JPH0319239B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel amino-oligosaccharide derivative that specifically inhibits carbohydrate-degrading enzymes, and more particularly to an amino-oligosaccharide derivative represented by the following general formula []. (In the formula, m represents an integer of 0 to 12, n represents an integer of 1 to 13, and m+n represents an integer of 1 to 13.) Conventionally, aminooligosaccharide derivatives that inhibit carbohydrate degrading enzymes Examples include substances produced by strains belonging to the genus Actinoplanes (Japanese Patent Publication No. 54-39474 and Japanese Patent Application Laid-open No. 122342/1972), Streptomyces S.
Substances produced by the P.A2396 strain (Feikoken Bacterium No. 4275) (Japanese Unexamined Patent Publication No. 1983-92909), amylostatin (Japanese Unexamined Patent Publication No. 1983-21596, Unexamined Japanese Patent Publications No. 51-54990, and Unexamined Japanese Patent Unexamined Publications No. 1983) -68694), Streptomyces calbus varietus TM-521 strain
4283) (Japanese Patent Application Laid-open Nos. 123986/1986 and 125398/1982) and oligostatin (The Journal of Antibiotics Vol. 34, No. 11, p. 1429)
1981) are known. The present invention provides an amino-oligosaccharide derivative represented by the above general formula [], which is significantly different in chemical structure from these known substances and has a useful carbohydrate-degrading enzyme inhibitory effect. The derivative of the present invention [] has the following structural formula [] which has a platelet aggregation inhibiting effect when heated using 2.0-4.0 N dilute hydrochloric acid. It has the characteristic that it is hydrolyzed into the compound represented by (hereinafter tentatively referred to as NSO) and glucose. Typical compounds included in the derivatives of the present invention [] are illustrated below. The names given at the beginning of each exemplified compound represent the tentative names of those compounds in this specification, and m, n and m+n are the general formulas []
means what is expressed in . NS1; O-[4,6-bisdesoxy-4-
(2,3-epoxy-3-hydroxymethyl-4,
5,6-trihydroxycyclohexane-1-ylamino)-α-D-glucopyranosyl]-(1→
4) -α-D-glucopyranose (compound where m is 0 and n is 1) NS2;O-[4,6-bisdesoxy-4-
(2,3-epoxy-3-hydroxymethyl-4,
5,6-trihydroxycyclohexane-1-ylamino)-α-D-glucopyranosyl]-(1→
4) -o-α-D-glucopyranosyl-(1→4)
-α-D-glucopyranose (compound where m is 0 and n is 2) NS3; O-{4,6-bisdesoxy-4-
[5,6-dihydroxy-2,3-epoxy-4
-(o-α-D-glucopyranosyl-(1→4))-
3-Hydroxymethylcyclohexane-1-ylamino]-α-D-glucopyranosyl}-(1→4)
--α-D-glucopyranose (m is 1, n is 1
compound) NS4; O-[4,6-bisdesoxy-4-
(2,3-epoxy-3-hydroxymethyl-4,
5,6-trihydroxycyclohexane-1-ylamino)-α-D-glucopyranosyl]-(1→
4) -O-α-D-glucopyranosyl-(1→4)
-O-α-D-glucopyranosyl-(1→4)-α
-D-glucopyranose (compound where m is 0 and n is 3) NS5; O-{4,6-bisdesoxy-4-
[5,6-dihydroxy-2,3-epoxy-4
-(O-α-D-glucopyranosyl-(1→4))-
3-Hydroxymethylcyclohexane-1-ylamino]-α-D-glucopyranosyl}-(1→4)
-O-α-D-glucopyranosyl-(1→4)-α
-D-glucopyranose (compound where m is 1 and n is 2) NS6;O-[4,6-bisdesoxy-4-
(2,3-epoxy-3-hydroxymethyl-4,
5,6-trihydroxycyclohexane-1-ylamino)-α-D-glucopyranosyl]-(1→
4) -O-α-D-glucopyranosyl-(1→4)
-O-α-D-glucopyranosyl-(1→4)-O
-α-D glucopyranosyl-(1→4)-α-D-
Glucopyranose (compound where m is 0 and n is 4) NS7; O-{4,6-bisdesoxy-4-
[5,6-dihydroxy-2,3-epoxy-4
-(O-α-D-glucopyranosyl-(1→4))-
3-Hydroxymethylcyclohexane-1-ylamino]-α-D-glucopyranosyl}-(1→4)
-O-α-D-glucopyranosyl-(1→4)-α
-D-glucopyranose (compound where m is 1 and n is 3) NS8;O-[4,6-bisdesoxy-4-
(2,3-epoxy-3-hydroxymethyl-4,
5,6-trihydroxycyclohexane-1-ylamino)-α-D-glucopyranosyl]-(1→
4) -O-α-D-glucopyranosyl-(1→4)
-O-α-D-glucopyranosyl-(1→4)-O
-α-D-glucopyranosyl-(1→4)-O-α
-D-glucopyranosyl-(1→4)-α-D-glucopyranose (compound where m is 0 and n is 5) NS9; O-{4,6-bisdesoxy-4-
[5,6-dihydroxy-2,3-epoxy-4
-(O-α-D-glucopyranosyl-(1→4))-
3-Hydroxymethylcyclohexane-1-ylamino]-α-D-glucopyranosyl}-(1→4)
-O-α-D-glucopyranosyl-(1→4)O
-α-D-glucopyranosyl-(1→4)-O-α
