JP2001286292A - New compound f-90558 and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new compound which exhibits N-acetylglucosamine transferase V inhibitory activity and is useful as a metastasis inhibitor. SOLUTION: This compound is the compound shown by the following formula (I) or (II). The above compound can be extracted and purified from the cultured cell of Aspergillus flavipes SANK 10700 strain (FERM BP-7102).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD [0001] The present invention relates to a novel compound having an activity of inhibiting N-acetylglucosamine transferase V and useful as a drug, particularly as a cancer metastasis inhibitor, and a method for producing the same.

[0002]

2. Description of the Related Art The metastatic property of cancer cells is a very important factor that largely determines the success or failure of solid cancer treatment. Numerous attempts have been made to find a drug that inhibits cancer metastasis, but at present there is no drug that has been clinically put to practical use.

[0003] The cell surface is covered with sugar chains existing in a form bound to proteins and lipids, and it has recently been revealed that these sugar chains are deeply involved in interactions such as recognition and adhesion between cells. Have been. It has been reported that cell carcinogenesis also changes the structure of cell surface sugar chains in association with canceration and transformation with a cancer virus, resulting in changes in cell adhesion and cell immunogenicity. . Furthermore, for example, the inhibitor of Golgi α-mannosidase II involved in the biosynthesis of the glycoprotein asparagine-linked sugar chain, swainsonine and its derivatives have an inhibitory effect on cancer metastasis at the experimental animal level, and clinical tests have been conducted on solid cancer patients. (Clinical Cancer Res., 1, 935-944 (199
5)), it has been shown that cancer metastasis can be controlled by modifying the structure of cell surface sugar chains (Protein / Nucleic Acid / Enzyme, Vol. 37, No. 11, pp. 1747-1752) , 1992
Year, etc.). However, even in such an attempt to suppress cancer metastasis by using a sugar chain biosynthesis inhibitor, no practically usable compound has been obtained so far.

[0004] As shown in the following formula (III), N-acetylglucosamine transferase V is a glycoprotein asparagine-conjugated complex using uridine diphosphate N-acetylglucosamine (abbreviated as "UDP-GlcNAc") as a sugar donor. Catalyzes the reaction of transferring N-acetylglucosamine to the 6-position of the α1-6-linked mannose residue of the type sugar chain with a β1-6 bond:

[0005]

Embedded image (In the formula, GlcNAc represents N-acetyl-D-glucosamine, Man represents D-mannose, and Asn represents asparagine.) As a structural change of a cell membrane glycoprotein sugar chain accompanying canceration of a cell, Galβ1- is often used. 4GlcNAcβ1-
6 (Galβ1-4GlcNAcβ1-2) Man structure (Gal represents D-galactose, GlcNAc represents N-acetyl-D-glucosamine, and Man represents D-mannose) as a partial structure, such as a complex triantenna type or tetra structure. There is a marked increase in antenna-type sugar chains (Glycoproteins and Disease, 211-227, Elsevier (19
96) etc.). N-acetylglucosamine transferase V is responsible for the biosynthesis of this structure, and the activity of this enzyme and the cell surface expression level of the sugar chain synthesized thereby are highly positively correlated with the enhanced metastatic potential of cancer cells. Has been shown in some cancer cells (Science 236, 582-585 (1987), Cli
n. Exp. Metastasis 12, 47-54 (1994), etc.). Further N
-Modification of cancer cell surface sugar chains by genetic engineering techniques using sugar chain biosynthetic enzyme genes such as acetylglucosamine transferase V gene, and analysis of their effects on metastasis and tumor formation in nude mice The results of these studies suggest that this enzyme or the sugar chains biosynthesized by this enzyme make some significant contribution to the acquisition of high metastatic potential in cancer cells (Proc. Natl. Acad. Sci. USA 92 ,
8754-8758 (1995), J. Cell. Biol. 130, 383-392 (199
Five)).

[0006] From the above findings, a selective inhibitor of N-acetylglucosamine transferase V can be a cancer metastasis inhibitor having a new mechanism of action, but no inhibitor for the enzyme has been found yet.

On the other hand, compounds structurally similar to the compounds of the present invention include the following formulas (IV) or (V):

[0008]

Embedded image Fomecin A (Can. J. Chem. 42, 1595 (19
64) and Chem. Pharm. Bull. 30, 2860 (1982)), however, for this compound, findings on the cancer metastasis inhibitory effect include the activity of inhibiting N-acetylglucosamine transferase V, Not reported at all.

[0009]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a novel compound which inhibits the activity of N-acetylglucosamine transferase V having a physiological function important for cancer cell metastasis and is useful for preventing cancer metastasis. An object of the present invention is to provide a method for producing the compound and a pharmaceutical composition containing the compound as an active ingredient.

