JPH08280398A - New anthracycline antibiotic substance - Google Patents

Abstract

PURPOSE: To obtain a new anthracycline antibiotic substance, capable of manifesting proliferation suppressing actions on a murine leukemic cell (L1210) and useful as an antitumor agent, etc., by culturing a specific microorganism belonging to the genus Streptomyces in a culture medium. CONSTITUTION: A new anthracycline antibiotic substance of the formula (R is H or OH). This antibiotic substance is capable of manifesting proliferation suppressing actions on a murine leukemic cell (L1210) and useful as an antitumor agent. The antibiotic substance is obtained by inoculating a microorganism belonging to the genus Streptomyces [e.g. Streptomyces violaceus SE2-2385 strain (FERM P-8165)] into a culture medium, cultured at 30 deg.C for 2 days under shaking to prepare a seed culture, which is then transplanted into a jar fermenter and cultured at 28 deg.C for 138hr with aeration. The prepared culture solution is then regulated to pH1.0 and the regulated solution is treated at 65 deg.C for 2hr and filtered to provide a filtrate, which is subsequently passed through an adsorbent resin column and adsorbed thereon. The adsorbed substance is then eluted and the resultant eluate is concentrated and precipitated in n-hexane to separate the precipitate. Thereby, the antibiotic is obtained.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to Streptomyces (S)
The present invention relates to a novel anthracycline antibiotic produced by a microorganism belonging to the genus Treptomyces.

[0002]

2. Description of the Related Art Known anthracycline antibiotics are daunomycin (US Pat. No. 3,616,242) and adriamycin (US Pat. No. 3,590,028), which are conventionally obtained from a culture solution of actinomycetes. These compounds have a broad anticancer spectrum against experimental tumors and are widely used clinically as cancer chemotherapeutic agents. However, daunomycin and adriamycin are quite unsatisfactory, although they have fairly strong anti-cancer effects. Therefore, attempts have been made to create various related compounds by various means such as fermentation, semi-synthesis, and microbial conversion. Furthermore, some anthracycline antibiotics, such as acracinomycin A and B (Japanese Patent Publication No. 51). -34915), 4-demethoxy-1.
1-deoxydaunomycin (JP-A-57-26494)
No.), rhodomycin group antibiotics (JP-A-56-1529)
No. 9) is proposed.

[0003]

As described above, as anthracycline antibiotics as antitumor agents, various related compounds have been proposed, and some of them are already widely used clinically. Some are also used in clinical trials. However, none of them are satisfactory in both toxicity and anticancer activity. In addition, since the results of in vitro tests and animal tests cannot always be reflected in human anticancer effects of antitumor agents, multifaceted research is required. Therefore, regarding anthracycline antibiotics, which are tentatively evaluated as antitumor agents, it is desired to propose compounds belonging to a new class.

[0004]

Means for Solving the Problems The inventors of the present invention have conducted extensive studies to propose a novel compound that can be a more useful anthracycline antibiotic or a synthetic intermediate thereof. Streptomyces viol
Aceus) A262 mutant strain was found to produce a novel anthracycline antibiotic, and the present invention was completed.

The novel anthracycline antibiotics provided by the present invention have the general formula (I)

Embedded image (In the formula, R represents a hydrogen atom or a hydroxyl group).

[0006] These compounds are novel antibiotics having no β-rhodomycinone anthracyclinone skeleton and β-isorhodomycinone anthracyclinone skeleton, which have not been published in the prior art.

Among the compounds represented by the general formula (I), the present inventors have selected A26 as the antibiotic represented by the formula (Ia).
2-6, the antibiotic represented by the formula (Ib) was designated as obermycin H.

[0008]

Embedded image

[0009]

[Chemical 4] Hereinafter, in the present specification, each compound will be described using the above names.

The above-mentioned compound of the present invention has a growth inhibitory effect on cultured leukemia cells L1210 and is useful as an autologous cancer agent. (Growth inhibitory effect on mouse leukemia L1210 cultured cells) L1210 cells were added to RPMI1640 medium (Rose Wellberg Laboratories) containing 10% calf serum at a concentration of 5 × 10 ^4.
Cells / ml and similarly inoculated with 0.005-1
It was added at a concentration of 0 μg / ml and cultured in a carbon dioxide incubator at 37 ° C. to determine the 50% growth inhibitory concentration with respect to the control group. The 50% growth inhibitory concentrations (IC ₅₀ ) of A262-6 and obermycin H against mouse leukemia L1210 cultured cells were 0.27 μg / ml and 0.
It was 33 μg / ml.

The above-mentioned production of anthracycline antibiotics is carried out by using, for example, ultraviolet rays or N-methyl-N'-nitro as a mutagen for a soil isolate or a known strain having the ability to produce a rhodomycin antibiotic and its related compounds. It can be carried out by culturing a strain producing the compound of the present invention, which is easily isolated by a usual mutagenesis treatment using -N-nitrosoguanidine (NTG), in a medium containing an appropriate nutrient source. Specific examples of these production strains include β-rhodomycin-producing strain Streptomyces
s violaceus) A262 strain is subjected to mutation treatment with ultraviolet light to obtain a mutant strain SE2-2385. The strain was deposited as FERM P-8165 at the Institute of Microbial Science and Technology of the Agency of Industrial Science (currently the Institute of Biotechnology, Institute of Industrial Science and Technology) on March 28, 1985, and further dated March 26, 1986. At FERM BP-
It has been transferred to international deposit as 1004.

The mycological properties of the SE2-2385 strain are shown below. (1) Morphology A well-branched basal mycelium forms spiral aerial hyphae, and no limbus branches are observed. 10 to 5 mature spore chains
A sequence of 0 spores is observed. The surface of spores is spiny.

(2) Growth state in various media (28 ° C.) Regarding the description of color, the standard shown in () is H. D. Tr
esner & E. J. Backus, System
of color wheels for Stre
ptomycete taxonomy (J. App
l. Microbiol. vol. 11,335-33
8 (1963)) and supplementarily, "Color Standard" published by Japan Color Research Institute.

(2-1) Sucrose / Nitrate Agar Medium Growth is good. The basal hyphae are dark red to dark reddish purple, and are slightly dotted with aerial hyphae in the air. It produces a purple soluble pigment. (2-2) Glucose / asparagine agar medium Growth is good. The aerial mycelium is a bright yellow pink color (7ca), and the basal mycelium exhibits a yellowish red to a deep red color. It produces a pink soluble pigment. (2-3) Glycerin / asparagine agar medium Growth is good. Aerial hyphae are light yellowish pink (7ca), and basal hyphae are dark red to dark magenta. It produces a purple soluble pigment. (2-4) Starch / inorganic salt agar medium Growth is good. Aerial hyphae are light yellowish pink (7ca). The basal hyphae are initially light yellowish pink (5 ca) and then reddish.
It produces a pink soluble pigment.

(2-5) Tyrosine agar medium Growth is good. Aerial hyphae are very pale purple (11
ca), and the basal hyphae exhibit a red to dark red color. It produces a purple soluble pigment. (2-6) Nutrient agar medium Growth is good. Aerial hyphae are very pale purple (11
ca), and the basal hyphae have a dark magenta color. It does not produce soluble pigment. (2-7) Yeast / malt agar medium Growth is good. The aerial hyphae are light pink at the periphery and the basal hyphae are dark magenta. It produces a pink soluble pigment. (2-8) Oatmeal agar medium Growth is good. The aerial mycelia are grayish yellowish pink (5cb to 6ec), and the basal hyphae are pink. It produces a pink soluble pigment.

(3) Physiological properties (3-1) Growth temperature range: using yeast-malt agar medium at pH 6.0, 20 ° C, 28 ° C, 30 ° C,
When experiments were conducted at 37 ° C. and 42 ° C., growth was observed at each temperature from 20 ° C. to 37 ° C. It did not grow at 42 ° C. (3-2) Liquefaction of gelatin: Glucose / peptone / gelatin medium was used and cultured at 20 ° C, it was positive. (3-3) Starch hydrolysis: When the culture was performed using starch / inorganic salt agar medium, it was positive. (3-4) Coagulation of skim milk and peptone formation: Initially all were negative, and after 15 days of culture, peptone formation began. (3-5) Production of melanin-like pigment: Tryptophan-yeast-broth, peptone-yeast-iron-agar, and tyrosine agar medium were used for culturing, and all the media were positive.

(4) Availability of various carbon sources: Fridham
When various carbon sources were added to the GOTORYB agar medium and growth was observed, L-arabinose, D-xylose, D-glucose, D-fructose, sucrose, inositol, L-rhamnose, raffinose and D-mannite were selected. Both carbon sources could be used.

Cultivation of the antibiotic-producing 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 the carbon source, glucose, glycerin, sucrose, starch, maltose, animal and vegetable oils can be used, and as the nitrogen source, soybean flour, meat extract, yeast extract, peptone, corn stapler, cottonseed meal, fish meal. Organic substances such as, and inorganic nitrogen such as ammonium sulfate, ammonium chloride, sodium nitrate, and ammonium phosphate can be used. If necessary, salt, potassium chloride, phosphate,
Others Mg ⁺⁺ , Ca ⁺⁺ , Co ⁺⁺ , Zn ⁺⁺ , Fe ⁺⁺ , Cu
It is possible to add divalent metal salts such as ⁺⁺ , Mn ⁺⁺ or Ni ^++, as well as amino acids and vitamins.
In order to suppress foaming during fermentation, for example, silicone-
An antifoaming agent such as KM75 (trade name: manufactured by Shin-Etsu Chemical Co., Ltd.) can be added.

Fermentation conditions such as temperature, pH, aeration and fermentation time may be selected so that the strain used accumulates a maximum amount of the compound. For example, the temperature is 20-40
C, preferably 30 C, pH 5-9, preferably 6-
In 7, the fermentation time is advantageously 1 to 10 days, preferably 7 days.

From the culture, the antibiotic A262-6 of the present invention
In order to isolate and collect Obermycin H, the culture after fermentation is added as it is, or concentrated hydrochloric acid, concentrated sulfuric acid and concentrated phosphoric acid are added to adjust the pH to 1.0 to 2.0, preferably p.
After adjusting to H1.0, the mixture is stirred at room temperature or under heating for about 1 to 10 hours. This is centrifuged or filtered in the presence of a suitable filter aid such as diatomaceous earth to separate the cells from the supernatant or the filtrate.

The supernatant or the filtrate is extracted with an organic solvent such as chloroform, toluene, ethyl acetate, butanol or the like at pH 7 to 9 or a suitable adsorption carrier such as synthetic adsorption resin (for example, Diaion HP-20 (commercial product) Name: manufactured by Mitsubishi Plastics Co., Ltd.)), treated with an ion exchange resin to be adsorbed, and then extracted with hydrated acetone or methanol.
On the other hand, if necessary, the cells are extracted with an organic solvent such as acetone, methanol, ethanol or butanol. All of them were concentrated to dryness to collect the crude powder,
By using a column using silica gel and thin layer chromatography or high performance liquid chromatography alone or in combination, the antibiotic A26
2-6 and Obermycin H can be purified and isolated.

The present invention will be described in more detail with reference to the following examples.

[0023]

【Example】

Example 1 Streptomyces violaceus
ces violaceus) SE2-2385 strain (F
ERM P-8165) YS (yeast extract 0.3%,
Soluble starch 1.0%, agar 1.5%, pH 7.2) 1 platinum loop was taken from the slant culture, and seed mother medium (soluble starch 0.5
%, Glucose 0.5%, Essan meat (trade name: manufactured by Ajinomoto Co., Inc.) 1.0%, yeast extract 0.1%, salt 0.1%, potassium diphosphate 0.1%, magnesium sulfate. Heptahydrate 0.1%, adjusted to pH 7.4, 120
The mixture was inoculated into a 500 ml Erlenmeyer flask containing 100 ml (heat sterilization at 15 ° C. for 15 minutes). This was shake-cultured at 30 ° C. for 2 days to prepare a seed mother.

Next, a production medium having the following composition (Essan meat (soybean flour: manufactured by Ajinomoto Co., Inc.) 2.5%, soluble starch 4.0%, yeast extract 0.1%, salt 0.25%, calcium carbonate 0. 3%, mineral mixture 0.1%, pH 7.
(Adjusted to 0) 15 liters were added, and 450 ml of the above culture solution was added and inoculated to two 30 liter jar fermenters heat-sterilized at 120 ° C. for 20 minutes. However, the mineral mixed liquid contains copper sulfate, ferrous sulfate, manganese chloride, and zinc sulfate at 1 g, 0.16 g, 1.3 g, and 0.3, respectively.
2 g was dissolved in 1000 ml of distilled water.

