JPH1121263A - BE-45985 antitumor substances - Google Patents

Abstract

(57) [Summary] [Problem] To create a new antitumor agent. Kind Code: A1 Structural formula [I] [Wherein, R ¹ represents a hydrogen atom or a methyl group, and R ² represents an oxygen atom or an imino group], or a pharmaceutically acceptable salt thereof.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention is useful in the field of medicine, and more specifically, a novel compound which inhibits proliferation of tumor cells and exerts an anticancer effect, a method for producing the compound, its use, and the present compound. New Streptomyces (S
(Treptomyces).

[0002]

2. Description of the Related Art In the field of cancer chemotherapy, many compounds have already been put into practical use as pharmaceuticals. However, its effect on various types of tumors is not always sufficient, and its clinical applicability is complicated as the phenomenon of resistance of tumor cells to these drugs is clinically revealed [47] Article of the General Meeting of the Japanese Cancer Society, 12
Pp. 15-15 (1988) etc.]. Under such circumstances, the development of new antitumor substances is always required in the field of cancer treatment.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel antitumor substance which can satisfy the above demand. That is, an object of the present invention is to find a compound having an excellent anticancer activity targeting various cancers in which existing anticancer substances cannot sufficiently exert an effect.

[0004]

Means for Solving the Problems In order to solve the above-mentioned problems, the present inventors screened a wide range of secondary metabolites of microorganisms for substances having antitumor activity, and found that the structural formula [I] and the structural formula [ The present invention was completed by finding that the compound represented by the formula [II] exhibits excellent antitumor activity. That is, the present invention provides a novel structural formula [I]

[0005]

Embedded image Wherein R ¹ represents a hydrogen atom or a methyl group, and R ² represents an oxygen atom or an imino group, or a pharmaceutically acceptable salt thereof, or a structural formula [II]

[0006]

Embedded image A compound represented by the above structural formula [II] and a compound represented by the structural formula [III]

[0007]

Embedded image The present invention relates to a microorganism belonging to the genus Streptomyces having the ability to produce the compound represented by the formula: The physical properties of the novel antitumor substances BE-45985 according to the present invention are shown below.
Here, the compound represented by the structural formula [III] is referred to as BE-4
5985A _1, R ¹ is hydrogen atom structural formula [I], R
² is an oxygen atom the compound BE-45985A _2, R ¹
Is a methyl group and R ² is an imino group.
-495985A ₃ , a compound in which R ¹ is a hydrogen atom and R ² is an imino group was converted to BE-451985A ₄ by the structural formula [I
The compound represented by I] is referred to as BE-45985X.

[0008] The compounds of the present invention can exist in the form of pharmaceutically acceptable salts. Such salts include:
For example, inorganic acids such as hydrochloric acid, sulfuric acid or acetic acid, citric acid,
And addition salts with organic acids such as tartaric acid and maleic acid. When the compound of the present invention contains an acidic group,
The acidic group may be present in the form of a salt with an organic base such as an alkali metal salt such as a potassium salt and a sodium salt, an alkali metal salt such as a magnesium salt and a calcium salt, an ethylamine salt and an arginine salt. N below
The meaning of the symbol in MR measurement is shown. s: singlet d: doublet t: triplet q: quartet m: multiplet br: broad J: coupling constant Hz: physicochemical properties properties of hertz BE-45985A _1; red-brown amorphous solid or crystalline molecular formula; C ₂₄ H ₁₉ O ₇ Cl mass spectrometry; [HRFAB-MS] (M + H) ⁺ m / z
As: Actual value 455.0899 Calculated value 455.0898 Ultraviolet absorption spectrum; λ _max (CHCl ₃ , nm): 3
10,434,510 (sh) infrared absorption spectrum; ν (KBr, cm ⁻¹ ): 308
1,960,1724,1626,1541,1,
500, 1410, 1371, 1350, 1280,
1220, 1169, 1126, 1078, 1039,
883, 719 ¹ H-NMR spectrum (CDCl ₃ , 500 MHz) δ
ppm: 12.1 (1H, s), 11.5 (1H,
s), 7.97 (1H, s), 7.42 (1H, s),
7.35 (1H, brs), 3.98 (3H, s),
3.84 (3H, s), 3.04 (2H, q, J = 7.
3 Hz), 2.54 (3H, d, J = 0.9 Hz),
1.27 (3H, t, J = 7.3 Hz) ¹³ C-NMR spectrum (CDCl ₃ , 125 MHz)
δ ppm: 192.9 (s), 183.4 (s), 16
6.5 (s), 160.4 (s), 156.3 (s),
153.8 (s), 141.1 (s), 140.5
(S), 138.6 (s), 137.9 (s), 13
7.0 (s), 133.3 (s), 126.6 (s),
122.7 (d), 119.4 (s), 119.3
(S), 118.9 (d), 117.6 (d), 11
5.5 (s), 61.4 (q), 52.5 (q), 2
1.1 (q), 20.3 (t), 13.8 (q) Solubility; soluble in organic solvents such as ethyl acetate. Distinguishing between acidic, neutral and basic substances; neutral substances Color reaction; sulfuric acid reaction Positive Rf value: 0.77 [Merck Kieselgel 60F
₂₅₄ used, developing solvent: hexane / ethyl acetate (2:
1) color reaction; physicochemical properties properties of sulfuric acid reaction positive BE-45985A _2; red-brown amorphous solid or crystalline molecular formula; C ₂₃ H ₁₇ O ₇ Cl mass spectrometry; [HRFAB-MS] (M + H) + m /
As z: Found 441.0739 Calculated 441.0741 UV absorption spectrum; λ _max (MeOH, nm): 2
32, 304, 435, 505 (sh) infrared absorption spectrum; ν (KBr, cm ⁻¹ ): 293
3,1716,1629,1558,1409,13
75, 1276, 1240, 1165, 1124, 1
081, 1041, 887, 729 ¹ H-NMR spectrum (CDCl ₃ , 400 MHz) δ
ppm: 12.1 (1H, s), 11.5 (1H,
s), 8.09 (1H, s), 7.43 (1H, s),
7.35 (1H, s), 3.85 (3H, s), 3.1
2 (2H, q, J = 7.4 Hz), 2.54 (3H,
s), 1.29 (3H, t, J = 7.4 Hz) ¹³ C-NMR spectrum (CDCl ₃ , 100 MHz)
δ ppm: 193.1 (s), 183.6 (s), 16
8.2 (s), 160.5 (s), 156.5 (s),
153.9 (s), 141.4 (s), 141.3
(S), 138.8 (s), 137.1 (s), 13
6.7 (s), 133.5 (s), 126.9 (s),
122.9 (d), 119.6 (s), 119.5
(D), 119.4 (s), 117.8 (d), 11
6.0 (s), 61.4 (q), 21.2 (q), 2
0.4 (t), 13.8 (q) Solubility; soluble in organic solvents such as dimethyl sulfoxide and methanol. Acidic, neutral, basic substance; acidic color reaction; sulfuric acid reaction positive Rf value: 0.43 [Merck Kieselgel 60F
₂₅₄ used, developing solvent: chloroform-methanol (3:
1)] Physicochemical properties of BE-45985A ₃ ; purple amorphous solid or crystal molecular formula; C ₂₄ H ₂₀ O ₆ NCl mass spectrometry; [HRFAB-MS] (M + H) ⁺ m / z
As: actual value 454.1044 calculated value 454.1057 UV absorption spectrum; λ _max (MeOH, nm): 2
44, 305, 575 infrared absorption spectrum; ν (KBr, cm ⁻¹ ): 332
3,2950, 1730, 1610, 1456, 13
57,1267,1243,1076,1022,8
81, ¹ H-NMR spectrum (400 MHz, DMSO-
d ₆ ) δ ppm: 16.3 (1H, s), 12.5 (1
H, brs), 11.9 (1H, s), 7.62 (1
H, s), 7.51 (1H, s), 7.38 (1H,
s), 3.89 (3H, s), 3.62 (3H, s),
2.89 (2H, q, J = 7.3 Hz), 2.48 (3
