JPH05252969A - Production of oxazoline derivative - Google Patents

Abstract

PURPOSE:To obtain the subject compound having remarkable sucrase-inhibiting activity and useful as an antiobestic agent and an antidiabetic agent by culturing a specific microbial strain belonging to the genus Micromonospora or Amycolatopsis. CONSTITUTION:A microbial strain belonging to the genus Micromonospora or Amycolatopsis and capable of producing the compound of formula [e.g. Micromonospora sp. SANK62390 (FERM BP-3521)] is cultured and the objective compound is separated from the culture product. The culture is preferably carried out at 28 deg.C for several days under shaking in the case of a small-scale culture. In a large-scale culture, it is preferably carried out at 28 deg.C under aeration and agitation using a tank provided with a stirrer and an aeration apparatus.

Description

Detailed Description of the Invention

[0001]

[Object of the Invention]

[0002]

TECHNICAL FIELD The present invention relates to a method for producing a novel oxazoline derivative having sucrase inhibitory activity.

[0003]

2. Description of the Related Art Conventionally, AO-128, acarbose and the like have been known as substances having a strong inhibitory activity against sucrase, and they have been reported as useful substances for antiobesity and antidiabetes (Reference: Satoshi Horii et al., Journal of Medical Chemistry
edicinal Chemistry), 29, 1038-1046, 1986
Year; Toshihisa Goda, “Nutrition and Food”, Volume 34, No. 2, 139-1
43, 1981). Therefore, compounds having sucrase inhibitory activity are expected to be useful as antiobesity agents and antidiabetic agents.

The compound represented by the following formula (I) of the present invention and another method for producing the same have already been described in Japanese Patent Application No. 4-140186.

[0005]

[Constitution of the invention]

[0006]

The present invention provides a compound of formula (I)

[0007]

[Chemical 4]

The present invention relates to a method for producing a compound represented by (hereinafter referred to as compound (I)).

More specifically, the present invention comprises culturing a bacterium producing a compound (I) belonging to the genus Micromonospora or the genus Amycolatopsis, and collecting the compound (I) from the culture. Manufacturing method.

The compound (I) obtained by the production method of the present invention has the following characteristics. 1) Color and shape: basic white powder 2) Solubility: Soluble in water. Insoluble in acetone and chloroform. 3) Molecular formula: C ₇ H ₁₂ N ₂ O ₅ 4) Molecular weight: 204 (measured by FAB-MS spectrum) 5) Specific rotation: [α] _D ²⁵ + 10.0 ° (c 0.51, water) 6) UV absorption Spectrum: λ _max nm (E 1 cm · 1%) The UV absorption spectrum measured in an aqueous solution does not show a characteristic absorption on the longer wavelength side than 210 nm. 7) Infrared absorption spectrum: ν _max (KBr) cm ^-1 3358, 1668, 1528, 1398, 1066 8) ¹ H-nuclear magnetic resonance spectrum: δppm TPS (sodium trimethylsilylpropionate) is used as an internal standard in heavy water. The measured nuclear magnetic resonance spectrum (500 MHz) is as follows. 3.73 (1H, d, J = 11.72Hz), 3.82 (1H, d, J = 12.21Hz), 3.97 (1
H, d, J = 4.4Hz), 4.23 (1H, dd, J = 4.4 and 2.44Hz), 4.
37ppm (1H, d, J = 8.79Hz), 5.03 (1H, dd, J = 8.79 and 2.4
4Hz) 9) High performance liquid chromatography separation column; Asahi Pack ES-502 C (manufactured by Asahi Kasei Corporation) Mobile phase: 20 mM ammonium acetate (pH 8.5) + 50 mM
Saline flow rate: 1 ml / min Detection wavelength: 210 nm Temperature: 25 ° C Hold time: 8.39 minutes.

The compound (I) of the present invention can be converted into a salt according to a conventional method. Examples of such salts include hydrofluorides, hydrochlorides, hydrobromides, hydrohalides such as hydroiodides, nitrates, perchlorates, sulfates and phosphates. Inorganic acid salts; lower alkane sulfonates such as methane sulfonate, trifluoromethane sulfonate, ethane sulfonate, aryl sulfonates such as benzene sulfonate, p-toluene sulfonate, Huma Mention may be made of organic acid salts such as-phosphates, succinates, citrates, tartrates, oxalates, maleates and the like and amino acid salts such as glutamate, aspartate. It is preferably a pharmacologically acceptable salt.

The compound (I) of the present invention has various isomers. In compound (I), all of these isomers and a mixture of these isomers are represented by a single formula. Therefore, the present invention includes all of these isomers and a mixture of these isomers.

Examples of strains belonging to the genus Micromonospora used in the method for producing the compound (I) of the present invention include Micromonospora sp.
The SANK62390 strain can be mentioned.

The mycological properties of the SANK 62390 strain are as follows.

1. Morphological characteristics SANK 62390 strain grows normally or slightly poorly on agar medium for strain identification at 28 ° C for 7 to 14 days. The basal hyphae swell and branch well, showing a bright orange, orange to dark brownish gray, but nocardia (Nocardi).
a) No genus strain-like breakage or zigzag extension is observed. The aerial hyphae are slightly formed in the original trace and show white to brownish white. Although spores are observed only on the basal hyphae, they are spherical at the tips of relatively short spores. The surface of spores is smooth. No special organs such as sporangia, sclerotium, and axle bifurcation are found.

2. Properties on various culture media Table 1 shows the properties after culturing on various culture media at 28 ° C for 14 days. The display of the color tone indicates the color chip number of the "Standard color table" for the Japan Color Research Institute.

[0017]

[Table 1] ──────────────────────────────────── Type of medium Item Properties of SANK 62390 strain ─ ─────────────────────────────────── Sucrose G: Not very good, flat, light orange (3 -9-6) Nitrate agar AM: Not formed R: Light orange (3-9-6) SP: Not produced ─────────────────────── ────────────── Glucose / G: Not very good, flat, yellowish orange (12-7-7) Asparagine agar AM: Not formed R: Light orange (6-8- 7) SP: Not produced ──────────────────────────────────── Glycerin G: Not very good , Flat, bright orange (8-7-6) asparagine agar AM: not formed (ISP 5) R: bright brown ash 2-6-6) SP: Not produced ───────────────────────────────────── Starch / inorganic Salt agar G: Good, flat, bright orange (8-7-6) (ISP 4) AM: Not formed R: Ash (N-5) SP: Not produced ──────────── ───────────────────────── Tyrosine agar G: Not very good, flat, dull orange (6-8-6) (ISP 7) AM: Slightly formed, original traces, white R: Brown white (2-9-7) SP: Not produced ────────────────────────── ─────────── Nutrient agar G: Not very good, flat, yellow-orange (10-8-7) (DIFCO) AM: Not formed R: Dull yellow orange (8-8-7) ) SP: Not produced ──────────────────────────────────── Yeast extract G: Good, flat , Dark tea taste ash (1-4-6) Malt extract agar AM: Slightly formed, traces of original, tea taste white (1-8-6) (ISP 2) R: Tea taste black (1-2-6) SP: Not produced ──────────────────────────────────── Oatmeal agar G: Good, flat, orange (10 -7-6) (ISP 3) AM: Slightly formed, original traces, white R: Orange (12-7-6) SP: Not produced ──────────────── ───────────────────── Water agar G: Not very good, flat, light yellowish orange (2-9-9) AM: Not formed R: Ash (N-5) SP: Not produced ───────────────────────────────────── Potato extract G : Good, flat, yellowish ash (1-9-10) Ginseng extract agar AM: Slightly formed, original traces, white R: Brownish white (2-9-7) SP: Not produced ──────────────────────────────────── G: Growth, AM: Aerial mycelium, R: Backside, SP : Soluble dye.

3. Physiological properties SANK 62 observed for 2 to 21 days after incubation at 28 ° C
The physiological properties of strain 390 are shown in Table 2.

