JPH0819148B2 - New substances DC-92B and DC-92D - Google Patents

Description

TECHNICAL FIELD The present invention relates to an antitumor antibiotic having an anthraquinone structure.
Regarding DC-92B and DC-92D materials.

BACKGROUND ART Conventionally, mitomycins, anthracyclines, anthraquinones, etc. have been known as antitumor antibiotics having a quinone structure (see C.I. Handbook of Antibiotic Compounds (CRC Handbook)).
Book of Antibiotic Compounds, Vol. 3, 1981, CRC Press, USA. Among the above-mentioned known anthraquinones, the following compounds are listed:

Anticancer antibiotics used in cancer chemotherapy include mitomycin C, adriamycin, and bleomycin. However, there are cancers for which these drugs are ineffective, or for which resistance is an issue, and therefore there is a constant demand for substances with even more effective antitumor activity.

Disclosure of the Invention According to the present invention, a novel substance having antitumor and antibacterial effects is obtained by culturing a microorganism belonging to the genus Actinomadura and capable of producing DC-92B and/or DC-92D.
You can get a DC-92B and/or a DC-92D.

The physicochemical properties of DC-92B are shown below.

(a) Molecular weight: 776 (Mass spectrometry: SI-MS m/z 777 [M+H] ⁺ ) (b) Color of substance: dark red (c) Ultraviolet absorption spectrum: shown in Figures 1 and 2.
(Fig. 1 shows the results of measurement in MeOH, and Fig. 2 shows the results of measurement in alkaline MeOH with NaOH.) (d) Infrared absorption spectrum: shown in Fig. 5. (CHCl ₃
(e) Solubility: Soluble in methanol, ethanol, acetone, chloroform, ethyl acetate, toluene, and dimethyl sulfoxide, but practically insoluble in water and hexane.

(f) ^1H -NMR spectrum (400MHz, measured in _CHCl3 , internal standard TMS) δ (ppm) 13.88 (1H, br s), ca. 9.7 (1H, br), 8.34 (1H, br s),
8.04(1H,d,J=0.6Hz),6.53(1H,s),5.50(1H,br d,J
=11.0Hz),5.45(1H,br dd,J=10.2,1.4Hz),3.56(1
H,dq,J=8.6,6.0Hz),3.54(1H,s),3.22(1H,br dd,J
=9.8,8.6Hz),3.17(1H,d,J=4.9Hz),3.02(3H,d,J=
0.6Hz), 2.86 (1H, ddd, J = 13.2, 9.8, 3.4Hz), 2.72 (1H,
d,J=4.9Hz),2.61(6H,br s),ca.2.6(1H),2.32(6
H,s),2.19(1H,m),1.91(3H,s),1.59(3H,s),1.44
(3H,d,J=6.0Hz),1.38(1H,br dd,J=11.1,3.8Hz),
1.35 (3H, s), 1.12 (3H, s), ca.1.1 (1H, m), 1.08 (3H,
s) (g) ¹³ C-NMR spectrum (100 MHz, measured in CDCl ₃ ,
Internal standard TMS) δ (ppm) 187.8,183.2,178.3,164.9,159.3,156.0,150.0,140.0,13
8.8,137.5,132.6,126.2,126.1,126.0,119.1,115.6,110.
9,100.0,79.0,77.5,75.2,71.7,67.3,63.6,62.9,59.9,5
9.0,58.6,58.2,43.1,41.0,40.4,28.5,25.2,24.2,24.1,1
9.9, 19.7, 18.8, 14.3 (h) Thin-layer chromatography: Toluene:
The Rf is 0.54 in the acetone (6:4 v/v) development system.

(i) Weakly basic substance The physicochemical properties of DC-92D are shown below.

(a) Molecular weight: 774 (Mass spectrometry: SI-MS m/z 775 [M+H] ⁺ ) (b) Color of substance: dark red (c) Ultraviolet absorption spectrum: shown in Figures 3 and 4.
(Fig. 3 shows the results of measurement in MeOH, and Fig. 4 shows the results of measurement in alkaline MeOH with NaOH.) (d) Infrared absorption spectrum: shown in Fig. 6. (CHCl ₃
(e) Solubility: Soluble in methanol, ethanol, acetone, chloroform, ethyl acetate, toluene, and dimethyl sulfoxide, but practically insoluble in water and hexane.

(f) ^1H -NMR spectrum (400MHz, measured in _CDCl3 , internal standard TMS) δ (ppm) 13.68 (1H, br), ca. 9.65 (1H, br), 8.02 (1H, br s), 7.
84(1H,d,J=0.5Hz),6.54(1H,s),5.46(1H,br d,J=
11.0Hz), 4.97 (1H, d, J = 2.1Hz), 4.33 (1H, dq, J = 9.
5,6.3Hz),3.66(1H,dd,J=9.5,8.4Hz),3.49(1H,dd,J
=8.4,2.1Hz),3.48(1H,s),3.17(1H,d,J=5.0Hz),
3.02 (3H, d, J = 0.5Hz), 2.74 (1H, d, J = 5.0Hz), ca.2.6
(1H),2.60(6H,br s),2.42(6H,s),1.91(3H,s),
1.55 (3H, s), 1.42 (3H, d, J=6.3Hz), 1.34 (3H, s),
1.33 (1H, dd, J=14.7, 11.5Hz), 1.11 (3H, s), 1.09 (3
H, s) (g) ¹³ C-NMR spectrum (100 MHz, measured in CDCl ₃ ,
Internal standard TMS) δ (ppm) 188.0,181.0,178.3,164.9,159.8,156.5,156.1,150.3,13
8.7, 137.3, 136.4, 129.4, 128.8, 126.1 (2), 119.0, 116.
3,111.0,100.0,95.5,79.0,76.7,69.2,66.1,63.6,62.6,5
9.8, 59.0, 58.6, 58.2, 43.3, 41.0, 40.9, 25.2, 24.25, 24.1
6, 19.8, 19.7, 17.7, 14.4 (h) Thin-layer chromatography: Toluene:
The Rf is 0.20 in the acetone (6:4 v/v) development system.

(i) Weakly basic substances: The above-mentioned physicochemical properties (a), (c), (d),
Based on (f) and (g), the planar structural formulae of DC-92B and DC-92D were determined as follows:

The biological properties of DC-92B and DC-92D are as follows:

Antibacterial activity was measured by mixing 3g of Bactotryptone (Difco), 3g of meat extract, 1g of yeast extract, 1g of glucose, and 16g of agar.
The measurement was carried out by the agar dilution method using a medium (pH 7) prepared by dissolving the compound in water.

(B) Acute toxicity The acute toxicity value _LD50 of DC-92B in intraperitoneal administration to mice was approximately 0.31 mg/kg. The _LD50 of DC-92D was approximately 5.6 mg/kg.
/kg.

(C) Anticancer activity (1) Therapeutic effect on sarcoma 180 tumors. Sarcoma 180 tumor cells (5 x ¹⁰⁶ cells) were implanted subcutaneously in the axilla of six male ddy mice weighing approximately 20 g. Twenty-four hours after implantation, 0.2 ml of various concentrations of DC-92B or DC-92D in phosphate buffered saline (PBS) was administered intraperitoneally once. PBS consisted of 0.8 g/dl NaCl, 0.02 g/dl KCl, and _{1.5 g} NaHPO4.
15g/dl, KH ₂ PO ₄ 0.02g/dl, pH 7.2.

As a comparative example, a group was set up in which 24 hours after tumor cell transplantation, 0.2 ml of PBS containing mitomycin C was administered intraperitoneally. The average tumor weight ( ^mm3 ) and T/C (T: average tumor volume in the test example, C: 0.2 ml of PBS administered intraperitoneally) 10 days after transplantation are shown below.

(2) Therapeutic Effect on Lymphocytic Leukemia P388 Tumor Lymphocytic leukemia P388 tumor cells (1 × 10 ⁶ cells) were intraperitoneally transplanted into groups of 5 male CDF1 mice weighing approximately 22 g. 24 hours after transplantation, DC-
0.2 ml of a PBS solution of 92B or DC-92D was administered intraperitoneally once.

As a comparative example, a group was set up in which 24 hours after tumor cell transplantation, 0.2 ml of PBS containing mitomycin C was intraperitoneally administered. The mean survival time after transplantation and T/C (T: mean survival time of test cases, C: mean survival time of control cases) are shown below.

Next, the manufacturing method of DC-92B and DC-92D will be explained.

DC-92B and/or DC-92D are produced by culturing DC-92B and/or DC-92D producing strains belonging to the genus Actinomadura in a nutrient medium;
DC-92B and/or DC-92D can be obtained by accumulating them in a culture and then harvesting them from the culture. Any strain that belongs to the genus Actinomadura and has the ability to produce DC-92B and/or DC-92D can be used as a DC-92B and/or DC-92D-producing strain. Furthermore, artificial mutation of these strains, such as by ultraviolet irradiation, has also been found to produce DC-92B and/or DC-92D, such as strain DO-92.

The mycological properties of the DO-92 strain are as follows:

(1) Morphological characteristics Aerial mycelium: Branched but not divided Substrate mycelium: Not divided Spores: Attached to the tips of simple branches from aerial mycelium as short, curved chains Spore surface: Smooth Spore shape and size: Elliptical, approximately 0.6 x 1.1 μm Spore motility: None No formation of sclerotia or spore spores is observed.

(2) Color Aerial mycelium: Beige Substrate mycelium: Beige to yellowish brown Soluble pigments: Cream to yellowish brown Melanin pigments: None (3) Chemical properties Three-dimensional form of diaminopimelic acid: Meso-form Total cell sugars: Trace amounts of madurose are present.

(4) Growth in various media The results of culturing DO-92 strain in various media at 28°C for 3 weeks are shown in Table 1. The colors are indicated according to the Color Harmony Manual (Container Corporation).
In the table, G indicates growth, AM indicates the degree and color of aerial mycelia, SM indicates the color of substrate mycelia, and P indicates the production or color of soluble pigments.

(5) Physiological characteristics: Growth temperature: 2 days after growth, 28°C after growth for the effects on skim milk and cellulose, and 1 month after growth.
The results are shown after two weeks at 28°C.

Growth temperature range: 20℃ to 37℃ Optimum temperature range: 28℃ to 32℃ Liquefaction of gelatin: Negative Decomposition of cellulose: Negative Coagulation of skim milk: Negative Peptonization of skim milk: Positive Production of melanin pigment: Negative (6) Assimilation of carbon sources (7) Identification of DO-92 strain The DO-92 strain contains meso-diaminopimelic acid in its cell wall, and madurose was detected as a characteristic sugar in the total cell sugars. Therefore, according to the classification by Rechevalier et al. (Int. J. Syst. Bacteriol. 20, 435-443, 1970), it is a strain with cell wall type III and total cell sugar type B.

On the other hand, morphologically, this strain forms aerial mycelia, has simple branches, and produces short spore chains at the tips of the branches. Therefore, this strain is classified into the genus Actinomadura within the order Actinomycetales.

Therefore, strain DO-92 was identified as Actinomadura
a) and named it Actinomadura sp. DO-92.
It has been deposited at the Fermentation Research Institute of the Agency of Industrial Science and Technology under the accession number 1019 (original deposit date: April 23, 1986).
day).

In the present invention, the culture medium may be any medium containing an appropriate amount of carbon, nitrogen, inorganic substances, etc., and may be any medium suitable for culturing actinomycetes.
As carbon sources, glucose, starch, glycerol, mannose, fructose, sucrose, molasses, etc. can be used alone or in combination. Furthermore, hydrocarbons, alcohols, organic acids, etc. can also be used depending on the assimilative ability of the bacteria.
Nitrogen sources include ammonium chloride, ammonium sulfate,
Inorganic and organic nitrogen-containing compounds such as ammonium nitrate, sodium nitrate, and urea, as well as natural nitrogen sources such as peptone, meat extract, yeast extract, dry yeast, corn steep liquor (CSL), soybean flour, and casamino acids, can be used alone or in combination. In addition, inorganic salts such as salt, potassium chloride, ferrous sulfate, zinc sulfate, manganese sulfate, copper sulfate, nickel sulfate, calcium carbonate, and phosphates can be added as needed, as well as organic and inorganic substances that promote the production of DC-92B and/or DC-92D, such as biotin and vitamins. Liquid culture, particularly submerged agitation culture, is the most suitable culture method. The culture temperature is between 20°C and 35°C.
The optimum temperature is 37°C, especially 28-32°C, and the pH of the medium should be adjusted to 4-10, especially 6-8, by adding ammonia water or ammonium carbonate solution.
After culturing for 1 to 7 days, the target substance is produced and accumulated in the culture medium. When the amount of production in the culture medium reaches a maximum, the culture is stopped.

To isolate and purify DC-92B and/or DC-92D from the culture medium,
Common separation and purification methods are used to isolate microbial metabolic products from their culture broth. For example, the culture is filtered, centrifuged, or otherwise removed, and the resulting liquid is passed through a nonionic porous resin, such as HP-20 (Mitsubishi Chemical), to adsorb the active components, followed by elution with methanol. The eluate is concentrated to dryness and then extracted with ethyl acetate. The extract is concentrated to dryness and purified using a gel filter, silica gel (Wako gel C-200, Wako Pure Chemicals), or the like, to obtain a crude product. This is further purified using silica gel (Wako gel LC _NH2, Wako Pure Chemicals), or the like, to obtain DC-92B and/or DC-92D. The purity of the DC-92B and/or DC-92D thus obtained can be further increased by recrystallization, HPLC, or other methods.

The behavior of DC-92B and/or DC-92D during the cultivation and purification procedures is measured by bioassay using Bacillus subtilis No. 10707.
The ultraviolet absorption of DC-92B and DC-92D can be used as a guide to track the reaction.

DC-92B and DC-92D can be used as antitumor agents, optionally together with at least one pharmaceutical diluent, adjuvant, or carrier.
DC-92B is administered intravenously at a dose of 0.002 to 0.22 mg/kg, and DC-92D is administered intravenously at a dose of 0.04 to 4 mg/kg, dissolved in saline, glucose, lactose, or mannitol injection.
Furthermore, intra-arterial, intraperitoneal, and intrathoracic administration is also possible at similar dosages. It may also be freeze-dried in accordance with the Japanese Pharmacopoeia, or may be a powder injection containing sodium chloride. It may also contain well-known pharmaceutically acceptable diluents, adjuvants, and/or carriers, such as salts that meet pharmaceutical uses. When used as an injection, it may be preferable to use an adjuvant to increase solubility. The dosage can be increased or decreased as appropriate depending on age and symptoms. The administration schedule can be varied depending on symptoms and dosage, for example, once a week or once every three weeks.
It is administered intermittently, such as once a day. Oral and rectal administration is also possible using the same dosage and method. For oral administration, it can be administered as tablets, powders, granules, syrup, or suppositories together with appropriate adjuvants.

The above antitumor agents are effective against chronic lymphocytic leukemia, chronic myeloid leukemia, breast cancer, stomach cancer, liver cancer, colon cancer, rectal cancer, lung cancer, pancreatic cancer,
It is expected to be used in the treatment of uterine cancer, head and neck tumors, etc. The content of DC-92B or DC-92D in the antitumor agent is 0.01 to 20 mg in 20 to 50 ml when used as an injection, and 0.00 mg in tablets, capsules, powders, granules, or suppositories.
A suitable range is 1 to 85% by weight.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the ultraviolet absorption spectrum of DC-92B in methanol.

FIG. 2 shows the ultraviolet absorption spectrum of DC-92B in alkaline methanol containing sodium hydroxide.

FIG. 3 shows the ultraviolet absorption spectrum of DC-92D in methanol.

FIG. 4 shows the ultraviolet absorption spectrum of DC-92D in alkaline methanol containing sodium hydroxide.

FIG. 5 shows the infrared absorption spectrum of DC-92B (measured with CHCl ₃ ).

FIG. 6 shows the infrared absorption spectrum of DC-92D (measured with CHCl ₃ ).

Examples The following are examples and reference examples of the present invention.

Example: Actinomadura sp. DO-92 strain was used as the seed culture. The strain was cultured in a 50 ml wide test tube containing 10 g/l of soluble starch and 5 g/l of Bacto Tryptone (manufactured by Difco).
l, yeast extract 5g/l, meat extract 3g/l, glycol 10g/l,
The bacteria is inoculated into 10 ml of a seed medium (pH 7.2 before sterilization) containing 2 g/l of calcium carbonate, and cultured at 28°C for 48 hours with shaking (200 rpm).

The resulting seed culture was then transferred to a 2-liter flask containing 30 ml of the same medium.
The resulting seed culture was inoculated into 15 L of a fermentation medium of the following composition in a 30 L jar fermenter, and cultured with aeration and agitation (rotation speed 250) at 28°C for 24 hours.
The culture was carried out at 1000 rpm (15 L/min aeration).

Fermentation medium composition: glucose 15g/l, cottonseed flour 20g/l, KH ₂ P
O ₄ 0.5g/l, MgSO ₄・7H ₂ O 0.5g/l (pH 7.0, NaOH before sterilization)
(adjusted with).

The pH of the medium during cultivation was maintained at pH 6.5 to 6.5 using ammonia water.
The culture is then adjusted to 7.5 and cultured for 90 hours. The bacterial cells are separated from the culture medium to obtain 15 L of liquid. This 15 L of liquid is passed through 2 L of non-ionic porous resin (HP-20; manufactured by Mitsubishi Chemical) to adsorb the active substance, and then washed with water and then with a 50% methanol solution to remove impurities. The resin is then eluted with methanol. The eluted fraction is concentrated and
The pH was adjusted to 10 and extracted with ethyl acetate. The ethyl acetate layer was concentrated and then filtered through molecular sieves, Sephadex LH20 (Pharmacia Fine Chemical
The active fraction was then applied to a column (manufactured by Icals) and eluted with methanol to obtain the active fraction.
The column was chromatographed on a 40 μm silica gel column (Baker, USA). The column was eluted with toluene-acetone (7:3) to obtain crude DC-92B and DC-92D. These were then further eluted on a chemically bonded silica gel (Wako gel LC
The mixture was applied to a column containing NH2 _1OH (Wako Pure Chemical Industries, Ltd.) and eluted with toluene-acetone (7:3). The fractions containing the active substance were concentrated to dryness to give 15 mg of DC-92B and 7 mg of DC-92D, respectively. DC-92B and DC-92D thus obtained exhibited the physicochemical and biological properties described above.

The behavior of DC-92B and DC-92D during the cultivation and purification procedures was monitored by bioassay using Bacillus subtilis No. 10707 and by measuring the ultraviolet absorption of DC-92B and DC-92D by thin-layer chromatography.

Reference Example 1 Injection 4 g of DC-92B was dissolved in 20 liters of ethanol, and then the solution was sterilized by filtering through a Millipore filter (pore size 0.22 μm) under pressure.
5.0 ml of the resulting sterile solution is dispensed into brown vials and lyophilized in a conventional manner to obtain 0.2 mg/vial of a lyophilized agent.

Reference Example 2 Tablet DC-92D 130 mg, lactose 90 mg, cornstarch 40 mg
Tablets are prepared from 100g of 100mg ... polyvinyl alcohol, 28mg of Avicel, and 1mg of magnesium stearate in a conventional manner.

Continued from the front page Examiner Hiroshi Taniguchi

Claims (1)

[Claims] Claim 1: DC-92B or DC-92D, whose planar structural formula is represented by the following formula I: (wherein R represents a group represented by formula II or III)