JPS6216495A - 3'-deamino-3'-(4-morpholinyl) derivative of anthra-cycline compound and use thereof - Google Patents

Abstract

NEW MATERIAL:The compound of formula I or its salt having the following properties. Appearance, yellow powder; melting point, 132-135 deg.C; molecular weight, 553 (FD-MS); molecular formula, C30H35O9N; element analysis (%), C 64.78, H 6.53, O 26.1, N 2.59; solubility, soluble in acidic and basic water, methanol, acetone, etc., and insoluble in water, (cyclo)hexane, etc. USE:An antitumor agent exhibiting antitumor activity especially against malignant tumor. PREPARATION:An anthracycline compound R20Y5 is sued as a starting raw material, and is made to react with the compound of formula II in a solvent (preferably a mixture of acetonitrile, water and chloroform) usually in the presence of a reducing agent such as NaBH4, preferably at room temperature of thereabout to obtain the objective compound of formula I. The R20Y5 used as the raw material is produced preferably from the cultured product of Actinomadura roseoviolacae R20 strain (FERM P-7138).

Description

DETAILED DESCRIPTION OF THE INVENTION BACKGROUND OF THE INVENTION TECHNICAL FIELD The present invention i1 Anti-pulmonary anthracycline compound 11-deoxy-13-deoxo-carminomycin (
3'-deamino-3'- of (hereinafter referred to as R20Y5)
(4-morpholinyl) derivative.

Prior art anthracycline compounds include downmycin (U.S. Pat.
16,242) and adriamycin (
(see US Pat. No. 3,590,028), and these compounds are widely used clinically as antitumor agents (see US Pat. No. 3,590,028).
It's being used. However, although these compounds exhibit strong antitumor effects, they also have strong side effects, so they are not necessarily satisfactory (ゝ.

3'-deamino-3'-(4-morpholinyl) of the present invention
As a compound related to derivatives, Giusetsube-Cancinelli et al.
5, Streptomyces veusetaius pearl.
Streptonwcespeuceti
a mutant strain of Streptomyces var, Caecius), namely Streptomyces peucetius var aureus.
var4aureus) was obtained from the culture solution of
It has been reported that this has antitumor activity (Unexamined Japanese Patent Publication No. 5
5-76896).

Furthermore, various derivatives of adriamycin, daunomycin, and carminomycin have been synthesized as morphoriinyl fatty conductors of anthracycline compounds, and their antitumor activity has been reported (Japanese Patent Laid-Open No. 163393/1983, U.S. Patent No. 4,301 Specification No. 277, JP-A-59-212484, JP-A-59-212499
No. Publication, Mo5her, C1W, et, al, 1
J.

Med, Cherry, 25 l5-24 (198
2), Johnston, J.B.: Biochem.
ical PharmacoloH32(21) 32
55-3258 (1983), Acton, E.M.
:J, Fi'led, Chem, 27638645
(1984)).

However, to the best of the inventors' knowledge, these compounds are also not necessarily satisfactory in terms of antitumor activity or toxicity.

Since anthracycline compounds constitute a group of useful antitumor agents, there is a constant desire for better anthracycline compounds.

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

That is, 3 of the anthracycline compounds according to the present invention.
'-Deamino-3'-(4-morpholinyl) derivative is
It 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 as an active ingredient a 3'-deamino-3'-(4-morpholinyl) derivative of an anthracycline compound represented by the following formula or an acid addition salt thereof.

Detailed Description of the Invention 1) Chemical Structure The compound according to the present invention is an anthracycline compound R20.
Y5 is a 11-deoxy-13-deoxo-carminomycin, 03'-deamino-3'-(4-morpholinyl) derivative having the chemical structure shown by the above formula (1).

