JPH08134091A - New compound a-76202 and its production - Google Patents

Abstract

PURPOSE: To obtain the new subject compound having a microsome α- glucosidase-inhibiting activity and a maltase-inhibiting activity and useful as an antitumor agent, an anti AIDS agent, etc., by culturing an A-76202-producing bacterium belonging to the genus Rhodococcus. CONSTITUTION: This new compound A-76202 represented by the formula is white power, soluble in dimethyl sulfoxide, slightly soluble in water, methanol and acetone, has a molecular formula: C20 H18 O9 (measured with a high resolution mass spectrum) and a molecular weight of 402 (measured with an FAB-MS spectrum), and has a remarkable microsome α-glucosidase-inhibiting activity and a maltase activity-inhibiting activity exhibited when using sucrose-isomaltase complex, and is useful as an antitumor agent, an anti-AIDS agent, an antiobesic agent, an antidiabetic agent, etc. The compound is obtained by culturing an A-76202-producing bacterium [e.g. Rhodococcus SP. SANK61694 strain (FERM- BP-4751)] belonging to the genus Rhodococcus and subsequently collecting the product from the culture product.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel compound A-76202 having an endoplasmic reticulum α-glucosidase inhibitory activity and an activity inhibiting a maltase activity of a sucrose-isomaltase complex, and a method for producing the same.

[0002]

BACKGROUND OF THE INVENTION α-Glucosidase is an enzyme widely distributed in the living body.

Of these, α-glucosidases present in the endoplasmic reticulum, that is, α-glucosidases I and II, are processed during the maturation process of secretory glycoproteins, cell surface glycoproteins, and N-linked sugar chains on viral surface glycoproteins. It is a related enzyme. Specifically, it has an activity of cleaving three molecules of glucose existing in the terminal portion of the immature N-linked sugar chain. After that, another enzyme works to mature the sugar chain. When this enzyme is inhibited in virus-infected cells including HIV, the sugar chains of the surface glycoprotein of the virus particles produced remain in an immature state, resulting in suppression of the infectivity of the produced virus. In fact, 1-deoxynojirimycin, castanospermine and its derivatives, known as endoplasmic reticulum α-glucosidase inhibitors,
It has been reported to inhibit HIV transmission and proliferation in vitro (H. Shimizu et al., AIDS, Volume 4, 975-).
979, (1990); BD Walker et al., Proc. Natl.
Acad. Sci. USA, 84, 8120-8124, (1987)
etc).

Further, it has been reported that an endoplasmic reticulum α-glucosidase inhibitor inhibits metastasis and invasion of malignant tumor cells (MJ Humphries et al., Cancer Research, 46.
Volume, 5215-5222, (1986); G. Pulverer et al., J.
Cancer Res. Clin. Oncol., 114, 217-220, (1
988)). Therefore, the compound having the endoplasmic reticulum α-glucosidase inhibitory activity is useful as an anti-AIDS drug or an antitumor drug.

On the other hand, the sucrose-isomaltase complex existing on the brush border membrane of the small intestine is an enzyme that decomposes maltose, sucrose, isomaltose and the like, and produces monosaccharides such as glucose. Similarly, the monosaccharide is transported into the body by the action of the transport protein existing on the brush border membrane of the small intestine, and is transferred into the blood. In this way, the sucrase-isomaltase complex is involved in digestion and absorption of sugars. The sucrose-isomaltase complex has two catalytic sites capable of catalyzing the hydrolysis of sucrose and isomaltose in one molecule, and maltose is used as a substrate at both sites. The behavior of the molecule can be seen by measuring the maltase activity.

Conventionally, AO-128, acarbose and the like have been known as substances showing a strong inhibitory activity against the maltase activity of the sucrase-isomaltase complex, and they are useful for antiobesity and antidiabetes. (Satosi Horii et al., Journal of Medicinal Chemi
stry), 29, 1038-1046, 1986; Shigenori Fuj
ioka et al., International Journal of
Obesity (International Journal of Obesity),
15: 59-65, 1991; Toshihisa Goda et al., "Nutrition and Food", 34, 139-143, 1981).

Therefore, the compound having an activity of inhibiting the maltase activity of the sucrase-isomaltase complex is useful as an antiobesity agent or antidiabetic agent.

[0008]

The present inventors have found that endoplasmic reticulum α-glucosidase inhibitory activity is higher than that of microbial secondary metabolites.
, And a substance having an activity of inhibiting the maltase activity exhibited by the sucrose-isomaltase complex, and as a result, SANK belonging to the genus Rhodococcus isolated from soil
Production of a novel compound A-76202 having an endoplasmic reticulum α-glucosidase inhibitory activity and an activity of inhibiting a maltase activity of a sucrase-isomaltase complex, in a culture solution of a strain 61694 (FERM BP-4751) The present invention has been completed by finding

Still another object of the present invention is A-7620.
Providing an α-glucosidase inhibitor containing 2 or a salt thereof as an active ingredient, particularly an anti-AIDS agent, an antitumor agent, or an inhibitor of maltase activity exhibited by a sucrase-isomaltase complex, particularly an antiobesity agent and an antidiabetic agent To do.

[0010]

The present invention provides (1) a novel compound A-76202 represented by the following formula (I) or a salt thereof:

[0011]

Embedded image

(2) Rhodococcus
(us) a bacterium belonging to the genus A-76202 is cultured, and A-76202 is collected from the culture, (3) Rhodococcus (Rh)
The A-76202 producing bacterium belonging to the genus Odococcus is Rhodococcus sp.
sp. ) SANK61694 strain, the production method according to (2), (4) Rhodococcus sp.
ccus sp. ) SANK61694 strain.

