JPS6176498A - Anthracycline compound 20x2 and its use - Google Patents

Abstract

NEW MATERIAL:A compound shown by the formula. USE:An antitumoragent, and an antibacterial agent. PREPARATION:For example, Actinomadura roseoviolacea IR20 (FERM P-7138) belonging to the genus Actinomadura, capable of producing anthracycline compound 20X2, is inoculated into a medium containing polypeptone, molasses, meat extract, etc., and cultivated to give a yeast culture. Then, it is cultivated in a medium consisting of an aqueous solution containing glucose, soybean flour, dried yeast, calcium carbonate, etc., the culture solution is filtered, and the mold is separated from the filtrate. The filtrate is passed through a column of an adsorbing resin. R20X2 is adsorbed on it, eluted with methanol, the elute is concentrated, and purified to give a compound shown by the formula.

Description

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

As anticancer antibiotics, anthracycline compounds 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, so there is a constant desire for anthracycline compounds that differ from existing ones in the types of aglycone moieties and sugar moieties or substituents. SUMMARY OF THE INVENTION The present invention satisfies the above-mentioned desire.

That is, the anthratha icrin compound R according to the present invention
20X2 is expressed 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 anthracycline compound R20X2 represented by the following formula or an acid addition salt thereof as an active ingredient.

H2 1) Chemical structure The anthracycline compound 120X2 according to the present invention is:
It has a chemical structure represented by the above formula (A).

This chemical structure was determined as follows.

R20X2 to 0. By dissolving in IN hydrochloric acid and hydrolyzing at 90° C. for 30 minutes, an aglycone moiety and a sugar moiety were obtained. As a result of comparing each with standard substances by thin layer chromatography (TLO), it was found that the aglycone is β-nirodomycinone (β-Rhoclomycinone),
The sugar was consistent with Daunosamine. Furthermore, the structure of R20X2 was determined as shown in Formula A above from the results of ultraviolet/visible absorption spectra, infrared absorption spectra, nuclear magnetic resonance spectra, FD mass spectra, elemental analysis, etc. of R20X2 and the aglycon moiety.

2) Physicochemical properties (1) Appearance: Dark brown powder (2) Elemental analysis: Engineering H-9-(3) Molecular weight! 515.5 (4) Melting point! 112-115℃ (5) Optical rotation: [α), -352° (0-0,1
, (!H(d3-MθSnoreI”-121) (6) Ultraviolet-visible absorption spectrum (Figure 1) MeOH
λmax nm (B”%) 0m 232 (777), 250 (454), 290 (143
), 468 (235), 489 (262), 494 (2
86), 514 (217), 526 (196) 0.1N
H(31-MeOHλmaxnm (B”)10
m 232 (806), 252 (468), 290 (163
), 468 (252), 480 (276), 494 (3
01), 514 (225), 528 (198) 0, lN
NaOHλmax nm (Fi:':m)23
B (625'), 280 (161), 561 (202)
, 600 (16'1) (7) Infrared absorption spectrum (potassium bromide method) Figure 2 (8) Proton nuclear magnetic resonance spectrum (100 MHz, in deuterium chloroform) Figure 3 (9) FD mass spectrum No. 4 Figure (10) Soluble acidic water, basic water, methanol, ethanol, n-7'
Soluble in lovanol, acetone, ethyl acetate and chloroform.

Insoluble in water, hexane, cyclohexane, diethyl ether and petroleum ether.

R20X2 is brown in methanol solution, but turns blue-purple in alkalinity.

8! 2:0.05
0.16 Chloroform: Methanol: Acetic acid 8: 2: 0.05 0
．． 26 Chloroform: methanol; aqueous ammonia 8
+ 2 + o, os O, 4
4 Use Merck silica gel 60 plate, connect MeOH
indicates methanol.

Since this compound has a primary amino group in the sugar moiety, its acid addition salt may exist. Typical acids in this case include hydrochloric acid and sulfuric acid.

1) Overview The anthracycline compound R20X2 can currently only be obtained by culturing microorganisms, but it can also be produced by synthetic chemical or microbiological modification of related compounds, or by total synthetic chemistry. I can do it.