[0010]

Means for Solving the Problems The present invention provides (1) the following formula (I):

[0011]

Embedded image Or the following formula (II):

[0012]

Embedded image Or a salt thereof, (2) a compound having the following physicochemical properties or a salt thereof: 1) Property: pale yellow powder; 2) Solubility: soluble in dimethyl sulfoxide (DMSO) and methanol, in water Poorly soluble, insoluble in ethyl acetate and chloroform; 3) molecular formula: C ₉ H ₁₀ NO ₅ (determined by high-resolution mass spectrum); 4) molecular weight: 198 (determined by FAB-MS spectrum); 5) ultraviolet absorption spectrum: λ _max nm (ε) UV absorption spectrum measured in methanol solution is 20
6 (33402), 225 (sh. 8910), 269 (1782), 315 (186
6) Infrared absorption spectrum (KBr): The infrared absorption spectrum measured on a ν _max cm ^-1 KBr disc is as follows: 3439, 3323, 3257, 1635, 1509, 1432 ,
1395, 1298, 1113, 996, 923, 897, 812; 7) ¹ H-nuclear magnetic resonance spectrum: δ (ppm) Nuclear magnetic resonance spectrum (360 MH) measured in deuterated dimethyl sulfoxide solution (tetramethylsilane internal standard)
z) is as follows: 1.91 (3H, s), 4.65 (1H, d, 12.0 Hz), 4.88 (1H, d)
d, 12.0, 1.44 Hz), 6.26 (1H, d, 1.44 Hz); 8) ¹³ C-nuclear magnetic resonance spectrum: δ (ppm) Nuclear magnetic resonance measured in deuterated dimethyl sulfoxide solution (tetramethylsilane internal standard) Spectrum (90MH
z) is as follows: 11.8 (q), 70.0 (d), 99.9 (t), 107.4 (s), 117.8 (s), 12
9.6 (s), 132.3 (s), 139.0 (s), 145.1 (s); 9) High Performance Liquid Chromatography: Separation Column: Symmetric ODS Column (φ4.6 ×
Mobile phase: 15% acetonitrile-85% water (v /
v); flow rate: 1.0 ml / min; detection wavelength: 210 nm; retention time: 5.9 minutes; (3) culturing a bacterium producing the compound according to (1) or (2) belonging to the genus Aspergillus. A method for producing the compound according to (1) or (2), wherein the compound according to (1) or (2) is collected from the culture, and (4) Aspergillus (Aspergillus).
lus) belonging to the genus (1) or (2), wherein Aspergillus flavipes is produced.
vipes) SANK 10700 strain (FERM BP-7
102), the method according to (3), (5) (1)
Or, it relates to a drug containing the compound of (2) as an active ingredient, and (6) a cancer metastasis inhibitor composition containing the compound of (1) or (2) as an active ingredient.

The present inventors have intensively searched for a novel substance that inhibits N-acetylglucosamine transferase V. As a result, the present inventors found that N-acetylglucosamine transferase V was found in the culture of filamentous fungi.
Was found, and a novel compound having an activity of inhibiting N-acetylglucosamine transferase V was successfully extracted and purified from the culture solution. Furthermore, the present inventors elucidated the structure and physicochemical properties of the novel compound, and completed the present invention.

The compound of the present invention is represented by the following formula:
Under normal conditions, the structure represented by the formula (I) and the structure represented by the formula (I)
In equilibrium with the structure represented by I):

[0015]

Embedded image In the present invention, the compound represented by the formula (I) and the compound represented by the formula (II) are collectively referred to as “F-905”.
58 ".

The compound F-90558 of the present invention represented by the above formula (I) or (II) has various stereoisomers. In the present invention, all of these isomers are represented by a single formula, but the present invention includes all of these isomers including racemates and mixtures of these isomers. When a stereospecific synthesis method is used,
Alternatively, when an optically active compound is used as a starting compound, individual isomers may be directly produced, while
Once a mixture of isomers has been prepared, the individual isomers may be obtained by conventional methods.

Compound F-90558 of the present invention can be salted using methods well known to those skilled in the art. The present invention also encompasses such salts of F-90558. F-905
The salt of 58 is not particularly limited as long as it is used medically and is pharmacologically acceptable. In addition, F-905
There is no limitation when the salt of 58 is used for purposes other than medicine, for example, when used as an intermediate. Such salts are preferably sodium salts, potassium salts,
Alkali metal salts such as lithium salts; alkaline earth metal salts such as calcium salts and magnesium salts; metal salts such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts, cobalt salts; Inorganic salts; t-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N , N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt,
Amine salts such as organic salts such as piperazine salt, tetramethylammonium salt and tris (hydroxymethyl) aminomethane salt; and amino acid salts such as glycine salt, lysine salt, arginine salt, ornithine salt and asparagine salt. Can be. More preferably, those preferably used as pharmacologically acceptable salts, that is, sodium salt, potassium salt, lithium salt, calcium salt, magnesium salt and the like can be mentioned.

The compound of the present invention or a salt thereof may be a solvate. For example, by leaving it in the air or by recrystallizing, it absorbs moisture,
The solvate may be adsorbed water or form a hydrate, and such a solvate is also included in the present invention.

