JPH08283287A - New anthracycline antibiotic - Google Patents

Abstract

PURPOSE: To obtain a new anthracycline antibiotic produced by a bacterium belonging to the genus Streptomyces, showing inhibitory action on proliferation of a cultured cell of mouse leukemia (L1210), having low toxicity and high carcinostatic properties, useful as an autitumor agent, etc. CONSTITUTION: This anthracycline antibiotic is shown by the formula. The anthracycline antibiotic shows inhibitory action on the proliferation of a cultured cell of mouse leukemia (L1210) and is useful as an antitumor agent. The new antibiotic is obtained by inoculating a bacterium [e.g. Streptomyces coeruleorubidus T-373 strain (FERM BP-15), etc.] belonging to the genus Streptomyces into a medium, subjecting the bacterium to shaking culture at 28 deg.C for 2 days to prepare a seed bacterium, inoculating it into a medium in a tank, subjecting the bacterium to aerated stirring culture at 28 deg.C for 6 days, centrifuging the culture solution, collecting the supernatant liquid, passing it through a synthetic adsorption resin column to adsorb a product on it, eluting it with 60% aqueous solution of acetone, concentrating the eluted solution, extracting the solution with chloroform and purifying the extract by a silica gel column.

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 diligent research to propose a novel compound which can be a more useful anthracycline antibiotic or a synthetic intermediate thereof. As a result, a culture of a microorganism belonging to the genus Streptomyces is cultivated. We have succeeded in isolating a novel anthracycline antibiotic that has not been published in the literature from the liquid, and have found that the compound has a growth inhibitory action on experimental tumors, and completed the present invention.

The novel anthracycline antibiotic provided by the present invention is a daunomycin derivative represented by the following formula (I). The present inventors
I named it. Hereinafter, the compound will be described in the present specification using the above names.

[0006]

Embedded image

The above-mentioned compound 14A6 of the present invention has a growth inhibitory action 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 the substance of the present invention 0.001-1
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 concentration (IC ₅₀ ) of 14A6 against mouse leukemia L1210 cultured cells was 1.47 μg / m 2.
It was l. The compound of the present invention was dissolved in 0.02M acetic acid (pH 3.0) at a concentration of 1 mg / ml, and then D
ulbecco PBS (-) (Nissui Pharmaceutical Co., Ltd.)
Diluted with and added.

The compound 14A6 of the present invention can be produced by culturing a strain belonging to Streptomyces and having the ability to produce the compound in a medium containing an appropriate nutrient source. Specific examples of these producing strains include Streptomyces coerulerubidos (Streptomyces) known as anthracycline antibiotic daunomycin (daunorubicin) producing strain.
s coeruleorubidus) 3T-373 strain. The strain was July 13, 1982.
FERM BP-165 at the Institute of Microbial Engineering, Institute of Industrial Technology (currently, Institute of Biotechnology, Institute of Biotechnology)
Has been internationally deposited as.

The mycological properties of the 3T-373 strain are shown below. (1) Morphology Linear or hook-shaped aerial hyphae are formed from branched basal hyphae, and no limbus branches are observed. 10 mature spore chains
The number of spores is 0.4-0.8 × 1.2-
It is about 2.0 μm, and the surface of spores is almost smooth. Occasionally, traces of spinous protrusions are slightly observed on the surface of some spores.

(2) Growth condition in various media (28
Regarding the description of color, the standard shown in () is based on H.264. 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 Light orange or dark orange, or light brown (4ea-4)
On the development of gc), aerial hyphae of gray-yellowish pink (5cb) settled, and almost no soluble pigment was observed. (2-2) Glucose / asparagine agar medium gray yellow or bright orange yellow, or light orange (3e
c-3ea-4ea). Little aerial hyphae are formed and few soluble pigments are produced. (2-3) Glycerin / asparagine agar medium On the development of light orange (4 ea) or orange, or dark orange, gray-yellowish pink (5 cb) aerial mycelium grows, and the soluble pigment is slightly orange. (2-4) Starch / inorganic salt agar medium Bright orange yellow or light orange (3 ea to 4 ea) or reddish orange on the development of grayish yellow pink (5 cb) aerial hyphae, soluble pigment is pink , Or slightly orange. (2-5) Tyrosine agar medium On the development of light orange (4 ea) or yellow tea or reddish orange, aerial mycelia of purple-white (13 ba) or gray-yellowish pink (5 cb) are grown. Almost no soluble pigment is observed. It takes on an orange color over a long period of culture (about 20 days).

