JP2786267B2 - New substance DC116 - Google Patents

Description

The present invention relates to a novel antitumor antibiotic DC116 having an anthraquinone pyran structure and a method for producing the same.

2. Description of the Related Art Conventionally, many compounds such as anthraquinone-based compounds, anthracycline-based compounds, and mitomycin-based compounds have been reported as antitumor antibiotics having a quinone skeleton.

[CRC Handbook of Anti-Biotech Compounds, CRC Press (CRC
Handbook of Antibiotic Compounds, CRC Press) USA
1981].

DC116 has an anthraquinone pyran structure. Substances currently known as anthraquinone pyran compounds include the following formula (I) (Where R is Or Expressed by DC-92B or DC-92D (JP-B-62-504380) represented by the following formula (II) The DC116 is a novel substance distinctly different from these substances, for example, SF2587 (JP-A-1-193265).

SUMMARY OF THE INVENTION An object of the present invention is to provide excellent antibiotics and antitumor compounds.

Means for Solving the Problems The present inventors have developed an antitumor activity in a culture obtained by culturing a microorganism (hereinafter referred to as DO-116 strain) isolated from soil in Machida-shi, Tokyo in a medium. Was found to be produced.

This substance was isolated, purified, and examined for its physicochemical properties. As a result, the substance was found to be a novel substance, and was named DC116.

According to the present invention, the following general formula A novel substance DC116 having an antitumor effect and an antibacterial activity represented by the formula: is provided.

This compound is obtained by culturing a microorganism belonging to the genus Streptomyces.

 Hereinafter, the present invention will be described in detail.

 The physicochemical properties of DC116 are shown below.

Molecular weight: 462 Molecular formula: C ₂₅ H ₁₈ O ₉ Mass spec: SIMS 465 (M + 3) ⁺ , 464 (M + 2) ⁺ , 463
(M + 1) ⁺ specific rotation: ▲ [α] ²⁴ _D ▼ = +270 ゜ (c = 0.012, CHCl
₃ ) UV absorption spectrum: (measured in methanol) λ _max : 242 nm (ε = 63,000), 264 nm (sh, ε = 29,000), 380 nm (ε = 5,700), 506 nm (ε = 11,000), infrared absorption spectrum: ( Measured by KBr tablet method) (c
m ^-1 ) 3700−2400,3430,1755,1734,1654,1629,1585,1454,12
24, PMR spectrum: (measured in CDCl ₃ -CD ₃ OD, internal standard T
MS) ¹ H-NMR (500 MHz): δ (ppm) 8.18 (1 H, s), 7.39 (1 H, d, J = 9.3 Hz), 7.34 (1 H, d, J
= 9.3Hz), 6.47 (1H, s), 5.85 (1H, ddq, J = 11.2,1.0,7.
1Hz), 5.15 (1H, ddq, J = 11.2, 8.5, 1.8Hz), 4.40 (1H, d,
J = 16.6Hz), 4.36 (1H, d, J = 16.6Hz), 3.96 (1H, d, J =
8.5Hz), 1.99 (3H, s), 1.85 (1H, dd, J = 7.1, 1.8Hz). CMR spectrum: (CDCl ₃ -CD ₃ OD, internal standard TMS) ¹³ C-NMR (125 MHz): δ (ppm) 185.30, 185.28, 178.8, 172.5, 166.7, 157.89, 157.88, 15
6.7,144.1,137.0,134.4,130.8,129.1,126.9,126.5,122.
6,121.8,113.4,112.5,110.9,62.2,61.3,42.1,19.9,13.
8. Solubility: chloroform, dimethyl sulfoxide (DMS
O), soluble in methanol, ethyl acetate and acetone,
Poorly soluble in water and n-hexane.

Color reaction: positive for iodine reagent.

Color and properties of substance: Red acidic substance Thin layer chromatography: Silica gel thin layer (HPTLC pl
ate Art.15647 E. Merck) Rf 0.60 with a developing solvent of toluene: acetone: acetic acid (7: 2: 0.1 v / v / v).

