JP2003183249A - New substance having inhibitory action on neovascularization - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a compound having inhibitory actions on chemotaxis of vascular epithelial cells, capable of exhibiting inhibitory actions on neovascularization and resultantly useful as an active ingredient of a medicine, an antitumor agent, etc., for prophylaxis and/or treatment of various diseases, e.g. diabetic retinopathy or articular rheumatism in which excessive neovascularization as one of causes of onset participates. <P>SOLUTION: This compound is represented by formula (I) or its physiologically acceptable salt. The medicine such as a neovascularization inhibitor or the antitumor agent comprises the compound or its salt as the active ingredient. <P>COPYRIGHT: (C)2003,JPO

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 Recently, it is becoming clear that angiogenesis is an important physiological phenomenon in the growth and metastasis of various advanced solid cancers. Angiogenesis includes 1) angiogenic factors secreted by tumor cells [VEGF (vascular endothelial growth fa
ctor) 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 new blood vessels thus formed supply oxygen and nutrients to the tumor to promote growth, and also serve as a route of invasion and metastasis of tumor cells to other sites.

[0003] Among them, the vascular endothelial growth factor VEGF is not only angiogenic neoplasia, including vascular endothelial cell proliferation induction and survival maintenance, vascular permeability enhancement, platelet migration, and macrophage chemotaxis. Blood vessels
It has various functions such as coagulation system. 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. [Eur. J. Cance
r, 32A, 2534-2539 (1996), Nature Med., 1, 27-331 (1
995), Immunity, 12, 121 (2000)].

Furthermore, the angiogenesis inhibitor has a possibility as a therapeutic drug for diabetic retinopathy, which is one of diabetic vascular complications. At present, the treatment for diabetic retinopathy has a strong symptomatic aspect, and the treatment may be delayed and often blind, so that an effective preventive / therapeutic drug is desired. In recent years, angiogenesis inhibitors with various mechanisms of action are in the clinical trial stage, but new compounds that can serve as lead compounds are considered to be a constant desire.

[0005]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a novel compound which exhibits angiogenesis-inhibiting activity, antitumor activity, and metastasis-inhibiting activity by inhibiting chemotaxis of vascular endothelial cells. 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 the filamentous fungus Aspergillu
The inventors have found that the novel compound isolated from the culture solution of the microorganism belonging to the s genus has angiogenesis inhibitory activity and antitumor activity, and completed the present invention.

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

[Chemical 3] The present invention provides a compound represented by or a salt thereof. According to a preferred embodiment of the present invention, the following formula (II):

[Chemical 4] A compound represented by or a salt thereof is provided.

From another point of view, the present invention provides a medicament containing the above compound or a physiologically acceptable salt thereof 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 or a physiologically acceptable salt thereof as an active ingredient; migration of vascular endothelial cells containing the above compound or a physiologically acceptable salt thereof as an active ingredient Antitumor agent containing the above compound or a physiologically acceptable salt thereof as an active ingredient; and suppressing metastasis of malignant tumors containing the above compound or a physiologically acceptable salt thereof as an active ingredient An agent is provided. The angiogenesis inhibitor can be administered, for example, for the prevention and / or treatment of diabetic retinopathy.

[0009] From still another aspect, there is provided a method for producing the above compound or a salt thereof, which comprises a step of separating and collecting the above compound or a salt thereof from a culture obtained by culturing a bacterium that produces the above compound belonging to the genus Aspergillus. . A method for treating malignant tumor, comprising a step of administering a therapeutically effective amount of the above compound or a physiologically acceptable salt thereof to a mammal including human; Administering a effective amount of the above compound or a physiologically acceptable salt thereof to mammals including humans.
A method for inhibiting angiogenesis in a tumor tissue, comprising the step of administering an effective amount of the above compound or a physiologically acceptable salt thereof to a mammal including human; A method comprising the step of administering an effective amount of the above compound or a physiologically acceptable salt thereof to a mammal including human; a method for preventing and / or treating diabetic retinopathy, which comprises the above compound or physiology Comprising the step of administering a prophylactically and / or therapeutically effective amount of a pharmaceutically acceptable salt thereof to mammals including humans; a method of inhibiting chemotaxis of vascular endothelial cells, which comprises the above compound or physiologically A method is provided by the present invention which comprises the step of administering an effective amount of an acceptable salt thereof to a mammal, including a human.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The compound of the present invention represented by the formula (I) or (II) (hereinafter, either or both of them will be referred to as "the compound of the present invention") is an asymmetric carbon. There are stereoisomers such as optical isomers or diastereomers based on asymmetric carbons, but within the scope of the present invention, in addition to stereoisomers in pure form, any stereoisomers Mixtures or racemates are included. Further, the compound of the present invention has a plurality of olefinic double bonds, and there may exist 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. Preferred geometric isomers are compounds of formula (II).

The scope of the present invention includes the compounds of the present invention in free form as well as salts of the above compounds, preferably physiologically acceptable salts. The form of the salt is not particularly limited, but it may form a sodium salt with a hydroxyl group in the molecule. Furthermore, the scope of the present invention includes hydrates or solvates of the above compounds or salts thereof. Among the compounds of the present invention, preferred compounds are the compounds represented by formula (II). This compound is referred to herein as "RKB-3384HS-A".
Sometimes called.

