JPH0812666A - New antibiotic substance f483a - Google Patents

Abstract

PURPOSE:To obtain a new antibiotic substance having high antitumor activities. CONSTITUTION:This antibiotic substance F483A of the formula is obtained by culturing a microorganism, belonging to the genus Penicillium and capable of producing the antibiotic substance F483A in a culture medium containing a defoaming agent, etc., added to a carbon source such as starch or dextrin and a nitrogen source such as peptone or soybean flour, etc., under conditions of preferably pH 5-8, 20-28 deg.C culture temperature, 300-350 r.p.m. stirring speed, (1/4) to (1/2) vol./vol. min air flow rate and 70-120hr culture time and then collecting the resultant substance from the culture solution thereof. Furthermore, a method for collecting the substance is to purify the culture liquid or an extract solution of the microbial cell by combination of various ion exchange chromatographies, gel filtration chromatographies, adsorption chromatographies and cellulose partition chromatographies, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel antibiotic, a method for producing the same, and an antitumor agent containing the same as an active ingredient.

[0002]

2. Description of the Related Art Conventionally, compounds such as anthracyclines and mitomycins have been reported as antitumor antibiotics. Compounds having a xanthone skeleton have been isolated from the natural world, especially from the plant kingdom, and many have been reported, and some of them have been found to have antitumor effects (J. Ph.
arm.Sci. 62, 483-485 (1973), Phytochemistry 2
7, 2795-2800 (1988), Phytochemistry 34, 1413-1
420 (1993)), a compound with higher antitumor properties was sought.

[0003]

An object of the present invention is to provide a novel antibiotic having a high antitumor property and a method for producing the same.

[0004]

[Means for Solving the Problems] As a result of diligent efforts, the present inventors have found that Penicillium sp.
p. It was found that the antibiotic produced by (1) is a novel compound and has a high antitumor activity, and has completed the present invention. That is, the present invention provides an antibiotic F483A represented by the following formula.

[0005]

Embedded image

The present invention also provides a penicillium (Penicill).
culturing an antibiotic F483A-producing bacterium belonging to the genus
The present invention provides a method for producing the antibiotic F483A, which comprises collecting the antibiotic F483A from the culture. Furthermore, the present invention provides an antitumor agent containing the antibiotic F483A as an active ingredient.

Hereinafter, the present invention will be described in detail. The microorganism producing the antibiotic F483A of the present invention is a bacterial species belonging to the genus Penicillium and capable of producing the antibiotic F483A. As an example, Penicillium sp. AJ117291 (Penici) described in detail below
llium sp. (AJ117291) (hereinafter, "AJ117291
A strain ”). Also, AJ11
Natural and artificial variants of strain 7291 may be used. The above AJ117291 strain is a soil filamentous fungus isolated from soil collected in Ehime Prefecture, and has been deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology on June 10, 1994, and its microorganism deposit number is , FERM P-14363
Is.

The AJ17291 strain has the following mycological properties. (1) Growth form on various media Growth on malt extract agar medium (25 ° C) is rather slow,
It is 33 mm in 14 days. The surface of the colony is velvety, grayish green, and the back is yellowish brown. No dye leaching into the medium or formation of droplets was observed. Grows well on Tuapec agar (25 ° C) and grows to 58m in 14 days.
m. The surface of the colony is velvety and white with a slight yellowish color at the center. The back side is pale yellow. No dye leaching into the medium is observed, but the formation of pale yellow droplets is observed. Growth on potato-glucose agar medium (25 ° C.) was 39 mm for 14 days, and the colony shape was the same as that of the malt extract agar medium. (2) Morphological properties On malt extract agar medium, conidia peduncle develops from aerial hyphae and is relatively short (30 to 50 μm × 2.0 μm), and is colorless.
Usually 2 to 4 primary branches (metre) at the tip of conidia peduncle
Was observed, and 3-5 fear rides were sprinkled on it,
A large number of conidia are formed in a chain at the tip. The metre is 2.0 × 8.0 to 10 μm. Fear ride is 1.5 ~
It is 2.0 x 6.0 to 8.0 μm and has a pen-like shape with a thin tip. Conidia are pale green, subspherical, univesicular and rough (1.8-2.2).
μm). Also, no complete generation is observed in any of the above media. The growth temperature is 10 to 37 ° C, and the optimum temperature is 25 to 2
8 ° C. The growth pH is 3 to 10, and 4 to 8 is optimal. Based on the above mycological properties, this bacterium is the genus Penicillium (1979, Academi).
c press, JI. According to Pitt), it was clarified that it belongs to the subphylum Subdivision, incomplete filamentous fungi, and Penicillium genus, and this strain was named Penicillium sp. AJ117291 (Penicillium sp. AJ117291) strain.

