CS200173B2 - Process for derivatives of aminosugars - Google Patents

Abstract

New amino sugar derivatives of the formula wherein R is a glucose unit or an oligosaccharide chain of 2 to 7 glucose units glycosidically linked .alpha.-1:4 inhibit glycoside-hydrolases of the digestive tract. A representative embodiment is O-{4,5-bisdesoxy-4-{1 S-¢1,4,6/5)-4,5,6-tri-hydroxy-3-hydroxymethylcyclohex-2-en-1-ylamino!-.alpha.-D-gluco-pyranosyl}-(1?4)-O-.alpha.-D-glucopyranosyl-(1?4)-O-.alpha.-D-gluco-pyranosyl-(1?4)-D-glycopyranose. (R = 3 glucose units). In addition to this series of compounds, an isomeric series of amino sugars for compounds having three or more glucose units are simultaneously produced by Actinoplanacetes. The isomeric material having three glucose units is O-{4,6-bisdesoxy-4-{1 S-¢1,4,6/5)-4,5,6-dihydroxy-3-hydroxy-methyl-4-O-.alpha.-D-glycopyranosyl-(1?4)-cyclohex-2-en-1-ylamino!-.alpha.-D-glycopyranosyl}-(1?4)-O-.alpha.-D-glucopyranosyl-(1?4)-D-glucopyranose.

Description

BACKGROUND OF THE INVENTION The present invention relates to a process for the production of novel aminosugar derivatives which are used in medicine, particularly in diabetes and anti-obesity.

It is known that many Actionomycetes, especially from the Actionoplanaceae family, form inhibitors of glycoside hydrolase, especially those that break down carbohydrates in the digestive system. One group of these inhibitors is relatively heat-resistant, and at room temperature also resistant to acids and bases. These inhibitors belong chemically to the group of oligo * carbohydrates or polysaccharides and their derivatives.

Most inhibitors of this class are highly potent amylase inhibitors but only moderate or weak sucrase inhibitors. (NSR Unloading File No. 2,064,092).

It has now been found that novel aminosugar derivatives of the general formula can be obtained

up to 7 hexose units, characterized in that microorganisms of the family Actinoplanaceae strain SE 50/110 - CBS 674.73 are grown in an aqueous nutrient medium containing carbon sources, nitrogen and inorganic salts at pH 5, -01 to 8.5 and temperatures of 15 to 15 45 ^° C under aerobic conditions and the amino sugar derivative is isolated after the fermentation is completed, and optionally, in the manufacture of compounds in which R is 1 or 2 monosaccharide units, compounds in which R is more than 2 monosaccharide units are degraded by enzymatic incubation with hydro baths or by acid hydrolysis using aqueous solutions of inorganic acids.

Furthermore, novel amino-sugar derivatives have been found to be potent inhibitors of glycoside hydrolase in the digestive system. The lower members of this series in which R is an oligosaccharide chain with 1 or 2 hexose units mainly inhibit sucrose, the higher members mainly inhibit amylase.

It is surprising that the novel amino sugar derivatives of the invention have a higher specific activity against sucrose and amylase than the prior art inhibitors. It is particularly noteworthy that some of the novel amino sugars have several times higher potency in vivo than in vitro, where

290173

R is an oligosaccharide chain of 1

It is particularly surprising that the lower members of the homologous series of the invention act to inhibit glucose resorption in vivo. In view of these surprising properties, the novel aminosugar derivatives of the invention are a significant medical enrichment.

Microorganisms of the order Actionomycetales, of the family Actionoplanaceae, in particular Actinoplanes strains, are used for carrying out the process according to the invention. Examples include Actinoplanes SE 50 (CBS 961.70J, SB 18 (CBS 957.70) and SE 82 (CBS 615.71). These microorganisms are already described in South African Patent No. 71/8677 and are deposited under the numbers given in in brackets at Centralbureau vor Schlmmelcultures in Baarn, Holland.

Further, as described in South African Patent No. 71/8677, mutants or variants of the above strains can also be used to carry out the process of the invention. Particular preference is given to the overall yields of the novel aminosugar derivatives according to the invention and / or to the size of the residue R in the aminosugar derivatives mixtures formed during fermentation by strains SE 50/13 (CBS 614.71) and SE 50/110 '(CBS 674.73). Both strains correspond largely to the characteristics of the original SE 50 strain. These strains were obtained by natural selection from the SE 50 strain without the use of mutagens.

For carrying out the process according to the invention, a solid or liquid, in particular liquid, aqueous nutrient medium can be used which contains carbon sources, nitrogen sources, inorganic salts and an antifoam agent in suitable concentrations. These concentrations may vary within wide limits.

The carbon sources used are, in particular, carbohydrates, in particular starches, maltose, glucose or mixtures of two or three of these, or these complex mixtures as malt extract.

Complex mixtures commonly used in microbiology, such as casein hydrolyzate, yeast extract, pspton, fishmeal, maize extract, meat extract, fish meal solubles or mixtures thereof, as well as amino groups and / or ammonium salts, can be used from nitrogen sources. .

In the process of the invention, an aerobic shake culture is used with continuous aeration. Cultures in a solid fermenter can also be used. The type and concentration of the carbon source depends on the strain used and the desired end product, in particular the desired size of the residue R, to the extent that by selecting this source the individual members of the homologous series can be obtained in almost pure form. In addition, it has been found that, in nutrient media having a starch content higher than 2 · 0 / o, mainly amino-sugar derivatives with 4 to 7 hexose units are formed, the strain SE 50/13 (CBS 614.71) being particularly suitable for this type of fermentation. It is usually sufficient to add to a nutrient medium which already contains, for example, 3.5% glucose or a base source of carbon 0.1 to 3, preferably 0.5 to 2% starch to obtain a mixture of amino sugars containing 4 to 7 hexose units. Furthermore, it has been found that in the starch-free culture medium and especially after the addition of maltose, and in particular when using strain SE 50 (CBS 961.70), aminosugar derivatives containing in particular 2 to 3 hexose units are produced in particular. Nutrient solutions which contain glucose as the sole carbon source are particularly suitable for the preparation of the amino sugar derivatives of the invention in which R = glucose. If the nutrient solution contains glucose in excess, longer-chain amino-sugar derivatives are also formed during the further fermentation period. Apparently, during fermentation, nitrogen sources are depleted at the same time as glucose is depleted. When glucose is completely eliminated from the nutrient solution and maltose is used as the carbon source, predominantly aminosugar derivatives with 2 hexose units are obtained. In this case, pure maltose can be replaced by a cheaper mixture, for example Maltzin, a natural malt extract from Diamalt AG, in Munich. Since this preparation contains a greater amount of maltotriose, higher homologues are also formed in this case. Especially suitable for the production of lower homologs is the strain · SE 50/110, which can be used to obtain approximately twice the yield of lower homologues with optimal nutrient composition than with the strain SE 50/13.

Otherwise, during the fermentation, the composition of the nutrient solution, in particular the concentration of the nitrogen sources and the composition of the inorganic salts, can vary within wide limits.

The culture medium is sterilized by conventional means. The nutrient solution now has a pH in the range of 5.0 to 8.5, preferably in the range of 6.0 to 7.8. The fermentation temperature is in the range of 15 to 45 ° C, preferably in the range of 24 to 32 ° C. For the production of higher homologues using strains SE 50 and SE 50/13, the use of a higher temperature, for example 28 ° C, is preferable, for the production of lower homologues using strains SE 50 and SE 50/110, a lower temperature, for example 24 ° C. The fermentation takes 1 to 8 days, preferably 2 to 6 days. Longer fermentation times, especially when using excess carbohydrates, result in higher homologues. The end of fermentation is determined by determining the content and potency of the obtained products in enzymatic assays and thin layer chromatography to determine the composition of the nutrient medium.

