JPH05125090A - Novel anthracycline antibiotic - Google Patents

Abstract

PURPOSE:To obtain a novel anthracycline antibiotic useful as a carcinostatic agent. CONSTITUTION:An antibiotic of formula I (Y is formula, II formula III and the like) and a pharmacologically permissible adduct salt thereof. For example, the antibiotic of formula IV (14A5). The compound of formula I is obtained by fermentation of Streptomyces coeruleorubidus-3T-373 strain (FERM BP-165) in a culture medium containing, glucose, glycerol, sucrose or the like as carbon sources, soybean flour, bouillon, yeast extract as nitrogen sources, and when desired, common salt, such as potassium chloride, phosphate salt, Mg<2+>, Ca<2+>, amino acids, vitamins, preferably at 28 deg.C and a pH of 6 to 7 for 6 days.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel anthracycline antibiotic having a carcinostatic action.

[0002]

BACKGROUND OF THE INVENTION As carcinostatic anthracycline antibiotics, daunomycin (see US Pat. No. 3,616,242) and adriamycin (US Pat. 590,02
No. 8, etc.) are known, and these compounds have a broad anticancer spectrum against experimental tumors and are widely used clinically as cancer chemotherapeutic agents.

[0003] Although daunomycin and adriamycin show a considerably strong anticancer action, they have serious side effects such as severe cardiotoxic action and are not completely satisfactory as carcinostatic agents. Therefore, several anthracycline antibiotics have been proposed by various means such as a fermentation method, a semisynthetic method, and a microbial conversion method. For example, JP-B-51-34915 (Aclacinomycin A and B), JP-A-57-56494 (4-demethoxy-11-deoxydaunomycin, etc.), JP-B-60-
No. 23679 (rhodomycin group antibiotics) and the like.

[0004]

As described above, as an anthracycline antibiotic as a carcinostatic agent, various related compounds have been proposed, and some of them have already been clinically widely used. Some are also used in clinical trials. However, none of them are satisfactory in both toxicity and anticancer activity. Moreover, since the results of in vitro tests and animal tests of anticancer agents do not always directly correlate with human anticancer effects, multifaceted research is required. Therefore, regarding anthracycline antibiotics, which have been tentatively evaluated as anticancer agents, it is desired to propose compounds belonging to a new class that are more effective as clinical drugs.

[0005]

Means for Solving the Problems As a result of repeated studies to propose a novel compound which can be a more useful anthracycline antibiotic or its synthetic intermediate,
The present invention was found to be successful in the isolation of a novel anthracycline antibiotic not described in the literature from the culture solution of a microorganism belonging to the genus Streptomyces, and also to find that the compound has a strong growth inhibitory effect on experimental tumor cells. completed.

The novel anthracycline antibiotics provided by the present invention have the general formula:

[Chemical 6] Is a compound represented by. All of these compounds are novel antibiotics which have not been described in the conventional literature and have structural features in the so-called A ring of the anthracycline skeleton.

Among the compounds represented by the formula (I),
formula,

[Chemical 7] The antibiotic shown by 14A5, formula,

[Chemical 8] 14A9, the formula,

[Chemical 9] 14A10, the formula,

[Chemical 10] The antibiotic shown by is called 14A12.

The compound of the present invention markedly suppresses the growth of mouse leukemia cultured cells (L1210). RPMI1640 medium containing 20% calf serum (Rosewell Burk Research Institute) was inoculated with 5 × 10 ⁴ cells / ml of L1210 cells,
The compound of the present invention is added to this in an amount of 0.0005 to 10.0 μg /
It was added at a concentration of ml, and was added at 37 ° C in a carbon dioxide incubator to
After culturing for 8 hours, the 50% growth inhibitory concentration with respect to the control was determined. The addition of the substance of the present invention was carried out by adding M / 50 acetic acid (pH
3.0) at a concentration of 1 mg / ml and then Dulb
It was diluted with ecco PBS (-) (Nissui Pharmaceutical Co., Ltd.) and added. The results are shown in Table 1.

[0009]

[Table 1] As described above, each of these compounds has a high growth-inhibitory effect on mouse leukemia cultured cells (L1210), and is useful as an autologous cancer agent.

Production of these anthracycline antibiotics can be carried out by culturing strains belonging to the genus Streptomyces and having the ability to produce these antibiotics in a medium consisting of an appropriate nutrient source. Among these strains, specific examples include Streptomyces coeruleorubub known as anthracycline antibiotic daunomycin (daunorubicin) -producing bacterium.
idus) 3T-373 strain (FERMBP-165). The method of isolating the 3T-373 strain and the mycological properties are described in JP-A-59-21394, and therefore the description of the present invention is replaced by the reference.

Cultivation of the production strain of the present invention can be carried out in a medium composition which is commonly used as a nutrient source for actinomycetes and is known per se. For example, as the carbon source, glucose, glycerin, sucrose, starch, maltose, animal and vegetable oils can be used, and as the nitrogen source, soybean powder, meat extract, yeast extract, peptone, corn steep liquor, cotton meal, fish meal. And organic substances such as ammonium sulfate, ammonium chloride, sodium nitrate, ammonium phosphate and the like can be used. If necessary, sodium chloride, potassium chloride, phosphate, other Mg ²⁺ , Ca ²⁺ , Zn ²⁺ ,
^{Fe 2+, Cu 2+, Mn 2+} , may be added a divalent metal salts and amino acids and vitamins ^{Ni 2+} and the like.

Fermentation conditions such as temperature, pH, aeration and fermentation time are selected so that the strain used accumulates a maximum amount of the compound. For example, it is advantageous to carry out the fermentation at a temperature of 20 to 40 ° C., preferably 28 ° C., a pH of 5 to 9, preferably 6 to 7, and a fermentation time of 1 to 10 days, preferably 6 days. 14A5-14 from the culture
In order to isolate and collect the A12 substance, the culture after fermentation is centrifuged or filtered in the presence of an appropriate filter aid such as diatomaceous earth to obtain the bacterial cells and the supernatant or Separate into filtrate. The supernatant or filtrate is extracted with an organic solvent such as chloroform, toluene or ethyl acetate at pH 7-9. If necessary, the bacterial cells are extracted with an organic solvent such as acetone, methanol, ethanol or butanol.
Each is concentrated to dryness to obtain a red crude powder.

This is treated by chromatography using an adsorption carrier such as a nonionic porous styrene resin or silica gel, or a treatment using an anion exchange resin or a cation exchange resin may be used alone or in combination. The 14A5-14A12 substances can be collected in pure form by using them.

Since the compound of the present invention has an amino sugar as described above, an acid addition salt corresponding to the acid used can be easily produced by a salt-forming reaction known per se with an inorganic acid or an organic acid. be able to. Acids that can be suitably used include, for example, sulfuric acid, hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, acetic acid, propionic acid, maleic acid, oleic acid,
Examples thereof include palmitic acid, citric acid, succinic acid, tartaric acid, fumaric acid, glutamic acid, pantothenic acid, laurylsulfonic acid, methanesulfonic acid, naphthalenesulfonic acid and the like.

The obtained compound has an ultraviolet and visible light absorption spectrum (hereinafter UV) and an infrared absorption spectrum (hereinafter I
R) and 400 MHz high resolution proton nuclear magnetic resonance spectrum (PMR) and mass spectrum analysis, and from the results of performing spectral analysis of these by obtaining partial decomposed products such as aglycone and sugar moieties obtained by acid hydrolysis. The substance of the present invention has the above formula (Ia)
It was determined to be the compound represented by (Id).

[0016]

[0017]

[0018]

[0019]

Next, the present invention will be described in more detail by way of examples.

【Example】

Example 1 Streptomyces coelre orbidas (Strept
omyces coeuruleorubidus) 3T
-Strain 373 YS (0.3% yeast extract, 1% soluble starch, 1.5%, pH 7.2) One platinum loop was taken from the slope culture, and 100 ml of the seed mother medium described below was dispensed and sterilized in a 500 ml triangle. The flask was inoculated and shake cultured at 28 ° C. for 2 days on a rotary shaker (220 rpm) to prepare a seed mother.

Seed culture medium (W / V%) 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% Second Potassium phosphate 0.1% Magnesium sulfate (including 7H ₂ O) 0.1% Tap water pH 7.4
(Before sterilization) Then, the above seed culture solution was added to 2 200 liter tanks which were sterilized by adding 100 liter of the production medium having the following composition.
Inoculated with liter added.

Production Medium (W / V%) Taiwan Yeast 5.0% Soluble Starch 7.5% Yeast Extract 0.3% Salt 0.2% Calcium Carbonate 0.3% Mineral Solution * 0.125
% (However, V / V%) Tap water pH 8.0
(Before heat _{sterilization) * CuSO 4 · 5H 2 O2.8g} , FeSO 4 · 7H 2
O0.4g, MnCl ₂ .4H ₂ O 3.2g, ZnSO
The ₄ · _2H 2 _O0.8g dissolved in distilled water 500 ml.

Aeration rate 50 liters / minute, stirring 100 rp
When cultured at 28 ° C. for 6 days, the culture broth exhibits a deep reddish brown color. Therefore, fermentation was stopped, the culture broth was collected from two tanks (total volume: 180 liters), and centrifuged to separate the cells and the filtrate. The product in the filtrate was extracted with a total volume of 50 liters of chloroform. On the other hand, the product of the bacterial cell division has a total amount of 1
It was extracted with 00 liter of acetone, and the extract supernatant was concentrated under reduced pressure to about 1/3 volume. Then chloroform 20
120 g of crude powder was obtained by extracting twice with liter, combining with the chloroform extract from the above filtrate, and drying under reduced pressure.
Got

Example 2 100 g of the coarse powder obtained in Example 1 was dissolved in 6 liters of water adjusted to pH 2.0, and 3 liters of adsorbed resin (HP-
20, manufactured by Mitsubishi Kasei Kogyo Co., Ltd., and eluted stepwise with methanol-acidic water (1: 9 to 9: 1) to give 14A12 and 14A5, 14A9, 14A1.
Fractions containing 0 were obtained in this order. The 14A12 fraction was extracted with butanol and concentrated to dryness to obtain 1.4 g of a coarse powder. The fractions containing 14A5, 14A9, and 14A10 were adjusted to pH 8.0, extracted with chloroform, and concentrated to dryness to obtain 3.7 g of crude powder.

Example 3 1.4 g of the crude powder of 14A12 obtained in Example 2 was adsorbed on 10 g of silica gel (Wakogel C-200 (manufactured by Wako Pure Chemical Industries, Ltd.)) and placed on the upper end of column chromatography of 80 g of silica gel. Chloroform-methanol-water (100: 10: 0.5 to 50: 10: 1 to 20: 1)
Elution stepwise with 0: 1) gave a fraction containing 14A12. This fraction was concentrated, extracted with 1% aqueous sodium hydrogen carbonate solution, and washed with chloroform. After adjusting the pH to 2.5, extract and concentrate with butanol, and cool in a cool place (5 ° C).
Let stand for days. The precipitate is filtered off and dried, 14A12
11 mg of powder was obtained.

Example 4 3.7 g of the crude powder containing 14A5, 14A9 and 14A10 obtained in Example 2 was added to chloroform-methanol (1
0: 1), adsorbed on a column of 300 g of silica gel, and chloroform-methanol-water-acetic acid-triethylamine (50: 10: 1: 0.5: 0.01-40: 40).
10: 1: 0.5: 0.01 to 25: 10: 1: 0.
5: 0.01) to elute stepwise to obtain the above-mentioned three-component-rich fraction. 1% aqueous sodium hydrogen carbonate was added to this fraction, the organic layer was separated, and concentrated to dryness. 30% acetonitrile water (pH 2.0, pH with phosphoric acid)
Preparation) and use this solvent as a mobile phase for preparative HP
LC device (body: LC-908, manufactured by Nippon Analytical Industry Co., Ltd .; column: Capsule pack C18 (30 mmφ × 250 m
m), made by Shiseido Co., Ltd.)
Fractions of 14A5, 14A9 and 14A10 were obtained respectively. In addition, 15% acetonitrile water (pH
2.0, pH adjustment with phosphoric acid) was used. After washing each fraction with chloroform, the pH was adjusted to 8.0 and extracted with chloroform. The extract was dried over anhydrous sodium sulfate and concentrated, and then excess hexane was added to cause precipitation. The precipitate was filtered and then dried to obtain 18 mg, 4 mg and 3 mg of 14A5, 14A9 and 14A10 powders, respectively.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Tomio Takeuchi 5-1-11 Higashigotanda, Shinagawa-ku, Tokyo NEW FUJIMANSION 701

Claims (5)

[Claims] 1. A general formula: An anthracycline antibiotic or a pharmacologically acceptable addition salt thereof. 2. The formula: An anthracycline antibiotic or a pharmacologically acceptable addition salt thereof. 3. The formula: An anthracycline antibiotic or a pharmacologically acceptable addition salt thereof. 4. The formula: An anthracycline antibiotic or a pharmacologically acceptable addition salt thereof. 5. The formula: An anthracycline antibiotic or a pharmacologically acceptable addition salt thereof.