JPH0539242A - New anthracycline compound - Google Patents

Abstract

PURPOSE:To obtain a new anthracycline compound having low toxicity and excellent antitumor activity, useful as an antitumor agent as well as an intermediate for anthracycline based antibiotics. CONSTITUTION:A compound expressed by formula I (R is H or daunosaminyl) and salt thereof, e.g. a compound expressed by formula II. The compound can be produced (a) by culturing a microorganism of the genus Streptomyces having abilities capable of simultaneously carrying out 4-O-methylation and hydroxylation of ethyl group at 9 position to 1-hydroxyethyl group of an anthracycline based antibiotic, in a medium containing a compound expressed by formula III or (b) by culturing a microorganism of the genus Streptomyces having abilities capable of simultaneously carrying out 4-O-methylation hydroxylation of ethyl group at 9 position to 1-hydroxyethyl group and daunosaminylation at 1 position of the anthracycline based antibiotic, in a medium containing a compound expressed by formula IV, then collecting the compound expressed by the formula II from the cultured mixture of (a) or (b), and as necessary, hydrolyzing the collected compound.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel anthracycline compound having an antitumor effect. More specifically, the following formula

[0002]

[Chemical 10]

In the formula, R represents a hydrogen atom or a daunosaminyl group, and a compound represented by the formula:

[0004]

BACKGROUND OF THE INVENTION Conventionally, as anthracycline compounds having antitumor activity, daunomycin (see US Pat. No. 3,616,242) and adriamycin (US Pat. No. 3,590,028) have been known. Although these compounds are widely used clinically, they are not satisfactory as antitumor agents because of serious side effects such as cardiotoxicity and myelosuppression.

[0005] Therefore, there is a strong demand for the development of a drug having less such side effects and an excellent antitumor action, and conventionally, anthra produced by various means such as an acid fermentation method, a microbial conversion method, a chemical synthesis method and the like. Several cyclin compounds have been proposed. For example, aclacinomycins A and B (see Japanese Patent Publication No. 51-34914), rhodomycin group antibiotics (see Japanese Patent Publication No. 56-15299), 4-O.
-Tetrahydropyranyl adriamycin (Japanese Patent Publication Sho 57
For example, refer to Japanese Patent Laid-Open No. 13558). Furthermore, for the derivatives of daunomycin and adriamycin, Topics in Antibiotic C
chemistry, Vol. 12, 102-279
Page (published by Ellis Horwood Limited) and The Chemistry of Anti
tumor Antibiotics, Vol.
1, 63-132 (Wiley luterscien
c)).

As the anthracycline antibiotics as antitumor agents, various related compounds have been proposed as described above,
Some of them are already in clinical use and some are in clinical trials. However, they are not sufficiently satisfactory in terms of toxicity and antitumor action.

In addition, the results of in vitro tests and animal experiments of antitumor agents often do not always directly correlate with the antitumor effect on human cancers, and thus multifaceted research is required. Therefore, it is a fact that even for anthracycline antibiotics, which are tentatively evaluated as antitumor agents, the development of new drugs that are more effective as clinical drugs is strongly desired.

[0008] On the other hand, it is generally said that the toxicity of anthracycline antibiotics is reduced by methylating the hydroxyl group at the 4-position.
-Methylation is already known (JP-A-3-
17093). However, in β-rhodomycinone glycoside or β-rhodomycinone, 4-
It has not hitherto been known to convert the ethyl group at the 9-position into a 1-hydroxyethyl group simultaneously with O-methylation.

The inventors of the present invention have noticed that anthracycline antibiotics generally have a phenomenon that toxicity decreases when the hydroxyl group at the 4-position is methylated, and 4-O-methylation of various anthracycline compounds is taken into consideration. To the extent that the present inventors have conducted an intensive research on the above, some microorganisms belonging to the genus Streptomyces showed that 4-O-methylation of β-rhodomycinone or β-rhodomycinone glycoside was accompanied by 1-hydroxylation of the ethyl group at the 9-position. It was found that the compound has the ability to be converted into an ethyl group, and by using the microorganism, it succeeded in producing a compound of formula (I) having low toxicity and excellent antitumor effect, and completed the present invention. I arrived.

[0010]

DISCLOSURE OF THE INVENTION The compounds of formula (I) provided by the present invention include the following two compounds.

[0011]

[Chemical 11]

(Hereinafter, this compound is referred to as MC-7)

[0013]

[Chemical formula 12]

Among the compounds of formula (I) of the present invention, MC-
Since 7 contains a basic amino sugar, it can exist in the form of a pharmacologically acceptable acid addition salt, and examples of such an acid addition salt include sulfuric acid, hydrochloric acid, nitric acid and the like. Salts with inorganic acids; acetic acid, citric acid, succinic acid, tartaric acid, fumaric acid, maleic acid, propionic acid, malic acid, oxalic acid,
Examples thereof include salts with organic acids such as stearic acid, linoleic acid and lauryl sulfonic acid; salts with amino acids such as glutamic acid and aspartic acid; pantothenate and ascorbate.

The compound of the formula (I) of the present invention has excellent antitumor activity per se and is expected to be useful as an antitumor agent, and also has a sugar chain bond, a low molecular weight or a high molecular weight. It is also useful as an intermediate for synthesizing a highly active derivative by chemical modification such as binding with an active substance.

Among the compounds of the above formula (I) of the present invention, MC
-7 is, for example, the following formula

[0017]

[Chemical 13]

Using the compound represented by the formula as a substrate, a microorganism having the ability to hydroxylate the ethyl group at the 9-position to a 1-hydroxyethyl group at the same time as 4-O-methylation (hereinafter referred to as 4M9H-forming ability) is cultured. It can be manufactured.

The above formula (II) used as a starting material
, Ie, oxanomycin, is, for example, Streptomyces sp. Which has been proposed as an oxanomycin-producing bacterium.
D788, RPM-5 (FERM P-7703, FE
It can be prepared by culturing RM BP-811) (see JP-A-61-33194).

The microorganism capable of converting 4M9H is, for example, a microbial strain having the ability to produce daunomycin, carminomycin or paumycin belonging to the genus Streptomyces and its analogs (this is a strain newly isolated from soil. Or it may be a known strain) as a mutagen such as ultraviolet light or N-methyl-N-nitro-N-nitrosoguanidine (NTG).
4 of the compound of formula (II) above
It can be obtained by isolating a mutant strain capable of converting to M9H.

Specific examples of such microbial mutant strains having the ability to convert 4M9H include strept for producing the anthracycline antibiotics MC-1 and MC-4 described in JP-A-3-17093. Mrs. Streptomyces sp. D788, DKN
-1 strain (FERM P-10643) and the like.

Below, the above-mentioned Streptomyces sp. D
788 shows the mycological properties of the DKN-1 strain.

(I) Morphology Linear aerial hyphae are elongated from the branched basal hyphae, and no limbus branches are observed. The mature spore chain has a chain of 10 or more spores, and the size of the spore is 0.6 to 0.8 ×.
The surface of spores is smooth at about 0.9 to 2.5 microns.
Ascospores and terminal spores are not found.

(Ii) Growth conditions in various media are shown in Table 1 below. Regarding the description of color, the standard shown in parentheses is H. D. Tresner & E. J. Backku
s, System of Color Wheels
for Streptomyces Taxonom
y (J. Appl. Microbiol., 11
Vol., Pages 335-338, 1963) and supplementarily using "Color Standard" published by Japan Color Research Institute.

[0025]

[Table 1]

(Iii) Physiological properties (1) Growth temperature range (using yeast and malt agar medium,
20 ℃, 28 ℃, 33 ℃, 37 ℃, 42 ℃ at pH 6.0
Experiment at each temperature): Growth was observed up to 20 ° C to 37 ° C.

(2) Liquefaction of gelatin (using glucose, peptone and gelatin medium and culturing at 20 ° C.): Positive (3) Hydrolysis of starch (starch, inorganic salt agar medium): Positive (4) Degreased milk Coagulation and peptone formation: Slight coagulation and peptone formation are positive. (5) Formation of melanin-like pigment (tryptone, yeast extract, iron agar medium): Positive (iv) Utilization of various carbon sources (on prideham and madrieve agar medium) *: L-arabinose + D-xylose + D-glucose + D-fructose ± sucrose ± inositol + L-rhamnose + raffinose ± D-mannitol ± * + grows well ± slightly grows Formula (Ia) of the present invention The compound, namely MC-7, is
Using the above-mentioned microorganism capable of converting 4M9H, for example,
It can be directly prepared from the compound of the formula (II) as follows.

First, a strain having the ability to convert 4M9H, which has been cultured in YS agar slant medium and stored at about 6 to 7 ° C.,
For example, DKN-1 strain, for example, starch, glycerin, glucose, maltose as a carbon source; soybean flour, meat extract, yeast extract, peptone, corn steep liquor, cottonseed meal, nitrogen-containing organic matter such as fish meal as a nitrogen source and / or Or, inoculate into a medium consisting of inorganic nitrogen such as ammonium sulfate, ammonium chloride, sodium nitrate or ammonium phosphate;
℃, preferably about 28 ℃ 1-5 days, preferably about 3
Shaking or stirring culture is carried out for a day, and a methanol solution of the compound of the formula (II) is used as a substrate at a final concentration of 10
It is added at 500 μg / ml, preferably about 50 μg / ml, and shake culture is further continued for 1 to 3 days, preferably about 1 day to complete microbial conversion. In order to suppress foaming during acid fermentation, Adecanol (made by Asahi Denka Kogyo Co., Ltd.) and silicone (made by Shin-Etsu Chemical Co., Ltd.) are used as antifoaming agents.
Etc. can be added as appropriate.

To collect MC-7 from this culture,
For example, the culture solution is separated into bacterial cells and a filtrate, and a crude pigment containing the compound is extracted and purified from the bacterial cells and the filtrate. The extraction can be performed using, for example, acetone, methanol, chloroform, ethyl acetate, toluene, dilute mineral acid, acidic buffer solution, or the like. For purification, a column using silica gel, cross-linked dextran gel [for example, Sephatex LH-20 (manufactured by Pharmacia)], weakly acidic ion exchange resin, other synthetic adsorption resin [for example, HP-20 (manufactured by Mitsubishi Kasei)], etc. And thin-layer chromatography, liquid chromatography using an appropriate solvent, and countercurrent distribution method can be advantageously combined by appropriate combination.

Among the compounds of the above formula (I) of the present invention, MC-7 is, for example, the following formula:

[0031]

[Chemical 14]

Using β-rhodomycinone as a substrate, a microorganism having the ability to simultaneously perform 4-O-methylation, hydroxylation of 9-position ethyl group to 1-hydroxyethyl group, and 1-position daunosaminylation is cultured. It can also be manufactured by

Specific examples of the microorganism having such an ability include the above-mentioned microorganism Streptomyces sp.
eptomyces sp. ) D788, DKN-1 strain (FERM P-10643). The amount of substrate added, the method of addition, the method of culture, and the method of purifying the product can be the same as described above.

Β used as a starting material in the above method
-Rhodomicinone is a compound known per se and can be produced, for example, according to the method described in the following references.

(1) Journal of Anti
biotics, 39 (7), 902-909, 198.
6 (Oxaunomycin) (2) Journal of Antibiotics
s, 36 (8), 1080-1083, 1983 (Di
trisarubicins A, Band C) (3) Journal of Antibiotics
s, 40 (1), 116-121, 1987 (A447).
C and D) (4) Liebigs Ann. Chem. , (1
0), 949-953, 1988 (5) Chem. Ber. , 113 (9), 2976
~ 2993, 1980 (6) Chem. Ber. , 96, 1356-137
2,1963 Thus obtained compound of formula (Ia) (MC
-7) is, if necessary, by a method known per se,
The acid addition salt can be converted into the acid addition salt by a method of reacting with a non-toxic acid as described above in a suitable solvent and freeze-drying or by precipitation using a solvent in which the acid addition salt is only slightly soluble.

On the other hand, the compound of the formula (Ib) of the present invention is prepared by subjecting the compound (MC-7) of the formula (Ia) produced as described above to ordinary hydrolysis. It can be manufactured. For example, the compound of formula (Ia) is dissolved in dilute hydrochloric acid, heated and hydrolyzed, and then the resulting compound of formula (Ib) is dissolved in an organic solvent such as chloroform, ethyl acetate, toluene, or a chloroform-methanol mixture. It can be produced by extracting with. The above-mentioned hydrolysis can generally be carried out at a temperature of about 80 to 100 ° C, preferably about 80 ° C for about 0.5 to 2 hours.