2) Physicochemical properties (1) Outside appearance: yellow powder (2) Melting point: 132-135°C (3) Specific rotation: [α] I)' = + 195.2° (C = 0.125, (in methanol) (4) Molecular weight: 553 (FD-MS) (5) Molecular formula -
030H3509N (6) Elemental analysis: CHON calculated value 1%) 65.09 6,37 26.01
2.53 Analysis value (%) 64.78 6.53 26
．． 10 2.59 (Power Ultraviolet/visible absorption spectrum (see Figure 1) λmax in methanol (El, @) 229 (541,6), 257 (360,8), 289
(164,8), 429(186,8) λmix (14ri228(552
,8), 257 (370,8), 291 (169,2)
, 431(192,8) in alkaline methanol λmax (14ri237(
492,8), 252sh(375,6), 292(1
(10) ) Behavior development system of thin layer chromatography
R, f, value chloroform: methanol 1 (J: 1 0.6
7 ethylnia acetate 7 tons 1: 1 0.43 (
Merck silica gel plate fiJF254 used) (1
1) Production of compounds of the present invention soluble in acidic water, basic water, methanol, ethanol, propatool, acetone, ethyl acetate, chloroform, pyridine, dimethyl sulfoxide, insoluble in water, hexane, cyclohexane, diethyl ether 1) Overview 3'-deamino-3'-(4-morpholinyl), which is not necessary for the invention
The enzyme conductor is R20Y5 produced by culturing microorganisms.
can be produced by synthetic chemical modification of

2) R20Y5 R20Y5 can be obtained from a culture of Actinomadura roseoviolaceae R20 strain isolated by the present inventors. As mentioned above, R20Y5 is a known substance and can also be produced by the method described in JP-A-55-76896.

(1) R20 strain The R20 strain, which the present inventors have discovered as a strain of the genus Actinomadilla that has the ability to produce the anthracycline compound R20Y5, has the following content.

■ White rice and the deposited number R20 strain were isolated from soil collected from a vegetable field in Oaza Onodani, Kaho-machi, Kaho-machi, Fukuoka Prefecture, on July 5, 1997.
It was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology in Japan, and has the number ``Imitation Technology Research Institute Closing No. 7138''.

■ Mycological properties and physiological properties The characterization of this bacterial strain was carried out in accordance with the method manual of the International Committee on Streptomyces Nomenclature (ISP) as follows.

A) Morphology The substratum hyphae spread radially on the agar medium surface while branching, and no hyphal division was observed. Aerial hyphae have a long main axis, the short plates are branched (uniaxial branching) at right angles to the y (with respect to the main axis), and a small dense spiral consisting of 10 or more spores is formed at the tip of the short plate. Spore chain (1-3 turns, diameter 2.0-2
．． 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, length 0.7-1.1μ,
Exhibits a smooth surface. True spore chains, flagellated spores, sclerotia, etc. are W! Not enshrined. Since the whole cell hydrolyzate contains meso-type tomadulose diaminopimelate, the cell wall type is determined to be IB.

B) Culture properties The results of the culture properties (cultured at 27°C) 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 (1) has a cell wall type of B, (2) the spore chain consists of 10 or more spores, (3) forms pseudosporangia and spore masses, and ( 4) Since neither true sporangia nor flagellated spores were observed, it was determined that Actinomadura (A
ctinomadura).

Nonomura's search table [Rikou, Vol. 52, pp. 71-77, 19
Listed in 1974, Shikou, No. 49, pp. 904-902,
1971], the present strain is A, Roseoviolaceae (A
.

roseoviolaeea).

Therefore, the standard strain of Roseoviolaceae [KC
CA-145 (Nonomura A-5) 1 was cultured under the same conditions,
The main properties of both strains were compared.

The results are shown in Table 3, although there are slight differences in fungal coloration, underside color, and optimal growth temperature, they are taxonomically very similar (similar to each other.・Rose Oviolaceae (Aetinomadu)
ra roseoviolacea Nonomura
et 0hara1971).

(2) Culture/Production of R20Y5 Anthracycline R20Y5 can be produced by culturing R20Y5-producing bacteria belonging to the genus Actinomadura aerobically in an appropriate medium and collecting the target product from the culture.

The medium can contain any nutrient source available to the R20Y5 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 cheap liquor and inorganic materials such as ammonium salt free nitrates, such as ammonium sulfate, sodium nitrate, and ammonium chloride, can be used. In addition, inorganic salts such as common salt, potassium chloride, phosphates, and summer metal salts can be added as necessary. In order to suppress foaming during fermentation, suitable antifoaming agents, for example 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 5°C to 45°C, but Z7"C
~30" (preferably. In this method, the production amount of R20Y5 reaches its maximum in 6 to 7 days for both shaking culture and aerated agitation culture.

In this way, a culture enriched with R20Y5 is obtained. In the culture, a part of R20Y5 exists in the bacterial cells, or most of it exists in the culture P solution.

Any convenient method can be used to harvest R20Y5t from such cultures. One method is based on the principle of extraction, and specifically, for example, for R20Y5 in the culture P solution, it is extracted with a water-immiscible R20Y5 solvent, such as ethyl acetate or chloroform. , butanol, etc. (the extraction efficiency is good if the culture P solution is neutral or slightly basic), or for R20Y5 inside the bacterial cells, the aggregate obtained by filtration, centrifugation, etc. is extracted with chloroform, acetic acid, etc. It can be recovered by treatment with ethyl, butanol, methanol, ethanol, acetone, hydrochloric acid solution, acetic acid solution, etc. The culture itself can also be subjected to the above extraction operation without separating the bacterial cells. Countercurrent distribution methods using appropriate solvents can also be included in the scope of extraction.

Another method for collecting R20Y5 from cultures is based on the principle of adsorption, in which R20Y5 is already in liquid form.
20Y5-containing substances, such as culture P solution or extract obtained by performing the extraction operation as described above,
The target R20Y5 can be obtained by column chromatography, liquid chromatography, etc. using a suitable adsorbent such as activated carbon, alumina, silica gel, Diaion HP20J (manufactured by Mitsubishi Kasei Corporation), etc.
-' by adsorption and subsequent bath separation, R2
It is possible to get 0Y5. R obtained in this way
If the 20Y5 f6 liquid is concentrated to dryness under reduced pressure, R20Y5
A rough sample of .

In order to further separate and purify a purified product of R20Y5 from the crude sample of R20Y5 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 can be carried out in an appropriate combination. can. Specifically, for example, R20Y5
Dissolve the crude sample in a small amount of chloroform, develop with a suitable solvent using a silica gel column to wash out the active ingredient, concentrate the eluate under reduced pressure, further develop with TLC, scrape off and wash out. Since and R20Y5 are separated as a single substance, R20Y5 can be obtained by t-condensation and drying.

The physicochemical properties of R20Y5 obtained in this way are:
It is as follows. These values are based on Japanese Patent Application Laid-Open No. 55-7689.
The physicochemical properties are in good agreement with the physicochemical properties described in Publication No. 6.

Physicochemical properties of R20Y5 (1) Appearance: yellowish brown powder (2) Melting point = 148-153°C (3) Specific optical rotation: [α1. )' 〜4187.2゜(C
= 0.125 in methanol) (4) Molecule ii:
483 (FD-MS) (5) Molecular formula: c26tan08
N (6) Elemental analysis: CHON Calculated value 64,59 6.05 26.47 2.90
(%) Analysis value 63.82-6.32 27,04 2
, 82 (%) (power Ultraviolet/visible absorption spectrum λm
ix (E),! ) (see Figure 4) 228 (670,4), 257 (460,0), 290 in methanol
(202,8), 430 (216,4) in acidic methanol 228 (685,6), 259 (462,8), 289
(205,6), 430 (225,2) 235 (617,6), 251 (sh) (460,0) in alkaline methanol
, 290 (195,6), 520 (188,0) (8)
Infrared absorption spectrum (see Figure 5) (9) Nuclear magnetic resonance spectrum (Zoo MHz, in deuterated chloroform) (see Figure 6) 00) Behavior of thin layer chromatography o o $#A: Meta'-' “Acetic acid′ water 8
．． .

~401:1:1 0,50 (1) Soluble acidic water, basic water, methanol, ethanol, propatool, acetone, ethyl acetate, chloroform, pyridine, dimethyl sulfoxide, water soluble hexane, cyclohexane, diethyl Insoluble in ether 3) Synthetic chemical modification of R20Y5 3'-deamino-
The 3'-(4-morpholinyl) derivative can be produced by reacting R20Y5 or an acid addition salt thereof with a compound represented by the following formula (11). This compound (1) is converted from meso-erythritol to Carbohy.
drate motion 5eareh Vol, 35.195~2
02 (1974).

The reaction between R20Y5 or its acid addition salt and the compound of formula (1) 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, with a mixed solvent of acetonitrile-water-chloroform being preferred.

This reaction is usually carried out using reducing agents such as photolithium borohydride (NaBH4), sodium cyanoborohydride (
It is preferable to carry out the reaction in the presence of NaBH3CN) or the like. The amount of reducing agent used is not critical, and can be used in amounts of at least 1 mol, preferably 1 to 5 mol, per 1 mol of 120Y5.

The amount of the compound of formula (n) to be used is advantageously at least 1.5 mol, preferably at least 5 mol, particularly preferably from 8 to 15 mol, per 1 mol of R20Y5.

The reaction temperature is generally within the range of the freezing point of the solvent to be used, preferably around room temperature.

Under the above reaction conditions, the reaction for converting amine groups into morpholinyl groups can be completed in about 10 minutes to 2 hours.

The 3'-deamino-3'-(4-morpholinyl) derivative, which is the target compound of the present invention, is isolated and purified from the reaction mixture obtained by the reaction of R20Y5 with the compound of formula ([l)] by the method of the present invention. This can be carried out by a known purification method used in the field of producing anthracycline glycoside derivatives, such as chromatography using silica gel or the like.

The 3'-deamino-3'-(4-morpholinyl) derivative represented by formula (1) thus obtained can be prepared as an acid addition salt thereof, such as hydrochloric acid, sulfuric acid, etc., by a method known per se.
Inorganic acids such as phosphoric acid, or acetic acid, propionic acid, maleic acid, oleic acid, palmitic acid, citric acid, succinic acid, tartaric acid, fumaric acid, glutamic acid, pantothenic acid,
It can be converted into its acid addition salt by treatment with an organic acid such as lauryl sulfonic acid.

Uses of the compounds of the present invention The 3'-deamino-3'-(4-morpholinyl) derivatives of anthracycline compounds according to the present invention have strong anticancer activity and a high therapeutic index, and are useful compounds as pharmaceuticals. .

l) Physiological activity (1) Antitumor properties 3'-deamino-3'-(4-morpholinyl) of the present invention
The derivative showed significant antitumor activity against leukemia in experimental animals. An example is P388 for cDF□ mice.
A suspension of leukemia cells (lx106 cells/mouse) was intraperitoneally transplanted, and 3'-deamino-
The 3'-(4-morpholinyl) derivative was administered intravenously. Survival prolongation rates (effects are indicated by @) are as follows, with the number of survival days of mice in the control group to which physiological saline was administered being 100 days.

From the above results, the 3'-deamino-3'-(4
-morpholinyl) derivatives were found to have therapeutic efficacy comparable to or even greater than that of adriamycin.

The therapeutic coefficient (
The 3'-deamino-3'-(4
-morpholinyl) derivative has a therapeutic index of approximately 6.5, which is higher than that of adriamycin, which is approximately 3.4.
The compounds of the present invention are considered to be extremely promising as therapeutic agents.

In addition, R20 shown in Japanese Patent Application Laid-Open No. 55-76896
From the comparison of the antitumor effect of Y5, that is, 11-deoxy-13-deokyne-carminomycin, and the antitumor effect of the 3'-deamino-3'-(4-morpholinyl) derivative of the present invention, it was found that R20Y5 was -3'-(4-
-f-ruforinylation), it was found that the antitumor activity was significantly increased.

(2) Acute toxicity (LD5o) 3'-deamino-3'-(4-morpholinyl) of the present invention
The LD5 of the derivative obtained by intravenous injection in ICR mice was as follows, and the 3'-deamino-3'-(
It was observed that the acute toxicity of this parent compound was reduced by 4-morpholinylation.

LD5o 3'-deami/-3'-(4-morpholy=#)R20Y
5 65 (comparison data) R20Y51
According to Japanese Patent Application Laid-Open No. 55-76896.

2) Antitumor agent Thus, the 3′ antitumor agent of the anthracycline compound of the present invention
It has been revealed that -deamino-3'-(4-morpholinyl) derivatives exhibit antitumor properties against animal tumors, particularly malignant tumors.

Therefore, the 3'-deamino-3'-(4-morpholinyl) derivative of the present invention can be used as an antitumor agent or a tumor therapeutic agent.

The 3'-deamino-3'-(4-morpholinyl>in4 form) as an antitumor agent can be administered by any convenient route of administration and in a dosage form determined by the route of administration adopted. It is usually in a diluted form with a pharmaceutically acceptable carrier or diluent.

When actually administering a 3'-deamino-3'-(4-morpholinyl) derivative as an antitumor agent, the typical method is to dissolve it in distilled water for injection or physiological saline and inject it. It can be mentioned as one. 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 the 3-deamino-3-(4-morpholinyl) derivative shall be determined by considering the results of animal studies and various circumstances, and the total dosage shall not exceed a certain amount when administered continuously or intermittently. It is determined as follows. The specific dosage will 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. 52
Moreover, 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.