The lower homologues of the amino sugar derivatives of the invention can also be obtained by cleaving the hexose units from the higher homologs. This cleavage can be carried out either with an aqueous solution of acids, in particular 1 to 5 N inorganic acids at temperatures of 50 to 100 ° C, preferably at a temperature range of 90 to 100 ° C, for a reaction time of 10 to 180 minutes. The same homologues can also be obtained by incubation with enzymes (hydrolase), in particular with jamylases or α-amylases which are not

200 173 incubated or with amyloglucosidases of microbial origin, such as Bacillus subtilis α-amylase or by incubation with microorganisms which are capable of growing in compositions obtaining the amino sugars of the invention at a concentration of 1 to 10%, preferably 2 up to 5%, such as. for example Aspergillus niger (ATCC 11394).

The isolation and purification of the amino-sugar derivatives according to the invention, either from a microbial nutrient medium or with an acid hydrolyzate, or from incubation mixtures in which the enzymatic and / or microbial cleavage of the higher homologues of the amino-sugar derivatives is carried out are carried out by generally known methods.

The methods of treatment are generally governed by the molecular weight region of the substances to be isolated. For higher homologues, precipitation can be carried out after previous discoloration and evaporation of the solutions.

In contrast, lower molecular weight homologues may be preferably isolated by adsorption to activated carbon at neutral pH followed by desorption using a mixture of water and alcohol or acetone, preferably at a concentration of 50 to 80% acetone. Desorption at a pH in the range of 1.5 to 4, preferably 2 to 3, can be completely achieved.

If the starting solutions are very dark in color, they must first be decolorised before adsorption of the compounds according to the invention, with activated carbon at an acidic pH of 1 to 3 or with non-specific resins, for example Lewapol R Ca 9221 / 0.35 mm Grain diameter (Bayer AG), in the range of pH 2 to 7, preferably 2 to 3. Activated carbon binds primarily in the acid medium dyes, whereas Lewapol adsorbs the dyes without adsorption of the amino-sugar derivatives of the invention at alkaline pH.

The weakly basic nature of these components is used to separate the amino sugar derivatives of the invention from the inactive carbohydrates and other inactive compounds. Homologous amino-sugar derivatives can be bound under suitable conditions at a pH of 1 to 8, preferably 2 to 4, at a lower ionic strength of the solution which corresponds to a conductivity of less than 10 mS, preferably less than 2 mS. strongly acidic cation exchangers, for example. Dowex R 50 W (Dow Chemicals), in the H + form. Particularly well, the aminosugar derivatives according to the invention can be bound from an acetone solution containing 50 to 80% acetone at a pH of from 1 to 5, preferably from 2 to 4, to cation exchangers which, under these conditions, have a considerably higher capacity. Except where the solution contains less than 50% acetone, the compounds also bind to weak acidic ion exchangers, for example Amberlite IRC-50 in H + form.

Aqueous solutions of bases or acids, preferably ammonia or hydrochloric acid, are used for desorption of the aminosugar derivatives according to the invention from cation exchangers.

From the desorbate, the aminosugar derivative is obtained after neutralization with a corresponding weakly acidic or weakly basic ion exchanger or removal of the base or acid by evaporation under vacuum in the case of a volatile base or acid, or evaporation of the solution by lyophilization or precipitation with an organic solvent. up to 20 volumes of acetone.

Further, the low molecular weight aminosugar derivatives of the present invention can be separated from the inert saccharides by chromatography on cellulose-based ion exchangers, preferably phosphocellulose (Serva, Heidelberg). Buffers, preferably phosphoric buffers of low ionic strength, preferably 2 to 100 mM, in particular 5 to 10 mM, in the pH range of 2.5 to 8, preferably 5 to 6, are used for elution. salts in the preparation to be fractionated, while dyes are far less detrimental to fractionate.

Individual members of the homologous series of aminocoar derivatives according to the invention can be purified, for example, by chromatography of partially purified preparations on a suitable molecular sieve, for example using Bio-Gel R P-2, Bio-Rad, Munich, with individual fractions being monitored. ^ γι phii on a thin layer. Fractions containing pure derivatives of ammonium sugars are combined, re-chromatographed and, after evaporation, lyophilized or precipitated with organic solvents as described above.

The novel compounds of the invention belong, by their chemical nature, to the carbohydrate class. It forms homologous series, the basic member of which is the following amino-sugar derivative (C19H33O13N), in which R = hexose.

снрн H

OH OH

The higher members of the homologous series may be derived from the base member such that they additionally contain a carbohydrate unit, preferably hexose, in particular glucose.

All members of this homologous series are further characterized by the fact that complete acid hydrolysis produces component I (C13H19O7N) and the corresponding monoscharium. The following formula was derived for component I.

An example of the structure principle of the homologous series may be one in which the oligosaccharide chain consists of 1 to 7 glucose units. The following formula shows a chain with η = 1 to 7 glucose units.

The base member of the board is obtained if η = 1 and is called component II. Each member of the series differs from the nearest lower or the next higher missing or excess glucose unit.

Compared to component II, one higher glucose unit is referred to as component III, two glucose units higher as component IV, and so on.

All of these components have the common property of providing component I and glucose when fully acid hydrolyzed.

Component II can be described with respect to its physicochemical properties, structure and reactivity as follows:

Component II is a colorless, amorphous solid that is readily soluble in water, dimethylformamide, dimethylsulfoxide, and metal hydroxides. It is also soluble in ethanol in heat.

i Thin layer chromatography

Solvent: ethyl acetate, metal hydroxide and water 10: 6: 4.

Commercially available films are used to perform thin layer chromatography:

I. Silica gel films F 1500 from Schlecher + Schüll

RR values: II.46 maltose 0.50 glucose 0.65

II. The F 254 plates from Merek

RR -I0.47 maltose0.54 glucose0.66

Component II is best stained on silica gel plates with a mixture of silver nitrate and sodium hydrolyte. . The brownish-black color develops already at room temperature or after slight heating.

component II is silylated together with or. sucrose as an internal standard in a 1: 0.5: 1 mixture of pyridine, trimethylchlorosilane and N-methyl-trimethylsilyltrifluoroacetamide.

Column:

6-meter glass column filled with 3% SE 30 on WAW chromasorb

Temperatures:

injection block 300 ° C detector 300 ° C furnace 220 ° C until elution of the α- and β-D-glucose peak, then temperature program 15 ° C / min up to 300 ° C.

Detector:

FIG (flame ionization detector)

Gases:

carrier gas: - N2 40 ml / min combustion gas: air 80 · ml / min Hz 20 ml / min Do'ba - retention: α-D-glucose 2 min / S-D-glucose-α 4 min sucrose 12 to 13 min II 16 to 17 min

c) Angle of rotation

A sample of component II obtained by evaporating the methanol solution of component II in a non-crystalline state is dissolved in water and the specific rotation [α] _D = + 134.3 ° is determined.

d) Sj ^ (^ l ^ t] um in infrared light:

The spectrum in the form of KBr pellets is poorly distinguishable, * the main absorption bands are in the region of the O-H and C-O groups when the valences move.

b) Gas chromatography

To carry out the gas chromatography,

200173 9 10 e) NMR spectrum in CD5OD at 220 MHz: (Fig. 1) in ppm Multiplicity Relative intensity 1.3 doublets, J = 6.5Hz 3 H 2.3 triplets, Ji and J2 8—10Hz 1 H 3.15 triplets, Ji and J2 7—9Hz 1 H 3.3 - 3.9 signals cannot be individually assigned 12 H 4.13 AB-system, J = 12Hz 2 H 4.48) j doublets, J = 7Hz) ) doublets, J = 2.5Hz) 1 H 5,1) 4.9 singlet 11 H protons exchanged for deuterium 5.0 doublets, J = 2—3Hz (difficult to distinguish) 1 H 5.8 doublets, J = 3-4Hz (difficult to distinguish) 1 H

Η

(f) Acetylation of component II:

Component II is converted in a 1: 1 acetic anhydride / pyridine mixture at room temperature to a 903 molecular weight decaacetyl derivative. When the reaction is carried out in a 1: 1 mixture of ethyl acetate and acetic anhydride in the presence of catalytic amounts In addition to the decaacetyl derivative, the formation of undecaacetyl derivatives with a molecular weight of 945 can be detected by mass spectroscopy.

