JPH064668B2 - New anthracycline antibiotic - Google Patents

Description

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

(Prior Art) Anticancer anthracycline antibiotics include daunomycin (see US Pat. No. 3,616,242) and madriamycin (US Pat. No. 3,590,028), and these compounds have broad anticancer spectrum against experimental ulcer and are widely used clinically as cancer chemotherapeutic agents. However, daunomycin and Adriamycin show a fairly strong anticancer action, but also have serious side effects such as a severe cardiotoxic action, and are not satisfactory as carcinostatic agents. Therefore, a large number of anthracycline antibiotics have been proposed by various means such as a fermentation method, a semisynthetic method, and a microbial conversion method [for example, Japanese Patent Publication No. Sho 51-34915 (Aclacinomycin A and B),
See JP-A-57-56494 (4-demethoxy-11-deoxydaunomycin, etc.), JP-A-56-15299 (rhodomycin group antibiotics), etc.].

(Problems to be Solved by the Invention) As described above, as anthracycline antibiotics as antitumor agents, various related compounds have been proposed, and some of them have already been clinically widely used. It has been used for the test. However, neither toxicity nor anticancer effect is satisfactory. Moreover, since the results of in vitro tests and animal tests do not always correlate immediately with the anticancer activity of humans, antitumor agents require multifaceted research. Therefore, regarding anthracycline antibiotics that are tentatively evaluated as antitumor agents, it is desired to propose compounds belonging to a new class that are more effective as clinical drugs.

(Means for Solving Problems) The present inventors have conducted research to propose a novel compound that can be a more useful anthracycline antibiotic or a synthetic intermediate thereof, and as a result, produce daunomycin and its related compounds. Soil isolates or known strains having the ability are used as mutagens such as ultraviolet rays or N-methyl-N '.
The bacterium obtained by the usual mutation treatment means using -nitro-N-nitrosodaanidin (NTG) has a certain anticancer effect and has a functional group capable of leading to various derivatives, The present inventors have completed the present invention by finding that they produce anthracycline antibiotics that are also useful as synthetic intermediates.

Therefore, the present invention is The present invention provides a novel anthracycline antibiotic.

The compounds of the present invention are novel anthracycline antibiotics not previously mentioned in the literature, which are characterized by consisting of 4-O-methylaclavinone and daunosamine. Below, formula (I)
The compound represented by is referred to as D788-16.

(Action / Effect) D788-16 has a strong growth inhibitory action on cultured mouse leukemia cells L1210 and is useful as an autologous cancer drug.

The action can be easily confirmed by the following test.

Proliferation and Nucleic Acid Synthesis Inhibitory Effect on Mouse Leukocyte L1210 Cultured Cells For example, 5 × 10 ⁴ L1210 cells / ml were inoculated into RPM1164 medium (Rosewell Park Laboratories) containing 20% calf serum, and the substance of the present invention was added thereto It was added at a concentration of 0.001 to 0.25 μg / ml and cultured at 37 ° C. for 48 hours in a carbon dioxide gas incubator, and the 50% growth inhibitory concentration relative to the control was determined. In addition, the addition of the substance of the present invention is 1 m in M / 50 acetic acid (pH 3.0).
After dissolving at a concentration of g / ml, it was diluted with Dulbecco PBS (-) (Nissui Pharmaceutical) and added.

Furthermore, the above-mentioned L1210 cultured cells were added to RPM11 containing 10% fetal calf serum.
After suspending in 640 medium at 8 × 10 ⁵ cells / ml and culturing at 37 ° C. in a carbon dioxide incubator for 1.5 hours, various concentrations of the substance solution prepared above were added, After 15 minutes, ¹⁴ C-uridine (0.05 μCi / ml) or ¹⁴ C
-Add thymidine (0.05 μCi / ml) and add 6 at 37 ° C.
Incubated for 0 minutes. Cold 10% trichloroacetic acid (TC
A) was added to stop the reaction, and at the same time, the acid insoluble matter was precipitated, accumulated by centrifugation, washed twice with 2 ml of cold 5% TCA, dissolved in formic acid, and measured for radioactivity. 50% from the radioactivity uptake rate for the control group without additives
The uptake inhibitory concentration was determined. The results are shown in Table 1.

The antibiotic D788-16 of the present invention can be prepared by using, for example, ultraviolet rays or N-methyl-, as a modifier, a soil isolate or a known strain having the ability to produce daunomycin or carminomycin belonging to the genus Actinomyces and its related compounds. A mutant strain capable of producing the compound D788-16 of the present invention, which is easily isolated by a conventional mutagenesis treatment using N'-nitro-N-nitroguanidine (NTG), is cultivated in a medium containing an appropriate nutrient source. It can be done by doing. Specific examples of these mutants include:
New anthracycline antibiotic D788-5 (Japanese Patent Application No. 59-3
8626), Streptomyces species D788,4
A mutant strain obtained by treating the L-660 strain (Microtechnology Research Institute, No. 7459) with NTG and the resulting strain is YDK-18 strain.

This strain was deposited on September 3, 1986, at the Institute for Microbial Industry, Institute of Industrial Technology, as Micromachine Research Institute No. 8952 (FERM P-8952). The mycological properties of the YDK-18 strain are shown below.

(i) Morphology A linear aerial bacterium system extends from a branched basal bacterium system, and no limbus branches are observed. The matured spore chain has 10 or more spore chains, and the spore size is 0.6 to 0.8 ×.
The surface of spores is smooth at about 0.9 to 2.5 microns.
Ascospores and flagella spores are not found.

(ii) Regarding the growth state color description in various media The standard shown in () is HD Tresner &
EJBakus, aystem of color wheels for streptomyc
es taxonomy (J.Appl.Microbiol. 11 volumes, 335-33
8 pages, 1963) and supplementally, "The color standard is used" published by the Japan Color Research Institute.

(iii) Physiological properties (1) Growth temperature range (using yeast and malt agar medium, pH
Experiment was conducted at each temperature of 20 ° C., 28 ° C., 33 ° C., 37 ° C. and 42 ° C.): Growth was observed from 20 ° C. to 37 ° C.

(2) Liquefaction of gelatin (glucose, peptone, gelatin
Cultivated at 20 ℃ using medium. Positive (3) Hydrolysis of starch (starch, inorganic salt agar culture)
(Ground): Positive (4) Coagulation and peptonization of defatted milk: slight coagulation and peptization
Positive mutation (5) Growth of melanin-like pigment (tryptone, yeast extract)
, Iron agar): positive (iv) availability of various carbon sources (fridham, madrieve agar)
On the medium):^* L-arapinose +^*+ Grows well D-xylose + soil grows slightly D-glucose + D-fructose ± sucrose ± inositol + L-rhamnose + raffinose ± D-mannite ± The culture of the strain used for the production of D788-16 according to the present invention is , Free
A medium known per se that is usually used as a nutrient source for filamentous fungi
It can be done in composition. For example, as a carbon source,
Glucose, glycerin, sucrose, starch, maltose, animal
Vegetable oil can be used, and soybean powder, meat
Kiss, yeast extract, peptone, corn stapler
, Cottonseed meal, organic nitrogen sources such as fish meal, and ammonium sulfate
Aluminum, ammonium chloride, sodium nitrate, phosphoric acid
Inorganic nitrogen such as ammonium can be used. Also if necessary
Salt, potassium chloride, phosphate and other Mg ， Ca ，
Zn ， Fe ， Cu  Mu Or Ni Divalent metal salts such as
In addition to adding amino acids and vitamins, foaming during fermentation
To prevent this, use various commercially available antifoaming agents such as silicone.
It can also be added as appropriate.

Fermentation conditions such as temperature, pH, agitation and fermentation time are selected so that the strain used accumulates the maximum amount of the compound. For example, the temperature is 20-40 ℃, preferably 28 ℃, pH
Is 5 to 9, preferably 6 to 7, and the fermentation time is 3 to
It is advantageous to carry out the fermentation for 10 days, preferably 6 days.

In order to isolate and collect the D788-16 substance from the culture solution, the culture after the fermentation is separated by centrifugation or by passing it in the presence of an appropriate super-auxiliary agent such as diatomaceous earth. Separate into supernatant or liquid. The supernatant is extracted with an organic solvent such as chloroform, toluene, ethyl acetate, n-pentanol or the like at pH 7-9. If necessary, acetone,
Extract with an organic solvent such as methanol, ethanol or n-butanol. D788-16 is prepared by treating it with an adsorption carrier such as a synthetic adsorption resin or chromatography using silica gel, or by using anion exchange resin or cation exchange resin alone or in combination. The substance can be purified and collected.

Hereinafter, the present invention will be described in detail with reference to examples.

Example 1 YS (0.3% yeast extract, 1% of Streptococcus spysis D788, YDK-18 strain (Microtechnology Research Institute, No. 8952))
Soluble starch, 1.5% agar, pH 7.2) A platinum loop was taken from the slant culture, and 100 ml of the seed mother medium described below was dispensed and inoculated into a 500 ml Erlenmeyer flask, which was 28 ° C, rotary shaker (220 rpm). ) Was shake-cultured for 2 days to prepare a seed mother.

Seed culture medium Soluble starch 0.5% Glucose 0.5% Soybean flour (Essan Meat, Ajinomoto Co., Inc.) 1.0% Food salt 0.1% Dibasic potassium phosphate 0.1% Magnesium sulfate (including, 7H ₂ O) 0.1% tap water pH 7.4 (before sterilization) Then, a production medium 15 having the following composition was added and sterilized 3
0 volume jar fermenter-1 to 7 seed culture medium
50 ml (corresponding to 5%) was added and inoculated.

