JPS6293298A - Anthracycline compound and use thereof - Google Patents

Abstract

NEW MATERIAL:A compound shown by the formula I (R is group shown by the formula II or formula III). USE:An antitumor agent. PREPARATION:For example, actinomadura roseoviolacea R 20 strain (FERM P-7138) capable of producing 13-deoxocarminomycin (R20X) is aerobically cultivated, to collect R20X. Then, 1mol of R20X or its acid addition salt is reacted with 8-15mol compound shown by the formula IV (R1 and R2 are CH3 or R1 and R2 are bonded to form group shown by the formula V) usually in a solvent (e.g., mixed solvent of acetonitrile, water and methanol) in the presence of preferably 1-5mol reducing agent (e.g., NaBH4) preferably at room temperature for 1-12 hours.

Description

DETAILED DESCRIPTION OF THE INVENTION Background of the Invention The present invention is anthracycline compounds that are antitumor. The present invention relates to an N-(x",3"-alkylidenedioxy)propyl derivative of /3-deokynecarminomycin (hereinafter referred to as R20X).

As an anthracycline compound, daunomycin (U.S. Patent No. J, A
/A, λμ2) and adriamycin (see US Pat. No. 3, syO, 0Zlr) are known, and these compounds are widely used clinically as antitumor agents. However, although these compounds exhibit strong antitumor effects, they also have strong side effects, so they are not necessarily satisfactory.

Compounds related to the novel derivatives of the present invention (details described later) include Rlvola, G, et, al,
)teeth.

The leading compound R&X is Streptomyces vixetix var carminatus (S
treptomyces peucetlous va
r+aarmlnatus) (/j here4LSM
, 31jOZ ATCC.

4Lri, 2ta FRI, DRJ'/FI
Farmltillamlkroblogis
che 8ummlung) and reported that it has antitumor activity (West German Published Patent No. 3,012,1&j).

However, to the knowledge of the present inventor, this compound is not necessarily satisfactory in terms of antitumor activity or toxicity.

Therefore, there is currently a constant desire for better anthracycline compounds.

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

That is, one anthracycline compound according to the present invention is
It is expressed by the following formula. The invention also relates to acid addition salts of this compound.

The present invention also relates to the use of anthracycline compounds or acid addition salts thereof.

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

/) Chemical structure The anthracycline compound according to the present invention is an N-(J", 3"-flukylidenedioxy)propyl derivative of the anthracycline compound R2oX and has the above formula (I).
It has the chemical structure shown below. There are two types depending on the difference in substituent R.

-) Physical and chemical properties (1) Melting point: 101-102℃ (2) [α] is also 7 = + #j' (C = 0. here, chloroform) (3) Elemental analysis HN calculation value (4) 6 Ko, GJ &, 37 2. KoIr
21,72 analysis value (4) 42. <AD &, 4A
J 2, 2! 21.92(4) Infrared absorption spectrum (potassium bromide tablet). -1 34410,291rO12P! 0,2900. /41
0.1jro, tp-6o, 1u (Ao, haqO1i,
iro.

/370. /Body 0. /230,124AO,/ZOO
1//F0. /130. //20.1070,10hiro0
11020, rito, y2o, trIrθ, lr, 10
, 71rO17 reference 00 (fi) 1H-nuclear magnetic resonance spectrum (lθOMHz,
(in deuterated chloroform) δ (ppm) 13.6 hiro (/H, a, phenolic OH), /2. r (/H, s, phenolic oH), /2. J4A (
/H,l, phenolic OH), 7, 0(/H,
dd, H-/), 7.4j (/H.

t, H-z), 7. zt, (/H, dd,
H-J)%j,l' (/H,br s, H-/'
), , r, 22 (IH, brs, H-7), Hiroshi, a ~3. y (J HeH-4', H-ko", H-
3 “&), 3.6 Hiro (IH.

dd, H-Jb), 3. ! 4 (IH
, br a, H-Hiro'), 3.3~. 2j (j
H, H-10, H-J'.

H-/), 2.4co<IH,d, H-ra).

J/-1, j (J'H, H-1rb, H-1, 7.H-
2′.

), 1.31 (JH, d, H-A'), I, J4
((6) Appearance: Red powder +71 FD-MS: 61 Hiro (M+/) + (8) Solubility in acidic water, basic water, methanol, ethanol, propatool, acetone, ethyl acetate.