The present invention further includes all compounds which are metabolized in vivo and converted into F-90558, so-called prodrugs.

[0020]

BEST MODE FOR CARRYING OUT THE INVENTION The compound of the present invention can be obtained by extracting and purifying from a culture in which bacteria producing the compound of the present invention belonging to the genus Aspergillus are cultured.

As the strain belonging to the genus Aspergillus used in the production of the compound of the present invention, for example, Aspergillus flavipes SAN
K10700 strain. SANK 10
The 700 strains were isolated from the soil collected in the grass grass field of Aka Park in Akajima, Okinawa Prefecture in July 1998 by the dilution plate method.

1. Bacteriological properties To observe the mycological properties of the fungus in culture, click and pit literature (Klich, MA
& Pitt, JI 1988.A laboratory guide to common
Aspergillus species and their teleomorphs.CSRIO,
(Australia).

Medium composition: CYA medium dipotassium phosphate 1g Tuapec concentrate * 10 ml yeast extract 5 g sucrose 30 g agar 15 g distilled water 1000 ml MEA medium malt extract 20 g peptone 1 g glucose 20 g agar 20 g distilled water 1000 ml CY20S medium dipotassium phosphate 1 g Tuapec concentrate * 10 ml yeast extract 5 g sucrose 200 g agar 15 g distilled water 1000 ml * Tupec concentrate sodium nitrate 30 g potassium chloride 5.0 g magnesium sulfate heptahydrate 5.0 g iron (II) sulfate heptahydrate 0.1 g zinc sulfate -Hexahydrate 0.1g Copper sulfate-Hexahydrate 0.1g Distilled water 100ml The mycological properties of SANK 10700 strain are as follows. In addition, the display of the color tone is described in "Metune Handbook
Of Color "(A. Kornerup and JH Wanscher: M
ethuen handbook of color (3rd. edition) .Erye Meth
uen, London, (1978)).

The colonies on CYA reach a diameter of 18 mm after culturing at 25 ° C. for 7 days. It has a convex shape as a whole, bulges at the center, has radial wrinkles, is composed of a shallow and dense mycelium, the mycelium is intertwined, the surface is grayish orange (5B3), and the back is dark brown ( 6F
7) with radial wrinkles. Is the aerial mycelium short?
Or it is well-developed, the colony surface is velvety and fluffy in the center. A yellowish-brown soluble pigment (5E7) is produced. No leachate is produced.

The colonies on the MEA reach 18 mm in diameter after culturing at 25 ° C. for 7 days. The surface is flat and velvety, and in the center it becomes fluffy due to the production of conidia. The front surface is yellowish white (4A2) and the back surface is brownish orange (5C6), which is darker at the center. Soluble pigments are produced but are not clear.

The colonies on CY20S reach a diameter of 23 mm after culturing at 25 ° C. for 7 days.
Same as on YA. Colonies on CYA at 37 ° C, 1
It reaches 11 mm in a week. It is composed of tightly entangled hyphae, swells at the center and has radial wrinkles. Its color tone is similar to that on CYA.

The conidial head has a radial to loose column shape,
The handle is about 400-600 μm, sometimes up to 800 μm, and is colorless to light brown, and the surface is smooth. The diameter of the apical sac is 15-25 μm and is spatula to subspherical. The conidiophore is two-stage and the metre is 7-8 × 4-5μ
At m, cover the upper 2/3 of the apical sac. Fearride is 7-8
× 2.5-3 μm. Conidia are spherical and 2-3 in diameter
μm, smooth and colorless. Teleomorph is not observed.

The mycological properties of the SANK 10700 strain as described above are described in Crick and Pitt (Klich, MA & P).
itt, JI 1988.A laboratory guide to common Aspe
rgillus species and their teleomorphs.CSRIO, Aust
ralia) as described in Aspergillus flavipes. Therefore, this bacterium was identified as Aspergillus flavipes SANK 10700 strain.

In addition, Aspergillus flavipes SA
NK 10700 shares were acquired on March 2, 2000.
It was deposited on March 3 with the Research Institute of Biotechnology and Industrial Technology of the Ministry of International Trade and Industry, under the accession number FERM BP-7102.

As is well known, fungi are liable to be mutated in nature or by artificial operations (for example, ultraviolet irradiation, irradiation, chemical treatment, etc.), and the same applies to the SANK 10700 strain of the present invention. is there. The SANK 10700 strain according to the present invention includes all mutant strains. Also, among these mutants, genetic methods,
For example, those obtained by recombination, transduction, transformation and the like are also included. That is, SANK 10700 strain producing F-90558, mutants thereof, and strains that are not clearly distinguished therefrom are all SANK 107 strains.
00 strains.

2. To obtain the culture F-90558, the culture of these microorganisms is performed in a medium such as that used to produce other fermentation products. Such a medium contains a carbon source, a nitrogen source and inorganic salts that can be assimilated by the microorganism.

In general, as a carbon source, glucose, fructose, maltose, sucrose, mannitol,
Glycerin, dextrin, oats, rye, corn starch, potato, corn flour, cottonseed oil, molasses, citric acid, tartaric acid and the like can be used alone or in combination. In general, the amount is varied from 1 to 10% by weight of the medium volume.

As the nitrogen source, a substance containing protein and its hydrolyzate or an inorganic salt is generally used in the fermentation step. Suitable nitrogen sources include soybean flour, bran, peanut flour, cottonseed flour, casein hydrolyzate, pharmamine,
Fish meal, corn steep liquor, peptone, meat extract, yeast, yeast extract, malt extract, sodium nitrate, ammonium nitrate, ammonium sulfate and the like. The nitrogen source is used alone or in combination in the range of 0.2 to 10% by weight of the medium volume.

The nutrient inorganic salts to be taken into the medium are ordinary salts from which ions such as sodium, ammonium, calcium, phosphate, sulfate, chloride and carbonate can be obtained. It also contains trace metals such as potassium, calcium, cobalt, manganese and magnesium.

In the liquid culture, silicone oil, vegetable oil, surfactant and the like are used as antifoaming agents. SAN
The pH of the medium for culturing the K10700 strain to produce F-90558 can be changed to 5.0 to 8.0.

This strain grows well in the range of 20 ° C. to 30 ° C. Further, 20 ° C. to 26 ° C. is suitable for the production of F-90558. F-90558 is obtained by aerobic culturing, and aerobic culturing methods usually used, for example, a solid culturing method, a shaking culturing method, an aerated stirring culturing method, and the like are used.

In small-scale culture, it is preferable to carry out shaking culture at 23 ° C. for several days. The culture is initiated by one or more stages of seed development in an Erlenmeyer flask. The seed flask is shaken in a constant temperature incubator for several days, or until fully grown. The grown seed is used to inoculate part or all of the seed in the next stage seed or production medium. The flask containing the inoculated production medium is shaken at a constant temperature for several days, and after completion of the culture, the contents of the flask are separated by centrifugation or filtration. In the case of mass culture, it is preferable to culture in a suitable tank equipped with a stirrer and aeration device. According to this method, a nutrient medium can be prepared in a tank. Nutrient medium at 121 ° C
Heat until sterilization, and after cooling, inoculate the sterilized medium with seeds that have been grown in advance. Culture is performed at 23 ° C. with aeration and agitation. This method is suitable for obtaining large amounts of compounds.

Usually, the production of F-90558 reaches the maximum value after culturing for 100 hours to 150 hours.

3. Extraction and Purification After completion of the culture, F-905 existing in the liquid portion of the culture solution
58, the bacterial cells and other solid parts are separated by a filtration operation using diatomaceous earth as a filter aid or centrifugation, and the F-90558 present in the filtrate or supernatant is utilized by utilizing its physicochemical properties. It is obtained by extraction and purification. Further, F-90558 contained in the cells and other solid parts can be purified in the same manner after extraction with aqueous methyl alcohol, acetone, or the like.

For example, a solution containing F-90558 can be purified by extracting F-90558 with an organic solvent immiscible with water, for example, n-butyl alcohol, ethyl acetate or the like, alone or in combination. is there. In addition, Amberlite XAD-2, XAD-4 (manufactured by Rohm and Haas Co.), which is activated carbon or an adsorption resin, Diaion HP-10, HP-20, HP-
50, CHP20P (manufactured by Mitsubishi Chemical Corporation) and the like are used. The liquid containing F-90558 is passed through a layer of the adsorbent as described above to adsorb and remove impurities, or after adsorbing F-90558, a mixture of water such as methanol water, acetone water or the like and an organic solvent is formed. It is obtained by elution using a mixed solvent. As the ion exchange resin, Dowex 50Wx4, Dowex 1x2, Dowex SBR-P (all manufactured by Dow Chemical Co., Ltd.) and the like are used. The liquid containing F-90558 is passed through a layer of the ion exchange resin as described above to adsorb and remove impurities,
Alternatively, it is obtained by adsorbing F-90558 and then eluting with hydrochloric acid or aqueous ammonia.

The thus obtained F-90558
Further, adsorption column chromatography using a carrier such as silica gel or florisil, Avicel (manufactured by Asahi Chemical Industry Co., Ltd.), Sephadex LH-20 (manufactured by Pharmacia), Cosmosil 140C18 column (manufactured by Nakarai Tesque), etc. And high-performance liquid chromatography using normal-phase and reverse-phase columns.

The location of the purification of F-90558 of the present invention can be determined by the following methods.

1) N-acetylglucosamine transferase V
Method using inhibitory activity as an index N-acetylglucosamine transferase V inhibitory activity is measured in a system in which a substrate sugar chain having a fluorescent end labeled with a reducing end is used to generate a reaction product in which one molecule of N-acetylglucosamine is transferred. The activity can be measured as the activity of suppressing the production of the reaction product. That is, after a test sample is incubated for a certain period of time in a reaction solution containing the substrate sugar chain uridine 5′-diphosphate N-acetylglucosamine and N-acetylglucosamine transferase V, the unreacted substrate Analysis is performed by high performance liquid chromatography under conditions that allow separation of the reaction product from the reaction product. By comparing the chromatogram of the reaction solution under the condition where the test sample was added with the chromatogram of the reaction solution under the condition where the test sample was not added, the test sample in which the amount of the reaction product generated was reduced,
It shall include N-acetylglucosamine transferase V inhibitory activity. In addition, N-acetylglucosamine transferase V
For example, mouse melanoma cell line B16-BL
This enzyme produced in the culture solution of No. 6 was partially purified by ion exchange chromatography, and also purified from rat kidney (J. Biol. Chem. 267, 2920-2927 (1.
992)) or some human liver cancer cell lines (eg, H
epG2 cells (American Type Culture Collection HB8065)
-Acetylglucosamine A product obtained by a method of purification using the activity in the transfer reaction system as an index or the like can be used.

2) Method Using High-Performance Liquid Chromatography High-performance liquid chromatography is performed under the following conditions to collect fractions eluted at a retention time of about 5.9 minutes: Separation column: Symmetric ODS column (φ4.6) ×
Mobile phase: 15% acetonitrile-85% water (v /
v); Flow rate: 1.0 ml / min; Detection wavelength: 210 nm.

The compound of the present invention is a novel compound not described in any literature, has an inhibitory activity on N-acetylglucosamine transferase V, and is useful as a cancer metastasis inhibitor.

When the compound of the present invention is used as a cancer metastasis inhibitor, it can be administered in various forms. Examples of the administration form include oral administration such as tablets, capsules, powders, granules, syrups and the like. Injectables (intravenous, intramuscular, subcutaneous) and the like can be mentioned. These various preparations are commonly used in the pharmaceutical preparation technical field such as excipients, binders, disintegrants, lubricants, flavoring agents, solubilizing agents, suspending agents, coating agents, and the like, in accordance with the usual methods for the main drug. It can be formulated using known adjuvants.

For forming into tablets, those conventionally known in the art can be widely used as a simple substance, such as lactose, sucrose, sodium chloride, glucose, urea, starch,
Excipients such as calcium carbonate, kaolin, crystalline cellulose, and silicic acid; binding of water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone, etc. Ingredients: disintegrating agents such as dried starch, sodium alginate, agar powder, laminaran powder, sodium hydrogencarbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, starch, lactose, sucrose, stearin, cacao Disintegration inhibitors such as butter and hydrogenated oil; absorption promoters such as quaternary ammonium bases and sodium lauryl sulfate; humectants such as glycerin and starch; adsorbents such as starch, lactose, kaolin, bentonite and colloidal silicic acid ; Purification Torque, stearate, boric acid powder, lubricants such as polyethylene glycol can be exemplified. Further tablets are tablets coated with normal skin as needed,
For example, sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets or double tablets or multilayer tablets can be prepared.

In the form of pills, carriers conventionally known in the art can be widely used as carriers, for example, excipients such as glucose, lactose, cocoa butter, starch, hydrogenated vegetable oil, kaolin, and talc; Binders such as rubber powder, tragacanth powder, gelatin, and ethanol; disintegrators such as laminaran agar;

For shaping in the form of suppositories, those conventionally known in the art can be widely used as carriers, for example, polyethylene glycol, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semisynthetic glycerides and the like. Can be mentioned.

When prepared as an injection, the liquid preparation and the suspension are preferably sterilized and isotonic with blood. When forming these liquid preparations, emulsions and suspensions, they may be diluted. All agents commonly used in this field can be used, for example, water, ethanol, propylene glycol, ethoxylated isostearyl alcohol,
Examples thereof include polyoxylated isostearyl alcohol and polyoxyethylene sorbitan fatty acid esters. In this case, a sufficient amount of salt, glucose, or glycerin to prepare an isotonic solution may be included in the pharmaceutical preparation, and ordinary solubilizers, buffers, soothing agents, etc. May be added.

Further, a coloring agent, a preservative, a flavor, a flavoring agent, a sweetening agent and the like and other pharmaceuticals may be contained as required.

The amount of the active ingredient compound contained in the above-mentioned pharmaceutical preparation is not particularly limited and may be appropriately selected in a wide range, but is usually 1 to 70% by weight, preferably 1 to 30% by weight of the whole composition.
It is appropriate that the amount is contained by weight%.

The administration method of the above pharmaceutical preparation is not particularly limited, and is determined according to various preparation forms, age, sex and other conditions of the patient, degree of disease and the like. For example, tablets, pills, solutions, suspensions, emulsions, granules and capsules are orally administered. In the case of an injection, it may be administered intravenously, alone or as a mixture with a normal replenisher such as glucose or amino acid, and, if necessary, intramuscularly, intradermally, subcutaneously or intraperitoneally. In the case of suppositories, they are administered rectally.

The amount used varies depending on symptoms, age, body weight, administration method, dosage form and the like.
It is preferable to administer 1 mg to 1000 mg per day in one or several divided doses.

[0055]

EXAMPLES Next, the present invention will be described in more detail with reference to Examples and Test Examples, but the present invention is not limited thereto.

1) Culture A 80-ml culture medium having the following composition was placed in 37 500-ml Erlenmeyer flasks, and sterilized by heating at 121 ° C. for 20 minutes. After cooling them to 23 ° C., the medium in each flask was added to Aspergillus flavipes SANK 10
700 (FERM BP-7102) strain was inoculated with one platinum loop from the slant, and rotated at 210 rpm with a rotary shaker.
The cells were cultured at 3 ° C. for 120 hours.

Medium composition: soluble starch 2.0% (w / v) glycerin 3.0% (v / v) glucose 3.0% (w / v) gelatin 0.25% (w / v) soybean flour 1 0.0% (w / v) Yeast extract (Difco) 0.25% (w / v) Ammonium nitrate 0.25% (w / v) Defoamer (CB-442) 0.01% (w / v) or more Is dissolved in tap water, and the pH before sterilization is adjusted to 6.5.

2) Extraction and Purification 120 g of Celite 545 (manufactured by Johns Manville Product Corporation) was added to about 3 liters of the culture solution obtained in 1) above as a filter aid, and the mixture was filtered through filter paper. The pH of 5 L was adjusted to 3.8 with 1 N hydrochloric acid. This filtrate was filled with water previously adjusted to pH 3.8 with diluted hydrochloric acid and equilibrated with Diaion HP-2.
0 resin (manufactured by Mitsubishi Chemical Corporation) through a 500 ml column, and then 2 liters of deionized water adjusted to pH 3.8,
Next, the resin was washed with 1.5 liter of 20% aqueous methanol (pH 3.8) to remove unadsorbed components. Then, 6
The adsorbed components were eluted using 0% aqueous methanol (pH 3.8), and the eluate was collected in 20 ml portions. Analysis was carried out according to the method described in the following Test Example, and fractions from the 19th to 40th in which the inhibitory activity against N-acetylglucosamine transferase V was recognized were collected, concentrated under reduced pressure, lyophilized, and subjected to freeze-drying. 2.25 g of a coarse powder containing -90558 was obtained.

After dissolving the obtained powder in a solvent prepared by adjusting 1 liter of deionized water to pH 3.8 with diluted hydrochloric acid,
After passing through a 360 ml column of Amberlite XAD-2 resin (manufactured by Rohm and Haas Co.) that has been equilibrated by filling with water previously adjusted to pH 3.8 with dilute hydrochloric acid, deionized water is diluted with dilute hydrochloric acid to pH 3.8. The resin was washed with 1.4 liters of the adjusted solvent to remove unadsorbed components. Then
The adsorbed components were eluted using 20% aqueous methanol (pH 3.8), and 140 ml of the eluate was collected. Analysis was performed by the method described in the following Test Examples, and fractions from the fifth to the tenth in which the inhibitory activity on N-acetylglucosamine transferase V was recognized were collected. The Amberlite XAD-2 column was further eluted with 30% aqueous methanol (pH 3.8), and the eluate was fractionated in 20 ml portions. Similarly, the 15th to 60th fractions were collected. This was combined with the previously obtained 20% methanol-water (pH 3.8) elution fraction, concentrated under reduced pressure, freeze-dried, and crude powder 60 containing F-90558.
3 mg were obtained.

600 mg of the obtained powder was dissolved in 100 ml of 80% aqueous methanol (pH 3.8), and 80%
Sephadex LH-20 resin (Pharmacia) equilibrated by filling with methanol water (pH 3.8)
After passing through a 1.1-liter column, the adsorbed components were developed and eluted using 80% aqueous methanol (pH 3.8), and the eluate was collected in 20 ml portions. Analysis was performed according to the method described in the following test example, and N-acetylglucosamine transferase V was analyzed.
The fractions from the 63rd to the 68th, which showed the inhibitory activity against, were collected and concentrated under reduced pressure to 25 ml. This was left at room temperature to form a crystalline precipitate, which was filtered with filter paper to obtain 237 mg of a crystalline powder of F-90558.

Test Example The inhibitory activity of N-acetylglucosamine transferase V was measured by the following method.

I) Preparation of Substrate and Reagent A substrate sugar chain represented by the following formula (VI) (hereinafter referred to as “GnGn-b”) having a reducing end fluorescently labeled with 2-aminopyridine.
i-PA "):

[0063]

Embedded image (Where PA represents a 2-aminopyridyl group, GlcNAc
Represents N-acetyl-D-glucosamine and Man represents D-mannose, respectively) is the corresponding unlabeled oligosaccharide GlcNAcβ1-2Manα1-6 (GlcNAcβ
1-2Manα1-3) Manβ1-4GlcNAcβ
Starting from 1-4GlcNAc (manufactured by Oxford Glyco Systems), a literature (Agric. Biol. Chem.
54, 2169-2170 (1990)).
Was prepared.

O- (2-acetamido-2-deoxy-D-glucopyranosilidene) -amino-N-phenylcarbamate is described in the literature (Helv. Chim. Acta 68, 2254-).
2274 (1985) and Eur.J. Biochem. 197, 815-818 (199
1)) It was synthesized according to the method described.

Ii) Preparation of Enzyme N-acetylglucosamine transferase V was obtained by partially purifying the enzyme produced in a culture of mouse B16-BL6 melanoma by ion exchange chromatography according to the method described below. Was.

First, mouse B16-BL6 melanoma cells were cultured in Dulbecco's modified Eagle's medium (D-MEM) supplemented with 10% fetal bovine serum at 37 ° C. and 5% carbon dioxide until the cells became confluent in a culture vessel. Next, the culture was centrifuged at 3000 × g for 15 minutes,
320 ml of the supernatant (specific activity 0.5 nmol / mg protein / hour, protein amount 2020 mg) was added to buffer A (50 mM MES buffer, pH 6.5, 5 mM).
After dialysis against ethylenediaminetetraacetic acid (hereinafter referred to as "EDTA"), the inner solution of the dialysis was recovered, and Triton X-100 (manufactured by Sigma) was added to a final concentration of 0.1%. . This was applied to a CM-Toyopearl 650M column (Tosoh Corporation, φ6 × 50 cm) which had been equilibrated with buffer B (buffer A plus 0.1% Triton X-100) in advance. Charged. Elution was carried out with buffer solution B at a column temperature of 4 ° C. and a flow rate of 15 ml / hour, and 500 ml of each eluate was collected. The obtained eluted fraction was measured for N-acetylglucosamine transferase V activity by the method described in iii) below. As a result, the eleventh to seventeenth fractions showing enzymatic activity were collected. Thus, the specific activity is 150 nmol / m
3.5 mg of partially purified enzyme of g protein / hour was obtained.

Iii) Reaction A reaction product represented by the following formula (VII) obtained by transferring one molecule of N-acetylglucosamine to a substrate GnGn-bi-PA (hereinafter referred to as “GnGnGn-tri′-PA”):

[0068]

Embedded image (Where PA represents a 2-aminopyridyl group, GlcNAc
Represents N-acetyl-D-glucosamine, and Man represents D-mannose), and the enzymatic activity was determined as follows. That is, 30 μ
M-O- (2-acetamido-2-deoxy-D-glucopyranosylidene) -amino-N-phenylcarbamate, 5 μM pepstatin A (manufactured by Wako Pure Chemical Industries, Ltd.),
5 μM leupeptin (manufactured by Wako Pure Chemical Industries, Ltd.), 1.5
mg / ml of (p-amidinophenyl) methanesulfonyl fluoride hydrochloride (manufactured by Wako Pure Chemical Industries, Ltd.), 1.25
% Triton X-100, and 25 mM EDTA
20 μm of 300 mM 2- (N-morpholino) ethanesulfonic acid buffer (pH 6.3) containing disodium salt
1 μl of 200 mM uridine 5′-diphosphate N-acetylglucosamine (Sigma) solution, 400 μM
10 μl of a GnGn-bi-PA solution, 10 μl of the N-acetylglucosamine transferase V solution prepared in ii) above
and 10 μl of the test sample solution were added, and the mixture was kept at 37 ° C. for 3 hours. Thereafter, the enzyme reaction was stopped by boiling at 100 ° C. for 3 minutes.

Iv) Measurement and Evaluation After centrifuging the reaction solution obtained in iii) above, the supernatant was subjected to high performance liquid chromatography under the following conditions: Separation column: Cosmosil 5C18-AR (φ4.6 ×)
250 mm, manufactured by Nacalai Tesque, Inc.) Mobile phase: 100 mM ammonium acetate buffer (pH
4.0) /0.1% 1-butanol (isocratic) Flow rate: 1.0 ml / min Column temperature: 45 ° C. Excitation wavelength: 320 nm Detection wavelength: 400 nm In high-performance liquid chromatography under the above conditions, the substrate or reaction The fluorescence emitted from the 2-aminopyridyl group of the product can be detected, but the substrate GnGn-
At 11.8 minutes, bi-PA had its N-acetylglucosamine transferase V reaction product GnGnGn-tri'-P
A elutes at 8.1 minutes each. From the area ratio of the peak corresponding to the substrate GnGn-bi-PA and the peak corresponding to the reaction product GnGnGn-tri'-PA on the obtained chromatogram, the production of the N-acetylglucosamine transferase V reaction product was determined. The rate (A) was determined. Even when the test sample was not added, the reaction described in iii) was performed under the condition that only the solvent of the test sample solution was added, and the production rate (B) of the reaction product in the reaction solution was determined. N of test sample
The acetylglucosamine transferase inhibition rate was calculated using the following formula: 100-100 × A / B. The inhibition rate was determined by changing the concentration of the test sample, and the N-acetylglucosamine transferase V activity was determined to be 5%.
The test sample concentration that inhibits 0% was defined as IC ₅₀ .

F-F finally obtained in 2) of the above example was used.
90558 N-acetylglucosamine transferase V
Was 23 μg / ml as an IC ₅₀ value.

Formulation Examples Capsules F-90558 100 mg Lactose 100 mg Maize starch 148.8 mg Magnesium stearate 1.2 mg Total amount 350 mg After mixing the powder of the above formulation and passing through a 60 mesh sieve, put the powder into a gelatin capsule. And a capsule.

[0072]

As described above, the compound of the present invention has an inhibitory activity on N-acetylglucosamine transferase V and is useful as a cancer metastasis inhibitor.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07C 47/56 C07C 47/56 C07D 307/88 C07D 307/88 C12P 13/00 C12P 13/00 17/04 17/04 // C12N 9/99 C12N 9/99 (C12P 7/24 (C12P 7/24 C12R 1:66) C12R 1:66) (C12P 13/00 (C12P 13/00 C12R 1:66) C12R 1 : 66) (C12P 17/04 (C12P 17/04 C12R 1:66) C12R 1:66) (72) Inventor Tsuyoshi Hosoya 33, Miyukigaoka, Tsukuba, Ibaraki Prefecture Sankyo Co., Ltd. (72) Inventor Tomoko Takayama Tokyo 1-258 Hiromachi, Shinagawa-ku, Tokyo F-term (reference) 4B064 AC26 AE45 BA02 BA03 BH07 CA05 DA01 4C037 RA01 4C086 AA01 AA03 AA04 BA06 MA04 NA14 ZB26 ZC20 4C206 AA01 AA03 AA04 CB09 MA04 M A57 NA14 ZB26 ZC20 4H006 AA01 AA03 AB28

Claims (6)

[Claims] (1) The following formula (I): Or the following formula (II): Or a salt thereof. 2. A compound having the following physicochemical properties or a salt thereof: 1) Property: pale yellow powder. 2) Solubility: Soluble in dimethyl sulfoxide (DMSO) and methanol, poorly soluble in water, insoluble in ethyl acetate and chloroform. 3) Molecular formula: C ₉ H ₁₀ NO ₅ (determined by high-resolution mass spectrum) 4) Molecular weight: 198 (determined by FAB-MS spectrum) 5) UV absorption spectrum: λ _max nm (ε) UV measured in methanol solution Absorption spectrum is 20
6 (33402), 225 (sh. 8910), 269 (1782), 315 (186
1) The absorption maximum is shown at nm. 6) Infrared absorption spectrum (KBr): The infrared absorption spectrum measured on the ν _max cm ^-1 KBr disk is as follows: 3439, 3323, 3257, 1635, 1509, 1432,
1395, 1298, 1113, 996, 923, 897, 812 7) ¹ H-nuclear magnetic resonance spectrum: δ (ppm) Nuclear magnetic resonance spectrum (360 MH) measured in deuterated dimethyl sulfoxide solution (tetramethylsilane internal standard)
z) is as follows: 1.91 (3H, s), 4.65 (1H, d, 12.0 Hz), 4.88 (1H, d)
d, 12.0, 1.44 Hz), 6.26 (1H, d, 1.44 Hz) 8) ¹³ C-nuclear magnetic resonance spectrum: δ (ppm) Nuclear magnetic resonance spectrum measured in deuterated dimethyl sulfoxide solution (tetramethylsilane internal standard) (90 MH
z) is as follows: 11.8 (q), 70.0 (d), 99.9 (t), 107.4 (s), 117.8 (s), 12
9.6 (s), 132.3 (s), 139.0 (s), 145.1 (s) 9) High performance liquid chromatography: Separation column: Symmetry ODS column (φ4.6 ×
Mobile phase: 15% acetonitrile-85% water (v /
v); Flow rate: 1.0 ml / min; Detection wavelength: 210 nm; Retention time: 5.9 minutes. 3. culturing a bacterium producing the compound according to claim 1 or 2, which belongs to the genus Aspergillus;
3. The method for producing a compound according to claim 1 or 2, wherein the compound according to claim 1 or 2 is collected from the culture. 4. The bacterium according to claim 1, which belongs to the genus Aspergillus, wherein the bacterium producing the compound according to claim 1 or 2 is Aspergillus flavipes SAN.
The method according to claim 3, which is strain K10700 (FERM BP-7102). 5. A medicament comprising the compound according to claim 1 as an active ingredient. 6. A cancer metastasis inhibitor composition comprising the compound according to claim 1 as an active ingredient.