JPH0948781A - New antibiotic lactonamycin and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new antibiotic exhibiting antimicrobial activity against a Gram-positive bacterium containing a drug tolerance bacillus such as a methicillin-resistant bacterium and exhibiting a suppressing effect against propagation of a cancerous cell, useful as an antibacterial agent and an antitumor agent. SOLUTION: This compound (A) is expressed by the formula and obtained by culturing a producing bacterium of A belonging to genus Streptomyces (e.g. Streptomyces sp. MJ773-88K4 strain) in a liquid nutritive medium using a nutrient assimilative with a carbon source, a nitrogen source, an inorganic salt, etc., at pH6-8, especially 6.5-7.5 and 25-30 deg.C culturing temperature under an aerobic condition, removing bacterium by filtering, etc., the resultant cultured solution, making the filtered solution acidic, isolating the acidic solution and purifying by subjecting to extraction in an organic solvent or various chromatography.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel antibiotic lactonamycin (Lacto) having antibacterial activity and antitumor activity.
namycin) and a method for producing lactonamycin. Further, the present invention relates to an antibacterial agent and an antitumor agent containing lactonamycin or a salt thereof as an active ingredient. The present invention also includes the Streptomyces MJ773-88K4 strain as a novel microorganism having the property of producing the antibiotic lactonamycin.

[0002]

2. Description of the Related Art A large number of antibiotics having antibacterial activity and / or antitumor activity produced by microorganisms are known.

[0003]

The emergence of multidrug-resistant bacteria is a significant problem in chemotherapy of bacterial infections. It is always desired to discover or create a new compound having a chemical structure different from that of a known antibacterial compound which is conventionally known or used, and research for it has been desired. Is being done. In addition, many antitumor substances generally have strong toxicity, which greatly restricts their use as antitumor agents.
Therefore, it has always been desired to discover or create an antitumor substance having low toxicity and a novel chemical structure,
Research for that purpose is being conducted.

[0004]

DISCLOSURE OF THE INVENTION The present inventors have aimed to provide novel antibiotics having antibacterial activity and antitumor activity which can meet the above-mentioned demands, and thus, to provide novel antibiotics which have hitherto been useful, Has promoted research on development and commercialization. As a result, they succeeded in isolating a strain belonging to the genus Streptomyces from a soil sample as a novel microorganism, and found that this strain produces an antibiotic having a new structural skeleton. Furthermore, we isolated this novel antibiotic and named it lactonamycin (Lactonamycin), confirmed its physicochemical properties and succeeded in establishing its chemical structural formula. Moreover, this novel antibiotic lactonamycin is a drug-resistant bacterium (methicillin-resistant bacterium, etc.)
It has been found that it exhibits antibacterial activity against Gram-positive bacteria including bacterium and suppressive effect on the growth of cancer cells.

Therefore, according to the first aspect of the present invention, the following equation (I): There is provided a novel antibiotic lactonamycin or a salt thereof having an antibacterial activity and an antitumor activity, which is a compound represented by.

In addition, lactonamycin has the physicochemical properties summarized below. A) Appearance and properties: Yellow powder, weakly acidic substance B) Melting point: 168-171 ° C C) Specific rotation: [α] _D ²⁷ + 34 ° (c 0.27, methanol) D) Molecular formula: C ₂₈ H ₂₇ O ₁₂ N E) Rf value of thin layer chromatography: 0.25 (Merck Ar
_{t.13124, HPTLC RP-18WF 254:} CH 3 CN-H as a developing solvent
₂ O = 1: 1 is used) F) Mass spectrum (m / z): 570 (M + H) ⁺ , 568
(M-H) ^- G) High resolution mass spectrum: Found 570.1613 (M ⁺ H) + calcd 570.1612 H) absorption peak of the ultraviolet absorption spectrum: when measured in methanol solution λmax nm (ε) 228 (11300 ), 257 (13200), 300 (20000),
395 (6700), 412 (7500) 0.1N-NaOH-When measured in 90% methanol solution λmax nm (ε) 232 (11600), 250 (12900), 287 (12200),
323 (11300), 367 (10700), 398 sh, 448 sh I) Absorption peak value of infrared absorption spectrum (measured by KBr tablet method): ν _max 3410, 2950, 1810, 1690, 1640 sh,
1620, 1250 cm ^-1 .

The antibiotic lactonamycin according to the first aspect of the present invention is a weakly acidic substance, and its salt is a salt with an organic base such as a quaternary ammonium salt or a salt with various metals such as sodium. There are pharmaceutically acceptable salts with various alkali metals, and these salts also have the above-mentioned antibacterial activity and antitumor activity.