Soluble in chloroform, dichloromethane, insoluble in water, hexane, cyclohexane, diethyl ether, petroleum ether (91 Rf value (using Merck's silica gel plate AOF Koyohiro) Development system Rf value Chloroform: Methanol-10: / 0.j!
r(1) melting point iot ~ ior ”c(2) [α]
Mo”=s+1zi0(c=o, ko2.chloroform) +31 Elemental analysis calculation value bu, 3t 4jJ s, tu xi,
, y2 (%) analysis value 1. Sail IA2 4j7 2.03
26,1rlr (1) (41 infrared absorption spectrum (
potassium bromide tablets). -1 J'AOO12ri2o, xrro, /600, /IAI
AO1iuoo, 1xro, 1a3o, /15F (7,
1iso.

1lIO1lO7O1ri70,9.10%1r10%
71,0°720° (511H-nuclear magnetic resonance spectrum (100 MHz, in deuterated chloroform) δ (ppm) 13, jlr (/H,s, phenolic on)
.

12, Ir Hiro ('Hp8+ phenolic 0) 1).

lz, zlA (IH, s, phenolic OH), 7.9
0 (IH, dd, H-/), 7. Air (IH.

t, H-2),, 7.32 (IH, t, I(
-J),! , lAr (/ H, br @, H-/
'), j, 2/ (IH, brs, H-7), Hiroshi, J
~3f (3H, H-j', H-2", H-J"a)
, JjtA(/H.

dd, H-J“b), 3.!A (IH, br s
, H-Hiro'), J, J~2. s (s H, H-to
, H-J'.

H-/“), 2.140 (IH, d, H-r
a), ko, /~1, tA(/jH,H-1rb,)1
-/j, H-H-b')-1,OA (JH,d,H-
11 (6) Appearance: Reddish brown powder +71 FD-MS: m/Z As1A (M+/
) + (8) Soluble Soluble in methanol, ethanol, propatool, acetone, ethyl acetate, chloroform, dichloromethane Insoluble in water, hexane, cyclohexane, diethyl ether, petroleum ether Slightly soluble in acidic water, basic water (9) Rf value (Merck silica gel plate AOF
Since the anthracycline compound according to the present invention has an amino group, its acid addition salt exists. Examples of acids in this case include inorganic acids such as hydrochloric acid, sulfuric acid, and 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, Examples include organic acids such as lacrylsulfonic acid.

Production of anthracycline compound/) Overview of the anthracycline compound R of the present invention, N-(
=",3"-alkylidenedioxy)propyl derivatives can be produced by synthetic chemical modification of R m X produced by culturing microorganisms.

c) R2oX R:10X can be obtained from the culture solution of Actinomanula roseoviolaceae R20 strain isolated by the present inventors. Note that RIX is a known substance and is disclosed in West German Published Patent No. 3. As mentioned above, it can also be produced by the method described in the Olko, No. 66j publication.

(1) R strain R discovered by the present inventors as a strain of the genus Actinomadilla that has the ability to produce anthracycline compounds R and NX.
The a stock has the following contents.

■ Origin and deposit number The R1 strain was isolated from soil collected in a vegetable field in Oaza Onodani, Kaho-machi, Kaho-gun, Fukuoka Prefecture, July 1999.
It was deposited with the Institute of Microbial Technology of the Agency of Industrial Science and Technology in Japan, and has been given the number ``Feikoken Bibori No. 7/31''.

■ 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 basal hyphae spread radially on the agar medium surface while branching, and no hyphal division was observed. Aerial hyphae extend their main axis long and branch into short plates at almost right angles (with respect to the main axis) (single-ring branching), and at the tip of the hyphae there is a dense small spiral spore spore (with around 10 or more spores). /~3 turns, diameter 2.0-2
．． jμ), pseudosporangia (diameter Jj~3.!μ) and spore masses are formed.

Spore size is 0. covered by a cylindrical sheath of j~o, rμ,
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 1 width O0S~o, rμ, length 0.7~1,/μ,
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 IIIB.

B) Culture properties Observation results of culture properties (cultivated at 27°C) in polysaccharide medium are as shown in Table/.

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

D) Discussion and Identification This strain has (1) cell wall type I[IB;
2) A spore chain consists of 70 or more spores, D
, (3) It forms pseudosporangia and spore masses, and (4) true sporangia and flagellated spores are not observed, so it is judged that it belongs to the genus Aclinomadura.

Nonomura's search table [Rikou, Vol. n, 77-77m, /ri7
tA year] and described [Rikou, Volume ≠ 9, RiOl, L~Ri 12
p. 197/year], this strain is A. roseoviolaceae (A. roseoviolaceae).

roseoviolacea).

Therefore, this strain A, a standard strain of Roseoviolaceae (KC
CA-/Hiroj (Nonomura A-j):l was cultured under the same conditions and the main properties of both strains were compared. As shown in Table 3, although there are slight differences in bacterial flora color, back color, and optimal growth temperature, they are taxonomically very similar.Therefore, this strain is an actinoma. Actlnomadura ro
aeoviolacsa Nonomuraat 0b
ara /ri7/).

(2) Culture/Production of R20X The anthracycline compound R, 2QX can be produced by culturing R20X-producing bacteria belonging to the genus Actinomagella aerobically in an appropriate medium and collecting the target product from the culture. can.

The medium contains any nutrient source utilized by the R20X-producing bacteria. Specifically, for example, glucose and shakerose as a carbon source.

Maltose, starch, and fats and oils can be used.
Soy flour, cottonseed meal, and meat extract as nitrogen sources.

Organics such as hefton, dried yeast, 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. Also,
If necessary, inorganic salts such as common salt, potassium chloride, and monoheavy metal salts of phosphates can be added. In order to suppress foaming during groove formation, a suitable antifoaming agent such as silicone can also be added according to a conventional method.

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 H''C to 5℃, but 27℃
~30°C is preferred. In this method, the production amount of RrX reaches its maximum in 4 to 7 days for both shaking culture and aeration stirring culture.

In this way a culture enriched with R,26X is obtained. In the culture, a part of R#X exists in the bacterial cells, but most of it exists in the culture P solution.

Any convenient method can be used to harvest RJX from such cultures. One method is based on the principle of extraction.

Specifically, for example, R and X in culture P solution are extracted using a water-immiscible R2oX solvent, such as ethyl acetate, chloroform, butanol, etc. (Culture F solution is a neutral or slightly basic For R:wX inside the bacterial cells, the bacterial mass obtained by filtration, centrifugation, etc. is extracted with chloroform, ethyl acetate, butanol, methanol, ethanol, acetone, hydrochloric acid solution, or acetic acid. It can be recovered by processing with a solution or the like. The culture itself can also be subjected to the above extraction operation without separating the bacterial cells. Countercurrent distribution methods using suitable solvents can also be included in the scope of extraction.

One of the other methods of harvesting RJX from culture is.

Based on the principle of adsorption, RJX-containing substances that are already in liquid form, such as culture P solution or the above-mentioned 5
A suitable adsorbent for the extract obtained by performing the extraction operation.

For example, target R &

By subsequent elution, RJX can be obtained. The R,26X solution obtained in step 5 is concentrated to dryness under reduced pressure to obtain a crude sample of R,21)X.

From the crude sample of RJX obtained in this way, further R,
In order to separate and purify the 20X n product, the above extraction method and adsorption method may be combined as necessary and used as many times as necessary. For example, silica gel, weakly acidic ion exchange resin,
Column chromatography using adsorbents such as activated carbon or gel filtration agents.

Liquid chromatography using a suitable solvent and a countercurrent distribution method can be carried out in combination as appropriate. Specifically, for example, a crude RaX sample is dissolved in a small amount of chloroform, developed with a suitable solvent using a silica gel column to elute the active ingredient, concentrated under reduced pressure, and further developed with TLC. , when scraped and eluted, R,26X
is separated as a single substance, so if this is concentrated to dryness, R27X can be obtained.

The physicochemical properties of RaX obtained in this way are first order.

Physical and chemical properties of RJX (1) Appearance Customers Dark brown powder (2) Melting point 2/3/~13tA”c (decomposition) (
3) Specific optical rotation; [α] is also 5=+2.

(c=o, or, methanol) (4) Molecule t: ≠ Tata,! (5) Elemental analysis calculated value j, 2.J-, 2j, tr λ, ?0 2
1. l”! (*) Analysis value 61, ♂3j, 2 Tako, 7G, 2ri, ≠4 (wa (6) Ultraviolet and visible absorption spectrum See Figure 1 (Power Infrared absorption spectrum (potassium bromide tablets) ) See Figure 2 (8) Nuclear gas resonance spectrum (/ 00hrNh, 3i chloroform/heavy methanol mixture) See Figure 3 (9) Dissolved acidic water, basic water, methanol, ethanol, propatool, acetone, acetic acid ethyl.

Soluble in chloroform, insoluble in water, hexane, cyclohexane, diethyl ether, petroleum ether.

J) Synthetic chemical modification of RIX The anthracycline compound of the present invention can be prepared by reacting R, VX or an acid addition salt thereof with a compound represented by the following formula (n). .

This compound (I[) is a known compound and can be produced from D-mannitol by a known method. That is, 2. D-mannitol in N,N-dimethylformamide in the presence of p-toluenesulfonic acid. Kodimethoxypropane mata is treated with cyclohexanone dimethyl acetal and l2.2.! , 6-di-O-impropylidene-D-nannitol, or 1,2·t,4
Compound (II
) can be easily obtained.

The reaction of R, 26X or an acid addition salt thereof with a compound of formula (I[) is usually carried out in a solvent. Solvents that can be used include acetonitrile, methanol, ethanol, water, chloroform, dichloromethane, carbon tetrachloride,
Benzene, dioxane, tetrahydrofuran, etc., alone or in combination, are preferred, and a mixed solvent of acetonitrile-water-methanol is particularly preferred.This reaction is usually carried out using a reducing agent such as sodium borohydride (NaBHq), sodium cyanoborohydride ( It is desirable to carry out the reaction in the presence of NaBH3CN) or the like. The amount of reducing agent used is not critical, at least 1 mole per mole of RJX, preferably /~! It can be used in molar amounts.

The amount of the compound of formula (II) used is 3 mol or more, preferably 3 mol or more, per 1 mol of RX)X.

Particular preference is given to a ratio of r to /S mol.

The reaction temperature is generally between the freezing point of the solvent used and! It is suitable for layering within the range of θ°C, especially around room temperature.

Under the above reaction conditions, the reaction can be completed in about 7 to 72 hours.

Isolating the N-(2'',j''-alkylidenedioxy)propyl derivative, which is the target compound of the present invention, from the reaction mixture obtained by the reaction of R,2JX with the compound of formula (If) by the method of the present invention. , for purification, known purification methods used in the field of conductor production of anthracycle glycosides, such as chromatography using silica gel, etc.
Yes, you can do this.

N-(2“
The 13''-alkylidenedi-oxy)propyl derivatives can be converted into their acid addition salts by treatment with a suitable acid (described above) according to methods known per se.

Uses of Compounds of the Present Invention The anthracycline compounds of the present invention have strong anticancer activity and are useful compounds as pharmaceuticals.

/) Physiological activity (1) Anti-tumor properties The N-(71,3//-alkylidenedioxy)propyl derivative of the present invention showed significant anti-tumor activity against leukemia in experimental animals. For example, CDFI mice were intraperitoneally transplanted with a suspension of p3ir leukemia cells g/×lo6/mouse, and N-(,2",3"- Alkylidenedioxy)propyl derivatives were administered intraperitoneally or intravenously.

Observation was carried out for 30 days, and the effect was expressed as a survival rate (%), where the number of survival days of the mice in the control group to which physiological saline was administered was taken as ioo, and the results were as follows.

(2) Acute Toxicity (LD50) The LD5° of the N-(u'',3-alkylidenedioxy)propyl derivative of the present invention in rCR mice is as follows.

D5Q N-(co,3-isoprobilyN-(,2",3"-cyclohexy,2))Anthracycline compounds of the present invention, such as antitumor agents, are effective against animal tumors, particularly malignant tumors. It was revealed that it has anti-tumor properties.

Therefore, the N-(2'',3''-alkylidenedioxy)propyl derivatives of the present invention can be used as antitumor agents or tumor therapeutic agents.

The N-(u,J-alkylidenedioxy)propyl derivatives as antitumor agents 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 actually administering an N-(2",3"-alkylidenedioxy)propyl derivative as an antitumor agent, a typical method is to dissolve it in distilled water for injection or physiological saline and inject it. It is mentioned as one of the things. Specifically, in the case of animals, injection methods include intraperitoneal injection, subcutaneous injection, intravascular injection into a vein or artery, and local administration.

The dosage of the N-(,+,3-alkylidenedioxy)propyl derivative is determined by taking into consideration 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 be exceeded. The specific dosage may vary depending on the method of administration, the circumstances of the patient or treated animal, such as age, body weight, sex, susceptible diet, time of administration, concomitant drugs, and the degree 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. v 4 J.

Examples/(Production of R, yx) (1) Preparation of seed mother The medium used was prepared by dissolving the components of the following composition in /liter of water [)87. It is adjusted to -.

Polypeptone / 1 Molasses ground meat extract 1 Iooml of the above medium was dispensed into a soOml Erlenmeyer flask, sterilized, inoculated with 1 platinum loop of Actinomadura roseobilaceae R, 2Q woslant, and kept at 27℃! The seed mother was cultured in a rotary shaker (20 rpm) for one day.

(2) The culture medium used was prepared by dissolving the following components in 1 liter of water and adjusting the pH to 7.