H, s), 1.13 (3H, t, J = 7.3 Hz) ¹³ C-NMR spectrum (100 MHz, DMSO-d
₆ ) δ ppm: 184.1 (s), 170.2 (s),
167.4 (s), 163.5 (s), 154.9
(S), 153.4 (s), 142.7 (s), 13
8.7 (s), 136.5 (s), 136.1 (s),
134.0 (s), 132.1 (s), 124.9
(S), 122.7 (d), 119.3 (s), 11
8.2 (s), 115.7 (d), 112.5 (d),
111.3 (s), 60.8 (q), 52.4
(Q), 20.6 (q), 20.4 (t), 14.1
(Q) Solubility; soluble in organic solvents such as dimethyl sulfoxide and methanol. Distinguishing between acidic, neutral and basic substances; weakly basic Rf value; 0.32 [Merck Kieselgel 60F
₂₅₄ used, developing solvent: chloroform-methanol (1
0: 1)] physicochemical properties properties of BE-45985A _4; purple amorphous solid or crystalline molecular formula; C ₂₃ H ₁₈ O ₆ NCl mass spectrometry; [HRFAB-MS] (M + H) + m / z
As: Actual value 440.0896 Calculated value 440.0901 Ultraviolet absorption spectrum; λ _max (MeOH, nm): 2
37, 303, 535 infrared absorption spectrum; (KBr, cm ^-1 ): 314
7,2962,1716,1670,1616,16
00, 1456, 1407, 1356, 1338, 1
240, 1051, 887, 759 ¹ H-NMR spectrum (400 MHz, DMSO-
d ₆ ) δ ppm: 16.3 (1H, s), 13.3 (2
H, brs), 12.0 (1H, s), 7.61 (1
H, s), 7.49 (1H, s), 7.37 (1H,
s), 3.62 (3H, s), 2.92 (2H, q, J
= 7.3 Hz), 2.47 (3H, s), 1.13 (3
H, t, J = 7.3 Hz) ¹³ C-NMR spectrum (100 MHz, DMSO-d)
₆ ) δ ppm: 184.3 (s), 170.1 (s),
168.8 (s), 163.6 (s), 155.3
(S), 153.4 (s), 142.1 (s), 13
8.6 (s), 136.5 (s), 136.1 (s),
135.5 (s), 132.0 (s), 124.7
(S), 122.6 (d), 119.4 (s), 11
8.0 (s), 115.7 (d), 112.7 (d),
111.2 (s), 60.8 (q), 20.6 (q),
20.3 (t), 14.2 (q) Solubility; soluble in organic solvents such as dimethyl sulfoxide and methanol. Distinction between acidic, neutral and basic substances; amphoteric Rf value: 0.23 [Merck Kieselgel 60F
₂₅₄ used, developing solvent: chloroform / methanol (2:
1)] Color reaction; sulfuric acid reaction Positive physicochemical properties of BE-45985X ; dark brown amorphous solid or crystal molecular formula; C ₂₃ H ₁₂ O ₈ mass spectrometry; [HRFAB-MS] (M + 3H) ⁺ m /
As z: actual value 419.0076 calculated value 419.0767 ultraviolet absorption spectrum; λ _max (MeOH, nm): 2
31, 272, 454, 550; (HCl acidic MeO
H, nm): 205, 233, 263, 468; (Na
(OH basic MeOH, nm): 234, 281, 375
(S), 610 infrared absorption spectrum; (KBr, cm ^-1 ): 335
4,3296,1653,1558,1541,14
19, 1373, 1105, 1070, 608 ¹ H-
NMR spectrum (500 MHz, THF-d ₈ ) δp
pm: 2.34 (3H, s), 6.31 (1H, s),
6.85 (1H, s), 7.14 (1H, s), 7.6
6 (1H, s), 8.31 (1H, s), 9.76 (1
H, s), 12.4 (1H, s), 13.9 (1H,
s) ¹³ C-NMR spectrum (125 MHz, THF-
d ₈ ) δ ppm: 21.9 (q), 112.4 (d),
117.0 (d), 117.7 (s), 118.1
(D), 120.2 (s), 120.3 (d), 12
2.5 (s), 124.0 (d), 125.2
(S), 133.7 (s), 136.2 (s), 14
2.2 (s), 142.4 (s), 143.2 (s),
156.2 (s), 158.1 (s), 162.4
(S), 163.5 (s), 181.3 (s), 18
8.9 (s), 189.3 (s), 192.8 (s) Solubility: Easily soluble in dimethylsulfoxide, dimethylformamide, etc., slightly soluble in methanol and tetrahydrofuran, but hardly soluble in water. Distinguishing between acidic, neutral and basic substances; weakly acidic substances Rf value: 0.38 [Merck Kieselgel 60F
₂₅₄ used, developing solvent: chloroform-methanol (1
0: 1)] Biological activity (anti-tumor effect) of BE-45985 class In order to determine the anti-proliferative effect of the antitumor substance BE-45985 class on experimental mouse tumor cells, in vitro
The test was performed at o. In an antitumor test for mouse leukemia cell P388, after dissolving BE-45985 in dimethyl sulfoxide, RPMI1 containing 10% fetal calf serum was used.
Serially diluted with 640 medium (containing 20 mM 2-mercaptoethanol) and cell culture medium containing 2 × 10 ³ tumor cells (RPMI1640 medium containing 10% fetal bovine serum, containing 20 mM 2-mercaptoethanol) 50
50 μl was added per μl. 5 hours at 37 ° C for 72 hours
After culturing under CO ₂ , comparison was made with the control group by MTT measurement.