The method for producing the compound of the present invention is not particularly limited. For example, a method of separating and collecting from the culture of a microorganism capable of producing the compound of the present invention (extraction method), or a similar structure contained in the culture of the microorganism. A method (synthesis method or semi-synthesis method) of chemically synthesizing the above compound as a starting material can be mentioned. In the extraction method, the microorganism capable of producing the compound of the present invention is cultured using a medium and conditions used for usual fermentation production, and the target product can be separated and collected from the obtained culture. As the microorganism capable of producing the compound of the present invention,
For example, Aspergillus genus BAUA3384 strain can be mentioned. The microorganism has been deposited under the deposit number FERM P-18283 at the National Institute of Advanced Industrial Science and Technology's Patented Organism Depositary (Central 1-1-1, Higashi 1-1-Tsukuba City, Ibaraki Prefecture).

The medium for producing the compound of the present invention is not particularly limited, and either a synthetic medium or a natural medium can be suitably 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 sugars such as glucose, maltose, fructose, sucrose and starch, alcohols such as glycerol and mannitol,
One or two or more kinds may be appropriately selected from the general carbon sources such as amino acids such as glycine, alanine and asparagine, and oils and fats such as soybean oil and olive oil in consideration of the assimilation of microorganisms. Examples of the nitrogen source include soybean flour, 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, etc. Then, one kind or two or more kinds may be appropriately selected and used. 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. Also, an antifoaming agent,
For example, vegetable fat, polypropylene alcohol and the like can be added as necessary.

The culturing temperature can be appropriately changed within the range where the microorganism grows and the compound of the present invention is produced, but it is preferably 10 to 32 ° C, more preferably 20 to 28 ° C. The initial pH is preferably around 6-8, and the culture time is usually day-
Although it takes about several weeks, the culture may be terminated when the production amount of the compound of the present invention reaches a harvestable amount, preferably reaches the maximum. The culturing method is not particularly limited, and any commonly used method such as solid layer culture or normal stirring culture can be preferably used.

Separation and collection of the compound of the present invention from the culture broth can be carried out by appropriately utilizing the means usually 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 combination, or can be used repeatedly.

The compounds of the present invention have excellent anti-angiogenic activity and antitumor activity. Therefore, the compound of the present invention is useful as an active ingredient of a medicament for the prevention and / or treatment of various diseases in which excessive angiogenesis is involved as one of the causes of its development. For example, the medicament of the present invention is for the prevention and / or treatment of diabetic vascular complications, diabetic retinopathy, rheumatoid arthritis, rheumatoid arthritis, diabetes, myocardial infarction, ulcerative colitis, psoriasis, angiogenic glaucoma and the like. In addition to the above drug, it can be used as an antitumor agent or tumor metastasis suppressor in mammals including humans.

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 1 per adult per day. It is about g, preferably about 1 mg to 100 mg, and in the case of parenteral administration, it is usually about 0.01 mg to 100 mg, preferably about 0.1 mg to 20 mg per day for an adult. It is also possible to administer this dose once or several times a day. Also,
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 exhibiting an angiogenesis inhibitory effect using the above compound as a reagent is 0.3 to 30 μg / ml, but an appropriate amount depends on the type and purpose of the cultured cell line. Different,
It can be appropriately selected. If necessary, an amount outside the above range can be used.

[0020]

Example 1 Glucose 1.0%, Soluble starch 2.0%, Soybean flour 1.5%, Malt extract 0.5%, Vegetable extract 10%, Dipotassium phosphate 0.05%, Potato dextrose 2.6%, Magnesium sulfate A medium having a composition of 0.05% was inoculated with the strain BAUA3384 of the genus Aspergillus and cultured at 28 ° C. for 72 hours with shaking. 140 ml of this culture medium is used as a medium of the same composition.
15 liters were inoculated and shaking culture was performed at 28 ° C. for 72 hours.
The above culture solution was separated into bacterial cells and supernatant with a centrifuge, and the supernatant was
The pH was adjusted to 7.0 and the mixture was extracted with 15 liters of ethyl acetate.
After extraction, all the ethyl acetates were combined and concentrated under reduced pressure to obtain 13.6 g of brown syrup.

This syrup was dissolved in 10 ml of chloroform and a silica gel column (diameter 4
cm, length 60 cm). First, chloroform 5
After elution with 00 ml, chloroform was changed in proportion
-Methanol solution (100: 0, 100: 1, 50: 1, 20: 1, 10: 1,
5: 1, 1: 1) Elution was performed with 800 ml each. Compounds of the invention RK
B-3384HS-A was eluted in the fraction of chloroform-methanol solution (100: 1). By concentrating this fraction under reduced pressure,
1.1 g of brown syrup was obtained. Then this brown syrup
Dissolve 1.1 g in 11 ml of methanol and divide into several portions to reverse phase O
Using a DS column (diameter 2 cm, length 25 cm, PEGASIL ODS, Senshu Scientific Co., Ltd.), fractionation by high performance liquid chromatography (acetonitrile: water = 55: 45, flow rate 9.0 ml).
/ Min) to give RKB-3384HS-A. In addition, hexane /
Recrystallization from a mixed solvent of ethyl acetate / methanol gave pure light yellow powder substance RKB-3384HS-A (30 mg). The physicochemical properties of RKB-3384HS-A are shown below.

Properties: Light yellow powder Melting point: 167-170 ° C (decomposition) Specific rotation: -1.0 (c = 0.1, 23 ° C, methanol) Molecular formula: C ₂₃ H ₂₇ NO ₆ High resolution mass spectrometry (HR-FABMS) : (M + H) ⁺ experimental value (m / z): 414.1948 theoretical value (m / z): 414.1917 UV λmax nm (methanol) (ε): 235 (27560), 360 (665
30) R _f value (silica gel 60 F ₂₅₄ , manufactured by Merck): 0.62 (developing solvent; chloroform: methanol = 10: 1) IR λmax (neat) cm ⁻¹ : 3400, 1750, 1725, 1655, 158
5, 1240, 1105, 995 Color reaction (positive): 10% Sulfuric acid solubility: Easily soluble in methanol, acetone and dimethylsulfoxide. Insoluble in n-hexane and water.

[0023]

[Chemical 5]

The NMR data of RKB-3384HS-A are shown in Table 1 (solvent: heavy acetone, δppm, internal standard: TMS, ¹³ C: 125).
MHz, ¹ H: 500 MHz).

[0025]

[Table 1]

(Test Example 1) Inhibition of chemotaxis of vascular endothelial cells by RKB-3384HS-A Normal human umbilical vein vascular endothelial cells HUVEC cells cultured and maintained in HuMedia-EG2 (KURABO) medium were chemotaxel chambered. Was plated on the upper layer of the three-dimensional culture. The chemotaxis of HUVEC cells was induced by filling the lower layer with HuMedia-EG2 containing endothelial cell growth factor (VEGF). RKB-3384HS-A
Suppressed the chemotaxis of HUVEC cells induced by VEGF at a concentration of 1-30 μg / ml. This result shows that RKB-3384HS-A exerts an inhibitory effect on chemotaxis of vascular endothelial cells based on the anti-VEGF effect, and the compound of the present invention is an angiogenesis inhibitor, an antitumor agent, or It has been shown to be effective as a tumor metastasis inhibitor, etc.

(Formulation Example 1) Injection / drip solution RKB-3384HS-A 10 mg of powdered glucose to contain 10 mg
After adding g, the mixture was aseptically distributed to a vial, sealed, sealed with an inert gas such as nitrogen or helium, and stored in a cool dark place. This formulation was dissolved in ethanol before use and then charged to 0.
Add 100 ml of 85% saline to make an intravenous injection,
Depending on the symptoms, 10 to 100 ml per day can be administered by intravenous injection or infusion.

(Formulation Example 2) Granules RKB-3384HS-A (1 g), lactose (98 g) and hydroxypropylcellulose (1 g) were taken, mixed well, and then formed into granules by a conventional method, and dried well. To produce granules suitable for bottles and heat-sealed packaging. 100 mg to 10 g per day can be orally administered depending on the symptoms.

[0029]

The compound of the present invention has an inhibitory action on chemotaxis of vascular endothelial cells and can exert an angiogenesis inhibitory action. Therefore, the compound of the present invention is effective as a drug or antineoplastic agent for the prevention and / or treatment of various diseases in which excessive angiogenesis is involved as one of the causes of its development, such as diabetic retinopathy and rheumatoid arthritis. It is useful as an ingredient.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) // (C12P 17/10 C12R 1:66 C12R 1:66) (72) Inventor Hideaki Kakeya Wako Saitama 2-1-1 Hirosawa City, RIKEN (72) Inventor Hiroshi Konno 11-20-5 Ohanamachi, Omagari-shi, Akita (72) Inventor Susumu Kanazawa 6-1-A-307 Enokigaoka, Aoba-ku, Yokohama-shi, Kanagawa F term (reference) 4B064 AE48 BA05 BC03 BE09 BE12 BE19 BG02 BH01 BH02 BH05 BH06 BH07 BH10 CA05 DA05 4C069 AC28 BB02 BB03 BB17 BB22 CC22 4C086 AA01 AA02 AA03 BC08 MA01 MA04 NA14 ZA36 ZB26

Claims (6)

[Claims] 1. The following formula (I): Or a salt thereof. 2. The following formula (II): Or a salt thereof. 3. A medicament comprising the compound according to claim 1 or 2 or a physiologically acceptable salt thereof as an active ingredient. 4. The medicine according to claim 3, which is an angiogenesis inhibitor. 5. The medicine according to claim 3, which is an antitumor agent. 6. A step of separating and collecting the compound or salt thereof according to claim 1 or 2 from a culture obtained by culturing a bacterium that produces the compound according to claim 1 or 2 belonging to the genus Aspergillus. A method for producing the compound according to claim 1 or 2, or a salt thereof.