Glucose Jj Soybean Flour 1. Dried yeast
Five liters of the above 0.2% calcium carbonate (precipitated) O9 medium was placed in a 5(1) IJ liter fermenter and sterilized, and three of the above seeds were inoculated. Culture was performed at 27°C for 7 days at aeration rate/V, V, rQ, and rotation speed of 2007 minutes.

(3) RIX collection After culturing, the culture solution was passed through an r-filter to separate the bacterial cells and the r-liquid.

Adjust the pH of the r solution to 2 with IN hydrochloric acid, and add [Diaion Hp
x > JC Mitsubishi Kasei fR) column 10x was adsorbed and died. After washing with distilled water and JO-containing methanol, elution was performed with methanol. Concentrate the eluate and adjust the concentrated solution to pHr.
, j, and extracted three times with a mixed solution of aa form-methanol (li:l). After concentrating this extract, 6 times the amount of hexane was added and the resulting precipitate was dried to obtain a red powder xsom9.

This was further flattened with chloroform in a column (Fucco-
The red fraction was fractionated using nachloroform:methanol=io:i on Gel C-200, JX Lee). After drying both obtained fractions under reduced pressure, TLC (
It was developed with Merck's "Silica Gel AO") and had an Rf value of 0.
The red-orange fraction around ≠3 was scraped off. Both fractions thus obtained were eluted, concentrated, and then recrystallized in chloroform to obtain 10m9 of R,26Xl.

Example a Preparation of N-(z",3"-isopropylideneoxy)propyl R20X RX) -D-
Glyceraldehyde Hiroshi, Oml (0,3 mmol) and Sodium Cyanoborohydride, 7119
(O0l mmol) in acetonitrile/water (///)
was dissolved in a mixed solution of jTnl and added, and the mixture was reacted at room temperature for 6 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and the concentrate was extracted three times with chloroform somt.

The chloroform layer was washed with 30 ml of water. The chloroform layer was dehydrated with anhydrous sodium sulfate and concentrated to dryness. The obtained concentrate was subjected to 7 silica gel column chromatography (
The target product was obtained by applying it to Wakogel C-aOθ) and eluting it with a mixed solvent of chloroform:methanol (iOO:/). Furthermore, it was crystallized from chloroform/hexane to obtain the target product/4A, Pm9 (o, o244 mmol).
was obtained in a yield of 334.

Production of N-(x", 3"-cyclohexylidenedioxy)propyl RIX RIX 37. θm? (o, o7tA mmol) was dissolved in 1 ml of methanol and -2,j-0-cyclohexylidene-D-glyceraldehyde AJOm9 (0,3
7 mmol) and sodium cyanoborohydride, J
mg (o, is mmol) in acetonitrile/water (//
/) was dissolved and added to the mixture, and the reaction was left at room temperature for 5 hours. After the reaction was completed, the reaction solution was concentrated and extracted three times with 3 o ml of chloroform. Subsequently, the chloroform layer was washed with water somt, dehydrated with anhydrous sodium sulfate, and concentrated to dryness. The obtained dried product was subjected to silica gel column chromatography (Wako Gel C-200) and eluted with a mixed solvent of chloroform/methanol (100%) to obtain the desired product. Furthermore, crystallization in chloroform/hexane is performed to obtain the target product ii, /
my (o, 7 mmol) was obtained in a yield of 2J.

[Brief explanation of drawings]

FIG. 1 is a reproduction of the ultraviolet/visible absorption spectrum of RIX. /: Konicacid in MeOH 3: In alkaline MeOH Figure 2 is a reproduction of the infrared absorption spectrum of RIX. FIG. 3 is a reproduction of the 1H-nuclear magnetic resonance spectrum of RX)X. Applicant's agent: Sato - Male Procedural Amendment 4, 1985 1.1 Revision Office Director Michibu Uga 1 Indication of the case 1985 Patent application No. 231973 2 Name of the invention Anthra → Neuclin compound a3 and Its use 3. What is the relationship between the person making the amendment and the case? Patent applicant Rin Beer Co., Ltd. Rei 4
Column 8 of “Detailed description of the invention” in the agent’s specification Contents of amendment

Claims (1)

[Claims] 1. An anthracycline compound represented by the following formula (I) or an acid addition salt thereof. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (I) [In the formula, R represents ▲There are mathematical formulas, chemical formulas, tables, etc.▼ or ▲There are mathematical formulas, chemical formulas, tables, etc.▼. 2. An antitumor agent containing 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) [In the formula, R represents ▲There are mathematical formulas, chemical formulas, tables, etc.▼ or ▲There are mathematical formulas, chemical formulas, tables, etc.▼. ]