JPS6150991A - Anthracyline compound r20y1 and use thereof - Google Patents

Abstract

NEW MATERIAL:An antibiotic expressed by the formula having the following physico-chemical properties; Yellowish brown powder. Molecular weight; 465.5. Elementary analysis (%); C, 67.09; H, 5.84; N, 3.01; O, 24.06. [alpha]D<25>=+289 deg. (C= 0.1, CHCl3). Soluble in acidic water, basic water, methanol, ethanol, n-propanol, n-butanol, acetone, ethyl acetate and chloroform, and insoluble in water, hexane, cyclohexane, petroleum ether, etc. USE:An antitumor agent. PREPARATION:For example, an R20 strain (FERM-P No.7138) belonging to the genus Actinomadura is cultivated in a suitable culture medium by the aerobic liquid submerged culture method at 27-30 deg.C for 6-7 days to produce and accumulate the aimed antibiotic expressed by the formula.

Description

DETAILED DESCRIPTION OF THE INVENTION Background of the Invention The present invention relates to novel anthracycline compounds and their uses.

Anthracycline compounds as anticancer antibiotics occupy an important position as medicines, and various compounds have already been proposed.

In general, the physiological activity of a chemical substance largely depends on its chemical structure. Therefore, in the case of anthracycline compounds, the d slope fc of the aglycon moiety and sugar moiety is different from the existing one in terms of substitution. It can be said that there is a constant desire for this.

Summary of the invention The present invention meets the above needs.

That is, the athracytalline compound 4iR according to the present invention
20Y1 is represented by the following formula. The invention also relates to acid addition salts of this compound.

The antitumor agent according to the present invention contains an acid addition salt of the anthracycline compound Rzoy1 or 7'c represented by the following formula as an active ingredient.

H2 Detailed Description of the Invention 1) Chemical Structure The anthracycline compound R20Y1 according to the present invention is:
It has a chemical structure represented by the above formula (A).

This chemical structure was determined as follows.

R20Y1 was hydrolyzed by dissolving it in full-length sulfuric acid and standing at room temperature for 24 hours to obtain daunosamine as the aglycone and sugar moieties, fc.

On the other hand, the structure of R20Y1 was determined as shown below from the results of ultraviolet light absorption spectrum, infrared absorption spectrum, nuclear magnetic resonance spectrum, FD mass spectrum, elemental analysis, etc. of R20Y1.

^ 2) Physicochemical and raw state Some of the physical and chemical properties of R20Y1 are as follows.

(1) Appearance: yellowish brown powder analysis value 67.00 5.97 2. c+s 24.
08 calculated value 67.09 5.84 3.01 24.
06 (3) Molecular weight: 465.5 (4) Melting point: 145-148°C (5) Specific optical rotation: [αID −+ 289° (C = 0.1. CHCl3
) (6) Ultraviolet/visible absorption spectrum Figure 1 (a) M
eOHAz@z rim ('E” tag cm 223 (528), 262 (486), 292 (4
93), 447 (272) 469 (252), 536
(63)(')0.1NHC1-MeOHAm2z
'nm (female) 221 (J4), 242 (460)
, 258 (574), 292 (527), 1% (c)O, INNaOH-MeoI (λ, rlaxnm
(ElaTl) 220 (460), 246 (576)
, 266 (676), 276 (606), (7) Infrared absorption spectrum (potassium bromide method) Figure 2 (8) Proton nuclear magnetic resonance spectrum Figure 3 (100 MHz, deuterium chloroform A) (9) FD mass spectrum
Figure 4 a [Solubility
- Soluble in acidic water, basic water, methanol, ethanol, n-propertool, n-methanol, acetone, ethyl acetate, chloroform.

Insoluble in water, hexane, cyclohexane, petroleum ether.

R20Y1 is yellow in methanol solution, but turns blue-purple in alkalinity.

The Rf values of QllRf value R20Y1 in various solvents on a silica gel plate are as follows.

Chloroform:methanol:water=s:2:0.05
0.30 Chloroform:methanol:acetic acid = 8:2:0.05
0.39 Chloroform: Methanol: Aqueous ammonia 8: 2: 0.05
0.63 (Merck & Co., "Silica Gel 60" for Platera stools) Since this compound has a primary amino group in the sugar moiety, its acid addition salt may exist. The acid in this case is
Typical examples include sulfuric acid and sulfuric acid.

l) Overview At present, the anthracycline compound R20Yl can only be obtained by culturing Masao proboscis, but it can also be produced by sweet oxidative or microbiological modification of the cervix proboscis, or by total synthesis. It could also be produced chemically.