[0019]

[Table 2] ──────────────────────────────────── Hydrolysis of starch Clarification of gelatin Liquefaction of gelatin Nitrate reduction Implicit milk coagulation Positive milk peptoneation Positive melanin-like pigment productivity (medium 1) ^* Implicit (medium 2) ^* Implicit (medium 3) ^* Implicit substrate-degrading casein Explicit tyrosine Implicit Xanthine Implicit Growth temperature range (medium 4) ^* 17-42 ℃ Optimum growth temperature (medium 4) ^* 27-32 ℃ Salt tolerance 2% ─────────────────── ────────────────── *: 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).

[0020] In addition, the Pridham-Gotrieve agar medium (I
Observed after culturing at 28 ° C for 14 days using SP 9)
The carbon source assimilation capacity of SANK 62390 strain is shown in Table 3.

[0021]

[Table 3] ──────────────────────────────────── D-glucose use D-fructose use L-arabinose used L-rhamnose not used D-xylose used sucrose not used inositol used raffinose used D-mannitol not used control not used ────────────────── ─────────────────.

4. Regarding the cell components, the cell wall of the SANK 62390 strain can be analyzed by the method of B. Becker et al., Applied Microbiology, Volume 12, pages 421-423, 1984.
Year), meso-diaminopimelic acid and glycine were detected. In addition, the sugar components in the whole cell wall of the SANK 62390 strain were analyzed by the method of MP Lechevalier (MP Lechevalier, Journal of Laboratory and Clinical Medicine (Journal of Labo
ratory & Clinical Medicine), 71, 834, 1968
, Arabinose and xylose were detected. The presence of mycolic acid was not confirmed. The acyl type in the cell wall peptidoglycan was the glycolyl type. In addition, MK-10 (H ₆ ), M
K-10 (H ₄ ) and MK-10 (H ₈ ) were detected.

From the above, it was revealed that this strain belongs to the genus Micromonospora among actinomycetes. Therefore, Micromonospora sp. SANK 62390
Strains (deposit organization, Institute of Microbial Technology, Agency of Industrial Science and Technology: Deposit No., Microtechnical Institute Article 3521 (FERM BP-3521): Original deposit date, July 26, 1990).

Examples of strains belonging to the genus Amycolatopsis used in the method for producing the compound (I) of the present invention include Amycolatopsis sp. SANK 60791 strain.

The mycological properties of the SANK 60791 strain are as follows.

1. Morphological characteristics The SANK 60791 strain grows normally or slightly poorly on the agar medium for strain identification at 28 ° C for 7 to 14 days. The basal hyphae satisfactorily elongate and branch to be flat or wrinkle-like, and show brownish white, light yellowish brown or dull yellow. The aerial mycelium is slightly poor or slightly formed in the original trace, white,
Light yellow to light orange. In the latter half of the culture, fragmentation of basal hyphae and aerial hyphae may be observed, and rod-like structures may be observed in aerial hyphae. Nocardia of mycelium (Nocar
No dia-like Zig-Zag extension is observed.
No special organs such as sporangia, sclerotium, and axle bifurcation are found.

2. Properties on various culture media The properties after culturing on various culture media at 28 ° C for 14 days are shown in Table 4. The display of the color tone indicates the color chip number of the "Standard color table" for the Japan Color Research Institute.

[0028]

[Table 4] ──────────────────────────────────── Type of medium Item Properties of SANK 60791 strain ─ ─────────────────────────────────── Sucrose G: Good, flat, yellowish gray (1- 9-10) Nitrate agar AM: Not very good, white R: Yellowish ash (2-9-10) SP: Not produced ───────────────────── ──────────────── Glucose / G: Not very good, flat, light orange (3-9-6) Asparagine agar AM: Slightly formed, yellowish gray (2-9 -10) R: Light brown (3-8-6) SP: Not produced ───────────────────────────────── ──── Glycerin ・ G: Good, flat, brownish white (2-9-7) Asparagine agar AM: Traces formed, white (ISP 5) R: Light yellowish green tea (6-8-8) SP: Not produced ───────────────────────────────── ──── Starch / inorganic salt agar G: Not very good, flat, light yellowish green tea (4-8-9) (ISP 4) AM: Trace-like formation, light yellow (3-9-10) R: Thin Yellow tea (6-8-9) SP: Not produced ──────────────────────────────────── Tyrosine Agar G: Very good, wrinkled, brownish white (2-9-6) (ISP 7) AM: Slightly formed, light orange (3-9-6) R: Light orange (6-8-7) SP : Not produced ──────────────────────────────────── Nutrient agar G: Good, flat, light yellowish tea (4-8-8) (DIFCO) AM: Originally formed traces, white R: Light yellow (3-9-8) SP: Not produced ──────────────── ───────── ─────────── Yeast extract · G: Good, wrinkled, dull yellow (10-7-9) Malt extract agar AM: Traces formed, white (ISP 2) R: Dull yellow Taste orange (10-7-8) SP: Not produced ───────────────────────────────────── Oatmeal agar G: Not very good, flat, light yellowish green tea (4-8-9) (ISP 3) AM: Traces formed, white R: Light yellow (4-9-9) SP: Not produced ── ────────────────────────────────── Water agar G: Not very good, flat, yellowish gray (1- 9-10) AM: Not very good, white R: Yellowish ash (1-9-10) SP: Not produced ─────────────────────── ─────────────── Potato extract ・ G: Not so good, flat, yellowish ash (1-9-10) Ginseng extract agar M: Original traces formed, white R: Yellowish ash (2-9-11) SP: Not produced ───────────────────────── ──────────── G: growth, AM: aerial mycelium, R: back surface, SP: soluble pigment.

3. Physiological properties SANK 60 observed for 2 to 21 days after incubation at 28 ° C
The physiological properties of strain 791 are as shown in Table 5.

[0030]

[Table 5] ──────────────────────────────────── Starch hydrolysis Degradation Anionic gelatin Liquefaction Positive Nitrate reduction Positive milk coagulation Positive milk peptoneation Milk negative melanin-like pigment productivity (medium 1) ^* Negative (medium 2) ^* Negative (medium 3) ^* Negative substrate-degrading casein Negative tyrosine positive Xanthine Implicit salt tolerance (medium 4) ^* 3% ──────────────────────────────────── * : 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).

Further, the carbon source assimilating ability of the SANK 60791 strain observed after culturing at 28 ° C. for 14 days using the Pridham-Gotrieve agar medium (ISP 9) is as shown in Table 6.

[0032]

[Table 6] ──────────────────────────────────── D-glucose use D-fructose use L-Arabinose Weakly used L-Rhamnose Used D-xylose Used Sucrose Used Inositol Not used Raffinose Used D-mannitol Used Not used ──────────────── ───────────────────.

4. Cell components of the SANK 60791 strain were determined by the method of B. Becker et al. [B.
Becker et al., Applied Microbiology, 12: 421-423.
1984], meso-diaminopimelic acid was detected. In addition, the sugar component in the whole cell wall of the SANK 60791 strain was analyzed by MP Recheverier [MP Lech
Evalier, Journal of Laboratories and
Clinical Medicine (Journal of Laboratory & Cli
nical Medicine), 71, 834, 1968], and arabinose was detected. The presence of mycolic acid was not confirmed. The acyl type in the cell wall peptidoglycan was the acetyl type. In addition, MK-9 (H ₄ ) was detected as the major menaquinone component.

From the above, it is considered appropriate to judge that the present strain is an actinomycete belonging to the genus Amycolatopsis among actinomycetes.
colatopsis sp.) SANK 60791 strain (deposit institution, Institute of Industrial Science, Institute for Microbial Technology: Deposit No.
Issue 3 (FERM BP-3513): Original Deposit Date, August 14, 1991
Was named).

SANK 62390 strain and SANK 60791
The strains are identified by ISP [The International Streptomyces Project (The International Strept
omyces Project) Criteria, Bergey's Manual of Systematic Bacteriology (Bergey's Man)
ual of Systematic Bacteriology, Vol. 4, The Actinomycetes, Vol. 2, and recent literature on actinomycetes.

