JPS6320431B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a new antibiotic saphenamycin or a salt thereof obtained from the culture by culturing a microorganism belonging to the genus Streptomyces, and also relates to a method for producing saphenamycin or a salt thereof. The properties of the new antibiotic safuenamycin according to the present invention are as follows. Safenamycin is a yellow columnar crystal with a decomposition point of 200-202°C. Specific optical rotation [α] ²⁴ _D = 0°C (c1.0, CHC ₃ ). Elemental analysis values are C68.30%, H4.54%, N6.61%, O19.75%
and the theoretical value of C ₂₃ H ₁₈ N ₂ O ₅ (C68.65%,
H4.51%, N6.96%, O19.88%),
This molecular formula was verified by the EI mass spectrum (M ⁺ : m/e402) and FD mass spectrum (M+1 ⁺ : m/e403) of safuenamycin. The infrared absorption curve measured with potassium bromide tablets is as shown in FIG. As shown in Figure 2, the ultraviolet absorption curves are 254 nm (ε106000) in methanol, 252 nm (ε74000) in 90% methanol solution containing 0.1N hydrochloric acid,
and 90% methanol solution containing 0.1N caustic soda showed an absorption maximum at 255nm (ε107000), respectively. As shown in Figure 3, the visible absorption curve is 366 nm (ε15600) in methanol solution containing 0.1N hydrochloric acid.
369nm (ε15400) in 90% methanol solution, and
In 90% methanol solution containing 0.1N caustic soda
Each showed an absorption maximum at 365 nm (ε14500).
^{The 1H} nuclear magnetic resonance spectrum measured in deuterated chloroform was δ1.98 (3H, d), 2.72 (3H, s), 6.75
(1H, d), 6.82 (1H, d), 7.29 (1H, t), 7.47
(1H, q), 8.06-8.24 (4H, m), 8.59 (1H,
dd), 8.99 (1H, dd), 11.10 (1H, s), 15.38
A signal was shown at (1H, s). ^13C nuclear magnetic resonance spectrum (in deuterochloroform) is 22.2 (q), 24.4
(q), 70.1(d), 112.4(s), 115.9(d), 123.1(d),
124.9(s), 127.6(d), 127.8(d), 130.4(d), 132.9(d
),
134.4(d), 135.4(d), 137.7(d), 139.8(s), 140.1
(s), 141.0 (s), 141.2 (s), 141.3 (s), 142
.6
(s), 163.0 (s), 165.7 (s), and 170.7 ppm (s). Safenamycin is slightly soluble in chloroform and dimethyl sulfoxide, but almost insoluble in water, methanol, and acetone. Safenamycin was analyzed using silica gel thin layer chromatography, developed with a mixture of benzene and methanol (19:1) to obtain Rf0.48, and developed with a mixture of chloroform and methanol (19:1) to obtain Rf0.87. A single spot was shown. The structure of safuenamycin is the crystal shown in Example 4 (P21/a, a = 19.605 Å, b = 12.850 Å,
c = 15.799 Å, β = 99.544°). Safenamycin is similar to griseortein A produced by actinomycetes (Journal of Antibiotics, Vol. 12, p. 55, 1959), but is clearly distinct from other known antibiotics. is,
It was confirmed that it is a new antibiotic. Safenamycin; R ₁ = H, R ₂ = CH ₃ ,

[Formula] Griseortein A; R ₁ = OCH ₃ , R ₂ = H,
R ₃ =-CH ₂ OH Safenamycin can also be produced in the form of salts, typical salts include metal salts, such as alkali metal salts and alkaline earth metal salts, especially sodium salts. . The minimum inhibitory concentration of safuenamycin against various bacteria and molds on a nutrient agar medium is shown in Table 1, and it strongly inhibits the growth of mainly Gram-positive bacteria. The acute toxicity of a 2% suspension of safuenamycin in dimethyl sulfoxide when administered intraperitoneally to mice is 125 mg/Kg. In a therapeutic experiment for murine leukemia L-1210, a 19% survival effect was observed when administered intraperitoneally for 10 consecutive days from the day of L-1210 inoculation. Furthermore, safuenamycin inhibited the proliferation of cultured cells L-5178Y by 50% at 0.15 mcg/ml. Therefore, safuenamycin can be used as an antitumor agent, as well as as a treatment for infections caused by Gram-positive bacteria in humans and animals (Table 1-(1)), and as a treatment for fish (Table 1). −(2)) is expected to be used.

