JPH0149357B2 - - Google Patents

Description

[Detailed description of the invention]

BACKGROUND OF THE INVENTION The present invention relates to an antitumor anthracycline compound, 13-deoxocarminomycin (hereinafter referred to as 13-deoxocarminomycin).
R20X) and 3'-deamino-3'-(4-morpholinyl) of 13-deoxo-10-hydroxycarminomycin (hereinafter referred to as R20X2. Also, R20X and R20X2 are collectively referred to as the R20 substance)
Regarding derivatives. As anthracycline compounds, daunomycin (see US Pat. No. 3,616,242) and adriamycin (see US Pat. No. 3,590,028), which are obtained from the culture solution of actinomycetes, have been known. It is widely used clinically as a tumor agent. However, although these compounds exhibit strong antitumor effects, they also have strong side effects, so they are not necessarily satisfactory. Compounds related to the 3'-deamino-3'-(4-morpholinyl) derivatives of the present invention include Rivola,
G. et.al. used their leading compound, R20X, in Streptomyces peuceticus var.
carminatus (1524DSM, 31502ATCC, 4929FRI,
DR81FI Farmitalia mikrobiologische
Sammlung) and reported that it has antitumor activity (West German Published Patent No. 3012665). Furthermore, various derivatives of adriamycin, daunomycin, and carminomycin have been synthesized as morpholinyl derivatives of anthracycline compounds, and their antitumor activity has been reported (Japanese Patent Laid-Open Publication No. 163393/1983, U.S. Patent No. 4301277,
JP-A-59-212484 and JP-A No. 212499,
Mosher, CWet.al: J.Med.Chem. 25 18−24
(1982), Johnston, J.B.: Biochemistry.
Pharmacology 32 , (21), 3255−3258 (1983),
Acton, EM: J.Med.Chem. 27 638−645 (1984)
reference). However, to the best of the present inventors' knowledge, these compounds are not necessarily satisfactory in terms of antitumor activity or toxicity, and there is a constant desire for better anthracycline compounds. Good morning. SUMMARY OF THE INVENTION The present invention meets the above needs. That is, the 3'-deamino-3'-(4-morpholinyl) of the anthracycline compound according to the present invention
The derivative 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 is an anthracycline compound represented by the following formula, 3'-deamino-3'-(4
-morpholinyl) derivative or its acid addition salt as an active ingredient. (In the formula, R represents a hydroxyl group) Detailed Description of the Invention 3'-deamino- of anthracycline compound
3′-(4-morpholinyl) derivatives Compounds of formula () according to the invention are R20X2(13
-Deoxo-10-hydroxycarminomycin. That is, this corresponds to the case where R in formula () is -OH. ) is a 3'-deamino-3'-(4-morpholinyl) derivative of
It is called 3′-(4-morpholinyl)-R20X2. 3′-
One method for producing deamino-3'-(4-morpholinyl)-R20X2 consists of converting the 3'-amino group of R20X2 to a 4-morpholinyl group,
On the other hand, one of the production methods for R20X2 is a microbiological method, in which case R20X (13-deoxocarminomycin, i.e., corresponding to the case where R is -H in formula ()) is co-produced. . Therefore, in the following explanation, R20X2, R20X and
3'-deamino-3'-(4-morpholinyl)-
R20X is also mentioned for reference. 1) Chemical structure of the anthracycline compound according to the present invention
The 3'-deamino-3'-(4-morpholinyl) derivative has a chemical structure represented by the above formula (). 2) Physicochemical properties A 3'-deamino-3'-(4-morpholinyl)
-R20X (1) Appearance: Reddish brown powder (2) Elemental analysis: C H N O analysis value (%) 62.98 6.31 2.40 28.31 Calculated value (%) 63.26 6.19 2.46 28.09 (3) Molecular weight: 569.6 (4) Melting point: 143 -144°C (decomposition) (5) Specific rotation: [α] ²⁵ _D +76° (C = 0.05, methanol) (6) Ultraviolet/visible absorption spectrum As shown in Figure 1. (b) λmax nm in methanol (E1% 1cm) 234 (683), 252 (545), 292 (158), 464
(205), 492 (261), 508 (194), 524 (181),
575 (18) (b) In acidic methanol λmax nm (E
1% 1cm) 234 (783), 252 (612), 292 (192), 466
(233), 492 (315), 510 (227), 524 (202) (c) λmax in alkaline methanol
nm (E1% 1cm) 226 (422), 243 (653), 290 (166), 528
(126), 562 (192), 596 (162) (7) Infrared absorption spectrum (potassium bromide tablet)
As shown in FIG. (8) Proton nuclear magnetic resonance spectrum (100 MHz, in deuterated chloroform) as shown in Figure 3. (9) Rf value (Merck silica gel plate)
60F254 used)

[Table] (10) Solubility Soluble in acidic water, basic water, methanol, ethanol, propanol, acetone, ethyl acetate, and chloroform. Insoluble in water, hexane, cyclohexane, diethyl ether, and stone ether. B 3'-deamino-3'-(4-morpholinyl)
-R20X2 (1) Appearance: Brown powder (2) Elemental analysis: C H N O analysis value (%) 61.32 6.30 2.26 30.12 Calculated value (%) 61.53 6.02 2.39 30.06 (C ₃₀ H ₃₅ NO ₁₁ ) (3) Molecular weight: 585.6 (4) Melting point: 155-157℃ (decomposition) (5) Ultraviolet/visible absorption spectrum As shown in Figure 4. (b) λmax nm in methanol (E1% 1cm) 234 (821), 252 (478), 290 (153), 468
(241), 480 (263), 492 (295), 514 (216),
526 (196), 582 (17) (b) In acidic methanol λmax nm (E
1% 1cm) 234 (805), 252 (479), 290 (155), 468
(246), 480 (273), 492 (297), 512 (214),
526 (193) (c) λmax in alkaline methanol
nm (E1% 1cm) 242 (831), 292 (149), 534 (212), 564
(280), 600 (226) (6) Specific rotation [α] ²⁰ _D = +306° (C = 0.05, CHCl ₃ ) (7) Infrared absorption spectrum (potassium bromide tablet) As shown in Figure 5 It is. (8) Proton nuclear magnetic resonance spectrum (100 MHz, in deuterated chloroform) as shown in Figure 6. (9) Solubility Soluble in acidic water, basic water, methanol, ethanol, propanol, acetone, ethyl acetate, and chloroform. Insoluble in water, hexane, cyclohexane, diethyl ether, petroleum ether. (10) Rf value (Merck silica gel plate)
60F254 used)

