JPH09100261A - Antimicrobial substance be-44651 - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an antimicrobial substance BE-44651 which has excellent antimicrobial effect against a variety of pathogenic microorganisms including MRSA and is useful in the field of medicines. SOLUTION: This microbial substance BE-44651 is represented by formula I (R is H or a group shown by formula II) produced by a microorganism in Actinomadura. The compound of formula I is produced by inoculating a microorganism of Actinomadura sp. A44561 or its modified strain to a culture medium containing nutrients at a pH of 4-8, culturing it, preferably at 26-36 deg.C for 68-240 hours and separating and purifying the product.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention is useful in the field of medicine, and more specifically, a group of novel compounds exhibiting an antibacterial effect by inhibiting the growth of microorganisms, a method for producing the same, its use, and production of the compound. Pertaining to microorganisms that do.

[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: MRSA) and many other hospital-acquired infections by multidrug-resistant bacteria have become a serious problem clinically [see Oguri et al., Clinical and Microorganisms, Vol. 15, pp. 7-15 (1988)].

[0003] Under such circumstances, development of a drug effective against MRSA is required.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide a multi-drug resistant MRS in which existing antibacterial agents cannot sufficiently exert their effects.
An object of the present invention is to provide a novel compound having excellent antibacterial activity against A.

[0005]

DISCLOSURE OF THE INVENTION As a result of extensive screening of secondary metabolites of microorganisms for substances having antibacterial activity, the present inventors have found that compounds represented by the following general formula [I] exhibit excellent antibacterial activity. The present invention has been completed.

That is, the present invention provides a novel compound represented by the general formula [I]:

[0007]

Embedded image [In the formula, R is a hydrogen atom or a formula:

[0008]

Embedded image And a method for producing the compound, and the ability to produce the compound. Actinomadura characterized by the following formula:
ra) microorganisms belonging to the genus Ra.

The physicochemical properties of the compound of the present invention are shown below.

Here, R in the general formula [I] is represented by the formula:

[0011]

Embedded image The compound which is a group represented by is referred to as BE-44651A, the compound where R is a hydrogen atom is referred to as BE-44651B, and both are referred to as BE-44651.

The meanings of abbreviations in NMR measurement are shown below.
You. s: singlet d: doublet t: triplet q: quartet m: multiplet br: broad J: coupling constant Hz: hertzPhysicochemical properties of BE-44651A Property: Yellow amorphous solid or crystal Molecular formula: C₅₀H ₆₇O₂₀N mass spectrometry: [high resolution FAB-MS] (MH)^-age
: Calculated value 1000.4178; measured value 1000.41
74 Specific rotation: [α]²⁰ _D= -24.0 ° (c0.100,
CHCl_Three) UV absorption spectrum: λ_max(MeOH, nm
(Ε)) 223 (25200), 267 (3950)
0), 426 (10600) red external absorption spectrum: (KBr, cm^-1) 3417,
2935, 1633, 1574, 1446, 1365,
1207, 1168, 1120, 1070, 1009,
905, 854, 756¹ H-NMR spectrum (500 MHz, CDCl_Three, Δ
ppm) 1.05 (3H, d, J = 6.3Hz), 1.
13 (3H, d, J = 6.7 Hz), 1.19 (3H,
d, J = 6.7 Hz), 1.31 (3H, d, J = 6.
3Hz), 1.32 (3H, d, J = 6.3Hz),
1.50-2.15 (14H, m), 2.20-2.3
0 (2H, m), 2.98-3.14 (4H, m),
3.18-3.27 (2H, m), 3.42-3.60
(6H, m), 3.56 (3H, s), 3.93 (1
H, brq, J = 6.7 Hz), 4.07 (1H, br
q, J = 6.7 Hz), 4.33 (1H, brs),
4.38 (1H, d, J = 10.1Hz), 4.45
(1H, brd, J = 10.1Hz), 4.49 (1
H, m), 4.50 (1H, dd, J = 1.8, 9.8)
Hz), 4.75 (1H, brs), 4.82 (1H,
brs), 4.95 (1H, brs), 5.99 (1
H, s), 6.89 (1H, d, J = 7.8 Hz),
7.01 (1H, s), 7.14 (1H, d, J = 7.
8Hz), 7.53 (1H, t, J = 7.8Hz),
9.46 (1H, s), 9.62 (1H, brs), 1
5.2 (1H, brs), 18.3 (1H, brs)¹³ C-NMR spectrum (125 MHz, CDCl_Three,
δppm) 16.8 (q), 17.0 (q) x 2, 1
7.6 (q), 18.0 (q), 24.3 (t) x 3,
24.4 (t), 24.8 (t), 25.0 (t), 2
6.4 (t), 37.0 (t), 39.0 (t), 4
2.3 (d), 54.6 (q), 66.6 (d), 6
7.4 (d), 67.6 (d), 71.5 (d), 7
1.6 (d), 71.7 (d), 73.7 (d), 7
4.3 (d), 75.6 (d), 75.8 (d), 7
6.1 (d), 77.5 (d), 80.8 (d), 8
2.4 (s), 97.4 (d), 99.2 (d), 9
9.5 (d), 100.6 (s), 101.2 (d),
101.3 (d), 109.2 (s), 111.8.
(D), 112.5 (s), 118.3 (d), 11
8.5 (d), 132.8 (s), 133.6 (d),
139.7 (s), 158.1 (s), 166.6
(S), 173.6 (s), 190.2 (s), 19
3.9 (s), 195.6 (s) Solubility: chloroform, methanol, dimethyl sulfoxide
It is easily soluble in organic solvents such as sides and hardly soluble in water.

Thin layer chromatography: [Merck
-Selgel 60F₂₅₄] Rf value 0.78 [Developing solvent: chloroform-methanol
-Water (14: 6: 1)] Color reaction: sulfuric acid reaction positivePhysicochemical properties of BE-44651B Property: Yellow amorphous solid or crystal Molecular formula: C₂₀H₁₇O₈N mass spectrometry: [high resolution FAB-MS] (M)⁺As:
Calculated value 399.0954; Measured value 399.0956 Specific rotation: [α]²⁰ _D= + 52.0 ° (c0.100,
CHCl _Three) UV absorption spectrum: λ_max(MeOH, nm
(Ε)) 223 (23100), 267 (3730)
0), 426 (9400) red external absorption spectrum: (KBr, cm^-1) 3423,
1635, 1570, 1446, 1411, 1383,
1099, 1076, 1016, 991¹ H-NMR spectrum (500 MHz, CDCl_Three, Δ
ppm) 2.95 (1H, m), 3.32 (1H, d
d, J = 1.8, 16.5 Hz), 3.45 (1H, d
d, J = 3.7, 16.5 Hz), 3.59 (3H,
s), 3.78 (1H, s), 4.06 (1H, d, J
= 12.2 Hz), 6.16 (1H, brs), 6.8
7 (1H, d, J = 7.9 Hz), 7.13 (1H,
s), 7.16 (1H, d, J = 7.9 Hz), 7.5
1 (1H, t, J = 7.9 Hz), 9.44 (1H,
s), 9.45 (1H, s), 15.0 (1H, s),
18.3 (1H, s)¹³ C-NMR spectrum (125 MHz, CDCl_Three,
δppm) 24.9 (t), 43.0 (d), 54.8
(Q), 69.6 (d), 81.1 (s), 99.1
(S), 109.1 (s), 111.5 (d), 11
2.3 (s), 118.4 (d), 119.4 (d),
132.5 (s), 133.5 (d), 139.9
(S), 157.9 (s), 166.3 (s), 17
3.6 (s), 193.7 (s), 194.3 (s),
194.6 (s) Solubility: chloroform, methanol, dimethyl sulfoxide
It is easily soluble in organic solvents such as sides and hardly soluble in water.

[0014] Thin layer chromatography: Merck Kieselgel 60F _254] Rf value 0.78 [developing solvent: chloroform - methanol - water (14: 6: 1)] Color reaction: sulfuric acid reaction positive BE-forty-four thousand six hundred fifty-one acids Antibacterial activity The antibacterial activity of BE-44651 was measured against various pathogenic microorganisms, and the minimum inhibitory concentration was determined.

First results for BE-44651s
It is shown in the table.

[0016]

[Table 1] As shown in Table 1, BE-44651 is MRSA.
It shows a remarkable antibacterial activity against various pathogenic microorganisms including. Therefore, it is useful as an antibacterial agent against diseases caused by these pathogenic microorganisms.

A method for producing BE-44651 will be described.

The microorganisms used for the production of the antibacterial substance BE-44651 of the present invention are antibacterial substances BE-44651.
Any microorganism can be used as long as it produces the same kind, but for example, a microorganism having the following mycological characteristics, that is, A44651 strain, can be used, which is collected from a grassland in the Philippine University. 1. Morphology A 44651 strain forms aerial hyphae and basal hyphae that are well elongated and branched, and no division of the hyphae and hyphae is observed. The spore chain formed on the aerial mycelium is relatively short (~ 10
The number of spores is 1.3 to 1.0 × 0.
It is about 9 to 0.6 μm. The spores are oval in shape and the surface is wart-like. Special organs such as pseudosporum, sporangium, flagella spores and sclerotium are not observed.

[0019]

[Table 2]

[0020]

[Table 3]

[0021]

[Table 4]

[0022]

[Table 5] 6. Bacterial cell components Although meso-diaminopimelic acid and galactose were detected from the cell wall, glycine and arabinose were not found, suggesting that the cell wall type is type III. Although majurose and galactose were detected as the major sugar components of all the cells, arabinose and xylose were not detected and the sugar pattern was B type. Further, mycolic acid is not detected, the main menaquinone was MK-9 (H _6). Regarding phospholipids, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine and unknown glucosamine-containing phospholipids were not detected, phosphatidylinositol mannoside and phosphatidylinositol were found, and the phospholipid type was PI type.

[0023]

[Table 6]

[0024]

[Table 7]

[0025]

[Table 8]

[0026]

[Table 9]

[0027]

[Table 10]

[0028]

[Table 11] From the above mycological properties, the A44651 strain is considered to belong to the actinomycete Actinomajura genus.

Therefore, the strain A 44651 was designated as Actinomajura SP A 44651 (Actinom).
adura sp. A 44651).

This strain has been deposited at the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry, and the deposit number is FERM P-1509.

Actinomajura SP A of the present invention
44651 (Actinomadura sp. A)
44651) mutant strain, for example, irradiation treatment with X-rays or ultraviolet rays, for example, nitrogen mustard, azaserine, nitrous acid, 2-aminopurine or N-methyl-N'-nitro-N-nitrosoguanidine (NTG).
Treatment with a mutagen, etc., phage contact, transformation,
Actinomajura SP A 44651 by a commonly used bacterial species conversion treatment method such as transduction or conjugation.
(Actinomadura sp. A 4465
It can be obtained by mutating 1).

In producing the BE-44651s of the present invention, BE-44651-producing strains are inoculated into a nutrient source-containing medium to aerobically grow to obtain BE-4.
A culture containing 4651s is obtained. As a nutrient source,
Known sources of 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. As the nitrogen source, commercially available soybean flour, corn steep liquor, meat extract,
Yeast extract, dried yeast, cottonseed flour, peptone, wheat germ,
Fish meal, meat meal, defatted rice bran, defatted meat and bone meal, inorganic ammonium salts, sodium nitrate, etc. may be 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. In addition to these, BE-
Any of those useful for the production of 44651s, such as 3- (N-morpholino) propanesulfonic acid or sodium borate, can be used.

The culture method may be the same as the method for producing a general microbial metabolite, and may be 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 appropriately 19 to 43 ° C., preferably 26.
~ 36 ° C. The pH of the preferred medium is in the range of 4 to 8, and the culture time is 120 hours to 288 hours, preferably 1 hour.
68 hours to 240 hours. Target B from the culture
To collect E-44651s, a separation means usually used for collecting from metabolites produced by microorganisms is appropriately used.

The BE-44651s are present in the culture filtrate and the cells, so that they can be separated from the culture filtrate or the cells by a conventional means such as a solvent extraction method, an ion exchange resin method or an adsorption or partition chromatography method and gel filtration. The purification can be carried out by a single method or a combination of methods.

The following method is mentioned as an example of preferable separation and purification. First, the culture solution is filtered to obtain cells. The obtained cells are extracted using an organic solvent such as methanol or acetone. The crude extract thus obtained was partitioned between water and ethyl acetate, and the extract obtained after distilling off ethyl acetate was subjected to Sephadex LH-20 chromatography (eluted with chloroform / methanol) to give BE-44651. You can get the kind.

The compounds BE-44651 of the present invention inhibit the growth of pathogenic microorganisms and exert an antibacterial effect. However, when the compound of the present invention is used as an antibacterial agent, various administration forms can be selected. Examples include oral agents such as tablets, capsules, powders, granules or solutions, or sterile liquid parenteral agents such as solutions or suspensions.

The solid preparation can be produced as it is in the form of tablets, capsules, granules or powders, or can be produced using appropriate additives. Examples of such additives include 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 metasilicate aluminate or anhydrous calcium phosphate, for example, polyvinyl, and the like. Synthetic polymers such as pyrrolidone or 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 , Other,
Examples of commonly used additives such as water, gelatin, talc, vegetable oil, and gum arabic are included.

The solid preparations such as tablets, capsules, granules and powders are generally 0.1 to 100% by weight,
It preferably contains from 5 to 100% by weight of active ingredient.

The liquid preparation uses suspensions, syrups or injections which are prepared by using appropriate additives usually used in liquid preparations such as water, alcohols or plant-derived oils such as soybean oil, peanut oil or sesame oil. It is manufactured as a form such as.

In particular, when administered parenterally by intramuscular injection, intravenous injection or subcutaneous injection, suitable solvents include, for example, distilled water for injection, lidocaine hydrochloride aqueous solution (for intramuscular injection), physiological saline and glucose aqueous solution. , Ethanol, a liquid for intravenous injection (for example, an aqueous solution of citric acid and sodium citrate) or an electrolyte solution (for intravenous drip and intravenous injection), or a mixed solution thereof.

These injections may be pre-dissolved, or may be in the form of powder or dissolved with appropriate additives before use. These injections usually contain 0.1 to 10% by weight, preferably 1 to 5% by weight, of the active ingredient. Liquid preparations such as suspensions or syrups for oral administration contain 0.5 to 10% by weight of the active ingredient.

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

[0043]

BEST MODE FOR CARRYING OUT THE INVENTION

[0044]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to examples, but the present invention is not limited to these examples. Example 1 Method for producing BE-44651s: Actinomycete A 44651 strain inoculated on slant soft agar medium was 0.2% glucose, 2% dextrin, 0.5% meat meal, defatted rice bran.
5%, defatted meat-and-bone meal 0.1%, dried yeast 0.05%, magnesium sulfate 0.025%, sodium bromide 0.025
%, Sodium chloride 0.25%, dipotassium hydrogen phosphate 0.05%, ferrous sulfate 0.0002%, cupric chloride 0.00004%, manganese chloride 0.00004%, cobalt chloride 0.00004% , Zinc sulfate 0.00008
%, Sodium borate 0.00008%, and sodium molybdate 0.00024% (pH
7.2) Inoculate three 500 ml Erlenmeyer flasks containing 100 ml and incubate at 28 ° C for 72 hours on a rotary shaker (1 min / min).
(80 rotations). 500 ml Erlenmeyer flask 1 containing 100 ml of the above medium, 2 ml each of this culture solution.
The seeds were inoculated into 00 cells and cultured on a rotary shaker (180 rotations per minute) at 28 ° C. for 240 hours.

The culture broth thus obtained (about 10
After the cells were separated from L) by filtration, the cells were extracted by adding methanol (5 L × 2), the extract was concentrated under reduced pressure, and ethyl acetate (1.5 L × 2) was added. The obtained ethyl acetate extract was concentrated under reduced pressure, 100 ml of n-hexane was added to the residue, and 784.8 mg of an insoluble portion was obtained. Dissolve this insoluble part in 5 ml of methanol and use Sephadex L
Elution with methanol was performed using a chromatography column (3.0 × 76 cm) of H-20 (Pharmacia).
The obtained fraction containing BE-44651 was dissolved in a solvent of chloroform / methanol (1: 1), and the column for Sephadex LH-20 chromatography (3.0 × 76 cm).
Was eluted with chloroform / methanol (1: 1) to obtain fractions containing BE-44651A and BE-44651B, and each fraction was concentrated to dryness under reduced pressure to give BE-44651A and B. 64.8m each
g, 24.0 mg were obtained.

Formulation 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-44651A), 15 parts of heavy magnesium oxide and 75 parts of lactose are uniformly mixed to obtain 350
Powder or fine-grained powder having a particle size of μm or less. This powder was placed in a capsule container to obtain a capsule. Formulation Example 2 45 parts of this substance (BE-44651A), 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, then crushed and granulated and dried. , Then sieved and diameter 1410-177μ
m granules. 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 To 90 parts of the granules obtained by the method of Formulation Example 2, 10 parts of crystalline cellulose and 3 parts of calcium stearate were added and compression-molded to obtain a tablet having a diameter of 8 mm. Sugar-coated tablets were prepared by adding a mixed calcium carbonate suspension. Formulation Example 5 0.6 parts of this substance (BE-44651A), 2.4 parts of a nonionic surfactant and 97 parts of physiological saline are heated and mixed, then placed in an ampoule and sterilized to prepare an injection. It was made.

[0047]

The BE-44651s of the present invention are MRSA.
It shows a remarkable antibacterial activity against various pathogenic microorganisms including. Therefore, it is useful as an antibacterial agent against diseases caused by these pathogenic microorganisms.

[0048]

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI Technical display location C12P 13/02 C12P 13/02 19/26 19/26 // (C12N 1/20 C12R 1:01 ) (C12P 13/02 C12R 1:01) (C12P 19/26 C12R 1:01) (72) Inventor Katsuhisa Kojiri 3 Okubo, Tsukuba City, Ibaraki Prefecture, Mana Pharmaceutical Co., Ltd. Tsukuba Research Institute (72) Inventor Hiroyuki Suda 3 Okubo, Tsukuba City, Ibaraki Prefecture, Tsukuba Research Laboratory, Banyu Pharmaceutical Co., Ltd.

Claims (6)

[Claims] 1. A compound of the general formula [I] [Wherein R is a hydrogen atom or a compound represented by the formula: Represents a group represented by]. 2. Actinomadu
ra) and belongs to the general formula [I] [Wherein R is a hydrogen atom or a compound represented by the formula: A group represented by the formula [1]] is cultured, and the compound represented by the general formula [I] is collected from the culture solution and cells. A method for producing a compound represented by the formula [I]. 3. A compound represented by the general formula [I]: [Wherein R is a hydrogen atom or a compound represented by the following formula: The microorganism having the ability to produce the compound represented by the following formula is Actinomadura sp.
44651 (Actinomadura sp. A 4
The method according to claim 2, which is 4651) or a mutant strain thereof. 4. A compound represented by the general formula [I]: [In the formula, R is a hydrogen atom or the formula: An antibacterial agent containing a compound represented by the formula [1] as an active ingredient. 5. A compound represented by the general formula [I]: [Wherein R is a hydrogen atom or a compound represented by the following formula: [Showing a group represented by].] Actino majura (Acti) having the ability to produce a compound represented by
microorganism belonging to the genus Nomadura). 6. An actinomadura (Actinomadu)
The microorganism belonging to the genus ra) is Actinomadura sp. A 44651 (Actinomadura s).
p. A 44651) or a mutant strain thereof.
The microorganism according to the above.