-D-glucopyranosyl-(1→4)-α-D-glucopyranose (compound where m is 1 and n is 4) NS10; compound where m+n is 6 NS11; compound where m+n is 7 NS12; compound where m+n is 8 NS13 ; Compound where m+n is 9 NS14; Compound where m+n is 10 NS15; Compound where m+n is 11 NS16; Compound where m+n is 12 NS17; Compound where m+n is 13 The specific manufacturing method of the derivative of the present invention [] is detailed below. do. A method for producing coarse powder containing the derivative of the present invention []. A strain belonging to the species Streptomyces flavochromogenes is inoculated into a medium and cultured for 2 to 5 days, and then the resulting culture is transferred to a production medium and main culture is carried out for 3 to 8 days. Streptomyces
A representative strain belonging to the Flavochromogenes species is Streptomyces Flavochromogenes No. 280 (Feikoken Bacterial Serial No. 934), which is described in Japanese Patent Publication No. 11197/1983. In the medium used for seed culture and main culture, carbon sources include glucose, sucrose, lactose, maltose, oatmeal,
Starch, molasses or glycerin or a mixture of two or more of these, nitrogen sources include amino acids, cornstarch liquor, meat extract, wheat gluten, peptone, casein hydrolyzate, yeast extract, soy milk or inorganic ammonium salts. Alternatively, nitrates or a mixture of two or more of these are suitable. Magnesium, if necessary,
Inorganic salts such as phosphates, sulfates or hydrochlorides of calcium, potassium, sodium, iron, zinc or manganese may be added to the medium. Liquid culture is suitable for culture, and the composition of the medium is based on the carbon source mentioned above.
0.1-10.0% (w/v), nitrogen source 0.1-5.0%
(w/v) is appropriate. For both seed culture and main culture, the pH value of the medium at the start of culture is 6.5.
Carry out under aerobic conditions at a temperature of 25-40 °C, adjusted to ~8.5 °C. Appropriate culture methods include aerated agitation culture, reciprocating shaking culture, or rotary shaking culture. Centrifuge the culture obtained in the main culture described above,
Collect the supernatant. By this centrifugation, bacterial cells and unnecessary solid matter can be easily removed. The supernatant liquid obtained here was adjusted to pH 1.5 to 3.0 using mineral acids such as hydrochloric acid, sulfuric acid, or nitric acid, and activated carbon was added to this and stirred for 30 minutes to 2 hours, followed by vacuum or pressurization. Obtain the filtrate. The appropriate amount of activated carbon to be added is 1 to 10 g per 1 part of the supernatant liquid. The purpose of this treatment is to remove impurities and decolorize by adsorption. Next, after neutralizing the above liquid, activated carbon is added thereto again and stirred. After standing for 1 to 24 hours, the activated carbon is collected by centrifugation. Neutralization is carried out using a base such as sodium hydroxide, potassium hydroxide, sodium carbonate or ammonia. The appropriate amount of activated carbon is 20 to 50 g per liquid. Most of the derivative of the present invention [ ] is adsorbed on activated carbon. The collected activated carbon was washed with water, and then an organic solvent was added for 10 minutes.
The adsorbed derivative of the present invention [ ] is eluted using a water-containing organic solvent containing ~60%. Suitable organic solvents include methanol, ethanol, isopropanol, normal propanol, and acetone. In this specification, the mixing ratio of water and organic solvent in the water-containing organic solvent used is expressed as a percentage of the organic solvent volume to the total volume of the water-containing organic solvent, unless otherwise specified. Next, the eluate obtained in the above step is brought into contact with a strongly acidic cation exchange resin, either as it is or after being concentrated. As the contact method, either a batch method or a column method can be employed, but the column method is preferable. Amberlite IR- is a strong acidic cation exchange resin.
118, IR-120B, CG-120 (manufactured by Rohm and Haas) or Dowex 50W-X2
(manufactured by Dow Chemical Company) is suitable. The resin is then washed with water, and the adsorbed derivative of the present invention [] is eluted using a mineral acid, organic acid, or base, preferably an aqueous solution containing a mineral acid, and the eluate is collected. Mineral acids include hydrochloric acid, sulfuric acid, or nitric acid, organic acids include acetic acid, and bases include sodium hydroxide,
Potassium hydroxide, sodium carbonate or aqueous ammonia are suitable. The obtained eluate is neutralized, desalted, and dried under reduced pressure, and the residue is freeze-dried to obtain a coarse powder. Desalination is performed by adsorption/desorption treatment using activated carbon. Specific rotation of the coarse powder thus obtained: [α] ²⁰ _D is +
150 to +175° (C = 1.0, water), and the constituent sugar content based on the total weight of the powder was determined by the phenol sulfuric acid method (Biochemistry Experimental Structure 4 Chemistry of Carbohydrates (Part 2), page 370, May 10, 1976) (Published by Tokyo Kagaku Doujin) 58.3-81.6%
It was (W/W). In addition, the inhibitory activity of the powder against carbohydrate degrading enzymes, such as bacterial liquefaction α-amylase and glucoamylase, is 48 to 48%, respectively.
They were 438 units/mg and 315-4500 units/mg. Method for isolating and purifying the derivative of the present invention [] from crude powder. Compounds with m+n of 1 to 5 from coarse powder, e.g.
Isolation and purification of NS1-9 can be carried out by high performance liquid chromatography using a strongly basic anion exchange resin column or a reversed phase distribution column (hereinafter abbreviated as liquid chromatography). As a strong basic anion exchange resin column, Hitachi Gel #3011-N (manufactured by Hitachi, Ltd.)
Microbondapak CH (manufactured by Waters) is a reverse phase distribution column.
is appropriate. As the elution solvent, a mixture of 0.6 to 2.5 (volume ratio) of acetonitrile to 1 part of phosphate buffer having a pH of 7.0 or 65 to 80% aqueous acetonitrile is suitable. The fractionation of the eluate is determined by the retention time (hereinafter Rt) of each peak extracted on the detector recording paper.
It is advantageous to use the value (abbreviated as "value") as an index. Regarding NS1, 2, and 4, the method is to gel-filter the coarse powder (hereinafter abbreviated as gel-filtration method) or to acetylate the powder and then separate it by silica gel column chromatography, and deacetylate each acetylated product. It can also be isolated and purified by a method of oxidation (hereinafter abbreviated as acetylation method). The gel used in the gel filtration method is Biogel P-
2 (manufactured by Bio-Rad), but water is suitable as the elution solvent. In the acetylation method, the acetylation of the coarse powder is suitably carried out in pyridine using an acetylating agent such as acetic anhydride or acetyl chloride. Elution of the acetylated product adsorbed on the silica gel is carried out using a mixed solution of ethyl acetate and benzene, the mixing ratio of which is adjusted in stages. Specifically, first, the acetylated product of NS1 was eluted with a mixture of acetic acid ethyl ester and benzene at a mixing ratio of 1:3.0 to 3.5 (volume ratio; the same applies hereinafter), and then the acetylated product of NS2 was eluted with a mixed solution of ethyl acetate and benzene at a mixing ratio of 1:3.0 to 3.5. : Sequentially elute the acetylated product of NS4 with a mixture of 2.0 to 2.2 and 1:1.5 to 1.8. Deacetylation of the separated and eluted acetylated product is performed using methanol or ethanol saturated with ammonia. Each compound obtained by deacetylation is separately purified by column chromatography using a strongly acidic cation exchange resin. In addition, NS1 and 2 can be advantageously isolated and purified from crude powder by activated carbon column chromatography (hereinafter abbreviated as activated carbon method). For elution of NS1 and 2 adsorbed on activated carbon, water-containing normal butanol was used, and the mixing ratio was 3.
Linear concentration gradient elution method with an upper limit of %
Gradient Elution Method). In addition, in the above-mentioned liquid chromatography method, gel filtration method, acetylation method, and activated carbon method, the coarse powder is
Treatment with normal dilute hydrochloric acid or 1.0 to 4.0 normal trifluoroacetic acid aqueous solution to hydrolyze compounds with m+n of 6 to 13 in the coarse powder into compounds with m+n of 1 to 5 and neutral sugars such as glucose or maltose, and then If this is adsorbed and desorbed onto a strongly acidic cation exchange resin to be purified into a purified powder and then those methods are carried out, the isolation and purification of the target compound will proceed more advantageously. Next, a compound where m+n is 6 to 13, that is, NS10
The isolation and purification of ~17 involves adsorbing the crude powder onto activated carbon, thoroughly washing the activated carbon with 5-20% aqueous ethanol, and then eluting the adsorbed substance with 35-55% aqueous isopropanol or 40-60% aqueous ethanol. This can then be carried out by treating the eluted material with high performance liquid chromatography using a reverse phase partition column. By washing the activated carbon with 5 to 20% aqueous ethanol, it is possible to remove substances that are inconvenient in the next step of high performance liquid chromatography, such as compounds with m+n of 1 to 5 and some impurities. Isolation and purification of the target product can be advantageously achieved. Micro Bonder Pack is a reversed phase distribution column used in high performance liquid chromatography.
A CH column is suitable, and acetonitrile containing 55 to 70% water is suitable as the elution solvent. Fractionation of the eluate from the reverse phase distribution column is performed using the Rt value of each target compound as an index. The physicochemical properties of each compound obtained by the above production method were as shown in Table 1. Measurement of the molecular weight, Rt value, and carbohydrate degrading enzyme inhibitory activity of each compound, as well as a structure confirmation test for each compound, were performed as follows. Method for Measuring Molecular Weight The molecular weight of each compound was determined by using any of the methods shown in (a) to (c) below or by combining two or more methods and comprehensively judging the obtained results. (a) Mass spectrometry FD or EI mass spectra of the test compound or its methylated product were measured, and the molecular weight was calculated from the detected molecular ion peak. The methylated product of the test compound was determined using the Hakomori method (Journal of Biochemistry Vol. 55, 205).
p. 1967). (b) Degree of polymerization method Harada et al.'s method (Biochimica et Biophysica
AC-TA Vol. 237, p. 422, 1971) to determine the average degree of polymerization of the test compound.
was measured, and the molecular weight was determined from the degree of polymerization. (c) Gas chromatographic analysis method: Accurately weigh 5.0 mg of the test compound, add 2 ml of 2N diluted hydrochloric acid, and heat under reflux for 1.0 to 3.0 hours to hydrolyze the test compound into NSO and glucose. Dilute the reaction solution after hydrolysis with water to PH1.5
Adjust this to Dowex 50W−X2 (H ⁺ type)
column (diameter 0.6 x 8 cm), NSO is adsorbed on the stationary phase and glucose is allowed to flow out as is.
Glucose efflux is performed using 100 ml of water. NSO adsorbed on the stationary phase is completely eluted with a 3N ammonia aqueous solution. The number of moles of NSO in the resulting eluate is determined by gas chromatography. Quantification is performed using the absolute calibration curve method. On the other hand, the number of moles of glucose in the effluent is similarly determined by gas chromatography. The quantitative value of glucose is determined by measuring in advance the loss rate of glucose produced when a maltooligosaccharide corresponding to the test compound is treated under the above-mentioned hydrolysis conditions and column conditions using a gas chromatography method under the same conditions, and based on the loss rate. Corrections will be made. Conditions for gas chromatography are set as follows. Reagent: TMS-PZ (manufactured by Tokyo Kasei Kogyo Co., Ltd.; trimethylsilylating agent) Column: 3% SE-30, Chromosolve W-HP
(60/80) (Diameter 3.0 x 1000mm; Gascro Industries
Co., Ltd.) Column temperature: 130 to 230°C (by heating method of 10°C/min) Injection part temperature: 260°C Carrier gas: helium Flow rate: 40ml/min NSO and glucose obtained as above Their molar ratio was calculated from the number of moles of , and the molecular weight of the test compound was determined from this molar ratio. The molar ratio obtained here was used as part of the data for confirming the structure of the test compound. Rt value measurement method The Rt value was measured by high performance liquid chromatography using at least one of the following conditions A or B. Hitachi as a high performance liquid chromatograph
A model 638-50 (manufactured by Hitachi, Ltd.) was used, and a Shodex RISE-11 high-sensitivity differential refractometer (manufactured by Showa Denko K.K.) was used as a detector. The same equipment was used when high performance liquid chromatography was used as the isolation and purification method in the production examples described below. (a) Condition A Column: Hitachi #3011-N (diameter 2.6 x 500 mm) Column temperature: 50°C Elution solvent: Acetonitrile-0.02M phosphate buffer (PH7.0) mixture (volume ratio 3:2) Flow rate: 1.5 ml/min (b) Condition B Column: Microbondapak CH (diameter 3.9 x 300
mm) Column temperature: 20℃ Elution solvent: 65% aqueous acetonitrile Flow rate: 20ml/min Structural confirmation test method (a) Structure confirmation method for NS1 to 9 Methylation analysis method (Chemistry area special issue 132, page 29
(1981), test compounds were 1, 2, 3,
5,6-penta-O-methyl-4-mono-O-acetylglucitol, 2,3,4,6-tetra-
O-methyl-1,5-di-O-acetylglucitol or 2,3,6-tri-O-methyl-
The structure was confirmed by converting it into 1,4,5-tri-O-acetylglucitol or a mixture thereof and quantifying it. (b) Structure confirmation method for NS10-17 30 mg of the test compound was added to 40 mmol acetate buffer (PH
4.9) Dissolve in 1ml and add 10ml to 2ml of homogeneous buffer.
A solution containing mg of β-amylase (manufactured by Sigma) is added and reacted at 37°C for 6 hours. After the reaction, boil for 5 minutes on a hot water bath to inactivate β-amylase.
The reaction solution is then centrifuged to collect the supernatant.
Add dilute hydrochloric acid to this supernatant to adjust the pH to 1.5, and apply it to a Dowex 50W-X2 column (diameter 1.0 x 15
cm). After thoroughly washing the stationary phase of the column with water, the basic substance adsorbed on the stationary phase is eluted with a 2N aqueous ammonia solution. The resulting eluate is desalted by treatment with activated carbon and then dried under reduced pressure to obtain a residue. This residue was dissolved in 2 ml of 65% aqueous acetonitrile, and analyzed under the Rt value measurement method condition B described above. Measure and quantify relative weight ratios. The structure of the test compound was confirmed from this Rt value and relative weight ratio. Method for measuring carbohydrate degrading enzyme inhibitory activity (a) Method for measuring α-amylase inhibitory activity Add water or a test compound to 2 ml of 0.2 molar acetate buffer (PH5.5) containing 0.4% (W/V) soluble starch. After adding 1 ml of the aqueous solution and leaving it for 15 minutes at 37°C, add 1 ml of 0.2 M acetate buffer (PH5.5) containing 0.00004% (W/V) of bacterial liquefied α-amylase (crystal sample, manufactured by Sigma). Add and react at 37°C for 7.5 minutes. Next, add 5 ml of 1N diluted hydrochloric acid and stir at room temperature for 10 minutes.
Leave for a minute. Remove 1 ml from this reaction solution,
Add 10 ml of 0.0005N iodine aqueous solution and measure the absorbance at 660 nm. In a reaction system that does not contain the test compound, one unit is the amount of enzyme that reduces the iodine-induced coloration of soluble starch by 10% during the initial minute of the reaction, and one unit of α-amylase activity is expressed as one unit.
The activity of the test compound inhibiting 50% was defined as 0.5 unit (hereinafter abbreviated as AIU). (b) Method for measuring glucoamylase inhibitory activity Glucoamylase (crystal preparation derived from Rhizopus niveus, manufactured by Seikagaku Corporation) at 0.001%
(W/V) containing 0.2M acetate buffer (PH5.0)
Add water or 0.1 ml of an aqueous solution of the test compound to 0.4 ml,
Soluble starch 2.5% after reacting at 40℃ for 10 minutes
0.2N acetate buffer (PH5.0) containing (W/V)
Add 0.5 ml and react at 40°C for 15 minutes. This was then added with 3,5-dinitrosalicylic acid reagent (W.
Rick & H.P. Segbauer, Methods of Enzymatic
Analysis 885 pages 1975 Varlag Chemie,
Weinheim & Academic Press, New York,
London) to stop the reaction. The reaction solution was heated on a boiling water bath for 10 minutes, then cooled and heated for 10 minutes.
Add ml of water and measure the absorbance at 540 nm. In a reaction that does not contain inhibitors, the amount of enzyme that releases 0.1 mg of glucose per minute is expressed as 1 unit, and the activity of the test compound that inhibits 50% of 1 unit of glucoamylase activity is 0.5 units (hereinafter referred to as GIU).
(abbreviated as). The carbohydrate degrading enzyme inhibitory activity of the crude powder containing the derivative of the present invention [] was also measured by the method described here.

【table】

【table】

【table】

【table】

【table】

【table】

【table】

[Table] Next, the pharmacological effects of the derivative of the present invention [] based on the carbohydrate degrading enzyme inhibitory activity are shown using a test example of NS2. Hypoglycemic effect: Using 12 Wistar rats fasted for 24 hours, we investigated the effect of NS2 on the rise in blood sugar in rats after starch administration.
The inhibitory effect of The rats were divided into 2 groups of 6 rats each, one group received 2 g/kg of starch, and the other group received 2 g/kg of starch.
g/Kg and 250 mg/Kg of NS were orally administered, blood was collected after 2 hours, and the amount of glucose in the blood was measured by the orthotoluidine borate method (Clinical Pathology, Vol. 12, p. 434, 1964). The results were as shown in Table 2.

[Table] Lipid-lowering effect The lipid-lowering effect of NS2 was tested using 12 Wistar rats that had been fasted for 24 hours. 6 rats
Divide the fish into two groups, one with Triton WR-
1339 (manufactured by Sigma) 400 mg/Kg was administered subcutaneously, and on the other hand, Triton WR-1339 400 mg/Kg was administered subcutaneously, and 1 hour later, NS2 10 mg/Kg was administered orally.
Then, 24 hours later, the same amount of NS2 was orally administered again. Blood was collected 36 hours after administration of Triton WR-1339, and the total amount of cholesterol in the blood was measured using the Zak-Henly method.
American Journal of Clinical Pathology Volume 27
583, p. 1957). The results were as shown in Table 3.

[Table] As is clear from the above test examples, the derivative of the present invention [] is a compound useful as a therapeutic or preventive agent for diabetes, hyperlipidemia, obesity, etc. The present invention will be further explained using production examples and reference examples. Production example 1 Production of coarse powder Oatmeal 3.0% (W/V), peptone 1.0%
Streptomyces flavochromogenes No. 280 was inoculated into a sterile liquid medium adjusted to pH 7.8 containing (W/V) and 0.5% (W/V) sodium chloride. The seed culture was cultured with rotary shaking for 1 day to obtain 1630 ml of seed culture. This seed culture was transferred to production medium 150, which had been aseptically treated and had the same composition as the above-mentioned liquid medium, and main culture was carried out at about 30° C. for 6 days using a 300-capacity jar fermenter. At the start of main culture, the pH was 7.4, the aeration rate was 150/min, and the rotation speed was 250 rpm. Next, the culture obtained by main culture was centrifuged to obtain a supernatant liquid 133. This supernatant liquid was adjusted to pH 2.5 using 12N diluted sulfuric acid, 670g of activated carbon was added thereto, stirred for 1 hour, and filtered under pressure.
Liquid 131 was obtained. This liquid was neutralized with a 10 N aqueous sodium hydroxide solution, and 4600 g of activated carbon was added thereto and stirred.
It was left for 12 hours. After standing, activated carbon was collected by centrifugation. The activated carbon was washed with water, and then the adsorbent was added to the
Elute with % aqueous isopropanol, eluent 104
was collected. Dowex 50W-
Pass through an X2 (H ⁺ type) column (diameter 8.0 x 100 cm),
After thoroughly washing the stationary phase with water, the adsorbed substance was eluted with 2N diluted hydrochloric acid, and eluate 21 was collected. This eluate was neutralized using a 10N aqueous sodium hydroxide solution, treated with activated carbon to desalinate, dried under reduced pressure, and then freeze-dried to obtain the compound of the present invention []
148 g of pale yellow crude powder containing . The physical and chemical properties of the coarse powder were as follows. Specific optical rotation; [α] ²⁰ _D = +163° (C = 1.0, water) Constituent sugar content relative to total weight: 72.6% Carbohydrate degrading enzyme inhibitory activity: 309 AIU・mg 1412 GIU/mg Isolation and purification of NS1 to 9 (liquid Chloro method) 20.0 g of the above coarse powder was dissolved in 200 ml of 1N diluted hydrochloric acid, and the solution was stirred at about 128° C. for 10 minutes to perform hydrolysis.
The resulting reaction solution was diluted with water and adjusted to pH 1.5.
This was passed through a Dowex 50W-X2 (H ⁺ type) column (diameter 4.0 x 25 cm) and thoroughly washed with water. The substance adsorbed on the stationary phase of the column was eluted with a 1N ammonia aqueous solution, and this was dried under reduced pressure to obtain 7.2 g of purified powder. Approximately 200ml of this purified powder
of water, and add this to an activated carbon column (diameter 6.0 x
50cm). The stationary phase was washed with 5% aqueous ethanol, and then the substances adsorbed on the stationary phase were eluted with 40% aqueous ethanol. The obtained eluate was dried under reduced pressure to obtain 3.8 g of white powder. Dissolve this powder in 55 ml of 70% aqueous acetonitrile, and apply it using high performance liquid chromatography using a Microbondapak CH column (diameter 3.9 x 300 mm) (flow rate 2.0 ml/
min, elution solvent (70% aqueous acetonitrile)
Fractionation was performed using the Rt value as an index. The Rt values used as indicators were 4.8 minutes, 5.9 minutes, 6.9 minutes, 7.7 minutes, 8.9 minutes, and 9.9, respectively.
minutes, 11.2 minutes, 12.2 minutes and 14.5 minutes. Each of the obtained fractions was concentrated under reduced pressure to a volume of about 5 ml, and then centrifuged to collect the supernatant. These supernatants were dried under reduced pressure, and the resulting residues were vacuum-dried for one day and night.
mg NS2, 140mg NS3, 360mg NS4, 216mg
NS5, 204mg NS6, 128mg NS7, 150mg NS8
and 66 mg of NS9 were obtained as white powders. Production Example 2 Isolation and purification of NS1, 2 and 4 (gel filtration method) 700 mg of the crude powder obtained in the step of Production Example 1 was dissolved in 7 ml of 2N trifluoroacetic acid aqueous solution, and approximately
Hydrolysis was carried out at 120° C. for 20 minutes with stirring. The resulting reaction solution was diluted with water and adjusted to pH 7. This adjusted solution was passed through a Dowex 50W-X2 (H ⁺ type) column (diameter 2.0 x 10 cm), the stationary phase was thoroughly washed with water, and the substance adsorbed on the stationary phase was eluted with a 1N ammonia aqueous solution. The obtained eluate was dried under reduced pressure to obtain 360 mg of purified powder. Dissolve this purified powder in 2 ml of water and add it to Biogel P-2.
Gel filtration was performed using a (400 mesh) column (diameter 1.6 x 100 cm). Water was used as the elution solvent.
The flow rate was set to 2.0 ml/hour, and the column temperature was set to 55°C. Then, fractionated solutions whose Rt values were 3.3 minutes, 3.9 minutes, and 4.6 minutes, respectively, as measured under the conditions B of the Rt value measurement method described above, were collected. Each fraction collected was dried under reduced pressure and dried under vacuum overnight, resulting in 40mg of
NS1, 19 mg of NS2 and 10 mg of NS4 were each obtained as white powders. Production Example 3 Isolation and purification of NS1, 2 and 4 (acetylation method) 2.0 g of the crude powder obtained in the step of Production Example 1 was heated to approximately 95°C for 2 hours in 40 ml of a 2N aqueous trifluoroacetic acid solution. and hydrolyzed. The resulting reaction solution was diluted with water to pH 1.1 and applied to an Amberlite IR-118 column (diameter 2.0 x 20 cm).
After sufficient water washing, the substances adsorbed on the stationary phase were eluted with a 1N ammonia aqueous solution. This eluate was dried under reduced pressure to obtain 0.8 g of purified powder. This powder
40ml of acetic anhydride-pyridine mixture (volume ratio 1:
1) and left at room temperature overnight for acetylation. After 400 ml of ice water was poured into this reaction solution and stirred for 30 minutes, 150 ml of chloroform was added thereto. Then, separate the chloroform layer and add the layer to water.
It was washed successively with 300 ml, 300 ml of 0.05N diluted hydrochloric acid, and 300 ml of water, and dried over anhydrous sodium sulfate. The chloroform solution thus obtained was dried under reduced pressure to obtain 1.4 g of a purified powder acetylated product.
This acetylated product was dissolved in 10 ml of benzene and passed through a column using 100 g of silica gel (230-400 mesh) as a stationary phase. The substance adsorbed on the stationary phase was eluted with a mixture of acetic acid ethyl ester and benzene (volume ratio 1:3.0), and the resulting eluate was dried under reduced pressure to obtain 310 mg of acetylated NS1. Then, in the same manner, the adsorbed substances remaining on the stationary phase were sequentially eluted with a mixture of acetic acid ethyl ester and benzene prepared at a mixing ratio of 1:2.0 and 1:1.8, and the acetylated products of NS2 and 4 were each eluted with 113
mg and 73 mg were obtained. The acetylated product of each compound was deacetylated by stirring in an excess amount of ammonia-saturated methanol solution at room temperature for 12 hours. This reaction solution was dried under reduced pressure, and each of the resulting residues was dissolved in 3 ml of 0.05N diluted hydrochloric acid.
Dowex 50W-X2 each solution obtained separately.
(H ⁺ type) column (diameter 2.0 x 20 cm), and after thoroughly washing the stationary phase with water, the adsorbed substance was eluted with a 1N ammonia aqueous solution. When each eluate was dried under reduced pressure and dried under vacuum overnight, 165 mg of
NS1, 60 mg of NS2 and 38 mg of NS4 were each obtained as white powders. Production Example 4 Isolation and purification of NS1 and 2 (activated carbon method) 10.0 g of the crude powder obtained in the step of Production Example 1 was dissolved in 500 ml of 2N trifluoroacetic acid aqueous solution,
Hydrolysis was carried out by heating to about 95° C. for 2.5 hours while stirring.
Dilute the resulting reaction solution with water to a pH of 2.0,
Column of Amberlite IR-118 (diameter 4.0)
After thoroughly washing the stationary phase with water, the adsorbed substances were eluted with a 1N ammonia aqueous solution. This eluate was dried under reduced pressure, and the resulting purified powder was washed with water.
This was then passed through an activated carbon column (diameter 4.5 x 45 cm). The substances adsorbed on the column stationary phase were eluted with aqueous n-butanol at a mixing ratio of 3% by linear concentration gradient elution. Next, Rt under Rt value measurement method condition B mentioned above.
The fractions showing values of 3.3 minutes and 3.9 minutes were collected, respectively. Both collected fractions were separately dried under reduced pressure and dried under vacuum for a day and night, resulting in 510 mg of NS1 and 320 mg.
mg of NS2 white powder was obtained. Production Example 5 Isolation and Purification of NS10 to NS17 5.0 g of the crude powder obtained in the step of Production Example 1 was dissolved in 70 ml of water, and this was added to an activated carbon column (diameter 4.0
×60cm). The stationary phase was thoroughly washed with 10% aqueous ethanol, and the substance adsorbed on the phase was eluted with 50% aqueous isopropanol, which was dried under reduced pressure to obtain 4.1 g of white powder. 65% of this powder
Dissolve it in 82 ml of aqueous acetonitrile and apply it to a high performance liquid chromatography method (flow rate 2.0
ml/min, elution solvent (65% aqueous acetonitrile) using the Rt value as an index. The Rt values used as indicators were 7.5 to 8.8 minutes, 9.2 to 10.6 minutes, and 10.9 to 12.6, respectively.
minutes, 13.0-15.1 minutes, 15.7-18.0 minutes, 18.6-21.3 minutes,
They were 21.7 to 25.5 minutes and 26.2 to 30.6 minutes. Each fraction obtained was concentrated to a volume of approximately 5 ml,
Then, each supernatant was collected by centrifugation. These supernatants were dried under reduced pressure and dried under vacuum overnight, resulting in 245 mg of NS10, 271 mg of NS11, and 282 mg of NS11.
NS12, 310mg NS13, 174mg NS14, 69g
NS15, 42 mg of NS16 and 21 mg of NS17 were obtained as white powders, respectively. Reference Example Production of NSO 5.0g of the coarse powder obtained in the process of Production Example 1 was
Hydrolysis was carried out in 35 ml of 2.5N diluted hydrochloric acid by heating under reflux for 1.5 hours. This reaction solution was diluted with water and adjusted to pH 1.5.
This was passed through a column (diameter 3.0 x 30 cm) of Dowex 50W-X2 (H ⁺ type). After thoroughly washing the stationary phase of the column with water, the adsorbed substances were eluted with a 3N ammonia aqueous solution. This eluate was dried under reduced pressure,
The resulting residue was dissolved in 50 ml of water. This solution was passed through a column (diameter 3.0 x 20 cm) of Dowex 1-X2 (OH ^- type; manufactured by Dow Chemical Company), and the effluent was collected. Wash the stationary phase of the column with 800 ml of water,
The washing liquid and the effluent were combined and dried under reduced pressure to obtain 0.6 g of white powder. This powder was dissolved in 40 ml of acetic anhydride-pyridine mixture (volume ratio 1:1) and stirred at room temperature for 12 hours. This reaction solution was poured into ice water.
After pouring into 400 ml and stirring for 30 minutes, 150 ml of chloroform was added thereto. Then, separate the chloroform layer and add the layer to 900ml of water and 900ml of 0.05N diluted hydrochloric acid.
After sequentially washing with 900 ml of water and 900 ml of water, it was dried over anhydrous sodium sulfate. The obtained chloroform solution was dried under reduced pressure to obtain 1.1 g of residue. This residue was dissolved in 10 ml of toluene, passed through a column using 120 g of silica gel (70-230 mesh) as the stationary phase, and the substance adsorbed on the stationary phase was removed with a toluene-acetic acid ethyl ester mixture (volume ratio: 2: 1)
It was eluted. The obtained eluate was dried under reduced pressure,
490 mg of NSO hexaacetate was obtained. This compound was deacetylated by dissolving it in an excess amount of ammonia saturated methanol solution and stirring for 30 minutes. This reaction solution was dried under reduced pressure, and the resulting residue was
Dissolve in 10ml of 0.05N diluted hydrochloric acid and apply it to a Dowex 50W-X2 (H ⁺ type) column (diameter 2.0 x 20cm).
After washing the stationary phase thoroughly with water, remove the adsorbed substance by
It was eluted with a normal ammonia aqueous solution. This eluate was dried under reduced pressure and dried under vacuum for a day and night.
mg of NSO was obtained as a white powder. Elemental analysis (as C ₁₃ H ₂₁ NO ₈ ); Theoretical value (%) C, 48.90 H, 6.63 N, 4.39 Actual value (%) 48.88 6.78
4.38 Molecular weight; 319 EI mass spectrum m/z 319 M ⁺ FD mass spectrum m/z 320 (M+H) ⁺ Rt value (Rt value measurement method condition B); 2.6 min Specific optical rotation; [α] ²⁰ _D = +62° ( C=0.5, water) Ultraviolet absorption spectrum (aqueous solution); shows terminal absorption. Infrared absorption spectrum (KBr tablet, ν _nax cm ^-1 ); 3400, 2930, 1080 Solubility in solvents Easily soluble in water and methanol, soluble in ethanol and dimethyl sulfoxide, insoluble in benzene and normal hexane Basic, acidic, medium Melting point of hexaacetate of basic NSO; 204.5-206℃ Nuclear magnetic resonance spectrum of hexaacetate of NSO (200MHz, CDCl ₃ , ppm); 1.39 3H, d J = 6.34 2.03-2.10 18H 3.33 1H, d, d J ₁ = 8.30, J ₂ = 4.64 3.50 1H, d J = 2.44 3.74 1H, d, d J ₁ = 8.05, J ₂ = 2.44 3.84 1H, d J = 11.96 4.33 1H, d, d J ₁ = 8.05, J ₂ =7.32 4.43 1H, d J = 11.96 4.94 1H, d, d J ₁ = 6.34, J ₂ = 4.64 5.02 1H, d J = 3.91 5.30 1H, d, d J ₁ = 6.84, J ₂ = 3.91 5.32 1H , d, d J ₁ = 9.77, J ₂ = 7.32 5.57 1H, d, d J ₁ = 8.30, J ₂ = 6.84

Claims (1)

[Claims] 1. General formula (In the formula, m represents an integer of 0 to 12, n represents an integer of 1 to 13, and m+n represents an integer of 1 to 13.)