JPH06135980A - New physiologically active substance a-72363 - Google Patents

Abstract

PURPOSE:To provide a new compound useful as a metastasis inhibitor or carcinostatic. CONSTITUTION:The objective compound (salt) of the formula having the following physicochemical characteristics: (1) appearance: amphoteric water-soluble white powder; (2) molecular formula: C8H14N2O5; (3) molecular weight: 218 (determined by FAB mass spectrometry); high resolution mass spectrometry (M-H)<->: 217.0804 (measured), 217.0824 (calculated); (4) elemental analysis (%) (as C8H14N2O5.H2O): C 41.07, H 6.43, N 12.28 (measured); C 40.67, H 6.83 and N 11.86; (5) specific rotatory power: [alpha]D<27>: -59 deg. (C 0.66, water); (6) ultraviolet absorption spectra (determined in water): giving terminal absorption at <=220nm; (7) infrared absorption spectra (lambdamaxcm<-1>): 3351, 1673, 1590, 1525, 1447, 1394, etc.; (8) solubility: soluble in water, insoluble inorganic solvents such as acetic acid, pyridine and methanol. The compound of the present invention can be obtained from a culture fluid produced by culture of Streptomyces nobilis-S-ANK 60192 (FERM P-4000; BP4000).

Description

Detailed Description of the Invention

[0001]

The present invention relates to heparanase and β
-A novel compound A-72363 having inhibitory activity against glucuronidase and a method for producing the same.

[0002]

2. Description of the Related Art Conventionally, as metabolites of microorganisms, cystatin A (Siastatin A), which has a sialidase inhibitory activity and whose chemical structure is unknown, and Siastatin B (Siastatin B) (H. Umezawa) which is a 6-epimer of A-72363. ,
J. Antibiotics, Vol. 27, p. 963 (1974)), and it is reported to have antiviral and antibacterial actions (JP-A-1-294687).

However, a compound having the above structural formula (I) showing heparanase and β-glucuronidase inhibitory activity has not been reported until now.

Heparanase is an enzyme that decomposes heparan sulfate, which is one of the main components of basement membrane. Heparanase has been reported to be involved in angiogenesis during cancer growth, cancer invasion and metastasis (J. Cellular Bioch
em., 36, 157 (1988)). Therefore, heparanase inhibitors are useful for the prevention and treatment of cancer metastasis.

Heparin and its derivatives (Biochemistry, Vol. 25, 5
322 (1986)), a sulfated derivative of chitin (Cancer Re
s., 50, 3631 (1990)), and suramin (surami
n: J. Biol. Chem., vol. 266, p. 9661 (1991)). It has been found that these compounds suppress invasion or metastasis of cancer cells mainly in a cancer metastasis model using mice.

On the other hand, β-glucuronidase is considered to be involved in drug metabolism in the body, and particularly has an important role in converting an inactive carcinogen into an active form (Can.
cer, 28, 60 (1971)). In addition, it has been reported that administration of β-glucuronidase inhibitors suppresses carcinogenesis in rats caused by carcinogens (Cancer Res., 42, 331 (1982), Carciogenesi.
s, vol. 6, p. 679 (1985)). Therefore, β-glucuronidase inhibitors are useful as anticancer agents.

[0007]

DISCLOSURE OF THE INVENTION The present inventors have proposed a novel compound having heparanase and β-glucuronidase inhibitory activity from a culture of SANK 60192 strain belonging to the genus Streptomyces isolated from soil in Higashi-mura, Kunigami-gun, Okinawa Prefecture. A-
The present invention was completed by finding that 72363 was produced.

[0008]

A-72363 has the following structural formula and physicochemical properties. Structural formula

[0009]

[Chemical 2]

Here, A-72363 is (3S, 4
S, 5R, 6S) -6-Acetylamino-4,5-dihydroxy-piperidine-3-carboxylic acid.

1) Properties of substance: Amphoteric water-soluble white powder 2) Molecular formula: C ₈ H ₁₄ N ₂ O ₅ 3) Molecular weight: 218 (measured by FAB mass spectrum method) 4) High resolution mass spectrometry: Glycerin-water The accurate mass, [MH] ⁻ , in the high resolution negative ion FAB mass spectrum measured as the matrix is as shown below.

Measured value 217.0804 Calculated value: 217.0824 5) Elemental analysis (%): Measured value as C ₈ H ₁₄ N ₂ O ₅ .H ₂ O C 41.07, H 6.43, N 12.28 Calculated value: C 40.67, H 6.83, N 11.86 6) Specific rotation: [α] _D ²⁷ −59 ° (c0.66, water) 7) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in water shows a terminal absorption at 220 nm or less.

8) Infrared absorption spectrum: λ _max cm ^-1 potassium bromide (KBr) The infrared absorption spectrum measured by the tablet method is as shown below.

3351,1673,1590,1525,1447,1394,1372,130
0,1283,1267,1188,1139,1115,1085,1035,1008,965,889,
850,833,750,707.

9) ¹ H-nuclear magnetic resonance spectrum: (δ:
ppm) 1 normal deuterated ammonia water, water ( H DO = 4.75pp) as internal standard
¹ H-nuclear magnetic resonance spectrum (360 m)
MHz) is as shown below.

2.05 (s), 2.55 (dt, J = 4.4, 9.9 Hz), 2.76 (dd, J
= 4.4,13.0Hz), 3.06 (dd, J = 9.9,13.0Hz), 3.79 (t, J = 4.1H
z), 4.27 (br.t), 4.80 (d, J = 4.1Hz).

10) ¹³ C-nuclear magnetic resonance spectrum: (δ:
ppm) ¹³ C-nuclear magnetic resonance spectrum (90 MHz) measured using 1,4-dioxane (67.4 ppm) as an internal standard in 1N deuterated ammonia water is as shown below.

23.2 (q), 38.0 (t), 48.3 (d), 62.9 (d), 68.9
(d), 70.6 (d), 175.2 (s), 180.6 (s).

11) Solubility: Soluble in water. Acetic acid, pyridine,
Insoluble or insoluble in organic solvents such as methanol, ethanol, acetone, chloroform, ethyl acetate, benzene, ether, hexane and petroleum ether.

12) Color reaction: Positive with ninhydrin reagent.

13) High Performance Liquid Chromatography: Column; Asahi Pack ES502N (7.6 φ × 100 mm,
Asahi Kasei Co., Ltd. Solvent: 1 mM phosphate buffer (pH 6.5) Flow rate: 1.0 ml / min Detection wavelength: 210 nm Retention time: 11.5 minutes.

A-72363 has a completely different biological activity from that of cystatin B in one position.

That is, A-72363 inhibits heparanase (IC ₅₀ ; 2.7 μg / ml), but cystatin B has 50 μm.
It does not inhibit heparanase at concentrations up to g / ml. Conversely, A-72363 does not inhibit sialidase at all at concentrations up to 50 μg / ml, whereas cystatin B strongly inhibits sialidase (IC ₅₀ ; 3.6 μg / ml).

A-72363 has a β-glucuronidase inhibitory action 300 times stronger than that of cystatin B.

Although the physicochemical properties of cystatin A are unknown and the chemical structure is undetermined, this substance has a sialidase inhibitory activity about 10 times stronger than that of cystatin B, and β-
It has been reported that glucuronidase does not inhibit (J. Antibiotics, 27, 963 (1974)).

From such a difference in specificity of the enzyme inhibitory activity, it is clear that A-72363 is a different substance from siastatin A.

A-7 having the above structural formula (I) of the present invention
2363 can be converted into a salt according to a conventional method. Examples of such salts include alkali metal salts such as lithium, sodium and potassium; alkaline earth metal salts such as calcium and barium; aluminum salts; basic amino acid salts such as lysine and arginine; ammonia. Amine salts such as, methylamine, dimethylamine;
Salts of inorganic acids such as hydrofluoric acid, hydrobromic acid, hydroiodic acid, hydrochloric acid, nitric acid, perchloric acid, carbonic acid, sulfuric acid, phosphoric acid;
Lower alkyl sulfonic acids such as methane sulfonic acid, trifluoromethane sulfonic acid, ethane sulfonic acid; benzene sulfonic acid, allyl sulfonic acid such as p -toluene sulfonic acid; acetic acid, fumaric acid, tartaric acid, oxalic acid, maleic acid, apple Examples thereof include salts of organic carboxylic acids such as acid, succinic acid and citric acid; salts of acidic amino acids such as glutamic acid and aspartic acid. Preferably, it is a pharmacologically acceptable salt.

The above SA for producing A-72363 of the present invention
The NK 60192 strain was collected from soil in Higashi-mura, Kunigami-gun, Okinawa, according to a conventional method and separated from a glycerin-asparagine agar plate.

SANK 60192, a bacterium producing A-72363
The mycological properties of the strain are as follows. 1. Morphological characteristics After culturing on an agar medium specified by ISP [International Streptomyces Project] at 28 ° C for 14 days, the basal hyphae of this strain are well elongated and diverged by observation under a microscope. It shows a reddish orange or orange, but the rupture or zigzag extension of Nocardia (Nocardia) strain belonging to the genus like is not observed. The aerial mycelium branches in the shape of an axle. The spore chain morphology is linear or curved and forms a chain of approximately 10 to 50 spores. When observed by a scanning electron microscope, the spores have an elliptical shape and their surface structure shows a smooth shape. Spores are formed only on the aerial mycelium. No special organs such as sclerotia, hyphal tears, sporangia were observed. 2. Properties on various culture media Properties after culturing on various culture media at 28 ° C. for 14 days are shown in Table 1. The color tone is displayed by the Japan Color Research Institute version,
"Standard color chip" color chip number (saturation-brightness-
Hue).

[0030]

[Table 1] ----------------------------------------------------------------------------------------------------------------- Type of medium Item Properties ----- −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Sucrose ・ G: Good, flat, bright reddish orange (12-7-5 ) Nitrate agar AM: not very good, velvety, white R: bright reddish orange (10-7-5) SP: not produced ---------- Glucose G: Good, flat, reddish orange (12-5-4) Asparagine agar AM: Well formed, fluffy, pink ( 6-8-2) R: Bright yellow red (12-6-3) SP: Not produced ----------------------------------------- −−−−−−−−−− Glycerin ・ G: Not so good, flat, orange (8-7-5) Asparagine agar M: Slightly formed, white (ISP 5) R: Dull orange (6-7-6) SP: Not produced −−−−−−−−−−−−−−−−−−−−−−−− −−−−−−−−−−−−−− Starch / inorganic salt agar G: Very good, flat, reddish orange (12-5-4) (ISP 4) AM: Well formed, fluffy, thin Orange (3-9-4) R: Yellowish red (12-5-3) SP: Not produced −−−−−−−−−−−−−−−−−−−−−−−−−− −−−−−−−−−−− Tyrosine agar G: Good, flat, orange (14-7-6) (ISP 7) AM: Slightly formed, white R: Orange (12-7-6) SP: Not produced -------------------------------------- Nutrient agar G: Very good, raised, Light yellowish tea (6-8-8) (DIFCO) AM: Not very good, velvety, white R: Bright tea (10-6-6) SP: Not produced ------------- −−−−−−−−−−−−−−−−−−−−−−−−−−− Yeast extract ・ G: Very good, bumpy, bright reddish orange malt extract agar (12-7- 5) (ISP 2) AM: not very good, velvety, white R: dull reddish orange (10-6-5) SP: not produced −−−−−−−−−−−−−−−−−− −−−−−−−−−−−−−−−−−−−− Oatmeal agar G: Very good, flat, reddish orange (12-5-4) (ISP 3) AM: Not very good, Velvety, light orange (2-9-5) R: Yellowish red (12-5-3) SP: Not produced ------------------------------- −−−−−−−−−−−−−−−− Water agar G: Not very good, flat, light yellowish orange (2-9-9) AM: Slightly formed, white R: Light yellowish orange ( 2-9-9) SP: Not produced −−−−−−−−−−−−−−−−−−−−−−−−−−−−− −−−−− Potato extract G: Not very good, flat, light yellow (4-9-9) Ginseng extract agar AM: Slightly formed, white R: Light yellow (4-9-9) SP: Produced No ------------------------------------------ G: growth, AM: aerial mycelium, R: back surface, SP: Soluble pigment Further, 0.05N hydrochloric acid and 0.05N sodium hydroxide were added to the basal hyphae of this strain, but no change in color tone due to these was observed.

3. Physiological Properties The physiological properties of this strain observed for 2 to 21 days after culturing at 28 ° C. are shown in Table 2.

[0032]

[Table 2]

Further, as shown in Table 3, the carbon source assimilation of this strain was observed after culturing at 28 ° C. for 14 days using Prideham-Gotrieve agar medium (ISP 9).

[0034]

[Table 3]

4. Regarding the cell components, the cell wall of this strain is the method of B. Becker et al.
al., Applied Microbiology, Vol. 12, 1984], and as a result, LL-diaminopimelic acid was detected, and it was confirmed to be cell wall type I. In addition, the sugar components in the whole cells of this strain can be analyzed by the method of MP Rechevalier [MP Lechevalier, Journal of Laboratory Clinica
l Medicine, 71, 934 (1968)],
No characteristic pattern was observed.

From the above-mentioned mycological properties, it was revealed that this strain belongs to the genus Streptomyces among actinomycetes. Description of ISP strain by Shirring and Gottlieb [(EB Shirling and D. Gottlieb), International Journal of System Bacteriology
atic Bacteriology), Vol. 18, pp. 68-189 (1968), Vol. 18
Volume, Pages 279-392 (1968), Volume 19, Pages 391-512 (1969), Vol.
22: 265-394 (1972)], Waxman, The Actinomycetes, SAWaksman; The 2nd
Volume, Buchanan and Gibbons, Vergise Manual
(REBuchanan and NEGibbons, Bergey's Manual of D
eterminative Bacteriology) 8th Edition (1974), Williams, Sharp and Holt, Vergise Manual
(Bergey's Manual of Systematic Bacteriology) Volume 4
(1989) and the strains described in the recent literature on Streptomyces actinomycetes, Streptomyces nobilis ( Streptomyces nobilis
ces nobilis ). However, Streptomyces Nobilis (Streptomyce
s nobilis) points nitrate reduction resistance is positive, the point salt resistance is of 7% and L- arabinose assimilation was observed difference in that it is positive. This strain having such bacteriological characteristics is apparently a new strain different from Streptomyces nobilis ( Streptomyces nobilis ), but it is not possible to distinguish the species with these differences, so this strain Streptomyces nobilis ( Str
eptomyces nobilis ) and SANK 601 as a preservation number
92 (Deposit institution, Institute of Microbial Science and Technology, Agency of Industrial Science and Technology: Deposit number, Microtechnology Research Institute Article 4000 (FERM BP 4000): Original deposit date, 1
(September 2, 992).

The SANK 60192 strain has been described above,
It is well known that the properties of actinomycetes are not constant and easily change naturally and artificially, and the strain that can be used in the present invention is A- which belongs to the genus Streptomyces.
It includes all strains producing 72363.

In order to obtain the novel compound A-72363 of the present invention, the cultivation of these microorganisms is carried out in a medium as is usually used for producing other fermentation products. Such a medium contains a carbon source, a nitrogen source and an inorganic salt that can be assimilated by the microorganism.

Generally, as carbon sources, glucose, fructose, maltose, sucrose, mannitol, glycerol, dextrin, oats, rye,
Corn starch, potato, corn flour,
Soybean powder, cottonseed oil, molasses, citric acid, tartaric acid, etc. can be used alone or in combination. Generally, the amount is variable at 1-10% by weight of the medium amount.

As the nitrogen source, a substance containing a protein is generally used in the fermentation process. Suitable nitrogen sources include soybean flour, bran, peanut flour, cottonseed oil, 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 sources are used alone or in combination in the range of 0.2-6% by weight of the medium amount.

The nutrient inorganic salts to be incorporated in the medium are usual salts capable of obtaining ions such as sodium, ammonium, calcium, phosphate, sulfate, chloride and carbonate. It also contains trace amounts of metals such as potassium, calcium, cobalt, manganese, iron and magnesium.

In liquid culture, defoaming agents such as silicone oil, vegetable oil, and surfactant are used.

Streptomyces nobilis
ces nobilis ) SANK 60192 strain and the pH of the medium for producing A-72363 can be changed to 5.0-8.0.

The growth temperature of the bacterium is 12 ° C. to 35 ° C., but the range of 23 ° C. to 30 ° C. is preferable.

A-72363 is produced in this temperature range, with 26 to 30 ° C being preferred.

A-72363 can be obtained by aerobically culturing, and a commonly used aerobic culturing method, for example, a solid culturing method, a shaking culturing method, an aeration and stirring culturing method or the like is used.

For small-scale culture, it is preferable to carry out shaking culture at 28 ° C. for several days. The cultivation is started in a Erlenmeyer flask equipped with a baffle (water flow control wall) by 1-2 stages of seed development process. The medium at the seed development stage is
A carbon source and a nitrogen source can be used together. The seed flask is shaken in a constant temperature incubator at 28 ° C. for 5 days, or until it grows sufficiently. The grown seed is used to inoculate a second seed medium, or production medium. If an intermediate development step is used, it is grown in essentially the same way and partially used to inoculate the production medium. The inoculated flask is shaken at constant temperature for several days, and after the incubation is complete, the contents of the flask are centrifuged or filtered.

In the case of large-scale culture, it is preferable to culture in a suitable jar fermenter or tank equipped with a stirrer and an aerator. According to this method, the nutrient medium can be prepared in a jar fermenter or a tank.
The nutrient medium is sterilized by heating to 125 ° C., after cooling, the sterile medium is inoculated with the pre-grown seeds. The culture is performed by aeration and stirring at 28 ° C. This method is suitable for obtaining large amounts of compounds.

A-723 produced with the progress of culture
The change with time of the amount of 63 can be measured by the inhibitory activity against heparanase. Usually, the maximum production of A-72363 is reached after 72 to 120 hours of culture.

After the completion of the culture, A-72363 present in the liquid component in the culture medium was separated from the bacterial cells and other solid parts by a filtration operation using diatomaceous earth as a filter aid or centrifugation, and the filtrate or supernatant thereof. A-72363 present in
Is obtained by utilizing its physicochemical properties to extract and purify. A-72363 is an unstable compound, and a good purification yield can be obtained by carrying out purification under low temperature conditions, preferably at 4 ° C. For example,
Extraction of A-72363 contained in the filtrate or supernatant after decolorization with activated carbon under basic conditions, adsorption on a strongly acidic cation exchange resin under acidic conditions, and elution with 1 to 3 normal ammonia water It is obtained by performing a purification operation.

A-72363 thus obtained
Further, it can be further purified under a low temperature condition by strong basic and weak basic anion exchange resins, high performance liquid chromatography using a cation or anion exchange type column, strong acidic cation exchange resin and the like.

A-72363 can be obtained by repeating the above-mentioned separation and purification means alone or in appropriate combination.
Can be separated and purified.

[0053]

The A-72363 of the present invention is a novel compound which has not been published in the literature, and is used in animals (eg, humans, dogs, cats, rabbits, etc.).
In, it shows inhibitory activity against heparanase and β-glucuronidase, and is useful as a cancer metastasis suppressor or anticancer agent.

[0054] Peparanaze activity is ³ H labeled heparan sulfate, but is measured by radioactivity of the degradation product, the Nakajima et al in order to separate the substrate and the reaction products effectively immobilized substrate method (Anal. Biochem., 157, 162 (1986)) is modified and used, and its inhibition can be observed by adding A-72363 to this reaction solution.

The β-glucuronidase activity can be determined by measuring the activity of degrading the substrate phenolphthalein-glucuronic acid and releasing phenolphthalein at UV550 nm. In addition, inhibition of the enzyme activity can be observed by adding an inhibitor to the reaction solution.

When A-72363 of the present invention is used as a medicine, it is mixed with itself or an appropriate pharmaceutically acceptable carrier, excipient or diluent according to a conventional method, and powder, granules,
It can be safely administered orally or parenterally in the form of tablets, capsules, injections and the like. Although the dose varies depending on the disease to be treated, the route of administration, the number of administrations, etc., for example, for adults, it is preferable to administer 10 mg to 2000 mg daily in one or several divided doses depending on the symptoms.

[0057]

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

Example 1. A-72363 A) Culture Streptomyces nobilis
SANK 60192 strain was aseptically inoculated into a 2-liter Erlenmeyer flask with baffle containing 500 ml of medium having the following composition sterilized, one platinum loop was inoculated, and at 28 ° C, a rotary shaking culture machine at 200 rpm (rotation radius of 7 cm). The cells were cultured for 5 days.

15 liters of the same medium as in the seed culture was placed in each of two 30 liter stainless steel jar fermenters, and this was sterilized by heating at 120 ° C. for 30 minutes. Then, add 450 ml of the above-mentioned seed culture to this, 2
Agitating speed of 100-300 rpm to maintain the dissolved oxygen concentration at 5 ppm at an air flow rate of 15 l / min for 5 days at 8 ° C.
The content was automatically controlled within the range of, and the culture was performed with stirring. The medium composition is shown in Table 4.

[0060]

[Table 4]

B) Isolation 6 liters of pure water was added to 30 liters of the obtained culture solution, and Celite 545 (Jones Manville, USA) as a filter aid.
1.8 kg (manufactured by Project Corporation) was added and filtered to obtain 30 liters of filtrate. The filtrate was adjusted to pH 9 with 6 N sodium hydroxide, and then 3 liters of activated carbon (purified Shirasagi, Takeda Pharmaceutical Co., Ltd.) was added and stirred for 1 hour. By adding 1.5 liters of Celite 545, filtering and washing with water, 32 liters of a filtrate was obtained. The pH of this filtrate was adjusted to 2 with 6N hydrochloric acid and adjusted to 1 at 4 ° C.
After standing for a period of time, the resulting precipitate was removed by continuous centrifugation. This separated liquid was applied to a 10-liter column of strongly acidic cation exchange resin Diaion PK216 (H ⁺ ) (manufactured by Mitsubishi Kasei Co., Ltd.), and this column was washed with 30 liters of pure water. The active substance adsorbed on the column was eluted with 60 liters of 1N ammonia water, and the solution was concentrated under reduced pressure.
Lyophilization gave 43.5 g of brown crude powder.

43.1 g of the obtained brown crude powder was dissolved in 600 ml of 1 mM phosphate buffer (pH 6.5) to prepare a phosphate type in advance.
Weakly basic anion exchange cellulose DE53 (manufactured by Whatman) column 2.7 equilibrated with mM phosphate buffer (pH 6.5)
The mixture was poured into liters, developed using the same buffer, and the active fractions were collected, concentrated and freeze-dried to obtain 394 mg of a pale yellow powder.

394 mg of the obtained pale yellow powder was dissolved in 4 ml of 1 mM phosphate buffer solution (pH 6.5), and the solution was prepared in advance in phosphate form to 1 mM.
Weakly basic anion exchange cellulose DE52 (manufactured by Whatman) column 160 equilibrated with phosphate buffer (pH 6.5)
The mixture was applied to ml and developed using the same buffer, and the active fractions were collected, concentrated and freeze-dried to obtain 55 mg of a pale yellow powder.

The obtained powder was further fractionated by high performance liquid chromatography. Fractionation was performed under the following fractionation conditions, and peaks eluted during 18 to 24 minutes were collected.

Column: Asahi Pack ES502NP (21.
5 φ × 100 mm, manufactured by Asahi Kasei Co., Ltd.) Developing solution: 1 mM Phosphate buffer solution (pH 6.5) Flow rate: 5 ml / min Detection wavelength: 210 nm The solution finally obtained is a strongly acidic cation exchange resin A.
G50WX8 (H ⁺ ) (Bio-Rad) column 4 ml
After washing the column with 20 ml of distilled water, the adsorbed active substance was eluted with 60 ml of 1N aqueous ammonia.
By freeze-drying this eluate, A-72363
White powder of 5.4 mg was obtained as a pure product.

[0066]

【The invention's effect】

Test Example 1. Heparanase inhibitory activity The heparanase inhibitory activity was determined by the method of Nakajima et al. (Anal.
hem., 157, 162 (1986)). That is, heparanase was prepared from a cell extract of mouse melanoma cells. In addition, heparan sulfate (Seikagaku Corporation)
Was prepared by labeling with ³ H-acetic anhydride, fixed on appropriate beads and used as a substrate. The appropriate beads here refer to various carriers that are usually used in affinity chromatography.

This enzyme solution, substrate solution and A-72363
Was added to 20 mM D-saccharic acid-1,4-lactone (manufactured by Sigma) and 0.1 M sodium acetate buffer (p
H5.0) was added to the mixture, the final volume was 400 μl, and the mixture was reacted at 37 ° C. for 3 hours. Trichloroacetic acid to a final concentration of 5
% To stop the reaction, 4 ℃, 12,000 rpm
It was centrifuged for 5 minutes. Radioactivity was measured by adding 5 ml of Picoflow (manufactured by Packard) to a fixed amount of this supernatant.

As a result, the IC ₅₀ of heparanase inhibition of A-72363 was 2.7 μg / ml. Cyastatin B, on the other hand, showed no inhibition at concentrations up to 50 μg / ml.

Test Example 2 Sialidase Inhibitory Activity The sialidase inhibitory activity was determined by the method of Umezawa et al. (J. Antibioti
cs, 27, 963 (1974)). That is, sialidase (manufactured by Sigma) obtained from Clostridium perfrendin was added to 450 μl of 50 mM phosphate buffer (pH 6.0) containing 0.32 mM of substrate N-acetylneuramine lactose (manufactured by Sigma) and A-72363. Add 50 μl of the solution containing the mixture and incubate at 37 ℃ for 30 minutes, then add 0.02
The reaction was stopped by adding 250 µl of 5 M sodium periodate and 0.125 N sulfuric acid.

After completion of the reaction, N-acetylneuraminic acid liberated was reacted with the thiobarbitur reagent method (J. Biol. Chem., Volume 234, 1).
Page 971 (1959)).

As a result, the IC ₅₀ of cystatin B was 3.6 μ.
Whereas g-ml, A-72363 did not inhibit sialidase at all at concentrations up to 50 μg / ml.

Test Example 3 β-Glucuronidase Inhibitory Activity β-Glucuronidase inhibitory activity was determined by the method of Umezawa et al.
(J. Antibiotics, 27, 963 (1974)). That is, to 180 μl of 0.1 M sodium acetate buffer (pH 5.0) containing 0.5 mM substrate phenolphthalein-glucuronic acid and A-72363, 20 μl of a solution containing bovine liver β-glucuronidase (manufactured by Sigma) was added, and 37
After reacting at 0 ° C for 1 hour, 0.5 ml of 0.5 M glycine-KOH buffer (pH 10.5) was added and the mixture was centrifuged at 3000 rpm for 5 minutes. After centrifugation, the absorbance of the supernatant at 550 nm was measured to quantify the released phenolphthalein.

As a result, A-72363 and cystatin B
The IC ₅₀ of each was 0.15 μg / ml and 48.0 μg / ml, respectively.

From the above, the novel compound A-723 of the present invention
63 is useful as a cancer metastasis inhibitor or an anticancer agent.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yoshinori Ogawa, 389-4 Oigata, Shimokawa, Izumi-cho, Iwaki, Fukushima Prefecture Sankyo Co., Ltd. (72) Ryuzo Enokida 33 Miyukigaoka, Tsukuba City, Ibaraki Sankyo Co., Ltd. (72) Inventor Naoo Okazaki 33 Miyukigaoka, Tsukuba, Ibaraki Sankyo Co., Ltd. (72) Takeshi Kinoshita 1-25-2 Hiromachi, Shinagawa-ku, Tokyo Sankyo Co., Ltd.

Claims (4)

[Claims] 1. A-723 having the following structural formula (I):
63 or salt thereof. [Chemical 1] 2. A-723 having the following physicochemical properties:
63 or salt thereof: 1) Property of substance: amphoteric water-soluble white powder 2) Molecular formula: C ₈ H ₁₄ N ₂ O ₅ 3) Molecular weight: 218 (measured by FAB mass spectrum method) 4) High resolution mass spectrometry: glycerin- The precise mass [MH] ⁻ in the high resolution negative ion FAB mass spectrum measured using water as a matrix is as shown below. Measured value 217.0804 Calculated value: 217.0824 5) Elemental analysis (%): Measured value as C ₈ H ₁₄ N ₂ O ₅ .H ₂ O C 41.07, H 6.43, N 12.28 Calculated value: C 40.67, H 6.83, N 11.86 6 ) Specific rotation: [α] _D ²⁷ −59 ° (c0.66, water) 7) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in water shows a terminal absorption at 220 nm or less. 8) Infrared absorption spectrum: λ _max cm ^-1 potassium bromide (KBr) The infrared absorption spectrum measured by the tablet method is as shown below. 3351,1673,1590,1525,
1447,1394,1372,1300,1283,1267,1188,1139,1115,1085,
1035,1008,965,889,850,833,750,707. 9) ¹ H-nuclear magnetic resonance spectrum: (δ: ppm) 1 normal deuterated ammonia water, water ( H DO = 4.75 pp) as internal standard
¹ H-nuclear magnetic resonance spectrum (360 m)
MHz) is as shown below. 2.05 (s), 2.55 (dt, J = 4.4,
9.9Hz), 2.76 (dd, J = 4.4,13.0Hz), 3.06 (dd, J = 9.9,13.0H
z), 3.79 (t, J = 4.1Hz), 4.27 (br.t), 4.80 (d, J = 4.1Hz). 10) ¹³ C-nuclear magnetic resonance spectrum: (δ: ppm) ¹³ C-nuclear magnetic resonance spectrum (90 MHz) measured using 1,4-dioxane (67.4 ppm) as an internal standard in 1N deuterated ammonia water is , As shown below. 23.2 (q), 38.0
(t), 48.3 (d), 62.9 (d), 68.9 (d), 70.6 (d), 175.2 (s), 180.6
(s). 11) Solubility: Soluble in water. Acetic acid, pyridine, methanol,
Insoluble or insoluble in organic solvents such as ethanol, acetone, chloroform, ethyl acetate, benzene, ether, hexane, and petroleum ether. 12) Color reaction: Positive for ninhydrin reagent. 13) High Performance Liquid Chromatography: Column; Asahi Pack ES502N (7.6 φ x 100 mm, manufactured by Asahi Kasei Co., Ltd.) Solvent: 1 mM phosphate buffer (pH 6.5) Flow rate: 1.0 ml / min Detection wavelength; 210 nm retention time; 11.5 minutes. 3. A-72 belonging to the genus Streptomyces
A 363-producing bacterium was cultivated, and from the culture,
The method for producing A-72363, which comprises: 4. The A-72363-producing bacterium belonging to the genus Streptomyces according to claim 3, which is Streptomyces nobilis SANK 60192 (Microtechnical Research Institute No. 4000: BP-4000). Manufacturing method of -72363.