JP3109923B2 - Manufacturing method of new trehazolin derivatives - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a novel trehazolin derivative having β-glucosidase inhibitory activity.

[0002]

2. Description of the Related Art Castanospermine, deoxynojirimycin, and the like are known as substances having a strong inhibitory activity on β-glucosidase, and have been reported as useful substances for antitumor, anti-AIDS, and the like. Yes (RAGruter
s et al., Nature, 330, 74-77, (1987
MJ Humphries et al., Cancer Res., Volume 46,
5215-5222, (1986) etc.). Therefore, compounds having β-glucosidase inhibitory activity are expected to be useful as antitumor agents and anti-AIDS agents.

The compound represented by the following formula (I) having the β-glucosidase inhibitory activity of the present invention is represented by the formula (I)
II)

[0004]

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The compound is also obtained by hydrolysis of a compound represented by the following formula (Japanese Patent Application No. 4-27901, EP 0499489):
A).

[0006]

The present inventors hydrolyze a compound represented by the following formula (II) to obtain
The inventors have found that a compound represented by the following formula (I) is obtained, thereby completing the present invention.

[0007]

The present invention provides a compound of the formula (II)

[0008]

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Which comprises hydrolyzing a compound of the formula (I)

[0010]

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The present invention relates to a method for producing the compound represented by the formula (1). Hereinafter, the compound represented by the formula (I) is referred to as a compound (I), and the compound represented by the formula (II) is referred to as a compound (I
I).

The hydrolysis reaction of the present invention is performed in the presence of an acid or a base. As the acid or base used,
There is no particular limitation as long as it is used in a usual hydrolysis reaction.

When an acid is used, examples of the acid to be used include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid and the like; sulfuric acid; perchloric acid; phosphoric acid; Or lower alkylcarboxylic acids such as formic acid, acetic acid, oxalic acid and trifluoroacetic acid; lower alkanesulfonic acids such as methanesulfonic acid, ethanesulfonic acid and trifluoromethanesulfonic acid; arylsulfonic acids such as benzenesulfonic acid and p-toluenesulfonic acid Organic acids such as; Preferably it is an inorganic acid, especially hydrochloric acid.

The reaction is usually suitably carried out in the presence of a solvent. The solvent to be used is not particularly limited as long as it does not affect the reaction. For example, alcohols such as methanol, ethanol, propanol and butanol; sulfoxides such as dimethyl sulfoxide and sulfolane;
Organic acids such as acetic acid and propionic acid; water; and the like. Preferably, it is water or a mixed solvent of water and an organic solvent.

The amount of the acid to be added is generally 1 to 20 mol, preferably 5 to 10 mol, per 1 mol of the starting compound. The reaction temperature is from room temperature to 150 ° C, preferably from 90 ° C to 110 ° C. The reaction time varies depending mainly on the reaction temperature, the reaction reagent or the type of the solvent used, but is usually 1 hour to 2 days, preferably 20 hours to 30 hours.

When a base is used, the base used is, for example, an alkali metal hydroxide such as lithium hydroxide, sodium hydroxide or potassium hydroxide; an alkaline earth hydroxide such as calcium hydroxide or barium hydroxide. Metal hydroxides; alkali metal carbonates such as sodium carbonate and potassium carbonate; inorganic bases or ammonia such as alkali metal halides such as sodium iodide, sodium bromide and potassium iodide; alkylamines such as triethylamine; Morpholine, N-ethylpiperidine,
Organic bases such as heterocyclic amines such as pyridine;

The reaction is generally and preferably effected in the presence of a solvent. The solvent used is not particularly limited as long as it does not affect the reaction. For example, alcohols such as methanol, ethanol, propanol and butanol; ethers such as tetrahydrofuran and dioxane; sulfoxides such as dimethyl sulfoxide and sulfolane ;
Water; and the like. Preferably, it is water or a mixed solvent of water and an organic solvent.

The amount of the base to be added is generally 0.01 to 10 mol, preferably 1 to 5 mol, per 1 mol of the starting compound. The reaction is carried out at a temperature of 0 ° C to 120 ° C, preferably room temperature to 100 ° C. The reaction time is
It depends mainly on the reaction temperature, the reaction reagents and the type of solvent used, but is usually 0.5 hours to 2 days,
Preferably, it is 1 hour to 20 hours.

In order to isolate the compound (I) from the thus obtained reaction solution in a pure manner, a method usually used for separation and purification of an organic compound, for example, chromatography using various carriers, alone or in combination with these compounds can be used. It is obtained by repeatedly performing the combination as necessary.

For example, after completion of the reaction, the reaction solution is subjected to an ion exchange resin, for example, Amberlite IRC-50, CG-50 (manufactured by Rohm and Haas), Dowex 50W × 4, Dowex SBR-P (Dow. (Emical)) to remove impurities by adsorption or by adsorbing compound (I) and then eluting with aqueous ammonia or hydrochloric acid. Alternatively, as an adsorbent, for example, activated carbon or adsorption resin Amberlite XAD-2, XAD-4 (manufactured by Rohm and Haas Co.), or Diaion HP-10, HP-20, CHP-20, HP- 50
(Mitsubishi Kasei Co., Ltd.) is used. Compound (I)
Is passed through a layer of the adsorbent as described above to adsorb and remove impurities, or after adsorbing compound (I), a mixed solvent of water and an organic solvent such as methanol water and acetone water is added. It is obtained by eluting with

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

The compound (I) thus obtained has the following properties. 1) Color and shape: basic white powder 2) Solubility: soluble in water. Insoluble in acetone and chloroform. 3) Color test: ninhydrin positive 4) Molecular formula: C ₆ H ₁₃ NO ₅ 5) Molecular weight: 179 (measured by FAB-MS spectrum) 6) Specific rotation: [α] _D ²⁵ −3.7 ° (c 0.51, (Water) 7) Ultraviolet absorption spectrum: λ _max nm (E (1 cm, 1%)) The ultraviolet absorption spectrum measured in an aqueous solution shows no characteristic absorption at 210 nm or more. 8) Infrared absorption spectrum: The infrared absorption spectrum measured on the ν _max (KBr) cm ^-1 KBr disk is as follows. 3384, 1582, 1474, 1380, 1040 9) ¹ H-nuclear magnetic resonance spectrum: δ ppm ¹ H-nuclear magnetic resonance spectrum (400 MHz) measured in heavy water using TMS (tetramethylsilane) as an external standard.
Is as follows. 3.12 (1H, d, J = 7.1 Hz), 3.56 (1H, d, J =
11.98 Hz), 3.62 (1H, d, J = 12.2 Hz), 3.62
(1H, d, J = 6.8 Hz), 3.78 (1H, dd, J = 5.5 and 6.8 Hz), 3.90 (1H, dd, J = 5.5 and 7.1 Hz)
) 10) ¹³ C-Nuclear magnetic resonance spectrum: [delta] ppm heavy water, ¹³ C-nuclear magnetic resonance spectrum measured using TMS (tetramethylsilane) to the external reference is as follows. 57.8, 61.0, 73.6, 79.4, 81.5, 81.7 11) Thin layer chromatography: Rf value; 0.39 adsorbent; silica gel plate Art 5715 (manufactured by Merck) Developing solvent: acetonitrile: acetic acid: water = 6: 1: 3.

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 hydroiodates, nitrates, perchlorates, sulfates, phosphates and the like. Inorganic acid salts; lower alkane sulfonates such as methanesulfonate, trifluoromethanesulfonate and ethanesulfonate; arylsulfonates such as benzenesulfonate and p-toluenesulfonate; -Organic acid salts such as phosphate, succinate, citrate, tartrate, oxalate, maleate and the like and amino acid salts such as glutamate, aspartate. Preferably, it is a pharmacologically acceptable salt.

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

The compound (II) as the starting compound of the present invention can be obtained as a microorganism product. Compound (I
Microorganisms used in the production of I) include, for example, Micromonospora sp. SANK 6 belonging to the genus Micromonospora.
2390 strain or Amycolatopsis sp. Belonging to the genus Amycolatopsis
sp.) SANK 60791 strain.

Micromonospora SP SANK 6239
The microbiological properties of the 0 strain are as follows. 1. Morphological Properties The SANK 62390 strain grows normally or slightly poorly at 28 ° C for 7 to 14 days on an agar medium for strain identification. The underlying mycelium elongates and diverges well, showing a bright orange, orange to dark brownish gray, but nocardia (Nocardi
a) Genus strain-like rupture and zigzag elongation are not observed. The aerial mycelium is slightly formed in the original trace and shows white to brownish white. The spores are observed only on the basal hypha, but form one at the tip of a relatively short spore stem and are spherical. The spore surface is smooth. No special organs such as sporangia, sclerotium, and axle branching are found.

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

[0028]

[Table 1] 種類 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: pale orange (3-9-6) SP: not produced ────────────────────────グ ル コ ー ス Glucose G: not very good, flat, yellowish orange (12-7-7) Asparagine agar AM: not formed R: pale 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 trace, White R: Brownish white (2-9-7) SP: Not produced ─────────────────────────────────栄 養 Nutrient agar G: Not very good, flat, yellowish orange (10-8-7) (DIFCO) AM: not formed R: dull yellowish orange (8-8-7) SP: not produced 産生イ ー ス ト Yeast extract G: Good, flat, dark brown ash (1 -4-6) Malt extract agar AM: Slightly formed, original trace, brownish white (1-8-6) (ISP 2) R: brownish black (1-2-6) SP: not produced せ──────────────────────────────── Oatmeal agar G: good, flat, orange (10-7-6) (ISP 3) AM: Slightly formed, trace of original, white R: orange (12-7-6) SP: not produced ────────────────────── ───────────── Water agar G: not very good, flat, pale yellowish orange (2-9-9) AM: not formed R: gray (N-5) SP: production Without ─────────────────────────────────── Potato extract ・ G: Good, flat, yellowish ash ( 1-9-10) Ginseng extract agar AM: Slightly formed, trace of original, white R: Brownish white (2-9-7) SP: Not produced ────────── ──────────────────────── G: growth, AM: aerial mycelium, R: back, SP: soluble pigment.

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

[0030]

[Table 2] 水 Hydrolysis of starch Liquefaction of positive gelatin Positive nitrate Reduction of negative milk Coagulation of positive milk Peptonization of positive milk Positive melanin-like pigment productivity (Medium 1) ^* Negative (Medium 2) ^* Negative (Medium 3) ^* Negative substrate degradability Casein Positive Tyrosine Negative Xanthine Negative Growth temperature range (medium 4) ^* 17-42 ℃ Suitable 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).

In addition, Prideham Gottlieb agar medium (I
(SP 9) was used for 14 days after incubation at 28 ° C.
Table 3 shows the assimilation of the carbon source of SANK 62390.

[0032]

[Table 3] ─────────────────────────────────── D-glucose used D-fructose used 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. About the cell components The cell wall of the SANK 62390 strain was determined by the method of B. Becker et al. (Applied Microbiology, 12, 421-423, 1984).
As a result, methodiaminopimelic acid and glycine were detected. The sugar components in the whole cell wall of the SANK 62390 strain were analyzed by MP Lechevalier (MP Lechevalier, Journal of Laboratory and Clinical Medicine).
ratory & Clinical Medicine), 71, 834, 1968
As a result, arabinose and xylose were detected. The presence of mycolic acid was not confirmed. The acyl form in the cell wall peptidoglycan was the glycolyl form. In addition, MK-10 (H ₆ ) and M
K-10 (H ₄ ) and MK-10 (H ₈ ) were detected.

From the above, it was found that this strain belongs to the genus Micromonospora among actinomycetes, and therefore, Micromonospora sp.
(FERM BP-3521: Depositary date: July 26, 1990).

Amycolatopsis SP SANK 6079
The bacteriological properties of one strain are as follows. 1. Morphological characteristics The SANK 60791 strain grows normally or slightly poorly on an agar medium for strain identification at 28 ° C for 7 to 14 days. The underlying mycelium elongates and branches well and becomes flat or wrinkled, showing brownish white, light yellowish brown or dull yellow. The aerial mycelium forms slightly poor or slightly traces of the original traces,
Shows pale yellow to pale orange. In the late stage of the culture, the division of the base mycelium and the aerial hyphae may be observed, and the bacillus-like structure may be observed in the aerial hyphae. Nocardia of mycelium (Nocar
dia) -like Zig-Zag extension is not observed.
No special organs such as sporangia, sclerotium, and axle branching are found.

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

[0037]

[Table 4] 種類 Type of medium Item Properties of strain SANK 60791 ── ───────────────────────────────── 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, pale orange (3-9-6) Asparagine agar AM: Slightly formed, yellowish ash (2-9-10) R: Light brown (3-8-6) SP: Not produced ─────────────────────────────────── Glycerin / G: Good, flat, brownish white (2-9-7) Asparagine agar AM: Originally formed, white (ISP 5) R: Yellow tea (6-8-8) SP: Not produced ─────────────────────────────────── Starch / Inorganic salt agar G: Not very good, flat, light yellowish tea (4-8-9) (ISP 4) AM: Originally traced, light yellowish (3-9-10) R: Light yellowish tea (6-8- 9) SP: Not produced ─────────────────────────────────── Tyrosine agar G: Very good, Wrinkled, brownish white (2-9-6) (ISP 7) AM: slightly formed, pale orange (3-9-6) R: pale orange (6-8-7) SP: not produced ─── ──────────────────────────────── Nutrient agar G: Good, flat, light yellow tea (4-8-8) ( DIFCO) AM: Originally traced, white R: pale yellow (3-9-8) SP: not produced ─────────────────────── ──────── ─── Yeast extract ・ G: good, wrinkled, dull yellow (10-7-9) Malt extract agar AM: trace formation, white (ISP 2) R: dull yellowish orange (10-7-8) ) SP: Not produced ─────────────────────────────────── Oatmeal agar G: Not very good, flat , Light yellowish tea (4-8-9) (ISP 3) AM: Originally traced, white R: Light yellow (4-9-9) SP: Not produced ─────────── ──────────────────────── Water agar G: Not very good, flat, yellowish gray (1-9-10) AM: Not so good, White R: Yellowish gray (1-9-10) SP: Not produced ──────────────────────────────── ─── Potato extract G: Not very good, flat, yellowish ash (1-9-10) Ginseng extract agar AM: Originally formed, White R: Yellowish gray (2-9-11) SP: Not produced ──────────────────────────────── ─── G: growth, AM: aerial mycelium, R: back, 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.

[0039]

[Table 5] 水 Hydrolysis of starch Liquefaction of negative gelatin Positive nitrate Reduction of positive milk Coagulation of positive milk Peptonization of positive milk Negative melanin-like pigment-producing (medium 1) ^* negative (medium 2) ^* negative (medium 3) ^* negative substrate degrading casein negative tyrosine positive Xanthine negative salt tolerance (medium 4) ^* 3% ％ *: medium 1; Trypton 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).

Table 6 shows the assimilation of the carbon source of the SANK 60791 strain, which was observed after cultivation at 28 ° C. for 14 days using Prideham Gottlieb agar medium (ISP 9).

[0041]

[Table 6] ─────────────────────────────────── D-glucose used D-fructose used L -Arabinose weakly used L-rhamnose used D-xylose used sucrose used inositol not used raffinose used D-mannitol used control not used ──────────────── ──────────────────.

4. Bacterial cell components The cell wall of SANK 60791 strain was determined by the method of B. Becker et al.
Becker et al., Applied Microbiology, 12, 421-423,
1984], meso-diaminopimelic acid was detected. The sugar components in the whole cell wall of the SANK 60791 strain were analyzed by MP Recheverrier [MP Lech
Evalier, Journal of Laboratory 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 acetyl type. Furthermore, MK -9 (H ₄₎ was detected as the major menaquinone component.

From the above, it is considered appropriate to judge that this strain is an actinomycete belonging to the genus Amycolatopsis among actinomycetes, and Amycolatopsis sp.
colatopsis sp.) SANK 60791 strain (deposited organization, Institute of Microbial Technology, National Institute of Advanced Industrial Science and Technology: Deposit No.
Issue 3 (FERM BP-3513): Original deposit date, August 14, 1991
Day).

The SANK 62390 strain and SANK 60791
The strain was identified by ISP [The International Streptmyces Project
omyces Project) standards, Bergey's Manual of Systematic Bacteriology (Bergey's Man)
Manual of Systematic Bacteriology, Volume 4, The Actinomycetes, Volume 2, and recent literature on actinomycetes.

As is well known, actinomycetes occur in nature,
Alternatively, mutations are liable to be caused by artificial operations (for example, ultraviolet irradiation, irradiation, chemical treatment, etc.), and the same applies to the SANK 62390 strain and SANK 60791 strain of the present invention. SANK 62390 strain and SANK
The 60791 strain includes all its mutants. These mutants also include those obtained by genetic methods, for example, recombination, transduction, transformation, and the like. That is, the SANK62390 strain and the SANK 60791 strain, the mutant strain thereof, and the strains that are not clearly distinguished therefrom all produce the compound (II).
It is included in the SANK 60791 strain.

To obtain compound (II), the culture of these microorganisms is carried out in a medium such as that used for producing other fermentation products. Such a medium contains a carbon source, a nitrogen source, and inorganic salts that can be assimilated by microorganisms.

In general, glucose, fructose, maltose, sucrose, mannitol,
Glycerol, dextrin, oats, rye, corn starch, potato, corn flour, soy flour, cottonseed oil, molasses, citric acid, tartaric acid, and the like can be used alone or in combination. In general, 1
Varies by -10% by weight.

As a nitrogen source, a substance containing a protein is generally used in the fermentation step. Suitable nitrogen sources include soybean flour, bran, peanut flour, cottonseed oil, cottonseed flour, casein hydrolyzate, pharmamine, fishmeal, corn steep liquor,
Peptone, meat extract, yeast, yeast extract, malt extract, sodium nitrate, ammonium nitrate, ammonium sulfate and the like. The nitrogen source is used alone or in combination in the range of 0.2-6% by weight of the medium volume.

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

For liquid culture, silicone oil, vegetable oil, surfactant and the like are used as antifoaming agents.

The pH of the medium for culturing the SANK 62390 strain or the SANK 60791 strain to produce the compound (II) is 5.0-5.0.
Can be changed to 8.0.

The growth of the bacterium is preferably in the range of 22 ° C. to 38 ° C., and the production of the compound (II) is preferably in the range of 22 ° C.
C is preferred. Compound (II) can be obtained by aerobic culturing, and aerobic culturing methods usually used, for example, a solid culturing method, a shaking culturing method, an aerated stirring culturing method and the like are used.

For small-scale culture, it is preferable to perform shaking culture at 28 ° C. for several days.

The culture is started in a conical flask with baffles (water flow control walls) by a 1-2 stage seed development process. The medium at the seed development stage can use a carbon source and a nitrogen source together. Seed flasks in a constant temperature incubator
Shake at 7 ° C for 7 days or until it has grown sufficiently. The grown seed is used to inoculate a second seed or production medium. When using an intermediate development step,
It is used in part to grow and inoculate the production medium in essentially the same way. The inoculated flask is shaken at constant temperature for several days, and after the incubation is completed, the contents of the flask are centrifuged or filtered.

In the case of large-scale culture, it is preferable to culture in an appropriate tank equipped with a stirrer and 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, inoculated with seeds that have been previously grown on the sterile medium. Culture is 28
It is carried out by aeration and stirring at ℃. This method is suitable for obtaining large amounts of compounds.

The compound (I) produced as the culture proceeds
To know the time-dependent change in the amount of I), for example, compound (II) in the culture filtrate is adsorbed on Amberlite IRC-50 (NH ₄ ⁺ ), washed with water, eluted with a 0.5 N aqueous ammonia solution, and concentrated. The amount of the compound (II) in the lyophilized powder is measured by high performance liquid chromatography, or the compound (II) is acetylated and then measured by gas chromatography-mass spectrometry (GC / MS). . Usually 9
After 6 to 168 hours of cultivation, the production of compound (II) reaches a maximum.

After the completion of the culture, the compound (II) present in the liquid part (and the cells) in the culture solution is separated from the cells and other solid parts by a filtration operation using diatomaceous earth as a filter aid or by centrifugation. The compound (II) present in the filtrate or supernatant can be obtained by extraction and purification utilizing its physicochemical properties.

For example, compound (II) present in the filtrate or supernatant is converted to an ion exchange resin such as Amberlite I
RC-50, CG-50, Dowex 50W × 4, Dowex
It is obtained by passing impurities through an SBR-P layer to remove impurities or by adsorbing compound (II) and then eluting with ammonia water or hydrochloric acid.
Alternatively, as the adsorbent, for example, Amberlite XAD-2, XAD-4 or the like, which is activated carbon or an adsorption resin, or Diaion HP-10, HP-20, CHP-20, HP-50 or the like is used. The liquid containing the compound (II) is passed through a layer of the adsorbent as described above to adsorb and remove impurities, or after adsorbing the compound (II), water such as methanol water and acetone water and an organic solvent are mixed. By using a mixed solvent of

Compound (II) thus obtained
Are further separated by adsorption column chromatography using a carrier such as silica gel or florisil, distribution column chromatography using Avicel, Sephadex LH-20, etc., and high-performance liquid chromatography using normal phase, reverse phase columns, and ion exchange columns. It can be purified by chromatography or the like.

In culturing Micromonospora sp. SANK 62390, which is a bacterium producing compound (II), Trehazolin was added together with compound (II).
(JP-A-4-99792) is also produced.

[0061]

The compound (I) obtained by the production method of the present invention
Is a novel compound not described in the literature and has β-glucosidase inhibitory activity. Therefore, the present compound is useful as an antitumor drug, an anti-AIDS drug and the like. When the compound (I) obtained by the production method of the present invention is used as a medicament, it is mixed with itself or an appropriate pharmaceutically acceptable carrier, excipient or diluent according to a conventional method to obtain a powder, granules, tablets, It can be safely administered orally or parenterally in the form of capsules, injections and the like. The dosage varies depending on the target disease, the administration route and the number of administrations. For example, it is preferable to administer 1 to 1000 mg per day to an adult in one or several doses depending on the symptoms.

[0062]

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

Embodiment 1 Production of Compound (I) 16 mg of Compound (II) powder was dissolved in 2 ml of 6N hydrochloric acid, placed in an ampoule, sealed and hydrolyzed at 100 ° C. for 24 hours. Water is added to the hydrolyzed solution, and concentrated to dryness under reduced pressure.The hydrochloric acid is distilled off.
The adjusted to 6.0 Amberlite ^{_{CG-50 (NH 4 +)}} 20 m
The compound (I) in the reaction solution was adsorbed through a column of 1 l, washed with 60 ml of deionized water, and eluted with 0.2 N aqueous ammonia. Concentrate the eluate under reduced pressure to obtain a concentrate
5 ml were obtained. The resulting concentrated solution is Dowex 1 ×
2 (OH ^-) subjected to a column packed with 50 ml, it was washed with 200 ml of deionized water. Then 5m with 20% methanol water
Each fraction was eluted. Fraction Nos. 5 to 23 having β-glucosidase inhibitory activity were collected, concentrated under reduced pressure, and freeze-dried to obtain 6.2 mg of compound (I) as a white powder.

[0064]

[Reference example]

Reference example 1. Production of Compound (II) (A) Culture Amycolatopsis sp. SANK 60791 strain was inoculated from a slant with a platinum loop into two 500 ml Erlenmeyer flasks (with baffles) containing 80 ml of the medium represented by Medium Composition-1. The cells were cultured at 28 ° C. for 96 hours in a rotary shaker at 210 rpm. Medium composition-1 Glucose 1% Sucrose 1% Glycerin 1% Oatmeal 0.5% Raw yeast 1% Soybean flour 2% Casamino acid 0.5% CaCO ₃ 0.1% CB-442 0.01% ──────────── PH pH 7.0 before sterilization.

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, after cooling to 28 ° C., 75 ml of a 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.
The cells were stirred and cultured at 7.5 liters / minute at 28 ° C. for 144 hours. Medium composition 1% raw yeast 0.9% -2 2% glucose soluble starch Far 0.5% meat extract polypeptone _{0.5% 0.5% NaCl CaCO 3 0.3} % CB-442 0.01% ─────────────── PH pH 7.2 before sterilization.

(B) Isolation Celite was added as a filter aid to 25 liters of the obtained culture solution.
1.5 kg of 545 (manufactured by Johns Manville Product Corporation) was added, and the mixture was filtered to obtain 23 liters of filtrate. The filtrate adjusted to pH 6.0 using hydrochloric acid is passed through a column packed with 3 liters of Amberlite IRC-50 (NH ₄ ⁺ ) to adsorb compound (II), washed with 15 liters of deionized water, and washed with 0.5 N ammonia Elution was carried out 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. The concentrated solution was then passed through a column packed with 450 ml of Dowex 1 × 2 (OH ⁻ ) and eluted with deionized water. 800 ml of first effluent
After removing the effluent, the subsequent effluent was fractionated in 20 ml portions. Analysis was performed by the quantitative method described below, and fraction numbers 60 to 90 in which compound (II) was recognized were collected. The obtained fraction was concentrated under reduced pressure and lyophilized to give compound (I) as a white powder.
I) 16.6 mg were obtained.

The compound (II) was quantified by the following method. Quantitative method by high performance liquid chromatography Separation column; Asahi Pack ES-502C (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 retention time; 8.39 minutes.

Quantitative Method by GC / MS After a sample was made up to a fixed volume, 10 μl thereof was placed in a reaction vial for acetylation and dried. Add 30 μl of acetic anhydride and 50 μl of pyridine to the residue and add to 60 ℃
For 40 minutes. Excess reagent is distilled off with nitrogen gas,
A fixed amount of an internal standard substance (pentaacetyl-1-amino-1-deoxy-β-D-glucose) was added, dissolved in 100 μl of ethyl acetate, and used as a sample to be injected into GC / MS. The analysis conditions were as follows: a gas chromatography column with a fused silica capillary column DB-5 (15 mx φ
0.25 mm, 0.25 μm thin film, manufactured by J & W SCIENTIFIC
5 psi with helium as carrier gas
Flowed away. Injector and interface temperature
The temperature was set at 250 ° C. Inject 2 μl of the sample splitlessly and raise the column temperature from 60 ° C to 25 ° C / min.
The temperature was raised to 80 ° C.

Using a quadrupole mass spectrometer Trio-1 (manufactured by VG), negative ions were detected by a chemical ionization method using methane gas. That is, an internal standard substance of m / z 388 with a retention time of about 8.8 minutes detected at an ion source temperature of 180 ° C, an ionization energy of 70 eV, and an ion source pressure of 1x10 ^-4 Torr.
The negative ion peak of compound (II) (both pentaacetate) at m / z 413 at 9.6 minutes was used for quantification. The content of compound (II) was calculated by the internal standard method.

The obtained compound (II) has the following properties. 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 ultraviolet absorption spectrum measured in an aqueous solution shows no characteristic absorption on the longer wavelength side than 210 nm. 7) Infrared absorption spectrum: ν _max (KBr) cm- ¹ 3358, 1668, 1528, 1398, 10668 8) ¹ H-nuclear magnetic resonance spectrum: δ ppm In heavy water, TSP (sodium trimethylsilylpropionate) is used as an internal standard. The nuclear magnetic resonance spectrum (500 MHz) measured as follows 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.
37 (1H, d, J = 8.79Hz), 5.03 (1H, dd, J = 8.79 and 2.
44Hz) 9) High performance liquid chromatography Separation column; Asahi Pack ES-502C (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 retention time; 8.39 minutes.

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

Medium composition-3 Glucose 2% Yeast extract (difco) 0.5% Polypeptone 0.5% CaCO ₃ 0.1% CB-442 0.01% PH pH 7.2 before sterilization.

Medium composition 30 liters of the medium represented by -3 was charged into a 60-liter jar fermenter, and
Heat sterilized at ℃ for 30 minutes. This is then brought to 28 ° C
After cooling, 600 ml of the primary seed culture was inoculated. Then, this was stirred and cultured at 28 ° C. for 48 hours at a rotation speed of 165 rpm and an aeration rate of 15 liter / min to prepare a second-generation seed culture solution. 300 liters of the medium represented by Medium Composition-4 was charged into a 600 liter tank, and sterilized by heating at 120 ° C for 35 minutes. Then, after cooling it to 28 ° C,
15 liters of the second-generation seed culture was inoculated. Then, the rotation speed was adjusted to 82-14 to maintain the dissolved oxygen concentration at 2 ppm.
The culture was adjusted within a range of 2 rpm, and cultured with stirring at 150 L / min, an internal pressure of 0.5 kg / cm ² and 28 ° C for 144 hours.

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

(B) Isolation Celite was added to 300 liters of the obtained culture solution as a filter aid.
Add 15 kg of 545 (manufactured by Johns Manville Product Corporation) and filter to obtain a filtrate 290
Liters were obtained. 20 liters of the solution are adjusted to pH 6.
Adjusted to 0, adsorbed compound (II) through a column packed with 3 liters of Amberlite IRC-50 (NH ₄ ⁺ ),
After washing with 15 liters of deionized water, elution was carried out with 0.5 N aqueous ammonia. 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 ^{1 X 2 (OH -) 500} m
l was passed through a packed column and eluted with deionized water.
After removing the first liter of effluent,
Fractionation was performed at 0 ml. Reference example 1. Analysis was performed by the quantification method described in the above, and fraction numbers 58 to 80 in which compound (II) was recognized were collected. The active fraction was concentrated under reduced pressure and freeze-dried to obtain 9.6 mg of a crude powder of compound (II). The powder again, Dowex 1 X 2 (OH ^-)
Purification with a 100 ml column yielded a compound (I
I) 4.8 mg was obtained. The properties of the obtained compound (II) are shown in Reference Example 1. It was the same as that obtained in.

[0076]

【The invention's effect】

Test example Inhibitory activity of compound (I) on β-glucosidase β-glucosidase (From Almonds), p-nitrophenyl-β-D-glucopyranoside, deoxynojirimycin,
Castanospermine was purchased from Sigma. 100 μl of 20 mM citrate-40 mM disodium phosphate buffer (pH 5.6) containing 0.01 Unit / ml β-glucosidase and each sample of compound (I), deoxynojirimycin and castanospermine was added at 37 ° C. After holding for 15 minutes, p-
50 μl of a buffer solution containing 3 mg / ml of nitrophenyl-β-D-glucopyranoside was added, and reacted at 37 ° C. for 20 minutes. Add 1 M glycine / NaOH buffer (pH 10.4)
Was added and the amount of released p-nitrophenol was reduced to 405
Measured by absorbance at nm. 50% of each sample in the reaction solution
The inhibitory concentrations are shown in Table 7.

[0077]

[Table 7] β Inhibitor β-glucosidase 50% inhibitory concentration ── ───────────────────────────────── Compound (I) 1.0 μg / ml deoxynojirimycin 15 μg / ml castano Spermine 4.3 μg / ml─────────────────────────────────── From above, the compound (I) of the present invention Has remarkable β-glucosidase inhibitory activity and is useful, for example, as an antitumor agent, an anti-AIDS agent and the like.

────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Shuji Takahashi 1-258 Hiromachi, Shinagawa-ku, Tokyo Sankyo Co., Ltd. Sankyo Co., Ltd. (72) Inventor Akira Sato, 389-4 Shimokawa, Izumicho, Iwaki, Fukushima Prefecture Sankyo Inside (72) Inventor Yasuyuki Takamatsu 389-4 Otsugi, Shimokawa-shi, Iwaki-shi, Fukushima Prefecture Inside Sankyo Co., Ltd. (72) Inventor Ryuzo Enoda 33 Miyukigaoka, Tsukuba, Ibaraki Prefecture In-house Sankyo Stock Company (56) Reference JP-A-5-65251 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C07C 215/44 C07C 213/00 CA (STN) REGISTRY (STN)

Claims (1)

(57) [Claims] 1. A compound of the formula (II) Which comprises hydrolyzing a compound represented by the formula (I): A method for producing a compound represented by the formula: