JP2007097548A - New antibiotic material sf2847 substance, method for producing the same and medicinal composition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new antibacterial agent and a method for producing the same. <P>SOLUTION: This new antibiotic SF2847 substance is provided. The method for producing the same is characterized by culturing actinomyces, SF2847 strain producing the substance and collecting the objective substance from a cultured material of the same bacteria. Further, the medicinal composition containing the same is also provided, and in addition, the antibacterial agent containing the same is also provided. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a novel antibiotic SF2847 substance, a process for producing the same, and a pharmaceutical composition comprising the compound as an active ingredient.

  The treatment of infectious diseases by chemotherapy has made tremendous progress since the practical application of penicillin, so far many antibacterials including β-lactam antibacterials, aminoglycosides, macrolides and new quinolones Drugs have been developed and used to treat infections. On the other hand, due to the frequent use of these drugs, resistant bacteria that do not work with existing antibacterial drugs have emerged, and the development of new antibacterial drugs that do not have cross-resistance with existing antibacterial drugs is desired.

  Conventionally, compounds having a tetronic acid structure such as kijanimicin (Non-patent Document 1) or decatromicins (Non-patent Document 2) are known as compounds similar in chemical structure to the compound of the present invention. Have distinct physicochemical properties and are clearly distinguished.

Mallams et al., J. Chem. Soc. Perkin Trans. I, pp. 1497-1534, 1983 Momose et al., J. Antibiotics 52, 781-786, 1999

  An object of the present invention is to provide a new antibacterial drug having a chemical structure different from that of existing antibacterial drugs and effective against various resistant bacteria in order to overcome the intractable disease caused by resistant bacteria. .

  In order to find an antibacterial drug having a new chemical structure, the present inventors have focused on antibiotics produced by microorganisms and searched for antibiotics that can become new antibacterial drugs from microorganism products. As a result, the presence of substances having antibacterial activity was observed in the culture solution of actinomycetes belonging to the genus Micromonospora.

  Subsequently, in order to identify the active substance in the culture broth, the active ingredient was purified from the culture broth and the structure was analyzed. As a result, it was revealed that the novel compound represented by the formula (1) has antibacterial activity, and the present invention Was completed.

  That is, the present invention provides a novel antibiotic SF2847 substance represented by the formula (1). The present invention also provides a method for producing a compound of the present invention, which comprises culturing a strain, SF2847, which produces the compound of the present invention, and collecting the target compound from the culture solution. Furthermore, this invention provides the pharmaceutical composition containing this invention compound, and provides the antibacterial agent containing this invention compound in detail.

2.ミクロモノスポラ属に属し、上記１に記載の化合物を生産する能力を有する微生物を培養し、その培養物から前記化合物を採取する工程を含む、上記１記載の化合物の製造法；
3.上記１に記載の化合物又はその製薬学的に許容される塩を、薬学上許容し得る担体とともに含んでなる医薬組成物；
4.上記１に記載の化合物又はその製薬学的に許容される塩を、薬学上許容し得る担体とともに含んでなる抗菌剤；
5.ミクロモノスポラ属に属する菌であって、上記１記載の化合物を生産する特徴を有し、独立行政法人産業技術総合研究所特許生物寄託センターにおける寄託番号がFERM P-20626であるMicromonospora sp. SF2847株又はその変異株；
に関するものである。 That is, the present invention
1. a novel antibiotic SF2847 represented by formula (1) or a pharmaceutically acceptable salt thereof;

2. A method for producing a compound according to the above 1, comprising culturing a microorganism belonging to the genus Micromonospora and capable of producing the compound according to the above 1, and collecting the compound from the culture;
3. A pharmaceutical composition comprising the compound according to 1 or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier;
4. An antibacterial agent comprising the compound according to 1 or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier;
5. A micromonospora sp. Belonging to the genus Micromonospora, which has the feature of producing the compound described in 1 above, and whose deposit number is FERM P-20626 at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center. SF2847 strain or its mutant strain;
It is about.

  The novel antibiotic SF2847 represented by the above formula (1) of the present invention has an excellent antibacterial activity and is useful as a pharmaceutical ingredient for the prevention or treatment of bacterial infections.

Details of the present invention will be described below.
The novel antibiotic SF2847 substance (hereinafter referred to as SF2847 substance) represented by the above formula (1) of the present invention has the following physicochemical properties.
(1) Color and properties: colorless powder (2) Molecular formula: C ₃₂ H ₄₄ O ₆
(3) Mass spectrum (HRFAB-MS):
Actual value: m / z 525.3215 [M + H] ⁺
Calculated value: m / z 525.3216 [C ₃₂ H ₄₄ O ₆ + H] ⁺
(4) Specific rotation: [α] _D = −88.6 (c 0.5, CHCl ₃ , 25 ° C.)
(5) UV absorption spectrum λ _max , nm (ε): [MeOH + 0.01N HCl] 204 (9958), 234 (6498), 310 (4192),
[MeOH + 0.01N NaOH] 208 (17505), 248 (8280), 290 (6394)
(6) Infrared absorption spectrum (KBr tablet)
ν _max , cm ^-1 : 3450, 2930, 1759, 1624, 1456, 1086, 993
(7) ¹ H-NMR spectrum (CDCl ₃ , 400 MHz)
δ (ppm): 0.86 (3H, d), 1.03 (3H, d), 1.21 (3H, d), 1.25 (H, m), 1.26 (3H, s), 1.40 (H, ddd), 1.47 (H , ddd), 1.58 (H, m), 1.59 (3H, s), 1.59 (H, m), 1.68 (H, ddd), 1.72 (H, m), 1.73 (H, dd), 1.82 (H, m), 1.87 (H, m), 1.93 (H, ddd), 2.00 (H, m), 2.01 (H, m), 2.08 (H, m), 2.36 (H, dd), 2.38 (H, dd ), 2.74 (H, m), 2.83 (H, m), 3.83 (H, m), 4.00 (H, d), 4.34 (H, d), 5.01 (H, ddd), 5.22 (H, s) , 5.38 (H, dd), 5.45 (H, ddd), 5.50 (H, ddd)
(8) ¹³ C-NMR spectrum (CDCl ₃ , 100 MHz)
δ (ppm): 15.8, 18.9, 20.7, 23.1, 23.3, 24.6, 27.6, 28.7, 31.4, 32.7 (× 2), 39.1, 39.4, 39.8, 40.9, 41.8, 42.0, 42.1, 50.6, 65.0, 66.8, 86.0 , 107.2, 120.7, 124.9, 130.1, 130.4, 137.0, 144.3, 166.9, 201.8, 204.3
(9) Solubility: Soluble in methanol, chloroform, ethyl acetate and dimethyl sulfoxide, insoluble in hexane and water.

  The “pharmaceutically acceptable salt” of the SF2847 substance includes a salt with a pharmaceutically acceptable base. For example, a salt with an alkali metal such as sodium, potassium or lithium or an alkaline earth metal such as calcium can be used.

  The SF2847 substance can be produced by culturing a SF2847 substance-producing bacterium belonging to the genus Micromonospora, collecting the SF2847 substance from the culture, and purifying it. Examples of the SF2847 substance-producing bacteria include the SF2847 strain belonging to the genus Micromonospora newly isolated from the Hachijojima soil sample by the present inventors.

  The SF2847 substance-producing bacterium used in the present invention is not limited to the specific microorganism described in the present specification. Any bacteria may be used as the SF2847 substance-producing bacteria as long as it has the ability to produce the SF2847 substance. Examples of microorganisms that can be used include the SF2847 strain, its subcultures, artificial mutants, natural mutants, and genetically modified strains.

The bacteriological properties, culture method and purification method of SF2847 substance of this strain are as follows.
1. Taxonomic properties of SF2847 strain
The morphological properties, culture properties, physiological properties, chemical taxonomic properties of SF2847 strain and taxonomic analysis by analysis of 16S rRNA gene are described in "Classification and Identification of Actinomycetes" (Classification and Identification of Actinomycetes, Japan The method described in the Actinomycete Society, published by the Japan Society for Business Administration, 2001) was followed.

(A) Morphological properties
The SF2847 strain was cultured at 28 ° C. for 14 days in yeast extract / starch agar medium (yeast extract 0.2%, starch 1.0%, agar 18 g, purified water 1 L, pH 7.0), and observed with an optical microscope and a scanning electron microscope. Vegetative mycelium develops well with a diameter of 0.2-0.3 μm and branches irregularly but does not break. No formation of aerial hyphae is observed. Numerous spherical spores are formed in the vegetative mycelium alone, their diameter is 0.5 to 1.0 μm, and the surface is slightly rough.

(B) Culture properties
The growth of the SF2847 strain after culturing at 28 ° C. for 14 days, the color of the back of the colony, the presence or absence of soluble pigments, and the color tone are as follows. In addition, formation of aerial hyphae was not recognized by all the culture media. The color display was in accordance with the Color Harmony Manual (Color Harmony Manual, Container Corporation of America, 1958).
(1) Yeast / malt agar medium (ISP-2): The colony grows well, and the reverse side is ocher. No soluble pigment is produced.
(2) Oatmeal agar medium (ISP-3): Growth is normal and the back side is dark brown. No soluble pigment is produced.
(3) Starch / inorganic salt agar medium (ISP-4): The growth is good and the back side is brown. No soluble pigment is produced.
(4) Glycerin / asparagine agar medium (ISP-5): The growth is good and the back surface is ocher. No soluble pigment is produced.
(5) Peptone / East / Iron Agar (ISP-6): Growth is poor and the reverse side is amber. No soluble pigment is produced.
(6) Tyrosine agar medium (ISP-7): Growth is normal and the reverse side is ocher. No soluble pigment is produced.
(7) Starch yeast agar medium: The growth is good and the back surface is ocher. No soluble pigment is produced.
(8) Glucose / asparagine agar medium: Growth is normal and the reverse side is yellowish brown. No soluble pigment is produced.

(C) Physiological properties (1) Production of melanin-like pigments: No melanin-like pigments are produced on peptone, yeast, iron agar (ISP-6) and tyrosine agar (ISP-7).
(2) Liquefaction of gelatin: Gelatin is liquefied.
(3) Milk coagulation / peptonization: Skim milk does not undergo peptone formation and coagulation.
(4) Starch hydrolysis: Starch is hydrolyzed.
(5) Reduction of nitrate: Nitrate is not reduced.
(6) Growth temperature range: It grows weakly at 15 ° C and 42 ° C, and grows well at 28 ° C to 37 ° C. The optimum temperature for growth is around 28 ° C.
(7) Salt tolerance: When yeast extract / starch agar medium is used as a basic medium, it grows at a salt concentration of 2% and grows slightly at 3%.
(8) Use of carbon source: D-glucose, raffinose, D-xylose, D-fructose, sucrose, trehalose and cellobiose are used, but L-arabinose, myo-inositol, D-mannitol and L-rhamnose are used. do not do.

(D) Chemical taxonomic properties The presence of meso-diaminopimelic acid and glycine was confirmed in the cell wall, and arabinose and xylose were detected in the hydrolyzate of the whole cell. It was classified as cell wall chemistry type IID type by classification. The main menaquinones were MK-10 (H ₄ ) and MK-10 (H ₆ ), accounting for about 70% in total. Next, MK-9 (H ₄ , H ₆ ) was the most. Cellular fatty acids were mainly branched fatty acids of iso-C16: 0, iso-C15: 0, iso-C17: 0, and did not contain 10 methyl fatty acids or hydroxy fatty acids.

  These morphological and chemical taxonomic properties strongly suggested that the SF2847 strain belongs to the genus Micromonospora.

(E) 16S rRNA gene analysis
As a result of decoding the partial base sequence (577bp) of 16S rRNA gene of SF2847 strain and searching the database, the homology with Micromonospora purpureochromogenes (X92611) is the highest at 98%, and it is included in the cluster of Micromonospora genus It was.

  Based on the above, the SF2847 strain is considered to belong to the genus Micromonospora, and this strain was designated as Micromonospora sp. SF2847.

  This strain was deposited at the Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology under the accession number FERM P-20626 on August 19, 2005.

2. Culture of SF2847 substance-producing bacterium In the method of the present invention, SF2847 substance-producing bacterium, for example, SF2847 strain is cultured in a nutrient medium containing an appropriate carbon source and nitrogen source. The medium used may be either a natural medium or a synthetic medium. As the carbon source, carbohydrates such as glucose, fructose, sucrose, molasses, starch or starch hydrolyzate, or organic acids such as propionic acid are used. On the other hand, as the nitrogen source, usually peptone, meat extract, yeast extract, corn steep liquor, oatmeal, wheat germ, casein hydrolyzate, soybean meal or soybean meal hydrolyzate are used, but ammonium, ammonium sulfate, ammonium nitrate or phosphorus Inorganic nitrogen compounds such as ammonium acid, or organic nitrogen compounds such as urea or amino acids are also effective. These carbon sources and nitrogen sources can be used in combination.

  If necessary, add potassium phosphate, potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, or other inorganic salts to the medium. May be. Moreover, when a culture medium foams, liquid paraffin, animal oil, vegetable oil, mineral oil, or silicon can be added.

  The SF2847 substance-producing bacterium is cultured under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature can be appropriately changed within a range in which the SF2847 substance-producing bacterium produces the target substance, but preferably 25 to 37 ° C. The culture time is usually 1 to 14 days.

3. Collection and purification of SF2847 substance When collecting and purifying the SF2847 substance of the present invention from a microorganism culture solution, conventional separation means using its properties, for example, a solvent extraction method, an adsorption / desorption method using an adsorbent, A chromatographic method using a resin and a precipitation method can be used in appropriate combination.

  Specifically, acetone is added to the culture solution of the SF2847 strain to extract the SF2847 substance from the cells, and the cells are removed by filtration or centrifugation. Acetone is distilled off from the obtained extract, followed by extraction with ethyl acetate, followed by purification in order using a column such as silica gel, LH-20 and ODS. Finally, purification by HPLC or TLC can be performed to isolate the SF2847 substance.

  The SF2847 substance of the present invention or a pharmaceutically acceptable salt thereof has antibacterial activity as shown in Test Examples, and a pharmaceutical composition containing the SF2847 substance as an active ingredient is used for animals including humans. It is useful to administer as an antibacterial agent. The target bacteria when the SF2847 substance is used as an antibacterial agent is not particularly limited as long as the SF2847 substance exhibits an antibacterial activity, but exhibits an excellent antibacterial activity particularly against gram-positive bacteria.

  A pharmaceutical composition containing the SF2847 substance or a pharmaceutically acceptable salt thereof can be formulated in accordance with a conventional method, if necessary, together with a carrier according to various administration forms or use forms.

  Examples of the preparation for oral administration include tablets, pills, granules, capsules, powders, solutions, suspensions, syrups, sublinguals and the like. Examples of the preparation for parenteral administration include injections, transdermal absorption agents, inhalants, suppositories and the like. In formulating, pharmaceutical additives such as surfactants, excipients, stabilizers, wetting agents, disintegrating agents, solubilizing agents, isotonic agents, buffers, coloring agents, and flavoring agents are appropriately used.

  As the carrier, pharmaceutically acceptable ones can be used, and the type and composition can be appropriately determined depending on the administration route and administration method. For example, water, alcohol, soybean oil, sesame oil, or the like can be used as the liquid carrier. As solid carriers, sugars such as maltose and sucrose, amino acids such as lysine, polysaccharides such as cyclodextrin, organic acid salts such as magnesium stearate, and cellulose derivatives such as hydroxylpropylcellulose can be used.

  When SF2847 substance or a salt thereof is administered as a pharmaceutical composition, the dosage varies depending on the patient's age, body weight, type and degree of disease, and route of administration. In the case of intravenous administration, it can be administered within the range of 0.01-100 mg / kg.

  Examples of the present invention will be described below, but the present invention is not limited to these examples, and includes all modifications and modification means not shown here.

<Example>
1. Cultured glucose of SF2847 substance producing bacteria 1.0%, soluble starch 2.0%, yeast extract 0.3%, polypeptone 0.5%, wheat germ 0.6%, soybean cake 0.2% and calcium carbonate 0.2%, 6 N sodium hydroxide The pre-culture medium adjusted to pH 7.0 with 20 mL was dispensed into 12 100 mL Erlenmeyer flasks and sterilized at 120 ° C. for 20 minutes. To this, SF2847 strain (FERM P-20626) in agar plate culture was inoculated one by one and then shaken at 28 ° C. for 3 days, and used as a seed culture solution.
On the other hand, it contains 2.0% glucose, 1.0% soluble starch, 1.5% soybean meal, 0.8% wheat germ, 0.1% polypeptone, 0.1% sodium chloride, 0.2% calcium carbonate, and 0.001% zinc sulfate, pH 7.2 with 6N sodium hydroxide. 80 ml of the production medium prepared in the above was dispensed into 100 500 ml Erlenmeyer flasks and sterilized at 120 ° C. for 20 minutes. Thereafter, 1.6 mL of the seed culture solution obtained previously was aseptically inoculated and cultured at 28 ° C. for 5 days with agitation of 200 rpm.

2. Purification of substance SF2847 To the obtained culture (5.8 L), an equal amount of acetone was added and stirred, and then the cells were filtered. Acetone in the filtrate was distilled off under reduced pressure, adjusted to pH 3.0 with 1 N hydrochloric acid, and extracted with 3 L of ethyl acetate. The ethyl acetate layer was concentrated under reduced pressure to obtain 4.67 g of a crude extract. The crude extract was covered with a small amount of silica gel (Wakogel C300, Wako Pure Chemical Industries, Ltd.), dried under reduced pressure, and overlaid on 50 g of silica gel (same as above). Silica gel was washed with 500 mL each of hexane, acetone-hexane (20:80), chloroform, and methanol-chloroform (1:99), then methanol-chloroform (3:97, 10:90, 30:70 500 mL each) Eluted with. The eluates were combined and concentrated under reduced pressure to obtain 925 mg of residue. This residue was dissolved in a small amount of methanol and applied to a column of Cosmo Seal 75C ₁₈ -OPN (Nacalai Tesque, 10 g) packed with 80% aqueous methanol solution containing 0.01% TFA. Elute sequentially with 100 mL. The methanol eluate was concentrated under reduced pressure to obtain 97 mg of a residue. Dissolve this residue in a small amount of methanol, inject it into HPLC (column: Inertosyl ODS-2, ID 2 cm x 25 cm, GL Sciences), and apply a gradient of 40% to 100% acetonitrile / 0.01% phosphoric acid aqueous solution. And eluted. The eluate containing the SF2847 substance was collected and acetonitrile was distilled off under reduced pressure. After extraction with ethyl acetate, the ethyl acetate layer was dried under reduced pressure to obtain 17.3 mg of SF2847 substance.

<Test example>
The antibacterial activity of SF2847 substance was measured as the minimum inhibitory concentration (MIC) by the agar plate dilution method. Test bacteria cultured overnight in growth medium are adjusted to 10 ⁶ cells / ml. One platinum loop is inoculated into Mueller Hinton agar medium (Difco) containing SF2847 substance and cultured at 37 ° C for 18-20 hours. did. The results are shown in Table 1.

Claims (5)

A novel antibiotic SF2847 represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

The method for producing a compound according to claim 1, comprising a step of culturing a microorganism belonging to the genus Micromonospora and capable of producing the compound according to claim 1, and collecting the compound from the culture. A pharmaceutical composition comprising the compound according to claim 1 or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier. An antibacterial agent comprising the compound according to claim 1 or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier. A micromonospora sp. Belonging to the genus Micromonospora, which has the characteristic of producing the compound according to claim 1 and whose deposit number is FERM P-20626 at the National Institute of Advanced Industrial Science and Technology (AIST). SF2847 strain or a mutant thereof.