JPH10330360A - Tetramic acid compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new compound that has an antitumor action, for example, having the proliferation-inhibitory action against human promyelocytic leukemia cells and human nose and throat cancer cells. SOLUTION: This new compound is a kind of tertraminnic acid (TEL0010) having the chemical structure of the formula. This compound shows the following physical and chemical properties: appearance: pale yellow powder; melting point: 287-291 deg.C; molecular weight: 391; specific rotatory power: [α]D<25> : +69.7 deg. (c=2.0 in MeOH); solubility in solvents: soluble in methanol ethanol, dimethyl sulfoxide, slightly soluble in chloroform, ethyl acetate and insoluble in water, hexane, benzene, ether; color reactions: positive to iodine, sulfuric acid, ammonium molybdate sulfate, negative to ninhydrin, enthrone sulfate; distinction of basic, acidic or neutral: weakly acidic. This compound is obtained by aerobically culturing a microorganism capable of producing this substance, for example, Streptomyces sp. TA-035 [FERM BP-6268] at pH of 4-10, 25-35 deg.C for 2-5 days and isolating the product from the culture mixture.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel tetramic acid compound having an antitumor effect.

[0002]

2. Description of the Related Art As a compound having tetramic acid in its structure, several kinds of compounds such as Tenuazonic acid have been reported, but no compound having an antitumor effect is known.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel physiologically active substance having an antitumor effect.

[0004]

Means for Solving the Problems The present inventors have isolated a large number of strains from soil and plants to achieve the above object,
As a result of various studies on the metabolites of the strain, it was found that a certain strain produces a novel physiologically active substance having antitumor activity, and the present invention was completed.

That is, the present invention relates to the following formula (1)

[0006]

Embedded image

The tetramic acid compound represented by
Abbreviated as TEL0010. ).

The strain producing TEL0010 is a strain newly isolated by the present inventors from the natural world, and has a microorganism name " Streptomyces sp. TA-0358" and a microorganism deposit number "FE".
RM BP-6268 "has been deposited with the National Institute of Advanced Industrial Science and Technology.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The mycological properties of this strain are shown below. A. Morphological properties The basic mycelia grow well and are branched, but no division is observed. The formation of aerial hyphae and spores is good in oatmeal agar and 3 other media. No sporulation is observed in the agar medium. It forms a compact spirally branched chain of segmented spores of 6 to 10 turns (30 to 50 spores) simply branched from aerial mycelia. Spores have a length of 1.1-1.3 μm and a width of 0.7-
0.9 μm, cylindrical shape, smooth surface. No sporangia or sclerotium formation or spore motility is observed.

B. Cultural Properties Table 1 below shows the results of macroscopic observation when cultured on various media at 28 ° C. for 14 days. In addition, the color display quoted the system color name of the Japan Standards Association and JIS color name book (1985).

[0011]

[Table 1]

C. Physiological Properties The growth temperature test was observed after 1 week, the carbon source utilization test was observed for 2 weeks, and the others were observed after 3 weeks of culture. Growth temperature range: 12-38 ° C, optimal growth temperature: 26-
31 ° C Gelatin liquefaction: Positive Skim milk coagulation: Negative Skim milk peptone conversion: Positive Melanin-like pigment production: Positive Starch hydrolysis: Positive Carbon source availability (on Pridham-Gottlieve agar medium) L-arabinose: + L-rhamulose: ± D-xylose: + raffinose: + D-glucose: + inositol: + D-fructose: + D-mannitol: + sucrose: + (+: used ±: slightly used) D. Chemical taxonomic properties The presence or absence of diaminopimelic acid and the optical isomer type: LL-type diaminopimelic acid were detected. Menaquinones: MK-9 (H ₆₎ and MK-9 (H ₈₎ is detected as main components. The MK-9 (H ₄₎ was also detected in small amounts. From the above properties, the TA-0358 strain can be classified into actinomycetes of the genus Streptomyces , this strain Streptomyces sp.
It was named TA-0358.

The production of TEL0010 is almost the same as the case of producing a general fermentation product, and Stre is used in a medium containing various nutrients.
This is performed by culturing ptomyces sp. strain TA-0358 under aerobic conditions. The medium is mainly a liquid medium and is composed of a carbon source, a nitrogen source, and an inorganic salt. If necessary, vitamins, precursors and an antifoaming agent can be added.
Adjust back and forth. Examples of the carbon source include glucose,
Sucrose, dextrin, glycerin, starch and the like are used alone or in combination. As the nitrogen source, for example, meat extract, oatmeal, yeast extract, soybean powder, polypeptone, corn steep liquor, urea, ammonium salt, or the like is used alone or in combination. As the inorganic salt, for example, monopotassium phosphate, magnesium sulfate, sodium chloride, calcium carbonate and the like are used alone or as a mixture. Adecanol, silicon compounds, and the like can be used as the defoaming agent.

