JP3068929B2 - New anthracycline antibiotics - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel anthracycline antibiotic having an anticancer effect.

[0002]

BACKGROUND ART As anticancer anthracycline antibiotics, daunomycin (see U.S. Pat. No. 3,616,242) and adriamycin (U.S. Pat. 590,02
These compounds have a broad anticancer spectrum against experimental tumors and are widely used clinically as cancer chemotherapeutic agents.

[0003] However, daunomycin and adriamycin have rather strong anticancer effects, but also have severe side effects such as severe cardiotoxic effects, and are never satisfactory as anticancer agents. Therefore, some anthracycline antibiotics have been proposed by various means such as a fermentation method, a semi-synthetic method, and a microorganism conversion method. For example, JP-B-51-34915 (acracinomycins A and B), JP-A-57-56494 (4-demethoxy-11-deoxydaunomycin, etc.),
No. 23679 (rhodomycin group antibiotics) and the like.

[0004]

As described above, various related compounds have been proposed for anthracycline antibiotics as anticancer agents, and some of them have already been widely used clinically. Some have been subjected to clinical trials. However, there is no satisfactory both toxicity and anticancer effect. In addition, since the results of in vitro tests and animal tests of anticancer drugs do not always directly correlate with human anticancer effects, diversified research is required. For this reason, there is a demand for a proposal of a new class of compounds that are more effective as clinical drugs, with respect to anthracycline antibiotics that have been tentatively evaluated as anticancer agents.

[0005]

Means for Solving the Problems The present inventors have conducted studies to propose a more useful anthracycline antibiotic or a novel compound that can be a synthetic intermediate thereof.
A novel anthracycline antibiotic, which has not been described in the literature, was successfully isolated from a culture of a microorganism belonging to the genus Streptomyces, and the present invention was found to have a strong growth inhibitory effect on experimental tumor cells. completed.

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

Embedded image Wherein Y is a group

Embedded image And a compound represented by the formula: All of these compounds are novel antibiotics which have not been described in the conventional literature and have structural characteristics in the so-called A ring of the anthracycline skeleton.

Hereinafter, among the compounds represented by the formula (I),
formula,

Embedded image 14A9, an antibiotic represented by the formula:

Embedded image 14A10, an antibiotic represented by the formula:

Embedded image Is designated as 14A12.

[0008] The compound of the present invention remarkably suppresses the proliferation of cultured mouse leukemia cells (L1210). L1210 cells were inoculated at 5 × 10 ⁴ cells / ml into RPMI1640 medium (Rosewell Burke Institute) containing 20% calf serum,
The compound of the present invention was added thereto in an amount of 0.0005 to 10.0 μg /
at a concentration of 37 ml.
After culturing for 8 hours, the concentration of 50% growth inhibition relative to the control was determined. In addition, the addition of the substance of the present invention is performed using M / 50 acetic acid (pH
3.0) at a concentration of 1 mg / ml in Dulb
It was diluted with ecco PBS (-) (manufactured by Nissui Pharmaceutical Co., Ltd.) and added. Table 1 shows the results

[0009] As described above, each of these compounds has a high growth inhibitory effect on cultured mouse leukemia cells (L1210), and is useful as an autologous cancer drug.

[0010] The production of these anthracycline antibiotics can be carried out by culturing a strain belonging to the genus Streptomyces and having the ability to produce these antibiotics in a medium comprising an appropriate nutrient source. Among these strains, specific examples include Streptomyces coerleorubub, which is known as an anthracycline antibiotic daunomycin (daunorubicin) -producing bacterium.
idus) 3T-373 strain (FERM BP-165)
Can be mentioned. The isolation method and bacteriological properties of the 3T-373 strain are described in JP-A-59-21394.
In this case, the description is replaced with the description in the specification of the present invention by showing the reference.

The cultivation of the production strain of the present invention can be carried out in a medium composition known per se, which is usually used as a nutrient source for actinomycetes. For example, as a carbon source, glucose, glycerin, sucrose, starch, maltose, animal and vegetable oils and the like can be used, and as a nitrogen source, soybean flour, meat extract, yeast extract, peptone, corn steep liquor, cottonseed meal, fish meal And inorganic nitrogen such as ammonium sulfate, ammonium chloride, sodium nitrate and ammonium phosphate. If necessary, salt, potassium chloride, phosphate, Mg ²⁺ , Ca ²⁺ , Zn ²⁺ , F
^{e 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 the 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. 14A9-14 from the culture
The A12 substance can be isolated and collected by centrifuging the culture after completion of the fermentation or by filtering in the presence of a suitable filtration aid such as diatomaceous earth. Separate into the filtrate. The supernatant or the filtrate is extracted at pH 7 to 9 with an organic solvent such as chloroform, toluene and ethyl acetate. If necessary, the cells are extracted with an organic solvent such as acetone, methanol, ethanol, or butanol.
Each is concentrated to dryness to obtain a red coarse powder.

This is treated by chromatography using an adsorption carrier, for example, a nonionic porous styrene resin or silica gel, or a treatment using an anion exchange resin or a cation exchange resin alone or in an appropriate combination. By using, each of the 14A9 to 14A12 substances can be collected in a pure form.

Further, since the compound of the present invention has an amino sugar as described above, an acid addition salt corresponding to the acid used easily can be produced by a known salt-forming reaction with an inorganic acid or an organic acid. be able to. Acids that can be suitably used include, for example, sulfuric acid, hydrochloric acid, bromic acid, nitric acid, phosphoric acid, acetic acid, propionic acid, maleic acid, oleic acid,
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 / visible light absorption spectrum (hereinafter referred to as UV) and an infrared absorption spectrum (hereinafter referred to as I).
R) and 400 MHz high-resolution proton nuclear magnetic resonance spectrum (PMR) and mass spectrum analysis, and further, a partial decomposition product such as an aglycone portion and a sugar portion obtained by acid hydrolysis was obtained, and these spectra were analyzed. The substance of the present invention may be a compound of the formula (Ia)
It was determined to be a compound represented by (Ic).

The physicochemical properties of the compound of the present invention are described below.

1. (Ia) 14A9 substance (1) Melting point: 184 to 188 ° C (2) [α] ²⁰ _D : + 162 ° (c 0.02, CHC
l ₃ ) (3) Mass spectrum (FAB-MS) = m / z 58
6 (M + H) ⁺ (4) UV spectrum (λmax, nm (E ^1% _1cm ))
a, 90% MeOH 492 (256), 292 (145), 254 (41
3), 234 (720) b, 90% MeOH-0.01 N HCl 492 (254), 292 (145), 254 (41
3), 234 (715) c, 90% MeOH-0.01 N NaOH 604 (184), 564 (233), 296 (12
6), 240 (660) (5) IR spectrum (ν ^{cm -1} _max (KBr)) 3445, 2936, 1732, 1603, 1433,
1402, 1292, 1238, 1196, 1165,
1115, 1015, 986 (6) ¹ H-NMR spectrum (CDCl ₃ , TMS internal standard) 1.36 (d, 3H), 1.7 to 1.8 (2H), 1.
22 (dd, 1H), 2.27 (s, 3H), 2.44
(D, 1H), 2,59 (d, 1H), 3.10 (d,
1H), 3.15 (m, 1H), 3.48 (brs,
1H), 3.72 (s, 3H), 4.16 (q, 1
H), 4.40 (s, 1H), 5.15 (br d, 1
H), 5.46 (br s, 1H), 7.31 (d, 1
H), 7.70 (t, 1H), 7.87 (d, 1H)