Production medium Soluble starch 5% Maltose 3% Dry yeast 3% Soybean flour (essan meat, supra) 2% Yeast extract 0.2% Food salt 0.1% Magnesium sulfate (including 7H ₂ O) 0.1% Carbonate Calcium 0.2% Mineral solution ^* 0.05% Water water pH7.4 (before sterilization) Aeration rate 15 / min, stirring 350 rpm / min, 28 ℃, 6
Incubate for a day. The culture solution is collected from the jar fermenter and centrifuged to separate the cells and the supernatant. Acetone 8 was added to the cells, and the mixture was thoroughly stirred and filtered to obtain an acetone extract. After concentrating this to about 2 under reduced pressure, the pH was adjusted to 8.0 with 4N caustic soda and extracted with chloroform 4 (total amount). On the other hand, regarding the culture supernatant, after adjusting the pH to 8.0 with 4N caustic soda,
Extract twice with chloroform. Chloroform extraction from the cells and chloroform extract from the supernatant are mixed and concentrated to a small amount. Excess n-hexane was added thereto to precipitate the extract, and the extract was dried to obtain a crude powder (3.4 g) containing D788-16 substance.

Example 2 3.4 g of the crude powder obtained in Example 1 was dissolved in 500 ml of chloroform, and this was extracted 3 times with 300 ml of 0.1 M citrate buffer. Mix the extraction aqueous solutions and add 300 ml of this toluene.
It was washed twice with. Next, the pH of the water tank was adjusted to 8.0 by adding 4N-caustic soda, and the mixture was diluted with 300 ml of chloroform to 2 times.
Extracted twice. 983mg of partially dried yellow powder
Got

Example 3 200 mg of the partially purified powder obtained in Example 2 was subjected to preparative high performance liquid chromatography as described below. That is, the whole amount was dissolved in 9 ml of the following liquid developing solvent and chromatographed by dividing into 1.5 ml by 6 times.

High-performance liquid chromatography conditions Column: YMC-Pack S343 ODS I-15 (φ2.0mm × 250mm) Pump: Waters M-6000A Detector: Waters Model 440 (λ 285nm used) Developing solvent: Acetonitrile-0.01M camphor-sulfonic acid (PH 4.1) (40:60) Flow rate: 5 ml / min Fraction: 7 ml Fractions 3 to 14 were collected and mixed for 6 times. After adding 4N caustic soda solution to adjust the pH to 8.0, the total amount of chloroform is 5
It was extracted with 00 ml. After washing with water and concentration to dryness, 134 mg of D788-13 powder having a purity of about 90% was obtained.

Example 4 134 mg of the powder obtained in Example 3 were then purified by preparative silica gel thin layer chromatography. Preparative silica gel thin layer plate PF ₂₅₄ (Merck: 20 cm x 20 c)
m) A sample solution dissolved in chloroform-methanol (15: 1) over the entire width was applied to the bottom 2 cm of 20 sheets, air-dried, and then chloroform-water-acetic acid-ammonia water (120:
It was developed in the 50: 5: 1: 1) system. The D786-16 section was scraped off and eluted with a mixed solution of chloroform-methanol (4: 1). The solution was concentrated to dryness, 30 ml of 0.1 M acetic acid buffer (pH 3.0) was added thereto for dissolution, the insoluble matter was removed by centrifugation, and the mixture was washed twice with 10 ml of toluene. The pH was adjusted to 8.0 with 4N caustic soda and extracted with chloroform. The chloroform extraction layer was washed with water, dehydrated by adding Glauber's salt, and concentrated to a small amount. Approximately 10 volumes of n-hexane was added to cause precipitation, which was collected by centrifugation and vacuum dried to obtain 9
86 mg of 788% pure D788-16 was obtained.

The physicochemical properties of the D788-16 compound obtained in the above example are as follows.

A) Property: Yellow powder B) Melting point: 138-141 ° C (decomposition) C) ▲ [α] ²³ _D ▼: + 106 ° (C0.02 in chloroform) D) UV-visible absorption spectrum: E) Infrared absorption spectrum (KBr) (υ cm ^-1 ): 3430,2950,1740,1680,1640, 1595,1450,1390,1300,1255, 1130,1020,990,850,760 F) FD-MS spectrum: m / z 556 (M + H) ⁺ (m.w555 as C ₂₉ H ₃₃ O ₁₀ N) 〃 390 (M-daunosamine)

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Tomio Takeuchi 5-1-11 Higashigotanda, Shinagawa-ku, Tokyo (72) Inventor Hamao Umezawa 4-23 Toyohitakita, Nerima-ku, Tokyo Examiner Hiroshi Taniguchi (56) References JP-A-60-185797 (JP, A) JP-A-60-185796 (JP, A) JP-A-57-114547 (JP, A)

Claims (1)

[Claims] 1. A formula A novel anthracycline antibiotic represented by.