As the culturing method, aerobic culturing such as shaking culturing and aeration stirring culturing is suitable, and the pH is 4 to 10 and 25 to 35 ° C.
For 2 to 5 days, desirably at pH 6 to 7, and at 25 to 28 ° C for 4 days. The TEL0010 produced by this culture can be isolated according to a general method of collecting a fermentation product. For example, the following method is effective. That is, after completion of the culture, a culture filtrate is obtained by centrifugation or filtration, adsorbed on a polystyrene resin such as Diaion HP-20 (trade name, manufactured by Mitsubishi Kasei), and then eluted with an organic solvent such as lower alcohol or acetone. Let The cells are extracted with an organic solvent such as lower alcohol or acetone.
Then, the cell extract and the eluate from the adsorption resin were combined and concentrated under reduced pressure to remove the organic solvent.Then, the resulting solution was transferred to a water-insoluble organic solvent such as ethyl acetate, chloroform, n-butanol and concentrated. And make a syrup. This syrup was dissolved again in an organic solvent such as benzene, ethyl acetate, acetone, methanol, chloroform, and subjected to silica gel column chromatography, gel filtration column chromatography, and column chromatography packed with ODS for reversed phase partition and high performance liquid chromatography. By attaching, TEL0010 can be purified and isolated.

TEL0010 obtained by the above purification method
Is the molecular weight, ultraviolet absorption spectrum, ¹ H-NMR
By analysis of spectrum, ¹³ C-NMR spectrum, etc.,
Its structural formula was determined as in equation (1).

The physicochemical properties of TEL0010 are shown below. (A) Appearance: pale yellow powder (b) Melting point: 287 to 291 ° C (decomposition) (c) Molecular weight: 391 (d) Molecular formula: C ₂₂ H ₃₃ NO ₅ (e) HREI mass spectrum Measured value: 391.2381 Theory _{value: 391.2359 (C 22 H 33 NO} 5) calculated (f) EI mass spectrum ^{m / z 391 (M) +} (g) specific ^{_{rotation: [α] D 25: +}} 69.7 ° (c = 0 .2, MeOH) (h) UV absorption spectrum: λ max nm (ε) MeOH: 243 (10700), 284 (1610)
0) MeOH + HCl: 225 (5900), 286 (15
500) MeOH + NaOH: 244 (12600), 283
(15900) (i) Infrared absorption spectrum: The result measured by the KBr method is shown in FIG.

(J) ¹ H-NMR spectrum: The result measured in heavy pyridine at 500 MHz is shown in FIG.

(K) ¹³ C-NMR spectrum: FIG. 3 shows the result of measurement in heavy pyridine at 125 MHz.

(L) Solubility in solvent: Soluble in methanol, ethanol and dimethylsulfoxide Insoluble in water, hexane, benzene and ether (m) Color reaction: Positive: I ₂ , H ₂ SO ₄ , ammonium molybdate sulfate Negative: ninhydrin, anthron sulfate (n) Basic, acidic, neutral distinction: weakly acidic.

[0020]

As shown in Table 2, the compounds of the present invention contain H
Since it has a growth inhibitory effect on L-60 cells and KB cells, it is useful as an antitumor agent.

[0021]

EXAMPLES The present invention will be specifically described below with reference to examples and test examples. Soluble starch 2.0%, glucose 0.5
%, NZ case 0.3%, yeast extract 0.2%, fish meal 0.5%, calcium carbonate 0.2% (PH 7.2%)
100 ml of the liquid medium containing 0) was placed in a 500 ml Erlenmeyer flask, and sterilized at 120 ° C. and 2 atm for 20 minutes. Next, Streptomyces sp. TA-0358 strain was inoculated into this sterile medium, and the mixture was subjected to rotary shaking culture at 28 ° C. and 200 rpm for 2 days to obtain seed culture. Then, using one 50 l jar fermenter, 2.0% glycerol, 2.0% dextrin, 1.0% soybean flour, 0.5% Pharmamedia,
Polypeptone _{0.1%, MgSO 4 · 7H 2} O 0.05
%, CoCl ₂ .6H ₂ O 0.0005%, calcium carbonate 0.3% (pH 7.0), 30 l of sterile production medium
400 ml of the above seed culture solution was inoculated into
The cells were agitated and agitated at pm for 4 days.

