JP2003026696A - New angiogenesis inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel compound which exhibits angiogenesis inhibitory activity and antitumor activity by inhibiting chemotaxis of vascular endothelial cells. SOLUTION: The following formula (I): And a compound useful as an active ingredient of a medicament such as an angiogenesis inhibitor or an antitumor agent.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel compound useful as an active ingredient of medicines such as angiogenesis inhibitors and antitumor agents.

[0002]

2. Description of the Related Art Angiogenesis is 1) VEGF (vascular endotherial growth facto)
r), etc.], 2) detachment of pericytes and degradation / digestion of extracellular matrix such as basement membrane, 3) migration and proliferation of vascular endothelial cells, 4) lumen formation by basement cells, formation of basement membrane ,
It is completed through steps such as maturation of blood vessels by pericytes. In tumor angiogenesis, the newly formed blood vessels supply oxygen and nutrients to the tumor to promote growth, and at the same time, serve as a route of invasion and metastasis of tumor cells to other sites.

Among the angiogenic factors, vascular endothelial growth factor VE
GF has various functions such as blood vessel / blood / coagulation system as well as angiogenesis, including induction of proliferation and maintenance of survival of vascular endothelial cells, enhancement of vascular permeability, migration by platelets and chemotaxis to macrophages. is doing. Therefore, novel compounds that inhibit VEGF-induced chemotaxis of vascular endothelial cells can be expected as angiogenesis inhibitors, antitumor agents, metastasis inhibitors, antirheumatoid arthritis agents, etc. [E
ur. J. Cancer, 32A, 2534-2539 (1996); Nature Med.,
1, 27-331 (1995)]. Furthermore, the angiogenesis inhibitor has a potential as a therapeutic drug for diabetic retinopathy, which is one of diabetic vascular complications. Currently, the treatment for diabetic retinopathy has a strong symptomatic side, and the treatment is delayed and often blindness is inevitable. Although several angiogenesis inhibitors have been developed and studied in recent years, new compounds that can serve as lead compounds are considered to be a constant desire.

PC having a structure similar to that of the compound of the present invention
-766B substance is known, and it has been reported that this substance has a growth inhibitory effect on Gram-positive bacteria, molds, and yeasts, and an antitumor effect in a system in which mouse P388 leukemia cells are transplanted in vivo [ J. Antibiot., 46,
972 (1993); J. Antibiot., 46, 1139 (1993)].

[0005]

An object of the present invention is to provide a novel compound which exerts physiological actions such as angiogenesis inhibitory activity, antitumor activity and tumor metastasis inhibitory activity by inhibiting chemotaxis of vascular endothelial cells. To provide. Another object of the present invention is to provide a method for producing a novel compound having the above characteristics, and a medicine containing the compound as an active ingredient.

[0006]

[Means for Solving the Problems] As a result of intensive studies to solve the above problems, the present inventors have found that actinomycete RK97-56 strain produces a novel compound, and determined the structure of the compound. did. Further, they have found that the compound has angiogenesis inhibitory activity and antitumor activity and is useful as an active ingredient of a medicine. . The present invention has been completed based on the above findings.

That is, the present invention provides the following formula (I):

[Chemical 2] A compound represented by

From another point of view, the present invention provides a medicament containing the above compound as an active ingredient. This medicine can be used as a medicine for preventing and / or treating a disease associated with excessive angiogenesis or a disease based on excessive angiogenesis, treating malignant tumor, and suppressing metastasis of malignant tumor. Further, according to the present invention, an angiogenesis inhibitor containing the above compound as an active ingredient; an inhibitor of vascular endothelial cell chemotaxis containing the above compound as an active ingredient; an antitumor agent containing the above compound as an active ingredient; And a malignant tumor metastasis inhibitor containing the above compound as an active ingredient.

[0009] From still another aspect, there is provided a method for producing the above compound, which comprises a step of separating and collecting the above compound from a culture obtained by culturing a bacterium producing the above compound. A method for treating malignant tumor, comprising a step of administering a therapeutically effective amount of the above compound to a mammal including human; a method for suppressing metastasis of malignant tumor, comprising an effective amount of the above compound for human A method comprising a step of administering to a mammal; a method of inhibiting angiogenesis in a tumor tissue, comprising a step of administering an effective amount of the above compound to a mammal including a human; suppressing abnormal hypervascularization of angiogenesis. Method,
A method comprising the step of administering an effective amount of the above compound to a mammal including human; a method of inhibiting chemotaxis of vascular endothelial cells, the method comprising the step of administering an effective amount of the above compound to a mammal including human. Methods of including are provided by the present invention.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The compound represented by formula (I) has a plurality of asymmetric carbons, and stereoisomers such as optical isomers or diastereomers based on the asymmetric carbons exist. , Within the scope of the invention, in addition to the stereoisomers in pure form,
A mixture of arbitrary stereoisomers, a racemate and the like are included. Further, the compound of the present invention has a plurality of olefinic double bonds, and there may be geometric isomers based on the respective double bonds, but in addition to the pure form geometric isomer, Mixtures of geometric isomers are also included within the scope of the invention. Furthermore, the compounds of the present invention may exist as tautomers, but any tautomer or mixture thereof is also included in the scope of the present invention. The compounds of the present invention can exist in any crystalline form, and may exist as hydrates or solvates. It goes without saying that all of these substances are included in the scope of the present invention.

The compound represented by the formula (I) of the present invention can be produced by culturing actinomycetes, which is a bacterium producing the above compound. Actinomycetes RK97 is a bacterium that produces the above compound.
-56 shares. The microorganism was deposited on July 10, 2001 at the Patent Biological Depository Center, National Institute of Advanced Industrial Science and Technology (Central No. 1-1-1, Higashi 1-1-1, Tsukuba, Ibaraki, Japan) under the deposit number FERM P-18413. Has been done.

The compound represented by the formula (I) of the present invention is an actinomycete, preferably actinomycete RK97, which is a bacterium producing the compound.
-56 strains can be produced by separating and collecting from the culture. As a production medium for the compound represented by the formula (I) of the present invention, either a synthetic medium or a natural medium can be preferably used as long as it appropriately contains a carbon source, a nitrogen source and an inorganic salt. If necessary, vitamins or other nutritional substances may be added appropriately. Examples of the carbon source include glucose, maltose, fructose, sucrose, sugars such as starch, alcohols such as glycerol and mannitol, amino acids such as glycine, alanine and asparagine, oils and fats such as soybean oil and olive oil. One kind or two or more kinds may be appropriately selected and used in consideration of the assimilation property of the microorganism from the specific carbon source. Soybean powder, corn steep liquor,
Beef extract, peptone, yeast extract, amino acid mixture, organic nitrogen-containing compounds such as fish meal or ammonium salts,
Inorganic nitrogen compounds such as nitrates may be mentioned, and one or more may be appropriately selected and used in consideration of the assimilation of microorganisms. Examples of the inorganic salt include calcium carbonate, sodium chloride, potassium chloride, magnesium sulfate, copper sulfate,
Manganese chloride, zinc sulfate, cobalt chloride, and various phosphates may be added as necessary. In addition, an antifoaming agent such as vegetable fat and polypropylene alcohol can be added if necessary.

The culturing temperature can be appropriately changed within a range in which the microorganism grows and the compound represented by the formula (I) of the present invention is produced in the culturing, but it is preferably 10 to 32 ° C., and It is preferably 20 to 25 ° C. The initial pH of the culture is preferably around 6-8. The culturing time is usually about a day to several weeks, but the culturing may be terminated when the production amount of the compound of the present invention reaches a harvestable amount, preferably the maximum amount. The culturing method is not particularly limited, and any commonly used method such as solid-phase culturing and normal stirring culturing can be preferably used.

The method for separating / collecting the above compound is not particularly limited, and for example, it can be suitably carried out by the method described in Examples below. For example, the compound of the present invention can be obtained by an ordinary separation / purification means after obtaining a culture solution by an ordinary culturing method of actinomycetes. After completion of the culture, the compound of the present invention can be separated and isolated from the culture medium by appropriately utilizing the means generally used for collecting microbial metabolites. For example, various ion exchange resins, nonionic adsorption resins, gel filtration chromatography, or chromatography with adsorbents such as activated carbon, alumina, silica gel, or high performance liquid chromatography, or crystallization, vacuum concentration, or freeze-drying means. Can be used alone or in appropriate combination, or can be used repeatedly.

The compound of the present invention inhibits the chemotaxis of vascular endothelial cells as specifically described in the experimental examples described later, and exhibits excellent angiogenesis inhibitory action and antitumor activity. Therefore, the compound of the present invention is useful as an active ingredient of a medicament for preventing and / or treating a disease accompanied by excessive angiogenesis or a disease caused by excessive angiogenesis, or a medicament for treating a malignant tumor. is there. Specifically, the medicament of the present invention is a medicament for the prevention and / or treatment of diseases such as diabetic vascular complications, rheumatoid arthritis, diabetes, myocardial infarction, and ulcerative colitis,
And can be used as a medicament for the treatment of tumors.

A medicine containing the compound of the present invention as an active ingredient is
The administration method, dosage form, and dose can be appropriately determined according to the purpose of use. For example, the dosage form of a drug containing the compound of the present invention as an active ingredient may be oral administration or parenteral administration. Examples of the dosage form include oral administration agents such as tablets, powders, capsules, granules, extracts and syrups, and parenteral administration agents such as injections, infusions and suppositories. These formulations can be produced by known methods using pharmaceutically acceptable additives such as excipients and binders. The dose of a drug containing the compound of the present invention as an active ingredient varies depending on the age, body weight, type of disease, degree of symptoms, etc. of the patient, but when administered orally, it is usually 0.1 mg to 100 mg per day for an adult. The amount is about mg, preferably about 1 mg to 20 mg, and in the case of parenteral administration, it is usually about 0.01 mg to 10 mg, preferably about 0.1 mg to 2 mg per day for an adult. It is also possible to administer this dose once or several times a day. If necessary, an amount outside the above range can be used.

When the compound of the present invention is used as a reagent, it can be used by dissolving it in an organic solvent or a water-containing organic solvent. For example, direct administration to various cultured cell lines can suppress cell growth. Examples of usable organic solvents include methanol and dimethyl sulfoxide. Examples of the dosage form include solid agents such as powder, and liquid agents dissolved in an organic solvent or a water-containing organic solvent. Usually, the effective amount for exerting an angiogenesis inhibitory effect using the above compound as a reagent is 0.001 to 1 μg / ml, but an appropriate amount depends on the type and purpose of use of the cultured cell line. Different, it can be appropriately selected. If necessary, an amount outside the above range can be used.

[0018]

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to the following examples. Example 1 glucose 2%, soluble starch 1%, meat extract 0.3%,
Yeast extract 2.5%, salt 0.05%, dibasic potassium phosphate 0.
005%, calcium carbonate 0.05%, magnesium sulfate 0.05%
The actinomycete RK97-56 strain was inoculated into a medium (pH 7.2) having the composition consisting of and cultured with shaking at 28 ° C. for 48 hours. This culture 140
15 ml of a medium of the same composition was inoculated with ml, and shake culture was performed at 28 ° C. for 96 hours.

The above culture broth was separated into bacterial cells and supernatant by a centrifuge, the supernatant was adjusted to pH 3.5 and extracted with 15 liters of ethyl acetate. After extracting the cells with 10 liters of acetone,
The solution was concentrated under reduced pressure, the obtained aqueous layer was adjusted to pH 3.5 and extracted with 6 liters of ethyl acetate. Subsequently, all ethyl acetates were combined and concentrated under reduced pressure to obtain 24 g of brown syrup. This syrup was roughly purified by silica gel column chromatography (chloroform-methanol solution) to obtain 5.8 g of an active fraction. Subsequently, the obtained active fraction was purified by the second silica gel column chromatography (hexane-ethyl acetate solution) to obtain 174 m of the active fraction.
got g. Next, this active fraction was divided into several times and separated by high-performance liquid chromatography using a reversed-phase ODS column (diameter 2 cm, length 25 cm, PEGASIL ODS, Senshu Scientific Co., Ltd.) (methanol: water). = 8.5: 1.5 → methanol: water =
0:10; linear gradient, flow rate 8.0 ml / min) was performed to obtain an active fraction. Finally, the compound RK-9756A (16 mg) of the present invention was obtained by preparative thin layer chromatography (developing solvent; hexane: ethyl acetate = 1: 4). The RK-9756A is
Known compound PC-766B obtained at the same time by the above purification step
It can be converted under more usual methanolysis conditions.

The NMR data of RK-9756A are shown in Table 1 (solvent: heavy dimethyl sulfoxide, δppm, internal standard: TMS,
¹³ C: 125 MHz, ¹ H: 500 MHz). Property: Colorless powder Specific rotation: +2.7 (c = 0.183, 25 degrees, ethanol) Molecular formula: C ₄₄ H ₇₀ O ₁₂ High resolution mass spectrometry (HR-FABMS): (M + Na) ⁺ Experimental value (m / z) ): 813.4786 Theoretical value (m / z): 813.4765 UV λmax nm (methanol) (ε): 229 (28500), 235 (sh,
27650), 250 (sh, 19700), 285 (9400) IR νmax (KBr) cm ^-1 : 3450, 1690 R _f value (Silica gel 60 F ₂₅₄ , manufactured by Merck & Co., Inc.): 0.54 (developing solvent; hexane: ethyl acetate = 1: 4) Color reaction (positive): 10% Sulfuric acid solubility: Easily soluble in methanol, methanol and dimethylsulfoxide. Insoluble in n-hexane.

[0021]

[Chemical 3]

[0022]

[Table 1]

Test Example 1: Inhibition of chemotaxis of vascular endothelial cells Normal human umbilical vein vascular endothelial cells HUVEC cells cultured and maintained in HuMedia-EG2 (KURABO) medium were three-dimensionally cultured in a chemotaxel chamber. Sprinkled on the upper layer. The chemotaxis of HUVEC cells was induced by filling the lower layer with HuMedia-EG2 containing endothelial cell growth factor (VEGF). RK-9756A is
Each suppressed the chemotaxis of HUVEC cells induced by VEGF at a concentration of 0.01-0.1 μg / ml. This result shows that the compound RK-9756A of the present invention inhibits chemotaxis of vascular endothelial cells based on the anti-VEGF action, and is effective as an active ingredient of drugs such as angiogenesis inhibitors and antitumor agents. Indicates that

Formulation Example 1: Injection / drip solution RK-9756A 10 mg was added to powdered glucose, and the mixture was aseptically distributed into a vial and sealed, and an inert gas such as nitrogen or helium was sealed. And store in a cool and dark place. Before use, dissolve in ethanol and use 0.85% physiological saline 100 ml.
Is added to give an intravenous injection, and 10 to 100 ml per day is administered by intravenous injection or infusion depending on the symptoms.

Formulation Example 2: Granules RK-9756A (1 g), lactose (98 g) and hydroxypropylcellulose (1 g) were taken, mixed well, and then formed into granules by a conventional method, and the granules were thoroughly dried. We manufacture granules suitable for bottles and heat-sealed packaging. Oral administration of 100 to 1000 mg per day depending on symptoms is possible.

[0026]

INDUSTRIAL APPLICABILITY The compound of the present invention exerts angiogenesis inhibitory activity and antitumor activity by inhibiting chemotaxis of vascular endothelial cells, and therefore, is useful for the prevention and / or treatment of diseases associated with excessive angiogenesis. Therefore, it is useful as a drug for antitumor agent or an agent for suppressing metastasis of malignant tumor.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 9/10 A61P 9/10 29/00 101 29/00 101 35/00 35/00 35/04 35 / 04 43/00 105 43/00 105 C12P 19/44 C12P 19/44 F term (reference) 4B064 AF52 BA09 BE09 BE19 BH02 BH04 BH05 BH06 BH07 CA03 DA05 4C057 AA01 AA05 BB02 CC01 DD01 KK02 4C086 AA01 AA02 MA11 GA02 A04 MA02 A04 MA02 NA14 ZA36 ZA66 ZB15 ZB21 ZB26 ZC35

Claims (5)

[Claims] 1. The following formula (I): The compound represented by. 2. A medicament containing the compound according to claim 1 as an active ingredient. 3. The medicine according to claim 2, which is an angiogenesis inhibitor. 4. The medicine according to claim 2, which is an antitumor agent. 5. A method for producing the compound according to claim 1, comprising a step of separating and collecting the compound according to claim 1 from a culture obtained by culturing a bacterium that produces the compound according to claim 1.