JPS6338038B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention is based on the general formula [] (In the formula, R ₁ and R ₂ represent a hydrogen atom or an acetyl group. However, if R ₁ is a hydrogen atom,
R ₂ is an acetyl group, and when R ₂ is a hydrogen atom, R ₁ is an acetyl group. ) (Hereinafter, compounds in which R ₁ is a hydrogen atom and R ₂ is an acetyl group are referred to as tetrocalcin E-1, and compounds in which R ₁ is an acetyl group and R ₂ is a hydrogen atom are referred to as tetrocalcin E-1.
Set it to 2. ) and its salt, and a method for producing tetrocalcin E-1 and E-2. 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 are considered to be compounds having extremely similar structural formulas based on their physicochemical substances, and the present inventors later decided to collectively refer to them as tetrocalcins. DC-11, DC-11-A-2, DC
-11-A-3 and DC-11-B are named tetrocalcin A, tetrocalcin B, tetrocalcin C, and tetrocalcin D, respectively. DC―
Regarding No. 11, the same applicant has already filed a patent application in 1983-
45916 (Japanese Unexamined Patent Publication No. 138501) and patent application No. 1385-
153027 (Japanese Unexamined Patent Publication No. 53-79322).
DC-11-A-2, DC-11-A-3, DC-11-
Regarding B, a patent application filed in 17498, filed by the same applicant,
55-24924 (JP 56-115794, 56-122392, above two DC-11-A-2), 54-152253, 55-24925
(Japanese Patent Publication No. 56-75500, 56-122393, above two DC-
11-A-3), 55-24926 (Unexamined Japanese Patent Publication No. 56-122394, DC
-11-B). Furthermore, regarding tetronolides, Japanese Patent Application No. 55-80482
-7479). In the above, it was later revealed that tetrocalcins A and B have the following chemical structures. As a result of further research, the present inventors discovered and isolated new substances tetrocalcin E-1 and tetrocalcin E-2, 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 E-1 and tetrocalcin E-2 can be obtained by chemically decomposing tetrocalcin A, tetrocalcin B, and tetrocalcin D, either alone or as a mixture. Tetrocalcin E-1 and tetrocalcin E-2 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, it can be expected that it itself exhibits antitumor properties. The specific physical and chemical properties of these compounds are as follows. (1) Tetrocalcin E-1 Melting point: 207-210℃ Elemental analysis value Calculated value: C: 61.6% H: 7.0% N: 2.9% Actual value: C: 61.3% H: 6.9% N: 3.0% (C ₄₉ H ₆₆ N ₂ O ₁₇ ) Infrared absorption spectrum (KBr tablet method): 1st
Figure PMR spectrum (in CDCl ₃ , TMS reference):
Figure 2 CMR spectrum δ (ppm) (in CDCl ₃ , TMS
Standard) 206.1, 201.3, 192.4, 170.4, 166.5,
157.3, 149.4, 141.3, 136.3, 135.7,
126.6, 125.5, 123.3, 118.3, 100.9,
99.7, 96.4, 91.5, 85.6, 83.9, 77.8,
75.1, 69.4, 69.2, 65.5, 62.1, 54.1,
53.7, 52.8, 51.2, 44.8, 43.4, 41.7,
38.4, 35.9, 35.3, 34.2, 31.1, 30.7,
29.7, 25.3, 21.9, 21.1, 17.5, 16.9,
16.1, 15.0, 14.4, 14.3 Specific optical rotation [α] ²⁸ _D = -31.6° (c, 1.0, acetone) (2) Tetrocalcin E-2 Melting point: 205-208℃ Elemental analysis Calculated value: C: 61.6% H: 7.0% N: 2.9% Actual value: C: 61.4% H: 7.1% N: 2.9% (calculated as C ₄₉ H ₆₆ N ₂ O ₁₇ ) Infrared absorption spectrum (KBr tablet method): 3rd
Figure PMR spectrum (in CDCl ₃ , TMS reference):
Figure 4 CMR spectrum δ (ppm) (in CDCl ₃ , TMS
Standard) 206.2, 201.4, 192.4, 171.3, 166.5,
157.3, 149.5, 141.2, 136.2, 136.0,
126.1, 125.8, 123.0, 118.2, 100.8,
97.7, 96.4, 91.5, 83.9, 83.8, 77.7,
71.6, 69.7, 69.3 (2 pieces), 64.6, 54.2,
53.7, 52.7, 51.2, 44.8, 43.2, 41.5,
38.4, 35.9, 34.4, 33.1, 31.1, 30.6,
29.7, 25.3, 21.9, 21.4, 17.6, 16.9,
16.0, 15.0, 14.3, 13.9 Specific optical rotation [α] ²⁸ _D = -27.4° (c, 1.0, acetone) Next, tetrocalcin E-1, tetrocalcin E
Table 1 shows the behavior in chromatography of a thin layer of -2. Table 1 Silica gel plate (product name DC-
Fertigplatteu 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 Developer: Toluene: Acetone = 35:65 (Volume ratio) Substance Rf value Tetrocalcin A 0.55 Tetrocalcin E-1 0.64 Tetrocalcin E-2 0.60 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 Next, Tetrocalcin E-1, Tetrocalcin E
Table 2 shows the antibacterial activity of -2 against various microorganisms (using a pH7.0 medium).

[Table]
ATCC 9992
From the above physical and chemical properties, tetrocalcin E-
Not only can one distinguish between 1 and E-2, but also between these and other tetrocalcins. Next, the method for producing tetrocalcins of the present invention will be described. Tetrocalcin E-1, Tetrocalcin E-2
As mentioned above, can be produced by fermentation, or by hydrolyzing tetrocalcin A, tetrocalcin B, tetrocalcin D, etc. Tetrocalcin E-1 and Tetrocalcin E
-2 from a microbial culture, a microorganism belonging to the genus Micromonosvora and capable of producing tetrocalcin E-1 and/or tetrocalcin E-2 is cultured in a nutrient medium, and the compound is added to the culture. Tetrocalcin E-1 was accumulated from the culture.
And/or by collecting tetrocalcin E-2, tetrocalcin E-1 and/or tetrocalcin E-2 can be obtained. Any microorganism can be used as long as it belongs to the genus Micromonospora and has the ability to produce tetrocalcin E-1 and/or tetrocalcin E-2.・Chaarcea
KY11091 stock is mentioned. The strain has been deposited as FIKEN Bibori No. 4458 and NRRL11289, respectively. The mycological properties of this strain were disclosed in Japanese Patent Application 1986-4596.
(Japanese Patent Publication No. 54-138501), Patent Application No. 152253 (Japanese Patent Application
56-75500), patent application No. 55-17498 (Japanese Patent Application No. 1983-17498
115794), patent application No. 1157-24926 (Japanese Patent Application No. 1983-122394)
It is stated in the specification. Next, the culture method will be described. In the cultivation of the present invention, ordinary methods for culturing actinomycetes are generally used. Various nutrient sources can be used for culturing, as shown below. 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. Inorganic and organic nitrogen compounds include ammonium chloride, ammonium sulfate, ammonium nitrate, sodium nitrate, urea, etc. Natural nitrogen sources include peptone, meat extract, yeast extract, dried yeast, corn stew liquor, soy flour, and casamino. Acids and the like are used alone or in combination. In addition, salt, potassium chloride, magnesium sulfate, calcium carbonate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate,
Add inorganic salts such as ferrous sulfate, calcium chloride, manganese sulfate, zinc sulfate, and copper sulfate. Furthermore, trace components that promote the growth of the bacteria used and the production of tetrocalcin E-1 and tetrocalcin E-2, such as vitamin B ₁ and biotin, 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. Isolation and purification of tetrocalcin E-1 and tetrocalcin E-2 from the culture solution utilizes separation and purification methods commonly used to isolate metabolic products of microorganisms from the culture solution. For example, the culture product is separated into a culture solution and bacterial cells, and the culture solution (PH6.0) is passed through a nonionic porous resin (trade name "HP-20" manufactured by Mitsubishi Kasei, etc.) to detect antibacterial activity. After adsorbing the components that have
Desorb the adsorbed substance 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, and chromatographed using silica gel that has been suspended in chloroform and filled in a column. First, impurity yellow pigment is removed by passing chloroform through the column. Then chloroform:methanol (98:2,
The active substance can be eluted with a mixed solution (volume ratio). Here, tetrocalcin A, tetrocalcin B, tetrocalcin C, tetrocalcin D, tetronolide compound F-1, tetrocalcin E
Elute as a mixture of -1 and tetrocalcin E-2. To carry out these separations, the active fractions are concentrated to dryness and dissolved in a small amount of the lower solvent of a mixture of chloroform:methanol:water (3:1:1, by volume). This was passed through a silica gel column filled with the lower layer solvent, and when eluted with the same solvent, tetrocalcin E-1 and tetrocalcin E-
2. Tetronolide compound F-1, tetrocalcin A, tetrocalcin B, tetrocalcin D,
Tetrocalcin C is eluted in this order. Fractions containing a large amount of tetrocalcin E-1 and tetrocalcin E-2 are taken and each is further subjected to re-chromatography or treated with silica gel thin layer chromatography to obtain tetrocalcin E-1 and E-2.
-2 each. For thin layer chromatography, use a thin layer of silica gel (product name: DC
After developing with chloroform:methanol (9:1, volume ratio) using FertigPlatten Kieselgel 60F ₂₅₄ (E. Merck), a corresponding portion is scraped off, eluted with the developing solvent or acetone, and the eluate is concentrated to dryness. Furthermore, if necessary, means such as extraction and crystallization may be added. In addition, to complete the separation, repeat the same chromatography as above or use Sephadex LH-20 (Pharmacia Fine Chemicals).
Inc., Sweden) may be added. Next, tetrocalcin E-1, tetrocalcin E