[Table] Outline of production of 3'-deamino-3'-(4-morpholinyl) derivative The 3'-deamino-3'-(4-morpholinyl) derivative of the present invention is produced by culturing microorganisms.
It can be produced by synthetic chemical modification of R20 substances. R20 Substance R20 Substance can be obtained from a culture of Actinomadilla roseoviolaceae R20 strain isolated by the present inventors. Regarding R20X,
As mentioned above, it can also be produced by the method described in West German Published Patent Application No. 3012665. (1) Strain R20 Strain R20, which the present inventors have discovered as a strain of the genus Actinomadilla that has the ability to produce the anthracycline compound R20, has the following content. Origin and deposit number Strain R20 was isolated from soil collected from a vegetable field in Oaza Onodani, Kaho-machi, Kaho-gun, Fukuoka Prefecture, and was deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology on July 5, 1981. ``Microtechnical Research Institute
No. 945 (Fiber Engineering Research Institute No. 7138). Mycological 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) Morphological characteristics The basal hyphae spread radially on the surface of the agar medium while dividing, and no division of the hyphae was observed. Aerial hyphae extend their main axis long, branch short branches almost at right angles (to the main axis) (uniaxial branching), and at the tips of the branches are dense small helical spore chains consisting of around 10 or more spores ( 1
~3 turns, forming pseudosporangia (diameter 2.0-2.5μ), pseudosporangia (diameter 2.5-3.5μ), and spore masses. The spore chain is covered with a cylindrical sheath with a width of 0.5 to 0.8 μ, the surface of which is rough, and the individual spores are connected in a phalanx shape. The spore mass is amorphous, and the spore surface is covered with a sticky substance. Free spores are rarely observed and are cylindrical or oblong, 0.5-0.8μ wide and 0.7-0.7μ long.
1.1μ, exhibits a smooth surface. True sporangia, flagellated spores, sclerotia, etc. are not observed. Since the whole cell hydrolyzate contains meso-type diaminopimelic acid and majulose, the cell wall type is determined to be B. B) Culture properties Culture properties in polysaccharide medium (27°C culture)
The observation results are shown in Table 1. C) Physiological properties Physiological properties (including carbon source assimilability)
is as shown in Table 2. D) Discussion and Identification This strain (1) has cell wall type B, (2) spore chains consist of 10 or more spores, (3) forms pseudosporangia and spore masses, and (4) ) Since true sporangia and flagellated spores are not observed, it is judged that it belongs to the genus Actinomadura. Nonomura's search table [Rikou, Vol. 52, 71
-77 pages, 1974] [Shikou, Vol. 49,
904-912, 1971], this strain is judged to be most closely related to A. roseoviolacea. Therefore, we used the standard strain of this strain A. roseoviolaceae [KCCA-145 (Nonomura A-5)].
were cultured under the same conditions, and the main properties of both strains were compared. As shown in Table 3, the results are very similar taxonomically, although there are slight differences in bacterial flora color, underside color, and optimal growth temperature. Afterwards,
This strain is Actinomadura roseoviolacea.
Nonomura et Ohara 1971).

【table】

【table】

【table】

[Table] (2) Cultivation/Production of R20 substance Anthracycline R20 substance is produced by aerobically cultivating R20 substance-producing bacteria belonging to the genus Actinomadilla in an appropriate medium and collecting the target substance from the culture. Therefore, it can be manufactured. The medium can contain any nutrient source that can be used by the R20 substance-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 steep can be used as nitrogen sources. Organics such as liquors and 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 in a customary manner. The most suitable culture method is the aerobic liquid-liquid deep culture method, which is the same as the commonly used method for producing antibiotics. Culture temperature is 25℃~45℃
is suitable, but 27°C to 30°C is preferred. With this method, the production amount of R20 substance reaches its maximum in 6 to 7 days for both shaking culture and aerated agitation culture. In this way, a culture with accumulated R20 material is obtained. In the culture, some of the R20 substance exists in the bacterial cells, but most of it exists in the culture solution. Any convenient method can be used to harvest R20 material from such cultures.
One method is based on the principle of extraction, and specifically, for example,
For R20 substances, extract them with a water-immiscible solvent for R20 substances, such as ethyl acetate, chloroform, butanol (the extraction efficiency is good if the culture solution is neutral or slightly basic), or R20 substances inside the bacterial cells can be recovered by treating bacterial aggregates obtained by filtration, centrifugation, etc. with chloroform, ethyl acetate, butanol, methanol, ethanol, acetone, hydrochloric acid solution, or acetic acid solution.
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 category of extraction. Another method for collecting R20 substances from cultures is based on the principle of adsorption, which involves using R20 substance-containing substances that are already in liquid form, such as culture medium, or performing the extraction operation as described above. Column chromatography and liquid chromatography using suitable adsorbents such as activated carbon, alumina, silica gel, "Diaion HP20" (manufactured by Mitsubishi Kasei Corporation), etc. The R20 substance can be obtained by adsorbing the target R20 substance with E, etc. and then eluting it. By concentrating the R20 substance solution thus obtained to dryness under reduced pressure, a crude sample of the R20 substance can be obtained. In order to further separate and purify R20X and R20X2 from the crude sample of the R20 substance obtained in this way, the above-mentioned extraction method and adsorption method may be combined as necessary and used 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 are carried out in an appropriate combination. be able to.
Specifically, for example, a crude sample of R20 substance 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 TLC. When scraped and eluted, R20X and
Since each R20X2 is separated as a single substance,
By concentrating this to dryness, R20X and R20X2 can be obtained. The physicochemical properties of the R20 substance thus obtained are as follows.

【table】

[Table] (3) Synthetic chemical modification of R20 substance The 3'-deamino-3'-(4-morpholinyl) derivative of the anthracycline compound of the present invention is
A method of reacting an R20 substance or its acid addition salt with a compound represented by the following formula () (hereinafter referred to as method A), or a method of reacting an R20 substance or its acid addition salt with a compound represented by the formula (). ) can be produced by a method of reacting with ether in the coexistence of a dehydrohalogenating agent (hereinafter referred to as method B).

[Formula] (In the formula, X represents a bromine atom or an iodine atom.) In method A, this compound () is a meso-
Literature from erythritol (Carbohydrate
Research Vol 35, 195-202 (1974)). The reaction of the R20 substance or its acid addition salt with the compound of formula () is usually carried out in a solvent. Usable solvents include acetonitrile, methanol, ethanol, water, chloroform, dichloromethane, carbon tetrachloride, benzene, dioxane, tetrahydrofuran, etc. alone or in mixtures, and a mixed solvent of acetonitrile-water-chloroform is particularly preferred. This reaction is usually preferably carried out in the presence of a reducing agent such as sodium borohydride (NaBH ₄ ), sodium cyanoborohydride (NaBH ₃ CN), or the like. The amount of reducing agent used is not critical and can be used in an amount of at least 1 mol, preferably 1 to 5 mol, per mol of R20 material. Further, the amount of the compound of formula () to be used is advantageously 1.5 mol or more, preferably 5 mol or more, particularly preferably 8 to 15 mol, per 1 mol of the R20 substance. The reaction temperature is generally between the freezing point of the solvent used and 50°C.
Temperatures within the range of °C, especially around room temperature, are suitable. Under the above reaction conditions, the reaction of converting an amino group into a morpholinyl group can be completed in about 10 minutes to 2 hours. In method B, the conditions for reacting the R20 substance or its acid addition salt with the compound () in the coexistence of a dehydrohalogenating agent are essentially those described in JP-A-57-
It is the same as that described in Publication No. 163393. In addition,
What should be noted about this B method is that R20X2
When subjected to this reaction, 3′-deamino-3′-
(4-morpholinyl)R20X2 is obtained, whereas when R20X is subjected to this reaction, 3′-deamino-
This means that a 3'-(4-morpholinyl) derivative can be obtained at the same time (see Example 4 below). To isolate and purify the 3'-deamino-3'-(4-morpholinyl) derivative, which is the target compound of the present invention, from the reaction mixture obtained by the reaction of the R20 substance with the compound of formula () by the method of the present invention. This can be accomplished by known purification methods used in the field of anthracycline glycoside derivative production, such as chromatography using silica gel or the like. As shown by the formula () obtained in this way
The 3'-deamino-3'-(4-morpholinyl) derivative can be prepared as an acid addition salt thereof, for example, with an inorganic acid such as hydrochloric acid, sulfuric acid or phosphoric acid, or with acetic acid, propionic acid or maleic acid, according to a method known per se. ,
It can be converted into acid addition salts by treatment with organic acids such as oleic acid, palmitic acid, citric acid, succinic acid, tartaric acid, fumaric acid, glutamic acid, pantothenic acid, and lauryl sulfonic acid. 3′-deamino- of anthracycline compounds
Uses of 3′-(4-morpholinyl) derivatives 3′- of anthracycline compounds according to the present invention
Deamino-3'-(4-morpholinyl) derivatives are
It is a compound that has anticancer activity and is useful as a medicine. 1) Physiological activity (1) Antitumor properties a. Antitumor properties against leukemia The 3'-deamino-3'-(4-morpholinyl) derivatives of the present invention showed remarkable antitumor activity against leukemia in experimental animals. For example, a suspension of P388 leukemia cells (1×10 ⁶ cells/mouse) was intraperitoneally transplanted into a CDF ₁ mouse, and 3′-
Deamino-3'-(4-morpholinyl) derivatives were administered intraperitoneally. Observations were conducted for 30 days, and the survival rate (%), where the number of survival days of mice in the control group administered with physiological saline was taken as 100, was as shown in Table 5 below.
The same table also shows the therapeutic coefficient (dose with maximum survival rate/dose with 130% survival rate), which is an index showing the promise of clinical therapeutic effects.

【table】

[Table] *Administration on day 1 only Furthermore, a suspension of P388 leukemia cells (1×10 ⁶ cells/mouse) was intraperitoneally transplanted into CDF ₁ mice, and administration was performed on days 1 and 5 after transplantation. A 3'-deamino-3'-(4-morpholinyl) derivative was administered intravenously. Observations were conducted for 30 days, and the survival rate (%), where the number of survival days of mice in the control group administered with physiological saline was taken as 100, was as shown in Table 6 below. The table also shows the therapeutic coefficient.

[Table] *Administration on day 1 only Furthermore, a suspension of P388 leukemia cells (1×10 ⁶ cells/mouse) was intraperitoneally transplanted into CDF ₁ mice, and administration was performed on days 1 and 5 after transplantation. A 3'-deamino-3'-(4-morpholinyl) derivative was orally administered to each patient. Survival prolongation rate (%) based on 100 days of survival of mice in the control group that were observed for 30 days and administered physiological saline.
The effects are shown in Table 7 below. The table also shows the therapeutic coefficient.

[Table] *Administration on day 1 only Adriamycin, one of the most powerful anthracycline antitumor drugs, is effective when administered intravenously, but is ineffective when administered orally. On the other hand, aclacinomycin, which is another specific example of this class of antitumor drugs, is known to be effective when administered orally.
From the above results, the 3'-deamino-3'-(4-morpholinyl) derivative of the anthracycline compound of the present invention has a therapeutic effect comparable to or greater than that of adriamycin when administered intraperitoneally or intravenously. Also, even when administered orally, madriamycin is ineffective.
It was found to have a stronger effect than aclacinomycin. Therefore, the compounds of the present invention are considered to be extremely promising as therapeutic agents. (b) Antitumor properties against adriamycin-resistant tumors Furthermore, the 3'-deamino-3'-
The (4-morpholinyl) derivative showed the same antitumor effect on adriamycin-resistant tumor cells as on sensitive tumor cells. For example, adriamycin-resistant
P388 leukemia cells (P388/ADR) and susceptible P388 leukemia cells (P388/S) were each incubated at 5×10 ⁴ in RPMI1640 + 10% FCS medium.
A suspension was made into a suspension at a concentration of 100 mg/ml, and a diluted 3'-deamino-3'-(4-morpholinyl) derivative and other drugs were added thereto, followed by culturing at 37°C in a 5% CO ₂ atmosphere. Two days later, the cell number was counted, and the drug concentration (IC ₅₀ ) representing 50% cell number of the drug-free control was compared to determine efficacy.

[Table] is shown below.
(2) Toxicity a Acute toxicity ( _LD50 ) The _LD50 of the 3'-deamino-3'-(4-morpholinyl) derivative of the present invention obtained by intravenous injection into ICR mice was as follows. Drug LD ₅₀ (mg/Kg) 3'-deamino-3'- (4-morpholinyl) R20X 12.3 3'-deamino-3'- (4-morpholinyl) R20X2 3.55 b Cardiotoxicity Anthracycline antitumor drug, In particular, cardiac toxicity is a side effect of adriamycin. 3′-deamino- of the present invention
The cardiotoxicity of the 3'-(4-morpholinyl) derivative was lower than that of adriamycin. Each sample was dissolved in physiological saline from the femoral pulse of a golden hamster anesthetized with urethane.
3'-deamino-3'-(4-morpholinyl)
The derivative was administered, and electrocardiogram changes were examined by inserting electrocardiogram measurement electrodes into the limbs. Electrocardiogram: 1 minute before administration, 0.5, 1, 3, 5, 10, after administration.
I checked after 15 minutes. Measurement items were T-wave potential (decreased due to myocardial damage), QRS interval (increased due to intraventricular conduction failure), PQ interval (increased due to intra-atrial conduction failure), and heart rate. The results are shown in FIG. Note that * and * * in the figure represent the values of the student's test, and the values are as follows. *P<0.05 * *P<0.01 of the anthracycline compound of the present invention
3'-deamino-3'-(4-morpholinyl)
The effects of dosage on each measurement item for all derivatives were gentler than that of adriamycin, and the toxicity was less than 1/6 of that of adriamycin in terms of dosage. Adriamycin showed toxicity to the heart at the dose at which the antitumor effect was expressed (T/C = 130%), but all of the derivatives of the present invention showed toxicity even at the dose at which the maximum antitumor effect was expressed (T/C = max). Nakatsuta. 2) Antitumor agent Thus, it is clear that the 3'-deamino-3'-(4-morpholinyl) derivative of the anthracycline compound of the present invention exhibits antitumor properties against animal tumors, particularly malignant tumors. was made into Therefore, the 3′-deamino-3′- of the present invention
(4-morpholinyl) derivatives can be used as antitumor agents or tumor therapeutic agents. 3′-deamino-3′-(4-
The morpholinyl) derivatives can be administered by any convenient route of administration and in a form determined by the route of administration employed. The drug is usually in a diluted form with a pharmaceutically acceptable carrier or diluent. 3′-deamino-3′-(4-
When a morpholinyl derivative 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.
In humans, there are methods by injection such as intravascular injection into a vein or artery or local administration, and methods by oral administration and intraperitoneal administration. The dosage of the 3'-deamino-3'-(4-morpholinyl) derivative is determined by taking into account the results of animal studies and various circumstances, such that the total dose is a certain amount when administered continuously or intermittently. It is set not to exceed. The specific dosage may vary depending on the method of administration, the circumstances of the patient or treated animal, such as age, weight, sex, susceptibility, diet, time of administration, concomitant drugs, and the severity of the patient or his or her disease. Needless to say, the appropriate dose and frequency of administration under certain conditions must be determined by a specialist's appropriate dose determination test based on the above guidelines. In addition, the LD ₅₀ of 3′-deamino-3′-(4-morpholinyl)-R20X2 by intravenous injection in mice
(mg/Kg) is 1/2.8 of that of Adriamycin, so the intravenous injection dose of the compound of the present invention is 0.07 to 0.07 to that of Adriamycin.
It can be said that the amount is 0.8mg/Kg/day. Experimental Example In the following, "%" is "w/v%". Example 1 (Production of R20 Substance) (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% Dispense 100 ml of the above medium into a 500 ml Erlenmeyer flask, sterilize it, and prepare Actinomadilla roseoviolaceae.
One platinum loop of R20 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) Culture 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 (sedimented) 0.4% 2.5 liters of the above medium was placed in a 50 liter jar fermenter and sterilized, and the amount of the three seedlings above was inoculated. Culture was carried out at 27°C for 7 days at an aeration rate of 1 v.vm and a rotational speed of 200/min. (3) Collection of R20X After culturing, the culture solution was filtered to separate the bacterial cells and the solution. Adjust the pH of the solution to 2 with 1N hydrochloric acid, and apply it to column 10 of "Diaion HP20" (manufactured by Mitsubishi Kasei).