A-76202 of the present invention has the following physicochemical properties. 1) Properties; white powder. 2) Solubility; soluble in dimethyl sulfoxide. Insoluble in water, methanol, and acetone. 3) molecular formula; C ₂₀ H ₁₈ O ₉ (measured by high resolution mass spectrum) 4) molecular weight; 402 (measured by FAB-MS spectrum) 5) ultraviolet absorption spectrum; λ _max nm The ultraviolet absorption spectrum measured in an aqueous solution is: 259n
It has a maximum absorption at m and an absorption coefficient at 259 nm (E
^1% _{1 cm} ) is 687. 6) Infrared absorption spectrum (KBr); The infrared absorption spectrum measured by ν _max cm ^-1 KBr disk is as follows. 3348, 2943, 1627, 160
1, 1574, 1515, 1284, 1212, 107
8, 1036, 969 7) ¹ H-nuclear magnetic resonance spectrum; δ (ppm) Nuclear magnetic resonance spectrum (500 MHz) measured in a heavy dimethyl sulfoxide solution (dimethyl sulfoxide internal standard: 2.49 pm) is as follows. is there. 3.46 (1H, d
d, J = 5.5, 11.8Hz), 3.57 (1H, dd, J = 3.5, 11.8Hz), 3.8
4 (1H, dd, J = 3.8, 6.0Hz), 3.96 (1H, ddd, J = 3.5, 5.5,
6.0Hz), 4.28 (1H, dd, J = 1.0Hz or less, 3.8Hz), 5.60 (1H,
d, J = 1.0Hz or less), 6.81 (2H, d, J = 8.5Hz), 7.23 (1H,
d, J = 8.8Hz), 7.39 (2H, d, J = 8.5Hz), 7.53 (1H, d, J = 8.
8Hz), 8.37 (1H, s) 8) ¹³ C-nuclear magnetic resonance spectrum; δ (ppm) Nuclear magnetic resonance spectrum (125 MHz) measured in a heavy dimethyl sulfoxide solution (dimethyl sulfoxide internal standard: 39.5 ppm) is It is as follows. 61.14, 7
6.66, 81.34, 85.95, 107.16, 114.44, 114.9 (3C), 11
9.58, 122.41, 123.17, 130.05 (2C), 136.15, 146.03, 1
47.58, 153.01, 157.17, 175.15 9) High performance liquid chromatography separation column; Senshupack ODS-H-2151 (φ6 × 15)
0 mm, Senshu Kagaku Co., Ltd. Mobile phase: 16% acetonitrile-water Flow rate: 1.5 ml / min Detection wavelength: 210 nm or 259 nm Temperature: 25 ° C. Holding time: 16.22 minutes.

The compound A-76202 having the above structural formula (I) of the present invention 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; calcium,
Inorganic salts such as alkaline earth metal salts such as barium; magnesium salts; aluminum salts; or amine salts such as ammonia, methylamine, dimethylamine, dicyclohexylamine; basic amino acid salts such as lysine and arginine; Organic base salts and the like can be mentioned. Preferably, it is a pharmacologically acceptable salt.

The compound A-76202 of the present invention is
It has various isomers. In Compound A-76202, all of these isomers and mixtures of these isomers are represented by a single formula. Therefore, in the present invention, all of these isomers and a mixture of these isomers are also included.

The compound A-76202 of the present invention is
When left in the air or recrystallized, water may be absorbed, and adsorbed water may be attached, or a hydrate may be formed. The present invention also includes such hydrates.

Rhodococcus (Rhodococcu) used in the method for producing the compound A-76202 of the present invention.
Examples of strains belonging to the genus s) include Rhodococcus sp. SANK.
The 61694 strain (hereinafter referred to as "SANK 61694 strain") can be mentioned. The SANK 61694 strain was isolated from soil on the eastern flank of Mt. Tsukuba, Tsukuba City, Ibaraki Prefecture by the soil dilution method.

The mycological properties of the SANK 61694 strain are as follows:
It is as follows.

1. Morphological properties SANK 61694 strain is 28 on the agar medium for strain identification.
It grows normally or poorly in culture at 7 ° C for 14 days. The colony shows a rough type. The basal hyphae extend in a zigzag shape and branch. The basal hyphae show brownish white to light yellowish orange. Aerial mycelium does not settle. SANK 616
In the strain 94, the hyphal fragmentation was observed in yeast dextrose solution at 28 ° C. for 2 days, the fragment was approximately 0.6 to 0.8 × 0.8 to 3.5 μm, and the surface was smooth. No special organs such as sporangia, sclerotium and axle branching are found.

2. Various Properties on Various Culture Media Properties after culturing on various culture media at 28 ° C. for 14 days are as shown in Table 1. The color tone is represented by the Munsell method color chip number of the "Standard color table" for the Japan Color Research Institute.

[0021]

[Table 1] ─────────────────────────────────── Type of medium Item ^{* 1} Properties of SANK 61694 strain ──────────────────────────────── Sucrose G: Poor, flat, brownish white (2.5Y 9/1 ) ^{* 2} Nitrate agar R: Brown white (2.5Y 9/1) SP: Not produced ────────────────────────────── ─── Glucose / G: Not very good, flat, brownish white (10YR 9/1) Asparagine agar R: Brownish white (10YR 9/1) SP: Not produced ──────────── ──────────────────── Glycerin ・ G: Poor, flat, brownish white (2.5Y 9/1) Asparagine agar R: Brownish white (2.5Y 9/1 ) (ISP 5) SP: Not produced ───────────── ────────────────── Starch / inorganic salt agar G: Poor, flat, brownish white (2.5Y 9/1) (ISP 4) R: Brownish white (2.5Y 9/1) SP: Not produced ──────────────────────────────── Tyrosine agar G: Bad, flat, Tea taste white (2.5Y 9/1) (ISP 7) R: Tea taste white (2.5Y 9/1) SP: Not produced ───────────────────── ─────────── Nutrient agar G: Not very good, flat, brownish white (10YR 9/1) (DIFCO) R: Light yellowish orange (2.5Y 9/2) SP: Not produced ──────────────────────────────── Yeast extract ・ G: Good, flat, light yellowish orange (10YR 8/3 ) Malt extract agar R: Light yellow (2.5Y 9/4) (ISP 2) SP: Not produced ────────── ─────────────────────── Oatmeal agar G: Poor, flat, brownish white (2.5Y 9/1) (ISP 3) R: Light yellow Taste orange (2.5Y 9/2) SP: Not produced ──────────────────────────────── Water agar G: Bad, flat, brownish white (2.5Y 9/1) R: Brownish white (2.5Y 9/1) SP: Not produced ───────────────────── ──────────── Potato extract ・ G: Not so good, flat, brownish white (2.5Y 9/1) Ginseng extract agar R: Brownish white (2.5Y 9/1) SP: Produce ──────────────────────────────────── * 1 G: Growth, R: Back side, SP: Soluble pigment * 2 The color in parentheses in the property column is the Munsell color tone display.

3. Physiological properties SANK observed for 2 to 21 days after culturing at 28 ° C
The physiological properties of the 61694 strain are as shown in Table 2.

[0023]

[Table 2] ──────────────────────────────────── Starch hydrolysis Negative gelatin Liquefaction Negative Nitrate reduction False positive Milk coagulation Negative milk Peptone negative Negative melanin pigment productivity (Medium 1) * Negative (Medium 2) * Negative (Medium 3) * Negative substrate degrading casein negative Tyrosine negative xanthine negative Adenine negative hypoxanthine negative testosterone negative Urea Negative Acid Producing Adonitol Negative Arabinose Negative Cellobiose Negative Dorsitol Negative Erythritol Negative Galactose Negative Glucose Positive Glycerin Positive Inositol Negative Lactose Negative Maltose Negative Mannose Negative Melibiose Negative Raffinose Negative Rhamnose Negative Lanitrizarin Negative Tresylose Utilization of organic acids Benzoate negative Citrate negative Lactate negative Oxalate negative Succinate positive 50 ℃ Survival negative Growth temperature range (medium 4) * 7-34 ℃ Optimal growth temperature (medium 4) * 14-29 ℃ Salt tolerance 5% ─── ──────────────────────────────── *: Medium 1; Tryptone yeast extract broth (ISP 1) Medium 2; Peptone-Yeast Extract-Iron Agar (ISP 6) Medium 3; Tyrosine Agar (ISP 7) Medium 4; Yeast Extract-Malt Extract Agar (ISP 2) Also, Pridham-Gottlieb Agar Medium (ISP 9)
SAN observed after culturing at 28 ° C. for 14 days using
The carbon source assimilating ability of the K61694 strain is shown in Table 3.

[0024]

[Table 3] ────────────────────────────── D-Glucose + Sucrose-D-Fructose ++ Inositol-L-arabinose -Raffinose-L-Rhamnose-D-Mannitol-D-Xylose-Control-────────────────────────────── ++: Strong Use, +: Use,-: Do not use 4. Regarding cell components, the cell wall of the SANK 61694 strain can be analyzed by the method of B. Becker et al., Applied Microbiology, Volume 12, 421-423.
Page, 1964), meso-diaminopimelic acid was detected. In addition, the sugar component in the whole cell wall of the SANK 61694 strain was analyzed by the method of MP Lechevalier (MP Lechevalier, Journal of Laboratory and Clinical Medicine).
oratory & Clinical Medicine), 71, 834, 1968
, And arabinose and galactose were detected. The presence of mycolic acid was confirmed. The acyl group type of muramic acid in the cell wall peptidoglycan was glycolyl type. In addition, MK as the main menaquinone component
-8 (H2) was detected. The GC content of DNA is 69.
5%.

ISP [The International Strep
tomyces Project)] Criteria, Vergie's Manual
Of Systematic Bacteriology (Bergey's M
anual of Systematic Bacteriology) Vol. 4, SAWaksman, The Actinomycetes
Actinomycetes 2) and recent literature on actinomycetes were identified and it was determined that this strain belongs to the genus Rhodococcus among actinomycetes. Therefore, this strain was designated as Rhodococcus sp.
It was named SANK 61694 strain. This strain is 1
Deposited at Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry, Ministry of International Trade and Industry, on July 22, 994, with deposit number FERM BP-4
751.

As is well known, actinomycetes are naturally occurring in
Alternatively, mutation is easily caused by an artificial operation (for example, ultraviolet irradiation, radiation irradiation, chemical treatment, etc.), and the SANK 61694 strain of the present invention has the same point. The SANK 61694 strain referred to in the present invention includes all mutants thereof. Further, these mutant strains also include those obtained by genetic methods such as recombination, transduction, transformation and the like. That is, the SANK 61694 strain and its mutant strains and strains which are not clearly distinguished from them are all SAN-producing SAN.
K 61694 strain.

[0027]

BEST MODE FOR CARRYING OUT THE INVENTION

1. Cultivation of these microorganisms to obtain culture A-76202
Carried out in a medium such as that used to produce 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, glucose, fructose, maltose, sucrose, mannitol, glycerin, dextrin as a carbon source,
Oat, rye, corn starch, potato, corn flour, soybean flour, cottonseed oil, molasses, citric acid, tartaric acid and the like can be used alone or in combination. Generally, the amount varies from 1 to 10% by weight of the medium amount.

As the nitrogen source, a substance containing a protein and its hydrolyzate or an inorganic salt 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 source may be used alone or in combination of 0.2 to 1 of the medium amount.
Used in the range of 0% by weight.

The nutrient inorganic salts taken into the medium are ordinary 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, silicone oil, vegetable oil, surfactant and the like are used as an antifoaming agent. SAN
The pH of the medium for culturing the K61694 strain and producing A-76202 can be changed to 5.0 to 8.0.

This strain grows in the range of 7 ° C to 34 ° C, and particularly good in the range of 14 ° C to 29 ° C. Further, 22 ° C to 28 ° C is suitable for the production of A-76202. A-76202 can be obtained by aerobically culturing, but a commonly used aerobic culturing method, for example, a solid culturing method,
A shaking culture method, an aeration stirring culture method and the like are used.

For small-scale culture, it is preferable to carry out shaking culture at 28 ° C. for several days. Culturing is initiated in a Erlenmeyer flask by one or more stages of seed culture development steps. The seed culture flask is shake-cultured in a constant temperature incubator for several days or until it grows sufficiently. The grown seed culture solution is used to inoculate a part or all of the grown seed culture solution to the next-stage seed medium or production medium. Shake the flask containing the inoculated production medium at constant temperature for several days,
After completion of the culture, the contents of the flask are separated by centrifugation or filtration. In the case of large-scale culture, it is preferable to culture in a suitable tank equipped with a stirrer and an aeration device. According to this method, the nutrient medium can be prepared in the tank. The nutrient medium is sterilized by heating to 125 ° C., and after cooling, the sterilized medium is inoculated with the seed culture solution that has been grown in advance. The culture is performed by aeration and stirring at 28 ° C. This method is suitable for obtaining large amounts of compounds.

A-762 produced with the progress of culture
To know the change with time of the amount of 02, for example, the filtrate obtained by filtering the culture broth was acidified and extracted with ethyl acetate, concentrated,
The amount of A-76202 in the dried oil is measured by an endoplasmic reticulum α-glucosidase inhibition test or high performance liquid chromatography. Usually, the maximum production of A-76202 is reached after 96 to 168 hours of culture.

2. Extraction and purification A-762 present in the liquid part of the culture medium after the completion of culture
02, the microbial cells and other solid parts are separated by a filtration operation or centrifugation using diatomaceous earth as a filter aid, and A-76202 existing in the filtrate or supernatant is used for its physicochemical properties. It is obtained by extraction and purification.
For example, A-76202 present in the filtrate or supernatant can be replaced with an ion exchange resin, such as Amberlite IRC-50,
Impurities are adsorbed and removed by passing through a layer of CG-50 (manufactured by Rohm and Haas Co.), Dowex 50WX4, Dowex SBR-P (manufactured by Dow Chemical Co.) or adsorbed A-76202. After that, it is obtained by eluting with aqueous ammonia. Alternatively, as an adsorbent, for example, activated carbon or adsorbent resin Amberlite XAD-2, XAD-4 (Rohm and
Haas Co., Ltd.) and the like, Diaion HP-10, HP-2
0, HP-50, CHP20P (manufactured by Mitsubishi Kasei Co., Ltd.)
Etc. are used. A liquid containing A-76202 is passed through a layer of an adsorbent as described above to adsorb and remove impurities, or after adsorbing A-76202, water such as methanol water or acetone water is mixed with an organic solvent. It is obtained by eluting with a solvent. It can also be obtained by extraction with an organic solvent such as n-butanol, ethyl acetate or methylene chloride.

A-76202 thus obtained
Is an adsorption column chromatography using a carrier such as silica gel or Florisil, distribution column chromatography using Avicel (manufactured by Asahi Kasei Co., Ltd.), Sephadex LH-20 (manufactured by Pharmacia) and normal phase. , High-performance liquid chromatography using a reverse phase column, an ion exchange column, etc.

The location in the purification of A-76202 of the present invention can be measured by each of the following methods.

1) Endoplasmic Reticulum α-Glucosidase Inhibition Test (1) Using a Triton X-100 solubilization treatment solution of rat liver microsome fraction as an enzyme solution and vesicular stomatitis virus glycoprotein containing tritium-labeled glucose as a substrate The α-glucosidase reaction proceeds in the presence of the test sample. The reaction is stopped by adding an aqueous solution of trichloroacetic acid to insolubilize the protein, and the radioactivity of free glucose in the filtrate filtered with a membrane filter having a pore size of 0.65 μm is measured using a liquid scintillation counter. The inhibition rate of the enzyme activity of the test sample is calculated by comparing with the radioactivity of the reaction solution containing no inhibitor.

2) Endoplasmic Reticulum α-Glucosidase Inhibition Test (2) Using Triton X-100 Solubilization Treatment Solution of Rat Liver Microsomal Fraction as an Enzyme Solution and Using Paranitrophenyl α-D-Glucopyranoside as a Substrate The α-glucosidase reaction proceeds below. The reaction is stopped by adding a buffer solution of pH 10, and the absorbance at 415 nm is measured to determine the amount of free paranitrophenol. Compared with the amount of free paranitrophenol in the reaction solution containing no inhibitor,
The inhibition rate of the enzyme activity of the sample is calculated.

3) Method using high performance liquid chromatography Separation column; Senshupack ODS-H-2151 (φ6 × 150 m)
m, Senshu Scientific Co., Ltd. Mobile phase: 16% acetonitrile-water Flow rate: 1.5 ml / min Detection wavelength: 210 nm Temperature: 25 ° C. Retention time: 16.22 minutes.

A-76202 or a salt thereof of the present invention is
It is a novel compound that has not been published in the literature and has endoplasmic reticulum α-glucosidase inhibitory activity. Therefore, A-76202 or a salt thereof is useful for use as an anti-AIDS drug or an antitumor drug. A-76202 also has the activity of inhibiting the maltase activity exhibited by the sucrase-isomaltase complex. Therefore, A-76202 or a salt thereof is useful for use as an antiobesity agent or antidiabetic agent. The present invention particularly provides an α-glucosidase inhibitor containing A-76202 or a salt thereof as an active ingredient, particularly an anti-AIDS agent and an antitumor agent.

Examples of the dosage form include oral administration by tablets, capsules, granules, syrups, etc., and parenteral administration by injections (intravenous, intramuscular, subcutaneous), eye drops, suppositories, etc. You can These various preparations are known auxiliary agents that can be usually used in the technical field of known pharmaceutical preparations such as excipients, binders, disintegrating agents, lubricants, flavoring agents, solubilizing agents, coating agents, etc. Can be used for formulation. The amount used is a symptom,
Depending on age, weight, administration method, dosage form, etc.,
Usually, it is preferable to administer 1 mg to 1000 mg / day to an adult once or in several divided doses.

[0042]

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 Rhodococcus sp.
p. ) SANK 61694 strain (FERM BP-47
51) which contains 80 ml of the medium represented by medium composition-1
One platinum loop inoculation from a slant was inoculated into two 00 ml Erlenmeyer flasks, and was shaken at 210 rpm at 28 ° C for 12 hours.
After culturing for 0 hour, a seed culture solution was obtained.

Medium Composition-1 Glucose 1% Sucrose 1% Glycerin 1% Oatmeal 0.5% Soybean flour 2% Casamino acid 0.5% CaCO ₃ 0.1% CB-442 0.01% ───── ────────────────────── Before sterilization pH 7.0 Put the medium shown in Medium Composition-2 into 60 500-mL Erlenmeyer flasks, 100 ml at a time, and 120 ° C. And heat sterilized for 40 minutes. After cooling this to 28 ° C., seed culture solution 2
ml was inoculated. Then, it was cultured at 28 ° C. for 144 hours in a rotary shaking culture machine at 210 rpm.

[0045] 1.0% Medium Composition -2 Glycerin 2.5% 2.5% glucose production yeast Soy flour _{1.0% KH 2 PO 4 0.05%} MgSO 4 · 7H 2 O 0.05% CaCo 3 0 0.5% CB-442 0.01% ─────────────────────────── Before sterilization pH 7.0 (B) isolated Celite as a filter aid in 6 liters of the culture solution
0.3 kg of 545 (manufactured by Johns Manville Product Corporation) was added and filtered to obtain 4.65 liters of a filtrate. The filtrate adjusted to pH 4.0 with hydrochloric acid was used as Dowex 50WX4 (Dowex50WX4 (H ⁺ )).
A-76202 was applied to a column packed with 460 ml.
Absorbed and washed with 1.5 liters of deionized water,
Elution was performed with a 0.5N aqueous ammonia solution. First 60
Remove 0 ml and collect the subsequent 1.3 liters of eluate,
It was concentrated under reduced pressure to 15 ml.

Next, this concentrated solution was added to Amberlite CG-
It was applied to a column packed with 500 ml of 50 (NH ₄ ⁺ ), developed and eluted with deionized water, and the eluate was fractionated by 20 ml. Analysis was carried out by the method described below (α-glucosidase inhibition test (1), (2) and high performance liquid chromatography), and fractions 29 and 30 in which the presence of A-76202 was confirmed were collected. The active fraction was concentrated under reduced pressure and then freeze-dried to obtain 143.6 mg of a crude powder containing A-76202. This crude powder was dissolved in 20 ml of distilled water, pH was adjusted to 4.0 with hydrochloric acid, and Amberlite CG-50 (NH ₄ ⁺ ) 500 was added again.
Purified by column packed with ml, powder 48.2mg
I got Then, this powder was separated and purified using HPLC. HPLC column (SENSHUPACK ODS-H
-5251) was equilibrated with 16% acetonitrile-water,
To this, 0.3 ml of the sample solution in which the powder was dissolved in distilled water to make 1.5 ml was injected. Elution was performed with 16% acetonitrile-water as a mobile phase at a flow rate of 8 ml / min, and the retention time was 42 minutes to 44 when monitored at a detection wavelength of 210 nm.
Minute eluate was collected. The same operation was performed for the remaining 1.2 ml of the sample solution. The eluate obtained by preparative HPLC using a total of 5 times was concentrated and lyophilized to give A
1.1 mg of a white powder of -76202 was obtained.

A part of this white powder was taken and analyzed by high performance liquid chromatography, and the following results were obtained.

Separation column; Senshupack ODS-H-215
1 (6φ x 150 mm, Senshu Scientific Co., Ltd.) Mobile phase;
16% acetonitrile-water flow rate; 1.5 ml / min detection wavelength; 210 nm temperature; 25 ° C. retention time; 16.22 minutes.

Example 2. (A) Culturing 15 liters of the medium represented by Medium Composition-3 was charged into 4 jar fermenters having a volume of 30 liter, and the medium was kept at 120 ° C. for 3 hours.
Heat sterilized for 0 minutes. After cooling this to 28 ° C., Example 1. Seed culture solution (150 ml) prepared in the same manner as above was inoculated. Then, the rotation speed was adjusted within the range of 100 to 110 rpm so as to maintain the dissolved oxygen amount of 5 ppm, and the cells were stirred and cultured at 30 ° C. for 120 hours at an air flow rate of 15 liters / minute.

Medium Composition-3 Glucose 5% Pharmamedia 0.6% Soybean flour 0.75% (NH ₄ ) ₂ SO ₄ 0.5% Yeast extract 0.5% Polypeptone 0.5% KH ₂ PO ₄ 0. 1% CaCO ₃ 0.55% CB-442 0.02% ─────────────────────────── Before sterilization pH 7.0 (B ) Isolation To 56 liters of the obtained culture broth, 3 kg of Celite 545 (manufactured by Johnsmanville Product Corporation) as a filter aid was added and filtered to obtain 52 liters of a filtrate. The filtrate was adjusted to pH 4.0 with hydrochloric acid.
The column was filled with 6 liters of Dowex 50WX4 (H ⁺ ) to adsorb A-76202. After washing the column with 25 liters of deionized water,
Elution was performed with a 0.5N aqueous ammonia solution. Eluent is 3
Fractions were divided into liters, and active fractions 2 to 6 were collected and concentrated to 300 ml. Hydrochloric acid was added to this concentrated solution to adjust the pH to 7.0, and Amberlite CG-50 was used.
A column was filled with 9 liters of (NH ₄ ⁺ )
-76202 was adsorbed and developed and eluted with deionized water.
After removing the first 4.4 liters, 18 ml was fractionated. Fractions 30 to 85 containing A-76202 were collected, concentrated under reduced pressure, and lyophilized to give a crude powder 23.4.
g was obtained. This was dissolved in 500 ml of deionized water, the pH was adjusted to 2.5 with hydrochloric acid, and the mixture was extracted 3 times with 500 ml of ethyl acetate. The extract was washed with saturated saline and then concentrated under reduced pressure to 100 ml. After adding 130 ml of water to the concentrated solution, an aqueous sodium hydroxide solution was further added to adjust the pH to 9.5.
Adjust to 10 to back-extract A-76202, and add 70 ml of water layer.
The mixture was concentrated to pH 6.0 and adjusted to pH 6.9 with hydrochloric acid.

Next, 100 ml of a column packed with HP-20 was applied to the column for adsorption, washed with water, further washed with 300 ml of 10% acetone water, and then eluted with 50% acetone water to fractionate 100 ml each. Active fractions 2 and 3 were collected, concentrated under reduced pressure and freeze-dried to obtain 73.7 mg of powder. The powder 60mg was dissolved in water 15 ml, pre-5% acetonitrile - Cosmosil 140C ₁₈ -opn equilibrated with water (Nacalai Tesque Co.
It was adsorbed on a column filled with 40 ml of the product. After washing with 540 ml of 5% acetonitrile-water, elution was performed with 10% acetonitrile-water, and the eluate was fractionated in 20 ml portions.
Active fractions 36 to 44 were collected, concentrated under reduced pressure, and freeze-dried to obtain 20 mg of A-76202 as a white powder.

A part of this white powder was taken and analyzed by high performance liquid chromatography, and the following results were obtained.

Separation column; Senshupack ODS-H-215
1 (6φ x 150 mm, Senshu Scientific Co., Ltd.) Mobile phase;
16% acetonitrile-water flow rate; 1.5 ml / min detection wavelength; 210 nm temperature; 25 ° C. retention time; 16.22 minutes.

[0054]

EFFECT OF THE INVENTION Test Example 1. ER α-glucosidase inhibition test
1) Using a viral protein having a sugar chain containing tritium-labeled glucose as a substrate, the radioactivity of the released labeled glucose was measured to determine the enzyme activity.

15 g of liver of Wistar rat was cut into small pieces, and a potassium phosphate buffer solution (pH 7.0) containing 0.25 M sucrose and 0.5 mM dithiothreitol.
Homogenize in 60 ml using a Teflon homogenizer. This was subjected to centrifugation at 1,800 xg for 10 minutes to obtain a supernatant. This supernatant was added to 15,000 × g,
The supernatant was obtained by subjecting to centrifugation for 30 minutes.

The supernatant was subjected to centrifugation at 120,000 xg for 60 minutes to obtain a precipitate. This precipitate contains 10 mM glycerol, 2 mM MgCl ₂ , and 0.5 μM dithiothreitol in 10 mM potassium phosphate buffer (pH
7.0) Add 60 ml and homogenize and repeat 12
It was subjected to centrifugation at 50,000 xg for 60 minutes. 10% glycerol, 2 mM MgCl ₂ , 0.
60 ml of 10 mM potassium phosphate buffer (pH 7.0) containing 5 μM dithiothreitol was added and homogenized to obtain a liver microsome fraction.

This liver microsome fraction was homogenized using a Teflon homogenizer in the presence of 2% Triton X-100. 20 mM of bovine serum albumin was converted to a standard sample by protein assay (manufactured by Bio-Rad) to obtain a protein concentration of 3.4 mg / ml.
The enzyme solution was diluted with 100 mM potassium phosphate buffer (pH 6.8) containing ethylenediaminetetraacetic acid.

The method of Kang et al. (MS Kang et al., Ana
lytical Biochemistry, 184, 109-112 (1989
)) And labeled vesicular stomatitis virus with tritiated glucose. That is, BHK cells infected with vesicular stomatitis virus were inoculated into 18 culture bottles having a culture area of 150 cm ² and cultured for 24 hours. The culture supernatant collected from these 18 culture bottles was 100,0
It was subjected to a centrifugation treatment at 00 × g for 2 hours to obtain a virus-containing precipitate. This was dissolved in distilled water and then precipitated with trichloroacetic acid. This precipitate was used as a substrate solution in an amount of 7 ml of a solution adjusted to pH 7 in the presence of 0.5% sodium dodecyl sulfate and 5 mM dithiothreitol.

Hereinafter, 100 mM potassium phosphate buffer (pH 6.8) containing 20 mM ethylenediaminetetraacetic acid was used for operations such as dilution. To each well of a 96-well microtiter plate, 5 μl of test sample solution, 10 μl of enzyme solution, 10 μl of 5-fold dilution of substrate solution and 75 μl of the above buffer solution were added to make 100 μl in total.
The reaction started. After allowing the reaction to stand at 37 ° C. for 60 minutes, 10 mg / ml bovine serum albumin aqueous solution and 5
25 μl each of 0% trichloroacetic acid aqueous solution was dispensed to stop the α-glucosidase reaction. After standing still at 4 ° C. for 60 minutes, it was filtered with a membrane filter having a pore size of 0.65 μm using a multiscreen assay system (manufactured by Millipore). The radioactivity contained in 100 μl of the filtrate was measured using a liquid scintillation counter. The inhibition rate of the enzyme activity of the test sample was calculated by comparing with the radioactivity of the reaction solution containing no inhibitor. In this method, the concentration at which A-76202 inhibited endoplasmic reticulum α-glucosidase by 50% was 8.1 ng / ml.

Test Example 2. Endoplasmic reticulum α-glucosidase
Inhibition test (2) Test example 1. The rat liver microsome fraction prepared in the above step was homogenized in the presence of 2% Triton X-100 using a Teflon homogenizer. Bovine serum albumin was converted into a standard sample by a protein assay (manufactured by Bio-Rad), and 100 mM containing 20 mM ethylenediaminetetraacetic acid was used so that the protein concentration was 2.6 mg / ml.
The enzyme solution was diluted with mM potassium phosphate buffer (pH 6.8).

For operations such as dilution, 100 mM potassium phosphate buffer containing 20 mM ethylenediaminetetraacetic acid (p
H 6.8) was used. To each well of a 96-well microtiter plate, 5 μl of test sample solution, 1 enzyme solution
The reaction was started by adding 0 μl, 10 mM para-nitrophenyl α-D-glucopyranoside solution (50 μl) and a buffer solution (85 μl) to a total of 150 μl. 20 at 37 ° C
After allowing the mixture to stand still for the reaction to proceed, 20 μl of a 4 M glycine sodium buffer solution (pH 10) was dispensed to stop the reaction.

The amount of free para-nitrophenol was determined by measuring the absorbance at 415 nm and compared with the amount of free para-nitrophenol in the reaction solution containing no inhibitor to calculate the inhibition rate of the enzyme activity of the sample.

In this method, A-76202 inhibits endoplasmic reticulum α-glucosidase by 50% at a concentration of 4.7 ng.
/ Ml.

Test Example 3. Rat sucrose
Inhibition of maltase activity by the somaltase complex
Test Kessler et al. (M.Kessler et al., Biochimica et Biophysica Actor (Biochimica et Biophysica
Acta), 506, 136-154, 1978), and a small intestine brush border membrane enzyme solution was prepared from the small intestine of Wistar rat (male). This is the method of J. Kolnska and J. Kraml, Biochimica et Biophysica Acta, 284
Vol., 235-247, 1972), and after purification with pepsin, purification was carried out twice on a Sephadex G-200 column. Through this procedure, sucrose activity, isomaltase activity, and maltase activity were found in the same elution fraction. This result indicates that the sucrase-isomaltase complex simultaneously has maltase activity (J. Kolinska).
and J. Kraml, Biochimica et Biophysica Acta, 284, 235-247, 1
972) and adapted to the facts known from The thus obtained sucrose-isomaltase complex fraction was used as an enzyme solution.

4-aminoantipyrine is available from Sigma
Peroxidase (Grade I) and glucose oxidase (Grade I) purchased from Boehringer Mannheim were used. Hereinafter, for operations such as dilution, 20 mM citric acid-40 mM disodium phosphate buffer (pH 6.
2) was used. To each well of a 96-well microtiter plate, bovine serum albumin 0.2 mg, glucose oxidase 3 unit, peroxidase 0.1
32 unit, 4-aminoantipyrine 20 μg, phenol 40 μg, maltose 3 μmole 125 μl of the above-mentioned buffer and test sample solution 5 μl were added,
The total volume was 130 μl. Then, add enzyme solution 20
The reaction was started by adding μl (total activity 0.001 unit (international unit)). This was reacted at 37 ° C. for 20 minutes, and the concentration of released glucose was quantified by measuring the change in absorbance at 492 nm. The inhibition rate of the enzyme activity of the test sample was calculated by comparing with the glucose release amount of the reaction solution containing no inhibitor.

In this method, the concentration at which A-76202 inhibited maltase activity by 50% was 1.2 μg / ml.

As described above, A-76202 or a salt thereof of the present invention has a remarkable endoplasmic reticulum α-glucosidase inhibitory activity, and thus is useful as an anti-AIDS agent and an antitumor agent. Furthermore, since it has an activity of inhibiting the maltase activity of the sucrase-isomaltase complex, it is useful as an antiobesity agent, an antidiabetic agent or the like.

Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location C12N 1/20 A 8828-4B C12P 19/60 7432-4B // (C12N 1/20 C12R 1:01) ( C72P 19/60 C12R 1:01) (72) Inventor Yasuyuki Takamatsu 389-4 Oigata, Shimokawa, Izumi-cho, Iwaki-shi, Fukushima Sankyo Co., Ltd. (72) Takeshi Kinoshita 1-2-2 Hiromachi, Shinagawa-ku, Tokyo No. Sankyo Co., Ltd. (72) Inventor Hideyuki Haruyama 1-258 Hiromachi, Shinagawa-ku, Tokyo Sankyo Co., Ltd. (72) Inventor Nao Okazaki 33 Miyukigaoka, Tsukuba City, Ibaraki Sankyo Co., Ltd. ( 72) Inventor Ryuzo Enoda 33 Miyukigaoka, Tsukuba City, Ibaraki Prefecture Sankyo Co., Ltd.

Claims (8)

[Claims] 1. A novel compound A- represented by the following formula (I):
76202 or a salt thereof. : [Chemical 1] 2. A novel compound A-76202 or a salt thereof having the following physicochemical properties. 1) Properties; white powder. 2) Solubility; soluble in dimethyl sulfoxide. Insoluble in water, methanol, and acetone. 3) molecular formula; C ₂₀ H ₁₈ O ₉ (measured by high resolution mass spectrum) 4) molecular weight; 402 (measured by FAB-MS spectrum) 5) ultraviolet absorption spectrum; λ _max nm The ultraviolet absorption spectrum measured in an aqueous solution is: 259n
It has a maximum absorption at m and an absorption coefficient at 259 nm (E
^1% _{1 cm} ) is 687. 6) Infrared absorption spectrum (KBr); The infrared absorption spectrum measured by ν _max cm ^-1 KBr disk is as follows. 3348, 2943, 1627, 160
1, 1574, 1515, 1284, 1212, 107
8, 1036, 969 7) ¹ H-nuclear magnetic resonance spectrum; δ (ppm) Nuclear magnetic resonance spectrum (500 MHz) measured in a heavy dimethyl sulfoxide solution (dimethyl sulfoxide internal standard: 2.49 ppm) is as follows. is there. 3.46 (1H,
dd, J = 5.5, 11.8Hz), 3.57 (1H, dd, J = 3.5, 11.8Hz),
3.84 (1H, dd, J = 3.8, 6.0Hz), 3.96 (1H, ddd, J = 3.5,
5.5, 6.0Hz), 4.28 (1H, dd, J = 1.0Hz or less, 3.8Hz), 5.60
(1H, d, J = 1.0Hz or less), 6.81 (2H, d, J = 8.5Hz), 7.23 (1
H, d, J = 8.8Hz), 7.39 (2H, d, J = 8.5Hz), 7.53 (1H, d, J
= 8.8Hz), 8.37 (1H, s) 8) ¹³ C-nuclear magnetic resonance spectrum; δ (ppm) Nuclear magnetic resonance spectrum (125 MHz) measured in a heavy dimethyl sulfoxide solution (dimethyl sulfoxide internal standard: 39.5 ppm). Is as follows. 61.14, 7
6.66, 81.34, 85.95, 107.16, 114.44, 114.9 (3C), 11
9.58, 122.41, 123.17, 130.05 (2C), 136.15, 146.03, 1
47.58, 153.01, 157.17, 175.15 9) High performance liquid chromatography separation column; Senshupack ODS-H-2151 (φ6 × 15)
0 mm, Senshu Kagaku Co., Ltd. Mobile phase: 16% acetonitrile-water Flow rate: 1.5 ml / min Detection wavelength: 210 nm or 259 nm Temperature: 25 ° C. Retention time: 16.22 minutes 3. Rhodococcus
A-76202, which comprises culturing an A-76202-producing bacterium belonging to the genus and collecting A-76202 from the culture.
76202 manufacturing method. 4. Rhodococcus
A-76202 producing bacterium belonging to the genus Rhodococcus sp. SANK61
The production method according to claim 3, which is strain 694. 5. Rhodococcus sp.
ccus sp. ) SANK61694 strain. 6. A medicine containing A-76202 or a salt thereof as an active ingredient. 7. An α-glucosidase inhibitor containing A-76202 or a salt thereof as an active ingredient. 8. An anti-AIDS agent containing A-76202 or a salt thereof as an active ingredient. 8. An antitumor agent containing A-76202 or a salt thereof as an active ingredient.