JPH051276B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to novel anthracycline antibiotics, and more particularly to formulas In the formula, Y is a group

[Formula] or

The present invention relates to a novel anthracycline antibiotic represented by the formula: As anthracycline antibiotics, 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. has a broad anticancer spectrum against experimental tumors and is widely used clinically as a cancer chemotherapeutic agent. However, although daunomycin and adriamycin exhibit fairly strong anticancer effects, they are by no means satisfactory, and attempts have been made to create various related compounds by various means such as fermentation methods, semisynthetic methods, and microbial conversion methods. , some proposals have already been made [for example, Japanese Patent Publication No. 51-34915 (Aclacinomycin A and B), F. Arcamone, Topics in
Antibiotic Chemistry, Vol. 2, pp. 102-279,
Published by ELLIS HORWOOD LIMITED, 1983
-56494 (4-demethoxy-11-deoxydaunomycin, etc.), JP-A-56-15299 (rhodomycin group antibiotics), etc.]. The present invention was also completed as a result of extensive searches for products of soil-isolated bacterial strains or mutant-treated strains thereof, in order to provide useful anthracycline antibiotics. That is, the present inventors have consistently searched for new anthracycline compounds produced by microorganisms and continued research on their biosynthesis. The present inventors discovered and proposed the fact that a new anthracycline antibiotic, which the present inventors named carborubicin, is produced and accumulated in the precursor of anthracyclinone, which has a carboxyl group at the 10th position of the anthracyclinone bone nucleus. (Refer to the application specification of Application No. 117215/1983). The present invention produces 11-deoxycarborubicin, that is, Y in formula (1) is a

Streptomyces D788, a newly isolated daunomycin- and baumycin-producing strain from soil, from the viewpoint that it is possible to obtain the substance represented by the formula
(Streptomyces D788) strain, and as a result of repeated searches, one of the mutant strains was found to be based on the 11-deoxycarborubicin and Y in (1) above.

The present invention was completed by discovering that the substance represented by the formula can be simultaneously produced and accumulated. Among the compounds of formula (1) provided by the present invention, Y is a group

Compounds and formulas when representing [Formula] The compound shown is an anthracycline bone nucleus.
It has a structural feature in that it has a carboxyl group at the 10th position, and Y is a group.

Compounds and formulas when representing [Formula] The compounds shown are structurally characterized by having a double bond in the A ring of the anthracycline bone core, and both are novel antibiotics that have not been described in conventional literature. Hereinafter, in this specification,
The antibiotic of formula (1-a) is designated as D-788-3,
The antibiotic of formula (1-b) is designated as D-788-4. Substances D788-3 and D788-4 have a proliferation inhibiting effect on cultured leukocyte L1210 as described below, and are compounds that can be used as anticancer agents. In particular, D788-3 has a free carboxyl group in the anthracyclinone bone core, as described above, and therefore exhibits higher water solubility than related compounds such as daunomycin and adriamycin. Therefore, the present compound itself and the aglycone obtained by its hydrolysis treatment are compounds that are also useful as synthetic intermediates for new types of anthracycline antibiotic derivatives. According to the present invention, D788-3 and D788-4 are produced using a soil-isolated strain or a known strain belonging to the genus Actinomycetes and having the ability to produce daunomycin or carminomycin and related compounds thereof, as a mutagen. , for example, by culturing the D788-3 producing strain isolated by conventional mutagenesis treatment using UV or N-methyl-N'-nitro-N-nitrosoguanidine (NTG) in a suitable nutrient medium. I can do it. Suitable examples of these production strains include mutant strains obtained by mutating a soil-isolated strain, which the present inventors named Streptomyces D788, with NTG.
One example is Streptomyces D788ap.KL-330. This strain was deposited with the Institute of Microbiology, Agency of Industrial Science and Technology on February 20, 1982 as FERM P-7458. ing. The mycological properties of strain KL-330 are shown below. (i) Morphology Branched basal mycelia form straight or slightly wavy aerial hyphae. (Section
considered to be classified as Retinacu-liaperti). Whorled branches are not observed. More than 10 spores can be counted in a mature spore chain, and the size of the spores is about 0.6-0.8 x 0.8-2.6 microns, and the surface of the spores is smooth. Asci and flagellar spores are not observed. (ii) Growth status on various media Regarding color descriptions: Standards shown in parentheses are H.
D. Tresner &. System of color by EJ Backua
wheels for Streptomycete taxonomy (J.
Appl. Microbiol. Vol. 11, pp. 335-338, 1963), and supplementary "Color Standard" published by Japan Color Research Institute was also used. (iii) Growth status in each of the following media. (Culture at 28℃ unless otherwise specified)

[Table] (iv) Physiological properties (1) Growth temperature range: (using yeast/malt/agar medium, pH 6.0, 20℃, 28℃, 30℃, 37℃)
(experiments were carried out at temperatures of 20°C and 42°C) Growth was observed from 20°C to 37°C. It does not grow at 42℃. (2) Liquefaction of gelatin: Positive (cultured at 20℃ using glucose/peptone/gelatin medium) (3) Hydrolysis of starch: Positive (starch/inorganic salt agar medium) (4) Coagulation/coagulation of skim milk Peptonization: Both negative. (5) Production of melanin-like pigments: (tryptone, yeast, broth, peptone, yeast, iron,
(Using agar and tyrosine agar medium) Positive results in both media. (v) Availability of various carbon sources: (on Fridham-Godelive agar medium) 1 L-arabinose positive 2 D-xylose 〃 3 D-glucose positive 4 D-fructose 〃 5 Sucrose 〃 6 Inositol 〃 7 L-rhamnose negative 8 Raffinose 〃 9 D-Mannite Positive The cultivation of the D788-3 producing strain according to the present invention can be carried out in a culture medium composition known per se that is commonly used as a nutrient source for actinomycetes. For example, carbon sources include glucose, glycerin, sucrose,
Starch, maltose, animal and vegetable oils, etc. can be used, and nitrogen sources include organic substances such as soybean flour, meat extract, yeast extract, peptone, cornstap liquor, cottonseed meal, and fish meal, as well as ammonium sulfate,
Inorganic nitrogen such as ammonium chloride, sodium nitrate, and ammonium phosphate can be used. Also, add salt, potassium chloride, phosphate, etc. as necessary.
In addition to adding divalent metal salts such as Mg ⁺⁺ , Ca ⁺⁺ , Zn ⁺⁺ , Fe ⁺⁺ , Cu ⁺⁺ , Mn ⁺⁺ or Ni ⁺⁺ , amino acids and vitamins, to suppress foaming during fermentation. , for example, silicone (manufactured by Shin-Etsu Chemical KK, -
An antifoaming agent such as KM75 (trademark) can also be added as appropriate. Fermentation conditions such as temperature, PH, aeration agitation and fermentation time are selected such 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. To isolate and collect D788-3 from the culture,
After completion of fermentation, the culture is separated into bacterial cells and a supernatant or filtrate by centrifugation or by filtration in the presence of a suitable filter aid such as diatomaceous earth. If necessary, the bacterial cells are extracted using a water-miscible organic solvent such as acetone, methanol, ethanol, or butanol, and the concentrated solution is mixed with the filtrate. By treating this mixed solution by chromatography using an adsorption carrier such as a synthetic adsorption resin or silica gel, or by using treatment using an anion exchange resin or a cation exchange resin alone or in appropriate combinations, D788-3 can be harvested in pure form. On the other hand, D788-4 is
During the isolation and purification process of D788-3, it can be isolated as a minor component, but since it corresponds to the decarboxylation and dehydration product of D788-3, the lees preparation or purified product of D788-3 can be isolated using N,N-dimethylformamide. (DMF), dimethyl sulfoxide (DMSO), acetone, methanol, etc., or a solution containing 10 to 80% water, and adjust the pH to 2 to 9.
D788 by stirring for 0.5-4 hours at 20-60℃
-4, extracted from the reaction solution using an organic solvent such as chloroform, and if necessary, purified by treatment such as silica gel chromatography to obtain a pure product. Next, the specific usefulness of D788-3 and D788-4 according to the present invention will be described. This substance has the effect of suppressing the proliferation and nucleic acid synthesis of cultured mouse leukemia cells L1210. Contains e.g. 20% calf serum
To RPM11640 medium (Rose Wellberg Institute)
L1210 cells were inoculated at 5 x ¹⁰ cells/ml, this substance was added at a concentration of 0.002 to 0.25 μg/ml, and cultured at 37°C in a carbon dioxide gas incubator to 50% that of the control group.
The growth inhibition concentration was determined. Furthermore, the above L1210 cultured cells were added to RPM11640 medium containing 10% calf serum 5x.
After suspending at ¹⁰⁵ cells/ml and culturing in a carbon dioxide incubator at 37°C for 1 to 2 hours, this substance was added at various concentrations, and 15 minutes later, ¹⁴ C-uridine ( 0.05 μCi/ml) or ¹⁴ C-thymidine (0.05 μCi/ml) were added and cultured at 37° C. for 60 minutes. Add cold 10% trichloroacetic acid to the reaction solution to stop the reaction, and at the same time precipitate acid-insoluble materials.
After washing twice more with cold 5% trichloroacetic acid, it was dissolved in formic acid, radioactivity was measured, and the 50% uptake inhibition concentration was determined from the radioactivity uptake rate relative to the control group without additives. The results are shown in the table below.

[Table] As shown in the above results, D788-3 and
D788-4 inhibits the proliferation of murine leukemia cell L1210 at extremely low concentrations, and at the same time inhibits RNA and
It has the effect of inhibiting DNA synthesis, but compared to the typical anthracycline antibiotic daunomycin, both have a weaker growth inhibiting effect on murine leukemia cells L1210, but have a stronger nucleic acid synthesis inhibitory effect, especially D788-3. is a compound that has a stronger DNA synthesis inhibitory effect than RNA synthesis inhibitory effect, and is expected to be used as an anticancer drug. The present invention will be explained in more detail below with reference to Examples. Example 1 Streptomyces D788,
YS (0.3
% yeast extract, 1% soluble starch, 1.5% agar,
PH7.2) Take a loopful from the slant culture, inoculate it into a sterilized 500 ml Erlenmeyer flask by dispensing 100 ml of the following seed culture medium, and culture with shaking in a rotary shaker (220 rpm) at 28°C for 2 days. Created a mother. Seed medium Soluble starch 0.5% Glucose 0.5% Esunmeat (soybean flour, manufactured by Ajinomoto Co.) 1.0% Yeast extract 0.1% NaCl 0.1% K ₂ HPO ₄ 0.1% MgSO ₄・7H ₂ O 0.1% Tap water PH7.4 (sterilized) Next, 750 ml (equivalent to 5%) of the above-mentioned seed culture solution was added to each sterilized 30-volume jar fermenter to inoculate them into two sterilized 30-volume jar fermenters containing production medium 15 having the following composition. Production medium Taiwanese yeast 5% Soluble starch 7.5% Yeast extract 0.3% NaCl 0.2% CaCO ₃ 0.3% Mineral mixture * 0.06% Tap water PH8.2 (before heat sterilization) * CuSO ₄・5H ₂ O 2.8g, FeFO ₄・7H ₂ O 0.4
g, MnCl ₂・4H ₂ O 3.2g, ZnSO ₄・7H ₂ O 0.8
g dissolved in 500ml of distilled water. Aeration rate 5/min, stirring 450 rpm, 28℃,
After culturing for 100 hours, the culture solution turned dark yellow, and approximately 180 μg of D788-3 was accumulated in 1 ml of the culture solution. To prepare this substance in the culture solution, add 1 ml of 1M citrate buffer (PH3.5) to 1 ml of the culture solution, add 2 ml of acetone, stir, leave at room temperature for 1 hour, and collect the supernatant by centrifugation. Spot 10 to 30μ of the silica gel plate (F254, manufactured by Merck & Co., Ltd.) at a position 2 cm from the bottom edge of the plate, develop with chloroform/methanol/water/acetic acid (120/40/3/0.4), and remove the yellow color of D788-3. Spot (Rf value: 0.2) for thin layers, chromato scanner (Shimadzu CS)
-910 type) at a wavelength of 430 nm, and quantified by calculation from a standard curve. After 140 hours of culture, the fermentation is stopped, and the culture solution is collected from two jar fermenters (total volume 27), adjusted to pH 1.8 with concentrated sulfuric acid, and separated into bacterial cells and filtrate by centrifugation. The bacterial cell fraction is extracted with a total volume of 8 volumes of acetone, and the extracted supernatant is concentrated under reduced pressure to approximately 1/3 volume. After mixing this with the previous filtrate and adjusting the pH to 2.3 using 4N caustic soda, Diaion HP-20 (synthetic adsorption resin, manufactured by Mitsubishi Chemical Corporation)
column. Wash with pH2 acidic water and then elute the yellow colored fraction with 50% acetone water (PH2.5) at approximately 2.5. The eluate was concentrated to approximately 1/2 volume, the pH was adjusted to 8.5 with 4N caustic soda, and the solution was washed and extracted twice with 1 part of chloroform. Adjust the pH of the aqueous layer containing D788-3 to 2.5 with 6N hydrochloric acid,
Extraction was performed with the same volume of n-butanol, and the extract was concentrated to dryness under reduced pressure to obtain 6.23 g of D788-3 crude powder. Example 2 2.5 g of the crude powder obtained in Example 1 was dissolved in 20 ml of a chloroform-methanol-water (100:10:0.5) mixture, suspended in the same solvent, and packed in a silica gel column (φ40 mm; 80 g Wako gel). C-200 (manufactured by Wako Pure Chemical Industries, Ltd.)) and developed with approximately 300 ml of the same solvent system, and then the composition ratio of chloroform in the developing solvent was sequentially reduced to 90, 80, 70, and 60. Expanded to approximately 300ml. D788-3 is a composition ratio
It was eluted with a solvent system of 60:10:0.5, followed by thin layer chromatography (described above), and the eluted fraction containing only D788-3 was collected and concentrated to dryness under reduced pressure to obtain 2.1 g of powder. . This was dissolved in a dilute aqueous sodium bicarbonate solution, extracted and washed with chloroform, and then extracted with 6N
Adjust the pH to 2.5 with hydrochloric acid and extract with n-butanol. The residue was concentrated to dryness under reduced pressure and vacuum dried in a desiccator to obtain 1.56 g of pure D788-3. Example 3 The chloroform-washed extract containing D788-4 from Example 1 was washed with water and concentrated to dryness under reduced pressure, resulting in a yellow powder.
1.3g was obtained. This was dissolved in 10 ml of a chloroform/methanol/water (100/10/0.5) mixture.
This was suspended in the same solvent and packed in a silica gel column (20g Wakogel C-200 (described above)), developed with the same solvent system, and then chloroform/methanol/
D788-4 was isolated and eluted by sequential development with water (90/10/0.5) and (80/10/0.5). After washing the elution area with water, drying with Glauber's salt and concentrating to dryness under reduced pressure, 27mg of
A pure D788-4 product was obtained. Example 4 Dissolve 300 mg of D788-3 obtained in Example 2 in 250 ml of 50% acetone water, and PH with 4N sodium hydroxide.
7.0 and stirred and heated at 50℃ for 3 hours to form D788-4.
It was converted to . After concentrating the reaction solution under reduced pressure and distilling off the acetone, the pH was adjusted to 8.0 with 4N sodium hydroxide.
and extracted three times with 100 ml of chloroform.
The extracts were collected, washed with water, dried over Glauber's salt, and concentrated to dryness under reduced pressure to obtain 200 mg of a yellow crude powder of D788-4. Silica gel (7.5 g, Wakogel C-
200) Perform column chromatography and use the developing solvent chloroform/methanol/water (80/10/0.5).
The D788-4 fraction eluted was collected, washed with water, dried with Glauber's salt, and concentrated to dryness to obtain 55 mg of pure D788-4. Example 5 60 mg of D777-3 obtained in Example 2 was dissolved in 50 mg of 0.1N hydrochloric acid.
ml, heat decomposed at 85° C. for 15 minutes, extracted twice with 50 ml of chloroform, and concentrated to dryness to obtain a crude aglycone powder. Dissolved in a small amount of chloroform,
The aglycone was purified and isolated by adsorbing it on a silica gel column (Wako gel (described above) and developing with chloroform/methanol (100/1) and then with chloroform/methanol (70/1). The aglycone-containing elution fraction was washed with water and dried with Glauber's salt. After drying under reduced pressure, 18 mg of aglycone was obtained. D788-3 and D788 obtained in the above example
The aglycones of -4 and D788-4 exhibit the following physicochemical properties. D788-3 (A) Melting point 186-187℃ (decomposed) (B) [α] ²⁵ _D = +120° (C0.018, methanol) (C) Ultraviolet/visible absorption spectrum (in methanol) λmax nm (E1% 1cm ): 206 (399), 228
(594), 260 (406), 432 (205) (D) Infrared absorption spectrum (KBr) (cm ^-1 ) 1700, 1670, 1620, 1380, 1290, 1010, 980,
760. (E) Proton nuclear magnetic resonance spectrum (pyridine-D ₅ ) (δ, ppm) 1.29 (3H, t, J = 7.5 H-14) 1.48 (3H, d, J = 6.5 H-6') 1.75-2.2 ( 2H, m, H-13) 2.25-2.7 (4H, m, H-2', H-8) 3.98-4.2 (1H, m, H-5') 4.27 (1H, bs, H-4') 4.38 (1H, s, H-10) 5.17 (1H, m, H-1') 5.38 (1H, m, H-7) 7.3 (1H, d, J=8 H-3) 7.63 (1H, s, H -11) 7.63 (1H, t, J=8 H-2) 7.77 (1H, d, J=8 H-1) D788-4 (A) Melting point 113-115℃ (decomposition) (B) [α] ²⁵ _D = +299° (C0.019, methanol) (C) Ultraviolet/visible absorption spectrum (in methanol) λmax nm (E1% 1cm): 225 (537), 260 (466),
293 (479), 447 (298) (D) Infrared absorption spectrum (KBr) (cm ^-1 ) 1670, 1640, 1615, 1590, 1380, 1280,
1010, 980, 750. (E) Proton nuclear magnetic resonance spectrum (CDCl ₃ ) (δ, ppm) 1.20 (3H, t, J = 7.5 H-14) 1.32 (3H, d, J = 6.5 H-6') 1.4-1.7 (2H, m, H-2') 2.33 (2H, q, J=7.5' H-13) 2.5-2.7 (2H, m, H-8) 3.4 (1H, bs, H-4') 3.98 (1H, q, J=6.5 H-5') 4.22 (1H, m, H-3') 5.3 (2H, bs, H-7, H-1') 6.44 (1H, bs, H-10) 7.28 (1H, dd, J=8, 2 H-3) 7.6 (1H, s, H-11) 7.66 (1H, t, J=8 H-2) 7.82 (1H, dd, J=8, 2 H-1) D788-3 Aglycon (A) Melting point 160-162℃ (decomposed) (B) [α] ²⁵ _D = +230° (C0.014, methanol) (C) Ultraviolet/visible absorption spectrum (in methanol) λmax nm (E1% 1cm): 205 (531), 229 (935),
260 (658), 290 (291), 433 (340) (D) Infrared absorption spectrum (KBr) (cm ^-1 ) 1700, 1670, 1615, 1280, 1210, 1020, 750. (E) Proton nuclear magnetic resonance spectrum ( _CDCl3 - _CD3OD ) (δ, ppm) 1.12 (3H, t, J=7.5 H-14) 1.5-2.0 (2H, m, H-13) 2.2 (1H, dd, J=16, 1.5 H-8) 2.55 (1H, dd, J=16, 5 H-8) 4.03 (1H, s, H-10) 5.29 (1H, dd, J=5, 1.5 H-7 ) 7.28 (1H, dd, J=8, 2 H-3) 7.65 (1H, t, J=8 H-2) 7.71 (1H, s, H-11) 7.79 (1H, dd, J=8.2 H- 1)

Claims (1)

[Claims] 1 formula A novel anthracycline antibiotic, wherein Y represents a group [formula] or [formula]. 2 formulas A novel anthracycline antibiotic according to claim 1. 3 formulas A novel anthracycline antibiotic according to claim 1.