It was adsorbed to ×40cm. After washing with distilled water and 50% methanol, it was eluted with methanol. The eluate was concentrated, the concentrated solution was adjusted to pH 8.5, and extracted three times with a chloroform-methanol (9:1) mixture. After concentrating this extract, 6 times the amount of hexane was added and the resulting precipitate was dried.
250 mg of red powder was obtained (crude sample of R20 substance). this
Dissolve 250 mg of R20 substance crude sample in chloroform,
250 g of silica gel was placed on a 4 x 40 cm column equilibrated with chloroform, the column was thoroughly washed with chloroform, and then fractionated with chloroform:methanol = 10:1. After drying the obtained fraction under reduced pressure, it was developed on TLC (Merck "Silica Gel 60") using a solvent system of chloroform: methanol: acetic acid: water = 40:8:1:1, and the Rf value was 0.43.
The nearby red-orange fraction was scraped off. The fraction thus obtained was eluted, concentrated, and then recrystallized in chloroform to obtain 110 mg of R20X. (4) Collection of R20X2 After culturing, the culture solution was filtered to separate the bacterial cells and the solution. Adjust the pH of the solution to 2 with 1N hydrochloric acid, and apply it to column 10 of "Diaion HP20" (manufactured by Mitsubishi Chemical Corporation).
It was adsorbed to ×40cm. After washing with distilled water and 50% methanol, it was eluted with methanol. The eluate was concentrated, the concentrated solution was adjusted to pH 8.5, and extracted three times with a chloroform-methanol (9:1) mixture. After concentrating this extract, 6 times the amount of hexane was added and the resulting precipitate was dried.
250 mg of red powder was obtained (crude sample of R20 substance). R20
Dissolve 250 mg of the crude substance sample in chloroform, place 250 g of silica gel on a 4 x 40 cm column equilibrated with chloroform, wash the column well with chloroform, and then mix chloroform:methanol =
It eluted at 10:1. After drying the obtained fraction under reduced pressure, it was developed on TLC (Merck "Silica Gel 60") using a solvent system of chloroform: methanol: ammonia water = 8:2:0.05, and the Rf value was 0.44.
The nearby red-orange fraction was scraped off. The fraction thus obtained was eluted and concentrated, and then recrystallized in chloroform to obtain 10 mg of R20X2. Example 2 3'-deamino-3'-(4-morpholinyl)-
Production of R20X Dissolve 135 mg (0.27 mmol) of R20X in chloroform (15 ml) and add 320 mg of diglycolaldehyde.
mg (2.66 mmol) and 17 mg (0.27 mmol) of sodium cyanoborohydride dissolved in a mixed solvent of acetonitrile/water (1:1) were added.
React for 1 hour at room temperature. After the reaction is completed, the reaction solution is extracted three times with 50 ml of chloroform, and the chloroform solution is washed three times with 40 ml of water. The chloroform solution is dried over anhydrous sodium sulfate and then concentrated to dryness. This crude product was subjected to silica gel column chromatography (10 g of "Wako Gel C-200") and eluted with a mixed solvent of chloroform:methanol (200:1) to obtain the desired product. This was further crystallized from chloroform/hexane to yield 90 mg (58%) of the title compound. Example 3 3'-deamino-3'-(4-morpholinyl)
Preparation of R20X2 Dissolve 80 mg (0.16 mmol) of R20X2 in chloroform (10 ml) and dissolve 186 mg of diglycolaldehyde.
(1.6 mmol) and 9.8 mg (0.16 mmol) of sodium cyanoborohydride were dissolved in a mixed solvent of acetonitrile/water (1/1) and reacted for 1 hour at room temperature. After the reaction was completed, the reaction solution was dissolved in 50 ml of chloroform. Extract the chloroform solution three times with 40 ml of water.
Wash 3 times with The chloroform solution is dried over anhydrous sodium sulfate and then concentrated to dryness. This crude product was subjected to silica gel column chromatography (10 g of "Wako Gel C-200") and eluted with a mixed solvent of chloroform/methanol (200/1) to obtain the desired product. This was further crystallized from chloroform/hexane to obtain 42 mg (48%) of the title compound as a brown powder. Example 4 R20X (40 mg) was dissolved in DMF (4 ml) and bis-(2-iodoethyl)ether (260
mg) and triethylamine (32 mg) were added. This was stirred at room temperature for 4 days, DMF was distilled off by vacuum concentration, and the residue was dissolved in 100 ml of chloroform, which was further washed with 100 ml of water. Next, anhydrous sodium sulfate was added to the chloroform layer to dehydrate it and then concentrate it. This was treated with silica gel chromatography, and chloroform: methanol = 50:
It was eluted in system 1. Two colored fractions were obtained, but the 3'-deamino-3'-
(4-morpholinyl)R20X (5 mg) and 3'-deamino-3'-(4-morpholinyl) R20X2 (7 mg) were obtained from the subsequently eluted fraction.