The antibiotic F483A of the present invention can be produced by culturing an antibiotic F483A-producing bacterium belonging to the genus Penicillium and collecting the antibiotic F483A from the culture. The F483A-producing bacterium can be cultured according to a conventional method, and the form of culture may be liquid culture or solid culture. For culturing industrially advantageously, a bacterial suspension or culture solution of the above antibiotic-producing bacterium may be inoculated into the medium, and aeration stirring culture may be carried out. The nutrient source of the medium is not particularly limited, but the medium may contain a carbon source, a nitrogen source and the like which are usually used for culturing microorganisms. As the carbon source, starch, dextrin, glycerin, glucose, sucrose, galactose, inositol, mannitol and the like, and as the nitrogen source, peptone, soybean powder, meat extract, rice bran, malt, urea, corn steep liquor, ammonium salt. , Nitrates, and other organic or inorganic nitrogen compounds are used. In addition, inorganic salts, micronutrient sources, etc. may be added as appropriate. In order to suppress foaming during fermentation, an antifoaming agent or the like can be added as appropriate. The culturing conditions such as culturing temperature and culturing time are selected so as to be suitable for the growth of the bacterium to be used and to maximize the production of F483A. For example, a suitable pH of the medium is 4-9,
5-8 is preferable, and 15-35 degreeC is suitable for culture | cultivation temperature, and 20-28 degreeC is preferable. The stirring speed is 200-4
00 rpm is suitable, 300 to 350 rpm is preferable, and the aeration is 1/20 to 1/1 v / v · min, 1/4 to 1/2 v / v · min is preferable, and the culture time is 24 to 168 hours is appropriate, 72
~ 120 hours are preferred. However, the culture conditions such as the culture composition, medium pH, culture temperature, stirring speed, aeration rate, and culture time may give desired results depending on the type of strain to be used and external conditions. Needless to say, it should be adjusted appropriately.

F483A can be prepared by appropriately utilizing the means usually used for collecting metabolites from the above culture.
Can be collected. For example, there are means for utilizing the difference in solubility between F483A and other substances contained in the culture, means for utilizing the difference in ionic binding force, means for utilizing the difference in adsorption affinity, and means for utilizing the difference in molecular weight. Any of them can be used alone, in appropriate combination, or repeatedly used. Specifically, F48
The 3A culture liquid and the bacterial cell extract are combined with various ion exchange chromatography, gel filtration chromatography, adsorption chromatography, liquid chromatography, cellulose partition chromatography, etc., and purified to contain F483A and other active ingredients. Fractions are obtained. The solid matter obtained by concentrating this fraction under reduced pressure is further subjected to high performance liquid chromatography, developed and purified to obtain a pale yellowish white powder of F483A.

The physicochemical properties of F483A obtained as described above are as follows. Molecular weight: 272 (C15H12O5) UV absorption spectrum (Hitachi U-3210 detector, MeO
H): As shown in FIG. Solubility: Soluble in dimethyl sulfoxide, methanol,
Soluble in chloroform and ethyl acetate, sparingly soluble in hexane and water. Color reaction: ferric chloride reaction positive substance color: pale yellowish white 1H-NMR (400MHz, MeOH-d4): main spectrum δ 2.78 (3H, s), 3.86 (3H, s), 6.25
(1H, brs), 6.40 (1H, brs), 6.62 (2H, brs) 13C-NMR (100 MHz, MeOH-d4):
Main spectra δ 23.6 (q), 57.1 (q), 92.3
(D), 98.9 (d), 102.7 (d), 117.6 (d), 105.4
(S), 112.6 (s), 144.6 (s), 159.1 (s), 160.9
(S), 164.9 (s), 165.0 (s), 166.6 (s), 183.4
(S) HR-FABMS: Measured value 273.0756 Calculated value 273.0763 From the above physicochemical properties, it was revealed that the antibiotic F483A is a novel compound represented by the following formula.

[0012]

Embedded image

The antitumor agent containing the antibiotic F483A of the present invention as an active ingredient can be used by any of the administration routes of oral and parenteral administration, and when orally administered, soft or hard capsules or tablets or granules are used. It can be administered in the form of a drug, a fine granule, a powder, etc., and when administered parenterally, a subcutaneous or intravenous injection such as an aqueous suspension or an oily preparation, an infusion, a suppository, an ointment. , Can be administered in a dosage form such as an ointment.

Antibiotic F483 which is the active ingredient of the present invention
To formulate A, a surfactant, an excipient, a lubricant,
Adjuvants, pharmaceutically acceptable film-forming substances, coating aids and the like can be used as appropriate. For example, the surfactant may include alcohols, esters, sulfated fatty alcohols and the like. Further, as the excipient, sucrose, lactose, starch, crystalline cellulose, mannite, light anhydrous silicic acid, magnesium amine acid, magnesium aluminometasilicate, synthetic aluminum silicate,
Examples thereof include calcium carbonate, sodium hydrogen carbonate, calcium hydrogen phosphate, and carboxymethyl cellulose calcium. Examples of lubricants include magnesium stearate, talc, hydrogenated oil, and the like, and as adjuvants such as suspending agents and wetting agents, coconut oil, olive oil, sesame oil, peanut oil, calcium lactate, safflower oil, Examples thereof include soybean phospholipid. As the film-forming substance, as a carbohydrate derivative such as cellulose or saccharide, cellulose acetate phthalate, or an acrylic acid-based copolymer, a methyl acrylate / methacrylic acid copolymer as a polyvinyl derivative such as dibasic acid mole ester, Examples thereof include a methyl methacrylate / methacrylic acid copolymer.
Examples of coating aids include plasticizers such as phthalates. In addition to the above components, sweeteners, flavors, colorants, preservatives and the like may be added.

[0015]

EXAMPLES The present invention will be described below in greater detail by giving Examples, but the scope of the present invention is not limited thereto.

[Production Example] Production of F483A from AJ117291 strain Penicillium sp. AJ117291 as inoculum
(Penicillium sp. AJ117291) was used. One platinum loop of the strain was used as a production medium 100 in a 500 ml Erlenmeyer flask.
The solution was inoculated into ml and cultured at 25 ° C. with orbital shaking (180 rpm) for 4 days. (Production medium composition; malt extract (Difco) 20 g / l, oatmeal powder 10 g / ml, peptone 10 g / ml, glucose 10 g / ml, soluble starch 10 g / l (pH 6.0)) Acetone was added and the mixture was extracted at 5 ° C for 2 days. Then, the filtrate (18 L) from which cells were removed was concentrated under reduced pressure by centrifugation and filtration,
Acetone was distilled off. The residue was extracted by adding 800 ml of ethyl acetate. After dehydrating the ethyl acetate layer, concentrate under reduced pressure,
Silica gel column chromatography (CHCl ₃ -M
MeOH) and the CHCl ₃ elution fraction was collected. This fraction was concentrated under reduced pressure and then subjected to silica gel column chromatography (n-Hexane-EtOAc) again to give 8
The 0:20 fraction was collected. After concentrating this fraction under reduced pressure, Se
After pretreatment with p-pakC ₁₈ (Millipore), CAPC
Purified by high performance liquid chromatography using ELL PAK NH ₂ 80, fractionated with 0.5% acetic acid water: acetonitrile containing 0.5% acetic acid = 65: 35, and concentrated under reduced pressure to give 1 mg of F483A as a pale yellowish white color. Obtained as a powder.

[Test Example] Measurement of antitumor activity of F483A (A) Antitumor effect on HCT-15 cells RPMI 1640 medium (manufactured by SIGMA), 10% fetal bovine serum, 100 units / ml penicillin and 10
HCT-prepared at 5 × 10 4 cells / ml in a medium (medium A) having a composition of 0 μg / ml streptomycin.
Fifteen μl of 15 cells were dispensed into each well of a 96-well microtiter plate. After culturing the plate in a carbon dioxide cell incubator at 37 ° C. for 20 hours, 50 μl each of the specimens appropriately diluted with the medium A was added thereto, and cultured in the carbon dioxide cell incubator at 37 ° C. for 72 hours. MTT solution (5 mg / ml Dulbecco's PBS (-)) was added thereto in an amount of 10 µl each, and the cells were cultured at 37 ° C for 4 hours in a carbon dioxide cell incubator. Then, 0.01 N hydrochloric acid / 20% SDS was added by 50 μl each to dissolve the resulting crystals, and the absorbance at 570 nm was measured by a microplate reader. By comparing the absorbances of untreated cells and cells treated with a known concentration of the sample, the sample concentration (I
C50) was calculated. The results are shown in Table 1.

(B) Antitumor activity against K562 cells K56 prepared at 8 × 10 4 cells / ml in the above medium A
50 μl of each 2 cells was dispensed into each well of a 96-well microtiter plate. To this, 50 μl of each sample appropriately diluted with the medium A was added, and the IC50 was calculated in the same manner as in the case of the above HCT-15 cells. The results are shown in Table 1.
It was shown to.

[0019]

[Table 1]

As is clear from Table 1, the novel antibiotic F483A of the present invention has a high antitumor effect.

[0021]

According to the present invention, the novel antibiotic F483A
And a method for manufacturing the same. The antibiotic F48
3A has high antitumor activity, and thus is useful as an antitumor agent.

[Brief description of drawings]

FIG. 1 is the MeO of the antibiotic F483A of the present invention.
It is a figure which shows the ultraviolet absorption spectrum in H solution. The vertical axis represents wavelength and the horizontal axis represents absorbance.

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Toshihiko Yoshimura 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa Ajinomoto Co., Inc. Central Research Laboratory (72) Inventor Ryosuke Fudo 1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa -1 Ajinomoto Co., Inc. Central Research Center (72) Inventor Toshiyuki Kameyama 1-1, Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa Ajinomoto Co., Inc. Central Research Center

Claims (3)

[Claims] 1. An antibiotic F483A represented below. Embedded image 2. A method for producing the antibiotic F483A, which comprises culturing an antibiotic F483A-producing bacterium belonging to the genus Penicillium in a medium and collecting the antibiotic F483A from the culture. 3. An antitumor agent containing the antibiotic F483A as an active ingredient.