CH633043A5 - Process for preparing aminosugar derivatives - Google Patents

Description

The present invention relates to the production of new 35 amino sugar derivatives which are suitable in medicine for controlling the carbohydrate metabolism of humans and animals by inhibiting glycoside hydrolases, such as for the treatment of diabetes, obesity and hyperlipemia.

40 It is known that a number of microorganisms of the order Actinomycetales form inhibitors for glycoside hydrolases, inhibitors with predominant amylase or saccharase inhibitory activity being obtained depending on the breeding conditions (see US Pat. Nos. 3,876,766,

45 3 855 066 and 3 879 546).

The new amino sugar derivatives of the formula I have now been found

H-

cha oh

) - ^ L ^ -0

\ \ I vjn un nP ✓ \ d ch, 0h h

- 0

cha oh

—R-J

çh2oh in which ni stands for a digit from 1 to 8 and m for a digit from 0 to 8, but the sum of m + m always has a value of n = 1 to 8, with the proviso that in the case of m + m = 1 or 2, n2 is always 0.

Depending on the value of the sum of m + m they have

Amino sugar derivatives predominantly amylase or saccharase 65 inhibitory effect. Amino sugar derivatives that can be used specifically to inhibit amylase are those with ni + m = 4 to 8, in particular with m + m = 4 and 5. Amino sugar derivatives that are specifically used to inhibit saccharase

633043

4th

lawn can be used, those with m + m = 1 to 3, preferably with m + m = 2 to 3, particularly preferably with nt = 2.

It has also been found that, surprisingly, amino sugar derivatives with m + m = 1 to 3, in particular with 5 m = 2, show a strong inhibition of starch degradation in vivo.

According to the invention, the above-mentioned new aminosugar derivatives are produced by cultivating strains of the Actinomycetales order, in particular the Actino-planaceae family, in a manner known per se and by subsequent separation and isolation of the individual compounds in a manner known per se.

Aminosugar derivatives with values of the sum of m + m of 1 to 4 can furthermore be obtained by chemical or enzymatic hydrolysis of higher molecular weight aminosugar derivatives.

Suitable strains of the order Actinomycetales for the production of the new amino sugar derivatives are, for example, SE 50 (CBS 961.70), SB 18 (CBS 957.70) and SE 82 (CBS 615.71), and mutants or variants of these 20 strains. The strains SE 50/13 (CBS 614.71) and SE 50/110 (CBS 674.73) proved to be particularly well suited with regard to the yield of amino sugar derivatives obtained. The description of both strains largely corresponds to the parent strain SE50 (CBS971.60), from which these strains 25 are obtained by natural selection without the use of mutagens.

Solid or liquid, in particular liquid, aqueous nutrient media which contain the known starting materials for carbon 20 and nitrogen, salts and anti-foaming agents in the customary concentrations can be used to cultivate the microorganisms.

Mainly carbohydrates, especially starch, maltose, glucose and complex mixtures such as commercially available 35 malt extract are used as starting materials for carbon.

Suitable starting materials for nitrogen are the complex substance mixtures known per se, such as casein hydrolyzate, yeast extract, peptone, fish meal, corn steep liquor, meat extract and mixtures of these starting materials, as well as individual amino acids and / or ammonium salts can be used as nitrogen sources.

The microorganisms are grown aerobically, suitably in aerated shake cultures or in the usual «container cultures.

The type and concentration of the raw materials used for carbon in conjunction with the particular strain used for fermentation influences the type of the predominantly formed end product. so

For example, in nutrient solutions containing starch above 2% by weight, compounds of the formula I with m + m = 4 to 8 are primarily formed. The SE 50/13 strain (CBS 614.71) is particularly suitable for the preparation of these amino sugar derivatives with m + m = 4 to 8. 55

If the nutrient solution contains sufficient glucose (approx. 3.5% by weight), 0.1 to 3% by weight of starch is sufficient to obtain predominantly mixtures of amino sugar derivatives with m + m = 4 to 8. All of the aminosugar derivatives obtained in this way can be used as starting compounds for the production of the lower homologues by chemical or enzymatic hydrolysis.

Amino sugar derivatives of the formula I with m + m = 1 to 4 are predominantly obtained when working in starch-free nutrient solutions. The addition of maltose to the nutrient solution is particularly beneficial. For example, when adding maltose and using the strain SE 50 (CBS 961.70), mixtures of amino sugar derivatives (I) with predominantly m + nz = 2 to 3 are obtained. The formation of an amino sugar with m = 1 is achieved in particular as a result that only glucose is used as the starting material for carbon. It should be noted here, however, that with an excess of glucose and a longer fermentation period, the higher molecular weight amino sugar derivatives are also formed.

If nutrient solutions which do not contain glucose are used and maltose is added as the sole starting material for carbon, the amino sugar of the formula I with m + m = 2 is predominantly obtained. In a technically advantageous embodiment, the pure maltose can be replaced by a cheaper product , such as B. Maitzin, a natural commercial malt extract.

The strain SE 50/110 (CBS 674.73) has proven to be particularly suitable for the production of amino sugar derivatives which predominantly contain derivatives (I) with m + m = 1 to 3.

This strain gives yields of low molecular weight amino sugar derivatives which are about twice as large as those obtained with the strain SE 50/13 (CBS 614.71).

The microorganisms are generally grown at incubation temperatures between 15 to 45 ° C., preferably between 24 to 32 ° C. Thus, when using the strains SE 50 (CBS 961.70) or SE 50/13 (CBS 614.71) aminosugar derivatives (I) with m + n2 = 4 to 8 at a higher temperature, e.g. B. 28 ° C, while at low temperatures such. B. 24 ° C, for example the strains SE 50 (CBS 961.70) and SE 50/110 (CBS 674.73) predominantly form aminosugar derivatives (I) with m + n2 = 1 to 3.

The culture period is generally 1 to 8 days, preferably 2 to 6 days. A longer culture period, especially in connection with excess carbohydrates in the nutrient medium, favors the formation of aminosugar derivatives (I) with higher values of m + m.

The pH of the culture medium generally varies between 5.0 to 8.5, with pH values from 6.0 to 7.0 being preferred.

The end point of the fermentation is generally determined by determining the content of inhibitory activity in the enzymatic inhibition test and by determining the composition of the fermentation medium by thin-layer chromatography.

In the case of chemical hydrolysis of high molecular weight amino sugar derivatives for the production of low molecular weight as already mentioned above, the procedure is generally such that the higher molecular weight amino sugar derivatives are treated with 1 to 5 normal aqueous mineral acids at temperatures of 50 to 100 ° C, especially from 90 to 100 ° C for 10 to 180 minutes. The likewise possible enzymatic hydrolysis is generally carried out by incubation with enzymes (hydrolases), in particular with β-amylases or uninhibitable a-amylases or amyloglucosidases of microbial origin, such as, for example, a-amylases from B. subtilis, or by incubation with microorganisms which are in Nutrient solutions which contain the higher molecular weight amino sugar derivatives in contents of 1 to 10%, preferably 2 to 5%, as the sole carbon source, are able to grow, for example Aspergillus niger (ATTC11.394).

The separation and isolation of the individual aminosugar derivatives obtained according to the invention is based either on the microbiological culture broths or on the hydrolyzates or incubation mixtures in which the enzymatic and / or microbiological degradation of the higher molecular weight aminosugar derivatives has been carried out.

Depending on the values for m + m, the processing takes place in different ways. So one goes to the pure representation of the

5

633043

Aminosugar derivatives of m + n2 = 1 to 4 in general in such a way that, if necessary after prior separation of the mycelium, the culture broth in question or the hydrolyzate is treated with activated carbon at neutral pH, then the amino sugar thus bound to the activated carbon. Ker derivatives desorbed by treating the activated carbon with aqueous alcohols or acetone, preferably 50 to 80% acetone. Desorption is particularly successful at acidic pH values in the range from pH 1.5 to 4, preferably from pH 2 to 3.

If the culture broths or hydrolysates to be separated are very dark in color, it has proven to be advantageous to first use the activated carbon or absorption resins such as the commercially available Lewapol Ca 9221 (0.35 mm grain size, Bayer AG) at pH values of pH 2 to 6, preferably 2 to 3, to achieve decolorization. The activated carbon preferably binds only the dyes in the acidic range, but not the aminosugar derivatives.

The separation of the individual aminosugar derivatives obtained according to the invention from the activated carbon desorbate obtained as described above is then generally carried out in such a way that this desorbate is known via strongly acidic cation exchangers known per se, such as, for. B. Dowex 50 W (Dow Chemicals) conducts (pH 1 to 8, preferably pH 2 to 4; at low ionic strength, corresponding to a conductivity <10mS-cm- *, preferably <2mS * cm_1) -The individual aminosugar derivatives are bound to the cation exchanger. The amino sugar derivatives from acetone solution (50 to 80% acetone, pH 1 to 5, preferably pH 2 to 4) can be particularly advantageously bound to such cation exchangers.

The selective desorption of the aminosugar derivatives prepared according to the invention from these cation exchangers is then generally carried out using aqueous acids or bases as the eluent, ammonia or hydrochloric acid preferably being used in concentrations of 0.01 to 1 val / liter.

The aminosugar derivative in question is then converted from the individual desorbates after neutralization of the desorbate with a weakly acidic or basic exchanger or after removal of the base or acid by stripping in vacuo in the case of volatile bases or acids after concentration of the solution by lyophilization or precipitation obtained with organic solvents, preferably with acetone.

It is also possible to separate low molecular weight aminosugar derivatives from inert saccharides by chromatography on cellulose-based exchangers, preferably phosphorus-cellulose (from Serva, Heidelberg). Buffers, preferably phosphate buffers, of low ionic strength, preferably 2 to 100 mM, in particular 5 to 10 mM, in the pH range from 2.5 to 8, preferably 5 to 6, are used as eluents. The prerequisite for effective fractionation is the lowest possible salt content in the preparations to be fractionated, the dyes of which are less disruptive.

To prepare the individual amino sugar derivatives, the pre-cleaned preparations are then chromatographed over a suitable molecular sieve, e.g. B. Bio-Gel P-2 (Bio-Rad, Munich) and examines the fractions of the eluate by thin layer chromatography. Fractions which contain the compounds according to the invention in pure form are combined, re-chromatographed and finally lyophilized after concentration or precipitated using organic solvents, as described above.

All aminosugar derivatives are characterized by the fact that "component I" [C13H21O7N] and glucose are formed in the case of total acid hydrolysis. The following structural formula was derived for "Component I":

Ohh O

0h h-c-oh t

"Component I"

The initial member of the aminosugar derivatives of the present invention has a glucose unit. This compound is a colorless, amorphous substance with good solubility in water, dimethylformamide, dimethyl sulfoxide, methanol and hot ethanol. In thin layer chromatography using ethyl acetate, methanol and water in a ratio of 10: 6: 4, the compound showed an Rp value of 0.46 (maltose = 0.50 and glucose = 0.65) on F1500 silica gel foils [Schleicher & Schüll] and of 0.47 (maltose = 0.54 and glucose = 0.66) on F 254 silica gel plates [Merck, Darmstadt].

When using a silver nitrate / sodium hydroxide spray reagent, a brown-black color is obtained at room temperature or after slight heating.

The compound was silylated together with D-glucose or sucrose as standards in a mixture of pyridine (1), trimethylchlorosilane (0.5) and N-methyl-trimethyl-silyltrifluoroaceta-mid (1) and then gas chromatography in a 1 , 8 m glass column, filled with 3% SE 30 silicone elastomer [Packard] on Chromosorb WAW.