-2 The production method by hydrolysis will be described. Tetrocalcin A, B, D or a mixture thereof (the composition thereof does not matter) is hydrolyzed in a mixture of an aqueous acid solution such as hydrochloric acid or sulfuric acid and an organic solvent (such as acetone). Hydrolysis may be carried out under the conditions shown in Example 3, for example. After hydrolysis, the solvent is distilled off, the remaining aqueous solution is extracted with ethyl acetate, the ethyl acetate layer is concentrated to dryness, and then dissolved in chloroform. When this is passed through a silica gel column previously filled with chloroform and developed with chloroform:methanol (100:0.5, volume ratio), tetrocalcin E-1 and tetrocalcin E-2 are eluted in this order. This can be further purified by rechromatography, thin layer chromatography, etc. to obtain pure tetrocalcin E-1 and tetrocalcin E-2. Here, two substances, tetrocalcin E-1 and tetrocalcin E-2, have been obtained from a single substance. Subsequent experiments showed that tetrocalcin E-1 was originally contained in the parent compound, and that tetrocalcin -2 is thought to have been generated. On the other hand, tetrocalcin E-2 can also be converted to tetrocalcin E-1 by transfer of the acetyl group. Such acetyl group transfer occurs when pure tetrocalcin E-1 and tetrocalcin E-1 are converted into a solution, gradually converting into tetrocalcin E.
This was also proven by the fact that a mixture of -1 and E-2 was formed. Furthermore, it can be easily assumed that a similar reaction occurs in the culture solution. Next, examples will be given to show 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 Micromonospora charcea as seed fungus
KY11091 was used. The strain was grown in a seed medium [KCl 4g/MgSO ₄ .
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〕300ml
The cells were inoculated and cultured at 30°C for 48 hours with shaking (220 rpm). The seed culture thus obtained was mixed into a fermentation medium 15 with the following composition in a 30-volume jar fermenter.
The culture was carried out 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 O0.5
g/, CaCO ₃ 1g/, PH7.2 (before sterilization)
Adjust 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 separate the
Got 13. First, liquid 13 is passed through a column of nonionic porous resin 1 (product 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 E-1, Tetrocalcin E-1 and E-2 are mixed and eluted. This is concentrated to dryness and dissolved in a small amount of the lower solvent of a mixture of chloroform:methanol:H ₂ O (3:1:1, volume ratio). A silica gel column (500ml) filled with this lower layer solvent
When placed gently on the gel and developed with the same solvent, tetrocalcin E-1, E-2, tetronolide compound F
-1, tetrocalcin A, B, D, and C are eluted in this order. Further, rechromatography is performed in the same manner, and the fractions containing tetrocalcins E-1 and E-2 as main components are 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 E-1 or E-2 is scraped off, each is eluted with a developing solvent or acetone, concentrated to dryness, and dissolved in ethyl acetate. Shake this with 0.1N HCl, remove the solvent layer, and concentrate to dryness. A powder can also be obtained by redissolving the dried product in ethyl acetate and precipitating it with hexane. In this way, about 5 mg of tetrocalcin E-1 and about 3 mg of tetrocalcin E-2 were obtained. The physicochemical properties and antibacterial activity of the tetrocalcins E-1 and E-2 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 stew liquor 5g/,
K ₂ HPO ₄ 0.5g/, MgSO ₄・7H ₂ O0.5
g/, CaCO ₃ 1g/, PH7.0 (before sterilization)
Adjust with NaOH. The culture solution was treated in the same manner as in Example 1 to give about 4 mg and about 2 mg of tetrocalcin E-1 and E-2, respectively.
I got mg. These physicochemical properties and antibacterial activity were in good agreement with those obtained in Example 1. Example 3 3 g of a mixture of tetrocalcins A, B, and D (composition ratio: 8:1.7:0.3, weight ratio) was mixed with 50% acetone 2
After adjusting the pH to 20 with HCl, the solution was refluxed at 50°C for 17 hours for decomposition. The decomposed solution was concentrated under reduced pressure to remove acetone, and then extracted with 500 ml of ethyl acetate. The extract was concentrated to dryness, dissolved in chloroform, gently placed on a silica gel column (500 ml) filled with chloroform in advance, and developed with chloroform:methanol (100:0.5, volume ratio). Fractions containing tetrocalcin E-1 and E-2 were collected, concentrated, and precipitated with petroleum ether. In this method, tetrocalcin E-1, E-2
150mg and 160mg of each were obtained. These physicochemical properties, antibacterial activity, etc. were in good agreement with those obtained in Example 1.

[Brief explanation of the drawing]

FIG. 1 shows the infrared absorption spectrum of tetrocalcin E-1. Figure 2 shows tetrocalcin E-1
The PMR spectrum of FIG. 3 shows the infrared absorption spectrum of tetrocalcin E-2. Fourth
The figure shows the PMR spectrum of tetrocalcin E-2.

Claims (1)

[Claims] 1. General formula (In the formula, R ₁ and R ₂ represent a hydrogen atom or an acetyl group. However, if R ₁ is a hydrogen atom,
R ₂ is an acetyl group, and when R ₂ is a hydrogen atom, R ₁ is an acetyl group. ) Tetrocalcins and their salts.