(2-6) Nutrient agar medium On the development of red to dark red, aerial hyphae of gray-yellowish pink (5cb) to extremely light purple (11ca) are settled, and the soluble pigment is pink. (2-7) Yeast-malt agar medium On the development of nibu orange (4 gc) or yellow tea or red tea, aerial hyphae of gray-yellowish pink (5 cb) in the aerial mycelium were initially light, and after 14 days of culture. It has abundantly grown since then, and the soluble pigment has a slight orange tinge. (2-8) Oatmeal Agar Medium Bright red tea (5 gc) or red tea, or aqua-yellow pink (5 cb) aerial mycelium grows on the growth of tea, and the soluble pigment takes on an extremely slight pink color. (2-9) Glucose / peptone / gelatin medium A slight white aerial mycelium is slightly settled on the development of yellow tea or dark tea, and the soluble pigment is slightly brown. (2-10) Non-fat milk medium It grows on the surface and casein does not coagulate but causes peptone formation. The soluble pigment has a brownish taste.

(3) Physiological properties (3-1) Growth temperature range: 20 ° C, 25 ° C, 30 ° C at pH 6.0 using yeast-malt agar medium
Experiments at each temperature of 37 ℃, 46 ℃ and 50 ℃
Growth was observed at each temperature from 0 ℃ to 37 ℃, 46 ℃
And did not grow at 50 ° C. Optimum temperature is 30 ℃
It seems to be around 37 ° 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 defatted milk and formation of peptone: Peptone is formed although there is almost no coagulation. (3-5) Formation of melanin-like pigment: Tryptophan-yeast-broth medium and peptone-yeast-iron-agar medium clearly produce melanin-like pigment. Tyrosine agar produces little or no melanin-like pigment.

(4) Availability of various carbon sources: Fridham
When various kinds of carbon sources were added to GOTORYB agar medium and growth was observed, all carbon sources of L-arabinose, D-xylose, D-glucose, D-fructose, sucrose, inositol and D-mannitol were used. We were able to. Utilization of L-rhamnose and raffinose is slight and suspicious.

Cultivation of the antibiotic-producing 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 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 the maximum amount of the compound. For example, the temperature is 20-40
C, preferably 28 C, pH 5-9, preferably 6-
In 7, the fermentation time is advantageously 1 to 10 days, preferably 6 days.

To isolate and collect the antibiotic 14A6 from the culture, centrifuge the culture after fermentation or
By filtering in the presence of a suitable filter aid such as diatomaceous earth, the cells are separated from the supernatant or the filtrate. The supernatant is extracted with an organic solvent such as chloroform, toluene and ethyl acetate at pH 7-9. From the bacterial cells, if necessary,
Extract 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 with an adsorption carrier, for example, a nonionic porous styrene resin, chromatography using silica gel, or an anion exchange resin,
The antibiotic 14A6 can be collected in a pure form by using treatments using a cation exchange resin alone or in combination as appropriate.

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, palmitic acid, citric acid, succinic acid, tartaric acid, fumaric acid, glutamic acid. , Pantothenic acid, lauryl sulfonic acid, methane sulfonic acid, naphthalene sulfonic acid and the like.