CHCl ₃ : Rf 0.30 with acetic acid (100: 1 v / v).

After deployment, the spot of DC116 is Bacillus subtilis (B
acillus subtilis), hot sulfuric acid and ultraviolet absorption.

 Next, the biological activity of DC116 will be described.

(A) Antibacterial activity The minimum inhibitory concentration (MIC) for various kinds of bacteria was measured by an agar dilution method (pH 7.0). Table 1 shows the results.

(B) Acute toxicity (LD ₅₀ ) It was 6.9 mg / kg by intravenous administration to mice.

(C) in CDF ₁ 1 group 5 male mice of the therapeutic effect weighing approximately 22g for antitumor activity lymphoblastoid Sai tick Ryukemia P-388 tumor, lymphoblastoid Sai tick Ryukemia (Lymphocytic Leukemia) P-38
1 × 10 ⁶ tumor cells were implanted intraperitoneally. 24 hours after transplantation, 0.2m of phosphate buffered saline (PBS) solution containing DC116
l was administered intraperitoneally once. PBS composition is NaCl 0.8g / dl,
KCl 0.02 g / dl, pH 7.2. As a comparative example, 0.2 ml of a PBS solution containing mitomycin C 24 hours after tumor cell transplantation
Was administered intraperitoneally. T / C for survival benefit after transplant
The results are shown in Table 2 as [T: average number of surviving days of the test example, C: average number of surviving days of control (0.2 ml of PBS solution administered intraperitoneally)].

Therapeutic Effect on Sarcoma 180 Tumors 5 × 10 ⁶ sarcoma 180 tumor cells were subcutaneously implanted subcutaneously into 6 ddy male mice weighing about 20 g per group. 5 times every 24 hours from day 1 to day 5 after transplantation
0.2 ml of a PBS solution containing 116 was administered intraperitoneally.

As a comparative example, a group in which 0.2 ml of PBS containing mitomycin C was intraperitoneally administered 24 hours after tumor cell transplantation was established.

T / C 10 days after transplantation [T: average tumor volume of test example (mm ³ ),
C: Average tumor volume (mm ³ ) of control (0.2 ml of PBS solution administered intraperitoneally)] was measured.

 Table 3 shows the results.

Next, a method for manufacturing DC116 will be described.

DC116 belongs to the genus Streptomyces, a microorganism capable of producing DC116 is cultured in a medium, and DC11 is added to the culture.
6 can be obtained by accumulating and accumulating DC116 from the culture.

As a DC116 producing strain, any strain belonging to the genus Streptomyces and having a DC116 producing ability can be used. In addition, if any of these strains can be used in the present invention as long as they produce DC116 even in a mutant strain or a mutant strain naturally mutated by an artificial mutation method such as ultraviolet irradiation, X-ray irradiation, treatment with a mutagenic agent, etc. be able to. A representative strain is DO-116 strain.

The bacteriological properties of the DO-116 strain are described below. The identification of the property can be performed by using International Streptomyces.
Recommended methods for characterization of Streptomyces species [EB Schering (EBShirli)
ng) and D. Gottlieb et al., International Journal of Systematic Bacteriology (Int. J. Syst. Bacteriol.) 16 , 313-340 (196).
6)]. The isomer of diaminopimelic acid in whole cell hydrolysates was determined by the method of B. Becker et al. [Appl. Microbio
l.) 12: 421-423 (1964)]. The morphological examination was performed using an optical microscope, and the morphology of the spore surface was measured using a scanning electron microscope. Color names are assigned in the Color Harmony Manual [Container Corporation of America].
merica), 4th edition 1958]. The bacteriological properties of the DO-116 strain are as follows.

(1) Form Aerial hyphae: branched.

 Underlying hypha: Branches but does not break.

Spores: settle on aerial hyphae as long chains of segmented spores (10 to 30 or more). The form of the chain is bent or looped.

 Spore surface: Smooth Spore motility: None Spore shape / size: elliptical (0.5 × 0.7 μm) No sclerotia or spores are observed.

(2) Color tone Aerial hyphae: Gray Underlying hyphae: pale yellow to yellow brown Soluble pigment: Brown Melanin pigment: None (3) Chemical composition of cell wall Stereotype of diaminopimelic acid: LL type (4) Physiological properties Gender: assimilate: glucose, xylose, inositol,
Mannitol, arabinose, rhamnose, raffinose, lactose, sucrose, salicin, galactose Not assimilated: none Gelatin liquefaction: negative Starch hydrolysis: positive Coagulation of skim milk: negative Peptone property of skim milk: positive Fiber degradation: positive Growth temperature range: 16 to 37 ° C (optimal 28 to 32 ° C) The growth temperature range is 2 days later, and the effects on gelatin, skim milk and fibrin are 28 ° C and 1 ° C.
Monthly results and other properties are shown at 28 ° C for 2 weeks.

(5) Growth state on various agar media Table 4 shows the results of culturing DO-116 strain on various agar media at 28 ° C for 28 days.

(6) Identification of DO-116 strain Since the LL-type diaminopimelic acid is detected in DO-116 strain, MP Rechevalier and H.A.
MPLechevalier and HALechevalie
r) Classification of actinomycetes [International Journal of Systematic Bacteriology (Int. J. Sy
St. Bacteriol.), Vol. 20, 435-443 (1970)]. Further combining the morphological characteristics of the strain, it is appropriate to assign this strain to the genus Streptomyces.

In the identification of species in this genus, gray aerial hyphae,
Bent or looped spore chains, smooth spore surface,
Based on the characteristics of this strain, such as non-melanin-like pigment production, soluble pigment production, and carbon source assimilation patterns, a bacteriological name approval list [VBD Scarman (VB
D. Skerman) et al., Int. J. Syst. Bacteriol. 30, Vol.
20 (1980)], a species with similar taxonomic characteristics is described in the ISP [Int. J. Syst. Bacteriol. 18
Vol. 69-189, (1968), Journal, Vol. 18, 279-392, (196
8), same magazine, 19 volumes, 391-512, (1969), same magazine, 22 volumes, 2
65-394 (1972) and R.E. Buchanan (REB
uchanan) and NE Kibons (NEGibbons)
Barges Manual of Determinant
Bacteriology (Bergey's Manual of Deter-minati
ve Bacteriology) 8th edition].

As a result of search, Streptomyces nigra (Streptom
yces nigra) was named as a closely related species of the DO-116 strain, but it was difficult to identify the species, and the strain was named Streptomyces sp. DO-116.

Based on the Budapest Treaty, the strain was supplied to the Institute of Microbial Industry and Technology by the Ministry of Industrial Science and Technology, No. 2559 (FERM BP-255).
9) (Original deposit date: August 22, 1989)
Day).

 Next, the culture method will be described.

In the culture of the present invention, a usual method of culturing actinomycetes is generally used. As a medium, any of a synthetic medium and a natural medium can be used as long as the medium contains assimilable carbon sources, nitrogen sources, inorganic substances, and necessary growth and production promoting substances.

As a carbon source, glucose, starch, dextrin, mannose, fructose, sucrose, lactose,
Xylose, arabinose, mannitol, molasses and the like are used alone or in combination. Further, hydrocarbons, alcohols, organic acids and the like may be used depending on the assimilation ability of the bacteria.

As the nitrogen source, ammonium chloride, ammonium sulfate, ammonium nitrate, sodium nitrate, urea, peptone, meat extract, yeast extract, dried yeast, corn steep liquor, soybean powder, casamino acid and the like are used alone or in combination.

In addition, if necessary, salt, potassium chloride, magnesium sulfate, calcium carbonate, potassium dihydrogen phosphate,
Dipotassium hydrogen phosphate, ferrous sulfate, calcium chloride,
Add inorganic salts such as manganese sulfate, zinc sulfate and copper sulfate.

Further, a trace component that promotes the growth of the bacterium used and the production of DC116 can be appropriately added.

As a culture method, a liquid culture method, particularly a deep stirring culture method, is most suitable. The cultivation is carried out at a temperature of 16 to 37 ° C., preferably 25 to 32 ° C., pH 4 to 10, preferably 6 to 8, usually completed in 1 to 7 days, and the target substance DC116 is contained in the culture solution and the cells. Generate and accumulate.

Ammonia water, ammonium carbonate solution, or the like is used to adjust the pH of the medium.

The culture is stopped when the production in the culture reaches a maximum.

Isolation and purification of DC116 from the culture is performed according to a method commonly used for isolating and purifying a microbial metabolite from the culture. For example, the culture is separated into a culture solution and cells by filtration, and the cells are extracted with chloroform, acetone, or the like. Next, the extract and the culture solution are combined and passed through a polystyrene-based adsorbent such as Diaion HP20 (manufactured by Mitsubishi Kasei) to adsorb the active ingredient, and then eluted with ethyl acetate, acetone or the like. Concentrate the eluate,
A red powder of DC116 is obtained by column chromatography on silica gel, high performance liquid chromatography, or the like.

During the culture and purification operations, the trend of DC116
Bioassay using Subtilis No. 10707 or UV absorption of DC116 by thin layer chromatography can be followed as a guide.

 Hereinafter, examples of the present invention will be described.

Example 1. Streptomyces SB DO-116 is used as an inoculum. The strain was placed in a 2 volume Erlenmeyer flask.
Tribton (Difco) 5g /, Yeast extract 5g /, Meat extract 3g /, Soluble starch 10g /, Glucose 10g /,
Seed medium consisting of 5 g / calcium carbonate (pH before sterilization)
7.2) The cells were inoculated into 300 ml and cultured at 30 ° C. for 48 hours with shaking (200 rpm).

The obtained seed culture solution is transferred at a rate of 10% (volume) to a fermentation medium 15 having the following composition in a 30-volume culture tank, and cultured at 28 ° C. by aeration and agitation (200 rpm, aeration 15 / min). Was done.

Fermentation medium composition: glycerol 25g /, glucose 25g /
, Dried yeast _{15g /, KH 2 PO 4 0.5g} /, MgSO 4 · 7H 2 O0.5g
/, Calcium carbonate 5 g / (pH 7.0 before sterilization, adjusted with NaOH) During the culture, the culture was cultured for 150 hours without particular control of the pH of the medium. After adding n-propanol 15 to the culture and stirring, the cells and sediment were separated from the culture,
I got

After concentration, the solution was diluted with water and passed through a column of polystyrene adsorbent Diaion HP20 (10) to adsorb the active substance.

The active substance was eluted with ethyl acetate after eluting the impurities with deionized water and 50% methanol. After the active fraction was concentrated, it was added to water and passed through a column of Diaion HP20SS (2),
Active substance was adsorbed.

After eluting impurities with 50% methanol, the active substance was eluted with 90% methanol. The active fraction was concentrated and extracted with ethyl acetate.

After concentrating the ethyl acetate layer, it was applied to Sephadex LH20 and eluted with methanol. Concentrating the fraction containing the active substance,
The obtained substance was then used for reverse phase silica gel (YMC ODS SH-36).
High-performance liquid chromatography was performed using 80% methanol as a solvent, using a method described in "3-5, YMC".
The fraction containing DC116 was extracted with chloroform and concentrated to dryness to obtain 30 mg of DC116 red powder.

Effects of the Invention The present invention provides a novel fermentation product DC116 having antibacterial and antitumor activities.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI (C12N 1/20 C12R 1: 465) (C12P 17/16 C12R 1: 465) (58) Investigated field (Int.Cl. ⁶ , DB name) C12P 17/00-17/18 C07D 407/04 303 CA (STN) REGISTRY (STN)

Claims (1)

(57) [Claims] 1. A novel substance DC116 represented by the following general formula: