JP2001261668A - Antitumor substance j-124131 and method for preparing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a compound capable of exhibiting antitumor effect even against various tumors to which existing antitumor substances can not exhibit sufficient effect. SOLUTION: A compound expressed by the formula [I] produced by Streptomyces sp. A74169 or its mutant, and an antitumor agent containing the compound as an active ingredient.

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 the growth of tumor cells and exerts an antitumor effect, a method for producing the compound, its use, and the use of the compound. It relates to the microorganism to be produced.

[0002]

2. Description of the Related Art In the field of cancer chemotherapy, many compounds have been put into practical use as pharmaceuticals. Bleomycin, adriamycin,
Actinomycin and the like have been used. Compounds structurally similar to the compound of the present application include the following structural formula [I
I]

[0003]

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[0004] The compound represented by
No. 94 (hereinafter abbreviated as Document A). The compound represented by the structural formula [II] described in Document A has two rhinosyl group derivatives in the molecule similarly to the compound of the present invention, but the bonding position of the two rhinosyl groups is different from that of the compound of the present invention. Is different, and the structure of the rhinosyl group itself is also different from the compound of the present application.

Japanese Patent Application Laid-Open No. 2-289532 (hereinafter abbreviated as Document B) discloses the following structural formula [III]:

[0006]

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[0007] The compound represented by the above formula [III] has only one rhosinosyl group in the molecule, and further has a side chain structure. Obviously, it differs from the compound.

[0008]

The antitumor substances used heretofore are not always sufficient in terms of efficacy, toxicity, etc., and the clinical phenomenon of the resistance of tumor cells to these drugs has been elucidated. , Its clinical applicability is becoming more complex [The 47th Annual Meeting of the Japanese Cancer Association, 12-12
5 (1988) etc.]. Under such circumstances, development of a novel antitumor substance is required in the field of cancer treatment. An object of the present invention is to provide a novel antitumor substance that can meet the above demand. That is, an object of the present invention is to provide a compound that exhibits an antitumor effect even on various tumors in which existing antitumor substances cannot sufficiently exert an effect.

[0009]

Means for Solving the Problems In order to solve the above-mentioned problems, the present inventors have widely screened secondary metabolites of microorganisms for substances having antitumor activity, and as a result, a strain belonging to the genus Streptomyces is produced. Structural formula [I]
The present invention has been completed by finding that the compound represented by the formula (1) exhibits excellent antitumor activity.

That is, the present invention provides a novel structural formula [I]

[0011]

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Or a pharmaceutically acceptable salt thereof, a method for producing the same, an antitumor agent containing the compound as an active ingredient, and Streptomyces having the ability to produce the compound. It relates to a microorganism belonging to the genus. (The compound of the present invention represented by the structural formula [I] is hereinafter referred to as J-124131.)

The compound of the present invention can exist in the form of a pharmaceutically acceptable salt, and the salt can be produced by using a compound of the formula [I] according to a conventional method.
Examples of such salts include alkali metal salts such as potassium salts and sodium salts, and alkaline earth metal salts such as magnesium salts and calcium salts.

The physicochemical properties of the novel antitumor substance J-124131 according to the present invention are shown below. The meanings of the abbreviations in the nuclear magnetic resonance spectrum are shown below. s: singlet d: doublet t: triplet q: quartet m: multiplet br: broad J: coupling constant Hz: hertz