[0009] An antitumor test against mouse colon cancer cell colon 26 was carried out by dissolving BE-45985 in dimethyl sulfoxide and then adding RPMI16 containing 10% fetal bovine serum.
Cell culture medium containing 1 × 10 ³ tumor cells (RPMI 1640 containing 10% fetal calf serum)
(Medium) 100 μl was added to 100 μl. After culturing at 37 ° C. for 72 hours under 5% CO ₂ , the cells were fixed with 50% trichloroacetic acid, stained with 0.4% sulforhodamine B, and
Dye was extracted from cells using 0 mM Tris solution. 45
The absorbance at 550 nm was measured using 0 nm as a control wavelength and compared with the control group. As a result, BE-45985
Show strong growth inhibitory activity against both cancer cells, with 50%
The growth inhibitory concentrations (IC ₅₀ ) are as shown in Table 1. In addition, the antitumor activity of BE-45985s against human cancer cells was tested in vitro.

The cells used are human colon cancer cells DLD-1, human lung cancer cells PC-13 and human gastric cancer cells MKN-45, and the cell culture medium is RPMI1640 medium containing 10% fetal calf serum for all cancer cells. Was used. BE-45
985s were first dissolved in dimethyl sulfoxide, and then serially diluted with RPMI1640 medium containing 10% fetal bovine serum to obtain a test solution. Assay for inhibition of cancer cell growth was performed by dispensing 100 μl of a cell culture medium containing 1 × 10 ³ cancer cells into a 96-well microplate, and heating at 37 ° C. for 24 hours with 5% C
After culturing under O ₂ , 100 μl of the above test solution was added. Further, after culturing for 72 hours, the cells were fixed with 50% trichloroacetic acid, and compared with a control group using the same method as for colon 26 cells. As a result, BE-45985s showed strong growth inhibitory activity even in human tumor cells,
The percent growth inhibitory concentrations (IC ₅₀ ) are as shown in Table 1.

[0011]

[Table 1] As mentioned above, BE-45885s have a marked inhibitory effect on the growth of mouse and human tumor cells. Therefore,
The present invention is useful as an antitumor agent for mammals including humans. Next, a method for producing BE-45885 will be described. BE-45985, an antitumor substance of the present invention
The microorganism used for the production of the microorganisms or a mutant thereof may be any microorganism that produces the antitumor substance BE-45885, and examples thereof include microorganisms having the following mycological properties. 1. Form A45985 strain forms an aerial mycelium with a base mycelium that elongates and diverges well, and there is no division of the ring vegetation and the hypha. A long chain of spores (50 or more) is formed on the aerial hyphae, and its form is mainly linear to wavy, but also hook-to-loop. Spore surface is hairy and 1.0-0.7 in size
It is a cylinder and oval of about 0.7 to 0.6 μm, and no special organs such as spores, flagella spores and sclerotia are observed. 2. Table 2 shows the cultivation properties of the A45585 strain on various agar plate media.

[0012]

[Table 2] 3. Growth temperature (yeast / malt agar medium, cultivated for 14 days) 9 ° C; not growing 12 ° C; poor growth, no aerial mycelium formation 15 ° C; good growth, aerial mycelium formation only 18 ° C; good growth, good aerial mycelium formation 22 ° C; very good growth and aerial mycelium formation 26 ° C; very good growth and aerial mycelium formation 29 ° C; very good growth and aerial mycelium formation 35 ° C; poor growth and no aerial mycelium formation 38 ° C; 4. Physiological properties (1) Gelatin liquefaction positive (glucose / peptone / gelatin medium) (2) Starch hydrolysis positive (starch / inorganic salt agar medium) (3) Coagulation of skim milk powder negative (Skim milk medium) (4) 4. Peptone conversion of skim milk powder positive (Skim milk medium) (5) Melanin-like pigment formation negative (6) Salt tolerance Growing at a salt content of 4% or less (Yeast / malt agar medium) Ability to Use Carbon Source Pridham-Godlieve Agar was used as a basal medium, and the following various sugars were added thereto and cultured at 28 ° C. for 14 days. (+ Indicates that it is used, and-indicates that it is used.)