The compound of formula (Ib) thus obtained is useful as a synthetic intermediate for anthracycline antibiotics or a precursor for microbiological conversion, as described above.

For example, "Topics in Anti"
biotic Chemistry ", ed.by
P. G. Sammes, Vol. 2, Ellis
Horwood, Chichester, Engl
And, 99-239 (1978), glycosylation can be performed to induce various anthracycline lycosides, and such glycoside derivatives are
Due to the structural characteristics of the naphthacenedione skeleton with saturated rings, it is highly probable that it will be an excellent anticancer agent.

The compound of the formula (Ia) of the present invention has an excellent antitumor activity as will be apparent from the tests described below, and is expected to be useful as an antitumor agent.

Against mouse leukemia L1210 cultured cells
Proliferation and Nucleic Acid Synthesis Inhibitory Action L1210 cells were inoculated into RPMI1640 medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing 20% calf serum at 5 × 10 ⁴ cells / ml,
The test compound of the present invention was added to this at a final concentration of 0.005-1.
0.0 μg / ml was added, and the mixture was incubated at 37 ° C. in a carbon dioxide incubator (3.5% carbon dioxide gas-containing air) for 2 days, and the 50% growth inhibitory concentration for the control group without addition was determined. It was Furthermore, the above L1210 cultured cells were suspended in RPMI1610 medium containing 10% calf serum at 5 × 10 ⁵ cells / ml, and cultured at 37 ° C. for 1 hour in a carbon dioxide incubator. 0.1 to 100 μ of the compound of the present invention
g / ml, and 15 minutes later, ¹⁴ C-uridine (0.05 μC / ml) or ¹⁴ C-thymidine (0.05 μg / ml) was added, and the mixture was immediately added at 37 ° C. for 6 minutes.
Incubated for 0 minutes.

Then, cold 10% trichloroacetic acid (TCA) was added to the culture medium to stop the reaction, and at the same time, the cultured cells were precipitated as an acid insoluble substance. The precipitate was placed on two lines with cold 5% TCA, dissolved in formic acid, and the radioactivity was measured. Thus, the concentration that causes 5% uptake inhibition was determined from the radioactivity uptake rate relative to the control group without addition. The results are shown in Table 2.

[0042]

[Table 2] Next, the present invention will be described more specifically by way of examples.

[0043]

Example 1 Streptomyces sp. (Strep
tomyces sp . ) D788, DKN-1 (Microtechnology Research Institute No. 10643) YS (0.3% yeast extract,
1% soluble starch, 1.5% agar, pH 7.2) One platinum loop was taken from the slant culture, and 100 ml of the seed mother medium described below was dispensed and inoculated into a 500 ml Erlenmeyer flask.
Seed mother was prepared by shaking culture for 2 days on a rotary shaker (220 rpm) at 0 ° C.

Seed medium Soluble starch 0.5% Glucose 0.5% Soybean flour (Essan Meat, manufactured by Ajinomoto Co., Inc.) 1.0% Yeast extract 0.1% Salt 0.1% Potassium diphosphate 0.1 % Magnesium sulfate 0.1% Tap water pH 7.4 (before sterilization) Next, 50 ml of a production medium having the following composition was dispensed and sterilized 5
1 ml of the above seed culture solution was added and inoculated into a 00 ml Erlenmeyer flask.

Production medium Soluble starch 5.0% Maltose 3.0% Dry yeast 2.0% Cotton seed meal 2.0% Fish meal 1.0% Yeast extract 0.2% Salt 0.1% Magnesium sulfate (7 water content) Material) 0.1% calcium carbonate 0.2% mineral solution * 0.05% tap water pH 7.0 (before sterilization) * CuSO ₄ .5H ₂ O 2.8 g, FeSO ₄ .7H ₂ O
0.4 g, MnCl ₂ .4H ₂ O 3.2 g, ZnSO
The ₄ · 2H ₂ O 0.8g were dissolved in distilled water 500 ml.

170 flasks were prepared and kept at 28 ° C. for 3 hours.
After culturing for a day (220 rpm), oxanomycin (5 mg / ml) as a substrate was added thereto in an amount of 0.5 ml per flask (base mass 2.5 mg), and the culturing was continued for 1 day.

