NO144850B - ANALOGY PROCEDURE FOR THE PREPARATION OF IMMUNPOTENTATING GLUCOSAMINE DERIVATIVES - Google Patents

Abstract

Analogous method of preparation. of immunopotentiating glucosamine derivatives.

Description

The object of the invention is analog method for the production of new immunopotentiating glucosamine derivatives, especially glucosamino-3-alkanoyl dipeptides

with the general formula

in which X means a carbonyl or sulfonyl group, R means an alkyl group or a phenyl group optionally substituted with a lower alkyl group and if X means the carbonyl group, R can also mean an alkoxy or benzyloxy group, R-^ means hydrogen, alkyl or a benzyl group, R2 means hydrogen or a lower alkyl-0 group, Rjj and Rg means hydrogen, alkyl, a benzyl or an alkanoyl group, R^ means hydrogen, lower alkyl or hydroxy-methyl, Rg means COOH, CONH"2, CONH-lower alkyl, COO-lower alkyl , CONH-benzyl or CONH-CH2-CONH2, and Rg means COOH, CONH2, CONHCHj or COO-lower alkyl, with the caveat that when R2 means a methyl group and X is a carbonyl group, the alkyl group R has more than one carbon atom, and if X means a carbonyl group, R2 means hydrogen or methyl, R^ methyl or phenyl and Rg and Rg each a carboxyl group, as well as their salts.

In the following, the expression "lower" contains modified residues, radicals or compounds, if nothing is specifically indicated, preferably up to 7, primarily up to 4 carbon atoms.

Alkyl is especially lower alkyl, e.g. isopropyl, straight or branched, in the desired position bound butyl, pentyl, hexyl or heptyl and above all methyl, ethyl or n-propyl.

Penyl residues can possibly e.g. be mono-, di- or polysubstituted with lower alkyl groups.

If the radical X is a carbonyl group, R can also mean an alkoxy, especially lower alkoxy, such as methoxy or ethoxy or benzyloxy acid radical. The group -NH-X-R then forms the residue of an ester of carbamic acid.

Alkanoyl, is the acyl residue of an alkanecarboxylic acid above all with 2-18 carbon atoms, such as acetyl or propionyl.

As lower alkoxycarbonyl groups, -COO-lower alkyl, it is particularly worth mentioning methoxycarbonyl or ethoxycarbonyl, but also propoxycarbonyl or isopropoxycarbonyl groups.

In the above-mentioned compounds, in which R2 means an alkyl residue, the R2-acetic acid amide residue linked to the oxygen atom in the 3-position of the glucosamine residue is optically active, i.e. it exists in the D form. If R^ does not mean hydrogen, the R^-aminoacetic acid is in the L form.

Depending on the type of their substituents, the new compounds are neutral, acidic or basic precursors.

If excess acid groups are present, they form salts with bases such as ammonium salts or salts with alkali or alkaline earth metals, eg sodium, potassium, calcium or magnesium. However, if excess basic groups are present, they form acid addition salts.

Acid addition salts are particularly pharmaceutically usable, non-toxic acid addition salts, such as those with inorganic acids, e.g. hydrochloric, hydrobromic, sulfuric or phosphoric acids, or with organic acids, such as organic carboxylic acids, such as acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methyl-maleic acid, fumaric acid, malic acid, tartaric acid, citric acid, benzoic acid, caneic acid, mandelic acid, salicylic acid, it-amino-salicylic acid, 2-phenoxybenzoic acid, 2-acetoxy-benzoic acid, embonic acid, nicotinic acid or isonicotinic acid or organic sulphonic acids, e.g. methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid or naphthalene-2-sulfonic acid, also other acid addition salts such as e.g. can be used as intermediate products, e.g. for purification of the free compounds or in the preparation of other salts as well as for characterisation, such as e.g. those with picric acid, picrolonic acid, flavian acid, phosphortungstic acid, phosphormolybdic acid, chloroplatinic acid, Reinecke acid or perchloric acid.

The compounds produced according to the invention have valuable pharmacological properties, in particular a pronounced immunopotentiating effect. This can be demonstrated with the help of the experimental device described below.

1. Potentiation of cellular immunity in vivo:

Increase in late-type sensitivity to ovalbumin in guinea pigs.

•Pirbright guinea pigs are immunized on day 0 with 10 mg of ovalbumin in complete Freund's adjuvant by injection on each 0.1 ml of an antigen-adjuvant mixture in the two hind paws. 4 weeks later, skin reactions are triggered by intracutaneous injection of 100 µg ovalbumin in 0.1 ml buffered physiological salt solution and, due to 24 hours later, with the help of erythema surfaces and skin thickness increase, calculated reaction volume quantified. The antigen-specific increase in the reaction volume observed after 24 hours (late type reaction) applies as a measure of cell-mediated immunity. Ovalbumin is too weak an immunogen, alone or in a water-oil emulsion with incomplete Freund's adjuvant (10 parts ovalbumin solution in 0.9% NaCl mixed with 8.5 parts Bayol F

and 1.5 parts Arlacel Å) and induces a late-type reaction, but for effective immunization must be applied in complete adjuvant, to which mycobacteria are added (5 mg dead and lyophilized M butyricum per 10 ml Bayol F/arlacel A). For detection of the immunopotentiating effect of the test substances, these can reach

in place of mycobacteria, doses from 10 to 100 µg are added to the antigen-oil mixture.

The glucosamine peptides produced according to the invention are capable of imitating the effect of the mycobacteria in the mentioned experimental device and of surpassing it quantitatively.

A clear potentiation of the late typing activity

against ovalbumin can also be achieved by compounds of the mentioned type not being incorporated into the antigen-oil mixture, but administered subcutaneously in doses from B to lOO^ug per animals for a few days after immunization (e.g. at days 0, 1, 2, 5, 6 and 7)

in saline solution.

Thus, it is shown that the compound of the mentioned type is able to considerably increase cellular immunity, namely both in mixture with the antigen itself (adjuvant effect in the narrower sense), as well as when administered temporally and locally from the antigen injection (systemic immunopotentiation).

2. Potentiation of humoral immunity in vivo:

Increase in antibody production against 'bovine' serum albumin' (BSA) in mice.

NMRI mice are immunized by intraperitoneal (i.p.) injection of 10 µg precipitate-free BSA on day 0. 9, 15 and 29 days later, serum samples are taken and examined for their content of anti-BSA antibodies with a passive hemagglutination technique.

In the dose used, soluble BSA is subimmunogenic *for the recipient animals, i.e. it fails to trigger or only a very small' production of antibodies. Additional treatment of the mice with certain immunopotentiating substances before or after the antigen administration leads to an increase in the antibody titer in the serum. The effect of the treatment is expressed by the score value obtained, i.e. by the sum of the log2 titer difference on the three bleeding days.

The compound according to the invention is capable of intraperitoneal or subcutaneous (s.c.) application of 100-300 mg/kg/animal on five consecutive days (day 0 to 4) after immunization with BSA to markedly increase antibody production against BSA. *.

The immunostimulating effect of said compounds is, in contrast to the other bacterial immunoleptics (e.g. LPS from E. coli) antigen dependent:-. Injection of the new compounds resulted in an increase of the anti-BSA titer only in RSA-immunized, but not in non-immunized, mice. Remarkably, the subcutaneous administration of said compounds is as effective as the intraperitoneal application, i.e. the observed immunopotentiating effect is systemic and does not depend on the stimulus having to be administered via the same route as the antigen or mixed, with the antigen as is the case with classical adjuvants.

In the aforementioned experiments, it is shown that the compounds of the mentioned type also specifically manage to increase humoral immunity, that they improve the immunological irritation reaction and that their immunopotentiating effects are due to a systemic activation of the immune system.

3- Potentiation of humoral immunity in vitro: T-cell-substituting effect by antibody response of mouse spleen cells to. schaferythrocytes (SE).

For the induction of an antibody response, thymus-derived lymphocytes (T cells) are required in many cases. These cells cooperate with the precursor antibody-forming lymphocytes (B cells) and help them to respond to stimulation with so-called T-dependent antigens with proliferation, differentiation and antibody synthesis. Spleen cell suspensions of congenitally at-hymic nu/nu mice contain no functional T cells, and are capable of e.g. in vitro in the presence of SE fail to form any anti-SE antibodies. The compounds according to the invention are surprisingly able to functionally replace T cells in such cultures and to enable an antibody response to SE. Addition of these substances to nu/nu spleen cell cultures in the presence of SE leads within 4 days to a considerable increase in the number of antibody-forming cells. The finding shows that the aforementioned compounds manage to increase the humoral antibody formation in vitro and manage to compensate a. defect of the T-cell system. 4. Selective mitogenicity for B cells: Proliferation-promoting effect in B-lymphocyte cultures.

Suspensions of highly enriched B-lymphocytes (lymph node cells of congenitally athymic-nu/nu mice) as well as highly pure immature or mature T-lymphocytes (thymus cells or cortisone-resistant, i.e. 48 hours after a cortisone injection persistent thymus cells of balb/ c mice) are incubated in the presence of the test substances for 3 days. Incorporation of H^-thymidine into the lymphocytes during the last 18 hours of the culture period applies as a measure of the proliferation activity.

The compound according to the invention is mitogenic for B-lymphocytes, (ie for the precursor of antibody-forming cells), but not for T-lymphocytes.

They are thus able to encourage the proliferation of lymphocytes that are involved in the humoral immune response.

5. Tolerability.

Although compounds of the mentioned type exert their potentiating effect on guinea pigs, for example already after a single dose of 0.05 mg/kg s.c., on mice after application of 5 times 10 mg/kg subcutaneously is also observed when application of

5 times 300 mg/kg intraperitoneally on mice no toxic

effects. The aforementioned substances therefore have an excellent therapeutic range.

The compounds according to the invention have that ability

on the one hand by mixing with an antigen to increase its immunogenicity, on the other hand by systemic application to increase the immunological reactivity of the treated organism. Thereby, the substances mentioned are capable of both promoting cellular and humoral immunity and activating the lymphocytes responsible for antibody formation.

The new compounds can thus be used as adjuvants in a mixture with grafting substances to improve the grafting result and to improve the infection protection mediated by humoral antibodies and/or cellular immunity against

bacterial, viral and parasitic agents.