[0013]

[Table 3] 6. Cell wall composition LL diaminopimelic acid was detected. From the above mycological properties, it is considered that the A45985 strain belongs to the genus Streptomyces. Therefore, the A44985 strain was transformed into Streptomyces sp.
ptomyces sp. A45985). This strain has been deposited with the Ministry of International Trade and Industry at the National Institute of Advanced Industrial Science and Technology, and the deposit number is FER.
MP-14707. A mutant strain of a microorganism that produces the antitumor substances BE-45985 used in the present invention is:
Irradiation treatment with, for example, X-rays or ultraviolet rays, for example, treatment with a mutagen such as nitrogen mustard, azaserine, nitrous acid, 2-aminopurine or N-methyl-N′-nitro-N-nitrosoguanidine (NTG);
It is a microorganism obtained by mutating a BE-45885-producing bacterium by a commonly used bacterial species conversion treatment method such as phage contact, transformation, transduction or conjugation. BE-45 of the present invention
In producing 985s, a culture containing BE-45985 can be obtained by inoculating a production strain of BE-45585 into a nutrient-containing medium and growing aerobically. As a nutrient source, a known nutrient source for actinomycetes can be used. For example, as a carbon source, commercially available glucose, maltose, starch, sucrose, molasses, dextrin or the like is used alone or as a mixture. Nitrogen sources include commercially available soy flour, corn steep liquor, meat extract, yeast extract, dried yeast, cottonseed flour, peptone, wheat germ, fish meal, meat meal, defatted rice bran, defatted meat-and-bone meal, inorganic ammonium Salt or sodium nitrate is used alone or as a mixture. As inorganic salts,
Commercially available calcium carbonate, sodium chloride, potassium chloride, magnesium sulfate, sodium bromide, sodium borate or various phosphates can be used. In addition, if necessary, trace amounts of heavy metal salts such as iron, manganese, zinc, cobalt, and molybdic acid can be added. In the case of severe foaming, for example, vegetable oils such as soybean oil or linseed oil, higher alcohols such as octadecanol, various silicon compounds, and the like may be appropriately added as antifoaming agents. In addition to these, any one that can be used by the producing bacterium and is useful for the production of BE-45985, such as 3- (N-morpholino) propanesulfonic acid or sodium borate, can be used.

The culturing method may be the same as the method for producing a general metabolite of a microorganism, and may be a solid culture or a liquid culture. In the case of liquid culture, static culture, stirring culture,
Either shaking culture or aeration culture may be performed, but shaking culture or deep aeration stirring culture is particularly desirable. The culture temperature is suitably from 16 to 36 ° C, but preferably 18 to 36 ° C.
~ 29 ° C. The preferable pH of the medium is in the range of 4 to 8, and the culture time is 72 hours to 400 hours, preferably 96 hours.
Hours to 360 hours. The desired BE-
In order to collect 44985, a separation means usually used for collecting from metabolites produced by microorganisms is appropriately used.

Since BE-45985 is present in the culture filtrate and in the cells, it can be separated from the culture filtrate or the cells by a conventional means such as a solvent extraction method, an ion exchange resin method or an adsorption or partition chromatography method and gel filtration. It can be purified by performing the method alone or in combination. Preferred examples of separation and purification include the following methods. First, the culture solution is filtered to obtain cells. The obtained cells are extracted using an organic solvent such as methanol or acetone. The obtained crude extract is partitioned with water-ethyl acetate, and the extract obtained after distilling off ethyl acetate is subjected to reverse phase column chromatography. The fraction containing BE-45685 was concentrated under reduced pressure, and the residue was again subjected to reverse-phase column chromatography to obtain BE-4585.
985 types can be obtained. Compound BE-4 of the present invention
The 5985s inhibit the growth of tumor cells and exert an anticancer effect.However, various forms can be selected as the administration form when the compound of the present invention is used as an antitumor agent, for example, tablets, capsules, powders, An oral preparation such as a granule or a liquid preparation, or a sterile liquid parenteral preparation such as a solution or suspension can be used.

The solid preparation can be produced as it is in the form of tablets, capsules, granules or powders, but can also be produced using appropriate additives. Examples of such additives include sugars such as lactose or glucose, for example, starches such as corn, wheat or rice, fatty acids such as stearic acid, inorganic salts such as magnesium metasilicate aluminate or anhydrous calcium phosphate, for example, polyvinyl, and the like. Synthesis of synthetic polymers such as pyrrolidone or polyalkylene glycol, for example, fatty acid salts such as calcium stearate or magnesium stearate, for example, alcohols such as stearyl alcohol or benzyl alcohol, for example, methylcellulose, carboxymethylcellulose, ethylcellulose or hydroxypropylmethylcellulose Cellulose derivatives, other,
Examples of commonly used additives such as water, gelatin, talc, vegetable oil, and gum arabic are included.

These solid preparations such as tablets, capsules, granules and powders generally contain 0.1 to 100% by weight,
It preferably contains from 5 to 100% by weight of active ingredient. Liquid preparations use suitable additives usually used in liquid preparations such as water, alcohols or plant-derived oils such as soybean oil, peanut oil or sesame oil, and are used in the form of suspensions, syrups or injections. Manufactured as
Particularly suitable solvents for parenteral administration by intramuscular injection, intravenous injection or subcutaneous injection include, for example, distilled water for injection, lidocaine hydrochloride aqueous solution (for intramuscular injection), physiological saline, glucose aqueous solution, ethanol, Liquid for intravenous injection (for example, aqueous solution of citric acid and sodium citrate) or electrolyte solution (for intravenous drip and intravenous injection)
Or a mixed solution thereof.

These injections may be dissolved in advance, or may be in the form of powder or a solution to which an appropriate additive is added, which is dissolved at the time of use. These injections usually contain 0.1 to 10% by weight, preferably 1 to 5% by weight, of the active ingredient. Liquid preparations such as suspensions or syrups for oral administration contain 0.5 to 10% by weight of active ingredient.

It should be noted that the actual preferred dosage of the compounds of the present invention will vary with the type of compound used, the type of composition formulated, the frequency of application and the particular site to be treated, the host and the tumor. It is. For example, the daily adult dose is 10 to 50 for oral administration.
0 mg, and in the case of parenteral administration, preferably intravenous injection, 10 to 100 mg per day. The number of administrations varies depending on the administration method and symptoms, but is 1 to 5 times. Hereinafter, the present invention will be described specifically with reference to examples, but the present invention is not limited to these examples.

[0020]

BEST MODE FOR CARRYING OUT THE INVENTION

[0021]

【Example】

EXAMPLE 1 BE-45985A 0.1% glucose actinomycetes A45985 strain was inoculated to the production slant soft agar medium _1, dextrin 2%, 0.5% fish meal, corn gluten meal 1%, 0.1% yeast extract, Magnesium sulfate 0.05%, sodium chloride 0.1%, calcium chloride 0.05%, ferrous sulfate 0.0002%, cupric chloride 0.00004%, manganese chloride 0.000%
4%, cobalt chloride 0.00004%, zinc sulfate 0.0
Inoculate two 500 ml Erlenmeyer flasks containing 110 ml of a medium (pH 7.0) consisting of 0008%, sodium borate 0.00008%, and sodium molybdate 0.00024%, and rotate on a rotary shaker at 28 ° C. for 3 days. (180 rotations per minute). This culture solution was inoculated into 50 500 ml Erlenmeyer flasks each containing 110 ml of the above medium in an amount of 2 ml each and inoculated on a rotary shaker (1 minute per minute) at 28 ° C. for 5 days.
80 rotations).

The thus obtained culture solution (about 5 L)
Was added with 300 g of Celite (Wako Pure Chemical Industries, No. 545), and the mixture was filtered to separate cells. 2L of methanol
After stirring for several hours, the cells were filtered off to obtain a methanol extract. After the methanol extract was concentrated under reduced pressure, the residue was extracted with 600 ml of ethyl acetate and 300 ml of deionized water. The obtained ethyl acetate extract was concentrated under reduced pressure to obtain a black residue. 30 ml of ethyl acetate was added to the residue, followed by filtration, and the filtrate was distilled off under reduced pressure. The obtained residue is subjected to a silica gel column (Merck Kieselgel 6).
0, 2.0 cmφ × 40 cm) and sequentially eluted with an elution solvent of hexane / ethyl acetate (30: 1, 15: 1, 10: 1). 10 ml of methanol was added to the eluted active fraction.
Was added, and the mixture was concentrated to precipitate crystals. The solution containing the crystals was filtered under reduced pressure, to give 49mg of reddish brown powder of BE-45985A _1. Example 2 BE-45985A obtained in Example 1 of ₂ BE-45985A ₁ 12.5mg
To the mixture were added 0.5 ml of a 1N aqueous sodium hydroxide solution and 5 ml of methanol, followed by stirring at 60 ° C. for 2 hours. After the reaction, the reaction solution was concentrated under reduced pressure, and then 0.5 ml of 1N hydrochloric acid was added, whereby a brown precipitate was formed. The solution containing the precipitate was extracted by adding 5 ml of deionized water and 5 ml of ethyl acetate, and further washed with 10 ml of deionized water. After concentrating the ethyl acetate extract under reduced pressure, ethyl acetate 2 was added to the residue.
Then, 4 ml of methanol and 4 ml of methanol were added to dissolve and concentrate, and crystals were precipitated. The solution containing the crystals was filtered under reduced pressure, to give 7.7mg of the reddish brown powder of BE-45985A _2. 1 Example 3 BE-45985A BE-45985A ₁ obtained in Production Example 1 of ₃ 13 mg
2 ml of N-ammonia-methanol solution was added, and the mixture was stirred at room temperature for 24 hours. After the reaction, the mixture was neutralized with 1N hydrochloric acid and concentrated. The obtained residue was extracted by adding 50 ml of ethyl acetate and 100 ml of deionized water. The obtained ethyl acetate extract was concentrated to dryness under reduced pressure, and BE-45985A
_Thus , 12.7 mg of purple powder was obtained. Example 4 BE-45985A 2 in BE-45985A ₁ 10mg obtained in Example 1 of ₄
2 ml of 8% aqueous ammonia was added, and the mixture was stirred at room temperature for 4 hours. After 4 hours, the temperature was raised to 60 ° C in order for the reaction to proceed.
20 hours later, temperature 80 ° C., methanol 1 ml, 2
1 ml of 8% aqueous ammonia was added, and 40 hours later, 1 ml of 28% aqueous ammonia was added at 100 ° C. and the mixture was stirred for 48 hours. After the reaction, the reaction solution was distilled off under reduced pressure, and the residue was subjected to Sephadex LH-20 (manufactured by Pharmacia, 1.5 cm
Purification was performed through a chromatography column (φx30 cm, elution; methanol), and the desired fraction was concentrated under reduced pressure. 50 ml of ethyl acetate and 50 ml of deionized water are added to the obtained residue.
It was added extraction, and the resultant ethyl acetate extract was concentrated to dryness under reduced pressure, a purple powder BE-45985A ₄ 8mg
Obtained. Example 5 Production of BE-45985X The actinomycete A45598 strain inoculated on a slope soft agar medium was prepared using glucose 0.1%, dextrin 2%, fish meal 0.5%, corn gluten meal 1%, yeast extract 0.1%, and sulfuric acid. Magnesium 0.05%, sodium chloride 0.1%, calcium chloride 0.05%, ferrous sulfate 0.0002%, cupric chloride 0.00004%, manganese chloride 0.000%
4%, cobalt chloride 0.00004%, zinc sulfate 0.0
Inoculate one 500 ml Erlenmeyer flask containing 110 ml of a medium (pH 7.0) consisting of 0008%, sodium borate 0.00008% and sodium molybdate 0.00024%, and rotate at 28 ° C. for 7 days on a rotary shaker. (180 rotations per minute). This culture solution was inoculated into seven 500 ml Erlenmeyer flasks each containing 110 ml of the above medium in an amount of 3 ml and inoculated on a rotary shaker (18 minutes per minute) at 28 ° C. for 5 days.
0 rotation). Further, this culture was subjected to glucose 0.1%, dextrin 2%, MS- # 3600 (Nippon Shokuhin Kako Co., Ltd.) 2%, fish meal 0.5%, corn gluten meal 1%, yeast extract 0.1%, Magnesium sulfate 0.05%, sodium chloride 0.1%, calcium chloride 0.05%, ferrous sulfate 0.0002%, cupric chloride 0.00004%, manganese chloride 0.00004%, cobalt chloride 0. 00004%, zinc sulfate 0.00008
%, Sodium borate 0.00008% and sodium molybdate 0.00024% (pH
7.0) 200 500 ml Erlenmeyer flasks containing 110 ml were inoculated and cultured at 28 ° C. for 14 days on a rotary shaker (180 rotations per minute). 30 L of methyl ethyl ketone was added to about 20 L of the culture solution thus obtained, and the mixture was stirred for 1 hour, and then celite (Wako Pure Chemical Industries, No. 54).
5) 500 g was added and filtered. The upper layer of the filtrate was concentrated under reduced pressure, and methyl ethyl ketone was distilled off. The crude extract was adjusted to pH 4 using 6N HCl and left at 4 ° C. overnight. The resulting precipitate was sequentially washed with 150 ml of water, 150 ml of methanol and 150 ml of methyl ethyl ketone to obtain 7.03 g of a crude extract. This crude extract 0.7
Take 2 g, dissolve in 30 ml of dimethylformamide and 100 ml of toluene, charge a Wakogel C-300 (Wako Pure Chemicals, 4.0 cmf x 48 cm) column, and then toluene-dimethylformamide-methanol (100: 30: 5) every minute Elution was at 9 ml. The active fraction was collected, 400 ml of ethyl acetate was added, and the mixture was washed with 400 ml of 6 mM hydrochloric acid. 400 m of ethyl acetate layer
After washing with 1 l of water, the upper layer was concentrated under reduced pressure. After the ethyl acetate was distilled off, 100 ml of 3 mM hydrochloric acid was added and the mixture was slightly concentrated, and the resulting precipitate was collected by filtration to obtain a crude product 6
5.3 mg were obtained. Of these, 15.1 mg, 15.6 m
g was dissolved in 2 ml each of dimethylformamide and subjected to reverse phase chromatography. Column is YMC P
ack A343 S5 ODS (YMC, 2.0 cmφ × 25 cm), 20 mM ammonium formate buffer (pH 3.81) -acetonitrile (5
5:54) and eluted at a flow rate of 2 ml / min using the linear concentration gradient method so as to become (10:90) in 120 minutes. The obtained active fractions were collected, 200 ml of ethyl acetate was added, and the mixture was washed with 200 ml of water.
Washed with 0 ml. The upper layer is concentrated under reduced pressure and the water is about 1m
When the volume became 1, 3 ml of methanol was added, and the mixture was centrifuged at 3000 rpm for 10 minutes. 1 ml of water is added to the obtained precipitate.
And freeze-dried to give pure BE-449
85 x 6.6 mg were obtained as a dark brown powder. Formulation examples of the compound of the present invention are shown below, but the formulation of the compound of the present invention is not limited to this formulation example. Formulation Example 1 This substance (BE-45585) 10 (parts) heavy magnesium oxide 15 lactose 75 is uniformly mixed to prepare a powdery or fine granular powder having a size of 350 mm or less. This powder was placed in a capsule container to obtain a capsule. Formulation Example 2 This substance (BE-45585) 45 (parts) Starch 15 Lactose 16 Crystalline cellulose 21 Polyvinyl alcohol 3 Distilled water 30 After uniformly mixed, crushed and granulated, dried, and then sieved to obtain a diameter of 1410 to 1040. The granules were 177 mm in size. Formulation Example 3 After a granule was prepared in the same manner as in Formulation Example 2, 3 parts of calcium stearate was added to 96 parts of the granule, followed by compression molding to prepare a tablet having a diameter of 10 mm. Formulation Example 4 To 90 parts of the granules obtained by the method of Formulation Example 2, 10 parts of crystalline cellulose and 3 parts of calcium stearate were added and compression-molded to obtain a tablet having a diameter of 8 mm. Sugar-coated tablets were prepared by adding a mixed calcium carbonate suspension. Formulation Example 5 This substance (BE-45585) 0.6 (part) Nonionic surfactant 2.4 Physiological saline 97 is heated and mixed, then put into an ampoule, sterilized to prepare an injection. did.

[0023]

The BE-45985s described in the present invention,
Since it has a strong growth inhibitory effect on mouse and human cancer cells, it is useful as a therapeutic agent for cancer in the field of medicine.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C07C 69/95 C07C 69/95 251/22 251/22 C12N 1/20 C12N 1/20 A C12P 7/66 C12P 7/66 29 / 00 29/00 // (C12N 1/20 C12R 1: 465) (C12P 7/66 C12R 1: 465) (C12P 29/00 C12R 1: 465) (72) Inventor Shigeru Nakajima 3 Okubo, Tsukuba, Ibaraki, Japan Address Banyu Pharmaceutical Co., Ltd. Tsukuba Research Laboratories (72) Inventor Katsuhisa Kojiri 3 Okubo, Tsukuba City, Ibaraki Pref. Tsukuba Research Laboratories, a pharmaceutical company

Claims (9)

[Claims] (1) Structural formula [I] [Wherein, R ¹ represents a hydrogen atom or a methyl group, and R ² represents an oxygen atom or an imino group], or a pharmaceutically acceptable salt thereof. 2. A compound of the formula [II] Or a pharmaceutically acceptable salt thereof. 3. Structural formula [III] Producing a compound represented by the structural formula [III], which comprises culturing a microorganism having the ability to produce the compound represented by the formula or a mutant thereof, and collecting the compound represented by the structural formula [III]. Law. 4. A method of culturing a microorganism having the ability to produce the compound of claim 2 represented by the structural formula [II] or a mutant thereof, and collecting the compound represented by the structural formula [II]. A method for producing a compound represented by the structural formula [II]. 5. The method according to claim 2, wherein the microorganism or a mutant strain thereof is Streptomyces sp. 6. The method according to claim 1, wherein the microorganism is Streptomyces sp. A.
45585 (Streptomyces sp. 45
985) or a mutant thereof. 7. A Streptomyces sp. Capable of producing the compound according to claim 2 or 3.
omyces sp. ) Or a mutant thereof. 8. A Streptomyces sp. A45985 capable of producing the compound according to claim 2 or 3.
(Streptomyces sp. A44985)
Or a mutant thereof. 9. An antitumor agent comprising the compound according to claim 1 or 2 as an active ingredient.