JP2000086627A - Antimicrobial substance BE-54476 and method for producing the same - Google Patents

Abstract

(57) [Summary] [Constitution] The present invention provides a novel structural formula [I] With respect to the compound represented by [Effect] The compound of the present invention has a strong growth inhibitory effect on pathogenic microorganisms, and thus is useful as a therapeutic agent for bacterial infections in the field of medicine.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention is useful in the field of medicine, and more specifically, a novel compound which inhibits the growth of microorganisms and exerts an antibacterial effect, a method for producing the compound, its use, and production of the compound. It is about microorganisms.

[0002]

2. Description of the Related Art Staphylococcus aureus
Ccus aureus is known as a causative agent of purulent diseases, but especially in large hospitals for severely ill patients, methicillin-resistant Staphylococcus aureus (Methicillin).
resistant Staphylococcus
aureus: hereinafter referred to as MRSA), and many hospital-acquired infections with multidrug-resistant bacteria have become a serious clinical problem. [Oguri et al., Clinical and Microbial Studies, Vol. 15, pp. 7-15 (1
988)]. Under such circumstances, development of a drug effective against MRSA is required.

[0003]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a novel antibacterial substance which can meet the above-mentioned demands. That is, an object of the present invention is to provide a novel compound having excellent antibacterial activity against multidrug-resistant MRSA in which existing antibacterial agents cannot sufficiently exert their effects.

[0004]

Means for Solving the Problems In order to solve the above-mentioned problems, the present inventors conducted a wide screening of secondary metabolites of microorganisms for substances having antibacterial activity, and as a result, they were represented by the following structural formula [I]. The inventors have found that the compounds exhibit excellent antibacterial activity and completed the present invention.

That is, the present invention provides a novel structural formula [I]

[0006]

Embedded image And a method for producing and using the same, and a microorganism belonging to the genus Streptomyces, which has the ability to produce the compound.

The structural formula [I] is based on the antibacterial effect and the production strain Streptomyces sp. A54476.
Is designated BE-54476.

The physicochemical properties of the novel antibacterial substance BE-54476 according to the present invention are shown below. The meanings of the abbreviations in the nuclear magnetic resonance spectrum are shown below. s: singlet d: doublet dd: double doublet ddd: double double doublet t: triplet q: quartet m: multiplet br: broad J: coupling constant Hz: hertz

[0009]Physicochemical properties of BE-54476 Properties: White amorphous solid or crystal Molecular formula: C_{twenty two}H₃₃N O_Five Mass spectrometry; [HRFAB-MS] m / z: found: 414.2287 (M + Na)⁺ Calculated value: 414.2256 (C_{twenty two}H₃₃N O_FiveNa) Specific rotation; [α]²⁰ _D+ 132 ° (c0.1, CHC
l_Three) UV absorption spectrum; λ_max(MeOH) nm
(Ε): 245 (6800), 284 (11600) Infrared absorption spectrum;_max(KBr) cm^-1: 34
48, 2960, 2929, 1655, 1603, 14
52, 1373, 1300, 1277, 1232, 10
99,1065 nuclear magnetic resonance spectrum;¹H-NMR (500 MHz,
CDCl_Three): Δ 5.76 (1H, brs), 5.50
(1H, brd, J = 5.8 Hz), 4.03 (1H,
dd, J = 12.0, 9.8 Hz), 3.87 (2H,
m), 3.30 (3H, s), 3.21 (1H, dd,
J = 9.4, 4.6 Hz), 2.77 (1H, ddd,
J = 12.0, 4.6, 3.8 Hz), 2.52 (1
H, m), 2.28 (2H, m), 1.63 (3H,
s), 1.62 (1H, m), 1.49 (2H, m),
1.35 (3H, d, J = 7.0 Hz), 1.32 (1
H, m), 1.16 (1H, m), 0.98 (3H,
d, J = 7.0 Hz), 0.98 (1H, m), 0.7
8 (3H, t, J = 7.0 Hz)¹³ C-NMR (125 MHz, CDCl_Three): Δ19
5.3 (s), 193.5 (s), 175.3 (s),
134.5 (s), 127.2 (d), 101.3
(S), 82.1 (d), 69.7 (d), 57.8
(Q), 57.5 (d), 43.2 (d), 39.2
(D), 36.2 (d), 34.2 (t), 32.6
(D), 31.2 (d), 30.9 (t), 20.8
(Q), 17.7 (q), 16.9 (t), 14.4
(Q), 12.0 (q) Solubility; chloroform, methanol, dimethyl sulf
It is easy to dissolve in organic solvents such as foxides and hard to dissolve in water. Rf value: 0.6 [Kieselgel 60F manufactured by Merck & Co., Ltd.]
₂₅₄Use and developing solvent: chloroform-methanol-water
(10: 6: 1)] Color reaction; Sulfuric acid reaction positive