(1) Physicochemical Properties of Antibiotic Lactonamycin Lactonamycin has physicochemical properties such as the appearance and properties of A), the melting point of B), the specific rotation of C), and the molecular formula of D). The Rf value of E), the mass spectrum of F), the high resolution mass spectrum of G), the absorption peak of the ultraviolet absorption spectrum of H), and the absorption spectrum of the infrared absorption spectrum of I) are shown.

An ultraviolet absorption spectrum curve (measured in methanol) and the same curve (measured in 0.1N-NaOH-90% methanol) are shown in FIGS. 1 and 2 of the accompanying drawings, respectively. The infrared absorption spectrum curve (measured by the KBr tablet method) is shown in FIG. 3 of the accompanying drawings.

In addition, the ¹ H-NMR spectrum of lactonamycin (measured in CDCl ₃ at 500 MHz, trimethylsilane internal standard) and the ¹³ C-NMR spectrum (125 M in CDCl ₃ 125 M).
(Measured in Hz, trimethylsilane internal standard) are shown in Figures 4 and 5 of the accompanying drawings, respectively.

Lactonamycin is soluble in methanol, ethyl acetate and chloroform, but insoluble in hexane.

(2) Biological properties of the antibiotic lactonamycin The lactonamycin of the present invention exhibits excellent antibacterial activity against various Gram-positive bacteria and is a multidrug-resistant Staphylococcus aureus (MRSA). It also shows a high antibacterial activity against. A) Antibacterial spectrum The minimum inhibitory concentration of the antibiotic lactonamycin against various bacteria on eutrophic agar plates is shown in Table 1 below. This antibacterial spectrum was measured by the multiple dilution method on the Mueller Hinton agar medium based on the Japanese Society of Chemotherapy standard method.

B) Antitumor or Anticancer Activity The lactonamycin of the present invention has an anticancer activity or an antitumor activity because it exhibits a growth inhibitory effect (cytotoxicity) on various cancer cells. 50% proliferation of cancer cells using various cancer cells
Inhibiting concentration Lactobacillus Na hygromycin (IC ₅₀ values) the MTT method [Journal of Immunological Methods, 65, pp. 55-60
(1983)]. The results are shown in Table 2.

[0014]

C) Acute toxicity The lactonamycin antibiotic has low toxicity to mammals. As a result of examining the acute toxicity of lactonamycin to mice, no abnormality was observed in the mice when 50 mg / kg of lactonamycin was intraperitoneally administered.

The antibiotic lactonamycin of the present invention is obtained by inoculating a nutrient medium containing a carbon source and a nitrogen source with a microorganism belonging to the genus Streptomyces and capable of producing the lactonamycin, and then culturing the medium. It can be produced by producing and accumulating lactonamycin in, and collecting lactonamycin from the resulting culture.

Therefore, the second aspect of the present invention is characterized in that the above-mentioned bacteria producing the lactonamycin antibiotic belonging to the genus Streptomyces are cultured in a nutrient medium and the antibiotic is collected from the culture. , A method for producing a novel antibiotic lactonamycin is provided.

An example of a lactonamycin-producing bacterium that can be used in the second method of the present invention is Streptomyces sp. MJ7.
There are 73-88K4 strains.

This lactonamycin-producing bacterium
It is a strain of actinomycetes isolated from soil in Yokohama City, Kanagawa Prefecture at the Institute for Microbial Chemistry that was assigned the strain number of MJ773-88K4.

The mycological properties of the MJ773-88K4 strain are as follows. 1. Form MJ773-88K4 strain extends aerial hyphae that are relatively longer than the branched basal hyphae, and helix formation is observed. The mature spore chain links 10 to 50 oblong spores. The size of spores is about 0.5-0.6 × 1.0-1.4 microns, and the surface is smooth or slightly wrinkled. No limbus, fungal filaments or sporangium are found.

2. Growing condition 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 (27 ° C culture) Growth is pale yellow [1 1/2 ga, Butter Yellow], aerial hyphae do not grow, and soluble pigments are not observed. (2) Glucose / asparagine agar medium (27 ° C culture) light yellow [2gc, Bamboo] to light yellow tea [2le, Mustard]
On the development of A. hygrophytes, aerial hyphae of bright brown ash [3dc, Natural] were partially settled and no soluble pigment was observed. (3) Glycerin / asparagine agar medium (ISP-medium 5, 27 ° C culture) Light brown ash [3dc, Natural] ~ bright for the growth of light yellow [2ea, Lt Wheat] to nibu yellow [2lc, Gold] Ash [3f
e, Silver Gray] aerial hyphae are settled, and the soluble pigment has a faint brownish tinge. (4) Starch / inorganic salt agar medium (ISP-medium 4,27
C. Cultivation) On the development of colorless to light yellow tea [2ne, Mustard Gold], aerial hyphae of bright ash [3fe, SilverGray-5fe, Ashes] are grown, and no soluble pigment is observed.

