JPS6038391A - Anthracyclin compound r20p1 and its use - Google Patents

Abstract

NEW MATERIAL:Anthracyclin compound R20P1 shown by the formula or its acid addition salt. Having the following physical and chemical properties. Appearance: reddish purple powder. Elemental analysis (wt%): C=64.79, H=5.69, N=2.87, and O=26.65(C26H27NO3). Molecular weight: 481.5. Melting point: 143- 148 deg.C. USE:Useful as an antitumor agent and an antibacterial agent (having strong activity especially against Gram-positive bacteria). Has low toxicity. PREPARATION:A bacterium [e.g., Actinomadura roseoviolacea R20 strain (FERM-P 7138) belnging to the genus Actinomadura, capable of producing R20P1, is cultivated in a nitritive medium usually at 27-30 deg.C for 6-7 days, and 20P1 shown by the formula I is collected from the culture.

Description

DETAILED DESCRIPTION OF THE INVENTION CI) 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, 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. You could say that.

[■] Summary of the invention The present invention meets the above needs.

That is, the anthracycline compound R2 according to the present invention
0P1 is expressed by the following formula. The present invention does not cover acid addition salts of this compound.

The antitumor agent according to the present invention contains an anthracycline compound R20P represented by the following formula or an acid addition salt thereof as an active ingredient.

[■] Detailed description of the invention 1) Chemical structure The anthracycline compound R20P1 according to the present invention has the following:
It has a chemical structure represented by the above formula (A).

This chemical structure was determined as follows.

That is, R20P1 is 0. Dissolved in IN hydrochloric acid, 100
Hydrolysis by standing at <RTIgt;°C</RTI> for 1 minute gave daunosamine as an aglycone moiety and a sugar moiety.

The following structure (B) of the aglycon moiety was estimated by analysis of nuclear magnetic resonance spectrum and mass spectrum.

In addition, R20P1's ultraviolet/visible absorption spectrum, infrared absorption spectrum, nuclear magnetic resonance spectrum, FAB (F
Ast Atom Bombardment) From the results of mass spectra, elemental analysis, etc., the structural formula of R20P1 was determined as the above formula (A).

2) Physicochemical properties Some of the physicochemical properties of anthracycline compound R20P1 are as follows.

(1) Appearance: Reddish-purple powder (2) Elemental analysis: 64.79 5.69 2.87 26.65
Calculated value 64.86 5.65 2.91 26.58
(C26H2□N08) (3) Molecular weight: 481.5 (4) Melting point: 143-148°C (5) Ultraviolet/visible absorption spectrum As shown in Figure 1.

MaOHλrrlax nm (E 255 (472), 268 (548), 490 (238
), 513 (262), 550 (162) 0.01 N Na OH + Me 0T (251 (45
7), 265 (495), 518 (190), 555 (
238), 597(171) (6) Infrared absorption spectrum (potassium bromide method) As shown in FIG.

(7) Proton nuclear magnetic resonance spectrum (400 MHz, in eastern chloroform) as shown in FIG.

(8) Carbon-13 nuclear magnetic resonance spectrum (io megahertz, in deuterated chloroform) as shown in FIG.

(9) F A B mass spectrum As shown in FIG.

(10) Soluble Soluble in acidic water, basic water, methanol, ethanol, n-propertool, n-butanol, acetone, ethyl acetate, chloroform Insoluble in water, hexane, cyclohexane, petroleum ether Note that R20P1 is a methanol solution Although it is red inside,
Turns purple in alkalinity.

(11) Rf value The Rf values in various solvents on the silica gel plate of R20P1 are as follows.

Development system Rf value (25℃) Chloroform:methanol:benzene 0.19=7
:3:3 Chloroform:methanol:acetic acid 0.35=8:2
: 0.05 Chloroform: Methanol: Aqueous ammonia 0.57=
8: 2: 0.05 (using Merck's "Silica Gel 60 F254-1 plate") Since this compound has a primary amino group in the sugar moiety, its acid addition salt is possible.The acid in this case is hydrogen chloride. Typical examples include acids and sulfuric acid.

Production of R20P1 1) Overview The anthracycline compound R20P1 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 chemical production. You could also do that.

In the case of culturing microorganisms, the strain used is one belonging to Actinoma jula that has the ability to produce R20P1. Specifically, the present inventors have revealed that Actinomadura roseoviolaceae R20, which the present inventors isolated, produces R20P1; It is possible by law to separate what is appropriate from nature. Also, A.

There is also room to increase R20P1 production ability by subjecting R20P1-producing bacteria, including Roseoviolaceae R20, to irradiation or other treatments.

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 R20P1, has the following content.

(1) Origin and deposit number Strain R20 was isolated from soil collected in a vegetable field in Onoya, Kaho-machi, Kaho-gun, Fukuoka Prefecture, on July 5, 1939.
It was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology in Japan, and has the number ``Feikoken Bibori No. 7138''.

(2) Mycological properties and physiological properties The characterization of this bacterial strain according to the method manual of the International Committee for Actinobacteria Nomenclature (IMF) 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 have a long main axis, short branches are branched at right angles to the main axis (uniaxial branching), and at the tip there is a dense small spiral spore chain consisting of around 10 or more spores. (1 to 3 turns, diameter 2.0 to 2
．． 5μ), pseudosporangia (diameter 2.5-3.5μ), and spore masses are formed.

The spore chain is covered with 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 round, width 0.5-0.8μ, length 0.7-1.1μ,
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 BIB.

B) Culture properties % The observation results of the culture properties (cultivated at 27°C) in the persimmon medium are as 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
) forms pseudosporangia and spore masses, and (4) no true sporangia or flagellated spores are observed, so Actinomadura (
It is judged that it belongs to the genus Actlnomadura).

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
.

roseoviolacea).

Therefore, we compared this strain with A, a standard strain of Roseopiolaceae (K).
CCA-145 (Nonomura A-5)] 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, the strain is taxonomically very well classified (IJ, L).
Actinomadura roseoviolaceae (Actjnom)
a mountain + ra roseovlolacea Nonom
ura et 0hara1971).

3) Culture/Production of R20PI Anthracycline R20P1 can be produced by culturing R20P1-producing bacteria belonging to the genus Actinomadula aerobically in an appropriate medium and collecting the desired product from the culture.

The medium can contain any nutrient source that can be utilized by the R20P1 producing bacteria. Specifically, for example, glucose, sucrose, ma/)-su, starch, and fats and oils can be used as carbon sources, and soybean flour, cottonseed meal, meat extract, pezotone, dried yeast, and nitrogen sources can be used. Organics such as yeast extract and corn steep liquor as well as inorganics 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 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
~30'C is preferred. In this method, the production amount of R20P1 reaches its maximum in 6 to 7 days for both the agitation culture and the aeration agitation culture.

In this way a culture enriched with R20P1 is obtained. In the culture, a part of R20P1 exists in the bacterial cells, but most of it exists in the culture slurry.

Any convenient method can be used to harvest R20P1 from such cultures. One of the methods is based on the principle of extraction, and specifically, for example, for R20P1 in the Baimanto solution, it is extracted with a water-immiscible R20Pl solvent (see above), such as ethyl acetate.
Method of extraction with chloroform, citanol, etc. (extraction efficiency is good if the culture P solution is neutral or slightly basic)
, or for R20P1 inside the bacterial cells, collect the bacterial mass obtained by p-filtration, centrifugation, etc., by treating it with chloroform, ethyl acetate, butanol, methanol, ethanol, acetone, (15) hydrochloric acid solution, or acetic acid solution, etc. I can do it. It is also possible to subject the culture as it is 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 R20P1 from cultures is based on the principle of adsorption, in which R20P1 is already in liquid form.
A suitable adsorbent such as activated carbon, alumina,
Silica gel, V Diamond Ion Hp20j (manufactured by Mitsubishi Kasei Corporation), [Sephadex LH20j (manufactured by Pharmacia Corporation)]
R20P1 can be obtained by adsorbing target R20P1 by column chromatography, liquid chromatography, etc., and then eluting it. The R20P1 solution thus obtained is concentrated to dryness under reduced pressure to obtain a crude sample of R20P1.

Cut out the crude sample of R20P1 obtained in this way16
) For further purification, the above extraction method and adsorption method may be combined as necessary and carried out as many times as necessary. for example,
Column chromatography using adsorbents or gel filtration agents such as silica gel, "Sephadex LH added", weakly acidic ion exchange resins, and activated carbon, liquid chromatography using an appropriate solvent, and countercurrent distribution method are combined as appropriate. They can be implemented together. Specifically, for example, R20
P1 crude sample was dissolved in a small amount of chloroform, developed with a suitable solvent using a silica gel column to elute the active ingredient, and the eluate was concentrated under reduced pressure, and then added to Sephadex LH.
R20P1 is separated as a single substance when eluted with a 2 (moon column), so R20P1 can be obtained as a crystal by concentrating it and then crystallizing it from a suitable solvent.

The anthracycline compound R20P1 according to the present invention is
It is a useful compound as a medicine because it has anticancer and antibacterial activities and low toxicity.

■) Physiological activity (1) Antitumor properties R20P1 showed remarkable antitumor effects against leukemia in experimental animals. For example, P3 for CDFl mice
A suspension of 88 leukemia cells, 1×106 cells/mouse, was intraperitoneally transplanted, and R20P1 was administered on the 1st and 5th day after the transplantation. Survival prolongation rate (%) based on 100 days of survival of mice in the control group subjected to Sekiguchi observation and administered with physiological saline
The effects were as follows.

8.6 178 4.3 168 2.15 153 1.08 146 0.54 114 (2) Antibacterial R20P1 is a substance that exhibits antibacterial activity against various microorganisms, and its minimum inhibitory concentration (MIC) is Determined by plate dilution method.