The physicochemical properties properties of J-124131; red powder molecular formula; C ₃₁ H ₃₄ O ₁₀ mass spectroscopy; [HRFAB-MS] m / z Found: 589.2078 (M ⁺ Na) + calcd: 589.2050 (C ₃₁ H ₃₄ O ₁₀ Na) UV absorption spectrum; λ _max (MeOH) nm
(Ε): 259 (18,300), 286 (14,80)
0), 456 (8,400) λ _max [0.1N HCl-MeOH (1: 9)] n
m (ε): 259 (17,600), 285 (13,9
00), 450 (7,900) λ _max ([0.1N NaOH-MeOH (1:
9)] nm (ε): 285 (11,000), 321
(8, 200), 551 (4, 300) infrared absorption spectrum; ν _max (KBr) cm ⁻¹ : 34
60,2937,1620,1548,1358,12
36,1165,1122,1076,1014,98
4,937,851,802,768,712,61
9,536,465 nuclear magnetic resonance spectrum; ¹ H-NM
R (400 MHz, CDCl ₃ ): δ 1.10 (3H,
d, J = 6.3 Hz), 1.22 (3H, d, J = 6.3 Hz).
7 Hz), 1.22 (1H, m), 1.57 (1H,
m), 1.70-1.82 (3H, m), 1.93 (1
H, m), 2.18 (1H, m), 2.27 (1H,
m), 2.40 (3H, s), 3.52 (1H, br)
s), 3.68 (1H, brs), 3.92 (1H, b
rq, J = 6.3 Hz), 4.12 (1H, brq, J
= 6.7 Hz), 4.84 (1H, brs), 4.87
(1H, brs), 5.32 (1H, brs), 5.3
8 (1H, brs), 6.83 (1H, s), 7.17
(1H, s), 7.35 (1H, d, J = 7.6H
z), 7.66 (1H, t, J = 7.6 Hz), 7.7
9 (1H, d, J = 7.6 Hz), 9.54 (1H,
s), 12.0 (1H, s) ¹³ C-NMR (100 MHz, CDCl ₃ ): δ17.
0 (q), 17.3 (q), 21.8 (q), 23.0
(T), 23.2 (t), 25.3 (t), 26.0
(T), 60.8 (d), 66.2 (d), 67.0
(D), 67.0 (d), 67.3 (d), 70.5
(D), 92.9 (d), 95.0 (d), 112.9
(S), 114.0 (s), 118.0 (d), 12
0.0 (d), 121.0 (d), 125.7 (d),
132.4 (s), 136.4 (d), 139.2
(S), 141.0 (s), 142.6 (s), 14
4.6 (s), 155.9 (s), 161.1 (s),
188.9 (s), 188.9 (s) Solubility; easily soluble in chloroform, methanol, acetone, and dimethyl sulfoxide, and hardly soluble in toluene and water. Rf value: 0.39 [Merck Kieselgel 60F
₂₅₄ used, developing solvent: chloroform-methanol (2
0: 1)] Color reaction; Sulfuric acid reaction positive

Next, a method for producing J-124131 will be described. J-124131 represented by the structural formula [I] belongs to the genus Streptomyces, and a microorganism having an ability to produce the compound represented by the structural formula [I] according to claim 1 is cultured. It can be produced by collecting the compound represented by the formula [I] and converting it into a salt of the compound represented by the structural formula [I] by a conventional method, if necessary. The microorganism used for producing the antitumor substance J-124131 of the present invention may be any microorganism that produces J-124131. For example, a microorganism having the following mycological properties, that is, A74169 may be used. it can. 1. Form A74169 forms an aerial mycelium with a base mycelium that elongates and diverges well, and there is no division of the ring vegetation or hypha. A short chain of spores (10-20) is formed on aerial hyphae, and its morphology is cyclic to incomplete spiral. The surface of the spores is rough wrinkled to spicy and 0.5-0.7 × 0.6-0.7 in size
It is elliptical in the order of μm, and no special organs such as spores, flagella spores and sclerotia are observed. 2. Culture properties on various agar plate media The culture characteristics (cultivation at 28 ° C for 14 days) on various agar plate media were as shown in Table 1.

[0017]

[Table 1] Table 1 Culture properties of various agar plate media

3. Growth temperature Growth temperature (starch / inorganic salt agar medium, cultured for 14 days)
Was as shown in Table 2.

[0019]

[Table 2] Table 2 Growth temperature

4. Physiological properties Table 3 shows the physiological properties.

[0021]

[Table 3] Table 3 Biological properties

5. Utilization of carbon sources Utilization of carbon sources is shown in Table 4.

[0023]

[Table 4] Table 4 Availability of carbon sources

6. Cell components LL-diaminopimelic acid and glycine were detected from the cell wall.

[0025] Due to the above mycological properties, A74169 strain is Streptomyces streptomyces.
s) It is considered to belong to the genus. Therefore, A74169
Streptomyces sp. A74169 (St
A74169). 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-16924.

The Streptomyces sp A of the present invention
74169 (Streptomyces sp. A7
4169) may be subjected to irradiation treatment with, for example, X-rays or ultraviolet rays, for example, nitrogen mustard, azaserine, nitrite, 2-aminopurine or 1-methyl-
It can be prepared by treatment with a mutagenic agent such as 3-nitro-1-nitrosoguanidine (MNNG), for example, a commonly used bacterial species conversion treatment method such as phage contact, transformation, transduction or conjugation.

[0027]

Now, the method for producing the compound of the present invention will be specifically described. In producing the J-124131 of the present invention, a J-124131-producing strain is inoculated on a nutrient-containing medium and grown aerobically, whereby J-12 is produced.
A culture containing 4131 is obtained. As a nutrient source, a known nutrient source for actinomycetes can be used. As the carbon source, for example, commercially available glucose, maltose, starch, sucrose, molasses, dextrin, or the like is used alone or as a mixture. Examples of the nitrogen source include commercially available soy flour, corn steep liquor, meat extract, yeast extract, dried yeast, cottonseed powder, peptone, wheat germ, fish meal, meat meal, defatted rice bran, defatted meat-and-bone meal, and inorganic ammonium Salt or sodium nitrate is used alone or as a mixture. As the inorganic salt, for example, 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. Other than these, the producing bacteria utilize,
Useful for production of J-124131, for example, 3- (N-
Any of morpholino) propanesulfonic acid (MOPS) and sodium borate can be used.

The culturing method may be the same as the method for producing general microbial metabolites, and may be either solid culture or 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 cultivation temperature is suitably from 14 to 34 ° C., preferably 25 to 34 ° C.
-30 ° C. The preferred pH of the medium is in the range of 4-8, and the culture time is 192-312 hours, preferably 216 hours.
~ 264 hours. J-124 from culture
In order to collect 131, separation means commonly used for collecting from metabolites produced by microorganisms can be appropriately used.

Since J-124131 is also present in the cells, it can be used alone or in combination with conventional separation means such as a solvent extraction method, an ion-exchange resin method or an adsorption or partition chromatography method and a gel filtration method. Purification can be performed.

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, the ethyl acetate layer is distilled off, and the obtained residue is subjected to reverse phase column chromatography. J
The fraction containing -124131 was concentrated under reduced pressure, and reverse phase column chromatography was performed again to obtain J-12.
4131 can be obtained.

Next, in order to show the usefulness of the compound of the present invention, antitumor activity against mouse and human tumor cells was measured. The test methods and the results of the antitumor activity are shown in Table 5 below.

Biological activity of J-124131 (antitumor
Action)  Experimental tumor cells of mouse antitumor substance J-124131
Test in vitro to determine the growth inhibition
went. Antitumor test against mouse leukemia cell P388
Dissolves J-124131 in dimethyl sulfoxide
After that, serially dilute with dimethyl sulfoxide, then
10-fold dilution with RPMI 1640 medium containing 10%
Liquid. Assay for cell growth inhibition was 1 × 10^ThreeTumors
Cell culture medium containing cells (PRM containing 10% fetal bovine serum)
I1640 medium, 20 μM 2-mercaptoethanol
5 μl of the above test solution was added to 100 μl). 3
72% at 7 ° C, 5% CO_TwoAfter culturing under
[Analytical Communication Vol. 36, No.
No. 2, paragraphs 47-50, 1999 (Anal. C.
ommun. , 36 (2), 47-50 (1999)].
Was used to compare with the control group. As a result, J-12413
1 shows strong growth inhibitory activity on mouse tumor cells,
The 50% growth inhibitory concentrations are as shown in Table 5.

