JP3327982B2 - New antibiotic MI481-42F4-A related substance - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel antibiotic MI481-42F4
MI481-42F4-A1, a new antibacterial substance obtained by decomposing -A
The present invention relates to -A2 and -A3 substances and salts thereof, and to a method for producing those substances.

[0002]

BACKGROUND OF THE INVENTION Many antibiotics produced by microorganisms have been discovered so far, and their degradation products or derivatives are widely used for treating infectious diseases.

[0003]

In the treatment of infectious diseases, the emergence of resistant bacteria is unavoidable, and there is a constant need for new antibacterial antibiotics effective against the resistant bacteria.

[0004]

Means for Solving the Problems As a result of the previous studies, the present inventors have found that a strain belonging to the genus Amycolatopsis (FERM P-127) is used.
39) A new antibiotic MI481-42F4-A was obtained from the culture broth ( patent application No. 4-142045 and patent No. 3107455).
Was ). Now, we have discovered that the antibiotic MI481-42F4-A
[The Journal of Antibiotics, Vol. 47, No. 10, 1153 (1994
Is the same substance as amithiamycin A shown in October
The novel antibacterial agents by decomposing a method described later to that]
MI481-42F4-A1, MI481-42F4-A2 and MI481-42F4- A3 were successfully obtained. Furthermore, these antibacterial substances MI481-42
F4-A1, antibacterial substance MI481-42 F4-A2 and antibacterial substance MI481-42
The chemical structure of F4-A3 was studied, and as a result,
Compounds of Formula (Ia), Formula (Ib) and Formula (Ic)
Was found.

Therefore, according to the first aspect of the present invention, an antibacterial substance
MI481-42F4-A is obtained by decomposing and the following formula (Ia) A novel antibacterial substance MI481-42F4-A , which is a compound represented by
1, or a salt thereof is provided.

The antibacterial substance MI481-42F4-A1 according to the first invention obtained by decomposing the antibiotic MI481-42F4-A has the following physicochemical properties.

(1) Color and shape: colorless powder (2) Classification of acidic, neutral and basic: amphoteric substances (3) High-resolution mass spectrum: HR FAB-MS m / z 1183.228
5 (M + H) (4 ) Molecular _{formula: C 50 H 50 O 9 N} 14 S 6

Further, according to the second invention, the antimicrobial substance MI
481-42F4-A is obtained by decomposing and the following formula (Ib) A novel antibacterial substance MI481-42F4-A , which is a compound represented by
2, or a salt thereof is provided.

Second Antimicrobial Substance MI481-42F4-A According to the Present Invention
2 has the following physicochemical properties. (1) Color and shape: colorless powder (2) Acidic, neutral, basic classification: basic substance (3) Mass spectrum: FAB-MS m / z 1218 (M + H) ⁺

Further, according to a third aspect of the present invention, there is provided an antibacterial substance MI.
481-42F4-A is obtained by decomposition and has the following formula (Ic) A novel antibacterial substance MI481-42F4-A , which is a compound represented by
3, or a salt thereof is provided.

This antibacterial substance MI481-42F4-A3 has the following physicochemical properties.

(1) Color and shape: colorless powder (2) Mass spectrum: FAB-MS m / z 1200 (M + H) ⁺

The antibacterial substances MI481-42F4-A1, -A2 and -A3 of the present invention can be produced by hydrolyzing the antibiotic MI481-42F4-A, while A1 and A3 produce MI481-42F4-A substance. It is also present in bacterial cultures and cells.

The above hydrolysis is carried out, for example, by diluting hydrochloric acid or hydrochloric acid-methanol solution at room temperature at room temperature.
It can be performed by subjecting 42F4-A to a decomposition reaction for a predetermined time. These reactions are not particularly limited as long as they serve to generate the MI481-42F4-A1, -A2 and / or -A3 substances of the present invention, and any known decomposition reactions can be used.

The antimicrobial substances MI481-42F4-A1, -A2 and -A3 of the present invention can be collected by hydrolyzing MI481-42F4-A, cooling the reaction solution, and concentrating to dryness. Alcohol, extraction method using an organic solvent such as acetonitrile, adsorption chromatography, gel filtration chromatography,
It is possible to collect pure antibacterial substances MI481-42F4-A1, -A2 and -A3 by appropriately combining or repeating known methods such as thin-layer chromatography, counter-current distribution chromatography, and high performance liquid chromatography. it can.

The biological properties of the antimicrobial substances MI481-42F4-A and -A1 are as follows: Antibacterial substance MI481-42F4-A
Table 1 shows the inhibitory concentrations of -A1 against various bacteria on a nutrient agar medium (by the agar plate dilution method). Table 1
As is clear, the antibacterial substances MI481-42F4-A and -A1 had strong antibacterial activity against Gram-positive bacteria. In particular
MI481-42F4-A also strongly inhibited the growth of multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus and Staphylococcus aureus No. 5 (MRSA).

[0017]

The antibiotics MI481-42F4-A, -A2 and
-To evaluate the antimicrobial activity of A3, the antimicrobial substance MI481-42F4
The inhibitory concentrations (by liquid culture dilution method) of -A, -A2 and -A3 against various bacteria in nutrient liquid medium were measured. The results are shown in Table 2 below. As apparent from Table 2, the antibacterial substances MI481-42F4-A, -A2 and -A3 had strong antibacterial activity against Gram-positive bacteria. In particular, it also strongly inhibited the growth of multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus and Staphylococcus aureus No. 5 (MRSA).

[0019]

Further, according to the acute toxicity tests carried out in mice, each LD ₅₀ for antimicrobial MI481-42F4-A and -A1 substance is intraperitoneally low toxicity be at 100 mg / kg or more.

From the above, the antibacterial substance of the present invention
MI481-42F4-A1, -A2 and -A3 may be used as antibacterial agents in the treatment of bacterial infections in humans and animals.

The physicochemical and biological properties of the antibacterial substances MI481-42F4-A1, -A2 and -A3 are as described in detail above, and no compounds similar to such properties have been known so far. Because the antibiotics MI481-42F4-A1, -A2 and
-A3 is a new substance. The antibacterial substances MI481-42F4-A1 and
The antimicrobial substance MI481-42F4-A3 is described in the above `` The Journal of A
ntibiotics ”, Vol. 47, No. 10, page 1153.
(Amythiamicin) C and Amythiamicin B
Are the same.

The novel antibacterial substances MI481-42F4-A1, -A2 of the present invention
Antibiotic MI4 used as a raw material for the production of -A3
81-42F4-A is Ri substances der having the following characteristics, of the
Shown in The Journal of Antibiotics, Vol. 47, No. 10, page 1153
Same as Amythiamicin A
Quality, and the following equation (II) It is a compound represented by these.

(1) Color and shape: colorless powder (2) Melting point: 223-228 ° C (decomposition) (3) Elemental analysis: C 49.46%, H 4.79%, N 16.76%, O
12.23%, S 15.72% (4) UV absorption spectrum: Lambda maximum (nm) (in methanol) 203 309 221 350 (shoulder) 245 (shoulder) (in methanol-hydrochloric acid) 205 308 222 353 (shoulder) 250 (shoulder) (In methanol-sodium hydroxide) 205 310 221 353 (shoulder) 250 (shoulder) (5) Infrared absorption spectrum (KBr method): 3300, 3120, 298
0, 1660, 1550, 1500, 1250, 990, 940, 760 cm ^-1 (6) Specific rotation: [α] _D ²⁸ = + 133 ° (c 0.745, dimethyl sulfoxide) (7) Acid, neutral, base Classification of sex: basic substance (8) Molecular weight: 1182.44 (9) Molecular formula: C ₅₀ H ₅₁ O ₈ N ₁₅ S ₆

This antibiotic MI481-42F4-A is obtained by culturing MI481-42F4-A-producing bacteria belonging to the genus Amycolatopsis and isolating and collecting the antibiotic MI481-42F4-A from the culture. And obtained by a method for producing the antibiotic MI481-42F4-A.

MI481- belonging to the genus Amycolatopsis
As an example of a 42F4-A producing bacterium, the present inventors
Amycolatopsis sp. Strain isolated from a soil sample collected in Nerima-ku, Tokyo in June 1962.
) There is MI481-42F4. The bacteriological properties of this strain are as follows.

Mycological properties of MI481-42F4 strain Morphology Well-branched basal hypha often presents a zigzag shape,
In addition, division is recognized. Aerial hyphae are straight or irregularly curved, fragmented, and form cylindrical spores. The spores are about 0.6-0.7 x 0.7-1.2 microns in size and the surface is smooth. No helical formation, ring branches and spore sac are observed.

2. Growing state in various media About color description Standards shown in [] are the Color Harmony Manual (Container Corporation of America) of Container Corporation of America.
Color harmony manual).

(1) Sucrose / nitrate agar medium (30
Cultivation at ° C.) On the growth of pale yellow [1 ea, Canary Yellow], white aerial hyphae grow slightly, and no soluble pigment is observed. (2) Glucose-asparagine agar medium (cultured at 30 ° C) Aerial mycelia of light yellow [1 ca, Pale Yellow] grow on yellow [1 1/2 ia, Sunlight Yellow], and soluble pigment is Slightly yellowish. (3) Glycerin-asparagine agar medium (ISP-medium 5, cultured at 30 ° C) Aerial mycelia of light yellow [1 1/2 ca, Cream] were grown on light yellow [1 1/2 ga, Butter Yellow]. Settles and the soluble pigment is slightly yellowish. (4) Starch / inorganic salt agar medium (ISP-medium 4, cultured at 30 ° C) White aerial hyphae grow slightly on the growth of light yellow [1 1/2 ic, Lt Antique Gold ~ 2gc, Bamboo] No soluble dye is observed. (5) Tyrosine agar medium (ISP-medium 7, cultured at 30 ° C.) On growth of yellow [1 1/2 lc, Gold], brown white [2 ca, Lt Ivory]
And the soluble pigment takes on a yellow tinge.

(6) Nutrient agar medium (cultured at 30 ° C.) On growth of light yellow [2 gc, Bamboo], white aerial hyphae grow slightly and no soluble pigment is observed. (7) Yeast / malt agar medium (ISP-medium 2, cultured at 30 ° C.) White aerial hyphae were formed on the growth of light yellow tea [2 ne, Mastard Gold], and no soluble pigment was observed. (8) Oatmeal / agar medium (ISP-medium 3, cultured at 30 ° C) White aerial hyphae grow slightly on the growth of pale yellow [1 1/2 ic, Lt Antique Gold]. Slightly yellowish. (9) Glycerin / nitrate agar medium (cultured at 30 ° C.) White aerial hyphae grow slightly on the growth of pale yellow [1 ga, Lt Lemon Yellow], and no soluble pigment is observed. (10) Starch agar medium (cultured at 30 ° C.) White aerial mycelium slightly develops on the growth of yellow [1 la, Lemon Yellow]. No soluble dye is observed.

(11) Lime malate agar medium (cultured at 30 ° C.) On growth of light yellow [1 ga, Lt Lemon Yellow], white aerial hyphae grow slightly, and no soluble pigment is observed. (12) Cellulose (synthetic solution with filter paper added, cultured at 30 ° C) Growth develops light yellow and white aerial mycelia, and no soluble pigment is observed. (13) Gelatin stab culture In a 15% simple gelatin medium (cultured at 20 ° C), the growth develops slightly yellow and white aerial mycelia, and no soluble pigment is observed. Glucose / peptone / gelatin medium (27 ℃
In the case of (culture), the growth is pale yellow, aerial mycelia do not form, and no soluble pigment is observed. (14) Skim milk (cultured at 37 ° C and 30 ° C) No growth was observed in culture at 37 ° C. In the case of culturing at 30 ° C., the growth is pale yellow, no aerial mycelium is formed, and no soluble pigment is observed.

3. Physiological properties (1) Growth temperature range Glucose / asparagine agar medium (glucose 1.0
%, L-asparagine 0.05%, dipotassium phosphate 0.05
%, String agar 3.0%, pH 7.0), 6 ℃, 20 ℃, 24 ℃
℃, 27 ℃, 30 ℃, 37 ℃, as a result of testing at each temperature of 50 ℃,
It grows at any temperature except 6 ° C, 37 ° C and 50 ° C, and the optimum growth temperature seems to be around 27 ° C to 30 ° C.

(2) Liquefaction of gelatin (15% simple gelatin medium, culture at 20 ° C .; glucose / peptone gelatin medium, culture at 27 ° C.) In a 15% simple gelatin medium, liquefaction starts about 3 days after cultivation. After a week, it was completed. Glucose, peptone,
In the gelatin medium, liquefaction was slightly observed from about the 7th day after the culture, but progress was extremely slow, and the culture was not completed after one month of culture.

(3) Hydrolysis of starch (starch / inorganic salt agar medium and starch agar medium, all cultivated at 30 ° C.) In any medium, water degradability was observed from about 6 days after cultivation, and the action was moderate. Degrees. (4) Coagulation / peptone conversion of skim milk (defatted milk, culture at 37 ° C. and 30 ° C.) No growth is observed in culture at 37 ° C. In the case of culture at 30 ° C, peptone formation starts without coagulation from about 10 days after culture,
It was completed after about one month of culture. (5) Melanin-like pigment formation (trypton yeast,
Broth, ISP-medium 1; peptone yeast / iron agar medium, ISP-medium 6; tyrosine agar medium, ISP-medium 7; all cultured at 30.degree. C.) Neither medium is negative.

(6) Utilization of carbon source (Pridham-Godlieve agar medium, ISP-medium 9; culture at 30 ° C.) D-glucose, L-arabinose, D-xylose,
It grows using inositol, D-mannitol and lactose, and does not use sucrose. D-fructose, rhamnose, raffinose are likely to be utilized. (7) Dissolution of malate lime (maleic acid lime agar medium, 30
Solubility was observed from about 10 days after the culture, and the effect was moderate. (8) Nitrate reduction reaction (peptone water containing 0.1% potassium nitrate, ISP-medium 8, cultured at 30 ° C.) Positive. (9) Degradation of cellulose (Synthetic solution with filter paper added, cultivation at 30 ° C) No degradation was observed in the observation for 6 months.

Summarizing the above properties, the MI481-42F4 strain often shows a zigzag base mycelium due to its morphology, and fragmentation is observed. The aerial hyphae are straight or irregularly curved, fragmented, form cylindrical spores, and have a smooth surface. No helix formation, ring-shaped branches and spores are found. In various media, white to pale yellow aerial hyphae develop on light yellow to yellow development, and the soluble pigment is slightly yellowish or not observed. The formation of melanin-like pigment is negative, the starch has moderate water degradability, the nitrate reduction reaction is positive, and the proteolytic activity is moderate.

The bacterial cell component of the strain MI481-42F4 has 2,6-diaminopimelic acid contained in the cell wall in the meso form, and the reducing sugar in all the bacterial cells is the A form containing arabinose and galactose. Also, without mycolic acid, the phospholipids are of PII type (including phosphatidylethanolamine and free of phosphatidylcholine and unknown glucosamine-containing phospholipids), and the main menaquinone is MK-9 (H ₄ ),
MK-9 (H ₂ ) was also found.

From the above results, MI481-42F4 strain was obtained from Amycolatopsis (literature, International Jo
urnal of Systematic Bacteriology, 36, 29-37,
1986) It is considered to belong to the genus. Amycolatopsis orientalis ( Amycolatopsis orientalis , Reference 1, Reference Id .; Reference 2, International Journal of Systematic Ba
cteriology, 37, pp. 292-295 (1987)) and Amycolatopsis medi terran.
ei , references 1 and 2, ibid.) were mentioned as closely related species. Therefore, the MI481-42F4 strain and the above-mentioned two strains preserved in our laboratory are being compared and studied in practice. At present MI481-42F4
The strain was transformed into Amycolatopsis sp.
) MI481-42F4.

The MI481-42F4 strain was applied for deposit with the Institute of Microbial Industry and Technology of the Agency of Industrial Science and Technology, and was deposited on Feb. 3, 1992 as a micro-organizational deposit No. 12739. Original deposited MI481-42
The F4 strain was subsequently based on the Budapest Treaty from the original deposit
The request for transfer to the deposit was received on July 14, 1997.
Deposited under accession number FERM BP-6023.

The above-mentioned antibiotic MI481-42F4-A is produced by inoculating the above strain into a medium suitable for producing the antibiotic MI481-42F4-A (see Reference Example 1 described later). The medium may be a nutrient-containing medium used for cultivation of normal actinomycetes. The nutrients include, for example, commercially available peptone, meat extract, corn steep liquor, cottonseed flour, peanut flour, soy flour, yeast extract, MZ-amine, casein hydrolyzate, sodium nitrate, ammonium nitrate,
Nitrogen sources such as ammonium sulfate, and commercially available carbohydrates such as glycerin, sucrose, starch, glucose, galactose, mannose, molasses, or carbon sources such as fat, and inorganic sources such as salt, phosphate, calcium carbonate, and magnesium sulfate Salt can be used. In addition, if necessary, a trace amount of a metal salt, a dynamic / vegetable / mineral oil as an antifoaming agent, etc. can be added. Any of these can be used as long as they are used by the producing bacteria and are useful for the production of the antibiotic MI481-42F4-A, and all known culture materials of actinomycetes can be used.

For mass production of the antibiotic MI481-42F4-A, liquid culture is preferable, and the culture temperature can be applied within a range where the antibiotic MI481-42F4-A can be produced. The cultivation can be carried out by applying the conditions described above and appropriately selecting according to the properties of the MI481-42F4-A producing bacterium. The antibiotic MI481-42F4-A is mainly present in the cells and can be extracted with an organic solvent miscible with water, such as alcohol, acetonitrile, and acetone. It can be extracted from the bacterial cell extract with a relatively polar water-immiscible organic solvent such as butanol. In addition to the above-mentioned extraction method, pure collection can be performed by appropriately combining or repeating known methods such as adsorption chromatography, gel filtration chromatography, thin-layer chromatography, countercurrent distribution chromatography, and high-performance liquid chromatography. Can be.

[0042]

EXAMPLES Next, the present invention will be described with reference examples and examples, but the present invention is not limited to these examples.

Reference Example 1 Production of Antibiotic MI481-42F4-A Glycerol 2.0%, dextrin (Wako Pure Chemical Industries) 2.0
%, Polypeptone (Wako Pure Chemical) 1.0%, Yeast Extract (Wako Pure Chemical) 0.3%, Ammonium Sulfate 0.2%, Calcium Carbonate 0.2%, Antifoam (KM70, Shin-Etsu Chemical) 0.01% (pH 7.4) 110 ml was placed in a 500 ml Erlenmeyer flask with a waffle, sterilized at 120 ° C. for 20 minutes, and then added to MI481-42F4 belonging to the genus Amycolatopsis grown on the agar slope.
One loopful of the strain (FERM P-12739) was inoculated and shake-cultured at 27 ° C. for 3 days. 2 ml of this inoculum was transplanted to the same medium as described above, and cultured at 27 ° C. for 4 days to accumulate the antibiotic MI481-42F4-A.

The culture solution (pH 8.2, 10.1 liter) was centrifuged to collect the cells, which were extracted with 5 liters of methanol.
The concentrated extract was concentrated, methanol was distilled off, 5 liters of water was added, and the mixture was extracted with an equal volume of butanol. After concentration, methanol was added and the precipitate was collected to obtain 4.3 g of a dried product. This was dissolved in dimethylformamide, suspended in dimethylformamide and packed with Sephadex LH-20 (Pharmacia) (outer diameter 50φ × 100m
m), and the mixture was subjected to gel filtration with dimethylformamide.
The active fraction (a fraction exhibiting antibacterial activity against Bacillus thermophilus ) was concentrated under reduced pressure, dissolved in dimethylformamide, suspended in dimethylformamide, and packed with Toyopearl HW-40 (Toso) (outer diameter 40φ × 420 mm) The active fraction was concentrated under reduced pressure and dried to obtain 780 mg of a dried product.

This was dissolved in 15 ml of chloroform, this solution was charged into a column packed with 25 g of silica gel (Merck Art.7734) suspended in chloroform, and the column was washed with 200 ml of chloroform. The active fraction was eluted with a mixed solution of / 1, and concentrated under reduced pressure to dryness to obtain 232 mg of a pure antibiotic MI481-42F4-A as a colorless powder.

Example 1 Preparation of MI481-42F4-A3 and -A1 Substances 40 mg of the pure antibiotic MI481-42F4-A obtained in Reference Example 1
Is weighed into a techno-vacuum hydrolysis tube and 20 ml of 1N-H
The suspension was added with Cl, and the tube was capped and sealed. This vacuum hydrolysis tube was kept in an oil bath kept at 110 ° C. for 24 hours. Thereafter, the reaction solution was concentrated under reduced pressure and dried to obtain a dried product 45.
mg was obtained. This was dissolved in 10 ml of acetonitrile water and purified and fractionated by high performance liquid chromatography (HPLC).

The conditions for reverse phase HPLC are as follows. Column: Capsule cup (Capcell Pak _C18 , Shiseido) 4.6
φ × 250 mm; eluent: 40% acetonitrile water → 48% acetonitrile water (linear gradient for 17 minutes); flow rate: 1.4 ml / min; ultraviolet detector: 254 nm.

As a result of the above analysis, the retention time was reduced to 8.8 minutes by MI.
481-42F4-A3 was detected, and MI481-42F4-A1 was detected at 11.3 minutes.

The pure MI481-
2 mg of 42F4-A3 substance and 8 mg of MI481-42F4-A1 substance were obtained.

Example 2 MI481-42F4-A1, -A2 and -A3
Production of Substance 41 mg of antibiotic MI481-42F4-A was weighed into an eggplant-shaped flask, 10.8 ml of 1N HCl-MeOH was added, and the mixture was allowed to stand at room temperature for 1.5 hours, and the obtained reaction solution was dried under reduced pressure. The dried product is dissolved in 10 ml of methanol water and subjected to high performance liquid chromatography (HPLC).
And purified.

The conditions for the reverse phase HPLC are as follows.

Column: YMC-packed column (SH-343-5S
-5 ODS, Yamamura Chemical Laboratory) 20 × 250mm; Eluent: 80% MeOH
Water; flow rate: 10.0 ml / min; UV detector: 254 nm.

By the above fractionation, pure MI481-42 was obtained.
11 mg of F4-A2 material were obtained.

Further, active fractions other than those described above were collected and subjected to thin-layer chromatography (developing solvent, chloroform-methanol, 10: 1 (v / v)) to extract the UV absorption band. By this operation, 3mg of MI481-42F4-A1 substance and MI481-
3 mg of 42F4-A3 material was obtained.

[0055]

As described in detail above, the present invention provides a novel antibacterial substance and a method for producing the same.

[Brief description of the drawings]

FIG. 1 shows the ¹ H-NMR spectrum of the antimicrobial substance MI481-42F4-A1 measured at 400 MHz in deuterated dimethyl sulfoxide.

FIG. 2 represents the ¹³ C-NMR spectrum of the antimicrobial substance MI481-42F4-A1 measured at 100 MHz in deuterated dimethyl sulfoxide.

FIG. 3 shows the ¹ H-NMR spectrum of the antimicrobial substance MI481-42F4-A2 measured in heavy pyridine at 400 MHz.

FIG. 4 shows the ¹³ C-NMR spectrum of the antimicrobial substance MI481-42F4-A2 measured in heavy pyridine at 100 MHz.

FIG. 5 shows the ¹ H-NMR spectrum of the antimicrobial substance MI481-42F4-A3 measured at 400 MHz in heavy pyridine.

FIG. 6 shows the ¹³ C-NMR spectrum of the antimicrobial substance MI481-42F4-A3 measured at 100 MHz in heavy pyridine.

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-5-610766 (JP, A) JP-A-3-201993 (JP, A) JP-A-3-98593 (JP, A) JP-A-63-1988 174996 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12P 1/04 C07D 471/04 BIOSIS / WPI (DIALOG)

Claims (3)

(57) [Claims] 1. The following formula (Ia) An antimicrobial substance MI481-42F4-A1, which is a compound represented by the formula: or a salt thereof. 2. The following formula (Ib) An antimicrobial substance MI481-42F4-A2, which is a compound represented by the formula: or a salt thereof. 3. The following formula (Ic): An antimicrobial substance MI481-42F4-A3, which is a compound represented by the formula: or a salt thereof.