2. (Ib) 14A10 substance (1) Melting point: 180-184 ° C (2) [α] ²⁰ _D : + 250 ° (c 0.01, CHC
l ₃ ) (3) Mass spectrum (FAB-MS) = m / z 54
4 (M + H) ⁺ (4) UV spectrum (λmax, nm (E ^1% _1cm )) a, 90% Me0H 492 (228), 292 (131), 254 (37)
8), 234 (659) b, 90% MeOH-0.01N HCl 492 (230), 293 (131), 254 (37
9), 234 (657) c, 90% MeOH-0.01 N NaOH 604 (173), 564 (214), 296 (11
2), 239 (605) (5) IR spectrum (ν ^{cm -1} _max (KBr)) 3436, 2926, 1732, 1605, 1456,
1433, 1402, 1290, 1238, 1196,
1165, 1123, 1015, 986 (6) ¹ H-NMR spectrum (CDCl ₃ , TMS internal standard) 1.35 (d, 3H), 1.42 (s, 3H), 1.6
8 (dd, 1H), 1,78 (td, 1H), 2.23
(D, 1H), 2.34 (dd, 1H), 3.10 (b
rd, 1H), 3,47 (brs, 1H), 3.7
3 (s, 3H), 4.12 (q, 1H), 4.26
(S, 1H), 5.25 (br s, 1H), 5.48
(Br d, 1H), 7.31 (d, 1H), 7.70
(T, 1H), 7.87 (d, 1H)

3. (Ic) 14A12 substance (1) Melting point: 174 to 176 ° C (2) [α] ²⁰ _D : + 200 ° (c 0.02, MeO
H) (3) UV spectrum (λ ^MeOH max, nm
(E ^1% _1cm )) 209 (362), 226 (371), 257 (32
9), 497 (169), 514 (155) (4) IR spectrum (ν ^{cm -1} _max (KBr)) 3400, 2900, 1710, 1600, 1450,
1390, 1280, 1230, 1190, 1160,
1115, 1010, 980 (5) ¹ H-NMR spectrum (CDCl ₃ , TMS internal standard) 1.30 (d, 3H), 1.51 (s, 3H), 1.9
5 (br d, 1H), 2.06 (br t, 1H),
2.23 (d, 1H), 2.37 (dd, 1H), 3.
60 (br d, 1H), 3.67 (br s, 1
H), 4.17 (s, 1H), 4.30 (q, 1H),
5.10 (br d, 1H), 5,50 (br s, 1
H), 7.25 (br s, 1H), 7, 69 (br
s, 1H), 7.75 (br s, 1H)

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

EXAMPLES Example 1 Streptomyces coelle orvidus (Strept)
omyces coeruleubuidus) 3T
One loopful was taken from the YS (0.3% yeast extract, 1% soluble starch, 1.5%, pH 7.2) slope culture of -373 strain, and a 500 ml triangular volume sterilized by dispensing 100 ml of the seed culture medium described below. The flask was inoculated and cultured with shaking at 28 ° C. on a rotary shaker (220 rpm) for 2 days to prepare a seed mother.

Seed medium (W / V%) Soluble starch 0.5% Glucose 0.5% Soybean flour (Essan Meat, Ajinomoto Co.) 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) Next, 100 l of a production medium having the following composition was put into the two sterilized 200-liter tanks, and the seed culture was added to the two tanks.
One liter was inoculated.

Production medium (W / V%) Taiwanese 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 O}} 2.8g, FeSO 4 · 7H 2 O
0.4 g, MnCl ₂ .4H ₂ O 3.2 g, ZnSO ₄
The · 2H ₂ O 0.8 g was dissolved in distilled water 500 ml.

Aeration rate 50 liter / min, stirring 100 rpm
After culturing at 28 ° C. for 6 days, the culture broth turned dark reddish brown. The fermentation was stopped, the culture broth was collected from two tanks (total volume: 180 liters), and the cells were separated by centrifugation into bacterial cells and filtrate. The product in the filtrate was extracted with a total of 50 liters of chloroform. On the other hand, the product of the bacterial cell classification has a total amount of 1
Extraction was performed with 00 liter of acetone, and the extraction supernatant was concentrated under reduced pressure to about 1/3 volume. Then chloroform 20
Extract twice with liter, combine with chloroform extract from the filtrate, and dry under reduced pressure to obtain 120 g of coarse powder.
I 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 adsorbent solution (HP-
20, manufactured by Mitsubishi Kasei Kogyo Co., Ltd.) and eluted stepwise with methanol-acidic water (1: 9-9: 1) to obtain fractions containing 14A12 and 14A9, 14A10 in this order. . The 14A12 fraction was extracted with butanol and concentrated to dryness to obtain 1.4 g of a crude powder. The fraction containing 14A9 and 14A10 was adjusted to pH 8.0, extracted with chloroform, and concentrated to dryness to obtain 3.7 g of a coarse powder.

Example 3 1.4 g of the coarse 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 a column chromatography of 80 g of silica gel. , Chloroform-methanol-water (100: 10: 0.5 to 50: 10: 1 to 20: 1)
0: 1) to give a fraction containing 14A12. After concentration, the fraction was extracted with 1% aqueous sodium hydrogen carbonate and washed with chloroform. After adjusting the pH to 2.5, extract and concentrate with butanol and place in a cool place (5 ° C).
Let stand for days. After the precipitate was collected by filtration, it was dried and 14A12
11 mg of powder were obtained.

Example 4 3.7 g of the crude powder containing 14A9 and 14A10 obtained in Example 2 was dissolved in chloroform-methanol (10: 1), adsorbed on a column of 300 g of silica gel, and mixed with chloroform-methanol-water-. Acetic acid-triethylamine (5
0: 10: 1: 0.5: 0.01 to 40: 10: 1:
0.5: 0.01 to 25: 10: 1: 0.5: 0.0
Elution was performed stepwise in 1) to obtain a fraction having a high content of the above three components. 1% aqueous sodium hydrogen carbonate was added to this fraction, and the organic layer was separated and concentrated to dryness. This dried product is 30
% Acetonitrile water (pH 2.0, pH adjusted with phosphoric acid), and using this solvent as a mobile phase, a preparative HPLC device (main unit: LC-908, manufactured by Nippon Kagaku Kogyo Co., Ltd.); 250 mm), manufactured by Shiseido Co., Ltd.) for 7 times.
14A10 fractions were obtained respectively. In addition, 15% acetonitrile water (pH 2.0, pH adjusted with phosphoric acid) was used as an eluate. 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, concentrated, and then precipitated by adding excess hexane. The precipitate was filtered off, dried and dried.
4 mg and 3 mg of 9, 14A10 powder were obtained, respectively.

──────────────────────────────────────────────────続 き Continued on front page Examiner Mayumi Saito (58) Field surveyed (Int. Cl. ⁷ , DB name) C07H 1/00-23/00 C12P 19/00-19/64 A61P 1/00-43 / 00 BIOSIS (DIALOG) CA (STN) REGISTRY (STN) WPI (DIALOG)

Claims (5)

(57) [Claims] 1. A compound of the general formula: In the formula, Y is a group Or an anthracycline antibiotic or a pharmacologically acceptable addition salt thereof. 2. A compound of the formula: Or an anthracycline antibiotic or a pharmacologically acceptable addition salt thereof. 3. The formula: Or an anthracycline antibiotic or a pharmacologically acceptable addition salt thereof. 4. The formula: Or an anthracycline antibiotic or a pharmacologically acceptable addition salt thereof. 5. Streptomyces (Streptomyc)
es) A microorganism belonging to the genus and having the ability to produce the anthracycline antibiotic according to any one of claims 1 to 4 is cultured, and the anthracycline according to any one of claims 1 to 4 is cultured from the culture. The method for producing an anthracycline antibiotic according to any one of claims 1 to 4, wherein the antibiotic is collected.