The culture solution was recovered and the cells were separated by centrifugation. Acetone (5 l) was added to this, and the mixture was extracted with stirring for 30 minutes and then filtered to obtain an acetone extract. After concentrating to about 1.5 l, the pH was adjusted to 8.0 with 4N sodium hydroxide and extracted with 3 l of chloroform. The extract was concentrated to about 100 ml, and excess n-hexane was added for precipitation. The precipitate was dried under reduced pressure to obtain 120 mg of crude powder.

The total amount of the obtained coarse powder was changed to silica gel (Wak
ogel C-200, 25 g) column (Φ16.5).
mm), and chloroform-methanol (10:
1) and chloroform-methanol-water (100: 1
Elution was performed sequentially with a mixed solution of 0: 0.2 to 100: 30: 0.5). MC-eluting at 100: 30: 0.5
Fractions containing 7 were collected and concentrated to dryness. Then, the dry solid is dissolved in a small amount of a chloroform-methanol mixed solution (10: 1), and adsorbed in a band shape at a position of about 20 mm from the lower end of 10 preparative thin-layer silica gel plates (PF ₂₅₄ , manufactured by Merck). , Chloroform-methanol-water-acetic acid-concentrated aqueous ammonia solution (110: 50: 5: 1: 1). The MC-7 band was scraped off and eluted with a chloroform-methanol mixture (5: 1), and water and toluene were mixed with 1
0 ml each was added and mixed. Separate the aqueous layer and adjust the pH to 8.0
After adjusting to, the mixture was extracted with 10 ml of chloroform. The extract was washed with 5 ml of saturated saline, dried over sodium sulfate, and then concentrated. Excess isopropyl ether was added to the concentrated solution to precipitate it, and then it was dried to obtain 11 mg of MC-7.

Physicochemical properties of MC-7 substance (1) Properties Reddish brown powder (2) Melting point 187 to 189 ° C. (decomposition) (4) FDMS m / z 545 (M ⁺ ) (5) UV and visible absorption spectra 204 (334), 234 (703), 252 (369),
289 (141), 481 (210), 497 (205), 531sh (106) (6) Infrared absorption spectrum 3400,
2930, 1610, 1580, 1440,
1400, 1280, 1240, 1210, 1
120, 1065, 1000, 980, 88
0, 820 (7) ¹ H-NMR CDCl ₃ -CD ₃ OD (10: 1), δ (ppm): 1.
33 (d, 3H), 1.38 (d, 3H), 1.68 (dd, 1H), 1.78 (dt, 1H), 2.
21 (dd, 1H), 2.54 (d, 1H), 2.96 (br d, 1H), 3.48 (br s, 1
H), 4.03 (q, 1H), 4.08 (s, 3H), 4.10 (q, 1H), 4.96 (s, 1
H), 5.23 (d, 1H), 7.41 (d, 1H), 7.78 (t, 1H), 7.99 (d, 1H)

[0050]

Example 2 8 mg of MC-7 powder obtained in Example 1
Was dissolved in 0.1N hydrochloric acid and hydrolyzed at 85 ° C. for 1 hour, and then the aglycone was converted into chloroform-methanol (1
It was extracted with 0: 1). The extract was washed with water, concentrated, and added with excess n-hexane to cause precipitation, which was dried to give MC-7.
To obtain 3 mg of aglycone.

Physicochemical properties of MC-7 aglycone substance
Jo (1) Properties reddish brown powder (2) Melting point two hundred forty-five to two hundred and forty-eight ° C. (decomposition) (4) FDMS m / z 439 (M + Na) (5) UV and visible absorption spectra 233 (1304), 251 (706), 287 (261),
480 (386), 496 (385) (6) Infrared absorption spectrum 3370,
2905, 1610, 1580, 1440,
1375, 1285, 1240, 1210, 112
0, 1065, 1040, 990, 875,
820 (7) ¹ H-NMR CDCl ₃ -CD ₃ OD (5: 1), δ (ppm): 1.3
8 (d, 3H), 2.18 (dd, 1H), 2.45 (d, 1H), 4.03 (q, 1H), 4.09
(s, 3H), 4.96 (s, 1H), 5.30 (d, 1H), 7.44 (d, 1H), 7.81 (t,
1H), 8.02 (d, 1H)

[0052]

[Example 3] Streptomyces sp. (Strep
tomyces sp . ) D788, DKN-1 (Microtechnology Research Institute No. 10643) YS (0.3% yeast extract,
1% soluble starch, 1.5% agar, pH 7.2) Take 1 platinum loop from the slant culture, and 100 ml of the following seed mother medium
Inoculate into a 500 ml Erlenmeyer flask sterilized by dispensing.
2 at 8 ℃, rotary shaker (220rpm)
Seed mother was prepared by shaking culture for one day.