As is well known, actinomycetes naturally occur in
Alternatively, mutation is easily caused by artificial manipulation (for example, ultraviolet irradiation, radiation irradiation, chemical treatment, etc.), and the SANK 62390 strain and the SANK 60791 strain of the present invention have the same point. SANK 62390 strain and SANK referred to in the present invention
The 60791 strain includes all its mutants. Moreover, these mutants also include those obtained by genetic methods such as recombination, transduction, transformation and the like. That is, the SANK 62390 strain and SANK 60791 strain, the mutant strains thereof and strains which are not clearly distinguished from the strains producing the compound (I) are all SANK 62390 strains and
It is included in the SANK 60791 strain.

To obtain the compound (I), the cultivation of these microorganisms is carried out in a medium as 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, glucose, fructose, maltose, sucrose, mannitol,
Glycerol, dextrin, oats, 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, one volume of medium
Vary at -10% by weight.

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, malto extract, sodium nitrate, ammonium nitrate, ammonium sulfate and the like. The nitrogen sources are used alone or in combination within the range of 0.2-6% by weight of the medium amount.

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, etc. are used as an antifoaming agent.

The pH of the medium for culturing the SANK 62390 strain or the SANK 60791 strain and producing the compound (I) is 5.0-8.
Can be changed to 0.

The growth of the bacterium is good in the range of 22 ° C. to 38 ° C. Further, for the production of compound (I), 22 ° C. to 28 ° C.
Is preferred. Compound (I) 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.

In small-scale culture, it is preferable to carry out shaking culture at 28 ° C. for several days.

Cultivation is initiated by 1-2 stages of seed development in an Erlenmeyer flask equipped with baffles (water flow control wall). The seed developing medium can use a carbon source and a nitrogen source in combination. Seed flask in a constant temperature incubator 28
Shake at 7 ° C for 7 days or until full growth. The grown seed is used to inoculate a second seed medium or production medium. When using an intermediate development process,
It is used in part to grow and inoculate the production medium in essentially the same manner. The inoculated flask is shaken at constant temperature for several days and the contents of the flask are centrifuged or filtered after the incubation.

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. Sterilize the nutrient medium by heating to 125 ° C, cool and inoculate the sterile medium with the pre-grown seeds. Culture is 28
Performed by aeration stirring at ℃. This method is suitable for obtaining large amounts of compounds.

To determine the time course of the amount of compound (I) produced with the progress of culture, for example, compound (I) in the culture filtrate was adsorbed on Amberlite IRC-50 (NH ₄ ⁺ ). After washing with water, eluting with 0.5 N aqueous ammonia solution, concentrating, and measuring the amount of compound (I) in the freeze-dried powder by high performance liquid chromatography, or after acetylating compound (I), It is measured by gas chromatography-mass spectrometry (GC / MS). Usually, the maximum production of compound (I) is reached in 96 to 168 hours of culture.

After completion of the culture, the compound (I) existing in the liquid portion (and the cells) in the culture solution is separated from the cells and other solid portions by a filtration operation using diatomaceous earth as a filter aid or centrifugation. The compound (I) present in the filtrate or the supernatant can be extracted and purified by utilizing its physicochemical properties.

For example, the compound (I) present in the filtrate or the supernatant is treated with an ion exchange resin such as Amberlite IRC.
-50, CG-50, Dowex 50W x 4, Dowex S
It can be obtained by passing impurities through a BR-P layer to remove it, or by adsorbing compound (I) and then eluting with aqueous ammonia. Alternatively, as an adsorbent, for example, activated carbon or an adsorbent resin such as Amberlite XAD-2, XAD-4 (manufactured by Rohm and Haas Co.) and the like, Diaion HP-10, HP-20, HP-50, C
HP 20 (manufactured by Mitsubishi Kasei) is used. The liquid containing the compound (I) is passed through a layer of the adsorbent as described above to adsorb and remove impurities, or after adsorbing the compound (I), water such as methanol water and acetone water and an organic solvent are added. It is obtained by eluting with a mixed solvent of.

The compound (I) thus obtained is
Further, adsorption column chromatography using a carrier such as silica gel and Florisil, distribution column chromatography using Avicel (manufactured by Asahi Kasei Co., Ltd.), Sephadex LH-20 (manufactured by Pharmacia) and normal phase, reverse phase It can be purified by high performance liquid chromatography using a phase column or an ion exchange column.

In the culture of Micromonospora sp. SANK 62390 strain, which is a compound (I) -producing bacterium, trehazoline (Trehazoline (Treh) was added together with compound (I).
Azolin) (Japanese Patent Laid-Open No. 4-99792) is also produced.

[0052]

The compound (I) obtained by the production method of the present invention
Is a novel compound that has not been published in the literature and has sucrase inhibitory activity. Therefore, this compound is expected to be useful as an antiobesity agent and an antidiabetic agent. When the compound (I) obtained by the production method 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, tablets, It can be safely administered orally or parenterally in the form of capsules, injections and the like. Although the dose varies depending on the disease to be treated, the route of administration and the number of administrations, for example, for adults, it is preferable to administer 1 mg to 1000 mg daily in one or several divided doses depending on the symptoms.

[0053]

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 Inoculate the Amycolatopsis sp. SANK 60791 strain into two 500 ml Erlenmeyer flasks (with baffles) containing 80 ml of the medium shown in Medium Composition-1, and inoculate one platinum loop from a slant, and culture with shaking at 210 rpm. Incubated at 28 ° C for 96 hours.

[0055] Medium Composition -1 0.5% glucose 1% sucrose 1% Glycerin 1% oatmeal 0.5% raw yeast 1% soybean meal 2% casamino acids _{CaCO 3 0.1% CB-442 0.01} % ───────── ────────────────── pH before sterilization 7.0.

The medium represented by Medium Composition-2 was charged into two 30-liter jar fermenters, 15 liters each, and sterilized by heating at 120 ° C. for 30 minutes. Then, this was cooled to 28 ° C., and 75 ml of the seed culture was inoculated. Then, adjust the rotation speed in the range of 100-400 rpm to maintain the dissolved oxygen concentration at 2 ppm, and adjust the aeration rate.
The culture was carried out at 7.5 liter / min at 28 ° C for 144 hours with stirring.

Medium Composition-2 Glucose 2% Soluble Starch 1% Raw Yeast 0.9% Far Eastern Meat Extract 0.5% Polypeptone 0.5% NaCl 0.5% CaCO ₃ 0.3% CB-442 0.01% ───────────── ─────────────── Pre-sterilization pH 7.2.

(B) Isolation Celite as a filter aid was added to 25 liters of the obtained culture solution.
1.5 kg of 545 (manufactured by Johns Manville Product Corporation) was added and filtered to obtain 23 liters of a filtrate. The filtrate adjusted to pH 6.0 with hydrochloric acid was passed through a column packed with 3 liters of Amberlite IRC-50 (NH ₄ ⁺ ) to adsorb compound (I), washed with 15 liters of deionized water and washed with 0.5 N ammonia. Elution was performed with an aqueous solution. After the pH of the eluate became alkaline, 4.5 liters were collected and concentrated under reduced pressure to 200 ml. Then concentrate the Dowex 1X2 (OH ^-) was developed eluted with deionized water passed through a column packed 450 ml of. After removing 800 ml of the first effluent, the subsequent effluent was fractionated by 20 ml. Analysis was carried out by the quantitative method described below, and the fraction numbers 60 to 90 in which the compound (I) was recognized were collected. The obtained fraction was concentrated under reduced pressure and freeze-dried to give 16.6 mg of a white powder of compound (I).
was gotten.

The compound (I) was quantified by the following method. Quantitative method by high performance liquid chromatography Separation column; Asahi Pack ES-502 C (manufactured by Asahi Kasei Corporation) Mobile phase: 20 mM Ammonium acetate (pH 8.5) + 50 mM
Saline flow rate: 1 ml / min Detection wavelength: 210 nm Temperature: 25 ° C Hold time: 8.39 minutes.

Quantitative Method by GC / MS After making a sample a constant liquid volume, 10 μl of the sample was put into a reaction vial for acetylation and dried. To the residue were added acetic anhydride (30 μl) and pyridine (50 μl) at 60 ° C.
Heated at 40 minutes. Distill off excess reagent with nitrogen gas,
An internal standard substance (pentaacetyl-1-amino-1-deoxy-β-D-glucose) was added in a fixed amount, dissolved in 100 μl of ethyl acetate, and injected into GC / MS as a sample. The analysis conditions are as follows: Fused silica capillary column DB-5 (length 15 m
x ID 0.25 mm, 0.25 μm thin film, J & W SCIENTIFIC
Company) and helium as the carrier gas 5
Flowed at psi. The injector and interfuse temperatures were set to 250 ° C. 2 μl of the sample was injected splitless, and the column temperature was raised from 60 ° C to 280 ° C at 25 ° C / min.

Negative ions were detected by a chemical ionization method using methane gas using a quadrupole mass spectrometer Trio-1 (manufactured by VG). That is, an internal standard substance with a retention time of about 8.8 min, which is detected at an ion source temperature of 180 ° C, an ionization energy of 70 eV, and an ion source pressure of 1x10 ^-4 Torr, is about 8.8 minutes.
The negative ion peak of compound (I) at 9.6 min m / z 413 (both pentaacetate) was used for quantification. The content of compound (I) was calculated by the internal standard method.

Example 2. (A) Culture slant of Micromonospora sp. SANK 62390 strain
One tube is homogenized with 10 ml of physiological saline to prepare a bacterial suspension, and 1 ml of the suspension is inoculated into two 2-liter Erlenmeyer flasks containing 500 ml of the medium shown in Medium Composition-3. At 28 ° C on a rotary shaker at 210 rpm
It was cultured for 96 hours to obtain a primary seed culture.

[0063] 2% Medium Composition -3 glucose yeast extract (Difco) 0.5% polypeptone _{0.5% CaCO 3 0.1% CB-} 442 0.01% ───────────────────── ────── pH before sterilization 7.2.

30 liters of the medium represented by Medium Composition-3 was placed in a 60 liter jar fermenter, and
Heat sterilization was performed at 30 ° C for 30 minutes. Then add this to 28 ° C
After cooling to 600 ml, 600 ml of the primary seed culture was inoculated.
Next, this was rotated at 165 rpm and the ventilation volume was 15 liters /
The culture was stirred for 48 hours at 28 ° C for 48 minutes to prepare a second subculture.

300 liters of the medium represented by Medium Composition-4 was placed in a 600-liter tank and sterilized by heating at 120 ° C. for 35 minutes. Then, this was cooled to 28 ° C. and then inoculated with 15 liters of the second subculture liquid. Then, adjust the rotation speed so that the dissolved oxygen concentration is kept at 2 ppm.
Adjusted in the range of 82-142 rpm, air volume 150 liters /
Min, an internal pressure of 0.5 kg / cm ² , and stirring culture at 28 ° C. for 144 hours.

Medium composition-4 glucose 8% (separate sterilization 120 ° C, 15 minutes) Rastergen FK 2% Raw yeast 1.8% Far east meat extract 1% Polypeptone 1% NaCl 0.5% CaCO ₃ 0.3% K ₂ HPO ₄ 0.25% CB -442 0.02% ─────────────────────────── pH 7.2 before sterilization.

(B) Isolation In 300 liters of the obtained culture solution, Celite was used as a filter aid.
The filtrate was obtained by adding 15 kg of 545 (manufactured by Johns Manville Product Corporation) and filtering.
Got liters. Of this, 20 liters were added with hydrochloric acid to pH 6.
The compound (I) was adjusted to 0 and passed through a column packed with 3 L of Amberlite IRC-50 (NH ₄ ⁺ ) to adsorb the compound (I), washed with 15 L of deionized water, and then eluted with 0.5 N aqueous ammonia solution. After the pH of the eluate became alkaline, 4.5 liters were collected and concentrated under reduced pressure to 150 ml.
Then, the concentrated solution Dowex 1X2 (OH ^-) was developed eluted with deionized water passed through a column packed with 500 ml. Remove the first liter of effluent and then add 20 ml of subsequent effluent.
Fractionated one by one. Example 1. Analysis by the quantification method described in 1., the fraction number 58-
Collected 80. The active fraction was concentrated under reduced pressure and freeze-dried to obtain 9.6 mg of a crude powder of compound (I). This powder was purified again with a column of 100 ml of Dowex 1X2 (OH ⁻ ) to obtain 4.8 mg of a white powder of compound (I).

[0068]

Industrial Applicability The compound (I) of the present invention has a remarkable sucrase inhibitory activity and is useful as, for example, an antidiabetic agent, an antiobesity agent and the like.

Test Example 1. Inhibitory activity of compound (I) on rat sucrase The rat small intestine brush border membrane enzyme solution was prepared from M.K. Kessler et al. (M. Kessler et al., Biochimica et Biophysica Acta), 506
Vol. 136-154, 1978). That is, an enzyme solution was prepared from the small intestine of 3 Wistar rats (male) and suspended in 3 ml of physiological saline. 4-
Aminoantipyrine (A 4382) was purchased from Sigma as peroxidase (Grade I) and glucose oxidase (Gr
ade I) was purchased from Boehringer Mannheim. Hereinafter, 20 mM citric acid-40 mM disodium phosphate buffer (pH 6.2) was used for operations such as dilution.

To each well of a 96-well microtiter plate (Falcon), bovine serum albumin (Sigma, A 7906) 0.2 mg, glucose oxidase 3 unit
s, peroxidase 0.132 unit, 4-aminoantipyrine 20 μg, phenol 40 μg, sucrose 3 μmo
le and the measurement sample were added to make a total of 130 μl. Then, 20 μl of a 100-fold diluted solution of rat small intestinal brush border membrane enzyme solution was added thereto. 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.

Inhibition rates were calculated assuming that the glucose concentration when the measurement sample was not added during the enzyme reaction and when the enzyme solution was not added were 0% and 100% inhibition, respectively. Table 7 shows the 50% inhibitory concentrations of compound (I).

[0072]

[Table 7] ──────────────────────── Sample Sucrase 50% inhibitory concentration ───────────────── ──────── Compound (I) 14 μg / ml ──────────────────────── From Table 7, Compound (I) is remarkable. It showed excellent sucrase inhibitory activity.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI technical display location (C12P 17/14 C12R 1: 645) 7804-4B (C12P 17/14 C12R 1:29) 7804- 4B (72) Inventor Shuji Takahashi 1-58, Hiromachi, Shinagawa-ku, Tokyo Sankyo Co., Ltd. (72) Inventor Akira Sato 389-4 Oigata, Shimokawa, Izumi-cho, Iwaki-shi, Fukushima Sankyo Co., Ltd. (72 ) Inventor Yasuyuki Takamatsu 389-4 Oigata, Imo-cho, Iwaki-shi, Fukushima Sankyo Co., Ltd. (72) Inventor Ryuzo Enokida 33 Miyukigaoka, Tsukuba, Ibaraki

Claims (3)

[Claims] 1. A formula (I) belonging to the genus Micromonospora or Amycolatopsis. A method for producing a compound represented by the formula (I), which comprises culturing a bacterium producing the compound represented by the formula (1) and collecting the compound represented by the formula (I) from the culture. 2. The formula (I) belonging to the genus Micromonospora according to [claim 1] The production method in which the microorganism producing the compound shown by is the Micromonospora sp. SANK 62390 strain (Microtech Lab. No. 3521). 3. The compound of formula (I) belonging to the genus Amycolatopsis according to claim 1. The production method in which the compound-producing bacterium is Amycolatopsis sp. SANK 60791 strain (Microtechnology Research Institute, Article No. 3513).