(5) Tyrosine agar medium (ISP-medium 7, 27 ° C. culture) For the development of yellow tea [3 ng, Yellow Maple-3ni, Clove Brown], yellowish ash [2ba Pearl] -light brown ash [3dc, Nat
ural] aerial mycelium, and the soluble pigment exhibits dark brown. (6) Nutrient agar medium (27 ° C. culture) Growth light yellow tea [2le, Mustard to 3le, Cinnamon],
The aerial mycelium does not settle, and the soluble pigment exhibits tea. (7) Yeast-malt agar medium (ISP-medium 7, 27 ° C
(Culture) On the development of light yellow tea [3ne, Topaz], aerial mycelia of white to light brown ash [3dc, Natural] to light ash [3fe, Silver Gray] are settled, and the soluble pigment is brownish. . (8) Oatmeal agar medium (ISP-medium 3,27 ° C culture) On the growth of light yellow [2ca, Lt Ivory ~ 1 1/2 ea, Lt Yellow], aerial mycelia of bright ash [3fe, Silver Gray] was added. Once settled, the soluble dyes have a faint yellow tint. No change in development color tone or soluble pigment due to pH was observed. (9) Starch agar medium (27 ° C culture) Nibu yellow [1 1/2 lc, Gold] to light yellow tea [3ne, Topaz]
On the development of S. aeruginosa, white aerial mycelium slightly colonized and no soluble pigment was observed.

(10) Lime malate agar medium (27 ° C. culture) Growth is colorless to pale yellow [1 1/2 ca, Cream], aerial hyphae do not grow, and soluble pigments are not observed. (11) Gelatin stab culture On a 15% simple gelatin medium (cultured at 20 ° C), a slightly yellow growth and white aerial mycelium are slightly settled, and the soluble pigment becomes yellowish. Glucose / peptone / gelatin medium (27 ° C culture)
In the case of, the growth is pale yellow to dark brown, white aerial hyphae are slightly settled, and the soluble pigment exhibits brown. (12) Skim milk (37 ℃ culture) Growth is light tea to light pink, and aerial mycelium does not settle,
Soluble dyes have a brownish tinge.

3. Physiological properties (1) Growth temperature range Glucose / asparagine agar medium (glucose 1.0%,
Asparagine 0.05%, dipotassium phosphate 0.05%, string agar 3.5%, pH 7.0), 10 ℃, 20 ℃, 24 ℃, 27 ℃, 30 ℃
As a result of testing at each temperature of ℃, 37 ℃ and 50 ℃, it grows at any of the temperatures except 10 ℃ and 50 ℃. The optimum temperature for growth is
It seems to be around 27 ° C. (2) Starch hydrolysis (starch / inorganic salt agar medium, ISP-medium 4 and starch agar medium, 27
C. Culture) Starch hydrolyzability was observed from about 2 days after culturing in any medium, and its action was moderate. (3) Formation of melanin-like pigment (tryptone, yeast,
Broth, ISP-medium 1; peptone-yeast-iron agar medium, ISP-medium 6; tyrosine agar medium, ISP-medium 7; both cultures at 27 ° C) Positive in any medium.

(4) Utilization of carbon source (Pridham-Gotrieve agar medium, ISP-medium 9, 27 ° C. culture) D-glucose, L-arabinose, D-xylose,
It develops using D-fructose, sucrose, inositol, rhamnose and raffinose, and does not use D-mannitol. (5) Liquefaction of gelatin (15% simple gelatin medium, 20 ° C culture; glucose / peptone / gelatin medium, 27 ° C culture) Negative in any medium.

(6) Coagulation and peptone conversion of skim milk (skim milk, 37 ° C. culture) About 7 days after the culture, peptone formation started without coagulation but was not completed in 21 days of culture.

(7) Dissolution of lime malate (lime malate agar medium, 27 ° C. culture) Dissolution was observed around 14 days after culturing, and its action was moderate. (8) Nitrate reduction reaction (0.1% potassium nitrate-containing peptone water, ISP-medium 8, 27 ° C. culture) is positive.

In summary of the above properties, the MJ773-88K4 strain grows aerial hyphae that are relatively longer than the branched basal hyphae,
Has a spiral formation. The matured spore chain is linked with 10 to 50 oval spores, and its surface is smooth or slightly wrinkled. On various mediums, light brown ash to light ash aerial hyphae grow on the development of light yellow to light yellow tea, and soluble pigments other than melanin pigment are not observed. The optimum growth temperature is around 27 ° C. The production of melanin-like pigment is positive. Hydrolysis of starch is positive, liquefaction of gelatin is negative, skim milk is peptone without coagulation. The 2,6-diaminopimelic acid contained in the cell wall was LL-type.

From these properties, it is considered that the MJ773-88K4 strain belongs to the genus Streptomyces .
Searching for closely related known species, Streptomyces galilaeus [Reference, Shirling,
EB and D. Gottlieb, `` International Journal of S
ystematic Bacteriology '', Vol. 22, 298, 1972) and Streptomyces rishir
iensis ) [Reference, Shirling, EB and D. Gottlieb, "In
ternational Journal of Systematic Bacteriology '' 22
Vol., Pp. 344, 1972]. Therefore, we plan to compare the above-mentioned two strains preserved by this institute and the MJ773-88K4 strain in practice. At present, the MJ773-88K4 strain is referred to as Streptomyces sp. MJ773-88K4.

Incidentally, the MJ773-88K4 strain was applied for deposit at the Institute of Biotechnology, Institute of Biotechnology, Institute of Industrial Technology, April 25, 1995, FE
Commissioned as RMP-14906.

The nutrient sources contained in the nutrient medium used for culturing the lactonamycin-producing bacteria include those normally used as nutrient sources for microorganisms, for example, assimilable nutrient sources such as carbon source, nitrogen source and inorganic salts. Can be used. For example, carbohydrates such as glucose, maltose, molasses, dextrin, glycerin, starch, and carbon sources such as oils and fats such as soybean oil and peanut oil, peptone, meat extract, cottonseed flour, soybean powder, yeast extract, casein, corn Steep Liquor, NZ-
Nitrogen sources such as amine, ammonium sulfate, ammonium nitrate and ammonium chloride, and inorganic salts such as dipotassium phosphate, sodium phosphate, sodium chloride, calcium carbonate, magnesium sulfate and manganese chloride can be used. If necessary, trace metals such as cobalt and iron can be added.
As the nutrient source, any known nutrient source can be used as long as it can be utilized by the lactonamycin-producing bacterium for the production of the antibiotic lactonamycin.

The mixing ratio of the above nutritional sources in the medium is not particularly limited and can be varied over a wide range, and the optimum composition and mixing ratio of the nutritional sources depends on the lactonamycin-producing bacterium used. Can be easily determined by the parties by a simple small-scale experiment.

The nutrient medium containing the above-mentioned nutrients can be sterilized prior to culturing, and the pH of the medium can be adjusted to a range of 6-8, particularly a range of pH 6.5-7.5 before or after the sterilization. It is advantageous to adjust.

Culturing of the lactonamycin-producing bacterium in such a nutrient medium can be carried out according to the method usually used in the production of antibiotics by general actinomycetes. Usually, it is preferable to culture under aerobic conditions, and usually, it can be performed with stirring and / or aeration. Further, as the culture method, any of static culture, shaking culture, and liquid culture with aeration and stirring can be used, but the liquid culture method is suitable for mass production of lactonamycin.

The culture temperature that can be used is not particularly limited as long as the growth of the lactonamycin-producing bacterium is not substantially inhibited and the antibiotic can be produced, and it depends on the producing bacterium used. The temperature in the range of 25-30 ° C can be mentioned as a particularly preferable temperature.

Culturing can usually be continued until sufficient lactonamycin is accumulated. The culturing time varies depending on the composition of the medium, the culturing temperature, the working temperature, the producing strain used, etc., but usually the desired antibiotic can be obtained by culturing for 95 to 170 hours.

The amount of lactonamycin accumulated in the culture can be quantified by Staphylococcus aureus Smith by the cylindrical plate method used for quantifying ordinary antibiotics.

[0038] Thus, the antibiotic lactonamycin accumulated in the culture is, after the culture, if necessary, the bacterial cells are removed by a separation method known per se such as filtration or centrifugation, and the filtrate is acidified (pH 2). -4) Use solvent extraction using an organic solvent, especially ethyl acetate, chromatography using adsorption or ion exchange ability, gel filtration, chromatography using countercurrent distribution, alone or in combination. Can be isolated, purified, and collected. Activated carbon, silica gel, porous polystyrene-divinylbenzene resin, or various ion exchange resins can be used as a chromatography carrier having adsorption or ion exchange ability. Further, from the separated bacterial cells, lactonamycin can be extracted from the bacterial cells by a solvent extraction method using an appropriate organic solvent or an elution method by disrupting the bacterial cells, and isolated and purified in the same manner as above. Thus, a novel antibiotic lactonamycin with the above mentioned properties can be harvested.

[0039]

BEST MODE FOR CARRYING OUT THE INVENTION Next, the present invention will be described in more detail with reference to Examples.

[0040]

Example 1 Production of the antibiotic lactonamycin Glycerin 2%, dextrin 2%, bacto-soyton 1%, powdered yeast extract 0.3%, ammonium sulfate 0.2%,
After sterilizing a liquid medium containing 0.2% calcium carbonate and 1 drop of silicone oil (adjusted to pH 7.4) at 120 ° C for 20 minutes,
Actinomycete strain MJ773-88K4 (FERM
P-14906) was inoculated and cultured at 27 ° C. for 6 days with rotary shaking (180 rpm). The culture fluid thus obtained was centrifuged to separate the bacterial cells.

The culture filtrate (1.7 liter) was adjusted to pH with 6N-HCl.
After adjusting to 2, the mixture was extracted with 1.7 liters of ethyl acetate, and the ethyl acetate layer was dried with anhydrous sodium sulfate. Further, 1 liter of methanol was added to the cells, and the mixture was stirred and then filtered. The methanol solution was concentrated to dryness under reduced pressure, then 100 ml of 0.01N-HCl was added, and the mixture was extracted with 100 ml of ethyl acetate. The ethyl acetate layer was dried over anhydrous sodium sulfate. The two ethyl acetate layers were combined and concentrated under reduced pressure to dryness,
It was dissolved in 30 ml of chloroform and the insoluble matter was filtered. The chloroform layer was diluted to 50 ml and methanol-water (4: 1) 50 ml
After washing twice with, the chloroform layer was concentrated to dryness under reduced pressure.

The residue was dissolved in 50 ml of methanol and hexane was added.
After washing twice with 50 ml, the mixture was concentrated to dryness under reduced pressure to obtain 0.177 g of crude lactonamycin. The crude lactonamycin (0.059 g) was subjected to column chromatography on Sephadex LH-20 (200 ml) and eluted with methanol. 0.010 g of pure lactonamycin having the above-mentioned physicochemical properties was obtained as a yellow powder (melting point 168-171 ° C).

[Brief description of drawings]

FIG. 1 UV absorption spectrum curve of lactonamycin
(Measured in methanol) is shown.

FIG. 2 UV absorption spectrum curve of lactonamycin
(Measured in 0.1AN-NaOH-90% methanol).

Fig. 3 Infrared absorption spectrum of lactonamycin (KBr
(Measured by the tablet method) is shown.

FIG. 4 ¹ H-NMR spectrum of lactonamycin (C
(Measured in DCl ₃ at 500 MHz).

FIG. 5: ¹³ C-NMR spectrum (C
(Measured in DCl ₃ at 125 MHz).

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technology display area // (C12N 1/20 C12R 1: 465) (C12P 17/18 C12R 1: 465) (72) Inventor Sawa Riki 4-6-7 Ayanishi, Ayase City, Kanagawa Prefecture (72) Inventor Hiroshi Naganawa 3-17, Denenchofuhonmachi, Ota-ku, Tokyo (72) Inventor, Masashi Hamada 1 26, Naito-cho, Shinjuku-ku, Tokyo Hidekazu Residence No.405

Claims (5)

[Claims] 1. The following formula (I): A novel antibiotic lactonamycin having antibacterial activity and antitumor activity, which is a compound represented by: or a salt thereof. 2. The method according to claim 1, which belongs to the genus Streptomyces.
The method for producing the novel antibiotic lactonamycin according to claim 1, wherein the bacterium producing the antibiotic lactonamycin according to claim 1 is cultured in a nutrient medium and the antibiotic is collected from the culture. 3. An antibacterial agent containing the antibiotic lactonamycin according to claim 1 or a salt thereof as an active ingredient. 4. An antitumor agent comprising the antibiotic lactonamycin according to claim 1 or a salt thereof as an active ingredient. 5. Streptomyces MJ773-88 having the property of producing the antibiotic lactonamycin according to claim 1.
K4 stock.