[Brief explanation of drawings]

Figure 1 is a reproduction of the ultraviolet/visible absorption spectrum of 3'-deamino-3'-(4-morpholinyl)R20X. 1...in MeOH, 2...in 0.1NHCl-MeOH,
3...0.1NNaOH-in MeOH. Figure 2 is a reproduction of the infrared absorption spectrum of 3'-deamino-3'-(4-morpholinyl)R20X. Figure 3 shows 3′-deamino-3′-(4-
This is a reproduction of the ¹ H-NMR spectrum of (morpholinyl) R20X. Figure 4 shows 3′-deamino-
This is a reproduction of the ultraviolet/visible absorption spectrum of 3'-(4-morpholinyl)R20X2. 1...in MeOH, 2...in 0.1NHCl-MeOH,
3...0.1NNaOH-in MeOH. FIG. 5 is a reproduction of the infrared absorption spectrum of 3'-deamino-3'-(4-morpholinyl)R20X2. Figure 6 shows 3′-deamino-3′-(4
This is a reproduction of the ¹ H-NMR spectrum of R20X2 (morpholinyl). FIG. 7 is a reproduction of the ultraviolet/visible absorption spectrum of R20X. 1...in MeOH, 2...in 0.1NHCl-MeOH,
3...0.1NNaOH-in MeOH. FIG. 8 is a reproduction of the infrared absorption spectrum of R20X. Figure 9 shows ^1H -NMR of R20X.
This is a copy of the spectrum. Figure 10 shows
This is a reproduction of the ultraviolet/visible absorption spectrum of R20X2. 1...in MeOH, 2...in 0.1NHCl-MeOH,
3...0.1NNaOH-in MeOH. FIG. 11 is a reproduction of the infrared absorption spectrum of R20X2. Figure 12 shows ^1H of R20X2.
-This is a reproduction of an NMR spectrum. 1st
Figure 3 shows that when (a) 3'-deamino-3'-(4-morpholinyl)-R20X is administered (indicated by □ in the figure), (b) 3'-deamino-3'-(4-morpholinyl)
- This shows the effects on the hearts of golden hamsters (0.5 minutes after administration) when R20X2 was administered (indicated by △ in the figure) and (c) when Adriamycin was administered (indicated by ○ in the figure). Then, A is the T-wave potential and B is the QRS
C indicates the PQ interval, and D indicates the heart rate.

Claims (1)

[Scope of Claims] 3′-deamino-3′-(4-morpholinyl) of an anthracycline compound represented by the following formula ()
Derivatives or acid addition salts thereof. (In the formula, R represents a hydroxyl group) An antitumor agent comprising a 3'-deamino-3'-(4-morpholinyl) derivative of an anthracycline compound represented by the following formula () or an acid addition salt thereof. (In the formula, R represents a hydroxyl group)