Actual experience example The "chi" in Z below is (w/v%).

Example 1 (Manufacture of R20Y5) (1) Preparation of seed mother The culture medium used was prepared by dissolving the following components in 1 liter of water and adjusting the pH to 7.2.

Polypeptone 1 Zymolases 1% Meat Extract 1 100 ml of the above medium was dispensed into a 500 ml Erlenmeyer flask, sterilized, and 1 platinum loop of Actinomadura roseobilaceae R20 was inoculated from a slant, and at γ°C for 5 days using a rotary shaker (200 rpm). The seedlings were used as seeds.

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

Grape circulation 2.5% soy flour
1.5% Dry yeast 0.2% Calcium carbonate (sedimented) 0.4% Five liters of the above medium was placed in a sterilized jar fermenter and inoculated with the three seed germs. 1@11 was carried out for 7 days at aeration f1, v, m, rotational speed 200/min, valley °C.

After culturing, the culture solution was t-filtered to separate the bacterial cells and F solution.

Adjust the pH of the solution to 8 with IN hydrochloric acid, and add ``Diaion HP''.
20j (manufactured by Mitsubishi Kasei Corporation) column 10X401. After washing with distilled water and 50% methanol, elution was performed with methanol. Concentrate the eluate and convert the concentrate to pI (
Adjusted to 8.5, chloroform-methanol (9:1)
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 powder. Further, chloroform: methanol: water = 70 = 1
It was placed on a silica gel column (Merck Silica Gel 6L 5 x 40 am) equilibrated at 0=1, and the yellow fraction was fractionated. After drying the obtained fraction under reduced pressure, it was developed on TLC (Merck's "Silica Gel") using a solvent system of chloroform: methanol: acetic acid: water = 40:8:1 = 1, and R, f , the yellow fraction around 0.50 was scraped off. The fraction thus obtained was eluted and concentrated, and then recrystallized in chloroform to obtain 51.6 mg of R20Y.

Example 23'-deamino-3'-(4-morpholinyl)
-Preparation of R20Y5 599.5 mg (0.21 ml) of R20Y was dissolved in 5 ml of acetonitrile/water (1/1) mixture, and 246 mg (2.05 mmol) of diglycolaldehyde and sodium cyanoborohydride were added. 12.9
mg (0.21 mmol) and reacted for 4 hours at room temperature. After the reaction was completed, the reaction solution was diluted with 50 ml of water, and the resulting precipitate was removed using a glass filter.
Wash with 500 ml of water. Solution F was further extracted three times with 50 ml of chloroform, and the chloroform layer was further washed several times with 50 ml of water. The chloroform layer is dehydrated with anhydrous sodium sulfate and concentrated to dryness.

This and the precipitate are combined to form a crude product. This was further subjected to silica gel column chromatography (Wakogel C-
200) and chloroformimethanol (50:1).
) to obtain the desired product. moreover,
Crystallize from chloroform/hexa/ to obtain target product 42
．． 2 mg was obtained.

[Brief explanation of drawings]

Figure 1 is a reproduction of the ultraviolet/visible absorption spectrum of 3'-deamino-3'-(4-morpholinyl)R20Y5. 1, 2 in methanol, 3 in acidic methanol, and 2 in alkaline methanol.
This is a copy of the infrared absorption spectrum diagram of I/)R20Y5. FIG. 3 is a reproduction of the 'H-NMR spectrum of 3'-deamino-3'-(4-morpholinyl) R20Y5 in deuterated chloroform. FIG. 4 is a reproduction of the ultraviolet/visible absorption spectrum of R20Y5. 1 in methanol 2 in acidic methanol 3 in alkaline methanol FIG. 5 is a reproduction of the infrared absorption spectrum of R20Y5. Figure 6 shows "H-N" in R20Y50 deuterated chloroform.
This is a reproduction of an MR spectrum diagram. Applicant's agent Inomata Seishi Proceedings 1st edition 1O May 30, 1960

Claims (2)

[Claims] (1) A 3'-deamino-3'-(4-morpholinyl) derivative of an anthracycline compound represented by the following formula (I) or an acid addition salt thereof. ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) (2) An antitumor agent containing a 3'-deamino-3'-(4-morpholinyl) derivative of an anthracycline compound represented by the following formula (I) or an acid addition salt thereof as an active ingredient. ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I)