After completion of the culture, 30 l of the obtained culture was separated into cells and supernatant by centrifugation. The cells were extracted with 6 l of acetone and then with 6 l of methanol, and both extracted fractions were combined and concentrated. The concentrated solution was extracted with 4 L of ethyl acetate to remove the fat-soluble fraction, and further extracted with 6 L of n-butanol, and the n-butanol fraction was concentrated under reduced pressure. The culture supernatant was adsorbed with 2 l of Diaion HP-20 (trade name, manufactured by Mitsubishi Kasei Co., Ltd.) to adsorb the active substance, and then eluted with 6 l of acetone and then 6 l of methanol. The two eluates were combined, concentrated under reduced pressure, and further combined with a concentrate from the cells. The concentrated solution was extracted with ethyl acetate to remove the fat-soluble fraction, and then extracted with 4 l of n-butanol to concentrate the n-butanol layer.
Chloroform was added to the concentrated solution to precipitate the active substance, and 3.58 g of white powder was obtained.

The obtained powder was dissolved in methanol and subjected to silica gel column chromatography (volume: 600 ml) with a chloroform-methanol system. Elution with 2.1 L of 20% methanol and 2.0 L of 50% methanol yields 50
The active fractions eluted with% methanol were collected and concentrated under reduced pressure. This was dissolved in methanol and divided into two portions, and Sephadex LH-20 (trade name, prepared by methanol)
Gel filtration was performed using a column (manufactured by Pharmacia) (volume: 500 ml), and the active fraction was concentrated to dryness under reduced pressure.
71 g of TEL0010 was obtained.

The purity was confirmed by high performance liquid chromatography under the following conditions. Column size 4.6φ × 150 mm Carrier ODS silica gel (manufactured by YMC) Solvent composition 60% acetonitrile, 40% water (pH 3.5) Flow rate 1.0 ml / min Temperature 50 ° C. Detection wavelength 215 nm Device Waters M-600 Retention time 7. 2 minutes.

Test Example 1 HL-60 Cell Growth Inhibition Test (Specimen) DMS was adjusted so that TEL0010 was 10 mg / ml.
A solution dissolved in O and diluted with sterilized water to a target concentration was used. (Test cells) Human promyelocytic leukemia cells HL-60 (Test method) HL-60 cells cultured in RPMI-1640 medium were placed on a 12-well flat bottom plate at 1 x 10 ⁵ cel.
ls / ml was added, and then a sample prepared to have a target concentration was added. The test cells were at 37 ° C.
After culturing for 48 hours in a 5% CO 2 incubator, the viable cells were measured, and the 50% inhibitory concentration (IC
₅₀ values) were calculated. Adriamycin was used as a control drug. The results are shown in Table 2.

Test Example 2 KB cell growth inhibition test (Sample) TEL0010 was adjusted to 10 mg / ml in DMS
A solution dissolved in O and diluted with sterilized water to a target concentration was used.

(Test cells) Human nasopharyngeal cancer cells KB (Test method) KB cells (1
(0 ³ cells / 100 ul / well) was added, and the cells were cultured for 24 hours. After the addition of the target concentration of the specimen, the cells were further cultured for 72 hours. After culturing, MTT [3- (4,5-dimethylthiazol-2-y
l) Add 2,5-diphenyltetrazolium bromide] reagent and add 4
Allowed to react for hours. After completion of the reaction, the absorbance was measured, the ratio of the absorbance of the sample-treated group to the absorbance of the control was determined, and the 50% inhibitory concentration (IC ₅₀ value) was determined. Adriamycin was used as a control. The results are shown in Table 2.

[0028]

[Table 2]

[Brief description of the drawings]

FIG. 1 shows an infrared absorption spectrum of TEL0010 measured by a KBr method.

FIG. 2. TEL001 measured at 500 MHz in heavy pyridine
¹ shows a ¹ H-NMR spectrum of 0.

Figure 3: TEL001 measured at 125MHz in heavy pyridine
1 shows the ¹³ C-NMR spectrum of 0.

 ───────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Tsutomu Ando 3-24-1, Takada, Toshima-ku, Tokyo Taisho Pharmaceutical Co., Ltd. (72) Inventor Masafumi Shibuya 5374-18-601 Shiba, Kawaguchi City, Saitama (72) Inventor Tadayasu Okazaki 3-24-1, Takada, Toshima-ku, Tokyo Inside Taisho Pharmaceutical Co., Ltd.

Claims (1)

[Claims] 1. A tetramic acid compound having the following structure: