JPH06247900A - New atpase inhibitor - Google Patents

Abstract

PURPOSE:To obtain the subject new enzyme inhibitor having inhibitory action on stomach-type H<+>/K<+> ATPase and useful as an agent for the treatment of peptic ulcer, etc., by culturing a microbial strain belonging to the genus Fusarium and capable of producing aquastatin A and collecting the product from the cultured material. CONSTITUTION:A microbial strain belonging to the genus Fusarium and capable of producing a new compound aquastatin A expressed by formula [e.g. Fusarium aquaeductuum SANK 11089 (FERM BP-4203)] is inoculated to a medium and cultured by shaking culture with a rotary shaker at 26 deg.C for 3 days. The obtained seed culture liquid is inoculated into a medium and subjected to main culture at 26 deg.C for 7 days. The culture liquid is separated into filtrate and microbial cells by celite filtration, the filtrate is adjusted to pH 3 with hydrochloric acid and extracted with ethyl acetate, the extract is collected and concentrated and the concentrate is purified by high-performance liquid chromatography to obtain the new ATPase inhibitor expressed by the formula, having inhibitory action on stomach-type H<+>/K<+> ATPase and useful as an agent for the treatment of peptic ulcer, reagent for research, etc.

Description

Detailed Description of the Invention

[0001]

The present invention relates to H ⁺ / K present in the stomach
⁺ Novel compounds aquastatin A and aquastatin B having ATPase inhibitory activity, salts thereof, and a method for producing these compounds.

[0002]

2. Description of the Related Art Gastric H ⁺ / K ⁺ ATPase is localized in the parietal cells of the gastric mucosa and plays an important role in regulating the proton pump involved in the final step of the gastric acid secretory mechanism.
It is possible to suppress gastric acid secretion by lowering this enzyme activity, and therefore gastric type H ⁺ / K ⁺ A
The inhibitor against TPase has a gastric acid secretion inhibitory action,
It is useful as a therapeutic drug for peptic ulcer.

Conventionally, as inhibitors for gastric type H ⁺ / K ⁺ ATPase, scopadalthic acid and diacetylscopadol (J. Biolo. Chem. 265 , 22167-22173,
(1990)) and the like are known, and these are obtained from Paraguay crude drugs.

Omeprazole (J. Biolo. Chem. 260 , 13263-13268, (198
7)), dicoprazole (Nature 290 , 159-161, (1980)
), E-3810 (Biochem. Pharmacol. 29 , 661-66).
7, (1990)), SCH28080 (J. Pharmacol. The
r. 226 , 114-120, (1983)) and the like are known and used as therapeutic agents for peptic ulcer.

[0005]

DISCLOSURE OF THE INVENTION The present inventors have proposed a novel compound that strongly inhibits gastric H ⁺ / K ⁺ ATPase in a culture of a microorganism belonging to the genus Fusarium, which is newly separated from sap. The production of aquastatin A and further the cleavage reaction using β-galactosidase with aquastatin A as a substrate results in gastric H ⁺ / K
^The present invention was completed by discovering that a new compound, aquastatin B, which strongly inhibits ATPase is produced.

[0006]

The present invention provides the following formula (I): (1)

[0007]

[Chemical 6]

The novel compound aquastatin A or a salt thereof represented by: (2) The following formula (II):

[0009]

[Chemical 7]

The novel compound aquastatin B represented by: or a salt thereof, (3) the following formula (I) belonging to the genus Fusarium:

[0011]

[Chemical 8]

[0012] A method for producing aquastatin A, which comprises culturing an aquastatin A-producing bacterium represented by: and collecting aquastatin A from the culture, (4) The aquastatin A-producing bacterium is Fusarium aqua Aidudoumu SANK 11089 (Ministry of Mechanical Engineering, Article 4203)
(3) The production method according to (3), wherein (5) the following formula (I):

[0013]

[Chemical 9]

The following formula (II) is characterized in that β-galactosidase is allowed to act on aquastatin A represented by:

[0015]

[Chemical 10]

The present invention relates to a process for producing aquastatin B or a salt thereof.

The aquastatin A-producing strain SANK11089 isolated by the present inventors was obtained from sap of Karuizawa, Nagano Prefecture in July 1987, and its mycological properties are as follows. ..

Growth on PDA is 25 cm, 2.0 cm in 10 days
Reach The hyphae aggregate closely and form flat colonies. Almost no aerial hyphae are formed, but several filaments are bundled, and it is slightly observed that hyphae bundles are formed. The color is light orange, and the color is dark in the center. The back surface is pale orange.

Conidia are actively produced on the LCA medium to form conidia. Conidia are formed mainly from dentate holes formed in hyphae, and differentiated conidia-forming cells are rarely observed. Conidia-forming cells are thin, elongated, cuticle-shaped cells that are singly or in large numbers. The conidia formation mode is a monoblastic fiaro type. Conidia are gently curved, crescent-shaped to needle-like, and are 40 in size.
-58 x 3-4 μm. The cell wall is thin and the septum is obscure. It has a base with an ambiguous foot shape and a sharp tip.

On LCA medium, the morphology of conidia is PDA
Shows larger mutations than above. That is, single cells (rarely 2%) from newborn conidia similar to those formed on PDA.
Cellular, morphological changes leading to small conidia (4-31 X 1-2 μm) that are string-shaped and strongly curved (C-shaped) are observed. Full age was not observed on any of the media.

As a result of comparing the above-mentioned various traits with those of known strains, the various properties of this bacterium are roughly "Booth, C .: The gen".
us Fusarium. Commonwealth Mycological Institue.
Kew, Surrey, England. Pp.237 ( 1971) "described F. Aqu
Matched the aeductuum var. medium. But F. aquaedu
Fusarium aquaeduc containing ctuum var. medium
There are various theories about tuum species and sub- species classification systems, and the classification is not established. Therefore,
Suspending identification of subspecies , Fusarium aquaeductuum
It was identified as Lagerh. 4203) was added.

The novel compound aquastatin A of the present invention is, for example, a novel compound aquastatin A belonging to the genus Fusarium.
It is produced by culturing a producing bacterium and collecting aquastatin A from the culture.

The novel compound aquastatin A-producing bacterium used in the present invention is the above-mentioned Fusarium aquaeductuum.
SANK 11089 can be mentioned, but strains that belong to the genus Fusarium and have the ability to produce the novel compound aquastatin A are liable to change in their properties as seen in the case of strains of other general microorganisms, for example, ultraviolet rays, It can be easily mutated by artificial mutagenesis using high frequency, radiation, chemical mutagen, etc., and even with such a mutant, all strains having the desired activity can be used in the method of the present invention. it can. That is, in the present invention, all strains that produce aquastatin A and are not clearly distinguished from the SANK 11089 strain and its mutant strain are included in the SANK 11089 strain.

In the method of the present invention, the culturing can be carried out under the conditions generally used for culturing fungi. The culture medium containing nutrients that can be used by the microorganisms can be any known culture medium used for general microorganism culture, and is not limited to the medium described in the examples. Amount of 1 to 10% by weight), nitrogen source (usually
Either a natural medium or a synthetic medium can be used as long as it is a medium appropriately containing 0.2 to 6% by weight of the amount of the medium), inorganic substances and the like.

The carbon source is not particularly limited as long as it can be utilized by the bacterium as a carbon source. For example, glucose, fructose, maltose, lactose, sucrose, starch, mannitol, dextrin, glycerin, starch syrup, molasses. , Molasses,
Carbohydrates such as oat flour, rye flour, corn starch, potato, corn flour, soy flour, malt extract; oils and fats such as soybean oil, cottonseed oil, olive oil, cod liver oil, lard; citric acid, sodium ascorbate, malic acid , Organic acids such as acetic acid, fumaric acid, tartaric acid, succinic acid, gluconic acid; methanol, ethanol, n-propanol, isopropanol, n-butanol
Alcohols such as alcohol, isobutanol and t-butanol; and amino acids such as glutamic acid can be used alone or in combination.

The nitrogen source is not particularly limited as long as it can be utilized by the fungus as a nitrogen source. For example, bran, peanut flour, casein hydrolyzate, fish meal, malt extract, soybean meal, soybean casein, Organic nitrogen compounds such as casamino acids, pharma media, cottonseed flour, wheat germ, meat extract, peptone, corn steep liquor, self-digesting yeast, dry yeast, yeast extract, urea; aspartic acid,
Amino acids such as glutamine, cystine and alanine; inorganic nitrogen compounds such as ammonium salts such as ammonium sulfate, ammonium nitrate, ammonium chloride and ammonium phosphate, nitrates such as sodium nitrate and potassium nitrate, can be used alone or in combination. .

The inorganic salts taken into the medium are ordinary salts capable of obtaining ions such as sodium, potassium, magnesium, ammonium, calcium, phosphate, sulfate, chloride and carbonate, and molybdenum, boron, copper and cobalt. It also contains trace amounts of metals such as manganese and iron. Specifically, for example, sodium chloride, manganese chloride, cobalt chloride, potassium chloride, calcium chloride, calcium carbonate, potassium aluminum sulfate, manganese sulfate, copper sulfate, cobalt sulfate, sulfuric acid. Examples thereof include zinc, ferrous sulfate, magnesium sulfate, monopotassium phosphate, dipotassium phosphate, disodium phosphate, and ammonium molybdate, which can be used in appropriate combination.

When a sulfur compound that can be assimilated by bacteria is added to the medium, the amount of the desired product produced may increase. For example, zinc sulfate, copper sulfate, ferrous sulfate, sulfates such as ammonium sulfate, thiosulfates such as ammonium thiosulfate, inorganic sulfur compounds such as sulfites such as ammonium sulfite; cystine, cystine, L-thiazolidine-
Sulfur-containing amino acids such as 4-carboxylic acid, hypotaurine,
Organic sulfur compounds such as sulfur-containing peptides such as glutathione Heavy metals such as ferrous sulfate and copper sulfate, vitamin B
1. Vitamins such as biotin, bacterial growth promoting substances such as thiamine, etc. are also added as necessary.

Further, a defoaming agent such as silicone oil, polyalkylene glycol ether, vegetable oil, animal oil, or surfactant may be added to the medium (especially suitable for liquid culture). Experimental examples are specifically described in Examples as typical examples.

As a culture method suitable for growing the producing bacterium,
There is no particular limitation, as long as it is a culture method generally used for microorganisms, for example, a solid culture method, a stationary culture method, a stirring culture method, a shaking culture method, an aeration culture method can be used, but preferably, Agitation culture method, which is an aerobic liquid culture method,
Shaking culture method or aeration culture method, more preferably,
It is a shaking culture method. Incidentally, the aeration stirring culture method is industrially suitable.

The pH of the medium is usually 5.0 to 8.0, preferably 6.0.

The culture temperature suitable for the growth of the bacterium is usually 15
C. to 30.degree. C., preferably 26.degree.

The production of aquastatin A usually reaches its maximum in 4-7 days of shaking culture.

Since the product after the culture is present in the culture filtrate and the microbial cells, the microbial cells and other solid parts in the culture are
The supernatant can be separated into the bacterial cells by centrifugation or a filtering operation using diatomaceous earth as a filter aid. Aquastatin A present in the filtrate was converted to gastric type H ⁺ / K ⁺ ATPas.
By measuring the e-inhibitory activity, it can be extracted and purified by utilizing its physicochemical properties.

The enzyme inhibitory activity can be measured by the following method.

From pig gastric mucosa, the method of Im et al. (Biochim. B
iophys. Acta., 692 , 355-360, (1982)) was partially modified to purify H ⁺ / K ⁺ ATPase.
That is, after crushing the porcine gastric mucosa obtained from the slaughterhouse, the buffer solution (0.25 M sucrose, 2 mM MgCl ₂ , 2 mM HEPES, 1 mM
EGTA, (pH 7.4)) and homogenize to 100,000 × g
It was centrifuged at to obtain a membrane fraction. Furthermore, H ⁺ / K ⁺ ATPas was obtained by sucrose density gradient centrifugation (75,000 × g).
Collect a fraction rich in e and repeat this fraction 100,000 × g
It was centrifuged overnight at 1, and this was used as an enzyme sample for assay.

The activity of H ⁺ / K ⁺ ATPase is Naga
Ya et al. (J. Pharmacol. Exp.Ther. 248 , 799-80
5, (1989)).

Since H ⁺ / K ⁺ ATPase is activated by KCl, the difference in enzyme activity in the presence and absence of KCl was defined as H ⁺ / K ⁺ ATPase activity. Furthermore, the enzyme activity was used as an index of activity by colorimetrically quantifying the inorganic phosphate purified when ATP was decomposed by H ⁺ / K ⁺ ATPase.

Aquastatin A or Aquastatin B
Is dissolved in dimethyl sulfoxide, added to a buffer solution (5 mM MgCl ₂ , 70 mM Tris-HCl, (pH 6.8)) containing 10 mM KCl together with the enzyme preparation (20 to 40 μg protein), and pretreated for 60 minutes. After that, 2 mM ATP was added and reacted at 37 ° C for 20 minutes. The reaction was stopped by adding trichloroacetic acid containing 100 mg activated carbon, centrifuged and the supernatant was collected.
The amount of inorganic phosphoric acid was measured by the method of J. Biol. Chem. 66 , 375-400, (1925). That is, 0.15 M sodium acetate was added to the supernatant after the reaction was stopped, 2% ascorbic acid and 2% ammonium molybdate were added to develop color, and the absorbance was measured at a wavelength of 700 nm. As a control group, dimethyl sulfoxide was used instead of aquastatin A or aquastatin B. Also, the absorbance in the presence of KCl was separately determined as a blank. The enzyme activity inhibition rate (R) was calculated from the following equation.

R = (1-B / A) × 100 A: Absorbance of control group = (Absorbance in the presence of KCl-Absorbance of blank) B: Absorbance of the test substance treated group = (Absorbance in the presence of KCl -Absorbance of blank) After the filtrate is acidified, the novel compound aquastatin A is obtained as a crude product by extracting with an organic solvent immiscible with water, such as ethyl acetate, butyl acetate, butanol and concentrating the extract. can get. In addition, from the bacterial cells, an organic solvent miscible with water (for example, alcohols such as methanol and ethanol; acetone, etc.) so that the final concentration of the bacterial cells is 50 to 90%. Ketones; nitriles such as acetonitrile and isobutyronitrile;
Formamide, N, N-dimethylformamide, N, N
-Dimethylacetamide, N-methyl-2-pyrrolidone, N-methylpyrrolidinone, amides such as hexamethylphosphorotriamide), and extract from the cells,
After removing the solvent, the same treatment as the supernatant can be performed to obtain the novel compound aquastatin A in the same manner as the filtrate.

As a method for further purification, a method usually used for separation and purification of organic compounds, for example, Sephadex LH-20 (Pharmacia), Amberlite XAD-11 (Rohm and Haas). ),
A method using a synthetic adsorbent such as partition column chromatography using a carrier such as Diaion HP-20 (manufactured by Mitsubishi Kasei), or a normal / reverse phase column chromatography method using silica gel or alkylated silica gel (preferred) Is a high-performance liquid chromatography), and is separated by eluting with an appropriate eluent,
It can be purified. It can also be purified by an adsorption column chromatography method using a carrier such as silica gel, alumina, or magnesium-silica gel florisil.

The solvent used is preferably an aliphatic hydrocarbon such as hexane, heptane, ligroin or petroleum ether; an aromatic hydrocarbon such as benzene, toluene or xylene; methylene chloride, chloroform, Halogenated hydrocarbons such as carbon tetrachloride, dichloroethane, chlorobenzene and dichlorobenzene; esters such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate and diethyl carbonate; diethyl ether, diisopropyl ether, tetrahydrofuran, Dioxane,
Examples thereof include ethers such as dimethoxyethane and diethylene glycol dimethyl ether.

The elution of the target substance can be checked by HPLC or by measuring the enzyme inhibitory activity using H ⁺ / K ⁺ ATPase derived from pig stomach.

Aquastatin B can be obtained by causing β-galactosidase to act on aquastatin A or a salt thereof to enzymatically cleave the galactoside bond. The enzyme may be any of animals, plants and microorganisms,
Usually, commercially available β-galactosidase is used.

In the cleavage reaction, the pH is preferably in the range of 5-9, preferably 7-8.

As the reaction buffer, any buffer can be used as long as it is used in the above pH range, but a phosphate buffer, an acetate buffer, a Tris-HCl buffer, an imidazole buffer and the like can be used. Phosphate buffer is preferred and preferred.

The enzyme concentration in the reaction is 0.01 to 20 u /
It is desirable to be in the range of ml. The reaction temperature is
It is preferably in the range of 25 to 35 ° C, and preferably 30 ° C.
Is. The product can be isolated and purified by a method similar to that of aquastatin A.

In order to form the salt of the novel compound aquastatin A, it can be converted into a salt by a conventional method at the stage of the crude product, but it is preferably converted into a salt after isolation. Preferred salts include alkali metal salts such as sodium and potassium, alkaline earth metal salts such as calcium and magnesium, aluminum salts, other metal salts, ammonium salts, and known salts with amines.

The physicochemical properties of the substance obtained as described above are as follows.

[Aquastatin A] 1) Property of substance: acidic fat-soluble white powder 2) Molecular weight (determined by FAB-MASS method): 676 3) Molecular formula: C ₃₆ H ₅₂ O ₁₂ 4) Ultraviolet absorption spectrum: (Ethanol Medium λ max nm
(ε)) 213 (52933), 265 (19033), 307 (10733) 5) Infrared absorption spectrum: (νmax cm in KBr tablets
-1) 2923,2851,1665,1613,1247,1142,1070,711 6) ¹ H-nuclear magnetic resonance spectrum: measured in a mixed solvent of deuterated chloroform and deuterated methanol with tetramethylsilane as an internal standard. The δ value of is as follows.

6.56 (d), 6.53 (d), 0.88 (t), 1.3 (m), 1.64
(m), 2.93 (m), 6.68 (d), 6.59 (d), 2.63 (s), 4.97 (d), 3.85
(dd), 3.63 (dd), 3.99 (dd), 3.72 (dt), 3.81 (dd), 3.85 (dd) 7) ¹³ C-nuclear magnetic resonance spectrum: in a mixed solvent of deuterated chloroform and deuterated methanol. The δ value when measured with methylsilane as an internal standard is as follows.

60.6 (t), 75.0 (d), 68.2 (d), 73.0 (d), 70.4
(d), 99.9 (d), 163.9 (s), 111.6 (d), 147.8 (s), 105.9 (d), 16
4.0 (s), 101.3 (d), 36.4 (t), 31.8 (t), 13.4 (q), 168.8 (s), 1
53.3 (s), 115.7 (s), 143.8 (s), 110.7 (s), 164.1 (s), 107.8
(d), 172.9 (s), 23.3 (q) 8) High performance liquid chromatography: Column: Waters RADIAL-PAK CARTRIDGE 8NVC
184 Mobile phase: Acetonitrile / triethylamine-phosphate buffer pH3.2 (60:40) Flow rate: 2.0 ml / min Detection: UV 210 nm Retention time: 6.8 minutes [Aquastatin B] 1) Properties of substance : Acidic fat-soluble white powder 2) Molecular weight (determined by FAB-MASS method): 514 3) Molecular formula: C ₃₀ H ₄₂ O ₇ 4) Ultraviolet absorption spectrum: (λmax n in ethanol)
m) 213, 265, 307 5) Infrared absorption spectrum: (νmax cm in KBr tablets)
-1) 2921, 2850, 1652, 1612, 1458, 1313, 1244, 1141, 80
26) ¹ H-nuclear magnetic resonance spectrum: δ value when measured with tetramethylsilane as an internal standard in a mixed solvent of deuterated chloroform and deuterated methanol is as follows.

6.66 (d), 6.57 (d), 6.33 (d), 6.29 (d), 2.93
(m), 2.63 (s), 1.63 (m), 1.26 (m), 0.92 (t) 7) ¹³ C-nuclear magnetic resonance spectrum: tetramethylsilane as an internal standard in a mixed solvent of deuterated chloroform and deuterated methanol. The δ value measured in the following manner is as follows.

173.7, 170.0, 166.0, 164.7, 163.5, 15
4.0, 149.3, 144.5, 116.5, 112.2, 111.3, 108.6, 10
3.3, 101.4, 37.4, 29.9, 22.9, 14.2 8) High performance liquid chromatography column: Waters RADIAL-PAK CARTRIDGE 8NVC
184 Mobile phase: Acetonitrile / triethylamine-phosphate buffer pH 3.2 (80:20) Flow rate: 2.0 ml / min Detection: UV 210 nm Retention time: 7.5 minutes Digestion of aquastatin A and aquastatin B of the present invention When used as a therapeutic drug for sex ulcer, it is administered in various forms. Examples of the dosage form include tablets, capsules,
Oral administration by powders, granules, syrups, etc. or injections (intravenous, intramuscular, subcutaneous) and the like can be mentioned. These various preparations are based on the conventional method, the main ingredient is an excipient,
It can be formulated using known auxiliary agents that can be usually used in the field of known pharmaceutical preparations such as binders, disintegrants, lubricants, solubilizers, flavoring agents and coating agents. The amount used will vary depending on symptoms, age, weight, administration method, dosage form, etc., but is usually 50 mg to 1000 per day for adults.
mg can be administered.

[0055]

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

Example-1 A) Culture A GPMY medium having the following composition was used for both the seed culture and the production medium.

[0057]

[Table 1] Medium composition (GPMY medium) Glycerin 50 g Malto extract 5 g Yeast extract 5 g Potato 50 g ──────────────────── Ion-exchanged water 1000 ml pH was not adjusted. 100 ml of the GPMY medium having the above composition was placed in a 500 ml Erlenmeyer flask with a baffle and sterilized at 120 ° C for 20 minutes. For seed culture, Fusarium aq was added to two Erlenmeyer flasks containing this medium.
uaeductuum SANK 11089 slants were inoculated from the slant one by one platinum loop, and rotary shaker (200rpm, 5cm
The culture was performed with shaking. After that, the seed culture solution was inoculated into 24 flasks containing the above medium, 2 ml each, and the seed culture solution was incubated at 26 ° C for 7 minutes.
Main culture was carried out for one day.

B) Isolation The culture broth was separated by filtration through Celite into cells and cells, and 2 L of the filtrate was adjusted to pH 3 with hydrochloric acid, and then extracted twice with 2 L of ethyl acetate. The upper layers of each were collected and concentrated to give 3.2 g of an oil. This oily substance was converted into H under the preparative conditions shown below.
PLC fractionation was performed.

Column: Merck Art.11447 LiChroprep
RP-8 size B Mobile phase: Acetonitrile / triethylamine-phosphate buffer pH3.2 (65:35) Flow rate: 7.5 ml / min Detection: RI (Differential Refractometer) RI (Differential Refractometer) 10 times oil dissolved in acetonitrile At 50
The peak of aquastatin A having a retention time of 6.3 minutes was collected. After repeating this 6 times, the fractionation liquids for 6.3 minutes were combined, the acetonitrile was distilled off, and the mixture was extracted with ethyl acetate. After concentration and drying, 1.6 g of pure aquastatin A was obtained.

Example-2 30 ml of 30 mg of aquastatin A obtained in Example-1
Dissolved in a mixed solvent of dimethyl sulfoxide and water,
It was added to 40 ml of 1/10 mM phosphate buffer solution having a pH of 7.3 and containing 50 mg of bovine albumin (Sigma). Β-galactosidase (manufactured by E. coli Sigma) was added to this solution at a final concentration of 10 units / ml.

When reacted at 30 ° C. for 7 hours, about 90% of aquastatin A was converted to aquastatin B. The reaction solution was adjusted to pH 3 with 1N hydrochloric acid and extracted with n-butanol. The oily substance obtained after washing with water and concentration was subjected to preparative HPLC.
That is, using an ODS column (manufactured by ODS-5152-S Senshu Kagaku), 90% acetonitrile / pH
3.2 Triethylamine / phosphate buffer (1%) 6m
Flowed at 1 / min. The peak at 32 minutes was measured by a differential refractometer and acetonitrile was concentrated and removed under reduced pressure.
The remaining liquid was extracted with ethyl acetate, the upper layer was washed with saturated saline and then concentrated to dryness to obtain 24 mg of aquastatin B.

Formulation Example-1 Oral Capsule

[0063]

[Table 2] After mixing the powders of the above formulation and passing through a 30 mesh sieve, 350 mg of this powder is placed in a gelatin capsule,
It was used as a capsule.

[0064]

The effects of the present invention will be described with reference to test examples.

Test Example-1 Pig gastric mucosa method of Im et al. (Biochim. Biophys. Act)
a., 692 , 355-360, (1982)) was partially modified to purify H ⁺ / K ⁺ ATPase. That is,
After crushing the pig gastric mucosa obtained from the slaughterhouse, a buffer solution (0.2
5 M sucrose, 2 mM MgCl ₂ , 2 mM HEPES, 1 mM EGTA, (pH
7.4)) was added and homogenized, followed by centrifugation at 100,000 × g to obtain a membrane fraction. Furthermore, sucrose density gradient centrifugation (7
Fractions rich in H ⁺ / K ⁺ ATPase were collected at 5,000 × g), and this fraction was again centrifuged overnight at 100,000 × g to prepare an enzyme sample for assay.

The activity of H ⁺ / K ⁺ ATPase is Naga
Ya et al. (J. Pharmacol. Exp.Ther. 248 , 799-80
5, (1989)).

Since H ⁺ / K ⁺ ATPase is activated by KCl, the difference in enzyme activity in the presence and absence of KCl was defined as H ⁺ / K ⁺ ATPase activity. Furthermore, the enzyme activity was used as an index of activity by colorimetrically quantifying the inorganic phosphate purified when ATP was decomposed by H ⁺ / K ⁺ ATPase.

Aquastatin A or Aquastatin B
Is dissolved in dimethyl sulfoxide, added to a buffer solution (5 mM MgCl ₂ , 70 mM Tris-HCl, (pH 6.8)) containing 10 mM KCl together with the enzyme preparation (20 to 40 μg protein), and pretreated for 60 minutes. After that, 2 mM ATP was added and reacted at 37 ° C for 20 minutes. The reaction was stopped by adding trichloroacetic acid containing 100 mg activated carbon, centrifuged and the supernatant was collected.
The amount of inorganic phosphoric acid was measured by the method of J. Biol. Chem. 66 , 375-400, (1925). That is, 0.15 M sodium acetate was added to the supernatant after the reaction was stopped, 2% ascorbic acid and 2% ammonium molybdate were added to develop color, and the absorbance was measured at a wavelength of 700 nm. As a control group, dimethyl sulfoxide was used instead of aquastatin A or aquastatin B. Also, the absorbance in the presence of KCl was separately determined as a blank. The enzyme activity inhibition rate (R) was calculated from the following equation.

R = (1−B / A) × 100 A: Absorbance of control group = (Absorbance in the presence of KCl−Absorbance of blank) B: Absorbance of the test substance treated group = (Absorbance in the presence of KCl -Absorbance of blank) Aquastatin A is H ⁺ / K ⁺ ATPa in a concentration-dependent manner.
Se activity was inhibited and the IC ₅₀ value was 6.2 μM.

As described above, the novel compound of the present invention, aquastatin A or aquastatin B, is the gastric H ⁺ / K ⁺
Since it inhibits ATPase, it is useful as a therapeutic drug for peptic ulcer or a research reagent.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location // A61K 31/235 ACL 9283-4C 31/71 AED 8314-4C (C12P 7/62 C12R 1: 77) (C12P 19/44 C12R 1:77) 7804-4B (72) Inventor Keiichi Tabata 1-2-2 Hiromachi, Shinagawa-ku, Tokyo Sankyo Co., Ltd. (72) Inventor Takeshi Kinoshita Shinagawa-ku, Tokyo 1-2-2 Hiromachi Sankyo Stock Company

Claims (5)

[Claims] 1. The following formula (I): The novel compound aquastatin A represented by or a salt thereof. 2. The following formula (II): The novel compound aquastatin B represented by or a salt thereof. 3. The following formula (I) belonging to the genus Fusarium: Characterized in that the aquastatin A-producing bacterium represented by: is cultured, and aquastatin A is collected from the culture.
Production method of aquastatin A. 4. An aquastatin A-producing bacterium is Fusarium aquaductum SANK 11089
No. 03). 5. The following formula (I): Β-galactosidase is allowed to act on aquastatin A represented by the following formula (II): A method for producing aquastatin B or a salt thereof represented by: