JPH0141636B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a 17-membered ring lactone compound T-2636L having antibacterial activity and salts thereof, and this compound is useful for preventing and treating swine dysentery and promoting the growth of pigs. Japanese Patent Publication No. 1983-6071 describes the manufacturing method of antibiotics T-2636A, B, and D, and Japanese Patent Publication No. 1982
- Publication No. 20959 describes the manufacturing method of antibiotic T-2636C,
Japanese Patent Publication No. 1973-7355 describes antibiotic
- The manufacturing method for 2636E and F is described. Furthermore, Japanese Patent Publication No. 1974-74 has T.
T-2636A is produced by enzymatic reaction using -2636C as raw material.
Japanese Patent Publication No. 47-4691 describes a method for producing T-2636C using an enzyme reaction using T-2636A as a raw material. It is known that all of the above antibiotics T-2636, except for T-2636B, are 17-membered ring lactone compounds (Japanese Patent Publication No. 10317/1983). Under these circumstances, the present inventors isolated microorganisms from a large number of soils for the purpose of searching for new antibiotics, and as a result of separating and searching for the antibiotics produced by them, certain types of microorganisms were found to be novel. that the microorganism belongs to the genus Streptomyces; that by culturing the microorganism in an appropriate medium, a large amount of the new antibiotic can be accumulated in the culture; I learned that it is possible to take advantage of its physicochemical properties and collect it at any desired purity. And, the antibiotic is the above-mentioned antibiotic T-
While 2636A, B, C, D, E, and F are neutral fat-soluble substances, two new acidic fat-soluble substances 17
It was found that this compound is a ring-membered lactone compound that can be interconverted under certain conditions. The inventors have decided to refer to these compounds as T-2636K,L. The present invention has been completed based on this knowledge. That is, the present invention provides a 17-membered ring lactone compound T
-2636L or its salts. T-2636L of the present invention can be produced by using a bacterium belonging to the genus Streptomyces that produces at least one of the 17-membered ring lactone compounds T-2636K or L. For example, a strain called No. T-2636, which the present inventors isolated from soil in the Nukata region of Osaka Prefecture, is an example of a strain most advantageously used in the method of the present invention. The properties of No. T-2636 strain on the medium are as follows, for example. (1) Morphology Spore-forming hyphae exhibit simple branching or pseudo-whorled filaments (Pseudoverticillus), with loop-shaped or spiral-shaped tips and attach spores in a chain. The spores are oval or oval in shape, and the size is 0.1-
It is 1.0μ×0.9−1.5μ and its surface is smooth. (2) Culture characteristics Regarding culture findings, basal hyphae are generally colorless, aerial hyphae are brown or brownish gray, and in almost all media, soluble pigments are not produced and non-・It is chromogenic. In addition, this strain has a medium pH of 5 to 9 and a culture temperature of
It grows well between 20℃ and 45℃. Description showing the properties of this bacterium in various media
Symbols with Rdg. are those in Ridgiway's Standard Color Name Table. 1 Tuapetsk agar medium Growth: thinly spread, colorless Aerial mycelium: poor, powdery, white to light drab (light grayish brown) Rdg・XL, 17′′′′—
b) Back side: colorless Soluble pigment: none 2 Glucose Czapetsk agar medium Growth: thinly spread, colorless Aerial mycelium: poor, white or light drab (light grayish brown) (Rdg・XL, 17′′′′-b ) Back side: Colorless Soluble pigment: None 3 Glycerol/Tsapetsk agar medium Growth: Thin spread, colorless Aerial mycelium: Poor, powdery, white to light cinnamon drab (Rdg・XL, 13′′′′-b)
Or write drive (Rdg/XL
, 17′′′′-b) Back side: Colorless Soluble pigment: None 4 Glucose-asparagine agar medium Growth: Spreading and showing fairly good growth, colorless Aerial mycelium: Quite abundant, powdery, light drab (Rdg・XL, 17′′′′-b) to drab (Rdg・XL, 17) or drab gray (Rdg・XL, 17′′′′-d) and sometimes white. Back side: colorless to pale yellow Soluble pigment: None 5 Nutrient agar medium Growth: Spreading and showing fairly good growth, colorless to pale yellow Aerial mycelium: Poor, white, slightly light drab (Rdg・XL, 17' There is a part ``''-b). Back side: Colorless to light brown Soluble pigment: None 6 Glucose nutrient agar Growth: Spreading and wrinkled abundant growth, colorless to pale yellow Aerial mycelium: Poor, white to drab gray (Rdg・XL, 17' ′′′-d) Back side: Light brown Soluble pigment: None or light brown pigment may be produced. 7 Glycerol nutrient agar medium Growth: Abundant growth with wrinkles, colorless to light yellow Aerial mycelium: Considerable growth, white Back side: Light brown or dark brown in places. Soluble pigment: None or pale yellow 8 Meat Juice Growth: Quite abundant, showing colorless to pale yellow ring-shaped growth, with growth also visible on the bottom of the vessel. Aerial mycelium: None or slightly grown, white Soluble pigment: None 9 Peptone agar growth: Poor, thinly spread, colorless Aerial mycelium: Poor, white Back side: Colorless Soluble pigment: None 10 Starch agar Medium Growth: Poor, colorless Aerial mycelium: None Back side: Colorless Soluble pigment: None11 Yeast extract agar medium Growth: Abundant wrinkled growth, colorless to light brown Aerial mycelium: Quite abundant, white to drab gray ( Rdg・XL, 17′′′′-d) or Mouse Gray (Rdg・LI, 15′′′′) Back side: Yellowish brown Soluble pigment: None12 Whole egg medium (37℃) Growth: Spreading It grows profusely, starting from colorless and later spreading out as moist black spots from the upper part of the slope. Aerial mycelium: Abundant, white to pale olive gray (Rdg・LI, 23′′′′-f) or mouse gray (Rdg・LI, 15′′′′)
No, Drab Gray (Rdg・XL
, 17′′′′-d) Soluble pigment: None13 Potato section medium Growth: Abundant, spreading, wrinkled growth, colorless Aerial mycelium: Abundant, white to drab gray (Rdg/XL, 17′′ ′′-d) or Mouse Gray (Rdg・LI, 15′′′′) Soluble pigment: None14 Carrot section medium Growth: Abundant, spreading, wrinkled growth, colorless Aerial mycelia: Abundant, powdery , white to light drab (Rdg・XL, 17′′′′-b) to drab (Rdg・XL, 17′′′′) Soluble pigment: None15 Litmus milk (37℃) Growth: Ring-shaped growth colorless to pale yellow Aerial mycelium: poor, white Soluble pigment: none Peptonizes without coagulating. PH remains almost unchanged. 16 Leffler's serum medium (37°C) Growth: Spread and wrinkled growth, colorless aerial hyphae: absent or slightly grown, drab,
Gray (Rdg・XL, 17′′′′-d) Soluble pigment: None Hardly liquefies, but shows weak liquefaction. 17 Gelatin puncture (24℃, 1 month) Growth: Poor, ring-shaped growth, colorless Aerial mycelium: Poor, white or pale drab serpent (Rdg・XL, 17′′′′-f) Soluble pigment :None Shows liquefaction properties for liquids that hardly liquefy or are weakly liquefied. 18 Cellulose medium Growth: May not grow or may show slight colorless growth. Aerial mycelium: not growing or slightly growing, drab gray (Rdg・XL, 17′′′′-d) 19 Bennet's medium Growth: abundant, spreading, colorless Aerial mycelium: abundant, powdery, drab (Rdg・XL,
17′′′′′) Back side: Brown-gray Soluble pigment: None 20 Calcium malate medium Growth: Abundant, spreading and colorless, or may show poor, pale growth. Aerial mycelium: shows considerable growth, powdery, white to light drab (Rdg/XL, 17
′′′′-b) or poor and white Back side: Colorless to pale yellow Soluble pigment: None 21 Tyrosine agar medium Growth: Appropriate growth, spreading, colorless to cream buff (Rdg・,
19″-d) Aerial mycelium: poor, white Back side: colorless to light yellow Soluble pigment: None 22 Nitrate reduction It shows almost no reducing property in Czapetsk liquid medium, but it shows quite strong reducing property in peptone water. 23 Starch hydration Decomposition Growth area: 7-10mm Enzyme area: 26-30mm (3) Carbon source availability Method of Pridham and Gottlieb (J.
Bacteriol, Vol. 59, p. 107, 1948). Erythritol + Adonitol ± D-Sorbitol + Inositol + D-Mannitol Drusitol + D-Xyrose L-Arabinose L-Sorbose + D-Galactose D-Glucose D-Fructose Rhamnose + Melibiose + Maltose Syucrose + ~ Lactose Roughinose Trehalose Salicin Esquiulin + Inulin + Dextran Sodium acetate Sodium succinate Sodium citrate D-mannose Starch Glycerin Control +: Grows very well. :Grows well. +: Slight growth. ±: Very little growth. The above characteristics and the Actinomycetes by S.A. Watzman, Volume 2, The Williams & Wilkins Company (1961), Bird's Manual of Determinative Bacteriology, 7th Edition, The Williams. and Wilkins Company (1957), L. Etlinger et al. Archif Microbiology Vol. 31, 326-358.
Page (1958), it is considered to be similar to Streptomyces lotiei. Streptomyces lotiei has linear or spiral spore-forming hyphae that quickly liquefy gelatin and produce a pale yellow soluble pigment. The potato sections turn reddish brown. While the growth on starch medium turns brown, the spore-forming bacteria of No. T-2636 are rarely seen in a straight line, but generally in a loop or spiral shape. Slightly liquefies gelatin and produces no soluble dyes. Potato sections are hardly colored and growth on starch medium is colorless. However, since other culturing properties are very similar, No. T-2636 is considered to be a mutant of Streptomyces rothiei. So No.T-2636
is Streptomyces rochei var volubilis
It was named. This strain has been deposited with the National Institute of Microbiology, Agency of Industrial Science and Technology under application accession number 6155, and with the Fermentation Research Institute under IFO 12507. The following bacteria have been reported as producing neutral macrolide antibiotics and other antibiotics similar to T-2636, but No. T-2636 is different from any of them. Similar antibiotic-producing bacteria 1 Streptomyces frasiae (megacidin-producing bacteria) ``L.
Tile Ante Abiesenjaften Volume 88
989 pages (1958)] 2. Streptomyces violaceoniger (Lancamycin and lancasidin producing bacteria) [Igorman et al., Special Publication No. 16700] 3. Streptomyces bikiniensis (Calcomycin producing bacteria) [Razia philips. Frohart et al., Japanese Patent Publication No. 36-22650] 4. Streptomyces albogriseolus (cominomycin-producing bacterium) [Egoiman et al., German Patent No. 1110820 specification] 5. Streptomyces labentenillae mutant strain (aldogamicin-producing bacterium) ) [M. Beekunstmann et al. Anti-Microbial Age and Chemotherapy p. 87, 1964] 6 Streptomyces lavendeurae mutant strain (Alzia mycin strain antibiotic producing bacterium) [Toru Hasegawa et al. Takeda Research Institute Annual Report Vol. 25 15 pages (1966)] 7 Streptomyces goshikiensis (pandamycin producing bacterium) [Shinichi Kondo et al. Special Publication 1977-
Publication No. 26948] 8 Streptomyces rimosus (Niniutramycin-producing bacterium) [Dewielfuemin et al. [G.M., G., Sakamoto et al., Journal of Antibiotics, Vol. 15, A, p. 98 (1962)] Of course, the properties of radial bacteria, especially microorganisms belonging to the genus Streptomyces, may vary naturally or artificially. This is well known, and Streptomyces rothiei var volubilis is no exception. That is, Streptomyces lothiei var. volubilis may mutate naturally or artificially, resulting in bacteria with different findings on the culture medium. However, it goes without saying that even these strains can be used for the purpose of the present invention as long as they do not lose the ability to produce at least one of T-2636K or L. In the method of the present invention, for example, Streptomyces lothiei var. volubilis and its variants or mutant strains thereof (hereinafter referred to as T-2636 producing bacteria) are cultured on a medium. The medium may be either liquid or solid, but it is more convenient to use a liquid medium. In the medium, T-
2636 Assimilable carbon sources such as glucose, soluble starch, digestible nitrogen sources such as corn staple liquor, polypeptone, raw soy flour, other inorganic salts, heavy metal salts, antifoaming agents, etc. are included. To culture T-2636-producing bacteria in a medium containing these nutrients, an aerated agitation culture method using a liquid medium is advantageous, but shaking culture may also be used in some cases. For example, for strain No. T-2636, glucose 5%, glycerin 0.5%, polypeptone 1.0%, meat extract 0.5%, raw soybean flour 1.0%, magnesium sulfate.
0.1%, calcium carbonate 0.5% (PH7.0) soybean oil 0.3
When cultured for 30 to 72 hours in a medium containing T-2636K and L, T-2636K and L accumulate in the culture solution. Although T-2636A, B, C, D, E, and F are co-produced in the culture solution, these can be easily removed by the separation method described below. Target T from culture
-2636L can be obtained by appropriately using separation means commonly used to collect metabolites produced by microorganisms from cultures of the microorganisms. For example, T-2636K and L are acidic fat-soluble substances, while T-2636A, B, C, D, E, and F, which are co-produced at the same time, are neutral fat-soluble substances, so they can be collected by means that utilize this property. I can do that. On the other hand, T-2636K is an acetyl form of T-2636L, and can be separated by changing the ratio of both in the culture solution using the action of enzymes contained in the culture solution. That is, when the culture solution is extracted with a non-aqueous solvent that does not contain an acetyl donor, such as methyl isobutyl ketone or n-butanol, an extract that mainly contains T-2636L is obtained, whereas a non-aqueous solvent that contains an acetyl donor is obtained. When extracted with a water-soluble solvent such as ethyl acetate, mainly T-
An extract containing 2636K is obtained. When the extract thus obtained is extracted with a dilute alkaline solution, such as a dilute sodium bicarbonate solution, the target substance moves to the aqueous layer. Next, these aqueous layers are adjusted to pH 2 to 4 with a dilute acid such as dilute hydrochloric acid, and extracted with a non-aqueous solvent to obtain the target extract containing T-2636L as the main component. During these operations, neutral fat-soluble substances such as T-2636A, B, C, D, E, and F are efficiently removed. The obtained extract is washed with water and then concentrated to obtain a crude substance of T-2636L, which is the compound of the present invention. When these crude substances are subjected to column chromatography using an adsorbent carrier such as silica gel or alumina or a high porous resin, purified T-2636L is obtained as crystals. As mentioned above, T-2636K is an acetyl form of T-2636L, and the desired compound can be produced by converting T-2636K to L using an enzymatic reaction. This conversion production method usually involves adding T-2636K dissolved in an organic solvent (e.g., methanol, ethanol) that is well miscible with water to an aqueous solution of a culture of Streptomyces bacteria or its processed product. T-2636L by stirring at room temperature.
can be manufactured. In the above, the culture solution refers to the entire culture solution itself when culturing the T-2636K or (and) L producing bacteria. In addition, the treated culture solution is
Live bacterial cells, dried bacterial cells, ground bacterial cells, crude or purified enzymes, insolubilized enzymes, etc. obtained by centrifugation, filtration, washing, drying, grinding, extraction, insolubilization, and other appropriate treatments of the culture solution. -Contains an enzyme system involved in the reaction that converts 2636K to L. T- obtained in Reference Example 1 and Example 1 described later
The physicochemical properties of 2636K or L are as follows. T-2636K (1) Shape: Colorless crystal (ethyl acetate-acetone) (2) Melting point: 180℃±10℃ (decomposition point) (3) Specific rotation: [α] ³¹ _D −48゜±10゜ (C =0.5, ethanol) (4) Molecular formula: Estimated molecular formula C ₃₄ H _41~45 NO _11~12 (5) Elemental analysis value (%) (Sample dried under vacuum at 60°C for 8 hours over phosphorus pentoxide) C, 61.96± 1.0 N, 6.32±0.5 N, 2.37±0.5 O, 28.71±1.0 (6) Ultraviolet absorption spectrum: The spectrum measured in ethanol is as shown in Figure 1, and its maximum value is λ ^EtOH _nax 229±2 nm ( E1% 1cm=727±50) (7) Infrared absorption spectrum: The spectrum measured as a potassium bromide tablet is shown in Figure 2, and the main peaks (wavelengths) are as follows. 3360, 2950, 1755, 1710, 1660, 1550,
1460, 1375, 1310, 1260, 1190, 1150, 1080,
1050, 1020, 970, 930, 890, 810, 750, 715,
620 (8) Thin layer chromatography [Silica gel F ₂₅₄
(manufactured by Merck & Co.)] Solvent system Rf value Methyl ethyl ketone: ethyl acetate (2:8)
0.33 Benzene: Acetone: Acetic acid (50:50:2) 0.42 (9) Color reaction Colors blue-purple with concentrated sulfuric acid. (10) Solubility Easily soluble in acetone and alcohols, slightly soluble in chloroform, ethyl acetate, etc.
Insoluble in water. T-2636L (1) Shape: Colorless crystal (methanol-ethyl acetate) (2) Melting point: 176°C ± 10°C (decomposition point) (3) Specific rotation: [α] ²⁶ _D −38° ± 10° (C =0.5, ethanol) (4) Molecular formula: Estimated molecular formula C ₃₂ H ₄₁ NO _10-11 (5) Elemental analysis value (%) (Vacuum dried over phosphorus pentoxide at 60℃ for 8 hours) C, 61.80±1.0 H , 6.58±0.5 N, 2.12±0.5 O, 28.39±1.0 (6) Ultraviolet absorption spectrum: The spectrum measured in ethanol is as shown in Figure 3, and its maximum value is λ ^EtOH _nax 227±2 nm (E1% 1cm=813±50) (7) Infrared absorption spectrum: The spectrum measured as a potassium bromide tablet is as shown in Figure 4, and the main peaks (wavelengths) are as follows. 3400, 2950, 1755, 1720, 1665, 1520,
1450, 1380, 1250, 1165, 1140, 1070, 1010,
970, 880, 705, 620, 550 (8) Thin layer chromatography [Silica gel F ₂₅₄
(Manufactured by Merck & Co.) Solvent system Rf value Benzene:acetone:acetic acid (50:50:2) 0.22 (9) Color reaction Colors blue-purple with concentrated sulfuric acid. (10) Solubility Easily soluble in alcohols, slightly soluble in acetone, ethyl acetate, etc., insoluble in water. Next, the biological activities of T-2636K and L will be described. That is, bouillon agar is used as a test medium for general bacteria, glycerin bouillon agar for acid-fast bacteria, and glucose bouillon agar for yeast. The spectrum is as follows.

[Table] Next, the present invention will be explained in more detail with reference to Examples. In the following, percentages indicate weight/volume %. Reference example 1 Glucose 5%, glycerin 0.5%, polypeptone 1.0%, meat extract 0.5%, raw soybean flour 1.0%, magnesium sulfate 0.1%, calcium carbonate 0.5% (PH
7.0) into two Sakaguchi flasks to culture Streptomyces rothiei var. volubilis (IFO 12507, IFO 12507, FIKEN application accession number FIKEN Bacterium No. 6155) from the slant culture. 1
Platinum loop was inoculated and cultured on a reciprocating shaker at 37℃ for 40 hours.
Using this 1.5 culture solution as a seed, use the same medium for 30 minutes.
Ported into an injected 50 stainless steel tank.
Soybean oil was used as an antifoaming agent, and 30 g was used at the time of preparation. Cultivation was carried out for 20 hours with 100% aeration, temperature, 37°C, stirring at 180 rpm, and this culture solution 10 was transplanted as a seed into the next tank, and cultured at 37°C for 43 hours. In other words, when preparing the medium, add 100 g of soybean oil as an antifoaming agent to a 200 stainless steel tank filled with the same medium 100,
During culture, 500 g was used, and the culture was carried out with 100% aeration and stirring at 200 rpm. Add High Flow Super Cell (2%) to the obtained culture solution 100, and adjust the pH of the filtrate to 80.
7, 50% of ethyl acetate was added, and the mixture was stirred for 2 hours. Separate the upper ethyl acetate layer and wash with water.
The antibacterial fraction was transferred to the aqueous layer with % sodium bicarbonate solution (10 x 2). The pH of the transferred aqueous layer was adjusted to 2 to 4 with dilute hydrochloric acid, and extracted with ethyl acetate (5x2). The extract was washed with water and concentrated, and the concentrated solution was subjected to column chromatography on silica gel (0.8, manufactured by Merck & Co., Ltd.) using toluene:ethyl acetate (2:8,4).
), the antibacterial activity category was eluted. The active fraction was concentrated, and the concentrate was crystallized from a mixed solvent of acetone and ethyl acetate. T-2636K (10.3 g) was obtained as colorless crystals. Example 1 Culture solution obtained as in Reference Example 1 (75
) was adjusted to pH 7 and extracted with n-butanol (50). After washing the extract with water, the antibacterial fraction was transferred to the aqueous layer using 2% sodium hydrogen carbonate solution (10 x 2). Adjust the pH of the dissolved aqueous layer to 2 to 4 with dilute hydrochloric acid, and
-Extracted with butanol (5x2). The extract was washed with water and concentrated, and the concentrated solution was subjected to column chromatography on silica gel (0.5), and the active fraction was eluted with ethyl acetate (5). Concentrate the active fraction,
The concentrate was crystallized from acetone. T-2636L
(8.6 g) was obtained as colorless crystals. Example 2 T-2636K (10
Dissolve g) in methanol (300ml) and add T-2636.
J.Antibiotics, 24 , 23-28 from the culture solution of the producing bacteria.
(1971) and stirred at 25°C for 2.5 hours. The reaction solution was concentrated, and the concentrated solution (1) was adjusted to pH 2.5 and extracted with the same amount of n-butanol. The extract was washed with water and concentrated, and the concentrated residue was subjected to column chromatography on silica gel (40 g). Antibiotics were eluted and fractionated with a mixture of ethyl acetate and acetone. The effective fraction was concentrated and the concentrated residue was crystallized from methanol/ethyl acetate to give colorless crystals of T-2636L (6.38 g).
was gotten.

[Brief explanation of drawings]

Figures 1 and 2 show the ultraviolet absorption spectrum and infrared absorption spectrum of the 17-membered ring lactone compound T-2636K obtained in Reference Example 1, and Figures 3 and 4 show the spectrum of the 17-membered ring lactone compound T-2636K obtained in Example 1. The ultraviolet absorption spectrum and infrared absorption spectrum of the 17-membered ring lactone compound T-2636L are shown.

Claims (1)

[Claims] 1. A 17-membered ring lactone compound T- having the following properties:
2636L or its salt (1) Shape: Colorless crystal (2) Melting point: 176°C ± 10°C (decomposition point) (3) Specific rotation: [α] ²⁶ _D −38° ± 10° (C = 0.5, ethanol) (4) Ultraviolet absorption spectrum: λ ^C2H5OH _nax nm (E1% 1cm) = 227±2 (813±50) (5) Infrared absorption spectrum (KBr), main peak (cm ^-1 ): 3400, 2950, 1755 , 1720, 1665, 1520, 1450,
1380, 1250, 1165, 1140, 1070, 1010, 970,
880, 705, 620, 550. (6) Thin layer chromatography [Silica gel F ₂₅₄
(Manufactured by Merck & Co.)]: Benzene: Acetone: Acetic acid (50:50:2), Rf
=0.22 (7) Color reaction: Colors blue-purple with concentrated sulfuric acid. (8) Solubility: Easily soluble in alcohols, slightly soluble in acetone and ethyl acetate, insoluble in water. (9) Elemental analysis values (%) (materials vacuum dried over phosphorus pentoxide at 60℃ for 8 hours): C, 61.80±1.0 H, 6.58±0.5 N, 2.12±0.5 O, 28.39±1.0