Seed medium Soluble starch 0.5% Glucose 0.5% Soybean flour (Essan meat, Ajinomoto Co.) 1.0% Yeast extract 0.1% Salt 0.1% Potassium diphosphate 0.1 % Magnesium sulfate 0.1% Tap water pH 7.4 (before sterilization) Next, 50 ml of a production medium having the following composition was dispensed and sterilized 5
1 ml of the above seed culture solution was added and inoculated into a 00 ml Erlenmeyer flask.

Production medium Soluble starch 5.0% Maltose 3.0% Dry yeast 2.0% Cotton seed meal 2.0% Fish meal 1.0% Yeast extract 0.2% Salt 0.1% Magnesium sulfate (7 water content) Material) 0.1% calcium carbonate 0.2% mineral solution * 0.05% tap water pH 7.0 (before sterilization) * CuSO ₄ .5H ₂ O 2.8 g, FeSO ₄ .7H ₂ O
0.4 g, MnCl ₂ .4H ₂ O 3.2 g, ZnSO
The ₄ · 2H ₂ O 0.8g were dissolved in distilled water 500 ml.

Prepare 150 flasks, and set them at 28 ° C. for 3 hours.
After culturing for a day (220 rpm), β-rhodomycinone (5 mg / ml) as a substrate was added thereto in an amount of 0.5 ml per flask (base mass 2.5 mg), and culturing was continued for 1 day.

The culture solution was collected and the cells were collected by centrifugation. Acetone (5 l) was added to this, and the mixture was extracted with stirring for 30 minutes and then filtered to obtain an acetone extract. The mixture was concentrated to about 1.5 l, washed with 1 l of chloroform, adjusted to pH 8.0 with 4N sodium hydroxide and extracted with 3 l of chloroform. The extract was concentrated to about 100 ml, and excess n-hexane was added for precipitation. The precipitate was dried under reduced pressure to obtain 80 mg of crude powder.

The total amount of the obtained coarse powder was changed to silica gel (Wak
ogel C-200, 25 g) column (Φ16.5).
mm), and chloroform-methanol (10:
1) and chloroform-methanol-water (100: 1
Elution was performed sequentially with a mixed solution of 0: 0.2 to 100: 30: 0.5). Fractions containing MC-7 eluted at 100: 30: 0.5 were collected and concentrated to dryness. Then, the dried solid was dissolved in a small amount of a chloroform-methanol mixed solution (10: 1), and adsorbed in a strip shape at a position of about 20 mm from the lower end of eight preparative thin-layer silica gel plates (PF ₂₅₄ , manufactured by Merck). , Chloroform-methanol-water-acetic acid-concentrated aqueous ammonia solution (110: 50: 5: 1: 1). The MC-7 band was scraped off and eluted with a chloroform-methanol mixture (0: 1) to remove water and toluene to 10 m.
l were added and mixed. The aqueous layer was separated and the pH was adjusted to 8.0, followed by extraction with 10 ml of chloroform. The extract was washed with 5 ml of saturated saline, dehydrated with sodium sulfate and then concentrated. Excess isopropyl ether was added to the concentrated solution to cause precipitation, which was then dried to obtain 7 mg of MC-7.

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] November 13, 1991

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0005

[Correction method] Change

[Correction content]

[0005] Therefore, there is a strong demand for the development of a drug having less such side effects and an excellent antitumor action, and anthra produced by various means such as an acid fermentation method, a microbial conversion method, and a chemical synthesis method has been conventionally used. Several cyclin compounds have been proposed. For example, acracinomycin A and B
(See JP-B-51-34914), Rhodomycin group antibiotics (see JP-A-56-15299), 4
An example thereof is -O-tetrahydropyranyl adriamycin (see Japanese Patent Publication No. 57-13558). Moreover, for derivatives of daunomycin and adriamycin, Topics in Antibiotics
Chemistry, Vol. 12, 102-27
Page 9 (Ellis Horwood Limited
Issued) and The Chemistry of Ant
itumor Antibiotics, Vol.
1, pp. 63-132 ( Wiley Interscie
has been introduced to nce issue).

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0014

[Correction method] Change

[Correction content]

Among the compounds of formula (I) of the present invention, MC
Since -7 contains a basic amino sugar, it can exist in the form of a pharmacologically acceptable acid addition salt, and examples of such an acid addition salt include sulfuric acid, hydrochloric acid, nitric acid and the like. With inorganic acids; acetic acid, citric acid, succinic acid, tartaric acid, fumaric acid, maleic acid, propionic acid, malic acid, oxalic acid,
Examples thereof include salts with organic acids such as stearic acid, linoleic acid and lauryl sulfonic acid; salts with amino acids such as glutamic acid and aspartic acid; pantothenate and ascorbate .

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0020

[Correction method] Change

[Correction content]

Further, the microorganism having 4M9H conversion ability is
For example, daunomycin belonging to the genus Streptomyces,
A microbial strain having the ability to produce carminomycin or baumomycin and its related compounds (this may be a strain newly isolated from soil, or may be a known strain), for example, ultraviolet rays or N-
Methyl-N-nitro-N-nitrosoguanidine (NT
It can be obtained by subjecting it to a conventional mutagenesis treatment with G), and isolating a mutant strain that is non-anthracycline pigment-producing and has the ability to convert the compound of formula (II) to 4M9H.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0041

[Correction method] Change

[Correction content]

Next, cold 10% trichloroacetic acid (TCA) was added to the culture medium to stop the reaction, and at the same time, the cultured cells were precipitated as an acid insoluble matter. The precipitate was washed twice with cold 5% TCA, dissolved in formic acid, and the radioactivity was measured. Thus, the concentration that causes 5% uptake inhibition was determined from the radioactivity uptake rate relative to the control group without addition. The results are shown in Table 2.

[Procedure Amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0042

[Correction method] Change

[Correction content]

[0042]

[Table 2] Next, the present invention will be described more specifically by way of examples.

[Procedure Amendment 6]

[Document name to be amended] Statement

[Correction target item name] 0049

[Correction method] Change

[Correction content]

[0049]Physicochemical properties of MC-7 substance  (1) Properties Reddish brown powder (2) Melting point 187-189 ° C (decomposition)(4) FDMS m / z 545 (M⁺) (5) Ultraviolet and visible absorption spectra(6) Infrared absorption spectrum

[Procedure Amendment 7]

[Document name to be amended] Statement

[Correction target item name] 0051

[Correction method] Change

[Correction content]

[0051]Physicochemical properties of MC-7 aglycone substance
State  (1) Properties Reddish brown powder (2) Melting point 245-248 ° C (decomposition)(4) FDMS m / z 439 (M + Na) (5) UV and visible absorption spectra(6) Infrared absorption spectrum

Continuation of front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location C12R 1: 465) (72) Inventor Rokuro Okamoto 2-18 Hananoki, Fujisawa City, Kanagawa Prefecture

Claims (4)

[Claims] 1. The formula: In the formula, R represents a hydrogen atom or a daunosaminyl group,
And a salt thereof. 2. Anthracycline antibiotic 4-O
A microorganism belonging to the genus Streptomyces having the ability to hydroxylate the ethyl group at the 9-position to a 1-hydroxyethyl group at the same time as methylation is represented by the formula: Cultured in a medium containing a compound represented by The compound of formula (I) is taken, and if necessary, the compound of formula (Ia) is hydrolyzed to give a compound of formula The method for producing the compound of formula (I) according to claim 1, wherein the compound of formula (I) is used. 3. Anthracycline antibiotic 4-O
A microorganism belonging to the genus Streptomyces having the ability to simultaneously perform methylation, hydroxylation of the 9-position ethyl group to 1-hydroxyethyl group and 1-position daunosaminylation,
Formula The cells are cultured in a medium containing a compound represented by The compound of formula (I) is taken, and if necessary, the compound of formula (Ia) above is hydrolyzed to give a compound of formula The method for producing a compound of formula (I) according to claim 1, wherein the compound is changed to 4. The formula: A compound characterized by hydrolyzing a compound represented by: The manufacturing method of the compound shown by these.