[0010] Biological activity of BE-54476 (antibacterial activity)
For) The antibacterial activity of BE-54476 was measured against various pathogenic microorganisms, and its minimum inhibitory concentration (MIC) was determined. For MIC measurement, the Japanese Society of Chemotherapy standard method [Chemotherapy Vol. 29, No. 1,
76-79, 1981], and the presence or absence of bacterial growth was determined 24 hours later. B
The results for E-54476 are shown in Table 1.

[0011]

[Table 1] As shown in Table 1, BE-54476 has remarkable antibacterial activity against various pathogenic microorganisms including MRSA. Therefore, it is useful as an antibacterial agent against diseases which cause these pathogenic microorganisms as causative bacteria.

Next, a method for producing BE-54476 will be described. The microorganism used for producing the antibacterial substance BE-54476 of the present invention may be any microorganism that produces the antibacterial substance BE-54476. For example, a microorganism having the following mycological properties, that is, A54476 is used. be able to. 1. Form A54476 forms an aerial mycelium with a base mycelium that elongates and diverges well, and there is no division of the ring vegetation and the hypha. On the aerial hyphae, a long chain of spores (30-50) is formed, and the morphology is spiral. The surface of the spore is smooth or wrinkled and cylindrical with a size of about 0.7 to 1.2 × 1.0 to 1.5 μm. Special organs such as spore sac, flagella spore and sclerotium are Not observed. The formation of pseudospores of irregular size on tyrosine agar is observed. 2. Culture properties on various agar plate media The culture characteristics (cultivation at 28 ° C, 14 days) on various agar plate media were as shown in Table 2.

[0013]

[Table 2] 3. Growth temperature The growth temperature (east-malt agar medium, cultured for 14 days) is as shown in Table 3.

[0014]

[Table 3] 4. Physiological properties Table 4 shows the physiological properties.

[0015]

[Table 4] 5. Utilization of carbon source Utilization of carbon source is shown in Table 5.

[0016]

[Table 5] 6. Cell components LL-diaminopimelic acid and glycine were detected from the cell wall.

Based on the above mycological properties, A54476 is considered to belong to the genus Streptomyces. Therefore, A54476 was replaced by Streptomyces sp A
54476 (Streptomyces sp. A5
4476). This strain has been deposited with the Research Institute of Biotechnology, Industrial Technology Institute of the Ministry of International Trade and Industry, and the deposit number is FERM P-16839.

The Streptomyces sp A of the present invention
54476 (Streptomyces sp. A
The mutant strain of No. 54476) is irradiated with, for example, X-rays or ultraviolet rays, for example, a mutation such as nitrogen mustard, azaserine, nitrous acid, 2-aminopurine or 1-methyl-3-nitro-1-nitrosoguanidine (MNNG). Streptomyces sp. A5447 by a commonly used bacterial species conversion treatment method such as treatment with an inducing agent, for example, phage contact, transformation, transduction or conjugation.
6 (Streptomyces sp. A5447)
This is a microorganism obtained by mutating 6).

In producing the BE-54476 of the present invention, the BE-54476-producing strain is inoculated into a nutrient-containing medium and grown aerobically, whereby the BE-544476 is grown.
A culture containing 76 is obtained. As a nutrient source, a known nutrient source for actinomycetes can be used. For example, as a carbon source, commercially available glucose, maltose, starch, sucrose, molasses, dextrin, or the like is used alone or as a mixture. Nitrogen sources include commercially available full fat soybean powder, corn steep liquor, gluten meal, meat extract, yeast extract, dried yeast, cottonseed meal, peptone, wheat germ, fish meal, meat meal, defatted rice bran, defatted Meat-and-bone meal, inorganic ammonium salts or sodium nitrate are used alone or as a mixture. As the inorganic salt, commercially available calcium carbonate, sodium chloride, potassium chloride, magnesium sulfate, sodium bromide, sodium borate or various phosphates can be used. In addition, if necessary, trace amounts of heavy metal salts such as iron, manganese, zinc, cobalt, and molybdic acid can be added. In the case of severe foaming, for example, vegetable oils such as soybean oil or linseed oil, higher alcohols such as octadecanol, various silicon compounds, and the like may be appropriately added as antifoaming agents. Other than these, those useful for the production of BE-54476 by the producing bacterium, such as 3- (N-morpholino) propanesulfonic acid (MOP)
Any of S) and sodium borate can be used.