The injection and detector temperature was 300 ° C. The oven temperature was 220 ° C., isothermal until the a and β-D-glucose standards were eluted. The temperature was then raised to 300 ° C. at a rate of 15 ° C./minute.

A flame ionization detector was used, introducing nitrogen as the carrier gas at 40 ml / minute, air as the combustion gas at 80 ml / minute and hydrogen at 20 ml / minute. The compound had a retention time of 16-17 minutes; (a-D-glucose = 3 minutes, β-D-glucose = 4 minutes and sucrose = 12-13 minutes.

A non-crystalline sample of the compound obtained by concentrating a methanolic solution was dissolved in water and the specific optical rotation was determined to be [a] o = + 134.3 °.

The NMR spectrum in CD3OD at 220 MHz is shown in FIG. 1 (abscissa = ô ppm).

The following table 1 shows predominant features of the NMR spectrum:

Table 1

8 in ppm multiplicity relative intensity

1.3

Doublet; J = 6.5 Hz

3 H

2.3

"Triplet",] i and J2 8-10 Hz

IH

3.15

"Triplet"; Ji and J2 7-9 Hz

IH

3.3-3.9

Signals are not individual

12 h

assign!

4.13

AB system; J = 12 Hz

2 H

4.48

Doublet; J = 7 Hz

1 H

5.1

Doublet; J = 2.5 Hz

4.9

Single

11 h

Deuterium exchanged protons

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

633043

Ô in ppm multiplicity relative intensity

5.0

Doublet; J =

2-3 Hz (bad

1 H

resolved)

5.8

Doublet; J =

3-4 Hz (bad

1 H

resolved)

33 H.

If this compound is reacted in a 1: 1 mixture of acetic anhydride and pyridine at room temperature, a decaacetyl derivative (molecular weight: 903) is obtained. 2 shows the NMR spectrum of the decaacetyl derivative.

If the reaction in glacial acetic acid / acetic anhydride is carried out 1: 1 with catalytic amounts of H2SO4, the formation of an undekaacetyl derivative (molecular weight: 945) can be demonstrated in addition to the decaacetyl derivative.

The mass spectrum of the decaacetyl derivative shows a molecular peak at 903 (2.5% pure intensity) and a base peak at 843. Important fragment peaks in the upper mass range are 844 (55% rI), 784 (36% rI) 783 (34% rI), 759 (34% rI), 556 (36% rI), 496 (37% rI) and 436 (29% rI).

Methylation with CH3J / NaH in dimethyl sulfoxide according to Hakomori results in the main product being the decamethyl derivative, and in a small amount also the undecamethyl derivative.

The mass spectrum shows a molecular peak at 623 5 (6.1% pure intensity) and a base peak at 535. A second molecular peak is observed at 637 (0.2% pure intensity).

Spectroscopic data and chemical properties give the following structure for this compound ii a

15 ch2oh h 0 '

In particular, the NMR spectrum shows that this compound IIa has the structure of a 0-14,6-bisdeoxy4- [IS- (1,4,6 / 5) 4,5,6-20 trihydroxy-3-hydroxymethylcyclohex-2-ene -l-ylamino] -aD-glu-copyranosylH 1 - * 4) -D-glucopyranose of the conformative formula

HIGH

IIB

CH20H

comes to.

The next link in the series (m + m = 2) has the general composition C25H43O18N and is a readily water-soluble amorphous product. In thin layer chromatography it shows an Rr value of 0.35 on F1500 silica gel sheets and 0.33 on F 254 silica gel plates using the system described above.

The optical rotation in water [a] d is + 167.0 °.

The NMR spectrum in D2O at 220 MHz shows FIG. 3 and the 13C NMR spectrum shows FIG. 4.

The methylation, as described above, gives a 13-fold methylated compound, in a small amount also a 14-fold methylated product.

The mass spectrum of the methylation product shows a molecular peak at 827 (1.5% from left) and corresponds to a molecular formula C38H69NO18.

50

55

With 0.1% r.I. there is a second molecular peak at 841. The main fragment peaks are:

739 (27% r.I.)

592 (3.7% r.I.)

535 (30% r.I.)

388 (9% r.I.)

386 (13% r.I.)

284 (13% r.I.)

187 (12% r.I.)

171 (40% r.I.)

101 (34% r.I.)

88 (25% r.I.)

75 base peak

The chemical and spectroscopic properties give the following structure for this compound:

ch20h

CHoOl

IIIA

7

633043

this second member of the series of the new aminozuk-D-glycopyranosylH 1-4) -0-aD-glucopyranosyl- (1-4) -D-gluco-ker derivatives (I) has the structure of a 0-f4,6-bisdeoxy -4- [IS- (1,4,6 / pyranose of the conformative formula: 5> 4,5,6-trihydroxy-3-hydroxymethylcyclohex-2-en-l-ylamino] -a-

The next higher link in the series (m + m = 3) is obtained. The product, which is present in smaller quantities, is the vérin, two isomeric forms, one of the two isomeric formations 0- (4,6-bisdeoxy-4- [l S - (1,4,6 / S) -4,5,6-trihydroxy-3-men is predominantly formed.hydroxymethylcyclohexy-2-en-l -ylamino] -aD-glucopyranosyIl-

In the case of acidic partial hydrolysis, both isomeric compounds (1 - * 4) -Ó-aD-glucopyranosyl (1-4> 0-aD-glucopyranosyl-dungs can be divided into the first link in the series (m + m = 1) and glucose ( Split 1–4> D-glycopyranose of the conformative formula in a molar ratio of 1: 2. 35

633043 8

The isomeric product present in large quantities is 1 -ylamino] -aD-glycopyranosylH 1 -4) -0-aD-glucopyranosyl-the compound CM4,6-bisdeoxy-4- [1 S- (1,4,6 / 5 ) 4,5,6-trihydroxy- (1-4) -D-glucopyranose of the conformative formula 3-hydroxymethyl-4-0-aD-glucopyranosyl- (1-4> cyclohex-2-en-

CH20H

hchs> i — 0

noJk '\

HIGH. a-0H

HO— ^ N.

CH20H

CH20H

These isomers show Rducose values of 0.41 to 0.46 on F-1500 plates using n-butanol, ethanol and water in a ratio of 50:30:20. The presence of the isomer with the structure shown in formula V can be demonstrated by analyzing the products from the hydrogenative degradation (palladium / activated carbon). These breakdown products include:

3-hydroxymethyl4,5,6-trihydroxy- [4-0-a-D-glucopyranosyl- (1 »cyclo-hexane, 0- (4-amino4,6-bisdexoxy-a-D-glucopyrano-

sylHl--4) -0-a-D-glucopyranosyl- (l - <4) -D-glucopyranose and glucose.

Since in this degradation the C-N bond in the alkyl position is cleaved and the hydrogenation is hydrogenated, it is clear that this compound, which is isomeric to the compound of the formula IV, is structurally related to the compound of the formula III A or III B; however, it is characterized by the presence of a further glucopyranosyl group in the 4-position of the cyclo-hexene ring.

CH.0H CHS 0H N 0 0 OH

OH OH OH OH OH 0H

Pd / C / H «t

CH * OH CHa OH

H3

HO OH

CHS

CHa0H CHÄ0H

- 0 + 0

OH OH

OH OH

CHa0H

> - 0 -O-OH + IHO - ^^ - OH

OH OH OH OH \ OH OH

The presence of the isomer with the structure shown in Formula IV is determined by the formation of Validatol and

0- <4-amino4,6-bisdeoxy-a-D-glucopyranosylH 1 -4) -0-a-D-

9 633043

glucopyranosyKl - *> 0-a-D-glucopyranosyl- (l - * 4) -D-glucopy-ranose proven.

cha oh i chj cha oh cha oh cha0h oh 0h | oh oh oh oh oh oh oh oh cha0h chs cha0h cha oh

+ han -? ^ - 0 0 0

0 oh óh0h oh oh oh oh oh oh

-Oh

In further studies, the isomers with m + m = 3 were methylated, hydrolyzed, reduced with sodium borohydride, acetylated and analyzed by gas chromatography using known methods. While the compound of formula IV gives only 1,4,5-trio-0-acetyl-2,3,6-tri-0-methyl-D-glucitol, the compound of formula V gives 1,4,5-tri- 0-acetyI-2,3,6-tri-0-methyl-DO-glucitol and l, 5-di-0-acetyl-2,3,4,6-tetra-0-methyl-D-glucitol in Molar ratio of 2: 1. The isomers with m + m = 3 of the formulas IV and V are not degraded by β-amylase.

The isomeric compounds of the formulas IV and V were separated by chromatography on an acidic exchange resin using 0.025 N hydrochloric acid as the eluent.

The product with m + n2 = 4 was hydrogenated at the Pd / C contact. The non-basic hydrogenolysis products were separated off using acidic ion exchangers and separated by preparative thin-layer chromatography. After acetylation, the following two pseudotrisaccharides were identified as the main breakdown products by mass spectrometry:

ch2oh ch2oh ce2oh.

HO OH OH OH OH OH (as deca acetate with the molecular peak at m / e = 906)

This structure also follows as a result of the degradation with β-amylase, in which the major part of the component with m + m = 4 is degraded to the compound of formula III B and maltose.

30 The component with m + m = 5 behaves as follows when degraded by ß-amylase:

About 90% are degraded to a compound of formula V and a corresponding amount of maltose, from which it follows that the isomer with a maltotriose unit which is glycosidically bonded to the cyclitol residue 35 is the main constituent of the product; approx. 10% are not degraded by ß-amylase.

The higher links in the series, which contain 4 to 8 glucose units with molecular weights from 969 to 1617, are less able to inhibit saccharase. 40 thin layer chromatography using n-butanol, ethanol, water in the ratio of 50:30:20 on F 1500 silica gel plates gives RGiUcose values of 0.30-0.34 4 glucose units 0.21-0.23 5 glucose units 45 0, 14-0.16 6 glucose units 0.09-0.11 7 glucose units

As with the lower links in this series, the total acid hydrolysis gives “component I” and glucose in discrete molar ratios, namely 1: 4.1: 5.1: 6.1: 7 and 1: 8 (the glucose percentages being 74.4 %, 79.7%, 83.3%, 86.5% and 89.1%, respectively).

In thin-layer chromatography using F-1500 silica gel plates with n-butanol, ethanol, water in the ratio of 45:35:20 as solvent, the following Rr 55 values are observed in the triple development:

ch2oh ch.

çh2oh

- £ KK}

ho oh oh oh oh oh

(as nona acetate with the molecular peak at m / e = 848)

The predominant main constituent of component 5 is therefore the isomer of the conformative formula VI, 4: 1 with a maltose unit which is glycosidically bonded to the cyclitol radical. 5: 1

relationship

Glucose: component I

default

RrValue

glucose

Maltose

Maltotriose

Maltotetraose

Maltopentaose

Maltohexaose

0.77 0.65 0.51 0.39 0.27 0.25 0.21 0.18

633043

10th

Ratio standard Rp value

Glucose: component I

Maltoheptaose 0.15

6: 1 0.13

Maltooctaose 0.11

7: 1 0.09

8: 1 0.07

The catalytic hydrogenation described above also shows that some but not all of the glucose units of these higher members are bound in the 4-position of the cyclohexene group.