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【table】

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[Table] The second gist of the present invention is to culture safuenamycin-producing bacteria belonging to the genus Streptomyces to accumulate safuenamycin or its salts,
The present invention relates to a method for producing safuenamycin or a salt thereof, in which safuenamycin or a salt thereof is collected from the culture. According to the invention, safuenamycin or a salt thereof is obtained in pure or crude form, in solution or in solid form. An example of a safuenamycin-producing bacterium is an actinomycete isolated from the soil at the Institute of Microbial Chemistry in February 1980, and was given the strain number MG314-hF8. Mycological properties of the MG314-hF8 strain 1 Morphology Under the microscope, the MG314-hF8 strain forms slightly curved aerial hyphae that are relatively longer than the branched basal hyphae, and the tips of the aerial hyphae are hook-shaped or shaped. It has a spiral shape (Spirales, Retinaculiaperti), and whorled branches are not recognized. A mature spore chain consists of 10 or more spores, and the size of the spores is 0.4~0.8×1.2~
The spores are about 1.6 microns in size and have a smooth surface. 2. Growth status in various media Regarding color descriptions: The standards shown in [ ] are based on Container Corporation of America's Color Harmony Manual (Container Corporation of America).
Corporation of America's Color harmony
manual) was used. (1) Sucrose/nitrate agar medium (cultured at 27℃) Light yellow aerial mycelia are grown on the growth of light yellow daisies [2ea, Lt Wheat] to moss green [24lg.Moss Green], and soluble pigments are grown on them. It is not allowed. (2) Glucose-asparagine agar medium (27℃ culture) Aerial mycelium of light yellow daisies [4ca, Flesh Pink] was attached to the growth of light yellow daisies [2gc, Bamboo] to dark yellowish green [24pi, Dk Leaf Green]. The soluble pigment is slightly brownish. (3) Glycerin-asparagine agar medium (ISP-
Medium 5, cultured at 27°C) White or brownish aerial mycelia were grown on the olive green [241/2pl, Dk Olive Green], and no soluble pigment was observed. (4) Starch/inorganic salt agar medium (ISP-medium 4,
(Cultivated at 27℃) Aerial mycelia of pale yellow to dark green [23pl, Dk Green] or dark yellowish green [23pi, Dk Grass Green] are grown, and aerial mycelium of pale daisy [4ca, Flesh Pink] grows on them, producing soluble pigments. It is not allowed. (5) Tyrosine agar medium (ISP-Medium 7, cultured at 27°C) Brown-white aerial mycelia were grown on the pale yellow to dark green [23pl, Dk Green] growth, and no soluble pigment was observed. (6) Nutrient agar medium (cultured at 27°C) A small amount of white aerial mycelium grows on the growth of light yellow tea [2ie, Lt Mustard Tan], and no soluble pigment is observed. (7) Yeast/malt agar medium (ISP-Medium 2, 27
℃ culture) Light tea [3ie, Lt Amber, 3ne Topaz] is grown on light tea [3ca, Shell, 4ca Flesh]
Pink] aerial mycelium, and no soluble pigments are observed. (8) Oatmeal agar medium (ISP-Medium 3, 27℃
Cultivation) Aerial mycelium of light yellow tea [4ca, Flesh Pink] was added to the growth of light yellow tea [2ga, Colonial Yellow] to dark yellowish green [23pi, Dk Grass Green] or dark yellowish green [23pg, Deep Grass Green]. It is epiphytic and no soluble pigment is observed. (9) Glycerin/nitrate agar medium (cultured at 27°C) A small amount of white aerial mycelium grows on the growth of Nibu blue-green [22ig, Sea Green], and no soluble pigment is observed. (10) Starch agar medium (cultured at 27°C) Pale aerial mycelium grows on the dark green [23pl, Dk Green] growth, and no soluble pigment is observed. (11) Malate lime agar medium (cultured at 27°C) A small amount of white aerial mycelium grows on the growth of pale yellow dailies, and no soluble pigments are observed. (12) Cellulose (cultured at 27℃) A small amount of white aerial mycelium grows on the colorless growth, and no soluble pigment is observed. (13) Gelatin puncture culture In simple gelatin medium (cultured at 20℃) and glucose peptone gelatin medium (cultured at 27℃), a small amount of white aerial mycelium was grown on the pale yellow growth, and the soluble pigment was unacceptable. (14) Skimmed milk (cultured at 37°C) White aerial mycelium grows on pale yellow growth, and no soluble pigment is observed. 3 Physiological properties (1) Growth temperature range Yeast starch agar (yeast extract 0.2