The cultivation method may be the same as the method for producing general microbial metabolites, and may be either solid culture or liquid culture. In the case of liquid culture, static culture, stirring culture,
Either shaking culture or aeration culture may be performed, but shaking culture or deep aeration stirring culture is particularly desirable. The culture temperature is suitably from 10 to 37 ° C., preferably from 14 to 37 ° C.
-30 ° C. The preferred pH of the medium is in the range of 6 to 8, and the culture time is 80 to 160 hours, preferably 96 to 1 hour.
20 hours. BE-547 of interest from culture
In order to collect 6, a separation means commonly used for collecting from metabolites produced by microorganisms can be appropriately used.

Since BE-54476 is present in the cells, conventional separation means such as solvent extraction, ion exchange resin, adsorption or partition chromatography, and gel filtration are used alone or in combination. Can be purified.

Preferred examples of the separation and purification include the following methods. First, the culture solution is filtered to obtain cells. The obtained cells are extracted using an organic solvent such as methanol or acetone. About the obtained crude extract, Sephadex L
Perform H-20 column chromatography. BE-5
The fraction containing 4476 was concentrated under reduced pressure and then HP-2
OSS column chromatography gives B
E-54476 can be obtained.

The compound BE-54476 of the present invention inhibits the growth of pathogenic microorganisms and exerts an antibacterial effect. However, when the compound of the present invention is used as an antibacterial agent, various administration forms can be selected. Tablets, capsules, powders,
An oral preparation such as a granule or a liquid preparation, or a sterile liquid parenteral preparation such as a solution or suspension can be used.

The solid preparation can be produced as it is in the form of tablets, capsules, granules or powder, but it can also be produced using appropriate additives. Such additives include, for example, sugars such as lactose or glucose, for example starches such as corn, wheat or rice, fatty acids such as stearic acid, inorganic salts such as magnesium aluminate or anhydrous calcium phosphate, for example polyvinylpyrrolidone or Synthetic polymers such as polyalkylene glycols; fatty acid salts such as calcium stearate or magnesium stearate; alcohols such as stearyl alcohol or benzyl alcohol; synthetic cellulose derivatives such as methylcellulose, carboxymethylcellulose, ethylcellulose or hydroxypropylmethylcellulose; , Water, gelatin,
Commonly used additives such as talc, vegetable oil, gum arabic and the like can be mentioned.

These solid preparations such as tablets, capsules, granules and powders generally contain 0.1 to 100% by weight,
It preferably contains from 5 to 100% by weight of active ingredient. Liquid preparations use suitable additives usually used in liquid preparations such as water, alcohols or plant-derived oils such as soybean oil, peanut oil or sesame oil, and are used in the form of suspensions, syrups or injections. Manufactured as In particular, for parenteral administration by intramuscular injection, intravenous injection or subcutaneous injection, suitable solvents include, for example, distilled water for injection, aqueous lidocaine hydrochloride (for intramuscular injection), physiological saline, aqueous glucose, ethanol, Liquid for intravenous injection (for example, aqueous solution of citric acid and sodium citrate) or electrolyte solution (for intravenous drip and intravenous injection)
Or a mixed solution thereof. These injections may be dissolved in advance, or may be in the form of powder or a solution to which an appropriate additive is added, which is dissolved at the time of use. These injections are usually 0.1 to 10% by weight,
It preferably contains 1 to 5% by weight of active ingredient. In addition, a liquid preparation such as a suspension or a syrup for oral administration is 0.5 to 1%.
Contains 0% by weight of active ingredient.

It should be noted that the actually preferred dosage of the compounds of the present invention will vary with the type of compound used, the type of composition formulated, the frequency of application and the condition of the patient. For example, the daily dose for an adult is 10 to 500 mg for oral administration, and 10 to 500 mg per day for parenteral administration, preferably intravenous injection.
100 mg. The number of administrations varies depending on the administration method and symptoms, but is 1 to 5 times.

[0027]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described specifically with reference to examples, but the present invention is not limited to these examples.

Example BE-54476 Production Method Actinomyces A54476 inoculated on a slope agar medium were prepared with 0.1% glucose, 2% dextrin (MS-3600), 0.5% fish meal, 1% corn gluten meal, and yeast extract. 0.1%, MOPS 0.5%, magnesium sulfate 0.
05%, sodium chloride 0.1%, calcium chloride 0.
05%, ferrous sulfate 0.0002%, cupric chloride 0.0
Medium (pH 7.0) consisting of 0004%, manganese chloride 0.00004%, cobalt chloride 0.00004%, zinc sulfate 0.00008%, sodium borate 0.00008%, and sodium molybdate 0.00024%.
Three 500 ml Erlenmeyer flasks containing 110 ml were inoculated and cultured at 28 ° C. for 120 hours on a rotary shaker (180 rotations per minute).

The culture solution obtained by the above culture (about 30
0 ml) to obtain bacterial cells by filtration. Methanol (100 ml) was added to the cells, and the mixture was stirred and filtered. After this operation was repeated once, these methanol extracts were combined, concentrated to dryness under reduced pressure, dissolved in methanol (5 ml), and then dissolved in a column of Sephadex LH-20 (Pharmacia, 3.0φ). × 50 cm) and eluted with methanol. The eluate was concentrated to dryness and then HP-20
The BE-54 column was eluted with 90% methanol using an SS column (Mitsubishi Kasei, 2.0φ × 17 cm).
476 (11.0 mg) were obtained.

The preparation examples of the compound of the present invention are shown below.
The preparation of the compound of the present invention is not limited to this preparation example. Formulation Example 1 10 parts of this substance (BE-54476), 15 parts of heavy magnesium oxide, and 75 parts of lactose were uniformly mixed, and
m or less powder or fine-grained powder. This powder was placed in a capsule container to obtain a capsule. Formulation Example 2 45 parts of this substance (BE-54476), 15 parts of starch, 16 parts of lactose, 21 parts of crystalline cellulose, 3 parts of polyvinyl alcohol and 30 parts of distilled water are uniformly mixed, crushed and granulated and dried. And then sieved to a diameter of 355 to 1400 μm
Granules of the size Formulation Example 3 After a granule was prepared in the same manner as in Formulation Example 2, 3 parts of calcium stearate was added to 96 parts of the granule, followed by compression molding to prepare a tablet having a diameter of 10 mm. Formulation Example 4 90 parts of granules obtained in the same manner as in Formulation Example 2 were mixed with 10 parts of crystalline cellulose and 3 parts of calcium stearate.
The resulting mixture was compression-molded to give a tablet having a diameter of 8 mm, and a syrup gelatin and a precipitated calcium carbonate mixed suspension were added thereto to prepare a sugar-coated tablet. Formulation Example 5 0.6 part of this substance (BE-54476), 2.4 parts of a nonionic surfactant and 97 parts of physiological saline are heated and mixed, then put into an ampoule, sterilized, and an injection is prepared. Produced.

[0031]

The BE-54476 described in the present invention has M
It shows remarkable antibacterial activity against various pathogenic microorganisms including RSA. Therefore, it is useful as an antibacterial agent against diseases which cause these pathogenic microorganisms as causative bacteria.

[0032]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification FI FI Theme Court II (Reference) C12R 1: 465) (C12P 17/10 C12R 1: 465) (72) Inventor Hisao Kondo Okubo, Tsukuba City, Ibaraki Prefecture 3 Banyu Pharmaceutical Co., Ltd. Tsukuba Research Laboratories (72) Katsuhisa Kojiri 2-3-2 Nihonbashi Honcho, Chuo-ku, Tokyo Inside Banyu Pharmaceutical Co., Ltd. (72) Inventor Hiroyuki Suda 3 Okubo, Tsukuba, Ibaraki Pref. Address Banyu Pharmaceutical Co., Ltd. Tsukuba Research Laboratories F-term (Reference) 4B064 AE48 BA05 BE07 BE09 BE19 BG01 BG09 BH01 BH02 BH04 BH05 BH06 BH07 BH08 BH10 CA04 DA02 4B065 AA50X AC14 CA24 CA44 4C069 AC17 BA01 ABA22A04 BC04 BC06 MA01 MA04 NA14 ZB35

Claims (6)

[Claims] (1) Structural formula [I] A compound represented by the formula: 2. Streptomyces (Streptomyc)
es) culturing a microorganism or a mutant thereof belonging to the genus and capable of producing the compound represented by the structural formula [I], and collecting the compound represented by the structural formula [I] from the culture. A method for producing a compound represented by the structural formula [I]. 3. A microorganism capable of producing the compound represented by the structural formula [I] is Streptomyces sp.
A54476 (Streptomycesp. A5
4476) or a mutant thereof. 4. An antibacterial agent comprising the compound represented by the structural formula [I] according to claim 1 as an active ingredient. A microorganism belonging to the genus Streptomyces, which has an ability to produce the compound represented by the structural formula [I] according to claim 1. 6. Streptomyces (Streptomyc)
es) is a microorganism belonging to the genus Streptomyces sp. A54476 (Streptomyces sp.
A54476) or a mutant thereof.