Aeration rate of 15 l / min, stirring at 300 rpm for 2
The cells were cultured at 8 ° C for 138 hours. The culture solution was recovered, and concentrated hydrochloric acid was added to adjust the pH to 1.0. This was heated and stirred at 65 ° C. for 2 hours to extract the target compound into the liquid layer. Filter aid (Topco perlite: manufactured by Toko Perlite Industrial Co., Ltd.)
2% was added and filtered to obtain a filtrate. The pH of the filtrate was adjusted to 2.5 with a 4N sodium hydroxide solution, and Diaion H was previously suspended and filled in acidic water with hydrochloric acid (pH 2.5).
P-20 column (manufactured by Mitsubishi Kasei Co., Ltd.) (φ60m
m: amount of resin 3 l), adsorbed, and washed with 2 bed volumes of acidic hydrochloric acid water (pH 2.5). Then 80%
Acetone water (pH 2.0) was used for elution, and a reddish purple elution section (about 3.5 l) was collected. After concentration under reduced pressure to about half the volume, the pH was adjusted to 8.0 with 4N sodium hydroxide solution, and the mixture was extracted with chloroform (total volume 2.5 l). The chloroform extract was washed with saturated saline, anhydrous sodium sulfate was added, and the extract was dried. After sodium sulfate was filtered off, the solution was concentrated under reduced pressure to a small amount, and n-hexane was added to this to cause precipitation, which was collected by filtration. This is vacuum dried and antibiotic A
Crude powder containing 262-6 and obermycin H 5.8
g was obtained.

Example 2 5.8 g of the crude powder obtained in Example 1 was dissolved in chloroform and silica gel column chromatography (φ50 mm: Wakogel C20) pre-filled with chloroform.
0 (trade name: manufactured by Wako Pure Chemical Industries, Ltd.) 500 g). Chloroform-methanol-water-acetic acid-concentrated aqueous ammonia (200: 50: 2: 0.5: 0.5 and 15
0: 50: 5: 1: 1) mixed solution was developed and antibiotic A26
An elution fraction containing 2-6 and overmycin H was collected. The eluted fractions were collected, the same amount of water was added thereto, the pH was adjusted to 8.0 with a 4N sodium hydroxide solution, and the mixture was stirred and mixed. The solvent layer was separated, concentrated under reduced pressure to dryness, and the antibiotic A26
Partially purified powder 2 containing 2-6 and obermycin H
24 mg was obtained.

Example 3 224 mg of the partially purified powder obtained in Example 2 was completely dissolved in 15 ml of the following developing solvent, and the solution was purified by the following high-performance preparative liquid chromatography in 3 ml portions in 5 batches. Fractions of antibiotic A262-6 and obermycin H were each collected and mixed in 5 batches. After washing with half the amount of the chloroform, the solution was adjusted to pH 8.0 with 4N sodium hydroxide solution and extracted with chloroform. The extract was washed with water, dehydrated with anhydrous sodium sulfate, concentrated to a small amount, and excess n-hexane was added for precipitation. After filtration, it is dried to give 7 mg and 5 mg of powders of antibiotic A262-6 and obermycin H, respectively.
Obtained.

(High-performance liquid chromatography conditions) Column: CAPCELL PAK C18, SG12
0.5 μm Pump: LC-908 (Nippon Analytical Industry Co., Ltd.) Detector: UV detector 310AB (Nippon Analytical Industry Co., Ltd.) λ254 nm used Developing solvent: Acetonitrile-water (27:75) (pH 2.0 with phosphoric acid) Flow rate: 5 ml / fraction: 10 ml

The physicochemical properties of the antibiotics A262-6 and obermycin H of the present invention are shown below. (1) Physicochemical properties of A262-6 (1-1) Appearance: Orange powder (1-2) Melting point: 138 to 140 ° C (decomposition) (1-3) Specific optical rotation: [α] _D ²⁰ + 560 ° ( c
0.01, CHCl ₃ ) (1-4) molecular formula: C ₂₈ H ₃₃ NO ₁₀ (1-5) high-resolution FAB mass spectrum (m / z):
544.2195 ((M + H) ⁺ , Δ + 1.3 mmμ) (1-6) UV absorption spectrum: maximum absorption measured in 90% methanol solution is as follows (unit: n
m). λmax (E _1cm ^1% ): 235 (727), 254 (4
16), 294 (138), 496 (257) (1-7) IR absorption spectrum (KBr): The characteristic absorptions are as follows (unit: cm ^-1 ). νmax: 1599

(1-8) ¹ H-NMR spectrum (40
0 MHz, CDCl ₃ ): The main absorptions are: δTMS (ppm): 1.12 (3H, t, J = 7.3
4Hz), 1.35 (3H, d, J = 6.60Hz),
1.55 (1H, dd, J = 12.47Hz & J =
5.13 Hz), 1.70 (1H, td, J = 12.4)
7 Hz & J = 3.67 Hz), 1.74 (1H,
m, J = 7.34 Hz), 1.85 (1H, m, J =
7.34 Hz), 2.14 (1H, d, J = 14.67)
Hz), 2.21 (s), 2.29 (1H, dd, J =
14.67 Hz & J = 5.14 Hz), 3.66
(1H, br s), 3.90 (1H, q, J = 6.6
0Hz), 5.00 (1H, s), 5.24 (1H,
d, J = 5.14 Hz), 5.45 (1H, d, J =
3.67 Hz), 7.31 (1H, d, J = 7.33H)
z), 7.72 (1H, t, J = 8.07Hz), 7.
89 (1H, d, J = 6.60Hz)

(1-9) ¹³ C-NMR spectrum (40
0 MHz, CDCl ₃ ): The main absorptions are: δ (ppm): 6.51, 17, 21, 29.24, 3
0.83, 34.18, 42.01, 59.73, 6
2.62, 66.18, 66.59, 70.56, 7
1, 98, 97.11, 112.01, 112.07,
115.96, 119.84, 124.83, 133.
54, 137.31, 137.42, 138.26, 1
55.94, 157.77, 162.73, 186.0
6,191.00

(2) Physicochemical properties of obermycin H (2-1) Appearance: Purple powder (2-2) Melting point: 173 to 177 ° C. (decomposition) (2-3) Specific optical rotation: [α] _D ²⁰ -768 ° (c
0.02, CHCl ₃ ) (2-4) molecular formula: C ₂₈ H ₃₃ NO ₁₁ (2-5) high-resolution FAB mass spectrum (m / z):
560.2124 ((M + H) ⁺ , Δ-0.8 mmμ) (2-6) UV absorption spectrum: The maximum absorption measured in a 90% methanol solution is as follows (unit: n).
m). λmax (E _1cm ^1% ): 240 (842), 297 (1
34), 521 (292), 549 (272) (2-7) IR absorption spectrum (KBr): The characteristic absorptions are as follows (unit: cm ⁻¹ ). νmax: 1589

(2-8) ¹ H-NMR spectrum (40
0 MHz, CDCl ₃ ): The main absorptions are: δTMS (ppm): 1.11 (3H, t, J = 7.3
4Hz), 1.33 (3H, d, J = 6.60Hz),
1.56 (1H, dd, J = 12.47Hz & J =
5.14 Hz), 1.7 to 1.8 (2H), 1.83
(1H, m, J = 7.33Hz), 2.14 (1H,
d, J = 14.67 Hz), 2.26 (s), 2.35.
(1H, ddd, J = 12.48 Hz & J = 2.2
0 Hz), 3.70 (1H, br s), 3.92 (1
H, q, J = 6.60 Hz), 4.98 (1H, s),
5.21 (1H, dd, J = 5.14Hz & J =
1.47 Hz), 5.44 (1H, d, J = 2.94H
z), 7.31 (2H, s)

(2-9) ¹³ C-NMR spectrum (40
0 MHz, CDCl ₃ ): The main absorptions are: δ (ppm): 6.30, 16.91, 28.67, 3
0.70, 33.91, 41.83, 59.94, 6
2.17, 66.21, 66.83, 70.56, 7
1.93, 96.90, 112.00, 112.19,
112.61, 112.64, 129.41, 129.
58, 136.45, 138.34, 155.99, 1
56.72, 157.67, 189.11, 189.2
5

Claims (3)

[Claims] 1. A compound of the general formula (I) (In the formula, R represents a hydrogen atom or a hydroxyl group.) A novel anthracycline antibiotic. 2. The novel anthracycline antibiotic according to claim 1, wherein R in the general formula (I) is a hydrogen atom. 3. The novel anthracycline antibiotic according to claim 1, wherein R in the general formula (I) is a hydroxyl group.