(b) When the test bacteria are bacteria, Mueller-H1nton agar medium flu, / pH
7.3/37℃/21) Time (b) If the test bacteria is yeast, 5abooraud dextrose agar medium/pH
5,675℃/48 hours (c) If the test bacteria is mold, 8 hours dextrose agar medium/pH
5,615℃ for 15 days MIC is obtained by preparing a cell suspension or spore suspension of the test bacteria at 106 cells/ml and inoculating it in a microplanter.
Judgment was made based on the presence or absence of growth after culture.

(19) Minimum Inhibitory Concentration (MIC) of R20P1 Microorganism MI
C (μg AI) Staphylococcu aureus IFO
12732> 6.7 Bacillus 5ubtil
ls IFO31346,7M1crococeus
1uteus ATCC9341 (MS-1) 3.35
Pseudomonas aeruginosa II
IQ) 12582 > 6.7Sa1mone11a
typhln+++rlumIID971 (MS-1)
>6.7Egeherjchla colt IFO
12734>6.7Saccharor+yeasee
rev1glaeATCC9763>6.7Candi
da albicans No. Yu 1200 >
6.7penicI111+lTI chryaog
@num ATCJko 10002 > 6.7Tri
chophyton mantagrophytes
>6.7 As mentioned above, R20P1 of the present invention has an antibacterial effect, and in particular shows antibacterial activity against Durham-positive bacteria, so it can be used as an effective antibiotic against infections caused by these bacteria. I can do it.

(3) Acute toxicity (LD50) The LD50 of R20P1 by intraperitoneal injection in mice was (2
0) 100 (mgAg) or more.

2) Antitumor agent Thus, the anthracycline compound R20 of the present invention
It has been revealed that P1 exhibits antitumor properties against tumors in animals including humans, especially malignant tumors.

Therefore, R20P1 of the present invention can be used as an antitumor agent or a tumor therapeutic agent.

R20P1 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 carrier or diluent.

When R20P1 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, local administration, etc. There is a method of injection.

The dosage of R20P1 is determined in consideration of the results of animal studies and various circumstances (7) so that the total dosage does not exceed a certain amount when administered continuously or intermittently.Specific dosage the method of administration, the circumstances of the patient or treated animal, such as age, body weight, sex, sensitivity, diet, time of administration,
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° Experimental Example In the following examples, "chi" is [w, /v%-!

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 1 t Dispense 15 ml of the above medium into large test tubes, sterilize them, inoculate 1 platinum loop of Actinoma Jura Roseoviolaceae R20 from a slant, and shake at 37°C for 72 hours using a test tube shaker. (23Orpm') and use it 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 2.5% Soybean flour 1.5% Dry yeast 0.2% Calcium carbonate (sedimentation) 0.4%
Dispense 1 m of the above seed mother into sterilized Erlenmeyer flasks.
Add 1 liter of water and shake on a rotary shaker (230 rpm).
Rotary culture was carried out at 27'C for 7 days using the following.

(3) After collecting and culturing R20P 1, the culture solution is filtered to separate the bacterial cells and the fermentation liquid (23). The filtrate was adjusted to pn 2 with IN hydrochloric acid, and column 5X40 of "Diaion" fP-204 (manufactured by Mitsubishi Chemical Corporation) was added.
Adsorb to cm. Elute with distilled water and diluted alcohol and concentrate. The concentrated solution was adjusted to pH 8.5 and extracted three times with a chloroform-methanol/I/(9:1) mixture. After concentrating this extract, 6 times the amount of hexane was added and the resulting precipitate was dried, resulting in 100 m of red powder.
g (R20P1 crude sample).

Example 2 100 mg of the R20P1 crude sample obtained in Example 1 was dissolved in methanol and equilibrated with methanol.
After washing the column well with methanol,
Elute with 0.51 acetic acid-methanol. After the eluate is concentrated to dryness, it is dissolved in a chloroform-methanol (9:1) mixture and washed several times with distilled water. The organic solvent layer was dried over anhydrous sodium sulfate and concentrated to dryness to obtain 10 mg of R20P1 powder.

(24)

[Brief explanation of the drawing]

FIG. 1 is a reproduction of the ultraviolet/visible absorption spectrum of R20P1. ■...2-=0,01N Na in methanol (Mf-2nd in methanol
The figure is a reproduction of the infrared absorption spectrum of R20P1. Figure 3 is a reproduction of the H-NMR spectrum of R20P1. Figure 4 is a reproduction of the 13C-NMR spectrum of R20P1. Figure 5 is a reproduction of the F'AB mass spectrum of R20P1. This is a copy. Applicant's agent Kiyoshi Inomata

Claims (1)

[Claims] 1. Anthracycline compound R20P represented by the following formula
1 or its acid addition salt 2, anthracycline compound R20P represented by the following formula
1 or an acid addition salt thereof as an active ingredient.