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

[0020]

【Example】

Example 1 Streptomyces coelre orbidas (Strept
omyces coeuruleorubidus) 3T
-373 strain (FERM BP-165) YS (yeast extract 0.3%, soluble starch 1.0%, agar 1.5%, p
H7.2) 1 platinum loop was taken from the slope 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%, dibasic potassium phosphate 0.1
%, Magnesium sulfate heptahydrate 0.1%, pH 7.4
And sterilized (heat sterilization at 120 ° C. for 15 minutes). This was shake-cultured at 28 ° C. for 2 days to prepare a seed mother.

Next, a production medium (Taiwan yeast 5.0%, soluble starch 7.5%, yeast extract 0.3%, salt 0.2%,
0.3% calcium carbonate, 0.125% mineral mixture,
pH adjusted to 8.0, heat sterilization at 120 ° C for 15 minutes) 10
A 200 l tank in 0 l was inoculated with 2 l of the above seed mother. However, mineral mixture may, CuSO ₄ · 5H ₂ O and 2.8 g, the _{FeSO 4 · 7H 2 O 0.4g,} MnCl 2
・ 4H ₂ O 3.2 g, ZnSO ₄ .2H ₂ O 0.8 g
Is dissolved in 500 ml of distilled water.

Aeration rate 50 l / min, stirring 100 rpm, 2
The culture broth is dark reddish brown because it is a product when shake-cultured at 8 ° C for 6 days. Therefore, stop the fermentation, collect the culture broth, dilute it 4 times with water, adjust to pH 2.0 with phosphoric acid, and then centrifuge. To separate the cells from the supernatant. The product in the supernatant was used as Diaion HP-20 (synthetic adsorption resin, manufactured by Mitsubishi Kasei Co., Ltd.).
It was passed through a 10 l column. It was washed with acidic water of pH 2 and then eluted with 60% aqueous acetone (pH 2.0). The eluate was concentrated to approximately 1/2 volume, the pH was adjusted to pH 8.5 with 4N sodium hydroxide, and extracted with 20 l of chloroform. 20 L of 0.1N acetic acid in the chloroform layer
Was added, the acid was transferred, and the aqueous layer was washed with 10 l of chloroform. The pH of the aqueous layer was adjusted to pH 8.5, extracted again with 20 l of chloroform, concentrated to a small amount, and n-hexane was added to obtain a precipitate. After dissolving this precipitate in chloroform, 1N-hydrochloric acid-
Methanol was added slowly. The precipitate was removed, and the supernatant was concentrated to dryness to obtain 92 g of crude antibiotic 14A6 powder.

92 g of the obtained crude powder was dissolved in 8 l of water adjusted to pH 2.5 and passed through a column of 3 l of Diaion HP-20 (synthetic adsorption resin, manufactured by Mitsubishi Kasei Co., Ltd.),
Adsorbed. This was added to a methanol-citrate buffer solution (0.
A fraction containing antibiotic 14A6 was obtained by stepwise elution with 1N, pH 3.5) (40:60 to 70:30). The obtained fraction was adjusted to pH 8.0, extracted with chloroform, and concentrated to dryness to obtain 3.1 g of partially purified powder of antibiotic 14A6.

3.1 g of the partially purified powder of the obtained antibiotic 14A6 was added to a silica gel column (Wakogel C-200).
(Manufactured by Wako Pure Chemical Industries, Ltd.), 150 g) and adsorbed onto chloroform-methanol-water-acetic acid-triethylamine (80: 15: 0.2: 0.1: 0.02 and 60:
(15: 0.5: 0.2: 0.02) was eluted stepwise to obtain a fraction containing the antibiotic 14A6. 1 for this fraction
After washing with an aqueous solution of sodium hydrogen carbonate (%), the mixture was concentrated to dryness.

25% acetonitrile water (pH 2.
0, pH adjusted with phosphoric acid), using this solvent as the mobile phase, preparative high performance liquid chromatography (main unit: 80)
70P & P System, manufactured by Tosoh Corporation; Column: Ts
k gel ODS-120T (φ55 × 300m
m), manufactured by Tosoh Corporation) to obtain a fraction of antibiotic 14A6. After washing the fraction of the antibiotic 14A6 with chloroform, 1N sodium hydroxide was added,
After adjusting to pH 8.0, it was extracted with chloroform. The extract was dried over anhydrous sodium sulfate and concentrated under reduced pressure to a small amount. An excess amount of n-hexane was added to the precipitate, which was collected by filtration and vacuum dried to obtain 279 mg of a purified powder of antibiotic 14A6.

The analysis of the antibiotic 14A6 during purification was carried out by using the following high performance liquid chromatography. At this time, the holding time of 14A6 was 20.9 minutes. (High-performance liquid chromatography conditions) Column: Zorbax TMS (φ4.6 × 250 m)
m) Mobile phase: water-acetonitrile-methanol-phosphoric acid (5
40: 290: 170: 2) 1 g of SDS is dissolved in 1 l, and the pH is adjusted to 3.6 with 2N sodium hydroxide. Flow rate: 1.5 ml / min

The physicochemical properties of the antibiotic 14A6 of the present invention are shown below. (1) Appearance: Red powder (2) Melting point: 206-210 ° C (decomposition) (3) Specific optical rotation: [α] _D ²⁰ + 93 ° (c 0.0
1, CHCl ₃ ) (4) Molecular formula: C ₃₃ H ₄₀ N ₂ O ₁₂ (5) FAB mass spectrum (m / z): 657 ((M
+ H) ⁺ ) (6) UV absorption spectrum: maximum absorption measured in 90% methanol solution is as follows (unit: n
m). λmax (E _1cm ^1% ): 234 (580), 252 (3
86), 290 (134), 478 (190), 495
(190) (7) IR absorption spectrum (KBr): Characteristic absorptions are as follows (unit: cm ^-1 ). νmax: 1705, 1610, 1580

(8) ¹ H-NMR spectrum (400M
Hz, CDCl ₃ ): The main absorptions are: δTMS (ppm): 1.21 (3H, d, J = 6.6
Hz), 1.27 (3H, d, J = 6.6Hz), 1.
71 (2H, br d, J = 7.3 Hz), 1.76
(1H, br dt, J = 13.9 Hz & J = 3.
7Hz), 2.09 (1H, dd, J = 14.7Hz
& J = 3.7 Hz), 2.20 (1H, dt, J = 1)
3.9 Hz & J = 3.7 Hz), 2.30 (1H,
d, J = 14.7 Hz), 2.40 (3H, s), 2.
88 (1H, d, J = 19.1Hz), 3.01 (1
H, br t, J = 8.1 Hz), 3.08 (1H,
q, J = 4.4 Hz), 3.17 (1H, d, J = 1)
9.1 Hz), 3.38 (1 H, dd, J = 4.4 Hz)
& J = 2.2 Hz), 3.49 (1H, br
s), 4.07 (3H, s), 4.09 (1H, q, J
= 6.6 Hz), 4.42 (1H, qd, J = 6.6H)
z & J = 1.5 Hz), 4.89 (1H, br
t, J = 3.7 Hz), 5.24 (1H, br d, J
= 1.5 Hz), 5.48 (1 H, br s), 7.3
7 (1H, d, J = 8.8Hz), 7.76 (1H,
t, J = 8.1 Hz), 7.98 (1H, d, J = 7.
3Hz)

(9) ¹³ C-NMR spectrum (400M
Hz, CDCl ₃ ): The main absorptions are: δTMS (ppm): 15.86, 17.51, 24.
72, 32.65, 33.27, 34.50, 34.8
1, 46.40, 49.07, 56.59, 64.0
9,68.08,69.45,73.34,81.8
5,100.03,101.12,111.13,11
1.27, 118.33, 119.68, 120.7
8, 134.24, 134.30, 135.38, 13
5.60, 155.73, 156.33, 160.9
2, 186.51, 186.82, 211.89

Claims (1)

[Claims] 1. Formula (I): A novel anthracycline antibiotic represented by.