NMR spectrum of decaacetyl derivative: (Fig. 2)

Mass spectrum of decaacetyl derivative:

vertex for molecule vertex for · base

903 (2.5% relative intensity)

843

Important peaks for fragments in the indicated mass spectrum:

844 (55% relative intensity)

784 (36% relative intensity)

783 (34% relative intensity)

759 (35% relative intensity)

556 (36% relative intensity)

496 (37% relative intensity)

436 (29% relative intensity)

(g) methylation of component II:

Methylation of component II with a mixture of methyl iodide and sodium hydride in dimethylsulfoxide according to the method of Hakomori yields the decamethyl derivative of component II as a major product, and also a small amount of the undecamethyl derivative.

Mass spectrum:

peak for the molecule peak for the base

2nd peak for a molecule

623 (6.1% relative intensity)

535

637 (0.2% relative intensity)

According to spectroscopic data and chemical properties, component II has the following formula:

OH

(a) Thin-layer chromatography:

Solvent:

ethyl acetate, metal hydroxide and water in a ratio of 10: 6: 4.

I. Silica gel films F 1500 (Schleicher a

Schull):

Component II is a mixture of α- and d-forms, as evidenced in particular by the NMR spectrum.

Another member of the homology series is component III ('C25H43O18N), which can be characterized according to their physico-chemical properties, structure and reactivity as follows:

Component II is an amorphous solid, well soluble in water.

R-values of component III: 0.35 maltose .0.50

II. Silica gel plates F 254 (Merck):

Rf-values: component III0.33 maltose 0.54

III. is colored black-brown with a mixture of silver nitrate and sodium hydroxide already at room temperature or with slight heating.

(b) Infrared spectrum:

The spectrum in the infrared light in the form of KBr pellets is very difficult to read and difficult to read. The main absorption bands are in the range of the O-H and C-O valencies. (3700-3100 cm ^-1 and 1180-950 cm -1).

(c) Angle of rotation:

[a] n in water: ψ 147.2 °

d) The NMR-s, spectrum of component III in D0 at 220 MHz is shown in Figure 3.

(e) Hakomori methylation:

Methylation of component III with methyl iodide and sodium hydride in dimethylsulfoxide affords component III, methylated with 13-methyl groups, to a small extent also 14-methyl groups, as the main product.

(f) Mass spectrum of methylated product:

The peak for the molecule at 827 (1.5% relative intensity). corresponds to the general formula C38H69NO18. At 841 there is a second peak with 0.1% relative. intensity.

The most important peaks for fragments are:

739 (27% relative intensity)

592 (3.7% relative intensity)

535 (30% relative intensity)

38.8 - (9 ° / o - relative intensity)

386 (13% relative intensity)

284 (13% relative intensity)

187 (12% relative intensity)

171 '- (40% relative intensity)

101 (34% relative intensity) (25 - relative intensity)

- Top for principle

For component III, the following formula is in agreement with the chemical and spectroscopic properties:

Component IV is another higher member of the homologous series. This compound can be cleaved by partial acid hydrolysis to component II and glucose in a ratio of 1: 2.

Thin layer chromatography:

Solvent:

n-butanol, ethanol and water in a ratio of 50: 30: 20

Chromatography is performed with -silica gel-plates F 1500 (Schleicher - | + Schull):

Rf value for glucose: component IV: 0.41 to 0.46.

Higher homologues, particularly components V-VIII, inhibit far less sucrose and α-amylase from the pancreas-far more strongly than the lower members of the series (components II-IV).

In acid hydrolysis of the higher components of the homologous series, inferior members of the series can always be detected as intermediates and, in addition, glucose and maltose as cleavage products.

Thin layer chromatography:

Solvent:

n-butanol, ethanol, water in a 50: 30: 20 by volume ratio

Chromatography is carried out on silica gel F 1500 plates (Schleicher - ^ - Schull):

Rglucose:

Component V: 0.30- to 0.34 Component VI: 0.21 to 0.23 Component VII: 0.14 to 0.16 Component VIII: 0.09 to 0.11

The specific inhibitory effects of the aminosugar derivatives of the present invention can be ascertained by in vitro enzymatic assays.

Amylase test

An amylase inhibitory unit (1 AIE) is defined as the amount of inhibitor that inhibits 50% of two amylase units.

One amylase unit (AE) is the amount of enzyme enzyme that cleaves one minute of the starch glycosidic bond under the given conditions per minute. The microequivalent of the cleaved compound is determined polarimetrically as one microequivalent of the reducing sugar formed by dinitrosalicylic acid and is reported by means of a maltose calibration curve as the equivalent of the maltose microequivalent. To perform the test, mix 0.1 ml of a 20 to 22 AE / ml amylase solution with 0 to 10 µg inhibitor or 0 to 20 µl of the test solution in 0.4 ml of 0.02 M sodium glycerophosphate buffer / 0.001 M CaCl 2 pH 6.9 and the mixture was allowed to reach equilibrium for 10-20 minutes in a 35 ° C water bath. The mixture is then incubated for 5 minutes with 0.5 ml at 35 ° C of a pre-heated 1% starch solution (soluble starch from Merck, Darmstadt No. 1252) at 35 ° C and then mixed with 1 ml of dinitrosalicylic acid (P. Benfeld in Collo Wick-Kaplan, Meth Enzymol., Vol. 1, p. 149). To produce a color, the test is heated in a boiling water bath for 5 minutes, then cooled and mixed with 10 ml of distilled water. Extinction at 540 nm sc is measured against an amylase-free blank. The evaluation is carried out according to a predetermined amylase calibration curve by subtracting the amylase activity remaining after addition of the inhibitor and calculating the percent inhibition of the amylase used. This inhibition is applied as a function of the quotient ________________ / zg inhibitor + ________________ AE + + ^b relative to the dry matter ^{+ +} AE in the sample without inhibitor of the same series

The 50% inhibition point is read from the curve and the result is converted to AIE / mg inhibitor.

Sucrose test

A sucrose inhibition unit (SIE) is defined as that amount of inhibitor that inhibits two sucrose units by 50%. The sucrose unit (SE) is the amount of enzyme that cleaves 1 µm-1 sucrose to glucose and fructose per minute under these conditions. The micromoles of the glucose formed are quantitated by the glucose oxidase reaction under conditions in which the saccharase cannot be further cleaved by the action of sucrose. The test is carried out by mixing 0,05 ml per 0,12 SE treated sucrose solution with 0 to 20 micrograms inhibitor or 0 to 20 microlitres of the test solution and making up to 0,1 ml with 0,1 M buffer sodium maleate, pH 6.0. [Soluble saccharase from porcine small intestine mucosa prepared according to B. Brogstrom, A. Dahlquist, Acta Chem. Scand. 12, (1958), 1997. This sucrose is diluted to the corresponding SE content with 0.1 M sodium maleate buffer pH 6.0.]. The test is allowed to equilibrate for 10 minutes at 35 ° C and then mixed with 0.1 ml at 35 ° C of a pre-heated 0.05 M sucrose solution in 0.1 M sodium maleate buffer pH 6.0. The mixture was incubated for 20 minutes at 35 ° C and then sucrose, quenched by the addition of 1 ml glucose oxidase reagent and the mixture was further incubated for 30 minutes at 35 ° C. (Glucose oxidase reagent was prepared by dissolving 2 mg of glucose-oxidase [Boehringer # 15,423] in 100 ml of 0.565 M Tris-HCl buffer pH 7.0 and then adding 1 ml of detergent solution [2 g tritone X 100 + 8 g 95% analytically pure ethanol], 1 ml dianisidine solution (containing 2160 mg of o-decanisidine X 2 HCl in 20 ml of water and 0.5 ml of a 0.1% aqueous solution of peroxidase from Boehringer No. 15,302). 1 ml of 50% sulfuric acid is then added and the extinction is measured at 545 nm against the corresponding blank test. The percentage inhibition of sucrose used is calculated and the SIE / g or SIE / liter is calculated by means of a calibration curve.

Maltase test

A maltase inhibition unit (MIE) is defined as that amount of inhibitor that inhibits two maltase units to 50%. Maltase unit (ME) is the amount of enzyme which cleaves 1 micromole of maltose into 2 micromoles of glucose per minute under the specified conditions. The micromoles of glucose formed are determined quantitatively by the glucose oxidase reaction under conditions where maltose can no longer be broken down by maltase. . This test is carried out by mixing together 0,05 ml of maltase solution, adjusted to 0,060 to 0,070 ME, with 0 to 20 micrograms of inhibitor or 0 to 2-0 microlitres of the test solution and replenish the test. to a volume of 0.1 ml with 0.1 M sodium maleate buffer pH 6.0. The test is allowed to equilibrate at 35 ° C and then mixed with 0.1 ml at 35 ° C of a pre-heated 0.05 M maltose solution in 0.1 M sodium maleate buffer pH 6.0. (Solubilized pig small intestinal mucosa is used according to B. Borgstrom, A. Dahlquist, Acta Chem. Scand. 12, (1958), 1997. This maltase is diluted with 0.1 M sodium maleate buffer pH 6.0 for the corresponding ME content). The mixture is then incubated for an additional 20 minutes at 35 ° C, then the maltase reaction is stopped by adding 1 ml glucose-o-oxidase reagent and incubated for an additional 30 minutes at 35 ° C. (Preparation of the glucose oxidase reagent is performed as described in the sucrose assay.). Then 1 ml of 50% sulfuric acid is added and the extinction is measured at 545 nm against the corresponding blank test.

200

For evaluation, the percent inhibition of the maltase used is calculated and the amount that inhibits 50% is calculated using a glucose calibration curve and this amount is converted to MIE / g and M, respectively ^; IE / liter.

Results of enzyme inhibition tests

n - number of hexose units AIE / g amino sugar derivative o n = 1 300000 n = 2 3000000 n = 3 1 400 000 n = 4-6 17 500 000 n = 5-7 30 -000,000

¹⁶ .

in vitro for the individual homologues of the aforementioned homologous series and the efficacy of the individual homologues of the series of amino-sugar derivatives of the invention are shown in the following table, in which the hexose units are glucose units.

SIE / g MIE / g

0005 000

000 15 * 000

000 - -5 000

500—

500—

As can be seen from the table, the specific activity against α-amylase from the pancreas with increasing molecular weight in the homologous series increases very sharply. In the region of n = 5 to 7, a 100-fold increase in in vitro potency is observed over the potency of an aminosugar derivative in which n = 1 or n = 2. On the other hand, the inhibition of sucrose is most strongly expressed in η - 1 inhibits this activity by only half, and for higher derivatives the specific activity against sucrose decreases.

In vivo, the activity of the derivatives of the invention against sucrose occurs in a sucrose loading assay approximately parallel to the course of specific in vitro activity. An in vivo starch load on the organism has been found to have amino sugar derivatives in which n = -1,2 and 3 have 10 to 40 times greater potency compared to the anti-amylase activity in vitro. (These facts are detailed in Example 18, Tables 1, 3, 4, and 5.)

It is known that in animals - in humans - carbohydrate-containing foods are used, for example after ingestion of - carbohydrates, potato starch, vegetables, - fruit juice, beer or - chocolate to raise blood sugar, which is caused by rapid degradation of carbohydrate glycoside hydrolase, for example, small-intestinal and pancreatic amylases, maltases and -saccharases according to the scheme amylase maltase starch or glycogen -> maltose - glucose sucrose sucrose -> glucose + fructose

This increase in blood sugar is particularly strong in diabetics and lasts very long. In obese people, an increase in blood sugar levels after a meal causes particularly strong insulin secretion, which again results in increased fat formation and reduced fat breakdown. This increase in blood sugar often results in a lowering of the blood sugar level in both normal and obese people due to insulin secretion. It is known that this hypoglycemia promotes the production of gastric juice, which may cause inflammation of the gastric mucosa, gastric ulcer or duodenal ulcer or aggravate these diseases, if they have already occurred.

It has now been found that glycoside hydrolase inhibitors produced and isolated by the method of the invention both reduce elevated blood sugar levels after loading rats / humans with wheat insulin and consequently lower blood sugar levels after loading rats / humans with wheat starch, sucrose or maltose while promoting passage of these carbohydrates through the stomach. In addition, they reduce glucose absorption in the intestine. Furthermore, there is also a reduction or deterioration - conversion of carbohydrates to lipids of adipose tissue and impaired incorporation of fat, taken in food into the adipose tissue.

Furthermore, it is known that carbohydrates in the oral cavity, particularly sucrose, are cleaved by the action of microorganisms, resulting in tooth decay.

Thus, glycoside hydrolase inhibitors are useful for medical use in the following indications:

Obesity, elevated blood lipid levels (arteriosclerosis), diabetes, prédiabetes, gastritis - gastric ulcer, gastric ulcer, duodenal ulcer and dental caries.

Benefits

It is administered - 30 to 300,000 AIE / kg or 1 to 10,000 SIE / kg once or several times a day - before and / or between and / or after eating or drinking orally.

Pharmaceutical forms

Dragee, tablets, capsules, solutions, suspensions, granules, chewing gum, toothpaste or as an additive to food and / or foodstuffs containing carbohydrate.

The toxicity of the glycoside hydrolase inhibitors of the present invention and glucose uptake inhibitors is very low. The active ingredient of Example 9, as a 26,000 SIE / g crude inhibitor, was tolerated in the same way as the other Examples at 340,000 SIE / kg in both mice and rats without any symptoms. A dose of 10,000 SIE / kg without symptoms was tolerated in intravenous injections in mice.

Combinations of inhibitors of the invention with known oral antidiabetics (β-cytotropic sulfonylurea derivatives and / or biguanides that affect blood sugar) are very preferred.

Below is a more detailed explanation of the reagents and excipients used:

For example, commercially available products such as Amberlite IRA 410 Cl (anion exchanger), Amberlite IRC 120 in H + form (strong acid cation exchanger), Amberlite (НСО'з form) (anion exchanger), Amberlite IRA 410 OH ( Amberlite IRC 50 in H ⁺ form (weakly acidic cation exchanger), all products of Rohm and Ha as, Dowex 50 WX 4H ⁺ (strong acidic cation exchanger), product of Dow Chemical Co. Midland, Michigan, USA.

For example, DE AE-cellulose from Schleicher and Schull can be used from cellulose-based anion exchangers.

For example, Biogel-P-2 (Bio-Rad Laboratories, Richmond, California, USA) can be used as the polyacrylamide gel.

Maltzin is a natural malt extract from Diamalt AG, Munich (Germany).

Lewapol ^R is a non-specific adsorption resin of Bayer AG, Leverkusen (NSR).

Clarcel is a filtration aid from CECA, Velizy-Villacoublay (France).

SE 30 is a silicone elastomer from Hewlett Packard.

In the following examples, hexose units represent glucose units.

Example 1

In the examples, the starting material is a preparation which can be prepared as follows:

In a glass fermenter, 8 liters of nutrient solution containing 5.0% starch, 1.0% yeast extract, 0.2% K2HPO4 are inoculated with a 3 day old shaking culture of strain SE 50/13 (CBS 614.71) and the fermenter is then aerated 3. days at 28 ^Q C. with vigorous stirring to obtain a fermentation broth at a concentration of 105,000 А1Е / / mL.

liters of this fermentation broth are brought to pH after cooling to 20 ° C

2.5 using 50% nitric acid, 30 g of activated carbon (Carboraffin) are added to the broth and mixed for 10 minutes, then centrifuged at 10,000 rpm for 15 minutes and the clear, light yellow supernatant is collected. after neutralization with ammonia, evaporate to a volume of 500 ml. 500 ml of this concentrate is mixed with 200 g of Amberlite IRA 410 Cl for 45 minutes, filtered with suction and then mixed with 4/5 of its volume = 400 ml of methanol to precipitate the bulk of the high molecular weight products by starch degradation and activated carbon residues . The mixture is centrifuged at 5,000 rpm for 5 minutes and 850 ml of the supernatant thus obtained is added dropwise to 4 liters of anhydrous ethanol with vigorous stirring. The white, flaky precipitate was suction filtered, washed with 3X anhydrous ethanol and 2X ether and then dried in vacuo at 50 ° C. The yield was 36 g of a 10 * 10 ⁶ AIE / g white powder.

Example 2

To assess the resulting fermentation product and to determine the composition of this preparation, thin-layer chromatography is carried out by applying 1 microliter of fermentation medium or 1 microgram preparation to a silica gel film (Schleicher and Schull, Dassel, type F 1500) and develops 2 X in a 50: 30: 20 n-butano / ethanol / vcda system.

The developed and well dried plate was sprayed with enzymatic gel (20 ml using a 20 x 20 cm plate) to stain the sucrose and to determine its inhibition and the gel was allowed to solidify. Then, the plate is preincubated in a humid chamber at room temperature for 5 minutes and finally sprayed with substrate gel. After this second gel layer has solidified, the plate is transferred to a damp chamber and incubated at 40 ° C. Inhibition is shown by light spots on a reddish-brown background, these spots developed after 60 to 90 minutes. As soon as the coloring is optimal, the incubation is discontinued and the plate and agar layers are dried with hot air by the bitch.

Preparation of gel

Enzymatic gel: 1.5 g of agarose (I / Industrie Biologique France) are suspended in 100 ml of 0.2 M sodium maleate buffer, pH 6.0 and dissolved by boiling. The clear agar solution is cooled to 50 ° C and mixed with 250 microliters of a Triton X-100 solution (2 g Triton X-100 + 8 g analytically pure ethanol) and 0.5 ml dianisidine solution (20 mg dianisidine). 1 ml acetone) by inversion. 1 ml of GOD / POD reagent (12.5 mg glucose oxidase, grade I, Boehringer, No. 15423, and 2.5 mg peiroxidase, grade II, Boehringer, No. 15302, dissolved, were added immediately prior to gel usage. in 5 ml of maleic buffer) and 4 to 5 saccharase units from the small intestine of pigs, prepared as described above in the sucrose assay. The gel can be maintained at 50 ° C until sprayed, otherwise it will solidify in the nozzle.

Substrate gel: 0.5 g of agarose is suspended in 100 ml of sodium maleate buffer at pH 6.0 and dissolved by boiling. The mixture was then cooled to 50 ° C, treated with 100 microliters of Triton (2 g Triton · X-100 T 8 g analytically pure ethanol) and 1 g sucrose (Seeva # 35579) was added. After dissolving sucrose, the gel is ready for use.

To color the amylase inhibition, the developed and dried thin layer chromatography plate was sprayed with 20 ml amylase gel onto a 20 x 20 cm plate and allowed to solidify. Then the plate is preincubated for 5 minutes at room temperature and transferred to a 0, 5% starch solution (1 g starch Gauge No. 1252 in 200 ml of 0.2 M glycerophosphate buffer with 0.01 M CaCl 2, pH 6.9 dissolve by boiling], leave in this solution at 40 ° C for 2 minutes, then rinse well with desalinated water and transfer to undiluted starch to dilute iodine solution (4 ml of iodine base solution to 500 ml of water, base water). iodine solution consists of 2.2 g of I2 + 4.4 g of KI in 100 ml of water) After 1 minute the color is optimal, the plate is photographed immediately because the blue spots disappear quickly.

Preparation of amylase g agarose gel is dissolved in 100 ml of 0.2 M sodium glycerophosphate buffer and 0.01 M CaCl 3 buffer pH 6.9 at 100 ° C, after cooling to 50 ° C, 100 µl of Triton are added. X-100 2 g Triton X-100 + 8 g analytically pure ethanol). Just prior to spraying, 100 microliters of the crystalline amylase suspension (10 mg of pancreatic pancreas amylase / ml in Boehringer ammonium sulfate solution # 15017) was added.

Example 3

1 liter Erlenmeyr flask containing 120 ml of nutrient solution consisting of 4% starch, 2,4% glucose, 0,9% casein hydrolyzate and 0,9% yeast extract pH 7,6, adjusted Sodium hydroxide is mixed with 0.4% calcium carbonate and sterilized for 30 minutes at 121 ° C, then seeded with 3 ml of a pre-culture of strain SE 82, which was grown in a nutrient solution made of 2% starch. , 1% glucose, 0.5% casein hydrolyzate and 1% yeast extract, after mixing, the pH was adjusted to 7.2 with sodium hydroxide, then 0.4% calcium carbonate was added and the medium was allowed to stand for 30 minutes sterilized at 121 ° C, inoculated, and fermented for 5 days at 28 ° C on a rotary shaker to obtain a fermentation medium containing 122,000 AIE / ml. The mycelium was separated from the culture medium by centrifugation at 12,000 rpm, the pH of 300 ml of the culture filtrate was adjusted to 2.5 with 50% nitric acid, and the mixture was stirred with 2.5 g of charcoal for 10 minutes. After 'separating the activated carbon by centrifugation at 12 000 rpm', the solution is neutralized to pH 6 with 10 N potassium hydroxide and mixed with 300 ml of methanol, allowed to stand for a short period of time and the precipitate formed is separated by centrifugation at 12 000 rpm. minute. The supernatant was added dropwise to 3 liters of ethanol, the precipitate was isolated after short standing by centrifugation at 12,000 rpm and washed with 2 X absolute ethanol and once with ether, and then dried in vacuo to give 2.23 g. The product has a concentration of 1.45 X 1 (® AIE / g, the product also contains more than '95% higher homologues of amino-sugar derivatives on> 4%).

He did

In a 1 liter Erlenmeyer flask pour 120 ml of a nutrient solution of composition

3.5% glucose, 2% starch, 0.5% casein hydrolyzate, 1.3% yeast extract, 0.3% CaCO 3 and -0.3% CaHPO 3, pH adjusted to 7.8 prior to sterilization and content The flasks are then sterilized for 30 minutes at 121 ° C and seeded with 6 ml of a pre-culture of strain SE 50/110 in nutrient medium containing 3% soy flour, 3% glycerin and 0.2% CaCO 3, fermentation is carried out 3 to 5 hours. 4 days on a rotary shaker at 24 ° C to obtain a fermentation broth containing 153,000 AIE / ml and 1220 SIE / liter.

1 liter of this fermentation broth is adjusted to pH 2.5 with nitric acid, left with 5 g of activated carbon for 10 minutes and then centrifuged at 5,000 rpm for 30 minutes, then neutralized by adding 25 g of IRA 410 in OH- form.

Evaporate the neutral solution to a volume of 100 ml, mix with 100 ml of methanol and filter. The filtrate is stirred into 2 L of absolute ethanol, the precipitate formed is collected by suction filtration, washed with 3 X acetone and ether and dried in vacuo.

In this way, 14 g of a white powder having a concentration of 5 * 10 < ⁶ > AIE / g with predominantly homologues of >

Example 5

The procedure is as in Example 4, except that -0.5% starch is added to give a fermentation broth containing 40,000 AIE and 184 SIE / ml after fermentation for 4 days. The nutrient medium contains homologues in which n = 1.

Example 6

Pour 120 ml of 3% glucose nutrient solution, 0,6% casein hydrolyzate into a 1 liter Erlenmeyer flask,

1.6% yeast extract, 0.3% CaCC3, 0.3% K2HPO4, the pH is adjusted to 7.8 with sterile potassium hydroxide and the flask is seeded with a pre-culture of strain SE 50/110 according to Example 4 and fermentation is performed 4 days at 24 ° O on a rotary shaker to obtain a fermentation broth of 10,800 SJE / liter containing mainly O1 n = 1.

liters of culture filtrate separated from mycelium at 13,000 rpm was adjusted to pH 2.5 with 50 ^l % nitric acid and stirred for 15 minutes with 35 g Merck activated carbon and 2'00 g Clarcel. The mixture was filtered with suction, neutralized with concentrated ammonia to pH 7, and evaporated to volume

1.5 liters and precipitated with 5 times the amount of ethanol. The flocculent precipitate was collected in a flow centrifuge at 12,000 rpm, the yellowish supernatant was evaporated to 150 ml and centrifuged again. Apply 50 ml of the solution to a column of Amberlite IR-12 (30 X 300 mm, 30 ml of water per hour). When a total of 300 ml of eluate containing inert carbohydrates and a fraction of unadsorbed inhibitor is removed, the ion exchanger with 400 ml of water is beakers and concentrated ammonia are added to pH 11.5 After 30 minutes of further stirring, the ion exchanger is separated, the solution is evaporated to 1/20 by volume, filtered through a column of IRA 410 ' X 150 ml at a flow rate of 30 ml per hour, collecting 500 ml of the eluate and evaporating to give 1.3 g of crude product after lyophilization.

For further purification, the preparation was fractionated on Bto-gel P-2 100-200 mesh (Bio-Rad, Munich). A 50 x 450 mm column and water were used at a flow rate of 400 ml / hour, fractions were collected. Each fraction was assayed by anthron method for carbohydrates and with a charase assay for inhibitors. Fractions containing sucrose inhibitor were examined by thin-layer chromatography according to Example 2 for the contents of the individual components. They were combined, evaporated and lyophilized to give 35 mg of the on-1 amino sugar derivative at a concentration of 0.3 X 10 AIE / g and 30,000 SIE / g.

Example 7

120 ml of nutrient solution containing 5% starch, 1% yeast extract, 0.2% K2HPO4 are poured into a 1-liter Erlenmeyer flask, and the flask is inoculated with 2 ml of the pre-culture of Example 4, after which 3 ml are added. days of fermentation at 28 - 0 O obtains a fermentation medium with the following content of amylase inhibitor:

Strain AIE / ml

SE 50 37 000

SE 50/13 109 000

SE 50/110 53 500

Amylase inhibitors are predominantly derivatives of the amino sugar o-ng4.

Example 8

Into a 1 liter Erlenmeyer flask pour 120 ml of nutrient solution - composition of 1.3% maltose, 3.5% glucose, 0.5% casein hydrolyzate, 1.3% yeast extract, 0.3% OaOO3, 0.3% K2HPO4, and the flask was seeded with 2 ml of the pre-culture of Example 4 to give the following yields after rotating on a rotary shaker at 24 ° C for 4 days and using different strains:

Strain

SIE / ml

AIE / ml

SE 50

SE / 50/13

SE 50/110

25580

14.81 460

57.9755

The inhibitor is predominantly an amino sugar derivative of n < 4.

Example 9

Fermenter containing 100 liters of nutrient solution containing 3.5% glucose, 2.5% dried malt extract, 0.5% casein hydrolyzate, 1.3% yeast extract, 0.3% OaOO3, 0.3% of KaH-POa and 0.1% of antifoam are inoculated with 5 liters of the pre-culture of Example 4 and the fermentation is carried out with stirring and aeration for 5 days at 24 ° C to obtain a fermentation medium of 73,000 SIE. liter and - containing predominantly the derivative on = 2.

liters of fermentation broth are adjusted to pH 2.5 with concentrated nitric acid and 900 g (= 1%) of activated carbon are added with stirring (Gauge) to adsorb most of the dye formed. It is centrifuged at 300 rpm from the mycelium and most of the coal and the supernatant is filtered by adding 3 kg of Clarcel under pressure to obtain 65 liters of a yellow-brown clear filtrate of -60,000 SIE / liter.

The filtrate is adjusted to pH 7 with concentrated ammonia and the active substance is absorbed for 30 minutes to 1300 g (2%) of activated carbon (Merek), the mixture is filtered under pressure and the activated carbon is washed with 3 X 10 liters of distilled water. dried and stirred with 3 X 4 liters of 50% acetone at pH 2.5 for 15 minutes each time to desorption the active substance Acetone solutions were decanted by filtration and evaporated on a rotary evaporator to a volume of 250 ml, adding sc 250 ml of methanol and the mixture is again filtered, 480 ml of the filtrate are added dropwise with vigorous stirring to 5 liters of acetone, the precipitate formed is collected by suction filtration and washed 3 times with acetone and ether and dried under vacuum at 35 ° C. g of product of 8,500 SIE / g.

g of this crude product is dissolved in 1 liter of water and 300 g of Dowex 50'WX 4 H ⁺ : (200-400 mesh) is added, the mixture is stirred for 30 minutes, the resin is separated by filtration and washed with 3X2 liters of 0.001 N HCl. The washed resin was suspended in 500 ml of water and the suspension was adjusted to pH by adding 25% ammonia to 9.0. Desorption is then carried out with 2 X 500 ml of 0.6% ammonia, the desorbates are decanted and evaporated on a rotary evaporator to a volume of 100 ml. К decolorizing the concentrate were added 2 g of DEAE-celulczy (Schleicher and -Schtill no. 02085, _0, 6 mikrovalencí / g), the mixture was stirred for 5 minutes and then centrifuged. The light yellow supernatant was mixed with 100 mL of methanol and added dropwise to 2 L of acetone with vigorous stirring. The precipitate was collected by suction filtration, washed with acetone and ether and dried under vacuum at 35 ° C. 4.2 g of product of 26,000 SIE / g are obtained. For further purification, 4.0 g of the inhibitor is filtered in portions of 0.5 g on a Biogel Р-2 gel. To this end, 0.5 g of the product is dissolved in 10 ml of water each and the solution is applied to a Biogel P-2 column (200-400 mesh, Bio-Rad) of 5 X 95 cm. The column is developed with water at a rate of 80 ml per hour. Fractions of 12 ml are collected. For all fractions, the carbohydrate content, the sucrose inhibitor and the amylase inhibitor were determined by anthropic method and extinction at E620, and thin layer chromatography according to Example 2 was also carried out.

The fractions in which the amino-sugar derivatives on-4-6 are detected are pooled, evaporated to 10 ml in vacuo and precipitated by dropwise addition to 200 ml of absolute alcohol. The precipitate was collected by filtration, washed with acetone and ether and dried in vacuo. Yield: 4.0 g of crude inhibitor. 0.2 g of amino sugar derivatives on = 4 to 6 with a concentration of 17,5 X 10 ⁶ AIE / g and 8 500 SIE / g. The fractions containing the aminosugar derivative on = 3 are treated in the same way, precipitation is performed with 200 ml of acetone. The yield is 4.0 g of crude inhibitor, 0.1 g of the amino sugar derivative on = 3 at a concentration of 1.4 X 10 ³ AIE / g and 21 000 SIE / g. The fractions containing the aminosugar derivative on = 2 are treated in the same way, the precipitation is carried out with acetone, yielding 0.9 g of the aminosugar derivative on = 2 at a concentration of 0.3 X 10 ⁶ AIE / g and 68 000 SIE / g.

Example 10 of nutrient solution containing 7.5% dried malt extract, 0.3% casein hydrolyzate, 0.7% yeast extract, 0.3% CaCO 3, 0.3% K 2 HPO 4 and fermenters were seeded with 5% preliminary culture of strain SE 50/100 obtained according to Example 4, whereby after 5 days of fermentation at 24 ° C, a fermentation medium with 73 SIE / ml and containing predominantly an amino sugar derivative on = 2 is obtained. After centrifugation for 30 minutes at 3000 rpm 20.5 liters of a deep brown solution of 67,000 SIE / liter was obtained per minute to separate the mycelium. The pH was adjusted to 3.5 with nitric acid and 60 g of Lewapol (Ca 9221, 0.35 mm grain size, Bayer) / liter, totally 1.23 kg of Lewapol were added to decolorize. After stirring for 20 minutes, the mixture was filtered through a Seitz K 3 filter. The decolorized solution was neutralized with ammonia (18.5 L, 67,000 SIE / L). The active ingredient is then absorbed per 20 g of activated carbon / liter, a total of 370 g of charcoal and mixed for 30 minutes. The mixture is then filtered again through a Seitz K 3 filter, coated with a Clarcel layer. 17.5 liters of 3600 SIE filtrate / liter are discarded. The activated carbon was washed with 3X2 liters of distilled water. The desorption is carried out by stirring with 3 * 1 liter of 80% acetone, stirred for 15 minutes, the pH being adjusted to 2.5 with concentrated hydrochloric acid. 2.4 liters of 371,000 SIE / liter desorbate was applied to 46 g of Dowex H ⁺ (Dowex 50W 4 in H ⁺ form, from Serva, Heidelberg), stirred for 20 minutes, the resin separated by filtration (Dowex fraction). I) and washed with 75% acetone. 3 liters of filtrate and wash liquor at a concentration of 215,000 SIE / liter are mixed with 60 g of Amberlite IRA 410 in OH form (from Serva, Heidelberg) / liter up to pH 7. The mixture is filtered and 2.8 liters of filtrate are added. A concentration of 219,000 SIE / liter is mixed with 72 grams of Dowex H ⁺ form and the mixture is stirred for 20 minutes, the pH is adjusted by dipping a nylon bag with Amberlite IRA 410 resin in the OH-form, Keep pH at 3.0. Then Dowex is separated by filtration (Dowex II), 2.6 liters of 27,000 SIE / liter filtrate is discarded.

The Dowex I and II fractions were washed separately with 3X 75% acetone at pH

3.5, and then Fraction I was washed with 3 X 100 mL 0.6% ammonia and Fraction II with 3 X 150 mL solution. In the first desorption, in which ammonia is not sufficient to neutralize the resin, concentrated ammonia must be added and the pH adjusted to 9 using a pH meter. The desorbates from fractions I and II are decanted separately, evaporated to near dryness, mixed with 50 ml of water and adjusted to pH 3-4 with hydrochloric acid, then 50 ml of methanol are added. The solution is added dropwise to 1.5 liters of absolute acetone,

Three liters were charged with 8 liters each of the precipitate formed by filtration and washed with 3X acetone and 1X ether and dried in vacuo.

Fraction I 6.5 g 25,000 SIE / g

Fraction II 12.3 g 36,000 · SIE / g n = 2, small amount n = 3.

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200 µg of the preparation of Example 1 are dissolved in 940 ml of distilled water and 60 ml of concentrated sulfuric acid and refluxed for 4 hours. (Internal temperature. 98-100 ° C, oil bath temperature 140 ° C). The cooled black-brown solution was mixed with 10 g of activated carbon (Merck Art. 2186) and stirred for 1 hour. The charcoal is collected by suction filtration, washed with water, and the filtrate is adjusted to pH 7-8 with 250 ml of 10N KOH. The solution is stirred for 1 hour with 50 g of charcoal, the charcoal is collected by filtration, washed with 2 liters of water and discarded . For desorption, activated charcoal is allowed to stand with 2 liters of 30% alcohol overnight. The charcoal is then removed by suction filtration and the alcoholic solution is evaporated on a rotary evaporator to a 6.2 g residue which is dissolved in 500 ml of water and mixed gently with 30 g of Amberlite IR 120 in H + form for 1 hour. The ion exchanger is collected by suction filtration, washed with distilled water until the filtrate is neutral and free of glucose. The ion exchanger was stirred with 15 ml of 25% ammonia in 1000 ml of water overnight, collected by filtration and the filtrate was evaporated to a residue of 3.7 g.

For further purification, cellulose chromatography is performed. 4.5 of the material desorbed from the ion exchanger onto a 1 m X 2.5 cm cellulose column. Ethanol / water (5: 1) was first used as the solvent, and ethanol: water (3: 1) was first used to elute the aminosugar of n = 1 at a rate of 20 drops per minute. 14 ml and these fractions were subjected to thin layer chromatography. Fractions 47 to 85 yield, after evaporation, 1.6 g of a slightly brown-colored aminosugar derivative of n = 1. The polluting dye plays no role in this case. Derivatives can also be obtained without the addition of a dye when purification is carried out on. strongly acidic exchanger not after. individual quantities separately, but on a column.

Example 12

200 g of the preparation of Example 1 are dissolved in 940 ml of distilled water and 60 ml of concentrated sulfuric acid and the mixture is refluxed for 1/4 hour at an internal temperature of 98-100 ° C and an oil bath temperature of 140 ° C. The cooled brown-black solution was mixed with 10 g of activated carbon (Gauge, Art. 2186) and stirred for 1 hour. The charcoal is then filtered off with suction, washed with water and the filtrate is adjusted to 7-8 with 10N potassium hydroxide solution. The solution is stirred with 50 g of charcoal for 1 hour. The charcoal is collected by suction filtration, washed with 2 liters of water and the filtrate is discarded. For desorption, activated carbon was digested overnight with 2 liters of 30% alcohol. Finally, the charcoal is collected by suction filtration and the alcoholic solution is evaporated on a rotary evaporator to give 8.0 g of a residue.

The residue is mixed with 15 ml of water and applied to a 20.times.2.4 cm column packed with 50 g of Aml @ + IR 120 in H @ ⁺ form. The flow rate is 3 drops per minute, then the column is washed with water at a rate of 12 drops per minute to remove non-basic components. The basic components were then eluted with 0.5% ammonia at a rate of 12 drops per minute and the aqueous solution was evaporated on a rotary evaporator to give 4.1 g.

Dissolve g of this residue in a small quantity of water and apply the solution to a 200 × 3 cm column packed with Sephadex G 15. The column is eluted with water. Fractions of 2 ml are collected at a rate of 8 ml per hour. The individual fractions were monitored by thin layer chromatography. From fractions 85 to 94, 280 mg of the aminocugar derivative of n = 2 with a specific activity of 50,000 SIE / g are obtained.

Example 13 g of the preparation of Example 1 are incubated in 60 ml of 20 mmol of sodium glycerophosphate buffer pH 6.9 and 1 mmol of CaCl2 with 1 g of α-amylase from spec. Aspergillus (Serva No. 13418) with continuous stirring at 37 ° C for 120 hours, then heated to 100 ° C for 5 minutes and centrifuged at 4,000 rpm. per minute from the insoluble portion. After lyophilization of the solution, 1.9 g of product A 3500 SIE / g and 2 X 10B AIE / g are obtained. When examining the product by thin-layer chromatography and for the inhibition of sucrose, in particular an aminosugar derivative of n. = 1, 2 .and 3.

Example 14 g of the preparation of Example 1 are incubated in 30 ml of 20 mM acetate buffer at pH

4.8 and 1.25 mg mg topinambur β-amylase (Boehringer 15471) with stirring at 37 ° C for 120 hours, after which the mixture was heated to 100 ° C for 5 minutes, the insoluble matter was centrifuged at 4 ° C. 1000 rpm, after freeze-drying the solution, 1.5 g of product are obtained

800 SIE / g and 3.8 X .106 AIE / g. After testing this product by thin-layer chromatography and sucrase inhibition according to Example 2, it is apparent that in particular an aminosugar derivative of n = 2 and 3 is present.

Example 15

200 ml Erlenmeyer flasks containing 25 ml of nutrient solution containing 0,1% K2HPO4, 0,2% (NH4) 2SO4, 0,05% MgSO4, 0,05% KCl, 0,01% FeSCh % of the preparation of Example and a spore suspension of Asp strain. nger ATCC 11394 is subjected to fermentation at 28 ° C on a rotary shaker, where after 6 days the AIE titer decreases from 210 000 AIE / ml to 53 000 AIE / ml, after 10 'to 21 300 AIE / ml. At the same time, the SIE / ml content of the. 7.0 to 72 SIE / ml.

2B of the solution which was incubated with the spore suspension for 10 days was centrifuged for 30 minutes to separate the mycelium at 3000 rpm. minute. 15 ml. The supernatant of 72,000 SIE / liter was stirred for 30 minutes with 2 g of Amberlite IRC 50 in H + form and 1 g of Amberlite IRA 410 in 0H-form to remove salts. (Conductivity is less than 2 m Siemens), Filter the mixture and pass the filtrate at a rate of 5 mL / hour through a 1 X 10 · cm column containing Dowex in the H + form in 0.001 N hydrochloric acid. 0.001 N hydrochloric acid For desorption, a 10 ml ammonia solution was passed through the column at 0.6% w / v, fractions of 5 ml were collected. The fractions containing the sucrose inhibitor were pooled, evaporated to 2 ml in a rotary evaporator. The solution was adjusted to pH 3-4 and precipitated by dropwise addition to 100 ml of acetone, and the precipitate was collected by suction filtration, washed with acetone and ether, and dried in vacuo to give a solid. with 26 mg of the product of 28,000 SIE / g, the product consisting of the amino sugar derivatives on = 2 and 3. The derivative on = 2 can be obtained from this product by the method of Example 9 by gel filtration through a Bio-Gel P-2 column. 7 mg of the aminosugar derivative on = 2 s are obtained a specific efficiency of 60,000 SIE / g.

EXAMPLE 16 liters of fermentation broth obtained according to Example 6 by mycelium separation at 13,000 rpm with an efficiency of 13,000 SIE / g are mixed with 500 g of a 2.5 part Amber lite mixture to reduce the salt content (10mS filtrate conductivity) IRC-50 in H ⁺ form and 1 part Amberlite IRA-410 · OH-forine for 1 hour. The ion exchanger was separated, the solution was evaporated to less than 100 ml and the insoluble matter was removed by centrifugation at 20,000 rpm for 15 minutes. The supernatant is made up to 100 ml, has a conductivity of 3.5 mS and further purified on a 55 X 400 mm column containing P-cellulose (Serva No 45130, prepared in the conventional manner, equilibrium reached in 5 moles of phosphate buffer pH 5.5). The column is eluted with the indicated buffer at a rate of 90 ml / hour. Fractions of 18 ml are collected.

Fractions were assayed for carbohydrate content by the antimony assay and for sucrose inhibitors by the sucrose inhibition assay, the carbohydrate-free fractions (fractions 60-80) that were simultaneously effective as sucrose inhibitors were evaporated to 150 ml and passed through a 50 X 300 column. mm containing Amberlite IRA-410 in HCO3 form. For better control of deionization, the eluate is collected in fractions (J 10 ml in 20 minutes and fractions are assayed for carbohydrate content by an antimony assay to be almost negative, for a phosphate reagent with ascorbic acid and molybdenum reagent and for sucrose inhibition by an enzymatic assay. 3 to 30 · are combined, evaporated and lyophilized, redissolved and lyophilized again to give 280 mg of the crude inhibitor.

For further purification, the crude inhibitor is fractionated on Bio-Gel P-2 according to Example 6. The pure aminosugar derivative on = 1 is obtained from the fractions, after freeze-drying, 30 mg of 0.3 X 10 6 AIE / g and 35,000 are obtained. SIE / g.

Example 17

To obtain an aminosugar derivative of n = 5-7, for example, the preparation of Example 1 is used.

g of the extract of Example 1 is dissolved in 250 ml of water. The conductivity of this solution is 10 mS, pH 5.5. The solution is dehydrated by mixing with 60 g of Amberlite IRC 50 in the H + form (a weakly acidic cation exchanger that binds the amino sugar derivatives in aqueous solution only in traces) and 20 g of Amberlite IRA 410 in the OH form and the mixture was stirred for 20 minutes. The filtrate having a conductivity of 0.5 · mS at pH 3.5 is adjusted to pH 3.0 · 1 N HCl and then has a conductivity of 0.6 mS. The solution is passed through a column of 2.5 x 40 cm containing Dowex 50 · W in H + form with a grain size of 200 to 400 mesh, the solution being equilibrated at 0.001 N HCl. The column is eluted with 2 liters of 0.001 N HCl. After washing, elution is carried out with 1.2% aqueous ammonia and 10 ml fractions are collected. The active fractions were combined, the ammonia was evaporated in vacuo and the solution was then evaporated in vacuo to a volume of 30 ml. Precipitation is effected by dropwise addition of the solution to 600 ml of anhydrous alcohol, the precipitate is collected by suction filtration and washed with alcohol and ether and dried under vacuum to give 4.4 g of the product. G.

0.5 of each product are gently purified by preparative on the Bio-Gel P-2 column of Example 9. Fractions which, according to thin layer chromatography, contain an amino sugar derivative on · 5 to 7 are combined, evaporated in vacuo and precipitated from above. in this way anhydrous ethanol. The yield from 0.5 g of crude product is 0.2 g of the aminosugar derivative of n = 5-7 with 30 X 10 6 AIE / g and 2500 SIE / g.

Example 18

I. Design of an experiment to determine the efficacy of glycoside hydrolase inhibitors and to prevent glucose resorption in rats in humans

To induce alimentary hyperglycemia and hyperinsulination, waking rats (n = 6) or waking subjects are administered either

2.5 g sucrose, 2.5 g maltose, 1 g boiled · starch or 2.5 g glucose / kg · orally or 50 g sucrose, 50 g maltose, 50 g boiled starch or 50 g glucose per person in aqueous solution or a suspension. Other rats ι (· π - 6) receive the same amount of the above carbohydrates and, in addition, the active ingredient of any of the above doses. Healthy subjects, alternately, received only a carbohydrate or a carbohydrate with the active ingredient at intervals of 2 days. Blood sugar and insulin levels were determined at intervals of 5 minutes for up to 3 hours after carbohydrate + drug loading in the blood, with blood taken from retro-orbital venous plexus rats and from the elbow vein or capillary blood in test subjects. Blood sugar was determined by an Auto-Analyzer (Technicon ^R , according to Hoffman: J. biol. Chem. 120, 51 (1937)) or enzymatically by glucose oxidase and o-dianisidine hydrochloride, the determination of blood serum insulin by the method of Hales and Randle, Biochem. J. 88, 137 (1963).

II. Investigation of inhibition of carbohydrate to fat lipid conversion.

The rats (n = 6) received the carbohydrates of paragraph I in an inactive form together with a suitable dose of the same radioactive carbon labeled carbohydrate + drug of the same example by a swallowing probe. After a short time the animals were sacrificed and adipose tissue and / or perirenal adipose tissue were analyzed, lipids were extracted with a 2: 1 mixture of chloroform and methanol, and non-lipid metabolites were washed off. The radioactivity of the isolated lipids was measured on a liquid scintillation counter. Efficacy was reported in dpm / 1 grams of adipose tissue.

In the tables P indicates the probability of error and s the standard deviation.

Table 1 to Example 18

Blood glucose in mg% (mean + 1s in waking rats at different times after oral starch administration + active ingredient of Example 17, amino-sugar derivative with n = 5 to 7

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Blood glucose in mg% [mean + 1s in waking rats at different times after oral starch administration + active ingredient of Example 6.

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Example 19

An example for the acid desorption of the compounds of the invention.

A 1.5 cm diameter column is filled with 30 g of wet weight of Dowex ^R 50 WXX 4 in a 200-400 mesh H ⁺ form in 0.001 N HCl. Pass 500 ml of mixed 400,000 SIE / liter desorbate at pH 2.5 containing 60% acetone obtained from Example 10 through the column. The desorbate is passed through the column for approximately 1 hour, then the column is washed with 500 ml of 0.001 N HCl. Under these conditions, only small traces of inhibitor present are washed out. The desorption is then carried out directly in the column with 0.0125 N hydrochloric acid, and the conductivity of the eluate is determined. In addition, the SIE content is determined in the eluate. The active fractions # 74-100 were combined and neutralized by the addition of Amberlite IRA 410 in OH form, then evaporated to 5 ml, mixed with 5 ml methanol and precipitated by dropwise addition of the solution to 200 ml acetone. After washing with acetone and ether, the solution was evaporated in vacuo.

The yield is 1 g of the amino-sugar derivative on - = 2 containing 65,000 SIE / g.

Amino sugars derivatives in which n = 3 or 4 can be obtained from the active pre-fractions.

This procedure for acid desorption also enables, in contrast to alkaline deisorption, the fractionation of individual homologues of a number of amino-sugar derivatives.

Claims (1)

A process for the preparation of novel amino-sugar derivatives of the general formula wherein R is an oligosaccharide chain with 1 to 7 hexose units, characterized in that microorganisms of the family Actinoplanaceae strain SE 50/110 ~ CBS 674.73 are grown in an aqueous nutrient medium containing carbon sources, nitrogen and inorganic salts at pH 5.0 to 8, 5 and at a temperature of 15 to 45 ° C under aerobic conditions and the amino sugar derivative is isolated after the fermentation is completed, and optionally, in the preparation of compounds wherein R is 1 or 2 monosaccharide units, compounds wherein R is more than 2 mono- osaccharide units by an enzymatic method by incubation with hydrolase or hydrolysis in an acidic medium using aqueous solutions of inorganic acids.