When culturing microorganisms, a strain belonging to the genus Actinomadura and capable of producing R20!2 is used. Specifically, the present inventors, K., have revealed that Actinomadura roseoviolaceae R20, which the present inventors isolated, produces R20X2, but other bacterial strains have not been isolated. It is possible to separate suitable substances from nature by conventional methods. Furthermore, there is still room to increase R20X2 production ability by subjecting R20X2 producing bacteria, including A. roseoviolaceae R20, to radiation irradiation or other treatments.

2) R20 strain The R20 strain, which the present inventors have discovered as a strain of the genus Actinomagella that has the ability to produce the anthracycline compound R20X2, has the following content.

(1) Origin and deposit number The R20 strain was isolated from soil collected from a vegetable field in Oaza Onodani, Kaho-machi, Kaho-gun, Fukuoka Prefecture, and was transferred to the Institute of Microbial Technology, Agency of Industrial Science and Technology on July 5, 1981. Deposited “
The number is ``Feikoken Bibori No. 7138''.

(2) Mycological properties and physiological properties The characterization of this bacterial strain according to the method manual of the International Actinobacteria Nomenclature Committee (ISF) is as follows.

A) Morphology The basal hyphae spread radially on the surface of the agar medium while branching, and no hyphal division was observed. Aerial hyphae extend their main axis long, the short waves branch at right angles to the main axis (uniaxial branching), and at the tip there is a dense small helical spore chain consisting of around 10 or more spores ( 1 to 3 turns, diameter 2.0 to 2
．． 5μ) and pseudosporangia (diameter 2.5-3.5μ) and spore masses.

The spore chain is covered by a cylindrical sheath with a width of 0.5 to 0.8μ,
Its surface is rough, and the individual spores are connected like phalanges. The spore mass is amorphous, and the spore surface is covered with a sticky substance. Free spores are rarely observed, cylindrical or oblong, 0.5-0.8μ wide, 0.7-1.1μ long,
Exhibits a smooth surface. True sporangia, flagellated spores, and sclerotia are not observed. Since the whole cell hydrolyzate contains meso-type diaminopimelic acid and madulose, the cell wall type is determined to be mB.

B) Culture properties The observation results of the culture properties (27''C culture) in the polysaccharide medium are shown in Table 1.

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

D) Discussion and Identification This strain has (1) a cell wall type of IIIB, and (2)
) A spore chain consists of 10 or more spores, (3
) It forms pseudospore chains and spore masses, and (4) true sporangia and flagellated spores are not observed, so it is judged that it belongs to the genus Actinomadura.

Nonomura's search table [Rikou, Vol. 52, pp. 71-77, 19
1974] [Rikou, Vol. 49, pp. 904-912,
1971], the present strain is A, Roseoviolaceae (A
.

rOθeoviolacθa).

Therefore, the present strain A, the Kai semi-bacterial strain of Roseopiolaceae (K(
! OA-145 (Nonomura A-5)''l was cultured under the same conditions, and the main properties of both strains were compared.

As shown in Table 3, although there are slight differences in the bacterial flora color, underside color, and optimal growth temperature, taxonomically they are very similar (similar to each other. Therefore, this strain is an actinoma strain. Jura Roseoviolaceae (Actinomadu)
ra roseoviolacea Nonomura
et3) Culture/Production of R20X2 Anthracycline R20X2 can be produced by culturing R20X2-producing bacteria belonging to the genus Actenomagella aerobically in an appropriate medium and collecting the target product from the culture.

The medium can contain any nutrient source that can be utilized by the R20M2 producing bacteria. Specifically, for example, glucose, sucrose, maltose, starch, and fats and oils can be used as carbon sources, and soybean flour, cottonseed meal, meat extract, peptone, dried yeast, yeast extract, and corn starch can be used as nitrogen sources. Organic materials such as chief liquor and inorganic materials such as ammonium salts or nitrates such as ammonium sulfate, sodium nitrate, and ammonium chloride can be used. Furthermore, inorganic salts such as common salt, potassium chloride, phosphate, and heavy metal salts can be added as necessary. In order to suppress foaming during fermentation, suitable antifoaming agents such as silicones can also be added according to conventional methods.

The culture method is the same as the commonly used method for producing antibiotics (the aerobic liquid deep culture method is the most suitable. The culture temperature is 25'C to 45C, but
C to 30'C is preferred. In this method, the production amount of R20X2 reaches its maximum in 6 to 7 days for both shaking culture and aerated agitation culture.

In this way a culture enriched with R20X2 is obtained. In the culture, a portion of R20X2 exists in the bacterial cells, but most of it exists in the culture fluid f.

Any convenient method can be used to harvest R20X2 from such cultures. One method is based on the principle of extraction, and specifically, for example, for R20M2 in culture F, it is extracted with a water-immiscible 120X2 solvent (see above) such as ethyl acetate, chloroform, etc. , butanol, etc. (the extraction efficiency is good if the culture P solution is neutral or slightly basic), or for R20X2 in the bacterial cells, the bacterial mass obtained by filtration, centrifugation, etc. is extracted with chloroform. , ethyl acetate, butanol, methanol, ethanol, acetone, hydrochloric acid solution, acetic acid solution, or the like. The culture itself can also be subjected to the above extraction operation without separating the bacterial cells. Countercurrent distribution methods using suitable solvents can also be included in the scope of extraction.

Another method for collecting R20X2 from cultures is based on the principle of adsorption, in which R20X2 is already in liquid form.
20X2-containing substances, such as culture broth or extract obtained by performing the extraction operation as described above,
Column chromatography using a suitable adsorbent such as activated carbon, alumina, silica gel, Diaion HP2 (IJ (manufactured by Mitsubishi Kasei Corporation), etc.)
Purpose R20X by liquid chromatography, etc.
By adsorbing and then eluting R20
We can obtain X2. R2 obtained in this way
By concentrating the 0X2 solution to dryness under reduced pressure, a crude sample of R2 (1X2) can be obtained.

In order to further purify the crude sample of R20X2 obtained in this way, 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, liquid chromatography using an appropriate solvent, and countercurrent distribution method may be carried out in appropriate combinations. I can do it. Specifically, for example, R20X2 crude sample is dissolved in a small amount of chloroform, developed with a suitable solvent using a silica gel column to elute the active ingredient, concentrated under reduced pressure, and further developed with TLO. , R20X2 is separated as a single substance when it is scraped and eluted.
After concentrating this, it is crystallized from a suitable solvent, and R20
X2 can be obtained as a crystal.

It is a compound that has anticancer and antibacterial activities and is useful as a medicine.

1) Physiological activity (1) Antitumor properties R20X2 showed significant antitumor activity against leukemia in experimental animals. For example, P3 for 0DFl mice
A suspension of 88 leukemia cells, 1 x 10'/mouse, was implanted intraperitoneally and treated with R20X on the 1st and 5th day after implantation.
2 was administered. Observations were conducted for 30 days, and the survival rate (correlation), where the number of survival days of mice in the control group administered with physiological saline was set as 100, was as follows.

0.25 133 0.125 158 0.0625 135 (2) Antibacterial properties R20M2 is a substance that exhibits antibacterial activity against various microorganisms, and its minimum growth inhibitory concentration (MIO) was determined by the agar plate dilution method.

(b) When the test bacteria are bacteria, Mueyu3er-H1nton agar medium/11H7,
3737°C/20 hours) If the test bacteria is yeast, 5 abouraucl dextrose agar medium/]
IH5,6/25℃/48 hours (c) If the test bacteria is mold, 5abouraua dextrose agar medium/pH
5, 6/25℃ for 15 days MIC is determined by preparing a cell suspension or spore suspension of the test bacteria at 10 cells/ml and inoculating it in a microplanter.
Judgment was made based on the presence or absence of growth after culture.

Minimum Inhibitory Concentration (MIO) of R20X2 As mentioned above, R20X2 of the present invention has an antibacterial effect, especially against Gram-positive bacteria, so it can be used as an effective antibiotic against infections caused by these bacteria. Can be used as a substance.

(3) Acute toxicity (LD5o) LD5o after intraperitoneal injection of R20X2 in mice was 0.7.
It was 5 mg/kg.

2) Antitumor agent Thus, the anthracycline compound R20 of the present invention
It has been revealed that X2 exhibits antitumor properties against tumors in animals including humans, especially malignant tumors.

Therefore, R20X2 of the present invention can be used as an antitumor agent or a tumor therapeutic agent.

R20X2 as an anti-tumor agent can be administered by any convenient route of administration and in a dosage form determined by the route of administration adopted. The drug is usually in a diluted form with a pharmaceutically acceptable partner or diluent.

When R20X2 as an antitumor agent is actually administered, one typical method is to dissolve it in distilled water for injection or physiological saline and inject it. Specifically, in the case of animals, injection methods such as intraperitoneal injection, subcutaneous injection, intravascular injection into a vein or artery, and local administration are used, and in the case of humans, injection methods include intravascular injection into a vein or artery or local administration. There is a method of injection.

The dosage of R20M2 is determined in consideration of the results of animal tests and various circumstances so that the total dosage does not exceed a certain amount when administered continuously or intermittently. The specific dosage depends on the administration method, the situation of the patient or treated animal,
For example, age, weight, gender, sensitivity, diet, administration time,
Needless to say, this will vary depending on the concomitant drugs, the patient, and the severity of the disease, and the appropriate dosage and frequency of administration under certain conditions will be determined by a specialist's appropriate dosage determination test based on the above guidelines. (1° Experiment In the following examples, "%" is [w/v % j.

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 qb Molasses 1 meat extract 1 Dispense 100 ml of the above medium into a 500 ml Erlenmeyer flask, sterilize it, and add Actinomadura roseoviolaceae R20.
One platinum loopful was inoculated from a slant and cultured at 27°C for 5 days in a rotary shaker (200 rpm), which was used as a seed mother.

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

Glucose 2.5q6 Soybean flour 1.5 Dried yeast 0.2 Calcium carbonate (sedimentation) 0.4% Put 25 liters of the above medium into a 50 liter jar fermenter and sterilize it, add the above seed mother 3 The duty was inoculated. Air flow rate IV, V, m, rotation speed 200/min, 27℃
Culture was performed for 7 days.

(3) After collecting and culturing R20!2, the culture solution was thoroughly filtered to separate the bacterial cells and P solution. r
Adjust the solution to 1) H2 with IN hydrochloric acid, and
It was adsorbed onto a column 1010×40 of 2oj (manufactured by Mitsubishi Kasei Corporation). After washing with distilled water and 50% methanol,
Eluted with methanol. Concentrate the eluate and adjust the concentrate to pH
Adjust to s, s, chloroform-methanol (971)
Extraction was repeated three times with the mixed solution.

After concentrating this extract, 6 times the amount of hexane was added and the resulting precipitate was dried to obtain 250 mg of red powder (
120X2 rough sample).

Example 2 250 mg of the R20X2 crude sample obtained in Example 1 was dissolved in chloroform, and 250 g of Silikage/I was placed on a column 4 x 40 Cm equilibrated with chloroform. After washing the column thoroughly with chloroform, X
Elution was performed with methanol-10=1. After drying both of the obtained components under reduced pressure, they were developed using TLO (Merck "Silica Gel 60") using a solvent system of chloroform, hot methanol: aqueous ammonia - 812: 0.05, and the Rf value was 0.44.
The nearby orange fraction was scraped off. After eluting and concentrating both fractions obtained in this way, they were recrystallized in chloroform and
Obtained 20×210 mg.

[Brief explanation of the drawing]

FIG. 1 is a reproduction of the ultraviolet/visible absorption spectrum of R20X2. FIG. 2 is a reproduction of the infrared absorption spectrum of R20!2. FIG. 3 is a photograph of R20X2 (7)'H-NMR spectrum II. FIG. 4 is a reproduction of the FD mass spectrum of R20X2. 1--- In NaOH, 2...0.1N HOl-
in NaOH, 3...0, IN NaOH-in NaOH.

Claims (1)

[Claims] 1. Anthracycline compound R_2_ represented by the following formula
0X_2 or its acid addition salt ▲ Numerical formulas, chemical formulas, tables, etc. are available ▼ 2. Anthracycline compound R_2_ shown by the following formula
An antitumor agent containing 0X_2 or its acid addition salt as an active ingredient. ▲Contains mathematical formulas, chemical formulas, tables, etc.▼