Since these compounds contain linear oligoglucoside chains with 1-4 bonds, they can be used as substrates for certain carbohydrate-degrading enzymes. The range of enzymes is apparently limited to those which are not significantly inhibited by the compounds.

Bacterial and fungal a-amylases can be used to break down any oligoglucoside chain containing 3 or more glucose units and containing low molecular weight compounds and inert saccharide fragments such as e.g. B. Maltose and mal totriose result. This degradation method is applicable to the compounds with m + m ^ 3 and is further proof of the a, 1-4-bond. Compounds according to the invention with 4- to 7-glucose units are also partially degradable by β-amylase. Since β-amylase maltose units split off from the non-reducing end of a glucose chain with a, 1 - »4 bonds, careful analysis of the β-amylase degradation products provides valuable information about every oligoglucoside substituent attached to the 4-position of the cyclohexene ring, especially about its chain length, and the number of glucose in the chain attached to the bisdeoxyglucose, i.e. H. in the reducing end of the connection. However, the compounds containing 4,5,6,7- or 8-glucose units are not completely degradable by β-amylase. The resistance of some of the compounds is apparently due to insufficient structural conditions for the β-amylase attack.

The results of the β-amylase degradation can be summarized as follows.

Number of glucose units

Number of glucose units removed in the starting material in degradation products

Maltose units

4th

4th

0

2nd

1

5

5

0

3rd

1

6

6

0

4th

1

2nd

2nd

7

7

0

5

1

3rd

2nd

8th

8th

0

6

1

4th

2nd

2nd

3rd

If necessary, part of the starting material is recovered.

Regarding the ß-amylolytic degradation it should be emphasized again that ß-amylase is only able to remove maltose units and only from the non-reduced end of an oligosaccharide. It should be avoided that any theory leads to a limitation. Although the exact structure of these higher compounds is not entirely clear, it can be concluded from the above findings that the compounds with 4,5,6,7- and 8-glucose units include derivatives of the lower compound with the formula III A, which chains with Contain 2,3,4,5- and 6-glucose units, which are connected a, 1-4, the oligosaccharide chain in a-conformation being connected to the 4-position of the cyclohexene ring.

Methylation of the compounds with methyl iodide / sodium hydride in dimethyl sulfoxide, total hydrolysis, derivatization and subsequent gas chromatographic analysis gives the 2,3,6-trimethylglucose derivative, so that the glucose units are necessarily 1-4 connected in an exclusively linear structure.

A second methylation product, which, depending on the methylated compound, is present to varying degrees or may not even be present, is the 2,3,4,6-tetramethyl derivative. The presence of this derivative and its molar ratio to the trimethyl derivative depends on the substituent attached to the cyclohexene ring.

It is known that in animals and humans after eating carbohydrate-containing foods and beverages (e.g. cereal, potato starch, fruit, fruit juice, beer, chocolate), hyperglycaemia occurs, which is due to the rapid breakdown of carbohydrates by glycoside hydrolases (e.g. Saliva and pancreatic amylases, maltases, saccharases) according to the following scheme

Amylase maltase

Starch or glycogen * ■ Maltose »'glucose

Sucrose

Sucrose ** glucose + fructose can be effected. These hyperglycaemias are particularly strong and persistent in diabetics. In obese people, alimentary hyperglycaemia often causes a particularly strong secretion of insulin, which in turn leads to increased fat build-up and reduced fat loss. Following such hyperglycaemia, hypoglycaemia often occurs in healthy and obese people as a result of insulin secretion. It is known that both hypoglycaemia and gastric chyme promote the production of gastric juice, which in turn triggers or favors the development of gastritis, ulcer ventriculi or duodeni.

It has now been found that the aminosugar derivatives obtained according to the invention considerably reduce the alimentary hyperglycemia, hyperinsulinemia and hypoglycemia, in particular also the reactive postprandial hypoglycemia.

It is also known that carbohydrates, especially sucrose, are split in the oral cavity by microorganisms, thereby promoting caries formation.

The aminosugar derivatives produced according to the invention can be used to prevent or reduce such caries formation.

In vitro amylase test

An amylase inhibitor unit (1 AIE) is defined as the amount of inhibitor that inhibits two amylase units by 50%. An amylase unit (AE) is the amount of enzyme that 1 p. In one minute under the test conditions given below. Equivalent glucosidic bonds cleave in starch. The | j.Val cleaved bonds are determined colorimetrically as n-Val-forming reducing sugar with dinitrosalicylic acid and are given using a maltose calibration curve as (iVal-maltose equivalents. To perform the test, 0.1 ml amylase solution (20-22 AU / ml ) with 0-10 ng inhibitor or 0-20 ym 1 of the solution to be tested in 0.4 ml 0.02m sodium glycerophosphate buffer / 0.001m CaCh pH 6.9 and about 10-20 minutes in a water bath at 35 ° C equilibrated.

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

11

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Then it is incubated for 5 minutes with 0.5 ml of a 1% starch solution (starch, soluble, from Merck, Darmstadt, No. 1252) preheated to 35 ° C at 35 ° C and then with 1 ml of dinitrosalicylic acid reagent (after P. Bernfeld in Colowick-Kaplan, Meth. Enzymol., Volume 1, page 149). To develop the color, the batch is heated on the boiling water bath for 5 minutes, then cooled and mixed with 10 ml of distilled water. The absorbance at 540 nm is measured against a correspondingly set blank value without amylase. For evaluation, the amylase activity still effective after the addition of inhibitor is read from a previously recorded amylase oak curve and the percentage inhibition of the amylase used is calculated therefrom. The percent inhibition is a function of the quotient

[ig inhibitor *

AE **

* based on dry matter

** AE applied in the uninhibited batch of the same series, the 50% inhibition point read from the curve and converted to EIE / mg inhibitor.

In vitro saccharase test

A sucrose inhibitor unit (SIE) is defined as the amount of inhibitor that inhibits two sucrose units by 50%. A sucrose unit (SE) is the amount of enzyme that cleaves 1 nmole of sucrose into glucose and fructose in one minute under the test conditions given below. The p.mol of glucose formed is determined quantitatively with the aid of the glucose oxidase reaction under conditions in which further sucrose cleavage by the saccharase no longer takes place. To carry out the test, 0.05 ml of a saccharase solution adjusted to 0.12 SE [solubilized saccharase from pig small intestinal mucosa according to B. Borgström, A. Dahlquist, Acta Chem. Scand. 12 (1958), page 1997. Diluted with 0.1 M sodium maleate buffer pH 6.0 to the appropriate SE content.] With 0-20% inhibitor or 0-20 p.1 of the solution to be tested and 0.1 Make up to 0.1 ml in sodium 6.0 maleinate buffer. It is equilibrated for 10 minutes at 35 ° C. and then mixed with 0.1 ml of a 0.05 M sucrose solution preheated to 35 ° C. in 0.1 M sodium maleate buffer pH 6.0. The mixture is incubated at 35 ° C. for 20 minutes and the saccharase reaction is stopped by adding 1 ml of glucose oxidase reagent [The glucose oxidase reagent is obtained by dissolving 2 mg of glucose oxidase (Boehringer, grade I) in 100 ml of 0.565 M Tris-HCl buffer pH 7.0 and subsequent addition of 1 ml detergent solution (2 g Triton X 100 + 8 g 95% ethanol pa), 1 ml di-nisidine solution (260 mg o-dianisidine • 2 HCl in 20 ml H2O) and 0, 5 ml of 0.1% aqueous peroxidase solution (from Boehringer; lyophilisate, degree of purity II).] And incubated at 35 ° C. for a further 30 minutes. Then 1 ml of 50% H2SO4 is added and measured at 545 nm against a corresponding blank value. For the evaluation, the percentage inhibition of the saccharase used is calculated and converted from the 50% inhibition point to SIE / g or SIE / liter using a glucose calibration curve.

Malta test

A maltase inhibitor unit (MIE) is defined as the amount of inhibitor that inhibits two maltase units by 50%. A unit of maltase (ME) is the amount of enzyme which cleaves 1 pmol of maltose into 2 jiMoles of glucose in one minute under the test conditions given below. The glucose formed is quantified with the aid of the glucose oxidase reaction under conditions in which a further maltose cleavage by the maltase no longer takes place. To carry out the test, 0.05 ml of a maltase solution adjusted to 0.060-0.070 ME [solubilized maltase from small intestinal mucosa according to B. Borgström, A. Dahlquist, Acta Chem. Scand. 12, (1958), page 1997. Diluted to the appropriate ME content with sodium maleate buffer pH 6.0.] With 0-20 ml inhibitor or 0-20 ml of the solution to be tested and 0.1 m sodium maleate pH buffer 6.0 to 0.1 ml. It is equilibrated for 10 minutes at 35 ° C. and then mixed with 0.1 ml of a 0.05 M maltose solution preheated to 35 ° C. in 0.1 M sodium maleate buffer pH 6.0. The mixture is incubated at 35 ° C. for 20 minutes and the maltase reaction is stopped by adding 1 ml of glucose oxidase reagent [preparation of the glucose oxidase reagent: as described in the sucrose test, p. 30, footnote 2).] And incubated at 35 ° C. for a further 30 minutes. Then 1 ml of 50% H2SO4 is added and measured at 545 nm against a corresponding blank value.

For evaluation, the percentage inhibition of the maltase used is calculated and converted from the 50% inhibition point to MIE / g or MIE / liter using a glucose calibration curve.

The results of the in vitro enzyme inhibition tests for individual aminosugar derivatives according to the invention are summarized in Table II below:

Table II

For

Number of a-amylase

Sucrose

Maltase-Inhi

mel

Glucose unit

Inhibition inhibition bition

ten

AIE / g

Victory

MIE / g

IIB

1

300000

30000

5,000

HIB

2nd

300000

68,000

15,000

V

3rd

1,400,000

21000

5,000

4-6

17,500,000

8500

-

5-7

30000000

2,000

-

As can be seen from the table, the spec. In vitro inhibition activity against pancreatic a-amylase strongly increases with increasing molecular weight of the series; the compounds with 5-7 glucose units show a 100-fold greater inhibition of enzyme in vitro than the compound with 1 or 2 units. Saccharase inhibition is most pronounced in the 2-unit derivative, the 1-glucose unit derivative inhibits only half as much, and the higher link shows a further decrease in the specific sucrose inhibition activity.

In vivo, the effectiveness in the saccharase inhibition of the spec found in vitro. Inhibitory activity roughly parallel. On the other hand, surprisingly, in the starch load test in vivo for the compounds with 1, 2 or 3 glucose units, an efficacy was found to be 10-40 times higher than that of the amylase inhibition in vitro.

To produce alimentary hyperglycaemia and hyperinsulinemia, groups with 6 fasting rats each received a) 2.5 sucrose,

b) 2.5 g of maltose or c) 1 g of boiled starch p.os applied in aqueous solution or as a suspension. 6 other rats receive the carbohydrates mentioned in the same amount and additionally a glucoside hydrolase inhibitor in the dosage indicated.

In addition, 6 other rats receive an appropriate amount of saline.

Blood glucose and serum insulin were measured at short intervals in the blood from the retroorbital venous plexus. The blood glucose determinations are carried out in the Auto-Analyzer [Technicon® according to Hoffman:]. biol. Chem. 120, 51 (1937) or

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633043 12

enzymatically with glucose oxidase and o-dianisidine hydrochloride - the results of in vivo saccharase inhibition (sucrose

rid), the serum insulin determination according to the method of administration) in the fasted rat gives the following

Haies and Rändle: Biochem. J. 88, 137 (1963)]. Table III:

Table III

Blood glucose in mg% (mean + deviation)

Formula n dose (SIE) 15 min. 30 min. 45 min. 60 min

Saline solution

69 ± 5.2

84 ± 3.9

91 ± 5.9

-

Sucrose (control)

117 + 11

135 ± 8.1

152 ± 18

-

Sucrose + 50 YOU

105 ± 11

123 ± 5.2 *

128 ± 11 *

-

Sucrose + 100 YOU

89 ± 6.5 ***

111 ± 4.1 ***

116 ± 3.9 ***

-

Sucrose + 200 YOU

72 ± 4.6 ***

95 + 9.1 ***

104 ± 9.6 ***

-

Saline solution

55 ± 4.5

92 ± 8.5

95 ± 6.4

-

Sucrose (control)

113 ± 9.9

126 ± 20

127 ± 14

-

Saccarose + 25 YOU

71 ± 3.7 ***

100 ± 7.3 *

107 ± 2.4 **

-

Saccarose + 100 YOU

58 ± 5.9 ***

92 ± 4.0 **

98 ± 5.0 ***

-

Saline solution

66 ± 4.6

73 ± 4.3

76 ± 3.9

-

Saccarose (control)

122 ± 6.6

125 + 16.0

128 ± 14.8

-

Saccarose + 25 YOU

88 ± 11.4 **

101 ± 12.0 *

104 ± 7.4 **

Sucrose + 50 YOU

91 ± 9.4 **

106 ± 7.0 *

94 ± 9.0 ***

-

Sucrose +100 YOU

81 ± 7.2 **

90 ± 8.0 ***

97 ± 1.6 ***

-

Saline solution

58 ± 2.9

49 ± 3.3

54 ± 4.8

63 ± 5.0

Sucrose (control)

107 ± 11

121 ± 6.0

132 ± 16

132 ± 6.7

Sucrose + 25 YOU

109 ± 6.4

105 ± 10 **

112 ± 9.1 *

108 ± 8.1

Sucrose + 50 YOU

103 ± 4.1

107 ± 15

102 ± 7.7 **

101 ± 9.2 '

Sucrose + 100 YOU

85 ± 8.1 **

94 ± 9.9 ***

95 ± 8.5 ***

85 ± 6.4

Saline solution

58 ± 2.9

49 ± 3.3

54 ± 4.8

63 ± 5.0

Sucrose (control)

107 ± 11

121 ± 6.0

132 + 16

132 + 6.7

Sucrose + 75 YOU

102 ± 11

108 ± 9.6 *

109 ± 7.5 **

101 ± 7.9 '

Sucrose + 150SIE

92 ± 7.0 *

100 ± 8.3 ***

109 ± 3.7 **

102 ± 6.3 '

Sucrose + 300 YOU

95 ± 8.1 *

84 ± 8.7 ***

93 ± 9.0 ***

91 ± 4.5 '

* Probability against control with sucrose = <0.05

** Probability against control with sucrose = <0.01

*** Probability against control with sucrose = <0.001

As can be seen from the above data, the in vivo activity in saccharase inhibition runs parallel to the inhibitory activity found in vitro. The ED increases so, while the saccharase injection units per mg decrease. The results are summarized in Table IV below:

Table IV

Formula Glucoseein-saccharase inhibition in vitro in vivo

YOU / mg (EDso mg / kg)

IIB (1) 30 3.00

HIB (2) 68 0.21

45 V (3) 21 2.65

(4-6) 8.5 -15.30

(5-7) 2.5 -52.00

50

Surprisingly, the in vivo inhibition of starch degradation by the compounds of the formulas IIB, IIIB and V is much higher than would have been expected from the data found in vitro for the amylase inhibition and therefore does not correspond to the expected pattern. Table V below provides the in vivo data for the fasted rat after starch administration as determined above:

Table V

For gluco dose (AIE) blood glucose in mg% (mean ± deviation)

with units

5 min. 10 min. 15 min. 20 min. 30 min. 45 min.

IIB 1 saline solution 74 ± 6.2 74 ± 3.0

Strength (control) 101 ± 8.8 135 ± 13

Strength + 75 AIE 102 ± 2.9 130 ± 6.9

Strength-I-150 AIE 98 ± 8.7 116 ± 9.0 *

Strength + 300 AIE 87 ± 13 106 ± 1.8 **

82 ± 9.4 146 ± 4.8 128 ± 5.4 *** 128 ± 5.8 *** 110 ± 8.9 ***

71 ± 11 151 ± 9.5 143 ± 11 127 ± 4.8 *** 111 ± 2.1 ***

76 ± 5.4 148 ± 18 125 ± 11 * 115 ± 5.2 ** 108 ± 5.3 *

90 ± 9.2 156 ± 19 132 ± 18 * 137 ± 5.2 ** 121 ± 7.2 *

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For gluco dose (AIE) blood glucose in mg% (mean ± deviation)

with units

5 min. 10 min. 15 min. 20 min. 30 min. 45 min.

IIIB 2

Saline solution

61 ± 3.7

68 ± 3.9

83 + 5.3

75 ± 8.7

91 ± 5.2

84 ± 1.5

Strength (control)

85 ± 7.9

120 ± 7.7

132 ± 11

140 ± 12

135 ± 5.0

128 ± 14

Strength + 150 AIE

82 ± 7.5

105 ± 4.2 **

120 ± 6.0 *

124 ± 7.0 *

134 ± 6.1

125 ± 5.2

Strength + 300 AIE

79 ± 8.2

97 ± 2.7 ***

105 ± 10 **

118 ± 6.7 **

119 ± 9.9 **

130 ± 5.9

Strength + 600 AIE

70 ± 4.6 **

80 ± 8.8 ***

94 ± 6.9 ***

91 ± 16 ***

103 ± 9.2 ***

102 ± 4.2 ***

Strength + 1200 AIE

70 ± 7.0 **

84 ± 7.7 ***

88 ± 7.4 ***

97 ± 5.2 ***

97 ± 5.0 ***

100 ± 4.3 ***

V 3

Saline solution

52 ± 2.3

75 ± 7.9

57 ± 3.7

78 ± 9.6

82 + 6.5

70 ± 6.9

Strength (control)

93 ± 7.3

133 ± 12

127 ± 9.3

152 ± 15

156 ± 15

124 ± 7.7

Strength + 750 AIE

86 ± 8.5

107 ± 5.0 ***

117 ± 8.7 *

121 ± 12 **

126 ± 11 ***

114 ± 3.6 ***

Strength + 1500 AIE

77 ± 14 *

92 ± 9.6 ***

94 ± 9.5 ***

113 ± 9.7 ***

112 ± 8.8 ***

111 ± 4.2 **

Strength + 3000 AIE

65 ± 5.6 ***

101 + 3.3 ***

82 ± 11 ***

112 ± 4.5 ***

109 ± 6.4 ***

104 ± 2.5 ***

4-6

Saline solution

-

55 ± 4.8

63 ± 5.9

67 ± 5.8

72 ± 4.7

66 ± 5.0

Strength (control)

-

103 ± 11

117 ± 8.6

118 ± 8.5

128 ± 8.9

109 ± 11

Strength + 6000 AIE

-

99 ± 4.5

118 ± 5.8

104 ± 8.1 *

123 ± 11

107 ± 7.3

Strength + 12000 AIE

-

90 ± 5.2 *

98 ± 8.2 **

99 ± 4.1 ***

117 ± 6.7 *

104 ± 8.2

Strength + 24000 AIE

-

74 ± 5.5 ***

82 ± 2.9 ***

83 ± 3.7 ***

96 ± 6.6 ***

85 ± 6.4 **

5-7

Saline solution

-

55 ± 4.8

63 ± 5.9

67 ± 5.8

72 ± 4.7

66 ± 5.0

Strength (control)

-

103 ± 11

117 ± 8.6

118 ± 8.5

128 ± 8.9

109 ± 11

Strength + 6000 AIE

-

95 ± 4.3

107 ± 3.3 *

102 + 5.5 **

111 ± 3.0 **

102 ± 6.7

Strength + 12000 AIE

-

87 ± 8.6 *

94 ± 8.8 **

89 ± 8.5 ***

98 ± 8.3 ***

91 ± 4.3 **

Strength + 24000 AIE

-

74 ± 8.9 ***

84 ± 7.2 ***

84 ± 6.9 ***

85 + 6.5 ***

85 ± 4.9 **

* Probability against control with strength = <0.05 ** Probability against control with strength = <0.01 *** Probability against control with strength = <0.001

From the above data it can be seen that while the degree of saccharase inhibition proves to be a distinct function of the molecular weight both in vitro and in vivo, the amylase inhibition is dependent on the molecular weight only in vitro, while in vivo the starch degradation inhibition with the falling molecular weight does not decrease, but surprisingly remains constant.

Thus, although the in vivo amylase inhibitory activity decreases with the molecular weight, the ED 50 values of the starch digestion inhibition are essentially the same for all compounds according to the invention. This is summarized in the following Table VI:

Table VI

Table VII

dose

Blood glucose in mg% (mean ± deviation) 15 min. 30 min. 45 min.

72 + 4.6 78 + 1.3 142 ± 12 142 ± 12

Saline solution glucose 35 (control)

Glucose + 30 135 ± 13 128 ± 5.5 mg

Glucose + 60 125 ± 13 * 118 ± 2.9 * mg

87 ± 6.8 158 ± 19

132 ± 7.3

130 ± 9.0 ***

α-amylase inhibition

Starch digestion inhibitor

tion

For

Glucose in vitro in vivo mel units

AIE / mg

(EDso mg / kg)

IIB

(1)

300

0.76

IIIB

(2)

300

1.60

V

(3)

1400

1.61

-

(4-6)

17500

1.42

-

(5-7)

30000

1.00

* Probability against control with glucose = 0.05 ** Probability against control with glucose = 0.01

Purification of the mixture of the higher links of this series and application in pure form unexpectedly leads to even higher activity and specificity. This can be demonstrated by comparing the a-amylase and saccharase in vitro inhibition data given in Table II with the data for the pure compounds given in Table VIII below:

50

Table VIII

It is therefore surprisingly possible to simultaneously inhibit sucrose and starch digestion with the compounds IIB, HIB and V, with a precise, predictable and characteristic dose.

The compounds also have an advantageous effect with regard to glucose absorption. This is evident from the following data in Table VII (experiments with starving rats) for the compound of the formula HIB (m = 2).

60

Number of a-amylase inhibition

Saccharase inhibition

Glucose units

AIE / g

Victory

4th

67 000000

7000

5

57,000,000

3500

6

42 000000

1200

7

24 000000

60

8th

5,000,000

10th

The previous results show the high activity of the compound with 4 glucose units in the role of an a-amylase inhibitor. The compounds with 5 and 6 units show significantly higher specific inhibitory activity than was found in previously known preparations. A decrease in inhibitor activity occurs with increasing molecular weight, as with the compounds with 7 and 8 gluco-

633043 14

units can be seen, although these are still considerable inhibitors. The tablets are made, for example, by activity. In vitro saccharase inhibition appears to result in the production of a powder mixture, coarse or fine-grained, and inversely dependent on the molecular weight. Addition of a lubricant and disintegrator. From this hen. The compound with 4 glucose units has about half of the mixture one forms tablets. A powder mixture prepares specific activity which the compound with 2 glucose units is prepared by mixing the substance which has suitable properties, whereas the inhibitory activity of the compound has been comminuted and a diluent with 8 glucose units is only slightly supplemented. or another carrier as described above. Counter

The compounds can be used without dilution, e.g. B. as a powder add a binder: z. B. carboxymethyl

ver or in a gelatin shell or in combination with a cellulose, alginate, gelatin or polyvinylpyrrolidone, a

Carrier in a pharmaceutical composition 10 solution retarders, such as. B. paraffin, a resorption

be applied. accelerators, such as B. a quaternary salt and / or

Pharmaceutical preparations can be a larger adsorbent, such as. B. bentonite, kaolin or dicalcium

or contain a minor amount of the inhibitor, e.g. B. 0.1% to phosphate. The powder mixture can be granulated together.

99.5%, in combination with a pharmaceutically compatible with a binder, such as. B. syrup, starch paste, Aka-

Chen non-toxic, inert carrier material, the carrier material being acne mucus, or solutions made of cellulose or polymer

one or more solid, semi-solid or liquid dilution materials. The product is then pressed through a coarse medium, fillers and / or non-toxic, inert and pharmaceutical sieve. As an alternative to this, the powder mixture containing compatible formulation agents can be run through a tablet machine and the resultant

can. Such pharmaceutical preparations are preferably in the form of dosage units in the form of dosage units, ie. H. physically- 20 shred. So that the resulting grains do not get stuck in the discrete tray-forming nozzles containing a certain amount of the inhibitor, they can be used with

Units that add a lubricant to a fraction or multiple of the dose, e.g. B. stearic acid, stearate salt,

correspond to that to bring about the desired inhibitory talc or mineral oil. This lubricated mixture

Effect. The dosage units 1, 2, 3, 4 can then be pressed into tablet form. The active compounds can also be combined with more single doses or 'A,' A or 1U of a single dose with free-flowing inert carriers.

hold. A single dose preferably contains a sufficient dose which can be brought directly into tablet form with

Amount of active ingredient to be used in an application according to a presentation of the granulate or fragmentation steps. You can provide the dosage scheme of one or more products with a clear or opaque protective cover,

Dosage units to achieve the desired inhibitory effect. B. a coating of shellac, a coating of sugar len, a whole, a half, or a third or a four or 30 or polymer substances and a polished shell made of wax,

teas of the daily dose, usually once, twice, three times, or dyes can be added to these coatings to be administered four times a day. Different therapeutic agents differ between the different dosage units

tel can also be taken. Although the dose can be.

tion and the dosing schedule in any case carefully. The oral preparation forms, such as. B. should be weighed, using thorough professional solutions, syrups and elixirs, can be prepared in a dose-based judgment and taking into account age, weight, so that a certain amount of preparation and the condition of the patient, the type and the severity of the given amount of active ingredient. Syrup can be prepared disease, the dosage will usually be in a range that the active ingredient is dissolved in an aqueous solution containing between about 30 to about 3 x 105 AIE / kg and between about 1 suitable flavors; Elixirs are up to about 1 x 104 SIE / kg of body weight per day. In 40 cases using non-toxic, alcoholic carriers - in some cases, sufficient therapeutic substances will be obtained. Suspensions can be achieved by dispersing the effect with a lower dose, while in the compound in a non-toxic carrier, other cases, a larger dose will be required. len. Solubilizers and emulsifiers, such as. B. ethoxy

Oral application can be carried out using solid and liquid isostearyl alcohols and polyoxyethylene sorbitol esters, Konger dosage units, such as. B. powder, 45 serving, taste-improving additives such. B. tablets, dragées, capsules, granules, suspensions, solution peppermint oil or saccharin and the like can also gene and the like. be added.

Powder is comminuted by comminution of the substance in a suitable dosage size can be indicated on the capsule and a suitable size can also be comminuted. In addition, the dosage can be secured in such a way that nert pharmaceutical carrier is produced. Although such an active ingredient is released with a delay, e.g. B. by observing edible carbohydrate, such as. B. starch, lactose, sucrose of the active ingredient in polymer substances, wax or the like

or glucose normally used for this purpose.

finds and can also be used here, it is desirable- The toxicity of these compounds is very low. Also worth a non-metabolizable carbohydrate, such as. B. a without final cleaning, for example, the raw product

To use cellulose derivative. 55 Example 8 with an activity of 26,000 SIE / g without

Sweeteners, flavor additives, preservatives, occurrence of side effects can be tolerated if 340,000 sieves / kg of a peringant and coloring agent can also be used. Mouse and rat p. have been applied. Even with intravenous

the. injection, mice tolerate 10,000 SIE / kg without secondary

The capsules can be prepared by preparing the effects described above.

The powder mixture and by filling already formed 60 The pharmaceutical compositions can also

Gelatine casings are made. The powder mixture can contain other pharmaceutically active compounds, in particular

one before the filling process with lubricants, such as. B. silica gel, special other oral antidiabetics, such as. B. β-cytotropic sul-

Talc, magnesium stearate, calcium stearate or solid poly- fonylurea derivatives and bigua

add ethylene glycol. You can also mix with nide.

a disintegrator or solubilizer, such as. B. Agar-65 In addition to the pharmaceutical mentioned above

Add agar, calcium carbonate or sodium carbonate, these active ingredients can also be contained in compositions.

when the capsule is taken, make the inhibitor accessible; for example sugar, bread,

improve. Potato products, fruit juice, beer, chocolate and others

15

633043

Confectionery, and canned goods, such as. B. jam, with these products being given a therapeutically effective amount of at least one of the new derivatives as inhibitors.

Explanation of some of the reagents and auxiliaries used:

As an ion exchanger z. B. the commercially available products Amberlite IRA 410 Cl "(anion exchanger); Amberlite IRC 120 (H + form) (strongly acidic cation exchanger); Amberlite (HCCh'-form) (anion exchanger); Amberlite IRA 410 OH" (strongly basic anion exchanger ); Amberlite IRC 50 H + (weakly acidic cation exchanger) [trademark of Rohm and Haas Company] and Dowex 50 WX 4H + (strongly acidic cation exchanger) [trademark of Dow Chemical Co. Midland, Michigan, USA] can be used.

As a cellulose-based anion exchanger, e.g. B. DEAE cellulose from Schleicher and Schull can be used.

As a polyacrylamide gel z. B. Bio-Gel-P-2 [trademark of Bio-Rad Laboratories, Richmond, California, USA] can be used.

Maitzin is a natural malt extract from Diamalt AG, Munich (FRG).

Lewapol® is a non-specific adsorption resin from Bayer AG, Leverkusen (FRG).

Clarcel is a filter aid from CECA, Velizy-Vil-lacoublay, France.

SE 30 is a silicone elastomer from Hewlett Packard.

The microorganisms used below have been deposited with the American Type Culture Collection under the following numbers:

Strain ATCC no.

SE 50 (CBS 961.70) 31042

SE 18 (CBS 957.70) 31041

SE 82 (CBS 615.71) 31045

SE 50/13 (CBS 614.71) 31043

SE 50/110 (CBS 674.73) 31044

example 1

Inoculate a glass fermenter with 8 liters of nutrient solution of the composition 5.0% starch, 1.0% yeast extract, 0.2% K2HPO4 with a 3-day old shaking flask from strain SE 50/13 (CBS 614.71) and incubated with intensive stirring and aeration 3 days at 28 ° C and receives a culture broth with 105000 AIE / ml.

After cooling to 20 ° C., 6 liters of this fermentation broth are adjusted to pH 2.5 with half-concentrated HNO3, mixed with 30 g of activated carboraffin and stirred for 10 minutes. The mixture is then centrifuged at 10,000 rpm for 15 minutes and the clear, light yellow supernatant is concentrated to 500 ml after neutralization with NH3. The 500 ml concentrate was stirred for 45 minutes with 200 g of Amberlite IRA 410 Cl ", filtered off with suction and mixed with 4 / s vol. = 400 ml of methanol in order to precipitate out the majority of the higher molecular weight starch breakdown products (together with any remaining activated carbon residues). It takes 5 minutes Centrifuged at 5000 rpm. The 850 ml supernatant are dropped into 41 dry spirits with intensive stirring. The white, flaky precipitate is filtered off, washed 3 times with dry spirit and 2 times with ether and dried in vacuo at 50 ° C. Yield: 36 g of a white powder with 10 106 AIE / g. In some of the following examples this preparation is used.

Enzyme inhibition on thin film plates

To assess the end product of fermentations and the composition of preparations by means of thin layer chromatography, apply 1 (il of the fermentation broth or 1 p, g of the preparations on silica gel ready foils (Schleicher and Schüll, Dassel, type F1500) and develop 2 times in n-butanol / Ethanol / water = 50/30/20.

For saccharase inhibition staining, spray the developed and well-dried plate with enzyme gel (20 ml / 20x20 cm plate) and allow the gel to solidify. Then it is preincubated for 5 'in a moist chamber at room temperature and then sprayed with substrate gel. After this second gel layer has solidified, the plate is placed in a moist chamber and incubated at 40 ° C. The inhibition staining (light spots, red-brown background) develops in 60-90 '. At the time of the optimal color development, the plate is broken off and the plate with the agar layers on it is dried on a hairdryer with warm air.

Preparation of the gels

Enzyme gel: 1.5 g agarose (L'Industrie Biologique Français) is suspended in 100 ml 0.2 m Na-maleinate buffer pH 6.0 and then dissolved by boiling. The clear agarose solution is cooled to 50 ° C and with 250 jo.1 Triton X-100 solution (2 g Triton X-100 + 8 g ethanol pa) and 0.5 ml dianisi dinine solution (20 mg dianisidine / 1 ml acetone) staggered. Immediately before use of the gel, 1 ml GOD / POD reagent (12.5 mg glucose oxidase, purity grade I, Boehringer, and 2.5 mg peroxidase, purity grade II, lyophilisate, Boehringer, dissolved in 5 ml maleate buffer) and 4 to 5 units of small intestine saccharase were added. The gel must be kept at 50 ° C until spraying, otherwise it solidifies in the nozzles during the spraying process. Substrate gel: 0.5 g agarose is suspended in 100 ml Na maleinate buffer pH 6.0 and dissolved while boiling. It is then cooled to 50 ° C., mixed with 100 μl of Triton (2 g of Triton X-100 + 8 g of ethanol p.a.) and 1 g of sucrose (Serva No. 35579) is added. After the sucrose has been dissolved, the gel is ready for use.

To stain the amylase, the sprayed and dried TLC plate is sprayed with an amylase gel (20 ml / 20x20 cm plate) and allowed to solidify. After 5 'preincubation at room temperature, the plate with the gel layer is placed in a 0.5% starch solution (1 g starch, Merck No. 1252, dissolved in 200 ml 0.2 m glycerophosphate buffer 0.01 m CaCh pH 6.9 with boiling ) and leave it there for 2 'at 40 ° C while swirling the solution. Then the plate is distilled. Rinse the water well and immerse it in a dilute J2 solution (4 ml Jt stock solution to 500 ml H2O; J2 stock solution: 2.2 g J2 + 4.4 g K] dissolved in 100 ml H2O) to stain the undigested starch. The color is optimal after about 1 minute. You take pictures immediately because the bruises fade quickly.

Preparation of the amylase gel

1 g agarose is dissolved in 100 ml 0.2 m sodium glycerophosphate / 0.01 m CaCh buffer pH 6.9 at 100 ° C, after cooling to 50 ° C 100 (xl Triton X-100 (2 g Triton X -100 + 8g ethanol pa) 100 ml of an amylase crystal suspension (10 mg pork pan-kreas amylase / ml of total NH4 sulfate solution, Boehriner) are added directly before spraying.

Example 2

Inoculate 11 Erlenmeyer flasks with 120 ml of a nutrient solution consisting of 4% starch, 2.4% glucose, 0.9% casein hydrolyzate and 0.9% yeast extract, pH adjusted to 7.6 with NaOH, with 0.4% CaC03 added and sterilized for 30 min at 121 ° C, with 3 ml of a preculture of the strain SE 82 (CBS 615.71), grown in a nutrient solution consisting of 2% starch, 1% gluc

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633043

16

cose, 0.5% casein hydrolyzate and 1% yeast extract, pH adjusted to 7.2 with NaOH, mixed with 0.4% CaC03 and sterilized for 30 min at 121 ° C, and incubated for 5 days at 28 ° C on a rotary shaker, see above a culture solution with 122 000 AIE / ml is obtained. For working up, the mycelium is separated from the combined culture solutions by centrifugation at 12,000 rpm, and 300 ml of the culture filtrate are brought in with half-conc. HNO3 to pH 2.5 and stir for 10 min with 2.5 g activated carbon p.A. After the coal has been separated off at 12,000 rpm, the solution neutralized to pH 6 using 10N KOH is mixed with 300 ml of methanol, left to stand briefly and the precipitate is removed at 12,000 rpm. If the supernatant is then dripped into 31 ethanol, the precipitate is isolated after a short standing by centrifugation at 12,000 rpm, the precipitate is washed twice with abs. Ethanol and once with ether and dried in vacuo gives 2.23 g of a product with 7.45 x 106 AIE / g, which contains over 95% compounds with m + m = 4 and more glucose units.

Example 3

Inoculate 11 Erlenmeyer flasks with 120 ml of a nutrient solution of the composition 3.5% glucose, 2% starch, 0.5% casein hydrolyzate, 1.3% yeast extract, 0.3% CaCCh and 0.3% K2HPO4 before sterilization to 7.8 adjusted, sterilization 307121 ° C, with 6 ml of a preculture of the strain SE 50/110 in a nutrient solution consisting of 3% soy flour, 3% glycerol and 0.2% CaCCh and incubated for 3-4 days on a rotary shaker at 24 ° C, this gives a culture solution containing 153,000 AIE / ml and 12 200 SIE / 1.

11 culture solution was adjusted to pH 2.5 with HNO3, stirred 10 'with 5 g activated carbon and then centrifuged 30' at 5000 rpm. The mixture was then neutralized by adding 25 g of Amberlite IRA 410 (OH ~ form). The neutral supernatant was spun in to 100 ml, mixed with 100 ml of methanol and filtered. The filtrate was stirred into 21 dry spirits, the precipitate which formed was filtered off with suction and, after washing three times with acetone and ether, dried in vacuo

Yield 14 g of a white powder with 5 x 106 AIE / g, which mainly contains compounds with at least m + m = 4 glucose units.

Example 4

If one works according to Example 3, but with 0.5% starch, after 4 days of fermentation, a culture broth with 40,000 AIE and 184 SIE / ml is obtained. The culture broth contains a mixture of the compounds according to the invention which have at least one glucose unit.

Example 5

Inoculate 11 Erlenmeyer flasks, the 120 ml nutrient solution of the composition 3% glucose, 0.6% casein hydrolyzate, 1.6% yeast extract, 0.3% CaCCh, 0.3% K2HPO4, pH before sterilization with KOH to pH 7.8 adjusted, with a preculture of the SE 50/110 strain (CBS 674.73) according to Example 3 and incubated for 4 days at 24 ° C. on a rotary shaker, a culture broth with 10 800 SIE / 1 is obtained, which predominantly contains the compound according to the invention contains a glucose unit.

51 culture filtrate, separated from the mycelium at 13,000 rpm, were mixed with semi-concentrated HNO3 adjusted to pH 2.5 and stirred for 15 min with 55 g activated carbon («Merck») and 200 g Clarcel. After removing the solids by suction, neutralized with conc. Ammonia to pH 7, concentrated the solution to 1.51 and precipitated with five times the amount of ethanol. The resulting flocculent precipitate was separated off by means of a continuous rotor at 12,000 rpm and the yellowish supernatant was concentrated to 150 ml and centrifuged at low speed to remove small amounts of undissolved material. 50 ml of this solution were placed on an Amberlite IR-120 (H + form)

filled column (30x300 mm; 30 ml H2O per hour). After a total of 300 ml of eluate had been collected, which contains inert saccharides and a portion of non-adsorbed inhibitory components, the exchanger was transferred into a beaker with approx. 400 ml of H2O and concentrated while stirring. Ammonia until the pH reached 11.5. After stirring for a further 30 min, the mixture was separated off, the volume was reduced to V20, the mixture was filtered through a column (20 × 150 mm) with Amberlite IRA-410 (HC03 ′ form) and the mixture was caught at a flow rate of 30 ml / h approx. 500 ml of eluate, which was concentrated and gave 1.3 g of crude product after lyophilization.

For further purification, the crude preparation was fractionated on Bio-Gel P-2,100-200 mesh (from Bio-Rad, Munich). This was done using a column of 50 mm 0 and 450 mm in length, which was operated with H2O at a flow rate of 40 ml / h, collecting fractions of 10 ml each. All fractions were tested for carbohydrates using the anthrone test and for inhibitory active components using the saccharase inhibition test. The fractions containing saccharase inhibitor were further examined for their content of individual components by thin-layer chromatography according to Example 1. Those fractions containing the compound with a glucose unit were pooled, concentrated and lyophilized. 35 mg of the substance were obtained with 0.3 x 106 AIE / g and 30,000 SIE / g.

Example 6

If 11 Erlenmeyer flasks are inoculated, each with 120 ml of a nutrient solution of the composition 5% starch, 1% yeast extract, 0.2% K2HPO4, each with 2 ml of a preculture according to Example 3, culture solutions are obtained after 3 days of incubation at 28 ° C. with the following yield Amylase inhibitor:

tribe

AIE / ml

SE 50 (CBS 861.70)

37,000

SE 50/13 (CBS 614.71)

109,000

SE 50/110 (CBS 674.73)

53500

Which mainly consist of a mixture of the compounds with 4 or more glucose units.

Example 7

Inoculate 11 Erlenmeyer flasks with 120 ml nutrient solution each with the composition 1.3% maltose, 3.5% glucose, 0.5% casein hydrolyzate, 1.3% yeast extract, 0.3% CaC03.0.3% K2HPO4 with 2 ml each a preculture according to Example 3, the following yields are obtained after 4 days of incubation on rotary shakers at 24 ° C. with different strains:

tribe

YOU / ml

AIE / ml

SE 50 (CBS 961.70)

25th

580

SE 50/13 (CBS 614.71)

14.8

1460

SE 50/110 (CBS 674.73)

57.9

755

Which mainly consist of a mixture of the compounds with 4 or less glucose units.

Example 8

Inoculate a fermenter with 1001 nutrient solution of the composition 3.5% glucose, 2.5% corn acid, 0.5% casein hydrolyzate, 1.3% yeast extract, 0.3% CaCoî, 0.3% K2HPO4 and 0.1% anti-foaming agent with 51 a preculture according to Example 3 and incubated with stirring and aeration for 5 days at 24 ° C, see above

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

17th

633043

a culture solution with 73,000 SIE / L is obtained which predominantly contains the compound according to the invention with m + m = 2.

901 fermentation batch were with mycelium on the pH meter with conc. HNO3 is adjusted to pH 2.5 and 900 g (= 1%) activated carbon (Merck) are added with stirring to adsorb the majority of the dyes formed. The mixture was stirred for 15 minutes, separated from the mycelium and the bulk of the coal by means of a centrifuge at 3000 rpm, and the supernatant was then filtered through a pressure filter with the addition of 3 kg of Clarcel. 611 yellow-brown, clear filtrate with a SIE content of 60,000 SIE / 1 was obtained.

The filtrate was concentrated. NH3 adjusted to pH 7 and stirred for 30 'to adsorb the active ingredient with 1300 g (2%) activated carbon (Merck). It is filtered through a pressure filter and the activated carbon sediment is washed 3 times with 101 distilled water. The coal was then pressed dry and stirred in 3x4150% acetone at pH 2.5 for 15 'each in order to desorb the active ingredient from the coal. The acetonic desorbates were - after separation from the coal by filtration - combined. The combined desorbate was concentrated to 250 ml on a rotary evaporator, an equal volume (250 ml) of methanol was added and the mixture was filtered through a folded filter. The filtrate (480 ml) was dropped in 51 acetone with vigorous stirring. The precipitate was filtered off and washed 3 times with acetone and ether. The mixture was then dried in vacuo at 35 ° C. Yield 230 g with 8500 SIE / g.

25 g of the above crude product was dissolved in 11H2O and stirred with 300 g Dowex® 50 WX 4 H + (200-400 mesh) 30 '. The resin was filtered off and washed 3 times with 210.001 N HCl. The washed Dowex was then suspended in 500 ml of H2O and the suspension was adjusted to pH 9.0 on the pH meter by adding 25% NH3. The mixture was then desorbed twice with 500 ml of 0.6% NH3 each, the desorbates combined and concentrated to 100 ml on a rotary evaporator. To decolorize this concentrate, it was stirred with 2 g of DEAE cellulose (from Schleicher and Schull No. 02035.0.6 meq / g) for 5 min, then centrifuged. An equal volume (100 ml) of methanol was added to the light yellow supernatant and then added dropwise in 21 acetone with vigorous stirring. The precipitate was filtered off, washed with acetone and ether and dried in vacuo at 35 ° C. Yield 4.2 g with 26,000 SIE / g. For further fine cleaning, the 4.0 g inhibitor was filtered in 0.5 g portions over Biogel P-2. For this purpose, 0.5 g of the preparation was dissolved in 10 ml of H2O and applied to a Biogel P-2 column (200-400 mesh, Bio-Rad) with a diameter of 5 cm and a length of 95 cm. It was developed in water at a flow rate of 80 ml / h. 12 ml fractions were collected. The total carbohydrate content (in the form of the anthrone test as an absorbance at E620) and the content of saccharase inhibitor and amylase inhibitor were determined from all fractions. In addition, the fractions were checked by thin layer chromatography (enzyme inhibition staining according to Example 1).

The fractions containing the compounds with 4-6 glucose units were combined, concentrated in vacuo to 10 ml and precipitated by dropping in 200 ml of a dry syringe. The precipitate was centrifuged off, washed with acetone and ether and dried in vacuo; Yield from 4.0 g of crude inhibitor: 0.2 g of the compounds with 4-6 glucose units with 17.5 x 106 AIE / g and 8500 SIE / g. The fractions containing the compound with 3 units were worked up in the same way, the precipitation being carried out with 200 ml of acetone; Yield from 4.0 g of crude inhibitor: 0.1 g of the compound with ni + m = 3 with 1, 4x 106 AIE / g and 21,000 SIE / g. From the fractions containing the compound with m + n2 = 2 (precipitation with acetone), 0.9 g of the compound with m + m = 2 units with 0.3 x 106 AIE / g and 68,000 SIU / g were isolated.

Example 9

Inoculate 3 small fermenters with 81 each of the nutrient solution 7.5% corn acid, 0.3% casein hydrolyzate, 0.7% yeast extract, 0.3% CaC03.0.3% K2HPO4 with 5% of a preculture of the SE 50/110 strain ( CBS 674.73) (obtained according to Example 3), after 5 days of incubation at 24 ° C., a culture broth with 73 sieves / ml is obtained which predominantly contain the compound with m + m = 2. After centrifugation (30 ', 3000 rpm) to separate the mycelium, 20.51 of a deep brown culture solution with 67,000 SIE / 1 was obtained. The pH was adjusted to 3.5 with HNO3 and 60 g Lewapol (Ca 9221, 0.35 mm grit, Bayer) / L = 1.23 kg Lewapol were added for decolorization. After stirring for 20 minutes, the mixture was filtered through a Seitz K3 filter. The decolorized culture solution was neutralized with NH3 (18.5 L, 67,000 SIE / 1). 20 g of activated carbon / 1 = 370 g were then stirred in to adsorb the active ingredient, and Hess 30 'was stirred in. The mixture was then filtered through a Seitz K3 filter which was coated with a layer of Clarcel filter aid. The filtrate (17.51.3600 SIE / 1) was discarded. The coal residue was 3 times with 21 dest. H2O washed. To desorb the active ingredient from the coal, it was washed 3 times in succession with 1180% each. Stirred acetone for 15 ', the pH with conc. HCl was adjusted to pH 2.5. The desorbates were combined (2.41.371 000 SIE / 1). 20 g of Dowex H + / l (Dowex 50 WX 4, H + form, from Serva, Heidelberg) = 46 g of Dowex were introduced into this desorbate and stirred 20 '. The resin was then filtered off (Dowex fraction I) and washed with a little 75% acetone. The filtrate and washing liquid (31 = 215,000 SIE / 1) were stirred with 60 g of Amberlite IRA 410 (OH "form) (Serva Heidelberg) / 1 until pH 7 was reached. The mixture was then filtered off and the filtrate (2, 8 L, 219,000 SIE / 1) were mixed with 72 g of Dowex H + and stirred for 20 minutes. The pH was kept at 3.0 by hanging in a porous nylon bag filled with Amberlite IRA 410 OH ". The Dowex was then filtered off (Dowex fraction II) and the filtrate (2.61.27 000 SIE / L) was discarded.

Dowex fractions I and II were each washed 3 times with 75% acetone at pH 3.5 and then desorbed 3 times with 100 ml (Dowex fraction I) and 150 m (Dowex fraction II) 0.6% NH3. In the 1st desorption, in which the amount of ammonia was not sufficient to neutralize the Dowex resin, was added by adding conc. NH3 adjusted to pH 9 on the pH meter. The 3 desorbates from Dowex fractions I and II were combined on their own, almost evaporated to dryness on a rotary evaporator, taken up in 50 ml of H2O, adjusted to pH 3-4 on a pH meter with HCl and mixed with 50 ml of methanol. The solutions were added dropwise in 1.51 absolute acetone with stirring, the falling precipitate was filtered off with suction and washed 3 times with acetone and once with ether. It was dried in a vacuum. Yield: Fracton I 6.5 g 25,000 sie / g Fraction II 12.3 g 36,000 sie / g

Fractions I and II contain as inhibitory components mainly the compound according to the invention with m + m = 2 in addition to small proportions of the compound with m + m = 3.

The following table shows the saccharase inhibitory activities of the preparation in successive stages:

yield

volume

(1)

YOU / 1

YOU total

She from-

prey

(%)

1) Culture solution

20.5

67000

1373500

100

2) according to Lewapol

19.5

67000

1306500

95

Discoloration

3) after

18.5

3600

66600

(4.8

Activated carbon adsorption

muddled

fen)

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

633 043

18th

Volu

YOU / 1

YOU total

She from

men

prey

(1)

(%)

4) 1. Desorbate

0.7

742000

519400

37.8

5) 2. Desorbate

0.9

329000

296100

21.6

6) 3. Desorbate

0.8

85000

68000 883500

5.0 64.4

7) Mixed desorbate

2.4

371000

890400

64.8

(4-6)

8) after 1.

3.0

215000

645000

Dowex adsorption

9) after neutralisate.

2.8

219000

613200

with IRA OH "

10) after 2.

2.5

27000

67500

(4.9

Dowex adsorption

discarded)

11) united NH3

0.29

682000

197780

14.4

Desorbate from

Dowex fraction I

12) Desorbate from

0.45

1419000

638550

46.5

Dowex fraction II

60.9

13) Precipitation of fraction 1

: 6.5 g

25000 / g

162500

11.8

Precipitation fraction II

12.3 g 36000 / g

442800

32.2

44.0

Example 10

200 g of a preparation as described in Example 1 were concentrated in 940 ml of distilled water and 60 ml. H2SO4 dissolved and heated under reflux 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 activated carbon was then filtered off with suction, washed with water and the filtrate was adjusted to pH = 7 to 8 with about 250 ml of 10N KOH. The solution was stirred with 50 g of activated carbon for 1 hour. The coal was suctioned off, washed with 21 water and the filtrate was discarded. The coal was digested with 21 30% alcohol overnight for desorption. Finally, the coal was sucked off and the alcoholic solution was concentrated on a rotary evaporator. Residue: 6.2 g. This crude product (6.2 g) was dissolved in 500 ml of water and carefully stirred with 30 g of Amberlite IR 120 (H® formula) for 1 hour. The exchanger was suctioned off and washed with distilled water until the filtrate was neutral and free of glucose. The exchanger was then stirred with 15 ml of 25% NH3 in 1000 ml of H2O overnight, separated and discarded. The filtrate was concentrated on a rotary evaporator. Residue: 3.7 g.

Chromatography on cellulose was carried out for further purification. 4.5 g of the material desorbed by the exchanger were applied to a 1 m long and 2.5 cm wide column filled with cellulose. Ethanol / HîO 5: 1 was initially used as the eluent, and ethanol / HîO 3: 1 was initially used to elute the compound according to the invention with m = 1. At a drip rate of 20 drops per minute, fractions of 14 ml each were removed. The individual fractions were checked by thin layer chromatography. After concentration, fractions 47-85 yielded 1.6 g of a slightly brownish-colored compound according to the invention with a glucose unit. The coloring impurities play no role in terms of quantity. The compound with m + m = 1 is obtained as a colorless resin if the cleaning step with a strongly acidic ion exchanger is not carried out in a batch process, but on a column.

Example 11

200 g of a preparation as described in Example 1 were concentrated in 940 ml of distilled water and 60 ml. H2SO4 dissolved and heated under reflux for 14 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. 5 2186) and stirred for 1 hour. The activated carbon was then filtered off, washed with water and the filtrate was adjusted to pH = 7 to 8 with about 250 ml of 10N KOH. The solution was stirred with 50 g of activated carbon for 1 hour. The coal was suction filtered, washed with 21 water and the filtrate was discarded. The coal was digested overnight with 21 30% alcohol for desorption. Finally, the coal was sucked off and the alcoholic solution was concentrated on a rotary steamer. Residue: 8.0 g.

The residue was taken up in 15 ml of H2O and applied to a column filled with 50 g of Amberlite IR 120 (H + form) (height: 20 cm, 0 2.4 cm). The mixture was drawn in at 3 drops / minute and washed with water (12 drops / minute) until all of the non-basic constituents had been removed. The basic products were then eluted from the column with 0.5% NH3 20 (12 drops / minute) and the aqueous solution was dried on a rotary evaporator. Residue: 4.1 g.

2 g of this residue were dissolved in a little water and applied to a column filled with Sephadex G-15 (height: 200 cm; 0: 3.0 cm). It was eluted with water. Fractions of 2 ml each were removed at a flow rate of 8 ml / hour. The individual fractions were checked by thin layer chromatography. Fractions 85-94 gave 280 mg of the compound with m + m = 2 with a spec. Activity of 50,000 SIE / g.

30th

Example 12

Incubate 2 g of a preparation as described in Example 1 in 60 ml of 20 mM sodium glycerophosphate buffer, pH 6.9 and 1 mM of CaCL, with 1 g of Aspergillus 35 spec. (Serva No. 13418) with constant stirring for 120 h at 37 ° C, finally heated for 5 min at 100 ° C and centrifuged at 4000 rpm to remove 1.9% of a product with 3500 after lyophilization of the solution YOU / g and 2x 106 AIE / g. If this product is tested by means of thin-layer chromatography and saccharase inhibition staining as described in Example 1, it can be seen that it essentially contains the compounds according to the invention with m + m = 1, 2 and 3 on inhibitory compounds.

45 Example 13

Incubate 2 g of a preparation as described in Example 1 in 30 ml of 20 mM acetate buffer of pH 4.8 with 1.25 mg of β-amylase from sweet potato (Boehringer) with constant stirring for 120 h at 37 ° C., finally heating for 5 min to 100 ° C in this way and centrifuged at 4000 rpm to remove undissolved matter, after lyophilization of the solution 1.5 g of a product with 1800 sieves / g and 3.8 x 106 AIE / g are obtained. If this product is tested by means of thin layer chromatography and saccharase inhibition staining as described in Example 1, it can be seen that it essentially contains the compounds according to the invention with m + m = 2 and 3.

Example 14

60 Inoculate a 200 ml Erlenmeyer flask containing 25 ml of a nutrient solution with the composition 0.1% K2HPO, 0.2% (NH4) 2S04.0.05% MgS04.0.05% KCl, 0.01% FeS04.2% a preparation as described in Example 1 with a spore suspension of the strain Asp. niger ATCC11394 and incubated at

65 28 ° C on a rotary shaker, the AIE titer drops from 210,000 AIE / ml to 53,000 AIE / ml after 6 days and to 21,300 AIE / ml after 10 days. At the same time, the SIE / ml content increases from 7.0 to 72 SIE / ml.

19th

633043

20 ml of a solution incubated with the spore suspension for 10 days were centrifuged at 3000 rpm to separate the mycelium 30 '. The 15 ml of supernatant (72,000 SI / l) were desalted by stirring for 30 minutes with 2 g of Amberlite IRC 50 H + and 1 g of Amberlite IRA 410 OH "(conductivity less than 2 mS • cm-1). The mixture was filtered off and the filtrate was left run at 5 ml / h through a column (0 1 cm x 10 cm) equilibrated with Dowex H + in 0.001 N HCl. Then it was washed with 200 ml 0.001 N HCl. For desorption, 0.6% NH3 solution was pumped through the column ( 10 ml / h) and 5 ml fractions were collected The fractions containing the saccharase-inhibitory activity were combined, rotated to 2 ml on a rotary evaporator and mixed with 2 ml of methanol, this solution was adjusted to pH 3-4 and brought to drip in 100 ml of acetone for precipitation, the precipitate was filtered off with suction, washed with acetone and ether and dried in vacuo. Yield: 26 mg with 28,000 sieves / g, consisting of compounds with m + m = 2 and 3. The pure compound was obtained with m + m = 2 from this product as in example el 8 described by gel filtration through a column with Bio-Gel P-2. 7 mg of the compound with m + m = 2 of 60,000 SIU / g are obtained.

Example 15

Culture filtrate, which is obtained from a fermentation batch as described in Example 5 by spinning off the mycelium at 13,000 rpm and which has an activity of 13,000 sieve / g, was used to reduce the salt content (conductivity of the culture filtrate: approx. 10 mS • cm "1) stirred with 500 g of a mixture of 2.5 parts of Amberlite IRC-50 (H + form) and 1 part of Amberlite IRA-410 (OH 'form) for 1 h. The exchanger was separated, the solution to a little below Concentrated 100 ml and centrifuged for 15 min at 20,000 rpm to remove undissolved constituents The supernatant was made up to 100 ml and now had a conductivity of 3.5 mS * cm- 'and was placed on a column (55x400 nm) with P-cellulose (Serva No. 45130; prepared according to known methods and applied in 5 mM ammonium phosphate buffer; pH 5.5; equilibrated). The named phosphate buffer was used as the eluent; of 18 ml volume wu are collected.

After the fractions of the eluate had been checked for their content of carbohydrates (by means of the anthrone test) and of saccharase-inhibiting components (by means of the saccharase inhibition test), those fractions were combined which in the anthrone test were virtually free of carbohydrates and at the same time in the saccharase Inhibition test had proven to be particularly effective (fractions 60-170), narrowed to 1501 and filtered through a column (50 x 300 mm) with Amberlite IRA-410 (HCOî 'form). For better control of deionization, the eluate was collected in fractions (10 ml per fraction in 20 min) and on carbohydrate (by means of anthrone test: almost entirely negative), on phosphate (by means of ascorbic acid-molybdate reagent: consistently negative) and tested for saccharase inhibition (using an enzyme inhibition test). The inhibitory fractions (3-30) were pooled, concentrated and lyophilized, redissolved and lyophilized to obtain 280 mg of crude inhibitor.

For further purification, the raw inhibitor was fractionated on Bio-Gel P-2 as described in Example 6. After the lyophilization, 30 mg of a product were isolated from the fractions which contained the pure compound with m + m = 1 at 0.3 × 106 AIE / g and 35,000 SIE / g.

For this purpose, 30 g of the preparation according to Example 1 were dissolved in 250 ml of h2o. The conductivity of this solution is 10 mS • cm-1, the pH 5.5. For desalination, 60 g of Amberlite IRC 50 H + (weakly acidic cation exchanger, 5 which only binds the aminosugar derivatives from aqueous solution only in traces) and 20 g of Amberlite IRA 410 OH "were added and the mixture was stirred for 20 minutes. The filtrate (conductivity 0.5 mS * cnr1, pH 3.5) was adjusted to pH 3.0 with 1N HCl (conductivity 0.6 mS • cmr1) This solution was passed through a column filled with Dowex 50 W 10 (H +) at 42 ml / h ( 2.5 cm, height 40 cm, equilibrated in 0.001 N HCl) and then washed with 210.001 N HCl After washing the column, the mixture was eluted with 1.2% aqueous ammonia and 10 ml fractions were collected combined, the ammonia-15 removed in vacuo and the solution subsequently concentrated in vacuo to 30 ml, it was precipitated by dropping in 600 ml of dry spirit, the precipitate was filtered off and dried in vacuo after washing with alcohol and ether. Yield 4.4 g with 26.5 x 106 AIE / g.

20 0.5 g portions were applied to a preparative Biogel P-2 column as described in Example 8 for fine cleaning and developed. The fractions which, after DC <amylase inhibition staining) compounds with m + m = 5-7, were combined, concentrated in vacuo and precipitated with dry spirit as described above. Yield from 0.5 g raw product: 0.2 g aminosugar derivative émit m + m = 5-7 with 30x 106 AIE / g and 2500 SIE / g.

Example 17

30 This example shows how the compounds according to the invention can be eluted from the cation exchanger under acidic conditions.

A 1.5 cm diameter column is filled with 30 g (wet weight) of Dowex® 50 Wx 4, (H +) 200-400 mesh in 0.001 35 N HCl. Then 500 ml of mixed desorbate (400,000 SI / 1, pH 2.5.60% acetone) are pumped, obtained according to Example 9 (table, running number 7) through the column in about 1 hour and then washed with 500 ml of 0.001 n HCl after. Under these conditions, only trace activity is eluted. Then 40 was desorbed with 0.0125 N HCl, the conductivity or the refractive index of the column eluate being registered. The SIE content of the eluate was also tested. The active fractions 74-100 were combined and neutralized by adding Amberlite IRA 410 OH ", then concentrated to 5 ml, reacted with 5 ml of methanol and precipitated by dropping in 200 ml of acetone. After washing with acetone and ether, the mixture was dried in vacuo .

Yield 1 g of the compound with ni + m = 2 with 65,000 sie / g.

50 The compounds with ni + n2 = 3 and 4 glucose units can be obtained from the active pre-fractions.

In contrast to alkaline desorption, this process of acid desorption enables fractionation of the individual amino sugar derivatives in this series.

If the substance prepared as described above with 4 to 8 glucose units is subjected to this process, the neutralized eluate being merely lyophilized, the following individual higher fractions are obtained: m + m = 4 = 67,000 AIE / mg 60 m + m = 5 = 57,000 AIE / mg m + m = 6 = 42,000 AIE / mg m + m = 7 = 24,000 AIE / mg m + n2 = 8 = 5,000 AIE / mg

Example 16 65

To obtain the aminosugar derivatives with 5-7 glucose example 18 units, z. B. from a preparation as described in Be- The above-described ß-amylase degradation process is game 1 was described. carried out as follows.

633 043 20

100 mg of compound were dissolved in 1.9 ml of 20 mM sodium acetate example 20 buffer (pH 4.75) and with 0.1 ml of β-amylase from sweet. Purified preparations of the compounds with m + m =

potato (Boehringer; 5 mg / ml; 500 U / mg) was added. The mixture can be maintained by circulating gel permeation chromatography for 48 hours at 37 ° C and for 5 minutes on (recycling gel permeation chromatography) after the following

Heated at 100 ° C and then centrifuged at 4500 rpm, 5 examples are obtained:

In order to remove precipitated protein and other impurities, 6.0 g of the crude preparations obtained according to Example 8 or 17. The whole mixture was then placed on a column with compounds with m + m = 4 and 5 through Bio-Gel P 2 (diameter 22 mm; 100 cm long; at 65 ° C ther- dissolving the material in 1 nM ammonia and Apply this mostatized) and eluted with water in a flow solution on a column (diameter 50 ml; length 100 cm) at a speed of 25 ml / h. The eluate is freed of salt admixtures by an io filled with Sephadex G-10®. The conductometer and a highly sensitive refractometer with column are eluted with 1 nM ammonia and the eluate is passed using flow cells. Fractions of 2.5 ml each were collected using a flow refractometer and a conductometer. The fractions can be monitored on amylase or a flow cell. The eluate can also be tested for thin-saccharase inhibitory activity or by means of the anthrone test for layer chromatography on silica plates (n-buta carbohydrate content. The stemming from the buffer is noI: ethanol: water = 45:35:20). Fractions, dissolving electrolytes and the enzyme preparation with which the material to be cleaned are collected, the exclusion volume is collected and the degradation products are removed and lyophilized (yield: 4.4 g).

the molecular weight eluted. Complete separation of the 2.0 g of this material is used for further separation

Compounds whose molecular weights differ little under the column (diameter 50 mm; length 100 cm), filled with bio-gel, are applied by recycling chromatography under the 20 P-2®, 200 to 400 mesh. The column is carried out at 65 ° C conditions described above. Fractions, thermostatted and with water at a flow rate which contain the products to be isolated, are combined and eluted at a rate of 80 ml / h.

lyophilized The unusable fractions and those fractions that

Containing salt, which may still be present according to Sephadex chromatography, Example 19 25, are discarded. The system, best-

In order to separate the isomeric compounds with three glucomend from the column, feed pump, flow refractometer and units, 10 g of an isomer miniature conductometer dissolved in water are closed and the solution is added to a column filled with Dowex® 50 Wx4 (H +) circulated through the gel. Bear the progress of separation. The column was first washed with water until it was neutral by constantly monitoring the refractive index and the eluate, and then eluted with 0.025N hydrochloric acid. Frak- 30 conductivity checked. Adequate separation is achieved after 5 portions of 3 ml each and thin circulations are achieved. Then the column is checked again by means of layer chromatography. Thin-layer chromatogra solvent reservoir connected and the column eluted, phie was trapped on silanized silica gel plates (Merck, German - each with fractions with a volume of 16 ml on land) with 100: 60: 40: 2 ethyl acetate + methanol + water-25% . The fractions are carried out by thin-layer ammonia with triple development. The 35 chromatography was tested as described above and the Frak bond of the formula IV runs a greater distance from the start containing the pure compounds, and twofold than the compound of the formula V. The 6 g isomeres are with fractions collected and lyophilized. Fractions 215 to 272 containing the formula V and the 600 prey: 600 mg of the compound with m + m = 4; 420 mg of the Ver-mg Isomeres with fractions 288 containing formula IV to bond with m + m = 5 and 100 mg of a little separated Mate-294 were combined, with Amberlite IRA-410 (OH-) neutral from the intermediate fractions.

siert and restricted

The in vitro saccharase inhibitory activity of the isomer of formula IV isolated by this method is 19,000 sie / g.

G

4 sheets of drawings

Claims (8)

633043 2 chaoh h ch2oh oh in the m for a digit from 1 to 8 and m for a digit from 0 2. The method according to claim 1 for producing 0- (4,6- is 8 and the sum of m and m is always a 20 bisdeoxy4 [lS- {l, 4.6 / 5) 4,5,6-trihydroxy-3-hydroxymethylcyclo- Has a value from 1 to 8, where in the case of m + m = 1 or 2, hex-2-en-l-ylamino> a-D-glucopyranosyU- (l-4) -D-glucopyra- m is always zero, by aerobically cultivating a microorganism of the conformative formula ganism of the order Actinomycetales in a nutrient medium. high ho- ' n ch0oh (IIB) 2nd PATENT CLAIMS 1. Process for the preparation of compounds of the formula CHjuOH \ H- n h n0 / x 3 633043 3. The method according to claim 1 for the preparation of 0-14,6- 45 copyranosyKl - 4> D-glucopyranose of the conformative for-bisdeoxy-4- [l S- {1,4,6 / 5) 4,5,6-trihydroxy- 3-hydroxymethylcyme with clohex-2-en-l-ylamino] -aD-glycopyranosyl} - (1-4) -0-aD-glu- (IIIB) 0 ch. Oh 4. The method according to claim 1 for the preparation of 0- {4,6-pyranosyll- (1-4) -0-aD-glucopyranosyl) - (1-4) -D-glucopyra-bisdeoxy-4- [IS- ( l, 4,6 / 5) -4,5,6-trihydroxy-3-hydroxymethyl-4-0-nose of the conformative formula aD-glucopyranosyl- (l-4> cyclohex-2-en-l-ylamino) -aD -glyco- ch20h j ^ t « ch2oh ^ vQH 5. The method according to claim 1 for the preparation of compounds of formula I, wherein m + m = 4. 6. The method according to claim 1 for the preparation of compounds of formula I, wherein m + m = 5. 7. The method according to claim 1 for the preparation of compounds of formula I, wherein m + m = 6. 8. A process for the preparation of low molecular weight compounds of the formula I, in which m + m has the value from 1 to 4, characterized in that the process according to claim 1 is carried out and the culture broth formed before isolating the fermentation products or the isolated fermentation products of a chemical or subject to enzymatic hydrolysis.