%, soluble starch 1.0%, thread agar 3.0%, PH7.0)
As a result of testing at each temperature of 20℃, 24℃, 27℃, 30℃, 37℃, and 50℃ using I think that the. (2) Liquefaction of gelatin For 15% simple gelatin (cultivated at 20℃), after incubation
Liquefaction begins around the 6th day, and its effect is moderate to weak. Glucose, peptone, and gelatin (cultured at 27°C) begin to liquefy around the 11th day after cultivation, but the effect is weak. (3) Hydrolysis of starch (starch/inorganic salt agar medium and starch agar medium, both cultured at 27°C) Water decomposition is observed from around 5 days after cultivation in both media, and its effect is stronger. . (4) Coagulation and peptonization of skim milk (skimmed milk at 37℃)
Culture) Coagulation starts around the 5th day after culture, and peptonization starts immediately after the coagulation is completed around the 7th day. Peptonization is almost completed 3 weeks after culture. The effect is moderate to strong. (5) Production of melanin-like pigments (tryptone yeast broth ISP-medium 1; peptone yeast iron agar ISP-medium 6; tyrosine agar ISP-
Medium 7; all cultured at 27°C) No production of melanin-like pigments was observed in any of the media. (6) Utilization of carbon sources (Pridham-Gotlieb agar ISP-medium 9, cultured at 27°C) L-arabinose, D-xylose, D-glucose, D-fructose, inositol, L-
It grows well using rhamnose, raffinose, and D-mannitol, and may or may not use sucrose. (7) Dissolution of malate lime (malate lime agar, 27
(°C culture) From around the 5th day after culturing, malate lime around the growth is dissolved, and its effect is moderate to strong. (8) Nitrate reduction reaction (peptone water containing 1.0% potassium nitrate, ISP-medium 8, cultured at 27°C) Often negative, but sometimes positive. To summarize the above properties, the MG314-hF8 strain belongs to the genus Streptomyces (in addition,
(L-type diaminopimelic acid (LL-DAP) was detected in all bacterial components of this strain), spiral formation of aerial hyphae was observed, no whorled branches were observed, and the surface of the spores was smooth. In various media, it forms pale yellow to very distinctive dark yellow-green or olive-green growth, and the aerial mycelia are white or pale orange. Note that almost no soluble dye is observed. The production of melanin-like pigments is negative, and the proteolytic ability is moderate to strong for coagulating and peptonizing skim milk, but the liquefying ability of gelatin is weak. In addition, starch has strong water-dissolving properties. Regarding sugar utilization, results are mixed for sucrose, but all other sugars are utilized. When searching for known bacterial species based on these characteristics, Streptomyces canarius (Streptomyces canarius) was found.
Canarius, Reference 1); Journal of Systematic
Bacteriology, Volume 22, Pages 281-282,
(1972); Reference 2): Antimicrobial Agents and
Chemotherapy, 1964, pp. 75-79) is the most closely related species. Next, we actually obtained the Streptomyces canarius ISP5528 strain and compared the results, and the summary of the results is as follows.

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[Table] As seen in Table 2, the MG314-hF8 strain exhibits properties that closely match those of the Streptomyces canarius ISP5528 strain. The bright yellow color is the origin of the species name Canarius.
It is also agreed that the growth of canary yellow (canary yellow) and that it sometimes takes on a distinctive green color. Furthermore, they also show consistent properties in that the reducing properties of nitrates are either negative or positive. The only differences are the color of the aerial mycelia and the availability of sucrose, but these are not major differences either. From the above points, the MG314-hF8 strain was used as a strain of Streptomyces canarius.
Identified as MG314-hF8. The MG314-hF8 strain was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology for storage entrustment on October 6, 1980, and the application receipt number is No. 5743. In carrying out the method for producing safuenamycin of the present invention, safuenamycin can be produced by inoculating the safuenamycin producing strain Streptomyces MG314-hF8 strain into a nutrient-containing medium and growing it aerobically. A culture containing the following is obtained. As nutrients, those known as nutritional sources for actinomycetes can be used.
For example, commercially available peptone, meat extract, corn steep liquor, cottonseed flour, peanut flour, soybean flour, yeast extract, NZ-amine, casein hydrolyzate, fish meal, sodium nitrate, ammonia nitrate, ammonia sulfate, etc. Nitrogen sources and commercially available carbohydrates such as glycerin, sucrose, starch, glucose, maltose, and molasses, or carbon sources such as fats and inorganic salts such as common salt, phosphates, calcium carbonate, and magnesium sulfate can be used. In addition, trace amounts of metal salts may be added as necessary.
These materials can be used as long as they are useful for the production of safuenamycin and can be used by the producing bacteria, and all known culture materials for actinomycetes can be used. Liquid culture is preferred for mass production of safuenamycin, and the culture temperature can be applied within a range that allows the producing bacteria to grow and produce safuenamycin. Cultivation is usually continued until sufficient accumulation of safuenamycin occurs. For example, MG314 was cultured on an agar slant in a liquid medium (PH7.4) consisting of 1.5% glycerin, 1.5% starch, 1.5% fish meal, 0.5% soybean flour, and 0.2% calcium carbonate.
- When the hF8 strain was inoculated and cultured aerobically with rotational shaking at 27°C, accumulation of the target antibiotic was observed from the second day of culture. The quantitative determination of safuenamycin is carried out by a conventional paper disc method using Bacillus subtilis PCI219 strain as the test bacterium. Safenamycin crystals (1000 mcg (potency)/mg, below)
When a 25mcg/ml chloroform solution (expressed as mcg ^* /mg) is assayed using a thick filter paper with a diameter of 8 mm,
It usually shows a blocking circle with a diameter of about 25mm. Actinobacteria for growth of safuenamycin
When culturing the MG314-hF8 strain, in addition to the above-mentioned shaking culture, a jar incubator generally used for the aerated agitation culture of microorganisms, or a large stainless steel
Steel tank culture vessels are also used for mass production. In the case of mass culture, a culture solution obtained by shaking culture for 2 to 3 days in the above liquid medium is used as a seed culture solution,
It is preferable to inoculate 1.5 to 3.0% (volume) of this. Safenamycin is fat-soluble and mainly exists in the insoluble portion in the culture solution of safuenamycin-producing bacteria. Safenamycin in a culture solution or aqueous solution can be collected using various adsorbents. When a porous nonionic adsorption resin, Diaion HP-20 (manufactured by Mitsubishi Kasei) is used as an adsorbent, the safuenamycin adsorbed to HP-20 is eluted with water-containing solvents such as methanol water, propanol water, and acetone water. , it is eluted in an even better yield with anhydrous solvents. When safuenamycin is collected from the insoluble part of the culture solution, it is extracted with a water-containing solvent such as methanol water, propanol water, acetone water, etc., and extraction with an anhydrous solvent has a higher yield. For example, a safuenamycin solution extracted with methanol from the insoluble part of a culture solution can be concentrated to precipitate safuenamycin and be purified in high yield by repeating the procedure of removing impurities from the supernatant. Crude crystals of enamycin can be obtained.
It is effective to purify the crude crystals of safuenamycin obtained by the above method using column chromatography using silica gel. Commercially available silica gels include silica gel 40, 60, and 100 (manufactured by Merck & Co.). Wakogel C-100, C-200, C-300 (manufactured by Wako Pure Chemical Industries, Ltd.) are used, and a mixture of chloroform, benzene, methanol, etc. is used as the developing solvent. The most effective developing solvent is a mixed solvent of benzene and methanol in a mixing ratio of about 49:1. In addition, safuenamycin can be used by reversed-phase chromatography using commercially available silanized silica gel or by Sephadex LH, a gel filtration agent for organic solvents.
-20 (manufactured by Pharmacia) can also be used for purification. Safenamycin can be purified in pure form by appropriately combining or repeating the above extraction, separation, or purification methods. The final method for obtaining pure safuenamycin is to precipitate crystals from a mixed solvent solution of chloroform and hexane. Examples are shown below, but since the properties and chemical structure of safuenamycin have been clarified by the present invention, various methods for producing safuenamycin can be devised based on the properties. Therefore, the present invention is not limited to the Examples, and the present invention is not limited to the Examples, but can be made by applying known means based on the properties of safuenamycin revealed by the present invention, as well as by modifying the Examples. It encompasses all methods for producing, concentrating, extracting, and purifying. Example 1 Streptomyces MG314-hF8 strain (TERM-P5743) cultured on agar slant medium was incubated with 1.5% glycerin and 1.5% starch.
%, fishmeal 1.5%, soybean flour 0.5%, calcium carbonate 0.2
500ml containing 110ml of liquid medium (PH7.4) consisting of %
It was inoculated into an Erlenmeyer flask and cultured on a rotary shaker (180 revolutions per minute) at 27°C for 48 hours to obtain a seed culture. Then 30 containing 15 liquid medium with the same composition as above
Inoculate the seed culture solution (330 ml for 3 Erlenmeyer flasks) into a stainless steel jar incubator with a capacity of 2.7 cm.
The cells were cultured at ℃ for 64 hours (agitation: 250 revolutions per minute, aeration rate: 15 per minute). The culture fluids from two jar incubators were combined and the bacterial cells were separated using a continuous centrifuge (12,000 revolutions per minute). The obtained bacterial cells (wet weight 1.5 kg) were extracted twice with methanol from No. 3 to obtain a methanol extract of the bacterial cells No. 6 (560 mcg ^* /ml). 1 at 40℃ under reduced pressure.
After concentrating, the mixture was left in an ice chamber at 8°C overnight to obtain 5g (300mcg ^* /mg) of precipitate. Yield 44.6%. Example 2 5 g of the precipitate obtained in Example 1 was dissolved in 500 ml of methanol, concentrated to 100 ml under reduced pressure at 40°C, and left overnight in an ice chamber at 8°C to form a crude powder of safuenamycin.
3g (400mcg ^* /mg) was obtained. Yield 80.0%. Furthermore, the obtained crude powder was dissolved in 50 ml of chloroform, 10 ml of methanol was added, and the resulting precipitate was removed by leaving it in an ice room at 8°C overnight. The supernatant was concentrated and dried under reduced pressure, and 2.2 g of green powder was obtained. (52/mcg ^* /mg) was obtained. Yield 70.8%. Example 3 1.6 g of the green powder obtained in Example 2 was subjected to silica gel column chromatography using 300 g of Wakogel C-200 (column: inner diameter 4.8 cm, volume 700 ml).
The mixture was developed with benzene containing 2% methanol, and fractionated into 150 ml portions. Fractions 15 to 18 were combined and concentrated under reduced pressure to form a yellow-green powder of safuenamycin.
Obtained 1.2g. Next, add this powder to Wakogel C-200,
Subjected to silica gel column chromatography (column; inner diameter 5.0 cm capacity, 490 ml) using 190 g.
Fractionation was performed as described above. Combine fractions 11-14,
After concentrating to 1 ml under reduced pressure at 40°C, 5 ml of a mixed solvent of normal hexane and chloroform (1:1) was added, and the mixture was left in an ice chamber at 8°C overnight to obtain 700 mg (895 mcg ^* / mg) of precipitated crude safuenamycin crystals. Ta. yield
73.7%. Example 4 Crude crystals of safuenamycin obtained in Example 3 700
mg was dissolved in 6 ml of chloroform and 9 ml of n-hexane was added to obtain 577 mg (953 mcg ^* /mg) of precipitated yellow crystals of safuenamycin. 200 mg of this yellow crystal was recrystallized from a mixed solvent of chloroform and n-hexane 1:1 to obtain 110 mg of yellow columnar crystals of safuenamycin (1000 mcg ^* / mg).
I got it.

[Brief explanation of the drawing]

FIG. 1 shows an infrared absorption curve measured with safuenamycin potassium bromide tablets. FIG. 2 shows the ultraviolet absorption curves of safuenamycin measured in methanol solution, 90% methanol in 0.1N hydrochloric acid solution, and 90% methanol in 0.1N caustic soda solution.
FIG. 3 shows the visible absorption curve of safuenamycin measured in the same solution as in FIG.

Claims (1)

[Claims] Primary formula: The antibiotic safuenamycin or its salt represented by: 2. Growing safuenamycin-producing bacteria belonging to the genus Streptomyces in a medium containing a nutrient source, allowing the culture to produce safuenamycin or its salt, and collecting safuenamycin or its salt from the culture. A method for producing safuenamycin or a salt thereof, characterized by: