No. 1 synthetic agar medium (cultured at 28℃) Aerial mycelium of yellowish ash grows on the growing yellow tea.
It produces a soluble pigment that gives a slightly yellowish brown color.

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

(2) Liquefaction of gelatin (28℃ culture), degradability of cellulose, coagulation and peptonization of skim milk (37
℃ culture) was 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) are positive.

(3) Utilization of carbon sources (Pridham-Gotlieb agar medium, cultured at 28°C) Grows well using D-xylose, inositol, D-mannitol, D-fructose, L-rhamnose, sucrose, raffinose, and D-glucose. and cellulose,
L-arabinose is not used.

(4) Analysis of cell wall components L,L-diaminopimelic acid was detected in the whole bacterial cell hydrolyzate.

Based on these properties, known bacterial species were identified by Nonomura's Journal of Fermentation Technology 52 .
78-92 (1974), Bergey's Manual of Determinative Bacteriology, 8th edition, by George Pridham.
Determinative Bacteriology, 8th edition)
1974, and Handbook of Classification of Streptomycetes.
Streptomycetes), 1975.
The species most closely related to strain G0069 is Streptomyces brunneogriseus Furumai.
et Okuda) [Journal of Antibiotics, 21 , ~85~
90 (1968)].

A comparison of strain G0069 and Streptomyces bruneogriseus furumaieto okuda shows that 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. Based on these results, strain G0069 is considered to be a subspecies of Streptomyces bruneogriseus, and Streptomyces bruneogriseus furumaieto okuda subspecies Bannaensis G0069 (Streptomyces
brunneogriseus Furumai et Okuda subsp.
bannaensis).

Next, a case will be described in which the G0069C substance of the present invention is produced by culturing, for example, the above-mentioned G0069C substance-producing bacteria belonging to the genus Streptomyces in a medium.

The culturing method is basically similar to that of general microorganisms, but the stirring aeration culture method using a liquid medium is usually advantageous. The medium used for culture is
Any medium containing a nutrient source that can be used by G0069C substance-producing bacteria belonging to the genus Streptomyces may be used.

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, corn meal, cottonseed oil, Soy flour, corn steep liquor, dried yeast, ammonium phosphate, ammonium sulfate, urea, etc. are used. Also, for example, disodium hydrogen phosphate, potassium dihydrogen phosphate,
Inorganic salts such as metal phosphates, carbonates, and chlorides such as calcium carbonate and magnesium chloride are also added as necessary. In addition, if foaming occurs significantly during culture, 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 too.

G0069C substance-producing bacteria usually grow at a temperature of about 20 to 40°C. G0069C substance production is 25~40℃
It is preferable to do this within a certain range. 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 aerated aerated cultivation. For efficient growth of microorganisms, the amount of air used for tank culture should be approximately 0.3 to 0.6 volume parts per 1 volume part of the nutrient medium per minute, and the air flow rate should be 200 to 200 parts per minute.
It is best to stir at around 400 rpm. From the culture conditions described above, optimal conditions can be appropriately selected and applied depending on the characteristics of the production strain used.

G0069C thus accumulated in the culture
Since the substance is mainly contained in the culture solution, after removing the bacterial cells from the culture by centrifugation or filtration, the substance can be separated and purified from the solution by conventional means used in the production of general antibiotics. That is, vacuum concentration, freeze drying, solvent extraction, liquid exchange, treatment with resins such as cation exchange resins, anion exchange resins, nonionic adsorption resins, or activated carbon,
G0069C substances can be separated, purified, and collected by treatment with adsorbents such as silicic acid, silica gel, and alumina, or by using methods such as crystallization and recrystallization alone or in combination in any order, or repeatedly. can. In addition, to confirm the active ingredients,
For example, it can be carried out by a bioassay method using microorganisms such as Saccharomyces sake whose growth is inhibited by the G0069C substance.

The salts used in the G0069C substance refer to salts commonly used in medicine, such as alkali metal salts such as sodium, potassium, and lithium, alkaline earth metal salts such as calcium and magnesium, and organic amines such as trimethylamine and triethylamine. Examples of acids used include inorganic acid salts such as hydrochloric acid, nitric acid, and sulfuric acid, or formic acid, acetic acid, oxalic acid, maleic acid, succinic acid, tartaric acid, and para-
Examples include organic acid salts such as toluenesulfonic acid.

Example Next, a more detailed explanation will be given with reference to examples.

Example Fermentation medium (1.0% starch, soybean meal
2.0%, peptone 0.5%, sodium chloride 0.4%, ammonium sulfate 0.25%, glucose 2.0%, dipotassium hydrogen phosphate 0.002%, magnesium sulfate 0.025
%, corn staple liquor 0.25%, calcium carbonate 0.6%), pH 7.2 to 7.4, was dispensed into 10 500 ml Erlenmeyer flasks, sterilized in an autoclave at 120°C for 20 minutes, and then placed on a starch agar medium. Inoculate each flask with 0.5 cm ² of spores and mycelium of G0069C substance-producing bacteria (Feikoken Joyori No. 706) grown in
Rotary shaking culture (220 revolutions per minute, 10 cm) for 2 days at °C.
I did this.

Next, 10 ml of the above culture solution was inoculated as a seed culture into 100 500 ml Erlenmeyer flasks into which 100 ml of the fermentation medium was dispensed, and cultured with rotary shaking (220 revolutions per minute, 10 cm) at 27°C for 3 days. Summer. After the culture was completed, the cells were centrifuged, and the supernatant liquid was subjected to the following separation operation.

Culture solution 5 was added to MCI gel HP-20 (manufactured by Mitsubishi Kasei)
Adsorb it into the resin column No. 1. Active ingredient in water 0.5
After elution with Amberlite IRA-410 (ROHM &
(manufactured by Haas) 0.4. Aqueous solution containing active ingredients passed through Amberlite IRA-410
0.5 was freeze-dried to obtain 8 g of light brown powder.

1 g of the light brown powder was dissolved in 2 ml of water, and C ₁₈ −
Charge it to a column (15 x 500 mm) of ODS resin (manufactured by Yamamura Kagaku Kenkyusho, 30 μm), elute the active ingredient with water, and then freeze-dry the active eluate fraction.

Repeat the above operation to obtain 80% coarse powder of G0069C substance.
I got mg.

After dissolving 80 mg of the obtained coarse powder of the G0069C substance in 80 ml of water, it was charged into Micro Bonder Pack C ₁₈ (manufactured by Waters, 7.8 x 300 mm) in 50 μ increments.
The active ingredient is eluted with water as the mobile phase at a flow rate of 1.5 ml per minute, and the fraction with a retention time of 9.4 to 9.9 minutes is fractionated, and the fractionated aqueous solution is lyophilized to obtain G0069C substance.
Obtained 10 mg.

[Brief explanation of drawings]

Figure 1 shows the infrared absorption spectrum of G0069C substance.
Figure 2 shows the ultraviolet absorption spectra of the G0069C substance.

Claims (1)

[Claims] 1 formula G0069C substance represented by and its salt.