In the case of culture V for imitative cutting, one strain that produces R20Y1, which is associated with actinomadules, is used. Specifically, the present inventors' isolated Actinomadura roseoviolaceae R20 is R20Yl
Although the present inventors have revealed that the bacterial strain exhibits a biological physiological function, it is possible to isolate suitable strains from nature using conventional methods of antibiotic-induced isolation. In addition, A. Roseoviolaceae R20Y1-producing bacteria including R20 gold were subjected to radiation irradiation and other treatments,
There is also room to increase R20Y1 production capacity.

2) R20 strain The R20 strain, which the present inventors have discovered as a strain of the genus Actinomadura that has the ability to produce the anthracycline compound R20Y1, has the following content.

(1) Origin and deposit number The R20 strain was isolated from soil collected in a vegetable field in Oaza Onoya, Kaho-machi, 4ho-gun, Fukuoka Prefecture, and was produced on July 5, 1997 in Showa Monde.
It was deposited at the Agency of Industrial Science and Technology's Institute of Microbial Technology and Microbial Technology, and bears the number ``Microbiological Research Institute No. 7138.''

(2) Dental properties and physiological properties The characterization of this bacterial strain was performed in accordance with the method manual of the International Committee on Streptomyces Nomenclature (ISP) as follows.

A) Morphology The obstructed hyphae spread radially on the surface of the medium containing the cocoons while branching, and no division of the cocoon threads was observed. Aerial hyphae are mainly lll1l
The short branches are branched at right angles (with respect to the main axis) (uniaxial branches), and at their tips there are dense small spiral spores consisting of 10 or more spores (1 ~3 rotations,
diameter 2.0-2.5 μ) and pseudospore chain (diameter 2.5-3.
5μ) and form spore masses.

The spore stage is arranged in a circular cylindrical sheath with a width of 0.5 to 0.8 μm, the surface of which is rough, and the individual spores are connected in a phalanx shape. The spore mass is amorphous, its spore surface.

is wrapped in sticky material. 411 spores are rarely observed, cylindrical or oval, 0.5-0.8μ wide.
2. It has a length of 0.7-1.1μ and a smooth surface. True spore cranes, flagellated spores, single nuclei, etc. are not enshrined in one inkstone. Since the whole cell hydrolyzed breast contains meso-type diaminopimelic acid and madulose gold, the cell type is determined to be IB.

B) Culture properties Observation results of culture properties (cultivated at 27°C) in various media are shown in Table 1.

C) Physiological properties □ Physiological properties (including carbon source assimilation) are as shown in Table 2.

D) Discussion and Identification The present strain has (1) a spore wall type of B, (2) a spore chain consisting of 10+a or more spores, and (3) pseudosporangia and spores. It is judged that it belongs to the genus Actinomadura because (4) true spore feeding and flagellated spores are not observed.

Nonomura's search table [Rikou, Vol. 52, 71-77 Tei, 1
974] C'+l Engineering, Volume 49, 904-91
2, 1971], the present strain is A. azeoviolaceae (A.

rossoviolacja).

Therefore, this strain and A, standard strain of Roseoviolaceae (
KCCA-145 (Nonomura A-5)) was cultured under the same conditions, and the main properties of both bacterial cells were compared. The results are in Table 3
As shown in Figure 2, although there are slight differences in the color of the bacterial flora, underside color, and suitable growth, they are taxonomically very similar. Therefore, this 100 plants are Actinomadura roseoviolaceae (Actinomadura ro
sa'oviolacen Nonomura etO
h^ra1971).

3) Cultivation/Production of R20Y1 Anthracycline R20Yl is manufactured by culturing R20Y1-producing bacteria belonging to the genus Actinomadura aerobically in an appropriate medium and collecting the target product from the culture.
I can do that.

The medium can contain any nutrient source that can be utilized by the R20Y1 producing bacteria. Specifically, for example, lucose, sucrose, maltose, starch, and fats and oils can be used as carbon sources, and soybean flour, cottonseed meal, meat extract, hefton, dried yeast, yeast extract, and nitrogen sources can be used. Organic substances such as corn steep liquor and ammonium salts can be used, as well as nitrates such as ammonium sulfate, sodium nitrate, and inorganic substances such as ammonium chloride. In addition, inorganic salts such as common salt, potassium chloride, phosphorus salt, heavy metal salt, etc. can be added as necessary. In order to suppress foaming during fermentation, suitable antifoaming agents such as silicone gold can also be added according to conventional methods.

The most suitable culture method is the aerobic liquid deep culture method, which is the same as the commonly used method for producing antibiotics. The appropriate culture temperature is 5°C to 45°C, but 27°C
~Blade °C is preferred. With this method, the production amount of R20Yl is
Both rapid culture and aerated agitation culture reach their peak in 6 to 7 days.

In this way a culture enriched with R20Y1 is obtained. In the culture, a part of 120Y1 exists in the bacterial cells, but most of it exists in the culture P solution.

Any convenient method can be used to harvest R20Ylt from such cultures. One method is based on the principle of extraction, and specifically, for example, for R20Y1 in cultured Pi, it can be extracted with a water-immiscible R20Y1 solvent (see above), such as ethyl acetate. Method of extraction with chloroform, butanol, etc. (Culture P
'E! The extraction efficiency is good if L is neutral or slightly basic), or for R20Y1 inside the bacterial cells, the bacterial cell aggregate obtained by f-filtration, centrifugation, etc. is mixed with chloroform, ethyl acetate, butanol, methanol, ethanol, acetone,
It can be recovered by treatment with a hydrochloric acid solution or an acetic acid solution. It is also possible to subject the entire culture as it is to the above extraction operation without separating the bacterial cells. A countercurrent distribution method using a suitable solution field is also included in the extraction process.One of the other methods for collecting R20Y1 from cultures is based on the 11x principle of adsorption, which is already in liquid form. A suitable adsorbent such as activated carbon, alumina, silica, Kagel, etc. is used as a coating for the R20Y1-containing material, such as the culture P solution or the extract obtained by performing the extraction operation as described above. Column chromatography using sound, liquid chromatography, etc.
R20Y1 can be obtained by adsorbing the target R2.oyxt by others and then making i volume. The 'ttfcR20Y1 solution thus obtained is concentrated to dryness under reduced pressure to obtain 4 crude samples of R20Y-1.

In order to purify the R20Y1 thus obtained, the above-mentioned extraction method and adsorption method may be combined as necessary and carried out as many times as necessary. For example, column chromatography using an adsorbent or gel-filtration agent such as silica gel, weakly acidic ion exchange resin, or activated carbon, immersion chromatography using an appropriate solvent, and countercurrent distribution method are carried out in appropriate combinations. be able to. Specifically, for example, a crude sample of R20Yl is dissolved in a small amount of chloroform, developed with an appropriate solvent using a silica gel column, and the active ingredient is eluted, and the bath solution
After that, it is further developed with TLC, scraped and eluted, and Rzo
Since yti is isolated as mono-im, it is concentrated and now crystallized from a suitable solvent to obtain R20, Y1e.
Can be obtained as crystals.

-Uses of R20Y1 The anthracycline compound R20Y1 according to the present invention is
It is a useful compound as a medicine in that it has anticancer activity and antibacterial activity and has low toxicity.

1) Physiological activity (1) Antitumor properties 120Y1 showed a significant anti-sleep effect against leukemia in experimental animals. For example, P38 for CDF1 mice
A suspension of 8 leukemia cells was transplanted into the vagina of a mouse, and R20Y 1 was transplanted on the 1st and 5th day after transplantation.
was administered. The survival rate is calculated based on the total number of survival days of mice in the control group treated with physiological saline for 30 days (100 days).
The effects expressed in terms of %) were as follows.

Dose (mg/kg/day) Life extension rate T/C (system (2)
Monobacterial R20Y1 is of a quality that exhibits a heavy effect on the imitative proboscis of Yuzu, and the 7'C minimum growth inhibitory concentration [(MIC) determined by the agar plate dilution method was as follows. .

0) If the cutting fungus is bacteria, Mueller-Hinton N medium/pH 7.3
/37°C/20 hours (b) If the test bacteria is yeast, 5 hours dextrose agar medium/pH
5,6/ゐ℃/48 hours (c) If the test bacteria is mold, dextroae agar medium/p)
15. The MIC for 15 days at 15°C is 106 cells/ml by preparing a bacterial suspension or spore suspension of the test bacteria and inoculating it in a microplanter with R20.
Minimum Inhibitory Concentration (MIC) of Y1 As mentioned above, R20Y1 of the present invention has an antibacterial effect, and can be used as an antidental substance that exhibits antidental properties, particularly against Durham-positive bacteria.

(3) Acute toxicity (LD5o) The LD5g of R20Y1 by intraperitoneal injection of R20Y1 was 15
．． Atsuniwa at 9mg/kg. ″2) Anti-;toi<;karasufu♀j Such a trap, the anthracycline compound 120 of the present invention
It has been revealed that Y1 exhibits anti-intestinal properties against malignant and tumorous tumors, including humans.

Therefore, R20Y1 of the present invention can be used as an anti-tumor wound or morning bath therapeutic agent.

Antillll1! R20Y1 as a drug can be administered by any route of administration and in a dosage form determined by the route of administration employed. The drug is usually in a form diluted with a pharmaceutically acceptable carrier or diluent.

When MeR20Yl is actually administered as an anti-soul drug, one typical method is to dissolve it in distilled water for injection or injectable water and inject it.'' In the case of animals, injection methods such as intraperitoneal injection, subcutaneous orthogonal injection, intravenous injection, intravenous injection into the mountain range, and local administration are used; in the case of humans, intravenous injection or injection of blood into other areas
・There are two methods: internal injection or local injection.

The dosage of R20Y1 is determined based on the results of animal studies and various
In consideration of the in-circumstances situation, continuous or intermittent administration is determined so that the injection melt does not exceed a certain level. The specific dosage depends on the method of administration, method, condition of the patient or treated animal, age, body weight, sex, sensitivity, diet, time of administration, concomitant drugs, and the extent of the patient or his illness. Needless to say, the appropriate dose and frequency of administration under certain conditions must be determined by a specialist's own trial and error judgment based on the above-mentioned guidelines.

Experimental example In the following, "1-%" means "177%".

Example 1 (1) Preparation of Seed Mother The medium used was prepared by dissolving the following components in 1 liter of water and adjusting the pH to 7.2.

Polypeptone 1% Molasses 1% Meat Extract Part 1 100ml of the above medium 1 'fil - 500rr + 1 Erlenmeyer flask was sterilized under partial pressure, 1 platinum lug was added to Actinomadura roseoviolaceae R20Q slant, and heated at 27°C for 5 days in a rotary shaker (200 rpm) ) was used as the total seed mother.

(2) The culture medium used in the culture vessel was prepared by dissolving the following components in 1 liter of water and adjusting the pH to 7.4.

Glucose 2.5% Soybean flour 1.5 t Dried yeast 0.2% Calcium carbonate (sedimentation) 0.4 t Put 5 liters of the above medium into a 50 liter jar fermenter and sterilize it, add the above seed mother Three doses were inoculated. Ventilation - R1 m, v, m, rotation speed 200. costume, Z7'
Culture was performed for 7 days at C.

(3) After collecting and culturing R20Y1, the culture was carried out once, and the whole and P solution were separated. P
ill f! L: Adjust the pH to 2 with IN hydrochloric acid, and apply column 10 of "Diaion IP-20" (manufactured by Mitsubishi Kasei Corporation).
Adsorb it to x 40cm. After washing with distilled water and 50 timethanol, elution was performed with methanol. The eluate was reduced to ml, concentrated overnight, adjusted to pH 8.5, and extracted three times with a chloroform-methanol (9:1) mixture. After this extraction 7 sheet metal image #i, 6 times the amount of hexane was added and the resulting precipitate 1'! i 'IIIJ k dried 1, red powder 2
50 mg f was obtained (R20Yl crude sample).

5131 example 2 1 type in example 1! L7'c R20Yl rough sample 250
mg f tdWj in chloroform, silica gel 250
Column 4 X 4Q equilibrated with g t-chloroform
Wash the column thoroughly with chloroform.
7. It was dissolved with oaform:methanol=lO:1.

Both obtained fractions were completely dried to dryness under reduced pressure at 7'C, and then chloroform:methanol/aqueous ammonia=8. : 2 : 0.0
TLC (Merck & Co., Ltd. “Silica Gel 60
), and the yellow fraction around Rf O,6 was scraped off. After eluting and concentrating the fraction obtained in this 5K manner,
Recrystallization in chloroform gave 3 mg of R20Y1. −

[Brief explanation of the drawing]

FIG. 1 is a reproduction of the ultraviolet/visible absorption spectrum of R20Yl. The second □□□ is a copy of the infrared absorption spectrum of R20Yl. FIG. 3 is a reproduction of the 1H-NMR spectrum of R20Y1. FIG. 4 is a reproduction of the F'D mass spectrum of -R20Yl.

Claims (1)

[Claims] 1. Anthracycline compound R20Y represented by the following formula
1 or its acid addition salt ▲ Numerical formulas, chemical formulas, tables, etc. are available ▼ 2. Anthracycline compound R20Y represented by the following formula
1 or an acid addition salt thereof as an active ingredient. ▲Contains mathematical formulas, chemical formulas, tables, etc.▼