JPS6324000B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention is based on the general formula [] (However, in the formula, when R ₁ is -CHO group, R ₂ represents a hydrogen atom, and when R ₁ is monoCH ₂ OH or -COOH, R ₂ represents the following disaccharide residue:

[Formula]) (hereinafter, compounds in which R ₁ is a -CHO group and R ₂ is a hydrogen atom) are referred to as tetrocalcin F, and R ₁ is -
A compound in which R ₁ is a CH ₂ OH group and R 2 is the above-mentioned disaccharide residue is called tetrocalcin G, and a compound in which R ₁ is a —COOH group and R ₂ is the above-mentioned disaccharide residue is called tetrocalcin H. ) and its salts and tetrocalcin F, G and H
Concerning the manufacturing method. In order to find useful antibiotics, the present inventors obtained a large number of microorganisms from the natural world and conducted research on the production of antibiotics. As a result, a bacterial strain (referred to as KY11091) was isolated from soil in Sendai City, Miyagi Prefecture.
When cultured in a culture medium, a novel antibiotic appears in the culture.
DC-11, DC-11-A-2, DC-11-A-3,
It was discovered that models such as DC-11-B were produced.
All of these compounds are considered to have very similar structural formulas due to their physicochemical properties, and the present inventors later decided to collectively name them tetrocalcins. DC-11, DC-11-A-2, DC-
11-A-3, DC-11-B are tetrocalcin A, tetrocalcin B, tetrocalcin C, respectively.
It is named tetrocalcin D. Regarding DC-11, the same applicant has already applied for a patent application in 1983.
45916 (Japanese Unexamined Patent Publication No. 138501) and patent application No. 1385-
No. 153027 (Japanese Patent Application No. 55-79322). DC-11-A-2, DC-11-A-3, DC-11
-Patent application filed by the same applicant for B in 1982-
17498, 55-24924 (JP-A-115794, 56-
122392, above two cases DC-11-A-2), patent application 1977-
152253, 55-24925 (Unexamined Japanese Patent Publication No. 1983-75500, 56-
122393, above two cases DC-11-A-3), patent application 1982-
24926 (Japanese Unexamined Patent Publication No. 56-122394, DC-11-B). Further later, tetrocalcin E-1 and E-2 were discovered. Regarding these compounds, patent application No. 55-114343 (Japanese Unexamined Patent Publication No. 57-38796) filed by the same applicant
It is described in. Furthermore, tetronolides are described in Japanese Patent Application No. 55-80482 (Japanese Unexamined Patent Publication No. 57-7479). As a result of further research, the present inventors discovered and isolated novel tetrocalcins F, G, and H, which are different from the substances described in the previous application, in the above-mentioned bacterial strain culture. It has also been found that tetrocalcin G and H can be obtained by chemically treating tetrocalcin A. Tetrocalcins F, G and H thus obtained exhibit antibacterial activity. Furthermore, as is clear from their structures, they can be used as raw materials for the production of tetronolides and tetrocalcins and their derivatives, which have already been filed. Furthermore, they themselves can be expected to exhibit antitumor properties. The specific physical and chemical properties of these compounds are as follows. (1) Tetrocalcin F Melting point: 201-204℃ Specific optical rotation: [α] ²⁷ _D = -49.4° (c = 0.61, acetone) Elemental analysis value (%) (as C ₅₅ H ₇₆ N ₂ O ₁₉ ) C H N Calculated value 61.8 7.2 2.6 Actual value 61.5 7.4 2.6 IR (KBr): Figure 1 PMR (CDCl ₃ , TMS standard): Figure 2 (2) Tetrocalcin G Melting point: 194-198℃ Specific optical rotation: [α] ²⁵ _D = -68.7゜ (c=1.0, acetone) Elemental analysis value (%) (as C ₆₇ H ₉₈ N ₂ O ₂₄ ) C H N Calculated value 61.2 7.5 2.1 Actual value 61.0 7.6 2.1 IR (KBr): Figure 3 PMR (CDCl ₃ , TMS standard): Figure 4 (3) Tetrocalcin H Melting point: 211-216°C Specific rotation: [α] ²⁵ _D = -69.2° (c = 0.72, acetone) Elemental analysis value (%) ( C ₆₇ H ₉₆ N ₂ O ₂₅ ) C H N Calculated value 60.5 7.3 2.1 Actual value 60.2 7.4 2.1 IR (KBr): Figure 5 PMR (CDCl ₃ , TMS standard): Figure 6 Next, tetrocalcin F, G and Table 1 shows the behavior of H in thin layer chromatography. Table 1 Silica gel plate (product name DC-
Fertigplatten Kieselgel 60F ₂₅₄ , E. Merck). Developer: Chloroform:methanol = 9:1
(Volume ratio) Substance Rf value Tetrocalcin A 0.57 B 0.52 C 0.56 D 0.53 E-1 0.77 E-2 0.65 F 0.66 G 0.47 H 0.27 Developer: Toluene: Acetone = 3 5.65 (capacity ratio ) Substance Rf value Tetrocalcin A 0.55 Tetrocalcin E-1 0.64 Tetrocalcin E-2 0.60 Tetrocalcin F 0.66 Developer: Ethyl acetate:acetic acid = 20:1 (volume ratio) Substance Rf value Tetrocalcin A 0.42 Tetrocalcin E-1 0.67 Tetrocalcin E -2 0.62 Tetrocalcin F 0.64 Tetrocalcin G 0.27 Next, the antibacterial activity of tetrocalcin F, G and H against various microorganisms (using a pH 7.0 medium) was evaluated in the second
Shown in the table.

【table】

[Table] From the above physical and chemical properties, tetrocalcin F,
Not only can G and H be distinguished from each other, but also these can be distinguished from other tetrocalcins. Next, the method for producing tetrocalcins of the present invention will be described. As mentioned above, tetrocalcin F, G, and H can be used by fermentation, and tetrocalcin G and H can also be produced by chemically treating tetrocalcin A. To obtain tetrocalcin F, G, or H from a microbial culture, microorganisms belonging to the genus Micromonospora and capable of producing tetrocalcin F, G, or H are cultured in a nutrient medium, and the compound is accumulated in the culture. Tetrocalcin F accumulated from the culture,
By collecting G or H, tetrocalcin F, G or H can be obtained. As the microorganism that can be used, any microorganism that belongs to the genus Micromonospora and has the ability to produce tetrocalcin F, G, or H can be used. A suitable bacterium is Micromonospora tyarcea KY11091 strain. The strain has been deposited as FIKEN Bibori No. 4458 and NRRL11289, respectively. The mycological properties of this strain were disclosed in a patent application filed in 1983.
4596 (Japanese Unexamined Patent Publication No. 54-138501), Patent Application No. 54-152253 (Unexamined Japanese Patent Application No. 56-75500), No. 55-17498 (Unexamined Japanese Patent Application No. 56-1983)
115794) and 55-24926 (Japanese Unexamined Patent Publication No. 56-122394). Next, the culture method will be described. As the medium used for the culture of the present invention, both natural and synthetic media can be used as long as they contain appropriate amounts of assimilable carbon sources, nitrogen sources, inorganic substances, micronutrients, and the like. As the carbon source, glucose, starch, dextrin, mannose, fructose, sucrose, molasses, etc. are used alone or in combination. Furthermore, depending on the assimilation ability of the bacteria, hydrocarbons, alcohols, organic acids, etc. may also be used. Nitrogen sources include inorganic and organic nitrogen compounds such as ammonium chloride, ammonium sulfate, ammonium sulfur, sodium nitrate, urea, etc., but also nitrogen sources of natural origin, such as peptone, meat extract, yeast extract, dried yeast, corn steep.・Liquor, soybean flour, soybean meal powder,
Casamino acids and the like are used alone or in combination. As the inorganic substance, common salt, potassium chloride, magnesium sulfate, calcium carbonate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ferrous sulfate, calcium chloride, manganese sulfate, zinc sulfate, copper sulfate, etc. are used. Furthermore, micronutrients such as vitamin B ₁ and biotin, which promote the growth of the bacteria used and the production of tetrocalcins F, G, and H, can be appropriately added. As a culture method, a liquid culture method, especially a deep agitation culture method, is suitable. Culture temperature is 25-40
℃, especially 28 to 38℃, is optimal, and the pH of the culture medium is adjusted to PH4 to 4 by adding aqueous ammonia or ammonium carbonate solution.
10, preferably 6 to 8. When culture is carried out in liquid culture for usually 1 to 7 days, the target substance is produced and accumulated in the culture solution. When the production amount in the culture solution reaches the maximum, the culture is stopped, the bacterial cells are separated, and the target product is isolated and purified from the obtained culture solution. For the isolation and purification of tetrocalcins F, G, and H from the culture fluid, separation and purification methods commonly used to isolate metabolic products of microorganisms from the culture fluid are utilized. For example, a culture product is separated into a culture solution and bacterial cells, and the culture solution is passed as is (PH6.0) through a nonionic porous resin (trade name "HP-20" manufactured by Mitsubishi Kasei, etc.), and the antibacterial activity is After adsorbing the component having , the adsorbed substance is desorbed using methanol, acetone, ethyl acetate, etc. This desorption solution is concentrated to dryness, dissolved in water, and adsorbed onto activated carbon. The active substance is eluted from the activated carbon using an organic solvent such as acetone or ethyl acetate. This eluate is concentrated to dryness, dissolved in chloroform, suspended in chloroform, and then chromatographed using silica gel packed in a column. First, impurity yellow pigment is removed by passing chloroform through the column. The active substance can then be eluted with a mixture of chloroform:methanol (98:2, by volume). Here, tetrocalcin A,
It is eluted as a mixture of tetrocalcin B, tetrocalcin C, tetrocalcin D, tetronolide compound F-1, tetrocalcin E-1, tetrocalcin E-2, and tetrocalcin F. To carry out these separations, this active fraction is concentrated to dryness,
A small amount of chloroform:methanol:water (3:1:
1, volume ratio) of the mixture in the lower layer solvent. This was passed through a silica gel column packed using the lower layer solvent, and eluted with the same solvent: tetrocalcine E-1, tetrocalcine F, tetrocalcine E-2, tetronolide compound F-1, tetrocalcine A, tetrocalcine B, tetrocalcine. D and tetrocalcin C are eluted in this order.
Tetrocalcin F can be obtained by taking a fraction containing a large amount of tetrocalcin F, subjecting it to re-chromatography, and further processing with silica gel thin layer chromatography. Thin layer chromatography is performed using a thin layer of silica gel (product name: DC-Fertigplatten).
After developing with chloroform:methanol (9:1, volume ratio) using Kieselgel 60F ₂₅₄ (E.Merck), the corresponding portion was scraped off, eluted with the developing solvent or acetone, and the eluate was concentrated to dryness to obtain tetrocalcin F. can get. The fraction containing tetrocalcin G was eluted in column chromatography using the above-mentioned mixture of chloroform:methanol (98:2, volume ratio). It can be obtained by elution with a mixture of (volume ratio) This fraction is rechromatographed using the same system to obtain a fraction containing tetrocalcin G. After developing this with chloroform:methanol (9:1, volume ratio) using the same thin layer of silica gel as above, the corresponding portion is scraped off, eluted with the developing solvent or acetone, and the eluate is concentrated. This is further subjected to thin layer chromatography under a mixture of chloroform:acetic acid:water (2:1:1, volume ratio), and the portion corresponding to tetrocalcin G near Rf=0.52 is scraped off. Tetrocalcin G is obtained by eluting this with the developing solvent or acetone and concentrating the eluate to dryness. The fraction containing tetrocalcin H is obtained by eluting the fraction containing tetrocalcin G, and then eluting with chloroform:methanol (9:1, volume ratio). This fraction was similarly chromatographed again using the same solvent system, then thin-layer chromatography was performed using chloroform:methanol (9:1, volume ratio) in the same manner as before, the corresponding portion was scraped off, and the developing solvent or acetone was used. Tetrocalcin H is obtained by elution and concentrating the eluate to dryness. When obtaining these tetrocalcins F, G, and H, means such as extraction and crystallization may be added as necessary. In addition, to complete the separation, repeat the same chromatography as above or use Sephadex LH-
20 (Pharmacia Fine Chemicals Inc, Sweden) column may be added. Next, tetrocalcin A of tetrocalcin G and H
The chemical manufacturing method from Tetrocalcin A has the following structure. To synthesize tetrocalcin G from tetrocalcin A, tetrocalcin A is reduced with a reducing agent such as NaBH ₄ under the conditions shown in Example 3 (1), and after the reaction is completed, the produced tetrocalcin G is chromatographed on silica gel, etc. It can be obtained by the following purification method. Tetrocalcin H is obtained by oxidizing tetrocalcin A in the presence of an oxidizing agent such as Ag ₂ O under the conditions shown in Example 3 (2), and after the reaction is completed, tetrocalcin H is isolated by silica gel chromatography. obtained by doing. Next, examples will be given to illustrate specific methods for producing the compounds of the present invention. In the examples, the trend of substances was determined by bioassay using Bacillus subtilis No. 10707 or TLC chromatography scanner method (Shimadzu chromatography scanner CS910) (ultraviolet reflection method, double beam, single scan, wavelength sample 260 nm, reference 350 nm) was tracked using. Example 1 Micromonosvora charcea as a seed fungus
KY11091 was used. The strain was grown in a 2-volume Erlenmeyer flask as a seed medium [KC4g/, _MgSO4 .
7H ₂ O 0.5g/, KH ₂ PO ₄ 1.5g/, Ammonium sulfate 5.0
g/, sucrose 20g/, fructose
10g/, glucose 10g/, corn steep liquor 5.0g/, CaCO ₃ 20g/ PH7.0〕300
ml and shake at 30℃ for 48 hours (220r.pm)
Cultured. The thus obtained seed culture solution was added to a fermentation medium with the following composition in a 30-capacity jar fermenter.
15 at a ratio of 5% (volume), and cultured at 30°C using an aeration stirring method (rotation speed: 250 rpm, aeration rate: 15/min). Fermentation medium composition: soluble starch
60g/, soybean meal powder 10g/, peptone 10g/
, K ₂ HPO ₄ 0.5g/, MgSO ₄・7H ₂ O 0.5g/
, CaCO ₃ 1g/, pH adjusted to 7.2 (before sterilization) with NaOH. Culture was carried out for 72 hours without controlling the pH of the culture medium. Separate the bacterial cells and precipitate from the culture solution, and
Got 13. First, liquid 13 is passed through a column of nonionic porous resin 1 (trade name "HP-10" manufactured by Mitsubishi Kasei) to adsorb the active substance, and after washing with water, an additional 30%
(V/V) Wash with acetone aqueous solution to remove impurities. Then elute with acetone. The acetone fraction is concentrated to dryness and dissolved in a 30% (V/V) acetone aqueous solution. This solution is adsorbed onto a column packed with 500 ml of activated carbon. After washing with a 30% (V/V) acetone aqueous solution, the active fraction is eluted with acetone. This operation can remove most of the dyes present as impurities. The active fraction is concentrated to dryness and dissolved in a small amount of chloroform (approximately 10 ml). Silica gel filled with this chloroform solution in advance using chloroform as a solvent [Product name: Silica gel for chromatography (100-200 mesh) Kanto Kagaku, the same applies below]
(500 ml), washed thoroughly with chloroform (approximately 2 times), and then eluted with chloroform:methanol (98:2, volume ratio). Tetrocalcins A to D, tetronolide compounds F-1, Tetrocalcin E-1, F, and E-2 are mixed and eluted. This was concentrated to dryness and a small amount of chloroform:methanol:H ₂ O (3:1:1,
(volume ratio) of the mixture in the lower layer solvent. A silica gel column (500
ml) and developed with the same solvent, tetrocalcins E-1, F, E-2, tetronolide compound F-1, and tetrocalcins A, B, D, and C were eluted in this order. Further, rechromatography is performed in the same manner, and fractions containing tetrocalcin F as the main component are collected and concentrated to dryness. This was dissolved in acetone or chloroform and developed with chloroform:methanol (9:1, volume ratio) using a thin layer of silica gel. The portion corresponding to tetrocalcin F is scraped off, eluted with acetone, concentrated to dryness, and dissolved in ethyl acetate. Shake this with 0.1NHCl, remove the ethyl acetate layer, and concentrate to dryness. A powder can also be obtained by redissolving the dried product in ethyl acetate and precipitating it with hexane. To obtain tetrocalcin G, the column after elution with chloroform:methanol (98:2, volume ratio) is eluted with chloroform:methanol (95:5, volume ratio). A fraction containing tetrocalcin G is obtained, which is further subjected to the following treatments.
The fraction containing tetrocalcin G is concentrated to dryness and then dissolved in a small amount of chloroform:methanol (98:2, volume ratio). Gently place the solution onto a 100ml silica gel column packed with the same solvent.
After flowing 200 ml, elute with chloroform:methanol (95:5, volume ratio). The fraction containing tetrocalcin G is concentrated to dryness, dissolved in a small amount of acetone, and developed on silica gel thin layer chromatography using chloroform:methanol (9:1, volume ratio) as a developing solvent. Scrape off the portion corresponding to tetrocalcin G,
After elution with acetone, concentrate to dryness and dissolve in a small amount of acetone. This is further subjected to thin layer chromatography using the lower layer of a mixture of chloroform:acetic acid:water (2:1:1, volume ratio) as a developing solvent. A portion corresponding to tetrocalcin G with Rf=0.52 is scraped off, eluted with acetone, concentrated to dryness, and dissolved in ethyl acetate. this
Shake and mix with 0.1NHCl, remove the ethyl acetate layer, and concentrate to dryness. Tetrocalcin G powder can also be obtained by redissolving the dried product in ethyl acetate and precipitating it with hexane. To obtain tetrocalcin H, the column after elution with chloroform:methanol (95:5, volume ratio) is eluted with chloroform:methanol (9:1, volume ratio). A fraction containing tetrocalcin H is obtained, which is further subjected to the following treatments. The fraction containing tetrocalcin H is concentrated to dryness and then dissolved in a small amount of chloroform:methanol (95:5, volume ratio). The solution was gently placed on a 100 ml silica gel column packed with the same solvent, and after flowing 200 ml of the same solvent, it was eluted with chloroform:methanol (9:1, volume ratio). The fraction containing tetrocalcin H was concentrated to dryness, dissolved in a small amount of acetone, and subjected to thin layer chromatography using chloroform:methanol (9:1, volume ratio) as a developing solvent, and the portion corresponding to tetrocalcin H was scraped off. Elute with acetone, concentrate the eluate to dryness, and dissolve in ethyl acetate. Shake this with 0.1NHCl, remove the ethyl acetate layer, and concentrate to dryness. At this time, the dried product is redissolved in ethyl acetate and then precipitated with hexane to obtain a powder of tetrocalcin H. In this way, 2 mg, 2.1 mg, and 2.2 mg of tetrocalcin F, G, and H were obtained, respectively. The physicochemical properties and antibacterial activity of the tetrocalcins F, G, and H obtained here are as described above. Example 2 Culture was carried out in the same manner as in Example 1 except that the composition of the fermentation medium was changed to the following. Fermentation medium composition: soluble starch 40g/, soybean meal powder 30g/, dextrin 5g/, corn steep liquor 5g/, K ₂ HPO ₄ 0.5g/,
MgSO ₄・7H ₂ O0.5g/, CaCO ₃ 1g/, PH7.0
Adjust with NaOH (before sterilization). The culture solution was treated in the same manner as in Example 1 to give 1 mg, 1.5 mg, and 1.3 mg of tetrocalcin F, G, and H, respectively.
I got mg. Example 3 Chemical production method of tetrocalcin G and tetrocalcin H from tetrocalcin A (1) Process for producing tetrocalcin G 107 mg of tetrocalcin A and 52 mg of sodium borohydride are added to 5 ml of anhydrous tetrahydrofuran and stirred for 30 minutes in an ice bath. Add 3 ml of acetone and stir for 5 minutes, then add 150 ml of 0.1NHCl and extract three times with 50 ml of ethyl acetate. Wash the ethyl acetate layer with water,
After drying over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure. The residue was subjected to silica gel column chromatography (chloroform:methanol=9:1, volume ratio) to obtain 94 mg of tetrocalcin G. (2) Method for producing tetrocalcin H 50 mg of tetrocalcin A and 500 mg of silver oxide are refluxed in 3 ml of tetrahydrofuran for 24 hours with stirring.
Add 100 ml of ethyl acetate and 120 ml of 0.1NHCl to the reaction solution, and stir for 10 minutes. The solid is removed by suction and the aqueous layer is extracted twice with 30 ml of ethyl acetate. The ethyl acetate layer was washed twice with 50 ml of water, dried over anhydrous sodium sulfate, and then ethyl acetate was distilled off under reduced pressure. The desired product is separated from the residue using silica gel thin layer chromatography (chloroform:methanol=9:1, volume ratio) and eluted with the same solvent. After removing the solvent under reduced pressure,
4 mg of tetrocalcine H was obtained by dissolving in 30 ml of ethyl acetate and treating with 0.1 NHC1 aqueous solution.
I got it.

[Brief explanation of the drawing]

FIG. 1 shows the infrared absorption spectrum of tetrocalcin F. Figure 2 shows PMR of tetrocalcin F.
The spectrum is shown. FIG. 3 shows the infrared absorption spectrum of tetrocalcin G. FIG. 4 shows the PMR spectrum of tetrocalcin G. FIG. 5 shows the infrared absorption spectrum of tetrocalcin H.
FIG. 6 shows the PMR spectrum of tetrocalcin H.

Claims (1)

[Claims] 1. General formula (However, in the formula, when R ₁ is a -CHO group, R ₂ represents a hydrogen atom, and when R ₁ is mono-CH ₂ OH or -COOH, R ₂ represents a disaccharide residue shown below. [Formula ]) Tetrocalcins and their salts.