IE44803B1 - Amino-sugar derivatives - Google Patents

Description

The present invention relates to amino sugar deriva- fives, to processes for their production, to pharmaceutical compositions wherein said compounds are the active agent, and to methods of controlling carbohydrate metabolism in humans. and animals by* inhibiting glucoside hydrolase with such compounds, as in the treatment of diabetes, adiposity and hyperlipemia.

It is known that a number of microorganisms of the order Aotinomycetes produce materials which inhibit glyΙθ . coside hydrolases. Depending on the culturing conditions there-are obtained inhibitors which predominantly1exhibit saccharase or amylase inhibiting properties, (cf. U.S. Patents Nos. 3,876,766; 3,855,066 and 3,879,546). Our earlier German Offenlegungschriff No. 2347782 discloses a method of forming amino-sugar derivatives of the formula ' (Ait- in which R is an oligosaccharide chain containing 1 to 7 monosaccharide units.

The compounds of the formula A are prepared according to our earlier specification, by culturing in a nutrient medium a microorganism of the family Actinoplanaoeae and of the order' Aotinomyoetales. preferably a strain of the genus Actinbplanes such as Actinoplanes species SB 50 (CBS 961.70), SB 18 (CBS 957.70), SB 82 (CBS 615.71), SE 50/13 (CBS 614-.71) or SE 50/110 (CBS 674.73).

By selecting the conditions under whioh the micro- 2 *4803 organisms are cultured products containing compounds of the formula A having 1, 2, 3 or 4 monosaccharide units per molecule can be prepared substantially free from homologues of higher molecular weight. Patent Specification No. 1946/76 is concerned with such compounds and their preparation.

We have now found that, surprisingly, the formation of compounds of the formula A containing 3 or more saccharide rings per molecule by the process of our earlier specification is accompanied' by the formation of structural isomers as hereinafter defined of the compounds of the formula A. Moreover, we have now been able to separate the structural isomers of the compounds containing 3 or 4 saccharide rings per molecule from each other and to prepare amino sugar derivatives containing 3 to 8 monosaccharide rings in a form free from homologous compounds.

The present invention therefore provides amino sugar derivatives of the formula (I):- wherein n^ designates an integer of from 1 to 8 and n2 designates 0 or an integer of from 1 to 8, such that the sum of n^ and n2 equals from 3 to 8, the derivatives being substantially free from structural isomers as hereinafter defined when n1+n2=3 or 4 and substantially free from homologous compounds of the formula Cl) when n^+n2=5 or more.

By structural isomers as used herein and in the claims we mean compounds with the same number of hexose units i.e. n^+n2 is the. same, but in which the hexose units do not have the same arrangement i.e. n^ and n2 are different.

Depending pn the value of the sum of n^+n2 these amino sugar derivatives exhibit predominantly saccharase or predominantly amylase inhibiting effects. For example, amino sugar derivatives with n,+n? = 4 to 8, preferably with n^+n2 = 4 or 5 are effective amylase inhibitors, whereas amino sugar derivatives with η^+η2 = 3, are effective saccharase inhibitors. It was further found that amino sugar derivatives with n^+n^ = 3 show a surprisingly strong inhibition of starch digestion in vivo. The preparation of the amino sugar derivatives of the present invention is achieved by the cultivation of / strains of the order Aotinomycetales. preferably strains of the family Actinoplanaoeae in a manner known per se and by subsequent separation and isolation of the individual compounds in a manner known per se.

Amino sugar derivatives with values of the sum of n^+n2 up to 4 can further be prepared by chemical or enzymatic degradation of higher molecular amino sugar derivatives.

In order to prepare the compounds of the invention, a microorganism of the family Actinoplanaoeae and of the order Aotinomycetales, preferably a strain of the genus Actinoplanes such as Actinoplanes spec. SE 50 (CBS 961.70), SB 18 (CBS 957.70) and SE 82 (CBS 615.71), or mutants or variants thereof is cultured in a now known manner. Strains SE 50/13 (CBS 614.71) and SE 50/110 (CBS 674.73) have proved to be particularly suitable with regard to the total yield. The description of both strains corresponds'largely to that of the parent strain SE 50 (OBS 971.60) from which these strains have-been obtained by natural selection without - 4 using mutagens., A solid or liquid, especially liquid, aqueous nutrient media is used, with the addition of the usual sources of carbon, sources of nitrogen salts and antifoaming agents in customary concentrations. The carbon sources used are generally carbohydrates, especially starch, maltose, glucose and mixtures of two or three of these material complex mixtures, such as commercially available malt extract. The nitrogen sources include the customary complex mixtures, such as casein hydrolysate, yeast extract, peptone, fishmeal, fish solubles, corn steep liquor, meat extract and mixtures thereof, as well as amino acids and/or ammonium salts. The culture is carried out aerobically in aerated shaken flasks or in conventional culture containers.

As is known, the nature and concentration of the source of carbon, in combination with the particular strain used for the fermentation, influences the nature of the product.

In nutrient solutions which contain more than 2 wt. of starch, compounds with n^+n2 = 4 to 8 are predominantly formed and use of the strain SB 50/13 (CBS 614.71) in particular favours this type of production. Under certain Circumstances, as little as' 0.1 to 3 wt. % of starch in a nutrient solution which also contains adequate glucose (about 3.5 wt. 0) will produce mixtures of several amino sugars with n^+ng - 4 to 8. Such conditions yield the higher compounds of the present invention which are suitable as starting materials for the lower members upon subsequent hydrolytic treatment.

On the other hand, use of starch-free nutrients, especially with the addition of maltose when using strain SE 50 (CBS 961*70), produces mixtures of compounds with predominantly n^Ug = 3 and compounds of the formula A in _ 5 to* which R contains 2 monosaccharide units (i.e, lower molecular weight homologues of the compounds of the formula I in which n^+n2 = 2 and n2 =0),.

If the nutrient solution contains excess glucose, the longer-chain compounds are also formed if the duration of fermentation is prolonged. On the other hand, if the glucose is dispensed with entirely in the nutrient solutions and maltose is added as the source of carbon, material in which the compound has a value of n^ + n2 = 2 is obtained predominantly. The pure maltose can be replaced by cheaper material such as, for example, ’’Maltzin, a natural malt extract, and depending upon the content of maltotriose, the next-higher oligosaccharide material is also formed.

The strain SE 50/110 (CBS 674.73) has proved to be particularly suitable for the preparation of material rich in the lower molecular weight compounds with n^ + n2 = 1 to 3· This strain produced a yield of lower chain material about twice that produced by SE 50/13 (CBS 614.71).

Incubation temperatures generally lie between 15° and 45°C, preferably between 24° and 32°C. However longer chain material containing 4 to 8 glucose units are produced with SE 50 (CBS 961.70) ahd SE 50/13 (CBS 614.71) at a higher temperature, for example, 28°0. Shorter chain material containing 1, 2 or 3 glucose units are obtained using strains SB 50 (CBS 961.70) and SE 50/110 (CBS 674.73) at a lower . temperature, for example, 24°O. The duration of culture is generally 1 to 8 days, preferably 2 to 6 days and here again longer durations of culture, especially if an excess of carbohydrate Is used, favour the formation of the longer chain material. .

The pH of the culture medium will range from 5.0 to 8.5» generally 6.0 to 7.8. The end. point of the fermentation can be determined, by determining the inhibitory activity content in an enzymatic inhibition test and by determining the composition by thin layer chromatography.

Material rich in the shorter chain compounds can be obtained from the longer chain material by chemical or enzymatic hydrolysis of monosaccharide units. Chemical hydrolysis is carried out in 1 to 511 aqueous mineral acid at 50° to 100°C,, especially at 90° to 100°C., over a period of 10 to 180 minutes. Enzymatic hydrolysis is carried out by incubation with a suitable hydrolase, especially a β-amylase, an a-amylase of microbial origin such as from B. subtilis that is not inhibited by the compounds of the .invention, or an amylogluoosidase.

Hydrolysis, can also be carried out miorobially by culturing a suitable microorganism, for example, Aspergillus niger ATCC 11,594» in a nutrient medium containing 1 to 10% of the amino sugar as the sole carbon source.

The separation and isolation of the individual compounds of the invention thus starts either from microbiological oijd-ture brothB or from acid hydrolysates or I from incubation mixtures in which the enzymatic and/or microbiological restructuring or degradation of the higher members of the amino sugar derivatives has been carried out.

The longer chain material containing 4 to 8 gluoose units is initially separated, aft6r prior decolorizing and concentration of the solutions, by .direct precipitation.

This material is further processed as discussed hereafter.

The shorter chain compounds containing 3 or 4 448°3 ' < glucose units are initially isolated, by adsorption on active charcoal at a neutral pH, with subsequent desorption utilizing aqueous alcohols or acetone, especially 50 to 80% strength acetone. The desorption can be carried out com5 pletely at acidic pH values in the range of pH 1.5 to 4,. preferably pH 2 to 3. If the starting solutions are very dark in colour, they are decolourized prior to the adsorption· by means of active charcoal, utilizing acidie pH values (pH 1 to 3), or with nonspecific adsorption resins, for example, lewapol CA 9221/0.55 mm particle size (Bayer AG) in a pH range of 2 to 7, preferably 2 to J. The active charcoal preferentially binds coloured material in the acid range only, while lewapol does hot adsorb the amino sugar derivatives either at neutrality or in the acid range.

In order to separate the pure compounds of the present ? invention, their weakly basic character can be utilized.

Under suitable conditions, namely a pH 1 to 8, preferably pH 2 to 4, and at low ionic strength corresponding to a conductivity of less than 10 mS.cm~\ preferably less than 20 2 mS.cm'’i, .the compound's are bound by strongly acid cation exchange^ such as for example, Dowex (.. -.- Trade Mark) 50 W (Dow Chemicals) in the protonated form. The compounds can be bound particularly successfully from an acetone solution (50% to 80% acetone, pH 1 to 5» preferably 2 to 4) to cation exchangers, which, under these conditions, exhibit a substantially enhanced adsorptive capacity for the compounds, If the solution contains more than-50% of acetone, it is also possible to bind the compounds to weakly acid exchangers such as Amberlite (registered-Trade' Mark) IRC-50 (protonated form).

Aqueous solutions of acids or bases, preferably ammonia - .: < - 8 - .--. or hydrochloric acid, particularly in concentrations of 0.01 to 1 3g./l, are best used for desorbing the compounds of the invention.from the cation exchangers.

The desorbates are neutralized with a weak acidic or basic ion exchanger, or the base acid is stripped from the desorbates in vacuo, and the compounds are obtained, after concentration of the solution, by lyophilization or by precipitation with organic solvents such as acetone.

Furthermore, it has proved possible to separate the low-molecular compounds of the present invention from inert saccharides by chromatography on exchangers based on cellulose, preferably phospho-cellulose (Serva, Heidelberg). Buffers, preferably phosphate buffers, of low ionic strength, preferably 2 to 10 mM and especially 5 to 10 mM, and having a pH in the range of 2.5 to 8, preferably at pH 5 to 6, are used as running agents. A prerequisite for effective fractionation is that the salt contents in the preparation to be fractionated should be as low as possible.

To prepare the individual compounds of the present invention in a pure state, the pre-purified preparations, prepared as described above, are chromatographed using a suitable molecular sieve,such as for example, Bio-Gel (. .. . Trade Mark) P-2 (Bio-Had, Munich). Fractions of the eluate are examined by thin layer chromatography and those which contain the pure compounds of the present invention are combined, reohromatographed and finally lyophilized after concentration, or precipitated by means of organic solvents, as described above.

All the· compounds of the invention are characterized in that upon total acid hydrolysis, component I £’£80® (C^^H21ΟγΝ) and glucose are formed. Component I has been shown to have the structural formula: The conformational formulae of the lower molecular weight homologues of the compounds of the invention (i.e, compounds of the formula I in which n2=0 and n^ - 1 or 2) has been confirmed as follows.

The compound in which + n2 is 1 is a colourless, amorphous solid of good solubility in water, dimethylformamide, dimethylsulphoxide, methanol and hot ethanol.

On thin layer chromatography using 10:6:4 (v/v) ethyl acetate: methanol: water, the compound shows an Sf value of 0.46 (maltose = 0.50 and glucose = 0.65) on F 1500 silica gel films (Schleicher & Schull) and of 0.47 (maltose’= 0.54 and glucose = 0.66) on F 254 silica gel plates (Merck, Darmstadt). A brown-black colouration is obtained at room temperature or after slight warming upon application of silver nitrate/sodium hydroxide spray reagent.

This compound is silylated (together with a- and β-Dglucose or sucrose as the internal standard)' in a mixture of pyridine (l), trimethylchlorosilane (0.5) and H-methyl-trimethyl-silyl-trifluoroacetamide (l) and subjected to gas chromatography in a 6 ft. glass column filled with 3% SE 30 - 10 44803 silicon-elastomer (Hewlett Packard-registered Trade Mark) on Chromosorb (registered Trade Mark) WAW. The injection anil detector tpmnprature ie 300°C. The oven temperature is 220°C, isothermal, until elution of nand 3-D-glucose standards with subsequent temperature increases at the rate of 15°C/minute up to 300°0. A flame ionisation.detector is employed with nitrogen as the carrier gas being fed at 40 ml/minute and air as the combustion gas at 80 ml/minute and hydrogen at 20 ml/ minute. The compound showed a retention time of 16-17 minutes (α-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 methanol solution) dissolved in water showed a specific optical rotation, [a]D, of +134.3°.

The IR spectrum in potassium bromide is poor and rather inconclusive, main absorption band being in the 0-H and C-0 areas.

The HMR spectrum in CD^OD at 220 MHz is shown in Figure 1 (Abscissa = S ppm). Predominant features are shown in Table I which follows: Table I In ppm Multiplicity Relative Intensity 1.3 Doublet; J = 6.5 Hz 3 H 2.3 Triplet, J, and J_ 8-10 Hz 1 . 1 H 3.15 Triplet; J, and J_ 7-9 Hz 1 z • 1 H 3.3 - 3.9 Signals cannot be allotted 12 H individually 4.13 A B system ϋ = 12 Hz 2 H - 11 - Table I (cont/d) In ppm Multiplicity Relative Intensity ( (4,48 and Doublet; J = 7 Hz ) 1 H (5.1 ‘ Doublet; J = 2.5 Hz ) 4.9 Singlet 11 H protons replaced by deuterium 5.0 Doublet; J = 2-3 Hz (poor resolution) 1 H 5.8 Doublet; J = 3-4 Hz (poor resolution) IS H When this - compound is reacted in 1:1 acetic anhydride: Ιθ pyridine at room temperature, a decaacetyl derivative (m.w. 903) is obtained. The AMR. spectrum of the decaaeetyl derivative is shown in Figure 2. If carried out in lil glacial acetic acid: acetic anhydride with catalytic amounts of sulphuric acid, the formation of an undecaacetyl derivative (m„w. 945) in addition to the decaacetyl derivative can he detected by mass spectroscopy. MS spectrum of the decaacetyl derivative shows a molecular peak at 903 (2*5% relative intensity) and'a base peak at 843. · Important fragnent peaks in the upper mass range are 844 (55% relative intensity), 784 (36% relative intensity), 783 (34% relative intensity), 759 (54% relative intensity), 55-6 (36% relative intensity),. 496 (37% relative intensity) and 436 (29% relative, intensity). -12 448o3 Spectroscopic data and chemical properties show the following structure for this compound.

More particularly the NMR spectra demonstrates that e· this compound is 0-£4,6-bisdesoxy-4-[l S-(1,4,6/5)4,5,6-trihydroxy-3-h.ydroxymethyloyclohex-2-en-l-ylamino]-a~ B-glucopyranosylj-(l—>4)-D-glucopyranose of the conformational structural formula: The next lower molecular weight homologue of the compounds of the invention has the formula and is a readily water-soluble amorphous solid product.

On thin layer chromatography it demonstrates an Sf value of 0.35 on E 1500 silica gel films and 0.33 on 3? 254 silica gel plates using the system described above.

The compound has a rather inconclusive IR spectrum, of - 13 poor resolution, with, main absorption bands again being in the range of the Ό-H and C-0 valency vibrations (3,700-3,100 shown in Figure 4.

Methylationas above produces a compound methylated 13-fold, and small amounts of a product methylated 14-fold. . The mass spectrum of the methylation product show the molecular- peak at 827 (l.5% relative intensity) corresponds to an empirical formula Ο^θΗθθίϊΟ^. A seoond molecular peak of 0.1% relative intensity -is present at 841. The most important fragment peaks ares' 739 (27% relative intensity), 592 (3.7% relative intensity), 535 (30%relative intensity), 388 (9% relative intensity), 386 (13% relative intensity), 284 (13% relative intensity), 187 (12% relative intensity), 171 (40% relative intensity), 101 (34% relative intensity) and 88 (25% relative intensity) with a base peak of 75.

Chemical and spectroscopic properties show the fallowing structure for thiscompound: •OH More particularly, this compound is 0-,4,6-bisdesoxy-4[1 S-(l,4,6/5)-4,5,6-trihydroxy-3-hydroxymethyloyclohex-2en-l-ylamino]-α-D-glucopyranosyl^-(l-> 4)-O-a-D-glucopyranosyl-(l-^>4)-D-glucopyranose of the conformational structural Ομ*Ή.0Η The compound of the invention having n1 + n2 = 3 is obtained in two isomeric forms. On acid partial hydrolysis, both these .compounds can he split to give a compound of the following formula IIA and glucose in the molar ratio of 1:2.

The material present in lower amounts is the compound 0-^4,6-bisdesoxy-4-tl 8-(1,4,6/5)-4,5,6-trihydroxy-3hydroxy-methylcyclohex-2-en-l-ylamino]-a-D-glucopyranosyl|(l-> 4)-Ο-α-D-glucopyranosyl-(l->4)-O-a-D-glucopyranosyl-(l-> 4) -D-glycopyranose of the conformational structural formula: The isomeric material present in larger amounts is the compound 0-^4,6-bisdesoxy-4-[l 3-(1,4,6/5)-4,5,6trihydroxy-3-hydroxymethyl-4-O-a-D-glucopyranosyl-(l->4)cyclohex-2-en-l-ylamino]-α-D-glycopyranosylj -(l->4)-0-aD-glucopyranosyl-(l->4)“jD-glucopyranose of the conformational structural formula: These isomers show Rgiuoose values of 0.41 to 0.46 on F 1500 plates using 50:30:20 n-butanol:ethanol:water. The presence of the isomer having the structure shown in Formula V can be shown by analysis of the products of hydrogenolytic degradation using palladium on charcoal. The products of this degradation thus include 3-hydroxymethyl-4,5,6-tri~ hydroxy-[4-0-a-D-glucopyranosyl-(1)]-cyclohexane, 0-(4amino-4,6-bisdesoxy-a-D-glucopyranosyl)-(l->4)-0-a~L«gluco17 44303 pyranosyl-(,l 4)-D-glucopyranose, and glucose. Since the hydrogenation reduces the double bond and cleaves the C-U bond in the ally Imposition, it is clear this compound, which is isomeric to the compound of Formula IV is structurally related to the compound of Formula IIIA or IIIB characterized however by-the presence of a further glucopyranosyl group in the 4-position of the cyclohexene ring.

The presence of the isomer having the structure shown in formula IV is - demonstrated by the formation of validatol (S. Horii et all, journal of Antibiotics XXXV, 59 (1971)) and 0-(4-amino-4,6-bisdesoxy-a-D“glucopyranosyl)-(l->4)-0a-D-glucopyranosyl-(l->4)-0-4)-D-gluoopyranose. - 18 *4Sq3 In a second experiment the isomers with 3 glucose units were methylated according to well known methods, hydrolysed, reduced.with sodium borohydride, acetylated and analyzed by. gas chromatography. Whereas the compound with formula IV yields only l,4»5-tri-0-acetyl-2,3>6-tri-0-methyl-D-glucitol, the compound with formula V yields l,4,5-tri-O-aoetyl-2,3,6tri-O-methyl-D-gluoitol and l,5-di-O-acetyl-2,3,4,6-tetra-Omethyl-D-glucitol in a molar ratio of 2 : 1. The isomeric compounds with formulas IV and V were not degraded by βAmylase.

The product with n^ + n2 = 4 was hydrogenated using palladium on charcoal as a catalyst. The non-basic products of the hydrogenolytic degradation were separated off on an acidic ion exchanger and isolated by preparative thin-layer chromatography. Upon acetylation the following main components were identified by mass spectrometry.

HO· (The decaacetyl derivative shows a molecular peak at m/e = 906). - 19. 44803 (The nonaacetyl derivative shows a molecular peak at m/e = 848). .

It must he concluded that the prevailing component of 5 the material with n^ + n2 = 4 is the isomer .of the conformational structural formula: ' The degradation by β-amylaee upon which most of the component with + Hg = 4 is degraded to a compound of formula IIIB and in addition maltose confirm this result.

The isomeric compounds with formulas IV and V were separated by chromatography on a acidic ion exchange resin with 0.025 H hydrochloric acid as an eluant.

On the degradation with β-amylase the product with n.| + n2 = 5 yields 90% of a compound of formula V and a corresponding amount of maltose. It follows that the product with a maltosetriose unit bound via a glycosidic bond to the cyclitol rest is the main component. About 10% are not degraded by β-amylase.

This· is bonsistant with the following conformational formula:- 22 «4803 The higher members of the series containing 4 to 8 glucose units with molecular weights of from 969 to 1617 are less active saccharase inhibitors although in vitro their α-amylase inhibition is higher. On acid hydrolysis of these higher members, the lower components can, in each - 23 44© 03 case, be detected, as intermediate products together with glucose and maltose. Thin layer chromatography using 50:30: n-butanol:ethanol:water, on F 1500 silica gel plates give Rg3.ucose values of 0.30-0.34 (predominantly four glucose units); 0.21-0.23 (predominantly five glucose units); 0.14-0.16 (predominantly six glucose units); and 0.09-0.11 (predominantly seven glucose units).

As in the case of the lower members of this series, total acid hydrolysis yields Component I and glucose in discrete molar ratios, specifically 1:4, 1:5, 1:6, 1:7 and 1:8, (the percentages of glucose being 74.40» 79.70, 83*30, 86.50 and 89.10, respective.

Upon thin layer chromatography using F 1500 silica gel plates with 45:35:20 n-butanol:ethanol;water as the solvent, the following Rf values are observed upon threefold development: .

Ratio Glucose:Compound I Standard Rf value Glucose 0.77 Maltose 0.65 ' .· Maltotriose 0.51 Maltotetraose 0.39 Maltopentaose 0.27 4:1 .- 0.25 Maitohexaose 0.21 • 5:1 - 0.18 - 24 β' ’«Oa Ratio Glucose:Compound I Standard R.£ value Maltoheptaose 0.15 6:1 0.13 Maltooctaose 0.11 7:1 0.09 8:1 0.07 Similarly, catalytic hydrogenation as discussed above demonstrates that some but not all of the glucose units of these higher members are bound through the 4-position of the cyclohexene group.

Since these compounds contain oligoglucosidic linear chains with 3»4 linkages, they can serve as substrates for certain carbohydrate degrading, enzymes. The range of enzymes is obviously limited to those which are not substantially inhibited by the compounds. Bacterial and fungal a-amylases can be used to degrade any oligoglucosidic chain containing 2 or more glucose units and yielding compounds of lower molecular weight and inert saccharide fragments such as maltose and maltotriose. This degradation procedure is further proof for 1-M α-linkage. Compounds of the invention containing 4 to 8 glucose units are also degradable to some extent by β-amylase. Since β-amylase splits off maltose units from the non-reducing end of a glucose chain having l->4 α-linkages, careful analysis of β-amylase degradation products yields valuable information on any oligoglucosidic substituent bound to the 4-position of the cyclohexene ring, specifically its chain length, and the number of glucoses in the chain attached to the bisdesoxyglucose; i.e., the reducing end of the.compound. The compounds containing 4, 5, .6, 7- or 8 glucose Units are however not completely degradable by β-amylase, the resistance of some fragment of the compound apparently being due to. insufficient structural requirements for β-amylase attack.

The results of β-amylase degradation can be summarized as follows: Ho. of glucose units in starting' material No. of glucose units in degradation product(s) Maltose units removed; : 4 4 0 2 1 5 0 5 3 . 1 6 0 6 4 1 2 2 . 7 0 Ί ' 5 1 3 2 8 0 8 6 1 4 2 - 2 3 In some cases- a minor amount of starting material, possibly isomeric, is-recovered. As to that material which is degraded, it should again be emphasized that the β-amylase will successively remove only maltose units and only- from the 2o non-producing end of the oligosaccharide. Consequently while not wishing to be bound by any theory it appears the compounds containing 4, 5» 6, 7 and 8 glucose units include derivatives ' - 26 44803 of the lower members of Formula IIIA and IIIB containing chains of 2,3,4,5 and 6 ^lucost unlbs Joined 1-^4 a to each other with the last being joined la to the 4-position of the cyclohexene ring.

Methylation of the compounds with methyliodide/sodium hydride in dimethyl sulphoxide, subsequent total hydrolysis and derivatization, followed by gas chromatographic analysis yields only the 2, 3, 6-trimethyl glucose derivative, so that the glucose units are necessarily joined 1—^4 in an exclusi10 vely linear structure. A second methylation product, which is found to a varying degree or under certain Circumstances not at all, is the 2,3,4,6-tetramethyl derivative. The existance of this derivative and its molar ratio to the trimethyl derivative is dependent on the substituent attached to the cyclohexene ring.

The following isomers of the compounds of the formula (i) in which n^+n2 = 6 and 7 are considered to be predominantly produced by culturing microorganisms of the family Actinoplanaoeae under the conditions indicated previously as favouring the formation of higher molecular weight compounds of the formula (1):- 27 and. - 28 44803 It is known that in animals and man, hyperglycaemias occur after ingestion of foodstuffs and beverages containing carbohydrates (for example cereal starch, potato starch, fruit, fruit juice, beer or chocolate). These hyperglycaemias - 29 44803 are due to a rapid degradation of the carbohydrates by gLycoside-hydrolases (for example salivary and pancreatic amylases, maltases and saccharases) in accordance with the following equation: amylase maltase Starch or glycogen·-:maltose-)——glucose ailnffiRS 3aaC^ara3^-g-Tnfir)gia 4· fructose ' These hyperglycaemias are particularly pronounced and longlasting in the case of diabetics. With adipose subjects, alimentary hypergiycaemia frequently, leads to a particularly powerful secretic of insulin which in turn, leads to increased fat synthesis and reduced fat degradation. Following such hypergLycaemias; hypoglycaemia frequently occurs, due to the insulin secretion, both in metabolically sound and in adipose persons. Xt is known.that both hypogLycaemias and chyme remaining in the stomach promote the production of gastric juice which in turn initiates or favours the development of gastritis or of gastric or duodenal ulcers.

The inhibitors of glycoside-hydrolases according to the invention, obtained and isolated in accordance with the presently described methods, substantially reduce alimentary hypergiycaemia, hyperinsulinaemia and hypoglycaemia.: This can be observed after feeding rats and/or humans with starch, sucrose or maltose. The compounds accelerate the passage of carbohydrates through the stomach and also inhibit the absorption of gluoose from the intestine. The conversion of carbohydrates into lipids of the fatty tissue and the incorporation of alimentary fat into the fatty tissue depots is accordingly reduced or delayed.

It. is also known that carbohydrates, especially sucrose J . - 30 44803 are split by microorganisms in the mouth cavity and that this encourages caries iormation. The present inhibitors can be used to prevent or reduce such action.

The following describes the inhibitory profile of the present compounds.

In vitro amylase test One amylase inhibitor unit (1 AIU) is defined as the amount of inhibitor which inhibits two aaylase units to the extent of 50%. One amylase unit (AU) is the amount of enzyme which under the test conditions specified below splits 1 ^equivalent of glucoside bonds in the starch per minute. The ^equivalents of split bonds are determined colorimetrically as ^equivalents of reducing sugars formed, using dinitrosalicylic aoid, and are quoted as ^equivalents of maltose equivalents, using a maltose calibration curve. To carry out the test, 0.1 ml of amylase solution (20-22 AU/ral) are mixed with 0-10 pg of inhibitor or 0-20 pi of the solution to be tested in 0,4 ml of 0.02 M sodium glycerophosphate buffer/0.00, M 0aCl2, pH 6.9, and the mixture is equilibrated for 10-20 minutes in a water bath at 35°C. The mixture is then incubated for 5 minutes at 35°C with 0.5 ml of a 10 strength starch solution which has been pre-warmed to 35°C (soluble starch No. 1,252 from Merck, Darmstadt), and thereafter 1 ml of dinitrosalieylio acid reagent (according to P. Bernfeld in Colowick-Kaplan, Meth. Enzymol,, Volume 1, page 149) is added. To develop the colour, the batch is heated for 5 minutes on a boiling water bath and then cooled, and 10 ml of distilled water are added. The extinction at 540 nm is measured against a correspondingly made-up blank without amylase. For evaluation, the amylase activity which is still effective after addition of inhibitor is read off - 31 44803 a previously recorded amylase calibration curve and the percentage inhibition of the amylabe employed is calculated therefrom. The percentage inhibition is plotted as a function of the quotient Hg of inhibitor + AU ++ + relative to solid ++ AU in non-inhibited batch of the same series and the 50% inhibition point is read off the curve and converted to AlU/mg of inhibitor.

In vitro saccharase test One saccharase inhibitor unit (SIU) is defined as the amount of inhibitor which inhibits two saocharase units to the extent of 50%. One saccharase unit (SU) is the.amount of enzyme which under the test conditions specified splits 1 pmol of sucrose to glucose and fructose per minute.

The pmols of glucose formed are determined quantitatively by means of the glucose oxidase reaction under conditions under which a further splitting of the sucrose by the saccharase no longer takes place. To carry out the test, 0.05 ml of solubilized saccharase [from the mucous membrane of the small intestine of the pig, according to B. BorgstrOm, A. Dahlquist, Acta Chem. Scand. 12. (1958), page 1,997], diluted with 0.1 M sodium maleate buffer of pH 6.0 to a SU content adjusted to 0.12 SU. is mixed with 0-20 pg of inhibitor or 0-20 μΐ of the solution to be tested and brought up to 0.1 ml with 0.1 H sodium maleate buffer of pH 6.0. The mixture is equilibrated for 10 minutes at 35°C and 0.1 ml of an 0.05 M sucrose - 32 *4603 solution in 0.1 M sodium maleate buffer of pH 6.0, pre-warmed J ' to 35°C, is then added. The mixture is incubated for 20 minutes at 35°C, the saccharase reaction is stopped by addition of 1 ml of glucose oxidase reagent, and the incuhation is continued for a further 30 minutes at 35°C.

(The glucose oxidase reagent is prepared by dissolving 2 mg of glucose oxidase, Boehringer, Ho. 15,423, in 100 ml of 0,565®. tris-HCl buffer of pH 7.0 and subsequently adding 1 ml of detergent solution (2 g of Triton-registered Trade Mark)X 100 + 8 g of 95% strength analytical grade ethanol), 1 ml of dianisidine solution (260 mg of o-di-anisidine.2HC1 in 20 ml of HgO) and 0.5 ml of 0.1% strength aqueous peroxidase solution, Boehringer, Ho. ,302). Thereafter, 1 ml of 50% strength HgSO^ is added and a measurement carried out at 545 nm against a corresponding blank. To evaluate the results, the percentage inhibition of the saccharase employed is calculated and converted to SlU/g or SlU/liter from the 50% inhibition point, using a glucose calibration curve.

In vitro maltase test One maltase inhibitor unit (MIH) is defined as the amount of inhibitor which inhibits two maltase units to the extent of 50%. One maltase unit (MH) is the amount of enzyme which in one minute, under the test conditions .specified below, splits 1 pmol of maltose into 2 pmol of glucose. . The pmol of glucose formed axe determined quantitatively by means of the glucose oxidase reaction under conditions such that further splitting of maltope by the. maltase no longer takes place. To carry out the test, 0.05 ml of solubilized maltase [from the mucous membrane of the small intestine of the pig, according to - 33 B. Borgstrom, A. Dahlquist, Acta Chem, Scan. 12, (195S), page 1,997], diluted with-0,1 M sodium maleate· buffer, of pH 6.0 to 0.060-0.070 MU is mixed with 0-20 pg of inhibitor of 0-20 pl of the solution to be.tested and made up to 0.1 ml with 0.1 M sodium maleate buffer of pH 6.0. The mixture is equilibrated for 10 minutes at 35°C and 0.1 ml of an 0.05 M maltose solution in 0.1 M sodium maleate buffer of pH 6.0, pre-warmed to 35°C, is then added. The mixture is incubated for ‘20 minutes at 35°C and the maltase reaction is stopped by addition of 1 ml of the glucose oxidase reagent described above, and the incubation is continued for a further 30 minutes at 35°C. Thereafter, 1 ml of 50% strength sulphuric acid is added and a measurement carried out at 545 nm against a corresponding blank.

To evaluate the results, the percentage inhibition of the maltase employed is calculated and converted to MlU/g or MlU/liter from the 50%·inhibition point, using a glucose calibration curve.

The results of the in vitro enzyme inhibition tests for specific individual compounds and discrete ranges of higher 'members of the ’compounds of the present invention are summarized in Table II which follows ' « Table II Formula Humber of Glucose Units per molecule a-Amylase Inhibition AlU/g Saccharase Inhibition 7 SIU/g Maltase Inhibi- tion MU/g II B - ‘ 1 300,000 30,000 5,000 III B - 2 300,000 65,000 15,000 V ' 3 1,400,000 21,000 5,000 - 34 **80s Table II (cont/d) Formula , Number of . Glucose Units per molecule a-Amylase InnibiOion AIU/g Saccharase Inhibition SIU/g Maltose Inhibi- tion MIU/g 4-6 17,500,000 8,500 -·· 5-7 30,000,000 2,500 As can be seen from the above, the specific in vitro inhibitory activity towards pancreas-oc-amylase increases greatly with increasing molecular weight in the series; thus, the compounds with 5 to 7 glucose units show a 100-fold greater inhibition in vitro than does the compound with 1 or 2 units. Saccharase inhibition is mo8t pronounced for the derivative having two units, the derivative having one glucose unit showing inhibition only half much, and the higher member showing further decreases in saccharase inhibition.' In vivo./the activity in saccharase inhibition (the sucrose overfeeding test) runs approximately parallel to the specific inhibitory activity found in vitro. On the other hand, in vivo starch digestion (the starch feeding test) for the compounds having 1, 2 or 3 glucose units unexpectedly increases 10 to 40-fold in comparison to the amylase inhibition in vitro. The significance of this will be seen from the following.

To produce an alimentary hyperglycemia and hyperinsulinaemia, groups of 6 fasting rats are given (a) 2.5 g of sucrose, (b) 2.5 g of maltose or (c) 1 g of boiled starch orally, in aqueous solution or suspension. Six other rats - 35 ¢¢803 are given the same carbohydrates in the same amount and a glycoside hydrolase inhibitor in the amount indicated. In addition, six other rats are given an appropriate volume of saline. The blood glucose and the serum insulin are then measured at short intervals of time, in the blood from the retro-orbital venous plexus. Blood glucose determinations are carried out in the Auto-Analyser device (Technicon), according to Hoffmann; J. biol. Chem. 120, 51 (1937), or enzymatically by means of glucose oxidase and o-dianisidine hydrochloride and the serum insulin determinations are carried out according to the method of Hales and Randle: Biochem. J. 88. 137 (1963)· The results for in vivo saccharase inhibition (administration of sucrose) in the fasting rat are shown in Table . III which follows; - 36 *48θ3 I I I I I I I I ι ι ι ι I o CQ +Ϊ * * * * * * * * * * * * * fi * * * * CO *. * * fi Eh * στ vo sh sh O OT sh O VD Φ • * co • • • sh • • « sh • • K C ΙΑ H Ή KT στ VO H CM LA cn H c— στ H »Η +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 γΛ ΙΑ Η CM ΓΛ vo sh IA r- t— m VO co sh δό Sh στ LA CM O στ CM o OT t- CM o στ στ a H H H H rd H H H fi •Η * * * · * * * * * φ * * * * * O O * * CQ Οτ H CM H LA tn o tn • • o o Ο • • « « O • • • VO CM • • ο a ίΛ CO LA σ» co CM t> xj- sh H H co 5} •Η £ +1 +i +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 Ο Μ* IA tn r-l IA CM vo o CM tn IA rd VO s in co tn CM r-j στ στ CM o στ t> CM O o στ δ H H a H rd H rd r-i £ * * * * * * * * * * * * * * * * sh * * · CM in vo in στ C— στ vo vo • sh CM •ν • • « • • • • • r-i • • a LA fi H VO sh στ tn LA sh vo H στ C— ri a +1 +1 +1 +1 +1 +1 +1 +Ϊ +1 +1 +1 +1 +1 +1 LA στ. C*· in OT CM in tn H 00 vo CM 00 H h VO o 00 t* LA H c- LA vo CM tt) στ 00 fi H fi H r-s p p χ’-'κ P x—>. P !=> H fi 1= H r-i P P H o H CO 03 o H K3 i O H H CQ fc CQ fc CQ fc CQ P «Ρ Q n -p O •P O a Γ) e o fi in o fi LA o O o LA CM o CM H o CM LA H χ-%, o ϋ o ί=> + + + S-x + + '—s + + + Η CQ Φ Φ Φ o Φ Φ Φ Φ Φ Φ Φ «Μ»* φ CB 03 CQ CQ Φ CQ 03 CQ 52 CQ CQ CQ CQ fi O O o O a O O O fi O ϋ O O φ *3 fc a fc fc •H a fc fc •H fc fc fc fc CQ 0 υ 0 ϋ ri 0 0 q H ¢3 q 0 q Ο •w β fi fi ® fi fi a a a fi 3 a η • CQ CQ CQ CQ n CQ CQ CQ CQ CQ CQ CQ CQ CM £3 CM tn a n H H H LA o o β z—x H o A P β Φ CQ O CQ •P ω .5 >> •P H *3 § δ ii * » o II II * II * * * *48o3 As can be seen from the above, in vivo activity in saccharaseinhibition parallels that observed in vitro. Thus the observed ΒΒ^θ increases as saccharase inhibition units per milligram decreases. These are summarized in Table IV as follows: TABLE IV Formula Glucose units Saccharase Inhibition In vitro SlU/mg In vivo (ED50 mg/kg) IIB (1) 30 3.0 IIIB (2) 6Q .21 V (3) 21 2.65 - (4-6) 8.5 15.30 - (5-7) 2.5 52.00 Surprisingly the in vivo inhibition of starch digestion by the compounds of Formulas IIB, IIIB and V is much higher than would be expected from the in vitro amylase inhibition data and does not follow the anticipated pattern. In vivo data in the fasting rat following administration of starch, determined as described above, are presented in Table V which follows. - 39 ti in ti* + 1 CM +1 + l +l * * CM + l * CM σ ·- M3 03 CM © t> * ν- • cn ch in «ι- tn rf— tn in αι r- +1 a S © O O Cf ti o o $ + l +l + l +i * # +1 fc M- © CM tn M3 • © in in « m ·. o in Mt rfT· CM rf— rf— in o in tn rf— ν—· rf- 0 ❖ & rf fc + l s +I + l in + l + l fc co fc V— +— ¥4 « tn rf— • rf— β o c- ·+— in Ch rf- <(— CM M· rf— CM . CM V— rf* rf— » fc fc $ 3 +( +T + l * + l +( fc ««t © ort rf- © cn CM a kb ft ft 00 « o « tn © Ch rt\ M CM in CM in rf— ©T~ rf— rf— - ft . fc 3 + l +l + l Ή * + l fc w o ch o © rf- c in tn o • M3 « M3 β o t- tn m M3 v- Ch o rf— . rf— rf— ν— *“ *“ -— . rf +l + l Ή + l •h CM GO Ch e~ a o rf- 0 CM o © . · p- tn c*· M3 o GO o CM ch © © ν— in rf— © CQ O A ti jti o .ti to .ti B £ o Fi o M 9 aj 0 -P H « Ft f+ 3 ti 3 © O 8 •P 0 P in «Ρ ΙΛ += a Q E>. © T- m §5 SiS O Pm - 40 44803 + 1 +1 ιη < · 00 ·ν οο τ- oj >+1 +1 cm σι ιη · Ο · cm in tn in * + 1 * CM CM · ο <Ο · ο +ι Ή Φ η ο ο ο ο ιη Μ· 4 * * ' ri a $ * +1 + 1 •H + 1 * + 1 * + £ CM o T CTi CM o in • cn • tn • t*- • Ο σ\ ΙΛ tn 1Λ tn CO cn o cn cn ΙΛ tn Τ“· *- «Γ“ * * ri * * * + 1 + 1 +i * +1 * +1 Ϊ + # κ t> O * CM ΙΛ ο CM • co • r” co £> • ο Ι> co *— CM 5— co cn *— cn 4Λ CM w— T“ • £ * 3 +1 +1 + 1 * + 1 * +1 ί + 53 tn o * cn il· ΙΛ • CM o « ΙΛ o <· • s • ΙΛ co ΙΛ tn 5— CM co o T“ O'» co co c— Τ r· T* r* * * * ri 4( * * s •5 + 1 +1 + 1 * +1 * + 1 * + * · S3 (Λ c- CM c- oo c*» 00 • o • ΙΛ • r* • o • • ο CO tn CM t- o cn CM co 0» co e- Ί“ ί! Κ + 1 +1 +1 +1 +1 ί + # c- σ» ιλ cm co ο · in · oj · σι · ο · ο · Β *4 φ φ ο Ρ § 3 ο S ΙΛ -Ρ τ- ω tn to β! Xt H xt O <} o Q frj O 3 o Λ CM +3 o +3 Φ co Φ •r» • 03 Ο -Ρ Ο Α Η Η Η 44303 +1 +1 a © a o o a s> tJ o o s' in o t> +l O' • *3· VO CM c~ +l .· > w* ·* <0 * * +1 CM • 'it *ct O * IA a o tn cj Ή a o CM a •rt © S3 O ft . ts u-p Οή h ja ap al g δ + 1 CM 4*1 C— in 4*1 in e*· +t CM in Φ CM r· * * *- if +1 4*1 * 4*1 & Ή * 0) -ct VO in vo v* CM • O' in *“ CM φ-* ω o VO 4-1 • CM O'. LA * +l ★ cm tn * # £e. * in +1 ° £— tn cm •H * 4-1 & 4*1 # , fr- tn t * · CM v- CO O' O' co +1 « tn - CM fr* E- tn ««- o £ Ο» • CM in σ» +1 * ’ 4*1 ?! +i MP * * * m O' o tn * _ Ή - +1 +1 - +1 ί in m in * λο »m · ia · c— m . w m r· β mt- — io m en - 42 44803 £ 3E Φ α ο ο *3 ο cj Α 1 ΙΑ Μ- + 1 +1 +1 +1 , +1 ί Ο tA CM Tt 10 ‘ 0*1 *- C*- * *3 · LA · <0 ΙΑ ο ««“Ο t*· ο co CO Μ> ν— Ί-» Τ- • 5 X ο tA 2 Ή -Η +1 +1 * +1 * a- <α ο~ ό 04 · C0 · KS τ- fr- · ^0 · fr- < 04 οο 04 ν- ι- M3 Ch M3 V Τ «— • κ ο 04 * & + 1 „ +1 +1 * +1 * +1 * 00 ιη *— ν- C— t- · co · ν· · cn · tn « >« ιη ,- coo co σ> ί- οο tn Τ“ *- • •9 κ ΙΑ τ- * * •Μ +ϊ +1 +1 * Ή * cn vo co oj cn ΙΑ · fr* ·00 · 00 * 04 · Μ) ΙΛ ν CO *- ITs Ch <30 CQ CM *· * (3 ϊ; ο * * + 1 +1 +1 + 1 * + 1 * CO LA CM ΙΑ LA · Κ\ ** Ch · Ο · « ία -e ο *-σ> σι ία ο- ία Β *ί φ φ ο fl φ ΰ ο •g a! la Η ο h S ο ο S * ο αί * ·ρ Μ)' ο II -Ρ α . η ο+> 3Ή Η Ο ΰ,ρ ΙΟ I •e Bi *3 A 4®° 3 »d § •η g δ ί J8 'β * * 4-1 +1 + 1 4-1 * +1 * fcti o c** tA cn KO • cn 4“· CM v · LA • IA KO LA o o KO Ch Tj· ω 8 «Φ V— *“ +1 e. * 2 § $ +1 +1 Ή § +| * +1 S 0) £-· σ> o tA LA a CM • co 0 4 00 ° LA • o C* CV 00 v~ tA Oi 00 co KO fA V— w s o « ΙΛ b+1 Fi* m © ¢5 Ο «Ρ gg C&fc3 f§ 4-1 CO t> 0 KO LA 4*1 4*1 LA · CM CO o . * 4-1 * LA Hi , * 4-1 * LA LA CO 03 CO KO 4-1 ko + '?Λ t, * + 1 * ω •e oo o tn cn co +1 + w v · oo t. r4 tAHO OOO • · · OOO v W il II fi * 4-i Ή 4-1 4-1 * +1 * * 03 LA KO cn LA © SA τ- LA • r- · 0 LA Ο *5* cn co co e- co ΰ © *T LA gs § o r a. ΐ •g aje - >> s H '.§« s I δ II II II * # φ + * •44· *4803 It will be observed from the foregoing that while the level of saccharase inhibition is a characteristic function of molecular weight, both in vitro and in vivo, and amyla.se inhibition is conversely a direct function of molecular weight in vitro, in vivo inhibition of starch digestion does not decrease with decreasing molecular weight but surprisingly remains constant. Thus even though in vitro amylase inhibiting activity decreases with molecular weight, the for the present compounds are substantially the same as those of the higher members. This is summarized in Table VI which follows.

TABLE VI. α-Amylase Inhibition Starch Digestion Inhibition Formula Glucose Units In Vitro AlU/mg In Vivo (ed5o ms/ke) IIB (1) 300 0.76 IIIB (2) 300 1.60 V (3) 1,400 1.61 - (4-6) 17,500 1.42 - (5-7) 30,000 1.00 It is thus possible to achieve simultaneously inhibition of both sucrose- and starch digestion with the pure lower members and to do so at a precise, predictable and characteristic dosage level.

The compounds also appear to have an advantageous effect on. glucose absorption, as can be seen from the following data'in Table VII in fasting rats for the compound of Formula IIIB (n = 2).

Table VII Dose -.......·. . Blood Glucose in mg% (Mean £ £>D) 15 Min, 30 Min. 45 Min. saline I 72 + 4.6 78 £ 1.3 87 £ 6.8 glucose (control) 142 £ 12 132 £ 12 158 £ 19 glucose + 30 mg 135 £ 13 128 ± 5.5 132 +7.3 glucose + 60 mg 125 £ 13* 118 + 2.9** 130 + 9.0*** * = Probability against glucose (control) =<0.05 if* — II II II II = Purification of the mixture of higher members of this 15 series results in further unexpected increases both in activity and in specificity. This can be seen from a comparison of the α-amylase and saccharase in vitro inhibition data given in Table II above with the data in Table VIII below for the purified compounds: ' Table VIII Humber of Glucose Units a-Amylase Inhibition AIU/g Saccharase Inhibition MlU/g 4 67,000,000 '7,000 5 ’ 57,000,000 3,500 6 42,000,000 1,200 7 24,000,000 60 8 5,000,000 10 - 46 **so3 The above results show the high activity as an vamylase inhibitor of the compound having 4 glucose unite. The compounds having 5 and 6 units also exhibit considerably higher specific inhibitor activities than found with any previous preparation. There is of course an obvious decrease in inhibitor activity with increasing molecular weight, as can be seen from the compounds having 7 and 8 glucose units although these still show considerable inhibition. In vitro saccharase inhibition appears to be an inverse function of molecular weight. The compound with 4 glucose units exhibits about 1/10 of the specific activity of the compound having two glucose units whereas the inhibitory activity of the compound with 8 glucose units is only marginal.

The compounds can be administered without dilution, as for example as a powder or in a gelatin sheath, or in combination'with a carrier in a pharmaceutical composition.

The present invention provides a pharmaceutical composition containing as active ingredient a compound of the invention (i.e. a compound of the formula I substantially free from isomers thereof) in admixture with a solid or liquefied gaseous diluent or in admixture with a liquid diluent other than a solvent of a molecular weight less than 200 (preferably less than 350) except in the presence of a surface active agent.

The invention further provides a pharmaceutical composition containing as active ingredient a compound of the invention in the form of a sterile or isotonic aqueous solution.

The invention also provides a medicament in dosage unit form comprising a compound of the invention either alone or in admixture with a diluent.

The invention also provides a medicament in the form of tablets (including lozenges and granules), dragees, capsules, pills, ampoules or suppositories comprising a compound of the invention either alone or in admixture with the diluent. ' Medicament as used in this Specification means physically discrete coherent portions suitable for medical administration. Medicament in dosage unit form as used in this Specification means physically discrete coherent units suitable for medical administration each containing a daily dose Or a multiple (up to four times) or submultiple (down to a fortieth) of a daily dose of the compound of the invention in association with a carrier and/ or enclosed within an envelope. Whether the medicament contains & daily dose or, for example, a half, a third, T5 or a quarter of a dally dose will depend on whether the medicament ie to be administered once or, for example, twice, three times or four times a day respectively.

The pharmaceutical compositions according to the invention may, for example, lake the form of ointments, gels, pastes, creams, sprays (including aerosols), lotions, suspensions, solutions and emulsions of the active ingredient in aqueous or non-aqueous diluents, syrups, granules or powders.

The diluents to be used in pharmaceutical compositions (e.g. granulates) adapted to be formed into tablets, dragees, capsules and pills include the following: (a) fillers and extenders, e.g. Btaroh, sugars, mannitol, and silicic acid.; (b) binding agents, e.g. carboxymethyl . cellulose and other cellulose derivatives, alginates, gelatine and polyvinyl pyrrolidone; (c) moisturizing agents, e.g. glycerol; (d) disintegrating agents, e.g. agar-agar, - 48 *49o3 calcium carbonate and sodium bicarbonate; (e) agents for retarding dissplution e.g. paraffin; (f) resorption accelerators^, e.g. quaternary ammonium compounds; (g) surface active agents, e.g. cetyl alcohol, glycerol I monostearate; (h) adsorptive carriers, e.g. kaolin and i bentonite; (i) lubricants, e.g. talc, calcium and magnesium stearate and solid polyethylene glycols.

The tablets, dragees, capsules and pills formed from the pharmaceutical compositions of the invention can have the customary coatings, envelopes and protective matrices, which may contain opacifiers. They can be so constituted that they release the active ingredient only or preferably in a particular part of the intestinal tract, possibly over a period of time. The coatings, envelopes, and protective matrices may be made, for example, of polymeric substances or waxes.

The ingredient can also be made up in microencapsulated form together with one or several of the above-mentioned diluents.

The diluents to be used in pharmaceutical compositions adapted to be formed into suppositories can, for example, be the usual water-soluble or water-insoluble diluents, such as polyethylene glycols and fats (e.g. cocoa oil and high esters [e.g. C^-alcohol with C^g-fatty acid]) or mixtures of these diluents.

The pharmaceutical compositions which are ointments, pastes, creams and gels can, for example, contain the usual diluents, e.g. animal and Vegetable fets, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide or mixtures of these substances. - 49 Shr pharmaceutical compositions which are powders and sprays can, for example, contain the usual diluents, e.g. lactose, talc, silicic acid, aluminium hydroxide, calcium silicate, and polyamide powder or mixtures of these substances. Aerosol sprays can, for example, contain.the usual propellants, e.g. chlorofiuorohydrocnrbons.

The pharmaceutical compositions which are solutions and emulsions can, for example, contain the customary diluents (with, of course, the above-mentioned exclusion of solvents having a molecular-weight below 200 except in the presence of a surface-active agent), such as solvents, dissolving agents and emulsifiers; specific.examples of such diluents are water, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils [for example ground nut oil], glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitol or mixtures thereof.

For parenteral administration, the solutions and emulsions should be sterile, and, if appropriate, blood-isotonic.

The pharmaceutical compositions which are suspensions can contain the usual diluents, such as liquid diluents, e.g.' ,· water, ethyl alcohol, propylene glycol, surface-active agents (e.g. ethoxylated'Isostearyl alcohols, polyoxyethylene sorbite and sorbitane esters), microcrystalline cellulose, aluminium metahydroxide, bentonite, agar-agar and tragacanth or mixtures thereof.

All the pharmaceutical compositions according to the invention can also contain colouring agents and preservatives as well as perfumes and flavouring additions (e.g. peppermint, oil and eucalyptus oil) and sweetening agents (e.g. saccharin). ‘ ' - $0 - .

The pharmaceutical compositions according to the invention preferably contain about 0.1 to 99.5, more preferably from about 0.5 to 95% of the active ingredient by weight of the total composition.

In addition to a compound of the invention, the pharmaceutical compositions and medicaments according to the invention can also contain other pharmaceutically active compounds. They may also contain a plurality of compounds of the invention.

Any diluent in the medicaments of the present invention may be any of those mentioned above in relation to the pharma oeutical compositions of the present invention. Such medicaments may include solvents of molecular weight less than 200 as sole diluent.

The discrete coherent portions constituting the medicament according to the invention will generally be adapted, by virtue of their shape or packaging, for medical administration and may be, for example, any of the following! tablets, (including lozenges and granules), Λ pills, dragees, capsules, suppositories and ampoules. Some of these forme may be made up for delayed release of the active ingredient. Some, such as capsules, include a protective envelope which renders the portions of the medicament physically discrete and coherent.

The production of the above-mentioned pharmaceutical compositions and medicaments is carried out by any method known in the art, for example, by mixing the' active ingredient (s) with the diluent(s) to form a pharmaceutical composition (e.g. a granulate) and then forming the composition into the medicament (e.g. tablets).

This invention further provides a method of combating (including preventions relief and cure of) the abovementioned diseases in non-human animals, which comprises ad ministering to the animals a compound of the invention alone or in admixture with a diluent or in the form of a medicament according to the invention.

It is envisaged that these compounds will be administered, perorally, parenterally (for example intramuscularly, intraperitoneally or intravenously), rectally or locally, preferably orally. Preferred pharmaceutical Ιθ compositions and medicaments are therefore those adapted for oral administration.

Although the dosage and dosage regimen must in each case be carefully adjusted,'utilizing sound professional judgement and considering the age, weight and condition of-the recipient, and the nature and gravity of the clinical condition, generally the dosage will be from about 30 to about 3x10^ ΑΙϋ/kg and from about 1 to about 1x10^ SlU/kg of body weight per day. In some instances a sufficient therapeutic effect can be obtained at a lower dose, while in others, a larger 2q dose will be required.

The toxicity of these compounds is very low. Even without final purification, the crude preparation of Example 8 having an activity of 26,000 SIU/g is tolerated without side effects. This ,is true of the compounds of the present inven25 tion described herein which have been tolerated at a dosage of 340,000 SlU/kg upon oral administration to mice and rats without adverse effects. On intravenous administration, mice tolerated 10,000 SIU/kg without side effects.

In addition to the above pharmaceutical compositions and medicaments the invention further'provides a medicated human foodstuff Or animal fodder comprising a compound of the - 32 invention and a nutritious material. The foodstuffs include sugar, bread potato products, fruit juice, beer, chocolate and other confectionery, and preserves such as jam, to which a therapeutical!;/ effective amount of at least one inhibitor of the present invention has been added. The nutritious material suitable for incorporation in an animal feedstuff includes oil cake, grain (e.g. barley) fish meal, soya bean meal, exhausted sugar beet, silage, hay and skimmed milk.

The following examples will serve to further typify the nature of this invention. In those examples, representative ion exchange resins which can be used include AMBERLITE IRA 410 Cl” (anion exchanger); AMBERLITE IRC 120 (H+ form) (strongly acid ion exchanger); AMBERLITE (HCO^“ form) (anion exchanger); AMBERLITE IRA 410 OH” (strongly basic ion exchanger); AMBERLITE IRC 50 H+ (weakly acid cation exchanger) and DOVEX 50 WX4 H+ (strongly acid ion exchanger).

The microorganisms used herein have been deposited with the American Type Culture Collection under the following numbers! ' Strain , ATCC Ho.

SE 50 (CBS 961.70) 310 42 SB 18 (CBS 957.70) 310 41 SE 82 (CBS 615.71) 310 45 SE 50/13 (CBS 614.71) 310 43 SE 50/110 (CBS 674.73) 310 44 Example 1 A fermenter filled with 8 litres of nutrient solution containing 5.0% starch, 1.0% of yeast extract and 0.2% of is inoculated with a 3 day old shaken flask culture of the strain SE 50/13 (CB3 614.71) and the - 53 mixture is incubated with intensive stirring and aeration for 3 days at 28°C, giving a culture broth containing 105,000 AIU/ml. ' litres of this culture broth are cooled to 20°C, the pH is adjusted to 2.5 with half-concentrated HNO^, g of Carboraff inactive charcoal are added and the mixture is stirred for 10 minutes. it is then centrifuged at 10,000 rpm for 15 minutes and the clear light yellow supernatant liquid is neutralized with HH^ and then concentrated to 500 ml. The 500 ml of concentrate were stirred for 45 minutes with 200, g of Amberlite IRA 410 Cl, the latter ts&b filtered off and the filtrate was treated with 4/5 of its volume (= 400 ml) of methanol in order to precipitate the bulk of the higher-molecular starch degradation products (together with active charcoal residues still present). The mixture Is centrifuged for 5 minutes at 5,000 rpm. The 850 ml of supernatant liquid are added dropwise to 4 litres of dry spirit, with intensive stirring. The white flocculent precipitate is filtered off, washed 3 times with dry spirit and twice with ether and dried in vacuo at 50°0. Yield: 2® g g of a white powder containing 10 x 10 AIU/g. This preparation is referred to below in several of the following Examples.

Enzyme Inhibition on Thin layer Plates To assess-the end products of fermentation and the composition of the final preparation by means of thin layer chromatography, 1 μΐ of the fermentation broths or I pg of the preparations is applied to ready-to-use silica gel TLC films (Schleicher and Schull, Dassel, type E l.500) jq and the chromatogram is developed twice in n.-butanol/ - -54- *4803 ethanol/water = 50/30/20(v/v).

To produce a saccharase inhibition colouration, the developed and well-dried plate is sprayed with enzyme gel (20 ml/20 x 20 cm plate) and the gel is allowed to solidify. The system is then pre-incubated for 5 minutes in a moist chamber at room temperature and then generously sprayed with substrate gel. After this 2nd gel layer has solidified, the plate is introduced into a moist chamber and incubated at 40°C. The inhibition colouration (light spots, red-brown background) develops in 60*-90'. At the point in time of optimum colour development, the treatment is discontinued and the plate, with the agar layers thereon, is dried using a warm air blower. .tfe?.. figAai,, Enzyme gels 1.5 g of agarose (from 1*Industrie Biologique Erancaise) is suspended in 100 ml of 0.2 M Ha maleate buffer of pH 6.0 and then dissolved by boiling up. The clear agarose solution is cooled to 50°C and 250 'μΐ of Triton X-100 solution (2 g of Triton X-100 + 8 g of analytical grade ethanol) and 0,5 ml of dianisidine solution (20 mg of i I. dianisidine/l ώ of acetone) are added, with swirling, Directly before using the gel, 1 ml of GOD/DOD reagent (12.5 mg of glucose bxidase, degree of purity I, Boehringer, order Ho. 15,423 and 2.5 mg of peroxidase, degree of purity II, Boehringer, order Ho. 15,302, dissolved in 5 ml of maleate buffer) and 4-5 saccharase units from the small intestine of the pig are added. The gel must be kept at 50°C until it is sprayed^ since otherwise it solidifies- in the nozzles during the spraying process.

Substrate gels 0.5 g of agarose is suspended in 100 ml of Ha maleate buffer of pH 6.0 and dissolved while boiling. - 55 4 Ο® The solution is then cooled to 50°C and 100 μΐ of Triton (2 g of Triton X-100 + 8 g of analytical grade ethanol) are then added, followed by 1 g of sucrose (Serva Ho. ,579). After the sucrose has dissolved, the gel is ready to use.

For the amylase inhibition colouration, the developed and dried thin layer chromotography plate is sprayed with an amylase gel (20 ml/20 x 20 cm plate) and is allowed to solidify. After 5 minutes of pre-incubation at room temperature, the plate bearing the gel layer is introduced into an. 0.5% strength starch, solution (l g of starch, Merck No., 1,252, dissolved, with boiling, in 200 ml of 0.2 M glycerophosphate buffer 0.01M CaClg.pH 6.9) and is left therein for 2 minutes at 40°C while swirling the solution. The plate is then well rinsed with distilled water and dipped into a dilute I2 solution (4 ml of I2 stock solution per 500 ml of HgO; Ig stock solution: 2*2 g of I2 + 4.4 g of XX dissolved in 100 ml of HgO) in order to colour the starch which has not been degraded. After about 1 minute, the colouration is at an optimum. It Is photographed immediately since the blue spots fade rapidly. g of agarose is dissolved In 100 ml of 0.2 M sodium glyoerophosphate/O.Ol M Ca01g buffer of pH 6.9 at 100°0 and after cooling to 50°C, 100 μΐ of Triton X-100 (2 g of Triton X-100 + 8 g of analytical grade ethanol)~are added. Directly before spraying, 100 μΐ of an amylase crystal suspension (10 mg -of pig’s pancreas amylase/ml of saturated NH. sulphate solution, Boehringar, No. 15,017) are added.

If a I litre Erlemeyer flask containing 120 ml of a -5630 *4So3 nutrient solution consisting of 45- of starch, 2.4% of glucose, 0.9% of casein hydrolysate and 0,95- of yeast extract, pH adjusted to 7.6 with NaOH, mixed with 0.45' of CaCO^ and sterilized for 30 minutes at 121°C, is inoculated with 3 ml of a pre-culture of the strain SE 82 (CBS 615.71), grown in a nutrient solution consisting of 2% of starch, 1% of glucose, 0.5% of casein hydrolysate and 1% of yeast extract, pH adjusted to 7.2 with NaOH, treated with 0.4% of CaCO^ and sterilized for 30 minutes at 121°C, and the whole is incubated for 5 days at 28°C on a rotary shaking machine, a culture solution containing 122,000 AlU/ml is obtained.

For working up, the mycelium is separated from the combined culture solutions by centrifuging at 12,000 rpm, 300 ml of the culture filtrate are brought to pH 2.5 with'halfconcentrated HN0„ and the mixture is stirred for 10 minutes with 2.5 g of analytical grade active charcoal. After separating off the charcoal at 12,000 rpm, the solution is neutralized to pH 6 with 10 Ν KOH, 300 ml of methanol are added, the mixture is allowed to stand briefly and the precipitate is removed at 12,000 rpm, If the supernatant liquid is now added dropwise to 3 liters of ethanol and the precipitate is isolated, after brief standing, by centrifuging at 12,000 rpm and is washed twice with absolute ethanol and once with ether and dried in vacuo, 2.23 g of a product containing 7.45 x 10^ AlU/g are obtained, which contains more than 95% of compounds having from 4 glucose units upwards.

If a 1 1 Erlenmeyer flask with 120 ml of a nutrient solution of composition 3.5% of glucose, of staroh 0.5% of casein hydrolysate, 1.3% of yeast extract, 0,3% of CaCO^ - 57 and 0.3% ‘of EgHPO^, adjusted to pH 7.8 before sterilization and sterilized for 30 minutes at 121°C, is inoculated with 6 ml of a pre-culture of the strain SB 50/110 (CBS 674.73) in a nutrient solution consisting of 3% of soya flour, 3% of glycerol and 0.2% of CaCO^ and the mixture is incubated for 3-=4 days on a rotary shaking machine at 24°0, a culture solution which contains 153,000 AlU/ral and 12,000 SIV/liter, is obtained, liter of culture solution was adjusted to pH 2.5 with HHO^ and the mixture was stirred for 10 minutes with 5 g of active charcoal and then centrifuged for 30 minutes at 5,000 rpm. It was then neutralized by adding 25 g of Amberlite IRA 410 (OH form). The neutral supernatant liquid was concentrated to 100 ml on a rotary evaporator, mixed with 100 ml of methanol and filtered. The filtrate was stirred into 2 liters of dry spirit and the precipitate which separated out was filtered off, washed, 3 times with acetone and ether and dried in vaouo.

Yield 14 g of a white powder containing 5 x 10® AlU/g 2q and predominantly ooataining compounds having from 4 glucose units upwards.

If the procedure of Example 3 is followed but with the addition of 0.5% starch, a culture broth containing 40,000 . AIU and 184 SlU/ml is obtained after 4 days' fermentation. The culture broth contains a mixture of compounds of the invention having from one glucose unit upwards.

,' \ -: If a 1 liter Erlenmeyer flask which contains 120 ml of nutrient solution of composition 3% of glucose, 0.6% of casein hydrolysate, 1.6% of yeast extract, 0.3% of OaGQ~. - 58 44803 and 0.30 of KgHPO^, pH adjusted to 7.8 x-rith KOH before sterilization, is inoculated with a pre-culture of the _. strain SB 50/110 (CBS 674.73) according to Example 3 and incubated for 4 days at 24°C on a rotary shaking machine, a culture broth of 10,800 SlU/liter, which predominantly contains the compound of the invention having one glucose unit, is obtained. liters of culture filtrate, separated from the mycelium at 13,000 rpm, were adjusted to pH 2.5 with halfconcentrated HNOj and stirred for 15 minutes with 55 g of active charcoal (I-ferck) and 200 g of Clarcel (registered Trade Mark). After removing the solids by suction filtration, the filtrate was neutralized to pH 7 with concentrated ammonia and the solution was concentrated to 1.5 liter and precipitated with a five-fold amount of ethanol. The resulting flocculent precipitate was separated off using a continuousflow rotor at 12,000 rpm and the yellowish supernatant liquid was concentrated to 150 ml and centrifuged at low speed to separate off minor proportions of undissolved material. 50 ml of this solution were charged onto a column filled with Amberlite IR-120 (H+ form) (30 x 300 mm; ml of HgO per hour?. After a total of 300 ml of eluate, which contains inert saccharides and a proportion of nonadsorbed components having an inhibiting action, had been collected, the exchanger was transferred into a beaker with about 400 ml of H^O and concentrated ammonia was added, while stirring, until the pH had reached a value of 11.5. After stirring for a further 30 minutes, the exchanger was separated off, the liquid was concentrated to 1/20 of its volume and filtered through a column (20 x 150 mm) ¢803containing Amberlite IRA-410 (H00^“ form) and about 500 ml . of eluate were oollected at a flow speed of 30 ml/hour; the eluate was concentrated and after lyophilization gave 1.3 g of crude product.

: For further purification, the crude product was fractionated on Bio-Gel F-2, 100-200. mesh (Bio-Rad, Munich). A column of 50 mm diameter and 450 mm length was used- for. this purpose and tas operated with HgO at a flow speed of 40 ml per hour, fractions of 10 ml each being collected. All fractions were tested by means of the enthrone test for carbohydrates and by means of the saccharase inhibition test for -components having an inhibiting action. The fractions containing saccharase inhibitor were -further . . examined by thin layer chromatography, in accordance id th Example 1, for their content of individual components. The fractions whioh contained the compound having one glucose unit were combined, concentrated and lyophilized. 35 mg of material showing 0.3 x 10^ AlU/g and 30,000 SIU/g, were obtained.

If 1 1 Brl'enmeyer flasks each containing 120 al of a .nutrient' solution of composition 5% of starch, 1% of yeast extract and 0.2% of ZgHPO^ are each inoculated with 2 ml of a pre-culture according to Example 3 and incubated, for 3 days at28°0, culture solutions with the following yield of amylase inhibitor are obtained: SB 50 $®S 961.70) SB 50/13'(CBS 614.71) SE-(CBS 674.75) AIU/ml 37,000 109,000 53,50044803 The mixture consists predominantly of a mixture of compounds with four or more glucose units.

Example 7 If 1 1 Erlenmeyer flasks each containing 120 ml of nutrient solution of composition 1.3% of maltose, 3.5% of glucose, 0.5% of casein hydrolysate, 1.3% of yeast extract, 0.3% of CaCOj and 0.3% of KgHPG^ are each inoculated with 2 ml of a pre-culture according to Example 3, the following yields are obtained after 4 days' incubation with various strains on rotary shaking machines at 24°0; Strain SlU/ml ATU/ml SE 50 (CBS 961.70) 25 580 SE 50/13 (CBS 614.71) 14.8 1,460 SE 50/110 (CBS 674.73) 57.9 755 The products consist predominantly of a mixture of compounds having four or less glucose units.

If a fermenter containing 100 1 of nutrient solution of composition 3.5% of glucose, 2.5% of dry powdered malt extract, 0.5% of casein hydrolysate, 1.3% of yeast extract, 0.3% of OaCOj, 0.3% of EgHPO^ and 0.1% of anti-foaming agent is inoculated with 5 1 of a pre-culture according to Example 3 and incubated for 5 days at 24°0 with stirring and aeration, a culture solution of 73,000 SIU/l is obtained, which predominantly contains the compound of the invention with n = 2.

A 90 liter fermentation batch together with the mycelium is adjusted to pH 2.5 on a pH meter by means of concentrated BHOj and 900 g (= 1%) of active charcoal (Merck) are added while stirring in order to adsorb the bulk of the dyestuffs formed» The mixture is stirred for 15 minutes, - 61 q3 the mycelium and the bulk of the charcoal were separated off on a centrifuge at 3,000 rpm and the supernatant liquid, with addition of 3 kg of Clarcel, is finally filtered through-a pressure filter, 65 1 of yellow-brown clear filtrate of SIU content 60,000 SlU/litre are obtained.

The filtrate is adjusted to pH 7 with concentrated J · .

EHj and stirred vith 1,300 g (20) of active charcoal (Merck) for 30 minutes in order to adsorb the active substance. The mixture is filtered through a pressure filter and the active lo charcoal sediment was washed 3 times with 10 liters of distilled water. The charcoal is then thoroughly pressed dry and stirred with. 3 times 4 liters of 500 strength acetone at pH 2.5, in each ease for 15 minutes, so as to desorb the active substance from the charcoal. The acetone desorbates are combined after removing the charcoal by filtration. The combined desorbate is concentrated to 250 ml on a rotary evaporator, an equal volume (250 ml) of methanol is added and the mixture is filtered through a folded filter. The filtrate (480 ml) is added dropwise to litres of acetone, with vigorous stirring. The precipitate which separated out is filtered off and washed 3 times with acetone and ether. It is then dried in vacuo at 35°C.

Yield 230 g of crude product containing 8,500 SlU/g. g of the above crude product are dissolved in 1 liter of HgO and stirred with 300 g of Dowex 50 WX 4 H+ (200-400 mesh) for 30 minutes. The resin is filtered off and rinsed 3 timse with 2 liters of 0.001 N HCl. The washed Dowsx is then suspended in 500 ml of HgO and the suspension adjusted to pS 9.0 on a pH meter by addition of 250 strength HKy Thereafter 2 further desorptions ars carried out, each with 500 ml of 0.60 strength and the desorbates are - 62 44803 combined' and concentrated to 100 ml on a rotary evaporator.

To decolourize, this concentrate, it is stirred for 5 minutes with 2 g of DEAE-cellulose (Schleicher and Schull, No. 02035, 0.6 milliequivalent/g), and then centrifuged, The light yellow supernatant liquid is mixed with an equal volume (100 ml) of methanol and the mixture is then added dropwise to 2 litres of acetone, with intensive stirring.

The precipitate is filtered off, washed with acetone and ether and dried in vacuo at 35°C, yield 4.2 g of product containing 26000 siu/g.

For addit4onal fine purification, the 4.0 g of inhibitor are gel-filtered, in 0.5 g portions, through Biogel P-2. For this purpose, each 0,5 g of the preparation is dissolved in 10 ml of HgO and -ihe solution was charged onto a Biogel P-2 column (200-400 mesh, Bio-Rad) of 5 cm diameter and 95 cm length. The column is developed in water at a flow rate of 80 ml/hour. 12 ml fractions are collected. For all fractions, the total carbohydrate content (in the form of the enthrone test, as an extinction at Egg^) cOn^eil'*: of saccharase inhibitor and amylase inhibitor is determined.

In addition, the fractions are tested by thin layer chromatography (enzyme inhibition colouration according to Example 1).

The fractions containing the compounds with 4-6 glucose units are combined, concentrated to 10 ml in vacuo and precipitated by dropwise addition to 200 ml of dry spirit. The precipitate ie oentrifuged off, washed with acetone and ether and dried in vacuo; yield from 4.0 g of crude inhibitor; 0.2 g of compounds having 4 to 6 glucose units with activity of 17.5 x ΙΟ^ ΑΙΠ/g ,and 8,500 SlU/g. The fractions containing the compound with 3 units are worked up in the same manner, the 440°3, fractions, the total carbohydrate content (in the form of the anthrone test, as an extinction at B^q) an<^ content of saccharase inhibitor and amylase inhibitor is determined. In addition, the fractions are tested by thin layer ehromato5 graphy (enzyme inhibition colouration according to Example l).

The fractions containing the compounds with 4-6 glucose units are combined, concentrated to 10 ml in vacuo and precipitated by dropwise addition to 200 ml of dry spirit.

The precipitate is centrifuged off, washed with acetone and ether and dried in vacuo; yield from 4.0 g of crude inhibitor: 0)2 g of compounds having 4 to 6 glucose units with activity of 17.5 x 10° AlU/g and 8,500 SlU/g. The fractions containing the compound with 3 units are worked up in the same, manner, the precipitation being carried out with 200 ml of acetone; yield from 4.0 g of crude inhibitor: 0.1 g of compound of the invention with 3 units, containing 1,4 x 10® AIU/g and 21,000 SlU/g. 0.9 g of the compound of the invention with 2 units containing 0.3 x 10® AlU/g and 68,000 SlU/g Is isolated from the fractions (precipitation with acetone) containing the compound having 2glucose units.

Example 9 If 3 small fermenters each containing 8 liters of a nutrient solution with 7.5% of dry powdered malt extract, 0.3% of casein hydrolysate, 0,7% of yeast extract, 0.3% of CaCO^ and 0.3%’of EgHPO^ are inoculated with 5% of a preculture of the strain SE 50/110 (CBS 674.73) (obtained according to Example 3), 5 days’ incubation at 24°0 gives a culture broth of 73 SlU/ml which predominantly contains jq compound with two glucose units. After centrifuging (30’, 3,000 rpm) to separate off the mycelium, 20.5 liters -64-4430s of a deep brown culture solution containing 67,000 SIU/l were obtained. Thia solution is adjusted to pH 3.5 with HNOj and 60 g of lewapol (Oa 9221, 0.35 mm particle size, Bayer A.G.) / 1 = 1,23 kg of lewapol were added to decolourize the solution. After stirring for 20 minutes, the mixture is filtered using a Seitz E 3 filter. The decolourized culture solution is neutralized with HHj (18.5 1, 67,000 SIU/l). 20 g of aotive charcoal/l = 370 g are then stirred in to adsorb the active substance, and the mixture was stirred for 30 minutes. It is then filtered through a E 3 filter which is covered with a layer of the filter aid Clarcel. The filtrate (17.5 1, 3,600 SIU/l) is discarded. The oharcoal residue is washed 3 times with 2 1 of distilled HgO. To desorb the aotive substance from the oharcoal, the latter is stirred 3 times in succession, each time for 15 minutes, with 1 1 of 80% strength acetone at a time, the pH being adjusted to 2.5 with concentrated HCl. The desorbates are combined (2.4 1, 371,000 SIU/l). 20 g of Dowex H+/l (Dowex 50W x 4, H+ form, Serva, Heidelberg) = 46 g of Dowex were introduced into this desorbate and the mixture was stirred for 20 minutes. The resin is then filtered off (Dowex fraction I) and rinsed with a little 75% strength acetone. The filtrate and wash liquid (3 1 = 215,000 SIU/l) are stirred with 60 g of Amberlite IRA 410 (0H“ form) (Messrs. Serva, Heidelberg)/1 until pH 7 was reached. The mixture is then filtered and the filtrate (2.8 1, 219,000 SIU/l) is mixe.d with 72 g of Dowex H+ and stirred for 20 minutes. While doing so, the pH is kept at 3.0 by hanging a porous nylon pouch, filled with Amberlite 410 0H“, into the mixture. The Dowex is then filtered off (Dowex fraction II) and the filtrate - 65 10 (2.6.1, 27,000 SIU/l) is discarded.

The Dowex ’fractions I and II are each washed individually 3 times with 750 strength acetone at pH 3.5 and then each desorbed 3 times with 100 ml of 0.60 strength at a time (Dowex fraction I) or with 150 ml of 0.60 strength i]H- at a time (Dowex fraction II). During the first desorption, during which the amount of ammonia does not suffice to neutralise the Dowex resin, the pH is adjusted to 9 on a pH meter by addition of concentrated HH-. The three desorbates from Dowex fraction I and II are ; respectively combined, concentrated almost to dryness on a rotary evaporator, taken up in 50 ml of HgO, adjusted to pH 3-4 oh a pH meter by means of HCl, and mixed with 50 ml of methanol. The solutions are added dropwise to 1.5 1 of absolute acetone, while stirring, and the precipitate formed is filtered off and washed 3 times with acetone.and once with ether. It is. dried in vacuo.

Yields Fraction I 6.5 g 25,000 SIH/g Fraction II 12.3 g 36,000 SIH/g Fraction 1 and II mainly contain, as the inhibiting constitusats, ihe compound of the invention with 2 glucose units in addition to small proportions of the compound having 3 glueose units.

The following Table shows the saccharase-inhibiting activities of the preparation at successive stages. -66 4-180 3 ρ Η «Η C •tf 7) ft cn •tf 0) na R ti1, □ co •rl Ό ftvo ο Η ιη CM co VD o vo Th xjrt vo •cf rt rt CM tn ο ο ft ft > ft Ο Ο ft νο ο tn Ο Ο Ο Ο Ο QOO νο ^·ΗΟ νο cnvo <ο νο Η σ>νο ιη ου O ο ο O o O o O o ο ο O 00 [ft o O *4· ο CM in t> ft in CO fe fe fe ·<. «* * O m tn [> CO CM CM σ\ ·< rt vo σ'. tn VO Th ω νο VO rt vo rt Th bC bQ ο ο ο fe ί> νο ο α ο * C* νο ο ο νθ fe tn OOC coo ooo fe * fe cm cnm *a* o o fe rt o tn O O O fe in rH CM o o CM co VO o o <2 cn rt Th σ o o VO tn in in in • • o cn co CM rt rt f- cnco OOO Η Ο ft Φ ft ft +3 c o •rl P ft R CQ § τί· CM O tn ttf • tuo cn ft ω ft CM vr ft tn • a * « • CM CM o O VO CM rt VO ί Ή O Th P R *»-* S3 ti 5 O R ft Φ •rt 3 P P o φ ti 3 rt φ p ft rt O $ R 0 δ • »rt ft 0 CO CQ P R £ S 03 •rt 0 O Φ Φ· rtf CJ « ♦tf H Φ 3 R R T? P Φ Φ φ rt P P P rtf rd K rtf ti ft m β r •rt pH Φ O «Ϊ < rtCMft £ •rl X*\ r—»» iH rt CM m Thinvo r ti o •rt P ft R O A3 £ I ft P CQ rt rt H H S3 C o O •rt •rt P P ϋ O tf ti Sm R ft ft 1 W W Φ Φ o & £ O o £3 ft ft ft K 3 ft ft O o 0 ft •rt P P 03 m H *ri P4 o Φ £ Si P P ti o ti ti O si ti ft ft •rt o R R P •ri ti O O o P 03 CQ ti ti H Φ Φ R CQ Φ rtf rtf rt κ H S3 O •H P 0} rt o tn tn a, fe ti ft •to Φ Φ R ft J3, P P P5 *tf •ti ti ti ti no na P P φ CM Φ 0) •rt •rt ti ti ti ft ft R •rt •rt •rt •rt R Φ ft ft O ϋ Φ P g Φ Φ P ft 0 0 R R ft *aj o o ft ft /•fe Z“fe O rt CM tn cn rt rt v- - 07 10 Example 10 200 g of a preparation as described in Example 1 were dissolved ia 940 - ial of distilled water and 60 ml of concentrated HgSO^ and the mixture ras warmed under reflux for 4 hours (internal temperature; 98o-100°C; oil bath temperature: 140°C). 10 g of active charcoal (Merck Art. 2186) were added to the cooled black-brown solution and the mixture was stirred for 1 hour. The active charcoal was then filtered off and washed with water and the filtrate was adjusted to pH = 7 to 8 with about 250 ml of 10 Sf KOH.

The solution was stirred for 1 hour with 50 g of active charcoal. The charcoal, was-filtered off and washed with 2 1 of water and the filtrate was discarded, For desorption, the charcoal Was digested overnight with 2 1 of.30% strength alcohol. Finally, the charcoal was filtered off and the alcoholic solution was concentrated on a rotary evaporator. Residues 6.2 g. This crude product (6.2 g) was dissolved in 500 ml of water and the solution was gently stirred with 30 g of Amberlite IR 120 (H+ form) for 1 hour. The exchanger was filtered off and washed with distilled water until the filtrate was neutral and free from gluoose. The exchanger was then stirred overnight with 15 ml of 25% strength NH^ in 1,000 ml of HgO, separated off and discarded. The filtrate was concentrated on a rotary evaporator.

Residues 3»? For further purification, a chromatography on cellulose was carried out» 4·5 g of the material desorbed from the exchange? wers'applied to a 1 m long and 2.5 cm wide column filled wiW'^ellulose. The running agent used was initially 5:1. ethanol/KgO, and 3sl ethanol/HgO was initially used to - 68 30 443 03 elute the compound of the invention with n = 1. Fractions of 14 ml were collected at a drip speed of 20 drops per minute. She individual fractions were examined by thin layer chromatography. Fractions 47-95 gave, after concentration 1,6 g of a compound of the invention with one glucose unit exhibiting pale brownish discolouration. The discolouring impurities were quantitatively insignificant. The compound with one glucose unit was obtained, as a colourless resin if the purification step with a strongly aoid ion exchanger was carried out on a column and not by the batch process.

Example 11 200 g of a preparation as described in Example 1 were dissolved in 940 ml of distilled water and 60 ml of concentrated and the solution was warmed under reflux for l/4 hour (internal temperature; 98°-100°0; oil bath temperature; 140°C). 10 g of active charcoal (Merck, Art. 2186) were added to the cooled, black-brown solution and the mixture was stirred for 1 hour. The active charcoal was then filtered off and washed with water and the filtrate was adjusted to pH « 7 to 8 with about 250 ml of 10 N KOH.

The solution was stirred with 50 g of active charcoal for 1 hour. The charcoal was filtered off and washed with 2 1 of water and the filtrate was discarded. For desorption, the charcoal was digested overnight with 2 1 of strength alcohol. Finally, the charcoal was filtered off and the alcoholic solution was concentrated on a rotary evaporator. Residue: 8.0 g.

The residue was taken up in 15 ml of HgO and applied to a column (height: 20 cm, diameter 2.4 cm) filled with 6908 0 3 g of Amberlite IR 120 (H+ form). The solution was absorbed at 3 drops/minute and the column was rinsed with water (12 drops/minute) until all non-basic constituents had been removed. The basic products were then eluted from the column with 0.5% strength NH^ (12 drops/minute) and the aqueous solution was evaporated to dryness on a rotary evaporator. Residue: 4.1 g. g of this residue were dissolved in a little water and applied to a column (height: 200 cm; 0: 3.0 cm) filled with Sephadex G-15. The column was eluted with water. Fractions of 2 ml each were collected at a flow speed of 8 ml/hour. The individual fractions were examined by thin layer chromatography. Fractions 85-94 gave 280 tog of the compound having 2 glucose units and a specific activity of 50,000 SIU/g.

Example 12 If 2 g of a preparation as described in Example 1, in 60 ml of 20 sSI sodium glycerophosphate buffer of pH 6.9, containing 1 mM of OaClg, are incubated with 1 g of a-amylase from Aspergillus spec. (SERVA No. 13,418) for 120 hours at 37°C with constant stirring and finally heated to 100°C for 5 minutes, and undissolved matter is centrifuged off at 4,000 rpm, lyophilization of the solution gives 1.9 g of a product with 3,500 SlU/g and 2 x ΙΟθΑΙϋ/g. If this product is tested by thia layer chromatography and saccharase inhibition’discolouration as described in Example 1, it is found .that the; compounds having an inhibiting action which &fce present gsfe .eepiaatially the compounds of the invention with 1, 2' aa^:^.-'-gliasose- units. . ; ./. -.. ...•“70 ; ‘ ' Example 13 If 2 g of a preparation as described in Example 1, in 30 ml of 20 oK -acetate buffer of pH 4.8, are incubated with 1.25 mg of β-amylase from sweet potato (SOEIHilHOER 15,471) for 120 hours at 37°O, with constant stirring and finally heated to 100°0 for 5 minutes, and undissolved matter is centrifuged off at 4,000 rpm, lyophilization of ths solution gives 1.5 S of a product with 1,800 3IU/g and 3»8 x 10® AIU/g, If this product is tested by thin layer chromatography and saccharase inhibition discolouration as described in Example 1, it is found that the compounds having an inhibiting action which are present are essentially the compounds of the invention with 2 and 3 glucose units.

Example 14 If a 200 ml Erlenmeyer flask containing 25 ml of a nutrient solution of composition 0.1% KgHPO^, 0.2% of (HH4)2SO4, 0.05% of IigS04, 0.05% of E01, 0.01% of PeS04 and 2% of a preparation as described in Example 1 is inoculated with a spore suspension of the strain Asp. niger ATCC 11,394 and incubated at 28°0 on a rotary shaking machine, the AIU concentration falls from 210,000 AIU/ml to 53,000 AIU/ml after 6 day3 and to 21,300 AIU/ml after 10 days. At the same time the SlU/ml content rises from 7.0 to 72 SlU/ml, ml of a solution which has been incubated with the spore suspension for 10 days are centrifuged for 30 minutes at 3,000 rpm to separate off the mycelium. 15 ml of supernatant liquid (72,000 SIU/1) are desalinated by stirring for 30 minutes with 2 g of Amberlite IEO 50 H+ ana 1 g of Amberlite ISA 410 0H“ (conductivity less than 2 mS’cm-1).

I The mixture is filtered and the filtrate allowed to run at 4 8 Ο 3 the rate of 5 ml/hour through a column (1 cm x 10 cm) of Dowex H+ equilibrated in 0.001 N HCl. The column is then rinsed with 200 ml of 0.001 N HCl, For desorption, 0.60 strength NHj solution is pumped through the column (10 ml/ hour) and 5ml fractions are collected. The fractions containing the saccharase-inhibiting activity are combined, concentrated to 2 ml on a rotary evaporator and mixed with 2 ml of methanol. This-solution is adjusted to pH'3-4 and precipitated by adding it dropwise to 100 ml of acetone. The precipitate Is filtered off, washed with acetone and ether and dried in vacuo. Yields 26 mg containing 28,000 SlU/g and consisting of compounds with 2 and 3 glucose units. The . isolation of the pure compound with 2 glucose units from this product is effected as described in Example & by gel fil15 tration through a column containing Bio-Gel JP—2. 7 mg of the compound with 2 glucose units of 60,000 SIH/g, are obtained. liters of culture filtrate obtained from a fermentation batch as described in Example 5 by centrifuging off the 2Q mycelium at 13,000 rpm and having an activity of 13,000 SlU/g were stirred with 500 g of a mixture of 2.5 g parts of Amberlite lRC-50· (H+ form) and 1 part of Amberlite IRA-410 (OH· form) for 1 hour in order to reduce the salt content (conductivity of the culture filtrate: about 10 mS*cm^).

The exchanger was separated off and the solution was concentrated to a little less than 100 ml. and centrifuged for -15 minutes at 20,000 rpm toremove undissolved constituents. The supernatant liquid was made up to 100 ml; it now had a conductivity of 3.5 mS«cm^) and was further purified by applying it to a column (55 x 400 mm) of F-cellulose (SERVA No. 45,130, pre-treated according to known methods and . 7 - 72 4 4 8 Ο 3 equilibrated in 5 mM ammonium phosphate buffer, pH 5.5).

The abovementioned phosphate buffer served as the running agent; the flow· rate was 90 ml/hour and fractions of 18 ml volume were collected.

After the fractions of the eluate had been tested for their carbohydrate content (by means of the anthrone test) and for their content of saccharase-inhibiting components (by means of the saccharase inhibition test), the fractions which in the anthrone test had proved almost free of carbo10 hydrates and equally in the saccharase inhibition test had proved particularly active were combined (fractions 60-170), concentrated to 150 ml and filtered through a column (50 x 300 mm) containing Amberlite IRA-410 (HCO^“ form). For better control of the deionisation, the elute was collected in fractions (10 ml per fraction in 20 minutes) and tested for carbohydrate (by means of the anthrone test; in each case virtually negative), for phosphate (by means of ascorbic acid-molybdate reagent; in each case negative) and for saccharase inhibition (by means of the enzyme inhibition test), The fractions having an inhibiting action (3-30) were’combined, concentrated, lyophilized, redissolved and lyophilized so as to give 280 mg of crude inhibitor.

For further purification, the crude inhibitor was fractionated on Bio-Gel F-2 as described in Example 5.

From the fractions which contained, pure, the compound with one glucose unit, 30 mg of a product with 0.3 x 10 AlU/g and 35,000 SIU/g were isolated after lyophilization.

Example 16 To isolate the compounds with 5-7 glucose units the starting material used can be, for example, a pre“ 7344803 - paratibn such as described in Example 1.

For this purpose, 30 g of the preparation according to Example 1 were dissolved in 250 ml of HgO. The conductivity —Ιοί the resulting solution was 10 mS«cm and the pH was 5.5.

The solution was desalinated by adding 60 g of Amberlite IRC H+ (weakly acid cation exchanger which only binds traces of the amino-sugar derivatives from aqueous solution) and 20 g of Amberlite IRA 410 0H“ and stirring for 20 minutes.

The filtrate ('conductivity 0.5 mS.cm-3·, pH 3.5) was adjusted to pH 3,0 with 1 N HCl (conductivity 0.6 mS*cm“^). This solution xias pumped at the rate of 42 ml/hour through a column filled with Dowex 50 W x 4, 200-400 mesh. (H+) (0 2.5 om, height 40 cm, equilibrated in 0.001 N HCl) and the I Dowex was then rinsed with 2 1 of 0.001 N HCl. After washing the column, elution was carried out with 1.2% strength aqueous ammonia and 10 ml fractions were collected. The fractions haying an inhibiting action were combined, the ammonia was stripped off in vacuo and the solution wsb then concentrated in vacuo to 30 ml. The product was pre20 cipitated by dropwise addition to 600 ml of dry spirit and the precipitate was filtered off, washed with alcohol and ether and dried in vacuo. Yield 4.4 g, containing 26.5 s 106 AIU/g. 0.5 g portions were subjected to a fine purification by application to a preparative Biogel P-2 column, as described in Example 8, and development. The fractions which according to a·thin layer chromatogram (amylase inhibition colouration) contain compounds with 5 to 7 glucose units were combined„ concentrated ifi vacuo and precipitated with dry spirit as'.described above. Yield from 0.5 g of crude products 0.2 gof amino-sugar derivatives with 5 to 7 glucose - F - . 74 44803 £ units containing 30 χ 10 AlU/g and 2,500 SlU/g.

Example 17 This Example illustrates how the compounds of the invention can be eluted from cation exchanger under acid conditions.

A column of 1.5 cm. diameter is filled with 30 g. (wet weight) of Rowex 50 W x 4, (H+) 200-400 mesh in 0.001 n HCl. Finally 500 ml. of mixed desorbate (400 000 SIU/l), pH 2.5, 60% acetone), obtained according to Example 9 (Table, run No. 7) are pumped through the column in about 1 hour and washed finally with 500 ml of 0.001 N HCl. Under these conditions only trace activity is eluted. Finally desorption therefrom with 0.0125 N HCl was effected, the column elute being monitored by conductivity or refraotometry. The SIU content of the elute was aleo tested. The active fractions 74-100 were combined and neutralized by the addition of Amberlite IRA 140 0H“, then reduced to 5 ml, reacted with 5 ml of methanol, and precipitated by dropping into 200 ml acetone. After washing with acetone and ether vacuum-drying was effected.

Yield 1 g of the compound with two glucose units with 65,000 SIU/g.

From the active initial fraction the compounds with 3 and 4 glucose units could be obtained.

This process of acid desorption therefore makes possible in contrast to the alkaline desorption, fractionation, fractionation of the individual amino -sugar derivatives of this.series. Subjecting material prepared as above having 4 to 8 glucose units to this process but simply lyophilizing the neutralized elutes, the individual higher tgC>3I'· actions ar^ obtained as follows: glucoqe units = 67,000 AIU/mg glucose units = 57,000 AIU/mg glucose units = 42,000 AIU/mg glucose units = 24,000 AIU/mg glucose units = 5,000 AIU/mg Έ5 The β-amylase degradation procedure described above is performed as follows· 100 mg of compound ware dissolved in 1.9 ml of 20 mM sodium acetate buffer (pH 4.75) and 0.1 nil of sweet potato β-amylase (BQEHRING-ER No. 15471} 5 mg/ml} 500 U/mg) Were added. The mixture was incubated at 37°C for 48 hours, heated to 100°C for 5 minutes and then centrifuged at 4500 rpm to remove precipitated protein and other impurities.

The entire mixture was then applied to a column (22 mm diameter; 1,000 mm in length} thermostatedly controlled at 65°C) and eluted with water at a flow rate of 25 ml/hour. The elute is monitored by 'a oonductometer and a highsensitivity refractometer equipped with flow-through cells. Fractions of 2.5 ml each were collected. The fractions can ba tested for amylase or saccharase inhibiting activity or for carbohydrate content by means of the anthrone reaction. Electrolytes originating from the buffer and the enzyme preparation are eluted with the void volume and the degradation products are eluted according to-decreasing molecular weight. Complete separation of compounds with small differences in molecular weight is effected by recycling chromatography using the same conditions as des76 44so3 cribed above. Fractions containing products which are to be isolated are pooled and lyophilized.

Example 19 For the separation of the isomeric compounds having throe glucose units, 10 g of a mixture of isomers dissolved in water were applied to a column (25 by 500 mm) filled with Dowex - 50 W X 4 (H+). The column was first washed with water until the elute was neutral and then eluated with 0.025 N hydrochloric acid. Fractions of 3 ml each were collected and tested by thinlayer chromatography. Thinlayer chromatography was performed on silanized silica gel plates (Mer.ck, Germany) with 100 ; 60 : 40 : 2 ethylacetate + methanol + water + 25 % ammonia with threefold development. The compound with formula IV travels a wider distance from I the origin than does the compound with formula V. Fractions 215 through 272 containing 6 g isomer with formula V and fractions 288 through 294 containing 600 mg isomer with formula IV were pooled, neutralised with Amberlite IHA - 140 (0H“) and evaporated.

The in vitro saccharase inhibiting activity of the isomer with formula IV isolated by this procedure is 19.000 SIE/g.

Example 20 Furified. preparations of compounds with n^ + n^ = 4 may be obtained by recycling gel permeation chromatography according to the following example: 6.0 g of a crude preparation of compounds with n^ + n2 = 4 and 5 obtained as described in Example 8 or in Example 17 are separated from salt contaminants by dissolving the material in 1 HH^OH and applying the solution to a column (50 nm in diameter; 100 cm in length) filled with Sephadex ( · Trade Mark) - 77 ' 44®θ3 G-10. The column is eluted, with 1 mM NH^OH and the eluate monitored hy a flow-through refractometer and a conduotometer equipped with a flow-through cell. Thin layer chromatography on silica gel plates (n-butanol: ethanol: water = 45:35:20) is also an appropriate way for testing the eluate. Fractions containing the material to be purified are pooled and lyophilized (yield: 4.4s). 2.0 g of this material Is further separated by applying it to a column (50 mm in diameter; 100 cm in length) filled with Bio-Gel P-2^\ 200-400 mesh. The column is thermostated at 65°C and water is used as an eluant at a flow rate of 80 ml per hour.

The fractions containing the void volume together with the fractions containing salts which may still be present after Sephadex chromatography are discarded. The system consisting of the column, a peristaltic pump, a refractometer and a conduotometer of flow-through type is- closed and the solution is recirculated through the gel. The progress of separation is checked by. continuously monitoring the refractive index and the .conductivity. Sufficient separation is achieved after 5 cycles. At this time the column is reconnected to the solvent reservoir and the column eluted collecting fractions of 16 ml volume each. The fractions - are tested by thin layer ohromatography as described above and the fractions containing pure compounds as well as intermediate fractions are pooled and lyophilized, field: 600 mg of compound with: n1 + ng = 4» 420 mg of compound with n^ + n2 = 5? and 100 mg of poorly separated material from intermediate fractions.

Claims (20)

1. A compound, of the formula (1):- wherein n, designates an integer of from 1 to· 8 and Hg designates 0 or an integer of from 1 to 8 such that n^+ng equals from 3 to 8, the said compound being substantially free frcm structural isemers as hereinbefore defined when n^+n^ = 3 or 4 and being substantially free from homologous compounds of the formula (I) when n^+ng = 5 or more.

2. A compound according to claim 1 wherein n 1 is at least 2.

3. A compound according to claim 1 or claim 2 wherein η,+ng = 4.

4. A compound according to claim 1 or claim 2 wherein n-i+ng = 5. t

5. A compound according to claim 1 or claim 2 wherein n^+ng = 6.

6. 0-(4,6-bisdesoxy4-[l S-(l,4,6/5)-4,5,6-trihydroxy3-hydroxymethylcyclohex-2-en-l-ylamino]-α-D-glucopyranosylj(l-> 4 )-O-«-D-glucopyranosyl- (1-44) -O-a-D-glucopyranosyl(l-»4)-D-glyoopyranose of the conformational structural -7944803 substantially free from structural isomers as hereinbefore define^ ?.» 0-|4,6“bisdesoxy-4-[l 8-(1,4,6/5)-4,5,6trihydrogy-3-bydroxymethyl-4“0-a-D-glyoopyranosyl“(l->4)5 qyclo-hex-2-en-l-ylamiho]-a-D-gLycopyranoByl^-( It»4)-0-«-Dgluoopyranosyl-(li“4)-D->glnoopyranoae of the conformational structural formulas -80*4803 substantially free from structural isomers as hereinbefore defined.

7. 8. A compound of the formula -81448° 3 CM t OH substantially free from structural isomers as hereinbefore defined. ' 82 44803

8. 9. A compound of the formula. substantially free from homologues thereof of the formula (I). -834.8 ® 3

9. 10. A compound, of tha formula *4 8 q 3

10. 11. A compound of the formula substantially free from homologuee thereof of the formula. (I).

11. 12. A compound of the formula -8544803 substantially free from homologues thereof of the formula. (I). .

12. 13. A compound according to any one of ol&lina 1 to 12 5 Substantially as described in any one of Examples 16 to 20.

13. 14. A pharmaceutical composition containing as an active -86448o 3 ingredient a compound according to any one of claims 1 to 13 in adijixtdre with a solid or liquefied gaseous diluent or in admixture with a liquid diluent other than a solventof a molecular weight less than 200 except in the presence of a surface-active agent.

14. 15· A pharmaceutical composition containing as an active ingredient a compound according to any one of claims 1 tol3 in the form of a sterile or isotonic aqueous solution.

15. 16. A composition according to claim 14 or 15 containing from 0.5 to 95% hy weight of the said active ingredient.

16. 17. A medicament in dosage unit form comprising a compound i according to any one of claims 1 to 13.

17. 18. A medicament in the form of tablets, pills, dragees, capsules, ampoules, or suppositories comprising a compound according to any one of claims 1 to 13.

18. 19. A method of inhibiting glucoside hydrolases in the digestive tracts of non-human animals which comprises administering to the said animal an Inhibitory amount of a compound according to any one of olaims 1 to 13 either alone or in admixture with a diluent or in the form of a composition or medicament according to any one of claime 14 to 18.

19.

20. Human foodstuff or animal fodder comprising a compound according to any one of claims 1 to 13 and a nutritious material.