JPS635399B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel macrolide antibiotic exhibiting excellent antibacterial properties. For more details, please refer to Micromonospora sp., a novel macrolide antibiotic-producing actinomycete.
A11725 strain (Micromonospora sp. A11725) was cultured in a medium, and the new macrolide antibiotics A11725 and A11725 were extracted and isolated from the culture.
The present invention relates to A11725a, a new macrolide antibiotic in which the epoxy of A11725 is treated with chromium chloride in an organic solvent to form a double bond, and A11725a as a control. Novel macrolide antibiotic of the present invention A11725
a (hereinafter referred to as A11725a) and A11725a as a control (hereinafter referred to as A11725a) are basic nitrogen-containing compounds having the following physicochemical properties. A11725a Color of substance; White crystal Molecular formula; C ₃₇ H ₆₁ NO ₁₁ Elemental analysis; Actual value; C = 64.05 H = 9.10 N = 2.04 Calculated value; C = 63.86 H = 8.84 N = 2.01 Molecular weight; 695 (from mass spectrum) Melting point: 174-176°C [α] ²⁵ _D : +2.7° (C=1.0, methanol) Ultraviolet absorption spectrum: as shown in Figure 1 (23γ/ml in methanol), λmax 215nm (E
1% 1cm = 326.1) λmax 283nm (E
1% 1cm = 333.9) Infrared absorption spectrum; Figure 2 (KBr method)
3600, 2970, 2930, 2880, 2830, 2780, 1720,
1710, 1678, 1645, 1625, 1596, 1460, 1380,
1350, 1322, 1275, 1258, 1230, 1165, 1105,
1072, 1045, 980, 960, 930, 882, 858, 830cm
It has an absorption band at each wavelength around ^-1 . The unit of wavelength in Figure 2 is cm ^-1 . Nuclear magnetic resonance spectrum; Figure 3 (CDCl ₃ ,
100MHzTMS). The unit of numerical values in FIG. 3 is ppm. Color reaction: positive for potassium permanganate decolorization reaction, negative for ninhydrin reaction, Sakaguchi reaction, ferric chloride reaction Distinction between basic, acidic, and neutral; Solubility in basic solvents: acidic water, methanol, acetone , Soluble in ethyl acetate and benzene, Slightly soluble in basic water, Insoluble in hexane A11725a Color of substance: White crystal Molecular formula: C ₃₇ H ₆₁ NO ₁₂ Elemental analysis: Actual value: C=62.21 H=8.82 N=1.94 Calculated value: C=62.43 H=8.64 N=1.97 Molecular weight: 711 (from mass spectrum) Melting point: 148-150℃ [α] ²⁵ _D ; +18.7゜ (C=1.0, methanol) Ultraviolet absorption spectrum; As shown in Figure 4 (20.42γ/ml in methanol), λmax215nm (E
1% 1cm = 323.2) λmax280nm (E
1% 1cm = 323.2). Note that the unit of wavelength in FIG. 4 is nm. Infrared absorption spectrum; Figure 5 (KBr method)
3550, 2970, 2930, 2880, 2830, 2780, 1720,
1710, 1670, 1640, 1590, 1450, 1375, 1345,
1320, 1270, 1250, 1230, 1160, 1120, 1100,
1070, 1040, 980, 955, 925, 880, 850, 830,
It has absorption bands at each wavelength around 740 and 710 cm ^-1 . The unit of wavelength in FIG. 5 is cm ^-1 . Nuclear magnetic resonance spectrum; Figure 6 (CDCl ₃ ,
100MHzTMS). The unit of numerical values in FIG. 6 is ppm. Color reaction: positive for potassium permanganate decolorization reaction, negative for ninhydrin reaction, Sakaguchi reaction, ferric chloride reaction Distinction between basic, acidic, and neutral; Solubility in basic solvents: acidic water, methanol, acetone , ethyl acetate, benzene, sparingly soluble in basic water, insoluble in hexane Antibacterial spectrum of the antibiotic (free base) of the present invention determined by the agar dilution method (indicating the minimum inhibitory concentration)
is as shown in Table 1. [Table] [Table] Based on the above properties, this substance is a basic 16-membered ring macrolide containing one sugar each of desosamine and 6-deoxy-2.3-di-O-methyl-hexose. , and the structure as expression The new macrolide antibiotics A11725 and A11725 have the chemical structure represented by (where R ₁ represents a hydrogen atom or a hydroxyl group), do not have an aldehyde group in the molecule, and are the starting materials.
(hereinafter referred to as A11725 and A11725, respectively), the epoxy in the molecule of A11725a is converted into a double bond, and R ₁ of A11725a is a hydrogen atom.
a is each substance whose R ₁ is represented by a hydroxyl group,
Based on these facts, this substance does not correspond to any known substances and is judged to be a new substance. The substance of the present invention can usually be used in the form of salts with pharmaceutically acceptable mineral acids, organic acids, etc., such as tartrates, citrates, succinates, and the like. The antibiotics of the invention can be taken orally as tablets and powders, and can also be applied by intravenous injection. The amount used is approximately 400 to 2000 mg per day for an adult, and it is effectively used against respiratory infections caused by Gram-positive bacteria such as Staphylococcus. Furthermore, the antibiotic of the present invention can be used as an antibiotic for feed addition or for animal treatment. Also, A11725 and A11725 in the present invention
A11725 and A11725, for example, are considered new substances as there are no known antibiotics belonging to the basic macrolide group based on their physicochemical properties. Belongs to Micromonospora sp.
Obtained from the culture of A11725 strain. This bacterium, a new macrolide antibiotic-producing actinomycete, is an actinomycete isolated from the soil of a potato field in Unazuki-cho, Shimoshinkawa-gun, Toyama Prefecture, and is Micromonospora sp., which belongs to the genus Micromonospora. A11725, and this bacterium has been submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology with the microbial accession number FERM
−BP 705” [This strain is Micromonospora griseorubia A11725 (Micromonospora griseorubia
The strain name has been changed as A11725). Furthermore, the mycological properties of this bacterium are as follows. [] Morphological characteristics The basal hyphae are long, wavy, and simply branched, with a diameter of 0.6 to 0.8μ, and no hyphal divisions are observed.
Spores are formed individually at the tips of short sporophores produced from basal hyphae, and are spherical or oval in size.
It has a diameter of 1.0 to 1.5μ, and its surface has thorn-like protrusions, giving it a konpeito-like appearance. Also, on an agar medium, depending on the medium composition, undeveloped aerial mycelia may be formed, or a black spore layer may be formed on the colony surface. [] Growth status on various media The results of culturing at 30°C for 20 days on various media are shown in Table 2. Color display is based on Color Harmony Manual, 4th edition.
1958 (Container Corporation of America)
According to the color classification by [Table] [Table] [] Physiological properties The physiological properties are as shown below. 1 Assimilation of carbon sources [Table] [Table] -; Negative (The Prudham-Godelive agar medium was D-glucose, and the growth of this bacterium was poor.
A 0.5% yeast extract and 1.5% agar medium was used as the basal medium. ) 2 Growth temperature range: 15-45℃ 3 Liquefaction of gelatin: Positive for glucose/peptone and gelatin media 4 Hydrolysis of starch Positive for starch/inorganic salt agar media 5 Skimmed milk: Positive for both peptonization and coagulation 6 Melanin-like pigment Formation: Negative on tyrosine agar and peptone yeast iron agar medium 7 Salt tolerance [Inter.J.System.Bacteriol.21, 240
-247 (1971) method was followed. ] [Table] 8 Decomposition of cellulose: Negative 9 Production of nitrous acid [Inter.J.System.Bacteriol.
21, 240-247 (1971) was used. ]:
Negative As mentioned above, the A11725 strain of this fungus attaches one spore to the tip of the sporophore produced from branched basal hyphae, does not form true aerial hyphae, and is a mesophilic bacterium. Therefore, it is a strain belonging to the genus Micromonospora. Based on the above, this bacterial strain A11725 was isolated from Micromonospora sp.
sp.A11725). The parent antibiotic A11725 of the present invention is obtained as follows. That is, for example, the above-mentioned Micromonospora sp. A11725 strain is aerobically cultured in a medium containing medium components commonly used for culturing microorganisms. As the medium, a solid medium or a liquid medium can be used, and especially for mass production, a liquid medium, especially an aqueous medium, is suitable. As a medium component, suitable carbon sources include glucose, starch, glycerin, sucrose, molasses, and dextrin. Suitable nitrogen sources include peptone, meat extract, soy flour, and casein hydrolyzate, but cottonseed meal, corn steep liquor, nitrates, and ammonia salts can also be used. Other inorganic substances include sodium, potassium,
Substances containing cations such as magnesium, calcium, cobalt, manganese, iron, and/or anions such as chlorine, sulfuric acid, phosphoric acid, acetic acid, etc. can be used. Furthermore, dry yeast and yeast extract can be used as bacterial growth promoting factors. Calcium carbonate can also be added to the medium to adjust the pH of the medium. In addition, appropriate amounts of silicone resin, animal and vegetable oils, etc. can be added to the culture medium to suppress foaming during culture. A particularly suitable medium for carrying out the method of the present invention is a medium containing glucose, dextrin-defatted soy flour, calcium carbonate, and cobalt chloride as medium components. As the culture conditions, known culture conditions used for the production of known antibiotics can be used. The culture temperature ranges from 20°C to 37°C, with temperatures of 26°C to 30°C being particularly suitable. The number of culture days varies depending on the culture conditions, but
Usually it takes 4 to 5 days. Any known culture method can be used, but the aerated agitation culture method in a fermentation tank is particularly suitable for mass production. Antibiotic of the present invention A11725
In order to isolate and collect from the culture,
The most suitable method is to first remove the bacterial cells and other solid substances by filtration or centrifugation, and then extract the liquid by extraction with an organic solvent. Organic solvents used for extraction include chlorinated hydrocarbons such as chloroform, dichloroethylene, and trichlorethylene, and fatty acid esters such as ethyl acetate, butyl acetate, and amyl acetate. Any organic solvent that is difficult to mix with can be used. The organic solvent extract containing A11725 and
00 volume, and then this concentrated solution was diluted with hydrochloric acid, sulfuric acid,
After adjusting the pH to 1.0 to 3.0 with acetic acid, separate the aqueous layer.
Adjust the pH to 7.8 to 9.0 with an alkaline solution such as caustic soda, caustic potash, or ammonia, and extract again with an organic solvent. A crude substance containing A11725 and A11725 is obtained by distilling off the solvent of this extract under reduced pressure and drying it. This crude substance is fractionated using means such as column chromatography using silica gel, countercurrent distribution method, etc., and each fraction is subjected to silica gel thin layer chromatography to confirm its components.
Fractions containing pure A11725 and A11725 are collected, and the solvent is distilled off under reduced pressure to dryness to obtain the respective white powders. A11725 obtained in this way and
It is a basic nitrogen-containing chloride compound having the following physicochemical properties. A11725 Color of substance; white powder Molecular formula; C ₃₇ H ₆₁ NO ₁₂ elemental analysis; (actual value) C = 62.34 H = 9.25 N = 1.96 Molecular weight: 711 (from mass spectrum) Melting point: 103-107℃ [α] ²⁵ _D ; -40.0゜ (C=1.0, methanol) Ultraviolet absorption spectrum; as shown in Figure 7 (0.025 mg/ml in methanol), λmax 217 nm (E1% 1cm = 340) Shoulder λmax 240nm (E1% 1cm = 180 ) is shown. Note that the unit of wavelength in FIG. 7 is nm. Infrared absorption spectrum; Figure 8 (KBr method)
It is as follows. The unit of wavelength in FIG. 8 is cm ^-1 . Nuclear magnetic resonance spectrum; Figure 9 (CDCl ₃ ,
100MHzTMS). The unit of numerical values in FIG. 9 is ppm. Silica gel thin layer chromatography; CHCl ₃ :methanol (5:1) Rf=0.48 CHCl ₃ :methanol:7% ammonia water (40:
12:20) Lower layer Rf = 0.72 Color reaction; positive for potassium permanganate decolorization reaction, negative for ninhydrin reaction, Sakaguchi reaction, ferric chloride reaction Distinction between basic, acidic, and neutral; dissolution in basic solvents Soluble in acidic water, methanol, acetone, ethyl acetate, and benzene. Slightly soluble in basic water. A11725 Color of substance: White powder Molecular formula: C ₃₇ H ₆₁ NO ₁₃ Elemental analysis: (Actual value) C=60.57 H=8.95 N=1.96 Molecular weight: 727 (from mass spectrum) Melting point: 102-106℃ [α] ²⁵ _D ; -31.0゜ (C=1,0, methanol) Ultraviolet absorption spectrum; as shown in Figure 10 (0.025 mg/ml in methanol) λmax 217 nm (E1% 1cm = 337) Shoulder λmax 240nm (E1% 1cm = 180). Note that the unit of wavelength in FIG. 10 is nm. Infrared absorption spectrum; Figure 11 (KBr method)
It is as follows. The unit of wavelength in Figure 11 is cm ^-1
It is. Nuclear magnetic resonance spectrum; Figure 12 (CDCl ₃ ,
100MHz, TMS). The unit of numerical values in FIG. 12 is ppm. Silica gel thin layer chromatography; CHCl ₃ :methanol (5:1) Rf=0.40 CHCl ₃ :methanol:7% ammonia water (40:
12:20) Lower layer Rf = 0.56 Color reaction; positive for potassium permanganate decolorization reaction, negative for ninhydrin reaction, Sakaguchi reaction, ferric chloride reaction Distinction between basic, acidic, and neutral; dissolution in basic solvents Soluble in acidic water, methanol, acetone, and ethylbenzene acetate. Slightly soluble in basic water. Furthermore, the antibacterial spectra of A11725 and A11725 mentioned above are shown in Table 3. [Table] [Table] Based on these properties, A11725 and A11725
are each a 16-membered ring macrolide substance having one sugar each of desosamine and 6-deoxy-2,3-di-O-methyl-hexose, and have a structure of the formula It _has a chemical structure represented by (where R ₁ represents a hydrogen atom or a hydroxyl group) and does not have an aldehyde group in the molecule _. is a substance represented by a hydroxyl group, and based on these facts, A11725 and A11725, which are the starting materials of the present invention, are recognized as new substances. Next, the new macrolide antibiotics mentioned above
A11725a, which is the object of the present invention from A11725,
In addition, in obtaining A11725a, which is the object of the present invention, from A11725, which is a control, for example,
A11725, A11725 may be dissolved in a lower fatty acid solvent such as glacial acetic acid or propionic acid, then chromium chloride is added under cooling, and the reaction is further allowed to proceed at room temperature for 3 to 20 hours. Further, the amount of chromium chloride used may be at least 2 moles relative to A11725. Next, this reaction product is poured into water or ice water, and the solution is made weakly basic to about PH8.5 with an aqueous solution of a basic compound, such as sodium carbonate, and then extracted with a solvent such as ethyl acetate or benzene. Wash the extract with water, dry it,
The solvent is removed under reduced pressure to obtain a crude powder, which is further purified by silica gel chromatography using a developing solvent of chloroform: methanol: 28% aqueous ammonia (400:10:1), for example. A11725a, which is the object of the present invention,
Obtain A11725a. A11725a of the present invention thus obtained
is a double bond formed by converting the epoxy in the molecule of A11725 into a bonding group, and
A11725a is a derivative of A11725 in which the epoxy in the molecule is converted into a bonding group to form a double bond, and is a parent macrolide antibiotic with antibacterial properties. Next, the present invention will be specifically described with reference to Examples and Reference Examples, but the present invention is not limited thereto. Example 1 A11725 obtained in the same manner as Reference Example 1 described below
/g in 30 ml of glacial acetic acid and then cooled
Add 1.0g of chromium chloride and further heat at room temperature16
The reaction was stirred for a period of time. Then, the reaction solution was
Pour into 700 ml of ice water, adjust the solution to pH 8.5 with an aqueous sodium carbonate solution, extract three times with 400 ml of ethyl acetate, collect and combine the ethyl acetate layers, wash with water, and add anhydrous sulfuric acid. Add sodium and dry, then dry under reduced pressure.
Approximately 800 mg of A11725a-containing crude powder was obtained. This coarse powder was then passed through a column packed with silica gel (24×
55cm) using chloroform:methanol:28
Elute with a developing solvent of % ammonia water (400:10:1) and fractionate into 15 ml fractions.
The fractions No. 130 to 210 were collected, combined, and dried under reduced pressure to obtain 605 mg of purified A11725a. Example 2 In place of A11725 in Example 1, reference example 1 described below
Using A11725 obtained in the same manner as above, the same procedure as in Example 1 was carried out, and fractions Nos. 150 to 220 were collected using the silica gel column to obtain 612 mg of purified A11725a. Reference example 1 [A] 100 ml of a medium (PH7.0) containing 1% dextrin, 1% glucose, 0.5% casein hydrolyzate, 0.5% yeast extract, and 0.1% calcium carbonate was placed in a 500 ml Erlenmeyer flask. , 120℃,
Heat sterilized for 20 minutes. One platinum loop from a slant culture of Micromonospora sp. A11725 strain was inoculated into 10 bottles of this medium, and cultured with shaking at 30°C for 120 hours. This was then transferred to a 30-volume jar fermenter containing heat-sterilized medium 20 with the same composition as above, and incubated at 30°C and 300 rpm for 72 hours.
The culture was carried out with aeration and stirring under conditions of sterile air at 20 m/min. Next, dextrin 5%, glucose 0.5%,
The above culture 10 was transplanted into a 250-volume tank containing 200 ml of heat-sterilized medium (PH 7.2) containing 3% defatted soybean flour and 0.2% calcium carbonate,
Culture was carried out at 30° C. for 120 hours with aeration and agitation under sterile air at 250 rpm and 100 rpm to obtain culture number 190. [B] The above culture 190 was filtered to remove bacterial cells and other solid substances, and a liquid 160 was obtained.
This liquid was extracted with the same amount of ethyl acetate to obtain 160 ml of an ethyl acetate solution containing the target product. This was concentrated under reduced pressure to a concentration of 50% and then mixed with 20% of an aqueous solution of hydrochloric acid having a pH of 2.5 to transfer and dissolve the target product into the aqueous layer. Furthermore, the pH of the hydrochloric acid aqueous solution was adjusted to PH8.5 using concentrated ammonia, extracted with 20% chloroform, and the chloroform layer was concentrated to dryness to produce the crude product.
8.5g was obtained. [C] 8.5 g of the above crude product was dissolved in 50 ml of chloroform and adsorbed onto a silica gel column (3 cm x 55 cm) that had been filled with chloroform in advance.
Then, it was developed with a solvent consisting of chloroform-methanol-28% ammonia (20:1:0.1),
It was fractionated into 15 ml portions. Antibacterial activity using Bacillus subtilis and chloroform-methanol-7% ammonia water (40:
12:20: Lower layer) was used as a developing solvent to confirm the target product by thin layer chromatography, and fractions containing it were collected. The 78th fraction contained only the substance identified as A11725- from the 61st fraction, and this fraction was concentrated and dried to obtain 1.2 g of A11725-. The 160th fraction contains only the substance identified as A11725 from the 126th fraction, and this fraction is concentrated and dried to yield 1.7g of A11725.
I got it.

[Brief explanation of the drawing]

Figure 1 is the ultraviolet absorption spectrum of A11725a as a control, Figure 2 is the infrared absorption spectrum of A11725a, Figure 3 is the nuclear magnetic resonance spectrum of A11725a, Figure 4 is the ultraviolet absorption spectrum of A11725a of the present invention, Figure 5 shows the present invention.
Infrared absorption spectrum of A11725a, Figure 6 is nuclear magnetic resonance spectrum of A11725a of the present invention,
Figure 7 is the ultraviolet absorption spectrum of A11725, Figure 8 is the infrared absorption spectrum of A11725, and Figure 9 is the infrared absorption spectrum of A11725.
The figure shows the nuclear magnetic resonance spectrum of A11725, No. 10.
The figure shows the ultraviolet absorption spectrum of A11725.
The figure shows the infrared absorption spectrum of A11725, the 12th
The figure shows the nuclear magnetic resonance spectrum of A11725.

Claims (1)

[Claims] 1. Parent antibiotic A11725 having the following properties:
a and its pharmaceutically acceptable salts Color of substance White Molecular formula C ₃₇ H ₆₁ NO ₁₂ Molecular weight 711 Melting point 148-150°C [α] ²⁵ _D +18.7° (C = 1.0, methanol) Ultraviolet absorption spectrum ( (in methanol) λmax215nm, λmax280nm Infrared absorption spectrum (KBr method) 3550, 2970, 2930, 2880, 2830, 2780, 1720,
1710, 1670, 1640, 1590, 1450, 1375, 1345,
1320, 1270, 1250, 1230, 1160, 1120, 1100,
1070, 1040, 980, 955, 925, 880, 850, 830,
Has an absorption band at each wavelength around 740 and 710 cm ^-1 Color reaction Potassium permanganate decolorization reaction + ninhydrin reaction, Sakaguchi reaction, ferric chloride reaction − Distinction between basic, acidic, and neutral Basic Dissolution in solvent Acidic water, methanol,
Soluble in acetone, ethyl acetate, benzene, sparingly soluble in basic water, insoluble in hexane