JPH0586774B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD OF THE INVENTION The present invention relates to the DC1043 substance. The DC1043 substance has antitumor effects and is useful as an antitumor agent. Conventional technology Conventionally, it has been used as an antitumor antibiotic.

[Formula] [In the formula, R represents CH ₃ ildine M) or CH ₂ OH (ildine S)] Ildins are known [Merck Index]
10th edition, page 714]. Problems to be Solved by the Invention There is a constant need for substances with excellent antitumor effects. Means for Solving the Problems The present invention is based on the general formula

The present invention relates to substance DC1043, a novel substance having an antitumor effect represented by the formula: (wherein R represents CH ₃ or CH ₂ OH). In the above general formula, the compound where R=CH ₃ is called DC1043A, and the compound where R=CH ₂ OH is called DC1043B.
respectively. The physical and chemical properties of DC1043A and DC1043B are shown below. (1) Physical and chemical properties of DC1043A Elemental analysis values: C: 77.59%, H: 8.62%,
O: 13.79% Molecular weight: 232 Molecular formula: C ₁₅ H ₂₀ O ₂ Ultraviolet absorption spectrum (in MeOH) 300 nm
(Wavelength showing maximum absorption) Ultraviolet absorption spectrum (KBr tablet method): 1st
Figure PMR spectrum (in DMSO- _d6 , TMS standard) 0.32 (1H, m), 0.63 (1H, m), 0.81 (1H,
m), 0.98 (1H, m), 1.10 (3H, s), 1.13 (3H,
s), 1.18 (3H, s), 1.43 (3H, s), 2.43 (2H,
s), 4.89 (1H, s), 6.47 (1H, s) CMR spectrum (in DMSO-d ₆ , TMS standard) 5.0, 7.6, 14.5, 24.5, 27.9, 28.7, 31.5,
42.9, 44.0, 75.9, 126.6, 135.9, 136.0, 147.2,
199.8 Solubility: Easily soluble in chloroform, DMSO, methanol, ethyl acetate, acetone, sparingly soluble in water. Thin layer chromatography: Using a thin layer of silica gel (Kieselgel 60Art 5715, manufactured by E.Merck) with a developing system of toluene:acetone (7:3V/V).
Rf is 0.84, hexane:ethyl acetate (20:
1V/V) and Rf is 0.3. (2) Physical and chemical properties of DC1043B Elemental analysis values: C: 72.58%, H: 8.06%,
O: 19.35% Molecular weight: 248 Molecular formula: C ₁₅ H ₂₀ O ₃ ultraviolet absorption spectrum (in MeOH) 320 nm
(Wavelength showing maximum absorption) Ultraviolet absorption spectrum (KBr tablet method): 2nd
Figure PMR spectrum (in DMSO- _d6 , TMS standard) 0.32 (1H, m), 0.64 (1H, m), 0.81 (1H,
m), 1.02 (1H, m), 1.09 (3H, s), 1.20 (3H,
s), 1.44 (3H, s), 2.23 (1H, s), 2.59 (1H,
d), 3.30 (2H, m), 4.82 (1H, t), 4.86 (1H,
s), 6.48 (1H, s) CMR spectrum (in DMSO-d ₆ , TMS standard) 5.1, 7.7, 14.5, 23.9, 24.7, 31.6, 38.3,
50.4, 68.1, 76.1, 121.5, 136.2, 137.6, 144.9,
199.7 Solubility: Easily soluble in chloroform, DMSO, methanol, ethyl acetate, acetone, sparingly soluble in water. Thin layer chromatography: Using a thin layer of silica gel (Kieselgel 60Art 5715, manufactured by E.Merck) with a developing system of toluene:acetone (7:3V/V).
Rf is 0.64, hexane:ethyl acetate (7:
3V/V) development system, Rf is 0.3. Next, the antibacterial action, acute toxicity, and antitumor action of DC1043A and DC1043B will be explained. (1) Antibacterial action Minimum inhibitory concentration (MIC) against various bacteria
was measured by the agar dilution method (PH7.0), and the results are shown in Table 1.

[Table] (2) Acute toxicity DC1043A in intraperitoneal administration to mice
The acute toxicity values ( _LD50 ) of and DC1043B are 20 mg/Kg and 5 mg/Kg, respectively. (3) Antitumor effect experimental example 1 Therapeutic effect on Sarcoma 180 solid tumor Sarcoma 180
5×10 ⁶ ascites-type tumor cells were subcutaneously implanted in the axillary region. 24 hours after transplantation at the concentrations shown in Table 2.
DC1043A was administered for 5 consecutive days. DC1043A is almost insoluble in water, so
25 mg of Tween 80 was added per 10 mg of DC1043A and mixed well, and a phosphate buffered saline (PBS) solution was added thereto to prepare a suspension. This was diluted with PBS to prepare a solution with the required concentration. At this time
A similar solution containing Tween 80 only was used as a control and had no effect on the experimental animals. The composition of PBS is NaCl0.8g/dl, KCl0.02g/dl,
Na ₂ HPO ₄ 1.15g/dl, KH ₂ PO ₄ 0.02g/dl, PH7.2
belongs to. As a comparative example, after tumor cell transplantation,
0.2 ml of a PBS solution containing Irudin S was intraperitoneally administered once every 24 hours. Seven days after transplantation, T/C [T: average tumor volume (mm ³ ) of test examples, C: average tumor volume (mm ³ ) of control examples (intraperitoneal administration of 0.2 ml of PBS solution)] was measured. The results are shown in Table 2.

[Table] Experimental Example 2 Therapeutic effect on _Lymphocytic ryukemia P-388 tumor.
leukemia) P-388 tumor cells (1×10 ⁶ cells) were implanted intraperitoneally. solution of DC1043A 0.2 hours after transplantation
ml was administered intraperitoneally for 5 consecutive days. As a comparative example, Irudin S was administered in the same manner as DC1043A. Average survival days after transplantation and survival rate T/C (%) [Average survival days of test cases/Average survival days of control cases x 100
] was measured. The results are shown in Table 3.

[Table] Next, the method for producing the DC1043 substance will be explained.
Any microorganism that belongs to the genus Pleurotus and has the ability to produce the DC1043 substance can be used as the microorganism. A specific example is Pleurotus japonica (Pleurotus japonica).
japonicus) ATCC20195. As the medium used in the present invention, either a natural medium or a synthetic medium can be used as long as it contains a suitable amount of carbon source, nitrogen source, and inorganic substances. As the carbon source, glucose, starch, dextrin, mannose, fructose, sucrose, molasses, alcohols (methanol, ethanol, etc.), organic acids (acetic acid, formic acid, citric acid, malic acid, etc.), etc. are used. Nitrogen sources include ammonium chloride, ammonium sulfate, ammonium nitrate, sodium nitrate,
Urea, peptone, meat extract, yeast extract, dried yeast, corn stew liquor, soy flour, casamino acids, etc. are used. As the inorganic substance, sodium chloride, potassium chloride, ferrous sulfate, manganese sulfate, copper sulfate, potassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, calcium carbonate, etc. are used. Furthermore, if necessary, substances that promote the production of DC1043 substances, such as biotin and vitamins, may be added to the medium. As a culture method, liquid culture method, especially deep agitation culture method, is suitable. Culture temperature is 20-35
℃, preferably 22 to 30℃, and adjust the pH of the medium to 4 to 4 by adding aqueous ammonia, ammonium carbonate solution, etc.
8, preferably 5-7. When culture is carried out in liquid culture for usually 1 to 7 days, DC1043 substance is produced and accumulated in the culture solution. When the production amount in the culture solution reaches the maximum, the culture is stopped, the bacterial cells are separated, and the DC1043 substance is purified and isolated from the obtained culture solution. For isolation and purification of DC1043 substance from culture solution,
Separation and purification methods commonly used to isolate microbial metabolic products from their culture fluids (e.g. extraction methods, ion exchange resin methods, silica gel methods)
is used. Aspects of the present invention will be explained below using Examples. Example 1 Pleurotus japonica was used as the inoculum.
ATCC20195 was used. One platinum loopful of the strain was inoculated into 50 ml of a seed medium having the following composition in a 300 ml Erlenmeyer flask, and cultured with shaking at 25°C for 48 hours. Seed medium composition: Peptone 5g/, Ebios 5g/
, glucose 10g/, vegetable juice 200ml/
, CaCO ₃ 3g/, PH6.0 Seed culture solution was transferred at a rate of 5% (volume) to fermentation medium 18 with the following composition in a 30-volume jar fermenter, and heated at 25°C with aeration stirring method (rotation speed 350 r. pm
Culture was performed with an aeration rate of 18/min). Fermentation medium composition: Seuclose 50g/, dry yeast 20g/, KH ₂ PO ₄ 0.5g/, MgSO ₄ .
7H ₂ O 0.5 g/, CaCO ₃ 0.5 g/, PH 7.0 During culture, the PH of the medium was not controlled and cultured for 200 hours. Separate the bacterial cells and precipitate from the culture solution,
Liquid 15 was obtained. After adjusting the pH of the liquid to 5 with hydrochloric acid, the liquid was passed through a 0.8 nonionic porous resin (trade name "Diaion SP207" manufactured by Mitsubishi Chemical Corporation) to adsorb the active substance. The impurities were eluted with 3 parts of water:methanol (4:1 V/V), and then the active substance was eluted with methanol. Concentrate methanol elution section 6 to approximately 200ml,
It was sprinkled on a small amount of silica gel for chromatography (manufactured by Kanto Kagaku Co., Ltd.) to form a powder. This powder sample was suspended in toluene in advance and placed on a column of silica gel (100 ml) packed in the column, and impurities were removed by passing toluene through the column.
The fraction containing DC1043A was then eluted with toluene:acetone (50:1 V/V). The fraction containing DC1043B was then eluted with toluene:acetone (20:1 V/V). Each of these active fractions was further developed using silica gel (Lichroprep Si 60 manufactured by Merck) with hexane and ethyl acetate-based solvents while applying medium pressure. ), the DC1043A fraction was eluted.
Then hexane:ethyl acetate (7:3V/V)
The DC1043B fraction was eluted. Each fraction was collected and concentrated to obtain 30 mg of pure DC1043A and
120 mg of DC1043B was obtained. Example 2 The same procedure as in Example 1 was carried out except that the composition of the fermentation medium in Example 1 was replaced with the following.
15 mg of DC1043A and 80 mg of DC1043B were obtained. Fermentation medium composition: dextrin 50g/, soybean flour
20g/, CaCO ₃ 5g/, KH ₂ PO ₄ 0.5g/,
MgSO ₄ 7H ₂ O 0.5g/, PH 7.0 Effects of the Invention Substance DC1043 exhibits excellent antitumor activity.

[Brief explanation of the drawing]

Figure 1 shows the infrared absorption spectrum of DC1043A. Figure 2 shows the infrared absorption spectrum of DC1043B.

Claims (1)

[Claims] 1. A DC1043 substance represented by the general formula [Formula] (wherein R represents CH ₃ or CH ₂ OH).