Furthermore, the antitumor activity of the antitumor active substance J-124131 on human tumor cells was tested in vitro. An antitumor test against human colon cancer cell DLD-1
After dissolving J-124131 in dimethyl sulfoxide, it was serially diluted with dimethyl sulfoxide.
A 10-fold dilution with RPMI1640 medium containing 10% was used as a test solution. Assay for tumor cell growth inhibition was performed by dispensing 100 μl of cell culture medium containing 1 × 10 ³ tumor cells into a 96-well microplate, culturing at 37 ° C. for 24 hours under 5% CO ₂ , 5 μl of test solution was added. After further culturing for 72 hours, the cells were fixed with 16.7% trichloroacetic acid,
After staining with% sulforhodamine B, the dye was extracted from the cells using 10 mM Tris solution. The absorbance at 550 nm was measured using 450 nm as a control wavelength and compared with the control group. As a result, J-124131 showed a strong growth inhibitory activity even in human tumor cells, and its 50% growth inhibitory concentration was as shown in Table 5.

[0034]

Table 5 Antitumor activity of J-124131

As described above, J-124131 has a remarkable growth inhibitory effect on mouse and human tumor cells.
Therefore, the present invention is useful as an antitumor agent for mammals including humans.

The compound J-124131 of the present invention inhibits the growth of tumor cells and exerts an antitumor effect, but 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 powder, but it can also be produced using appropriate additives. Such additives include, for example, 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 aluminate or anhydrous calcium phosphate, for example polyvinylpyrrolidone or Synthetic polymers such as polyalkylene glycols; fatty acid salts such as calcium stearate or magnesium stearate; alcohols such as stearyl alcohol or benzyl alcohol; synthetic cellulose derivatives such as methylcellulose, carboxymethylcellulose, ethylcellulose or hydroxypropylmethylcellulose, etc. , Water, gelatin,
Commonly used additives such as talc, vegetable oil, gum arabic and the like can be mentioned.

The 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 are usually 0.1 to 10% by weight,
It preferably contains 1 to 5% by weight of active ingredient. In addition, a liquid preparation such as a suspension or a syrup for oral administration is 0.5 to 1%.
Contains 0% 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.

[0040]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to examples, but the present invention is not limited to these examples. Production method of Example J-124131 Actinomyces A74169 inoculated on a slope soft agar medium was glucose 0.1%, dextrin (MS-3600)
2.0%, fish meal 0.5%, corn gluten meal
0%, yeast extract 0.1%, MOPS 0.5%, humic acid 0.03%, magnesium sulfate 0.05%,
Sodium chloride 0.1%, calcium chloride 0.05
%, Ferrous sulfate 0.0002%, cupric chloride 0.0
0004%, manganese chloride 0.00004%, cobalt chloride 0.00004%, zinc sulfate 0.00008
%, Sodium borate 0.00008% and sodium molybdate 0.00024% (pH
7.0) Inoculate two 500 ml Erlenmeyer flasks containing 110 ml, and incubate at 28 ° C. for 48 hours on a rotary shaker (1 minute per minute).
80 rotations). 500 ml Erlenmeyer flask 5 containing 110 ml of the above medium in 2 ml portions of this culture solution
0 inoculated at 28 ° C. for 10 days on a rotary shaker (180 per minute).
(Rotation).

The culture solution obtained by the above culture (about 5
Cells were obtained from L) by filtration. Methanol (5 L) was added to the cells, and the mixture was stirred and filtered to obtain a methanol extract. The methanol extract was concentrated under reduced pressure, extracted twice with 2.5 L of ethyl acetate, and the ethyl acetate fraction was concentrated to dryness under reduced pressure. The dried product (1.0 g) was applied to a silica gel column.
(Keizergel 60, manufactured by Merck Ltd., 2.8φ × 12 cm), washed with hexane / ethyl acetate (1/1), and eluted with ethyl acetate. After the eluate was concentrated to dryness, it was separated by preparative thin-layer chromatography (Art 5744, manufactured by Merck, 20 × 2).
0 cm). Hexane / ethyl acetate (1
/ 1) and eluted with ethyl acetate. This was dissolved in methanol and then applied to Sephadex LH-20 (manufactured by Pharmacia, 1.8φ × 90 cm), and eluted with methanol to obtain 18.7 mg of powder of J-124131.

The preparation examples of the compound of the present invention are shown below.
The preparation of the compound of the present invention is not limited to this preparation example. Formulation Example 1 10 parts of the substance (J-124131), 15 parts of heavy magnesium oxide, and 75 parts of lactose were uniformly mixed to give 500 μl.
m or less powder or fine-grained powder. This powder was placed in a capsule container to obtain a capsule. Formulation Example 2 45 parts of the substance (J-124131), 15 parts of starch, 16 parts of lactose, 21 parts of crystalline cellulose, 3 parts of polyvinyl alcohol and 30 parts of distilled water are uniformly mixed, crushed and granulated and dried. And then sieved to a diameter of 355 to 1400 μm
Granules of the 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 90 parts of granules obtained in the same manner as in Formulation Example 2 were mixed with 10 parts of crystalline cellulose and 3 parts of calcium stearate.
The resulting mixture was compression-molded to give a tablet having a diameter of 8 mm, and a syrup gelatin and a precipitated calcium carbonate mixed suspension were added thereto to prepare a sugar-coated tablet. Formulation Example 5 0.6 part of this substance (J-124131), 2.4 parts of a nonionic surfactant and 97 parts of physiological saline are heated and mixed, then put into an ampoule, sterilized, and an injection is prepared. Produced.

[0043]

According to the present invention, J-124131 is
Since it has a strong growth inhibitory effect on mouse and human tumor cells, it is useful as a therapeutic agent for cancer in the field of medicine.

[0044]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // (C12N 1/20 (C12N 1/20 A C12R 1: 465) C12R 1: 465) (C12P 17 / 06 (C12P 17/06 C12R 1: 465) C12R 1: 465) (72) Inventor Hiroshi Nishioka 3rd Okubo Tsukuba, Ibaraki Pref. Inside Tsukuba Research Laboratory, Banyu Pharmaceutical Co., Ltd. (72) Inventor Shigeru Nakashima Tsukuba, Ibaraki No. 3 Okubo in the Tsukuba Research Laboratories of Manyu Pharmaceutical Co., Ltd. (72) Inventor Mioko Hirayama No. 3 in Okubo Tsukuba, Ibaraki Pref. 4B064 AE41 AE46 CA04 CD19 CD22 CE08 CE10 DA05 4B065 AA50X AC12 AC14 AC16 BA22 BB03 BB18 BB23 BB26 BC03 BC26 BD14 BD16 BD50 CA04 CA18 CA44 4C062 AA19 4C086 AA01 AA02 AA03 GA02 MA01 MA04 MA52 NA14 ZB26

Claims (5)

[Claims] (1) Structural formula [I] Or a pharmaceutically acceptable salt thereof. 2. Streptomyces (Streptomyc)
es) A microorganism belonging to the genus and capable of producing the compound represented by the structural formula [I] according to claim 1 is cultured, and the compound represented by the structural formula [I] is collected from the culture. A method for producing a compound represented by the structural formula [I] or a salt thereof. 3. A microorganism having an ability to produce the compound represented by the structural formula [I] according to claim 1 is Streptomyces sp A74169 (Streptomyces).
ssp. A74169) or a mutant thereof. 4. An antitumor agent comprising the compound represented by the structural formula [I] according to claim 1 as an active ingredient. 5. A microorganism belonging to the genus Streptomyces, which has the ability to produce the compound represented by the structural formula [I] according to claim 1, wherein the microorganism is Streptomyces sp. A74169 (S
A. Treptomyces sp. A74169) or a mutant thereof.