Finally, the mentioned compounds are suitable in admixture with a wide variety of antigens as adjuvants in the experimental and industrial production of antisers for therapy and diagnostics and in the induction of immunologically activated lymphocyte populations for the cell transfer procedure.

Moreover, the new compounds can also be used without simultaneous antigen delivery to promote already sub-threshold immune reactions in humans and animals. The compounds are therefore particularly suitable for stimulating the body's own defences, e.g. in chronic and acute infections or in selective (antigen-specific) immunological defects, as well as in congenital but also acquired general (i.e. not antigen-specific) immunological defect conditions, as they appear in age, in the course of stronger primary diseases and above all after therapy with ionizing rays or with hormones that have an immuno-suppressive effect. The above-mentioned substances can thus preferably also be administered in combination with anti-infective antibiotics, chemotherapeutics or other healing methods to counteract immunological damage. Finally, the mentioned substances are also suitable for general prophylaxis of infectious diseases in humans and animals.

The invention relates in particular to compounds of formula I, in which X means the carbonyl radical and R a lower alkyl radical or a phenyl radical optionally substituted with lower alkyl and . Rj> ^4' ^6' R7' R8 °^ ^9 have the above meaning as well as their salts.

Particularly valuable compounds in which R 2 means hydrogen and the other residues have the above-mentioned meaning as well as their salts.

To emphasize are especially compounds with the general formula

in which R means lower alkyl or phenyl, R-j_ means hydrogen or lower alkyl, R2 means hydrogen or methyl, Ry means hydrogen, lower alkyl or hydroxymethyl, Rg means carbamoyl and Bg means carboxyl with the precaution that the lower alkyl residue R has more than 1 carbon atom if R2 means methyl, as well as their salts.

In the first place, it is necessary to mention compounds of formula II, in which R means lower alkyl or phenyl, R-j_ means hydrogen, R2 means hydrogen or methyl, Ry means hydrogen, methyl or hydroxymethyl, Rg means carbamoyl and Rg means carboxyl with the precaution that the lower alkyl radical R has more than 1 carbon atom if R 2 means methyl, as well as their salts.

The new connections can be obtained when one is on i and for

in a known manner, a compound of formula condenses

in which X, R and R2 have the above meaning and R°, <R>jj and Rg stand for the residues R-^, R^ resp. Rg or an easily removable protecting group or a derivative thereof with a compound of formula

in which R°, Rg and R° have the meaning of Ry, B-3' and R^ with the precaution that in these residues present carboxyl and, if desired, free hydroxyl groups^ are protected with easily removable protective groups and optionally remove present protective groups.

Condensation thereby takes place e.g. so that one reacts compound III in the form of the activated carboxylic acid with amino compound IV or that one reacts the acid III with compound IV, if the amino group is present in activated form. The activated carboxyl group can for example be an acid anhydride, preferably a mixed acid anhydride such as an acid azide, an acid amide, such as an imidazolide, isoxazolide or an activated ester. As an activated ester, special mention should be made of: Cyanmethyl ester, carboxymethyl ester, p-nitrophenylthioester, p-nitrophenylester, 2,H,5-trichlorophenylester, pentachlorophenylester, N-hydroxysuccinimide ester, N-hydroxyphthalimide ester, 8-hydroxy-quinoline ester, 2-hydroxy-l,2 -dihydro-1-carboethoxy-quinoline ester, N-hydroxypiperidine ester or enol ester, which is prepared with N-ethyl-5-phenyl-isoxazolium-3'-sulfonate. Activated esters can also optionally be obtained with a carbodiimide with the addition of N-hydroxysuccinimide or an unsubstituted or e.g.

with halogen, methyl or methoxy substituted 1-hydroxybenzo-

triazole, 3-hydroxy-4-oxo-3, 4-dihydro-benzo_/~d7-1, 2,3-triazine.

The amino group is, for example, activated by reaction

with a phosphitamide.

Under the methods of the reaction with activated esters

it is particularly worth mentioning this with N-ethyl-5-phenyl-isoxazolium-3'-sulfonate (Woodward Reagent K) or 2-ethoxy-1,2-dihydro-1-carboethoxy-quinoline or carbodiimide.

Easily removable protecting groups are such as'

is known from peptide resp. sugar chemistry. For carboxyl groups, tertiary butyl, benzyl or benzhydryl should be mentioned in particular and for the hydroxy groups especially acyl residues, e.g. lower alkanoyl residues such as acetyl, aroyl residues, such as benzoyl and, above all, residues derived from carboxylic acid such as benzyloxycarbonyl or lower alkoxycarbonyl, or alkyl, especially tert.butyl,

optionally with nitro, lower alkoxy or halogen substituted benzyl or tetrahydropyranyl or optionally substituted alkylidene residues, which connect the oxygen atoms in the 4- and 6-position.

Such alkylidene residues are especially a lower alkylidene-, in the first

series of ethylidene, isopropylidene or propylidene residues or also an optionally substituted, preferably in p-position sub-

substituted benzylidene residue.

These protective groups can be split off in and

known manner. Thus, they can be removed hydrogenolytically, e.g. with hydrogen in the presence of a noble metal, such as palladium

or platinum catalyst or by acid hydrolysis.

The starting materials used are known or can be prepared in a manner known per se. Thus, one can obtain prefixes with formula III, e.g. at the turnover of the

corresponding in the 3-position to unsubstituted sugars with a halogen-R2~

acetic acid, R 2 having the above meaning and their ester in the presence of a strong base. Here, halogen is preferably bromine or primarily chlorine.

Another method for the production of the new glucosamine derivatives consists of condensing, in a manner known per se, a compound of formula V in which R, R^0» I^j Rij°> Rg° °£ Ry° have the above meaning, with a compound with formula

in which Rg and RQ have the above-mentioned meaning, with the ratio fjiiik O O O

The residues Ry, Rg and R^ are present carboxyl and, if desired, free hydroxy group protected with easily removable protective groups, and optionally remove present protective groups.

Condensation thereby takes place e.g. so that

one reacts compound V in the form of the activated carboxylic acid with the amino compound VI or that one reacts the acid V with the compound VI, whose amino group is present in activated form. This activated carboxyl group can, for example, be an acid anhydride, preferably a mixed acid anhydride, an acid amide or an activated ester. As such, the above-mentioned acid anhydrides, amides or esters are particularly relevant. The amino group is, for example, activated by reaction with a phosphitamide.

The easily removable protective groups also correspond to those already mentioned above. They can be split off in a manner known per se, e.g. hydrogenolytically, for example with hydrogen in the presence of a noble metal such as palladium or platinum catalyst or by acid hydrolysis.

The starting materials can be produced in a manner known per se. Thus, one can e.g. correspondingly react unsubstituted sugars in the 3-position with a halogen-Rg-acetamido-Ry0-acetic acid or react a compound of formula III with an amino-Ry-acetic acid, whose carboxyl group is protected in the above-mentioned manner and cleave off the protecting group.

A further method for introducing the side chain placed in the 3-position of the sugar residue consists in reacting a compound

where X, R, R-|_°> R^° and Rg° have the above meaning, and optionally hydroxy groups present therein are protected with an easily cleavable protecting group with a compound of formula

in which Z means a reactive esterified hydroxy group qg Ry , Rg° and Rg° have the above-mentioned meaning, and optionally cleaves off any protective groups present.

A reactive, esterified hydroxy group is, in particular, a hydroxy group esterified with a strong inorganic or organic acid, primarily one that is esterified with hydrohalogen acids such as hydrochloric, bromic or hydroiodic acid.

The easily cleavable protecting groups correspond to those already mentioned above. They can be split off in a manner known per se, e.g. hydrogenolytically, for example with hydrogen in the presence of a noble metal such as palladium or platinum catalyst or by acid hydrolysis.

The starting materials used in these process variants are unknown.

The new compound can also be obtained when, in a compound of formula in which R, R^, R^<0>, Rg° and R^0 have the above-mentioned meaning and-R^ is an alkylidene or cycloalkylidene group, the oxazoline and dioxolane ring is split acidically and optionally cleaves off the protective groups present and introduces the X-R residue into the optionally set free amino group in the 2-position of the sugar molecule.

The alkylidene is in particular the lower alkylidene such as isopropylidene and cycloalkylidene, primarily cyclopentylidene and cyclohexylidene.

This cleavage likewise takes place in a manner known per se, e.g. with an acidic ion exchanger, especially those with sulfonic acid groups such as "Amberlite" IR-120 (a styrene resin with strongly acidic sulfonic groups) or "Dowex" 50 (polystyrene sulfonic acids) or a strong inorganic or organic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid or a sulfonic acid , e.g. methanesulfonic acid or an optionally substituted phenylsulfonic acid in the aromatic ring, such as p-toluenesulfonic acid or trifluoroacetic acid. If you work therewith in the presence of water, you get a free hydroxy group in the 1-position, but if you work in the presence of an alcohol with the formula HO-R-^, where R-^ means an optionally substituted alkyl residue, you get 1-0- The R-^-f orb compound. If one of the carboxyl groups Rg and/or R^ is also esterified with an alcohol, especially a lower alkanol, it can be saponified especially at a higher temperature with aqueous acid.

However, it is also possible that this cleavage releases the amino group in the 2-position of the sugar molecule. In this case, the X-R residue must be introduced afterwards. -This takes place in the usual way by acylation or sulphonylation.

In the compounds formed, the protective groups on the peptide residue can then be cleaved off, e.g. by hydrogenolysis,

like for example. by catalytically energized hydrogen or hydrolysis.

The starting materials used thereby can e.g.

achieve, when one introduces the Rg-acetamidopeptide residue in one or more steps in a corresponding oxazoline with a free hydroxy group in the 3-position of the sugar residue.

The compounds formed can be known in and of themselves

way is transferred in their salts e.g. by reacting formed acid compounds with alkali or alkaline earth hydroxides or formed basic compounds with acids.

The methods mentioned above are carried out

according to per se known methods in the absence or preferably in the presence of diluents or solvents, if necessary, under cooling or heating, under elevated pressure and/or in an inert gas, such as nitrogen atmosphere.

Thereby, taking into account all substituents present in the molecule, if necessary, especially in the case of

be of easily hydrolyzable O-acyl residues and use particularly gentle reaction conditions, such as short reaction times, appli-

look at mild acidic or basic agents in low concentration, stoichiometric quantity ratio, choice of suitable catalysts, solvent, temperature and/or pressure conditions.

The compounds produced by the method according to the invention can be used in the form of pharmaceutical preparations, which contain the compound of formula I. Pharmaceutical preparations according to the invention are those for enteral, such as oral or rectal, as well as parenteral administration to warm-blooded animals and which contain the pharmacological active substance alone or together with a pharmaceutically usable carrier material. The dosage of the active substance depends on the type of warm blood, the age and the individual condition as well as the method of application.

The new pharmaceutical preparations contain from approx.

10% to approx. 95 fo, preferably from approx. 20% to approx. 90% of

the active substance. Pharmaceutical preparations according to the invention can e.g. present in dosage unit forms such as dragées, tablets, capsules, suppositories or ampoules.

The pharmaceutical preparations according to the invention are prepared in a manner known per se, e.g. by means of conventional mixing, granulating, coating, dissolving or lyophilization methods. In addition to the previously mentioned forms of application, pharmaceutical preparations can also be obtained, especially for oral use, by combining the active substance with solid carriers, a resulting mixture is optionally granulated and the mixture or the granulate, if desired or necessary after the addition of suitable excipients, is processed into tablets or dragon cores.

Suitable carriers are especially fillers such as sugar, e.g. lactose, sucrose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, e.g. tricalcium phosphate or calcium hydrogen phosphate, further binders such as starch pastes when using e.g. of corn, wheat, rice or potato starches, gelatins, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone and/or, if desired, disintegrants such as the above-mentioned starches, further carboxymethylstarch, cross-linked polyvinylpyrrolidone, agar , alginic acid or a salt thereof, such as sodium alginate, auxiliaries are primarily flow control and lubricants, e.g. silicic acid, talc, stearic acid or salts thereof, such as magnesium or calcium stearate and/or polyethylene glycol. Dragon cores are equipped with suitable, possibly stomach-juice-resistant coverings, where one e.g. uses concentrated sugar solutions, which possibly contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, varnish solutions in suitable organic solvents or solvent mixtures or for the production of gastric juice-resistant coatings, solutions of suitable cellulose preparations such as acetyl cellulose phthalate or hydroxypropyl methyl cellulose phthalate. Dyes or pigments can be added to the tablets or dragee coatings, e.g. for identification or for the characterization of different active substance doses.

The invention shall be explained in more detail by means of the following examples.

Example 1

A solution of 2.6 g of benzyl-3-O-|D-1-(L-i-D<->l<->carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl]-carbamoyl-ethyl^-2-deoxy-2- Propionylamino-α-D-glucopyranoside benzyl ester in 67 ml of 60 per cent acetic acid is stirred with 0.6 g of 5 per cent palladium on charcoal as a catalyst at normal pressure and room temperature until standstill (hydration duration approx. 20 minutes). The catalyst is filtered off, washed with a little 60 per cent acetic acid and the filtrate is evaporated to dryness in a water jet vacuum. The residue is recrystallized from ethanol/ether. One thus obtains benzyl-3-O-|p_-1-[L-1(D-1-carbamoyl-3-carboxy-propyl)-carbamoylethyl)-carbamoyl-ethyl^-2-deoxy-2-propionylamino-α-D-glucopyranoside of melting point 155 -160°C during cleavage and of optical rotation (a)^° = +105° +1° (dimethylformamide, c=0.58).

The starting material used benzyl-3-O-[j3-1-(L-1(D-1-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl)-carbamoyl-ethyl]-2-deoxy-2-propionylamino-α-D-glucopyranoside -benzyl ester can be prepared as follows: 4>1 g of benzyl-2-amino-4,6-0-benzylidene-3-0-(D-1-carboxyethyl)-2-deoxy-α-D-glucopyranoside is dissolved in 200 ml of methanol and 3 ml triethylamine and mixed dropwise with 0.95 ml of propionic acid chloride while stirring at room temperature. After stirring for 2 hours, the reaction mixture is evaporated to dryness and the residue is dissolved in 100 ml of water. This solution is adjusted with ice-cold 2N hydrochloric acid to pH 4> the separated product is sucked off, washed with water and dried in a vacuum over sodium hydroxide. The thus formed benzyl-4>6-0-benzylidene-3-0-(D-1-carboxyethyl)-2-deoxy-2-propionylamino-α-D-glucopyranoside can be recrystallized from methanol. Melting point 257°C, 0)q° +I32<0> +1° (dimethylformamide, c=1.086).

A solution of ^, 1 g of the compound formed in 150 ml of acetonitrile and 0.9 ml of triethylamine is mixed with 1.5 g of N-ethyl-5-phenyl-isoxazolium-3'-sulfonate (Woodwards Reagent K) and stirred until complete solution at room temperature (approx. 60 minutes). 2.2 g of L-alanyl-D-glutamic acid-l-amide-f-benzyl ester hydrochloride and 0.9 ml of triethylamine are now added and it is allowed to stir at room temperature for a further 18 hours. After distilling off the solvent in a water jet vacuum, the residue is mixed with water, the insolubles are sucked off, washed thoroughly with water and dried. The formed benzyl-4,6-0-benzylidene-3-0-|D-1-(L-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl)-carbamoyl-ethyl^-2-deoxy-2- propionylamino-α-D-glucopyranoside benzyl ester of Rf value 0.45

on silica gel thin-layer plates in the system methylene chloride/methanol 10/1 is dissolved in 200 ml of 60 per cent acetic acid and kept for 1 l/2 hours at 95-100 C. After cooling, the solvent is distilled off. The residue is taken up twice more in a little water and evaporated each time to dryness. The formed benzyl-3.-O-|D-1-(L-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl]-carbamoyl-ethyl^-2-deoxy-2-propionylamino-α-D-glucopyranoside benzyl ester crystallize from methanol Melting point l89°C, O) p° = +100° +1°

(dimethylformamide, c=1.268).

Example 2

A solution of 4 g of benzyl-2-acetamido-3-O-| -benzyl ester in 80 ml of methanol is hydrated with 0.4 g of 5 percent palladium on charcoal as a catalyst at normal pressure and room temperature until standstill. The catalyst is filtered off, washed with a little methanol and the filtrate is evaporated to dryness in a water jet vacuum. The residue is dissolved in 50 ml of distilled water and hydrated with 1 g of 5 percent palladium on charcoal as a catalyst at normal pressure and room temperature further to standstill. The catalyst is filtered off, washed with a little water and the filtrate is evaporated to dryness. The 2-acetamido-3-O-£QL-1-(D-1-carbamoyl-3-carboxyl-propyl)-carbamoyl-ethyl)-carbamoyl-methylj-2-deoxy-D-glucose is dried in Ca)p^ = -10° +1° (Water, c 0.930).

The starting material can be prepared as follows: A solution of 9>5 g of benzyl-2-acetamido-4,6-0-benzylidene-3-0-carboxymethyl-2-deoxy-oc-D-glucopyranoside in 400

ml of acetonitrile and 3 ml of triethylamine are mixed with 5.3 g of N-ethyl-5-phenyl-isoxazolium-3'-sulfonate (Woodwards Reagent K) and stirred at room temperature until a clear solution is formed. 7.15 g of L-alanyl-D-glutamic acid-l-amide-^-benzyl ester hydrochloride, 3 ml of triethylamine and 200 ml of acetonitrile are now added and the reaction mixture is stirred for a further 18 hours at room temperature.

temperature. The crystallized benzyl-2-acetamido-4,6-0-benzylidene-3-0-\(L-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl)-carbamoyl-methylj-2-deoxy-α-D The -glucopyranoside benzyl ester is filtered off with suction, washed with half-saturated sodium bicarbonate solution and water and dried, (cx) ^ = +8l° +1° (dimethylformamide, c=0.8l6). ..A solution of 8 g of this compound in /\. 00 ml of 60 percent acetic acid is kept for 1 hour at 80°C. After cooling, the solution is evaporated to dryness, the residue is mixed twice more with 50 ml of water and evaporated each time to dryness. The formed, crystalline residue is stirred with a little water, the crystals are sucked off and dried. Benzyl-2-acetamido-3-0-[L-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl)-carbamoyl-methyl]-2-deoxy-oc-D-glucopyranoside benzyl ester of mp 200-202°C, (a) ^° = +77<0> +1° (dimethylformamide, c=0.599).

Example

A solution of 0.9 g of benzyl-2-acetamido-3-0-(L-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl)-carbamoyl-methyl-2-deoxy-6-0-stearoyl -α-D-glucopyranoside benzyl ester in 40 ml of methanol is hydrogenated with 0.2 g of 5 percent palladium on charcoal as a catalyst at normal pressure and room temperature until 22.4 ml of hydrogen is absorbed. The catalyst is filtered off, washed with methanol and the filtrate is evaporated to dryness. One thus obtains benzyl-2-acetamido-3-0-(L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoyl-ethyl}-carbamoyl-methyl-2-deoxy-6-0-stearoyl-α-D-glucopyranoside, ( a)^° = +33<0> + 1° (chloroform, 0=1.046).

The starting material can be produced as follows:

A solution of 2.8 g of benzyl-2-acetamido-3-0-(L-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl)-carbamoyl-methyl-2-deoxy-α-D-glucopyranoside -benzyl ester in 30 ml of pyridine is mixed with stirring and moisture exclusion drop by drop over the course of 1 hour with a solution of 1.4 g of stearic acid chloride in 7.5 ml of methylene chloride and stirred for 48 hours at room temperature. The reaction mixture is poured into ice water and extracted with methylene chloride. The organic phase is washed with ice-cold 2N hydrochloric acid and water, dried over magnesium sulfate and evaporated to dryness. The residue is purified by elution with ethyl acetate column chromatography on silica gel.

Benzyl-2-acetamido-3-0-(L-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl)-carbamoyl-methyl-2-deoxy-6-0-stearoyl-α-D-glucopyranoside is thus obtained -benzyl ester of C°0d° = +30° +1° (chloroform, c=1.203).

Example

Analogous to example 2, benzyl-2-acetamido-4,6-0-benzylidene-3-0-(D-1-carboxy-propyl)-2-deoxy-α-D-glucopyranoside is condensed with L-alanyl-D-glutamic acid-1 -amide-7-benzyl ester hydrochloride and cleaves off the protecting group. 2-acetamido-3-O-[D-1-(L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoyl-ethyl)-carbamoyl-propyl]-2-deoxy-D-glucose is thus obtained.

The starting material can be produced as follows:

A solution of 60 g of benzyl-2-acetamido-4,6-0-benzylidene-2-deoxy-α-D-glucopyranoside in 600 ml of dimethylformamide is mixed with 10 g of sodium hydride and stirred for 1 l/2 hours in a nitrogen atmosphere at 45°C After cooling to 0°C, 75 ml of D,L-α-bromobutyric acid ethyl ester is added. The reaction mixture is stirred each time for 1 hour at room temperature and ^>0°C, neutralized with acetic acid and the solvent is evaporated in a water jet vacuum. The residue is distributed between methylene chloride and water, the organic phase is washed once more with water, this is dried over magnesium sulphate and the solvent is evaporated. The residue dried in high vacuum is purified by column chromatography on silica gel. Elution with methylene chloride/acetic ester (85/15) gives benzyl-2-acetamido-4'6-0-benzylidene-3-0-(D-1-carboxy-propyl)-2-deoxy-α-D-glucopyranoside ethyl ester of ( o)^ = +113° +1°

(chloroform, c=0.5), melting point 154°C (from methylene chloride/ether) and Rf value 0.21 and benzyl-2-acetamido-4»6-0-benzylidene-3-0-(L-1-carboxy -propyl)-2-deoxy-α-D-glucopyranoside ethyl ester of (oc)q° = +42° +1° (chloroform, c=0.511), melting point 240°C (from acetic ester) and Rf value 0.04.

A solution of 3^»1 g of benzyl-2-acetamido-4f6-0-benzylidene-3-0-(D-1-carboxy-propyl)-2-deoxy-α-D-glucopyranoside ethyl ester in 300 ml of methanol is mixed with 100 ml of IN caustic soda and kept for 1 hour at 60°C. The solution is then cooled, evaporated to approx. 150 ml, dilute with 400 ml of ice water and mix with 100 ml of an IN ice-cold hydrochloric acid. The crystallized product is filtered off, washed with water and dried. Benzyl-2-acetamido-4,6-0-benzylidene-3-0-(D-1-carboxypropyl)-2-deoxy-a-D-glucopyranoside of melting point 210-213°C and (a)g° = +H0° is obtained +1° (dimethylformamide, c=0.554)•

In an analogous manner, benzyl-2-acetamido-4,6-0-benzylidene-3-0-(L-1-carboxy-propyl)-2-deoxy-α-D-glucopyranoside of melting point 285°C and C°0j^ = + 71° +1° (dimethylformamide, c=0.589).

Example 5

3.77 g of 2-phenyl<1->4,5-(3-0-(D-1-carboxyethyl)-5,6-0-isopropylidene-D-glucofurano)-A-oxazoline in 60 ml of acetonitrile and 15 ml of dimethylformamide it is stirred together with 1.4 ml of triethylamine and 2.55 g of N-ethyl-5-phenyl-isoxazolium-3'-sulphonate for 1 l/2 hours at 0°C, whereby almost everything dissolves. 3.44 g of L-alanyl-D-glutamic acid-1-amide-3-benzyl ester hydrochloride and a further 1.4 ml of triethylamine are then added and allowed to stir for 24 hours at room temperature. Oil is evaporated in a vacuum to syrup and chromatographed on silica gel with a mixture of chloroform and acetone (8/2). You get a colourless, solid syrup which crystallizes when extracted with ether. Melting point 96-99°C»

(a)

The crystalline benzyl ester is hydrated with 5 percent palladium charcoal in dioxane at room temperature and normal pressure and, after evaporation in vacuum, gives the corresponding acid as a syrup.

It is stirred in water with 10 ml "Dowex" 50-H+ at room temperature for 15 hours. After filtration and freeze-drying, a colorless powder with a decomposition point of 140°C is obtained. The formed 2-benzoylamino-3-0-|-D-1-(L-1-(D-1-carbamoyl-3-carboxy-propyl)-1-carbamoyl-ethyl)-carbamoyl-ethylj-2-deoxy-α,β-D-glucose contains depending on the drying conditions varying amounts of crystal water, in the above case after drying at 60°C, 0.01 Torr, at 15 hours: 1/3 water.

Example 6

6.0 g 2-phen<y>1-4,5-(3-0-(D-1-carboxypropyl)-5,6-0-isopropylidene-D-glucofurano).- A -oxazoline, 4»08 g N ^Ethyl-5-phenyl-isoxazolium-3'-sulfonate and 2.25 m^-triethylamine are stirred in 100 ml of acetonitrile and 25 ml of dimethylformamide for 1 hour at 0-5°C, whereby everything dissolves. 5.55 g of L-alanyl-D-glutamic acid-1-amide-7-benzyl ester hydrochloride and a further 2.35 ml of triethylamine are then added and stirred for 48 hours at room temperature. One evaporates under oil vacuum and chromatographs on silica gel a mixture of chloroform and ethanol (19/10.

9 g of the colorless syrup thus formed is hydrated in dioxane with 5 1° of palladium charcoal, the catalyst is filtered off, evaporated in vacuo and hydrolyzed in a mixture of 40 ml of tetrahydrofuran

and 30 ml of water with 1.5 ml of trifluoroacetic acid at room temperature. The water is evaporated in a vacuum four times to dryness, dissolved in water and lyophilized. The formed 2-benzoylamino-3-O-£-E)-1-(L-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl)-carbamoyl-propyl^-2-deoxy-α,6- D-glucose crystallizes with 0.5 mol of water, melting point 114-152°C, (a)^° = +17<0> (in methanol).

Example 7

3.63 g 2-phenyl-4,5-f3-0-carboxymethyl-5,6-0-isopropylidene-D-glucofurano)-A-oxazoline, 3>43 g L-alanyl-D-glutamic acid—1 -amide-γ-benzyl ester hydrochloride, 1.21 g of N-hydroxy-succinimide, 2" in 16 g of dicyclohexylcarbodiimide and 1.45 ml of triethylamine are dissolved in 40 ml of dimethylformamide and stirred for 24 hours at room temperature. It is evaporated in an oil vacuum, taken up with dichloroethane and water, suction off precipitated dicyclohexyl urea and shake the organic phase twice with water and the aqueous phase twice with dichloroethane. After drying and evaporation of the organic phases, a solid syrup is obtained which is chromatographed on silica gel in a mixture of chloroform/ethanol (9/1).The peptide ester formed which crystallizes on expulsion with ether, melts at 167-168°C, (a)^° = -5° (chloroform).

4.5 g of the aforementioned ester are hydrated with dioxane with 5 percent palladium charcoal, filtered off from the catalyst and this is subsequently extracted with ethanol. The combined filtrates are evaporated and the residue is recrystallized from isopropyl alcohol. The acid formed melts at 200-207°C.

2.85 g of this is stirred in a mixture of 30 ml of water and 15 ml of tetrahydrofuran with 5 ml of "Dowex" 5^-H+ for 15 hours at room temperature, filtered through a hard filter and evaporated

in vacuo to dryness. When extracted with ether, a colorless powder is obtained, 2-benzoyl-amino-3-0-£(L-1-(D-1-carbamoyl-3-carboxypropyl)-1-carbamoyl-ethyl)-carbamoyl-methyl^-2- deoxy-α-B-D-glucose

of melting point 175-177°C (as hydrate).

In modification to that in Acta Chem. Scand. l8> 1^5 (I964) mentioned method allows the starting material to be prepared as follows: 100 g of 2-phenyl-4,5(5>6-0-isopropylidene-D-glucofurano) - A p-oxazoline is dissolved under the exclusion of moisture and carbon dioxide in 1 liter of acetonitrile and add portionwise 15.2 g of a 55 percent NaH mineral oil dispersion with good stirring and continue stirring for one hour at room temperature. Then, at 0°C, 42 ml of chloroacetic acid ethyl ester are added dropwise and after 1 l/2 hours a further 42 ml. After 1 l/2 hours, 11.4 S NaH dispersion is added again, stirred for half an hour and a further 42 ml of chloroacetic ester are added dropwise at 0°C. After a further 2 hours, it is allowed to warm to room temperature and evaporated in vacuum, towards the end under an oil vacuum, to a syrup. This is taken up with ether, shaken three times with water, the ether phase is dried over sodium sulphate and, after evaporation, 155 6 of a brown oil is obtained. This is dissolved in 150 ml of methanol and mixed with a solution of JO g of potassium hydroxide in 150 ml of water, extracted twice with ether and the ether phase is washed once with water. The water phases are freed from ether in a vacuum and adjusted with 1N hydrochloric acid on a pH meter at pH = 4"

The precipitated, crystalline 2-phenyl-4,5-(3-O-(carboxymethyl)-5,6-O-isopropylidene-D-glucofurano)-A-oxazoline is suctioned off, washed with water and dried. One obtains 107 g, 93% of the theoretical, of melting point 186-188°C, = -9° (CHCl^, c=0.9) and CoOq0 = -230 (CHC13 > c=3).

Example 8

2.5 g of 2-benzamido-2-deoxy-3-0-(L-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoylethyl)-carbamoylmethyl-D-glucopyranose are dissolved in a mixture of 17 ml of absolute pyridine and 4 ml of butyric anhydride. After 20 hours at room temperature and 2 hours at 50°C, the mixture is mixed with water and evaporated in a vacuum. The residue is dissolved in chloroform, shaken with IN hydrochloric acid and water, dried over sodium sulfate and the chloroform phase is evaporated. The oily residue is extracted several times with ether and thus a solid, amorphous mass of melting point 110-120°C is obtained, namely a,B-1,4,6-tri-butyroyl-2-benzamido-2-deoxy-3~0- (L-l-(D-l-carbamoyl-3-carboxy-

propyD-carbamoylethyl)-carbamoyl-methyl-D-glucopyranose, Rp =

0.52 in CHClyCH^OH = 7:2 (silica gel thin layer, Merck).

Example 9

2.3 g of 2-benzamido-2-deoxy-3-0-(L-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoylethyl)-carbamoylmethyl-D-glucopyranose are dissolved in 20 ml of pyridine and 5 ml of acetic anhydride. After 3 hours at room temperature, it is mixed with water and evaporated in a vacuum. It is purified on 70 g of silica gel with CHCloiCH^OH = 3:1 and a colorless powder is obtained with a melting point of 122-158°C, (a)^0 = +48° (CH^OH,

c = 1.074), a,B-1,4,6-triacetyl-2-benzamido-2-deoxy-3-0-(L-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoylethyl)-carbamoylmethyl-D-glucopyranose , Rp = 0.54, CHClyCH^OH = 3:2 (silica gel thin gout, Merck).

Example 10

6.33 g 2-phenyl-4,5-(3-0-carboxymethyl-5,6-0-isopropylidene-D-glucofurano)-A-oxazoline, 5.75 g 2-ethoxy-N-carbethoxy-1, 2-dihydroquinoline (EEDQ) and 9.3 ml of triethylamine are added to a solution of the trifluoroacetate of L-alanyl-D-glutamic acid dibenzyl ester (formed from 8.3 g of N-tert-butyloxycarbonyl-L-alanyl-D -glutamic acid dibenzyl ester with 5>1 ml of trifluoroacetic acid and 2.6 ml of dichloroethane during 4 hours of hydrolysis at 40°C)

in 70 ml of dichloroethane. The mixture is allowed to react for 15 hours at 40°C, diluted with chloroform, shaken twice with water and the aqueous phases once with chloroform. After drying over sodium sulfate and evaporation of the chloroform solution, 19.9 g of an oil is obtained, which is purified over 400 g of silica gel Merck by elution with ether, then with chloroform:acetone 17:3» Thus pure 2-phenyl-4,5- [3-O-(1-L-|1-D,3-dibenzyloxy-carbonyl-propyl]-carbamoylethyl)-carbamoylmethyl-5,6-0-isopropylidene-D-glucofurano)-N-oxazoline of melting point 113-116° C and ( a)^° = -47° (CHCl^,

c = 1.54).

7 g of the above compound is hydrated with 1.8 g

5 percent Pd/C in a mixture of 80 ml of tetrahydrofuran and 20 ml of water until standstill, aspirate off the catalyst, evaporate in a vacuum and expel the residue in ether. 4.9 g of the dicarboxylic acid is thus obtained as a colorless powder.

4.4 g of the above-mentioned dicarboxylic acid is stirred

20 hours at 40°C with 11 ml of ion exchanger "Dowex" 50 W x 4 in a mixture of 45 ml of tetrahydrofuran and 20 ml of water. After filtration and clarification with charcoal ("Darco" G 60), the solution is freeze-dried and colorless, amorphous 2-benzamido-2-deoxy~3-0-U--L-(1-D»3-dicarboxy-propyl) is obtained -carbamoyl-ethyl)-carbamoylmethyl-D-glucopyranose with optical rotation (aj ^<0> = +25° (HgO, c = 0.997).

Example 11

Analogously to example 10, 5.7 g of 2-phenyl-4,5(3_0-carboxymethyl-5,6-0-isopropylidene-D-glucofurano]-A-oxazoline is condensed with 4.9 g of L-seryl-D-glutamic acid α-amide-^-tert-butyl ester hydrochloride with the addition of 2.3 ml of triethylamine and 5.2 g of 2-ethoxy-N-carbethoxy-1,2-dihydroquinoline in 45 ml of dichloroethane After 18 hours at 40° C, a crystallisate has formed. It is mixed with an additional ^0 ml of dichloroethane, cooled in ice, filtered off with suction and the crystals are washed with cold dichloroethane. The analytically pure, colorless crystals of 2-phenyl-4,5-[3_0-(1- L-[1-D-carbamoyl-3-tert.butyloxy-carbonyl-propyl]-carbamoyl-2-hydroxyethyl)-carbamoylmethyl-5,6-0-isopropylidene-D-glucofurano]-[alpha]-oxazoline has a melting point of 187-188°C and (aj^° = + 7° (CH^OH, c = 1.125). 2 g of this compound are hydrolysed at room temperature for 20 hours with a mixture of 15 ml of methylene chloride and 5 ml of trifluoroacetic acid. It is evaporated under an oil vacuum, the residue is driven off with ether and gives 2-benzamido-2-deoxy-3-0-(1-L-(1-D-carbamoyl-3-carbox γ-propyl)-carbamoyl-2-hydroxyethyl)-carbamoylmethyl-D-glucopyranose as a beige powder of melting point 100-115°C fOp<0> = +23° (H2O, c = 0.886), which crystallizes with 2 mol of water and 1 mole of trifluoroacetic acid. Rj, = 0.28 CHClyCH^OH = 1:1 (silica gel thin layer, Merck).

Example 12

Analogous to example 10, 5.25 g of 2-phenyl-4,5-(3-0-carboxymethyl-5,6-0-isopropylidene-D-glucofurano]-A p-oxazoline and the trifluoroacetic acid salt of L-alanyl-D -glutamic acid-a-n-propylamide-y-benzyl ester (formed from 6.2 g of N-tert-butyloxy-carbonyl-L-alanyl-D-glutamic acid-a-n-propylamide-^-benzyl ester and 4.2 ml of trifluoroacetic acid in 2, 5 ml of dichloroethane for 6 hours at Q. 0oC)

in 60 ml of dichloroethane with the addition of 7.75 ml of triethylamine and 4.8 g of 2-ethoxy-N-carbethoxy-1,2-dihydroquinoline. After 20 hours at 40°C, dilute with 50 ml of chloroform, shake twice with water and the water twice with chloroform. After drying and evaporation of the chloroform phase, 15 g of an oil are obtained, which is purified

over 200 g of silica gel Merck, by elution with ether, then with chloroform:acetone = 7:3. 6.4 g of colorless, amorphous substance is obtained from RF = 0.35 CHClyAceton = 7:3 (silica gel thin gout, Merck).

This is hydrated with 1.8 g of 5% Pd/C in 80 ml of tetrahydrofuran and 20 ml of water until standstill, filtered from the catalyst and evaporated. The acid shows Rp = 0.58 CHClyCH^OH = 3:1 (Silica gel thin layer, Merck). 50 ml of tetrahydrofuran and 25 ml of water are then stirred with 10 ml of ion exchanger "Dowex" 5O-W x 4» for 15 hours at room temperature and 12 hours at 40°C.

After filtration, clarification with charcoal ("Darco" G-60), repeated filtration and freeze-drying, the colorless, amorphous 2-benzamido-2-deoxy-3-0-(l-L-(l-D-N-n-propyl-carbamoyl-3- carboxy-propyl)-carbamoylethyl)-carbamoyl-methyl-D-glucopyranose of melting point 65-140°C and fa) ^° = +28<0> (water, c = 1.03),

Rp = 0.48 CHClyCH^OH = 1:1 (silica gel thin gout, Merck).

Example 13

7.3 g of 2-phenyl<1->4,5-(3-0-carboxymethyl-5,6-0-isopropylidene-D-glucofurano)-A<2->oxazoline, 6.5 g of a-amino- isobutyroyl-D-glutamic acid-α-amide-7-tert.-butyl ester hydrochloride and 2.9 g of chloroformic acid isobutyl ester are dissolved in 25 ml of dimethylformamide and 50 ml of dichloroethane. For this, you drip at -15° to -10°C

during 30 minutes a solution of 6.1 ml of triethylamine in 20 ml of dichloroethane. It is then allowed to warm to room temperature and stirred for another 15 hours at room temperature. It is diluted with 50 ml of dichloroethane, shaken with water, twice with 0.5N NaOH and three times with water, the aqueous phase twice with dichloroethane, the organic phase is dried and, after evaporation, 16.6 g of oil are obtained. This is purified on silica gel, Merck, by elution with CHClyCgHj-OH = ig:1. 9.7 g of colorless, amorphous 2-phenyl-4,5_£3-0-(1-methyl-1-(1-D-carbamoyl-3-tert-butyloxycarbonyl-propyl)-carbamoylethyl)-5,6- 0-isopropylidene-D-glucofuranoJ -A^-oxazoline, optical rotation: {a)^° = + 6° (CHCl^, c = 1.027), mp = 75-89°C, Rp = 0.35, CHCl^ C₂OH = 9:1 (silica gel thin layer, Merck).

8.3 g of the above compound is allowed to stand for 15 hours at room temperature in a mixture of 20 ml of trifluoroacetic acid,

60 ml of methylene chloride and 2 ml of water. It is then evaporated in a vacuum and the residue is expelled with ether. The resulting pink powder is dissolved in 200 ml of water and treated with 0.5 g of "Darco" G-60 charcoal. After filtration and evaporation, the colorless, amorphous 2-benzamido-2-deoxy-3-0-fl-methyl-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoylethyl)-carbamoylmethyl-D-glucopyranose of m.p. 110-120°C, fa= + 31<0> (HgO, c = 0.88), Rp = 0.52 Acetone:ethanol = 1:1 (silica gel thin layer, Merck) which crystallizes with 0.6 mol of trifluoroacetic acid and 1 ,7. moles of water.

Example 14

Analogous to example 10, 2-benzamido-2-des-oxy-3-0-carboxymethyl-8-ethyl-D-glucopyranoside and the trifluoroacetic acid salt of L-alanyl-D-glutamic acid-α-amide-^-tert.- butyl ester with 2-ethoxy-N-carbethoxy-1,2-dihydroquinoline the corresponding glycopeptide with fa)^° = -23° (CH^OH, c = 1.107) and % = 0.47 (CH2C12:C2H^0H = 8 :2) and after hydration with 5 percent Pd/C

in tetrahydrofuran/water 2-benzamido-2-deoxy-3-0-(L-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl)-carbamoylmethyl-B-ethyl-D-glucopyranoside of m.p. 215-217°C, fa)^<0> = -22° (CH^OH,

c = 0.97), RH = 0.36 in CHClyCH^OH = 1:1 (silica gel thin gout, Merck).

The 2-benzamido-2-deoxy-3-0-carboxymethyl-P-ethyl-D-glucopyranoside used as starting material is obtained as follows: 2-phenyl<1->4,5-f3-0-carboxymethyl-5^6 -O-isopropylidene-D-glucofurano)-A-oxazoline dissolves mari in 0.1N HCl/C2H^0H and leaves it for 6 hours at room temperature. Neutralize with sodium ethylate in ethanol, evaporate to dryness and take up with acetone. The solution is filtered over a layer of Merck silica gel, the eluate is evaporated to dryness and the residue is extracted twice with ether at room temperature. After recrystallization from acetic ester, 2-benzamido-2-deoxy-3-0-carbethoxymethyl-B-ethyl-D-glucopyranoside is obtained with melting point I85-I88 C and fa) ^ = -35° (CH^OH, c = 1.121) .

9.4 g of this ester is saponified with a solution of 1.7 g of potassium hydroxide in 250 ml of ethanol and 25 ml of water for 2 hours at room temperature. The pH value is then adjusted with IN hydrochloric acid to 3.5 °g, evaporated in a vacuum.

The residue is first expelled with ether, then three times with 20 ml of ice water each time and aspirated. One thus obtains crystal

clay of 2-benzamido-2-deoxy-3-0-carboxymethyl-8-ethyl-D-glucopyranoside of melting point 205-210°C and (a)j<*>° = -40° (CH^OH,

c = 1.04).

Example 1* 5

2-benzamido-2-desoxy-3-0-((D-1-carbamoyl-3-carboxy-propyl)-carbamoylmethyl-J-carbamoylmethyl-D-glucose is obtained from 3 g of 2-phenyl-4,5-(3- 0-jD-1-carbamoyl^-carboxy-propyl-carbamoylmethylj -carbamoylmethyl-5,6-0-isopropylidene-D-glucofurano)-A^-oxazoline by hydrolysis with 1.5 ml of trifluoroacetic acid in a mixture of 15 ml of dimethoxyethane and 15 ml of water at 40°C during 3 hours. Evaporate to dryness in vacuo and extract the residue again with ether. The remaining powder is dissolved in water, treated with "Darco" G-60 charcoal, filtered and freeze-dried. You get thus a colorless, amorphous substance of melting point 115-155°C and 0)q° = +34<0> (water, c = 0.8l), Rp = 0.28 CHCl^CH^OH = 1:1 (silica gel thin layer , Merck).

The starting material for this is produced as

following:

8.0 g of N-tert-butyloxycarbonyl-glycyl-D-glutamic acid-α-amide-γ-benzyl ester is dissolved in a mixture of 6.3 ml of trifluoroacetic acid and 7 ml of dichloroethane and allowed to react for 2 days at room temperature and 3 hours at 45°C 12.1 ml of triethylamine, y.0 g of 2-ethoxy-N-carbethoxy-1,2-dihydroquinoline (EEDQ) and 8.1 g of 2-phenyl-4,5-(3 -O-carboxymethyl-5,6-O-isopropylidene-D-glucofurano)-A-oxazoline and 20 ml of dimethylformamide. After 20 hours at 40°C, the mixture is evaporated in an oil vacuum and the residue is distributed between methylene chloride and water. After drying and evaporation of the methylene chloride phase, a solid residue is obtained, which is extracted twice with ether and recrystallized from toluene. Yield 8.25 g, melting point 157°C, O)q° = +10°

(CHCl^, c = 1.48), RH = 0.35 (CHCl^ethanol = 9:1) (silica gel thin layer, Merck).

The benzyl ester thus formed is hydrated with 1 g

5 percent Pd/C in 100 ml tetrahydrofuran and 25 ml more water

standstill. After filtration from the catalyst and evaporation, the substance is chromatographed on 250 g of silica gel, Merck in CHClyCH^OH = 4:1. 5.8 g of colorless, amorphous 2-phenyl-4,5-[3-0-[(D-1-carbamoyl-3-carboxy-propyl)-carbamoylmethylj-carbamoylmethyl-5,6-O-isopropylidene-D-glucofurano are obtained )-A^-oxazoline of Rp = 0.43»

CHClyCH^OH = 3 = 2 (silica gel thin gout, Merck).

Example 16

Analogously to example 10, 9.5 g of 2-phenyl-4,5-(3-0-carboxymethyl-5,6-0-isopropylidene-D-glucofurano]-A-oxazoline is condensed with 6.25 g of L-alanyl-D -glutamic acid-α,γ-diamide hydrochloride with the addition of 3>4 ml of triethylamine and 7.95 g of 2-ethoxy-N-carbethoxy-1,2-dihydro-quinoline (EEDQ) in a mixture of 50 ml of dichloroethane and 150 ml of dimethylformamide. It is allowed to react with stirring for 2 days at room temperature and 4 hours at 40° C. It is evaporated under oil vacuum <p>g and the residue is extracted first twice with ether and then twice with ice water.

After drying, the product can be recrystallized from dichloroethane. Colorless crystals with melting point 170-184°C are thus obtained, (a)^° = +3<0> (DMSO, c = 1.43) RH = 0.64 CHClyCH^OH = 3:1 (silica gel thin layer , Merck).

6.1 g of this compound is hydrolysed with 13.5 ml of ion exchanger "Dowex" 50 in a mixture of 60 ml of dimethoxyethane and 60 ml of water for 15 hours at room temperature. After filtration and evaporation, the residue is taken up with water, clarified with charcoal "Darco" G-60, filtered off with suction and freeze-dried. Colorless, amorphous 2-benzamido-2-deoxy-3-0-(L-1-(D-1,3-dicarbamoyl-propyl)-carbamoylethyl]-carbamoylmethyl-D-glucopyranose of melting point 82-143°C, fa] is obtained ^° = +24° (HgO, c = 0.98), Rp = 0.45 CHCl^: CH^OH = 1:1 (silica gel thin layer, Merck). The substance crystallizes with 1.23 mol of crystal water. •

Example 17

Analogously to example 16, from ethyl-2-benzamido-2-deoxy-3-0-carboxymethyl-β-D-glucopyranoside and L-alanyl-D-glutamic acid-α,β-bis-methylamide hydrochloride, 6-ethyl-2- benzamido-2-deoxy-3-0-(L-1-(D-1,3-bis-N-methyl-carbamoyl-propyl)-carbamoylethyl)-carbamoylmethyl-D-glucopyranoside of melting point 233-240°C (a)^° = -20° (CH₂OH, c = 0.937), Rp = 0.38 in CHCl₄ethanol = 7:3 (silica gel, thin-layer plates, Merck).

Example 18

Analogously to example 16, from 2-benzamido-2-deoxy-3-0-carboxymethyl-D-glucopyranose, 2-benzamido-2-deoxy~3-0-(L-1-(D-1,3-bis-N-methylcarbamoyl- propyl)-carbamoylethyl)-carbamoyl-methyl-D-glucopyranose of melting point 125-132°C, U)q°= +24° .-

(H 2 O, c = 0.93) Rp = 0.26 CHCl/Ethanol = 7:3 (silica gel thin layer plates, Merck).

Example 19

Analogous to example 16, from 2-benzamido-2-deoxy-3-0-carboxymethyl-D-glucopyranose and the hydrochloride of L-alanyl-D-glutamic acid dimethyl ester, 2-benzamido-2-deoxy-3-0-(L-l -(D-1,3-bis-methoxycarbonyl-propyl)-carbamoyl-ethyl)-carbamoyl-methyl-D-glucopyranose as hydrate of melting point 80-90°C, (a)^° = <+>25° (CH^OH , c = 1.017), Rp value = 0.23 1 CHCl^ethanol = 9:1 (silica gel thin layer plates, Merck).

Example 20

Analogously to example 16, from ethyl-2-benzamido-2-deoxy-3-0-carboxymethyl-B-D-glucopyranoside, ethyl-2-benzamido-2-deoxy-3-0-(L-1-(D-1,3-bis- methoxy-carbonyl-propyl)-carbamoylethyl")-carbamoylmethyl-B-D-glucopyranoside of melting point 127-135°>

(a^Q0 = 17° (CH^OH, c = 1.024), Rp = 0.26 in acetic ester:acetone = 2:1 (silica gel thin-layer plates, Merck).

Example 21

Analogous to example 12, 2-benzamido-2-deoxy-3-0-[L-1-(D-1-N-benzyl-carbamoyl-3-carboxy-propyl)-carbamoylethyl)-carbamoylmethyl-D-glucopyranose is obtained.

The -y-carboxy group of the glutamic acid is released in this case from a tert-butyl ester by saponification with "Dpwex" 5O-H

in tetrahydrofuran-water.

Example 22

Analogous to example 12, 2-benzamido-2-deoxy-3-0-fL-1-(D-1-N-carbamoylmethyl-carbamoyl-3-carboxypropyl)-carbamoylethyl)-carbamoylmethyl-D-glucopyranose is obtained. The starting material for the peptide part is N-tert.-butoxycarbonyl-L-alanyl-D-glutamic acid-K-benzyl ester-α-glycinamide.

Example 23

Analogous to example 12, 2-benz-amido-2-deoxy-3-0-fL-l-(D-l- carbamoyl-3-carboxy-propyl)-carbamoylbutyl)-carbamoylmethyl-D-glucopyranose.

Example 24

Analogously to example 12, 2-benzamido-2-deoxy-3-0-(L-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoyl-propyl)-carbamoylmethyl-D-glucopyranose is obtained.

Example 25

Analogous to example 12, 2-benzamido-2-deoxy-3-0-(L-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoyl-2-methyl-propyl)-1-carbamoylmethyl-D-glucopyranose is obtained.

Example 26

A solution of 10.7 g of benzyl-2-acetamido-4>6-0-benzylidene-3-0-((D-1-carbamoyl-3-carboxy-propyl)-carbamoyl-methyl-carbamoylmethyl)-2-deoxy-α-D -glucopyranoside benzyl ester in 200 ml of glacial acetic acid and 100 ml of water is hydrated with 2 g of 5 percent palladium on charcoal at normal pressure and room temperature for 57 hours. The catalyst is filtered off, washed with water and the filtrate evaporated. The residue is taken up in water, filtered over 100 ml of ion exchanger "Amberlite" IR 120 (H+<-> form) and the filtrate is freeze-dried. 2-acetamido-3-0-((D-1-carbamoyl-3-carboxy-propyl)-carbamoylmethyl-carbamoylmethyl)-2-deoxy-D-glucose crystallizes from methanol/acetic ester and is dried under high vacuum. The product containing 1/4 mole of acetic acid shows the optical rotation (aj^<0> =

+27° +1° (Water, c = 0.944).

The starting material used can be prepared as follows: 8 g of N-t-butyloxycarbonyl-glysyl-D-isoglutamine benzyl ester are dissolved at room temperature and under moisture exclusion in a mixture of 18 ml of 1,2-dichloroethane and 8.4 ml of trifluoroacetic acid and allowed to stand for 16 hours. The reaction mixture is diluted with 200 ml of tetrahydrofuran, neutralized with external cooling with triethylamine and mixed with a solution of 8.3 g of benzyl-2-acetamido-4>6-0-benzylidene-3-0-carboxy-methyl-2-deoxy -α-D-glucopyranoside in 100 ml of tetrahydrofuran and 2.52 ml of triethylamine. After adding 5.5 g of 2-ethoxy-N-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ), the mixture is stirred for 24 hours at room temperature. The crystallized product is filtered off with suction, washed with tetrahydrofuran and ether and dried. The formed benzyl-2-acetamido-4,6-0-benzylidene-3-0-((D-1-carbamoyl-3-carboxypropyl)-carbamoylmethyl-carbamoyl-methyl]-2-deoxy-α-D-glucopyranoside benzyl ester has the optical rotation { a)^ ° = +66° +1° (N,N-dimethylformamide, c = 1.308).

Example 27

A solution of 4.5 g of methyl-2-acetamido-3-O-|(L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoyl-ethyl]-carbamoyl-methylj-2-deoxy-α-D-glucopyranoside benzyl ester in 125 ml 5% aqueous methanol is hydrated with 1.5% palladium on charcoal at normal pressure and room temperature to absorb I78

ml of hydrogen. The catalyst is filtered off and the filtrate is evaporated in a water jet vacuum. The residue is dissolved in 50 ml of distilled water and freeze-dried. Methyl-2-acetamido-3-O-£(L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoyl-ethylj-carbamoyl-methylj-2-deoxy-ot-D-glucopyranoside is thus obtained from (a) ^ = +49° 11° (water,

c = 0.939).

The starting material can be prepared as follows:

8.1 g of N-t-butyloxycarbonyl-L-alanyl-D-isoglutamine-benzyl ester are dissolved at room temperature and under moisture exclusion in a mixture of 8.1 ml of 1,2-dichloroethane and 8.1

ml of trifluoroacetic acid and leave for 16 hours. The reaction mixture is diluted with 200 ml of tetrahydrofuran, neutralized with external cooling with triethylamine and mixed with a solution of 7.62 g of methyl-2-acetamido-4,6-0-benzylidene-3-0-carboxymethyl-2- deoxy-α-D-glucopyranoside and 2.77 ml of triethylamine in 100

ml of tetrahydrofuran. After adding 5.0 g of 2-ethoxy-N-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ), the reaction mixture is heated to 40°C, stirred at this temperature for 30 hours and allowed to stand at room temperature for a further 24 hours. The precipitated product, methyl-2-acetamido-4,6-0-benzylidene-3-0-|(L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoyl-ethyl]-carbamoyl-methyl}-2- deoxy-α-D-glucopyranoside benzyl ester is filtered off with suction, washed with tetrahydrofuran and ether and dried, (α]p° = +58<0> +1° (N,N-dimethylformamide, c = 1.125).

A solution of 10.5 g of methyl-2-acetamido-4,6-0-benzylidene-3-0-£(L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoylethyl)-carbamoyl-methylj-2-deoxy- α-D-glucopyranoside benzyl ester in 320 ml of glacial acetic acid is diluted with stirring with 200 ml of water and the whole is stirred for 2 hours at 50-55°C. After cooling, the solution is evaporated to dryness, the residue is mixed four more times with 100 ml of water and each time evaporated to dryness. One thus obtains methyl-2-acetamido-3-O-£(L-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl)-carbamoyl-methylj-2-deoxy-α-D-glucopyranoside benzyl ester of [a)2° = +64° +1° (N,N-dimethylformamide, c = 1.268).

Example 28

A solution of 3% g of methyl-2-acetamido-3-0-|(L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoyl-ethyl)-carbamo<y>1-methylj-2-deoxy-6- O-stearoyl-α-D-glucopyranoside benzyl ester in 100 ml of ethanol and 100 ml of tetrahydrofuran is stirred with 0.6 g of 5 percent palladium on charcoal at room temperature and normal pressure, filtered off from the catalyst and evaporated to dryness in a water jet vacuum. The crystalline residue darkens to methyl-2-acetamido-3-0-£(L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoyl-ethylj-carbamoyl-methylJ-2-deoxy-6-0-stearoyl-α-D-glucopyranoside of taJ^ = +50° +1° (N,N-dimethylformamide,

c = 0.921).

The starting material is produced as follows:

A solution of 3>9^ g of methyl-2-acetamido-3-O-£(L-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl)-carbamoyl-methylj-2-deoxy-α-D-glucopyranoside -benzyl ester in 40 ml of absolute pyridine is mixed with stirring and exclusion of moisture at 0-5°C dropwise over 3 hours with a solution of 2.12 g of stearic acid chloride in 20 ml of 1,2-dichloroethane and allowed to stand for 18 hours at room temperature. The reaction mixture is diluted with chloroform, washed with water, ice-cold 2N hydrochloric acid and water, dried over magnesium sulfate and evaporated to dryness. The product, methyl-2-acetamido-3-0-|(L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoyl-ethyl)-carbamoyl-methyl)-2-deoxy-6-0-stearoyl-α-D- glucopyranoside benzyl ester is crystallized from ethanol/ether, [α)^ = +22° +1° (chloroform, c = 1.030).

Example 29

A solution of 6.8 g of benzyl-2-acetamido-3-O-£(L-1-(D-1,3-dicarbamoylpropyl)-carbamoylethyl)-carbamoylmethylj-2-deoxy-α-D-glucopyranoside in 200 ml of 50 percent aqueous methanol is hydrated with 5 percent palladium on charcoal at normal pressure and room temperature for 60 hours. The catalyst is filtered off and the filtrate is evaporated. The residue is taken up in 50 ml of water and lyophilized. One thus obtains 2-acetamido-3-O-£(L-1-(D-1,3-dicarbamoylpropyl)-carbamoylethyl)-carbamoylmethylj -2-deoxy-D-glucose which contains 1.24 mol of water, as white powder of (a) p^ = +7° +1° (water, c <= >0.514).

The starting material can be prepared as follows:

A solution of 9>1 g of benzyl-2-acetamido-4,6-0-benzylidene-3-0-carboxymethyl-2-deoxy-α-D-glucopyranoside in 100

ml of N,N-dimethylformamide and 2.77 ml of triethylamine are mixed with 5.0 g of L-alanyl-D-glutamic acid diamide hydrochloride and 5.1 g of 2-ethoxy-N-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) and set aside 48 hours at room temperature. After distilling off the solvent, the residue is extracted thoroughly with ether and water, dried and recrystallized from chloroform/methanol, C°0<d>° = +83<0> +1° (N,N-dimethylformamide, c = 0.531)•

A solution of 4 g of benzyl-2-acetamido-4,6-0-benzylidene-3-0-£(L-1-(D-1,3-dicarbamoylpropyl)-carbamoylethyl]-carbamoylmethylj-2-deoxy-α-D-glucopyranoside in 120 ml glacial acetic acid is diluted with 80 ml of water and stirred for 3 hours at 60° C. The reaction mixture is then cooled, evaporated, the residue is mixed three more times with 100 ml of water and this is distilled off each time.

The formed benzyl-2-acetamido-3-0-£[L-1-(D-1,3-dicarbamoyl-propyl)-carbamoylethyl]-carbamoyl-methylj -2-deoxy-α-D-glucopyranoside is recrystallized from methanol, melting point 223-225°C .

Example 30

A solution of 5.7 g of benzyl-2-acetamido-3-O-β-1-((D-1-carbamoyl-3-carboxypropyl)-carbamoylarethyl]-carbamoyl-propylj-2-deoxy-α-D-glucopyranoside benzyl ester in 100 ml of glacial acetic acid is hydrated in the presence of 5 percent palladium on charcoal at normal pressure and room temperature. The catalyst is filtered off and the filtrate is evaporated. The remaining 2-acetamido-3-0-£p_-l-((D-l-carbamoyl-3-carboxy- propyl)-carbamoylmethyl)-carbamoyl-propylj-2-deoxy-D-glucose is taken up in 50 ml of water and freeze-dried, [a]^° = +46<0> +1° (water, c = 0.630).

The starting material used can be prepared as follows: 5.1 g of N-t-butyloxycarbonyl-glysyl-D-isoglutamine benzyl ester is dissolved at room temperature and under moisture exclusion in a mixture of 5.1 ml of 1,2-dichloroethane and 5.1 ml of trifluoroacetic acid and allowed to stand for 16 hours . This solution is diluted with 100 ml of tetrahydrofuran, hypertralized under external cooling with triethylamine and a solution of 6.3 g of benzyl-2-acetamido-4,6-0-benzylidene-3~0-(D-1-carboxypropyl)-2- deoxy-α-D-glucopyranoside and 1.8 ml triethylamine in 100 ml tetrahydrofuran and 3.2 g 2-ethoxy-N-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ). After 24 hours, evaporate to dryness and distribute the residue between vinegar and water. The organic phase is washed with ice-cold 2N hydrochloric acid, water, a saturated sodium bicarbonate solution and water and evaporated. The remaining benzyl-2-acetamido-4,6-0-benzylidene-3-0-£D-1-((D-1-carbamoyl-3-carboxypropyl)-carbamoylmethyl)-carbamoylpropylj-2-deoxy-α-D-glucopyranoside benzyl ester is crystallized from tetrahydrofuran/ether, (a)<**>0 = +760 +1° (N,N-dimethylformamide, c = 0.457).

A mild acid hydrolysis of this product in 60 per cent aqueous acetic acid leads to benzyl-2-acetamido-3-0-£p_-1-((D-1-carbamoyl-3-carboxypropyl)-carbamoylmethyl)-carbamoyl-propylj -2-deoxy -α-D-glucopyranoside benzyl ester which crystallizes from methanol/ether, melting point 180-185°C, (.oc]^<0> = +87<0>

+1° (methanol, c = 1.035).

Ex empe l 31

4 g of N-t-butyloxycarbonyl-L-alanyl-D-isoglutamine benzyl ester are dissolved at room temperature and under exclusion of moisture in a mixture of 4 ml of trifluoroacetic acid and 4 ml of 1,2-dichloroethane and allowed to stand for 16 hours. This reaction mixture is now diluted with 10 ml of 1,2-dichloroethane, ultracentrized under external cooling with triethylamine and mixed with a solution of 3.7 g of benzyl-2-acetamido-3-0-carboxy-methyl-2-deoxy-α-D-glucopyranoside and 1.38 ml of triethylamine in 100 ml of tetrahydrofuran. After adding 2.6 g of 2-ethoxy-N-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ), it is allowed to stand for 24 hours at room temperature and evaporated to dryness. The residue is dissolved in chloroform/methanol 9/1, and this solution is washed with water, ice-cold 2N hydrochloric acid, water, a saturated sodium bicarbonate solution and water and the solvent is evaporated. The thus formed benzyl-2-acetamido-3-O-£(L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoyl-ethyl l -carbamoyl-methyl l -2-deoxy-α-D-glucopyranoside benzyl ester is recrystallized from ethanol, mp 208-212°C, (a)^° = +77° +1° (N,N-dimethylformamide, c = 0.546).

After the hydrogenolytic cleavage of the two benzyl residues as described in example 2, 2-acetamido~3-O-^(.L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoylethyl)-carbamoyl-methyl]-2-deoxy is obtained -D-glucose.

Example 32

A solution of J, 8 g of benzyl-2-acetamido-3-0-|(L-1-(D-1,3-dicarboxypropyl)-carbamoylethyl]-carbamoylmethylj - 2-deoxy-α-D-glucopyranoside dimethyl ester in 100 ml of methanol is hydrated in the presence of 5 percent palladium on charcoal at normal pressure and room temperature. After complete water hydrogen absorption, the catalyst is filtered off and the filtrate is evaporated to dryness. The residue is taken up in 70 ml of water and freeze-dried. The foam formed is 2-acetamido-3-0-L-l-(D-l, 3-dicarboxypropyl)-carbamoylethyl)-carbamoylmethylj - 2-deoxy-D-glucose-dimethyl ester with l°0^ = +23° +1° (water,

c = 0.814).

The starting material used can be prepared as follows: A solution of 12.9 g of benzyl-2-acetamido-4,6-0-benzylidene-3-0-carboxymethyl-2-deoxy-α-D-glucopyranoside and 4.0 ml of triethylamine in 100 ml of N ,N-dimethylformamide is mixed with 8.1 g of L-alanyl-D-glutamic acid dimethyl ester hydrochloride and 6.95 g of 2-ethoxy-N-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) and allowed to stand for 20 hours at room temperature. The solvent is then evaporated, the residue is taken up in chloroform and this solution is washed with water, ice-cold 2N hydrochloric acid, water, a saturated sodium hydrogen carbonate solution and water. After drying over magnesium sulphate, it is evaporated to dryness. The residue is extracted with hot ethanol, undissolved benzyl-2-acetamido-4,6-0-benzylidene-3-0-|(L-1-(D-1,3-dicarboxypropyl)-carbamoylethyl)-carbamoylmethyl)-2-deoxy-oc-D -glucopyranoside dimethyl ester is filtered off and dried, (a)2n 0 =

+22° +1° (chloroform, c = 1.160).

A solution of 15.6 g of benzyl-2-acetamido-4,6-0-benzylidene-3-0-^(L-1-(D-1,3-dicarboxypropyl)-carbamoylethyl J-carbamoylmethylj-2-deoxy-α-D-glucopyranoside dimethyl ester in 420 ml of glacial acetic acid and 280 ml of water are heated to 10° C. After 4 hours of stirring at this temperature, the reaction mixture is cooled and evaporated to dryness. The residue is mixed three more times with 100 ml of water and each time evaporated to dryness. The residue is also taken up in chloroform, this solution is washed with water, dried over magnesium sulfate and evaporated to dryness. This gives benzyl-2-acetamido-3-0-|(L-1-(D-1,3-dicarboxypropyl)-carbamoylethyl)-carbamoylmethylj -2-deoxy- α-D-glucopyranoside dimethyl ester as yellowish resin of l^x]^<0> = +31<0> +1° (chloroform, c = 1.070).

Example 33

A solution of 6.1 g of benzyl-3-O-|(L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoylethyl-carbamoylmethyl-2-deoxy-2-propionamido-α-D-glucopyranoside benzyl ester in 200 ml of tetrahydrofuran/water 2/l is hydrated in the presence of 0.6 g of 5 per cent palladium on charcoal at normal pressure and room temperature. After completion of hydrogen absorption, the catalyst is sucked off and the filtrate is evaporated. The residue is mixed with 150 ml of water and hydrated for a long time in the presence of 5 per cent palladium on charcoal until no more hydrogen is taken up. The catalyst is filtered off and lyophilized. Thus, 3_0-£(1:-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoylethyl).-carbamoylmethylj -2-deoxy-2-propionamido-D- glucose.

The starting material used can be prepared as follows: A solution of 9° g of benzyl-2-acetamido-2-deoxy- α-D-glucopyranoside in 900 ml of N,N-dimethylformamide is mixed with 0.3 ml of methanesulfonic acid while stirring, exclusion of moisture and ice water cooling. A solution of 60 ml of isopropenyl methyl ether in 240 ml of N,N-dimethylformamide is then added dropwise over the course of one hour, stirred for two hours at room temperature and made alkaline with triethylamine. The solvent is distilled off, the residue is taken up in acetic acid, this solution is washed with water, dried over magnesium sulphate and evaporated to dryness. The product, benzyl-2-acetamido-2-deoxy-4 > 6-0-isopropylidene-α-D-glucopyranoside is crystallized from ether, m.p. 136-137°C, (°0 o° =<+>103° +1° (chloro6form , c = 1.125).

52.5 g of benzyl-2-acetamido-2-deoxy-4>6-0-isopropylidene-α-D-glucopyranoside are dissolved in a solution of 225 g of potassium hydroxide in 75 ml of ethanol and 40 ml of distilled water, boiled for 4 l/2 hours during reflux. After cooling, the reaction mixture is evaporated to half and poured onto ice. One extracts with chloroform, washes the organic phase with water, dries and magnesium sulfate and evaporates to dryness. The residue, benzyl-2-amino-2-deoxy-4»6-0-isopropylidene-α-D-glucopyranoside is crystallized from ether, melting point 145-146°C, (a)^<0> = +117° +1°

(chloroform, c = 1.295).

A solution of 24>7 g of benzyl-2-amino-2-deoxy-4,6-O-isopropylidene-α-D-glucopyranoside in 192 ml of chloroform is mixed with a solution of 16.0 g of potassium bicarbonate in 192 ml of distilled water and cooled to 0°C. Now, while stirring, 8.16 g of propionic acid chloride are added dropwise over the course of 20 minutes and the mixture is stirred again at this temperature for a further 50 minutes. The organic phase is now separated, washed with water, dried over magnesium sulphate and evaporated to dryness. The product, benzyl-2-deoxy-4,6-0-isopropylidene-2-propionamido-α-D-glucopyranoside is crystallized from acetic ester/petroleum ether, mp 121-122°C, ( a)^ ° = +112° 11° (chloroform, , c = 0.977).

A solution of 9>1 g of benzyl-2-deoxy-4,6-0-isopropylidene-2-propionamido-α-D-glucopyranoside in $0 ml of acetonitrile is mixed with 1.25 g of sodium hydride. (Fluka) and stirred for 2 hours at /\. 0°G. It is then cooled to -5 to -10°C and mixed with 4.2 ml of bromoacetic acid. After a further 20 minutes, 10 ml of ethanol are added, the reaction mixture is neutralized with glacial acetic acid and evaporated to dryness. The residue is distributed between ether and water, the ether solution is washed with water, dried over magnesium sulphate. and evaporated. The product, benzyl-3-O-carboxymethyl-2-deoxy-4,6-0-isopropylidene-2-propionamino-α-D-glucopyranoside ethyl ester is crystallized from ether/petroleum ether, melting point 94-95°C> OJ^° =<+ >145 +1° (chloroform, c = 1.218).

A solution of 6.8 g of benzyl-3-0-carboxymethyl-2-deoxy-4,6-0-isopropylidene-2-propionamido-α-D-glucopyranoside ethyl ester in JO ml of methanol is mixed with 22.5 ml of 1N caustic soda. After completion of ester hydrolysis, 7.5 ml of 1N hydrochloric acid is added and evaporated to dryness. The product is dissolved in 50 ml of N,N-dimethylformamide and condensed with 15 mmol of L-alanine-D-isoglutamine benzyl ester trifluoroacetate in the presence of 3>72 g of 2-ethoxy-N-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ). It is then evaporated to dryness and the residue taken up in chloroform. This solution is washed with water, ice-cold 2N hydrochloric acid, water, a saturated sodium bicarbonate solution and water, dried over magnesium sulfate and evaporated to dryness. The product is crystallized from dilute ethanol, melting point 177-180°C, (a)^° = +710 +1° (chloroform, c = 1.047).

A solution of 8.1 g of benzyl-3-O-£(L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoylethyl]-carbamoylmethylj-2-deoxy-4,6-0-isopropylidene-2-propionamido-α-D- glucopyranoside benzyl ester in 150 ml of methanol is mixed with 15 ml of 1N hydrochloric acid and allowed to stand for one hour at room temperature. 15 ml of 1N caustic soda is now added and evaporated to dryness. The formed benzyl-3-0-|(L-1-(D-1 -carbamoyl-3-carboxypropyl)-carbamoyl-ethyl]-carbamoylmethylj-2-deoxy-2-propionamido-α-D-glucopyranoside benzyl ester is crystallized from methanol/water and dried, melting point 208-210°C,

-[cOp° = +75° +1° (N,N-dimethylformamide, c = 1.120).

Example 34-

By catalytic hydrogenation of benzyl-3-O-£(L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoylethyl)-carbamoylmethylj-2-caprinoylamido-2-deoxy-α-D-glucopyranoside benzyl ester in N,N -dimethylformamide in the presence of 5 percent palladium on charcoal gives J-0-^(L1-(D-1-carbamoyl-3-carboxypropyl)-carbamoylethyl)-carbamoylmethylj -2-caprinoylamido-2-deoxy-D-glucose as a white foam.

The starting material used can be prepared as follows: Analogous to Example 35, 24.76 benzyl-2-amino-2-deoxy-4.6-0-isopropylidene-α-D-glucopyranoside is added with 16.7 g of caprinoyl chloride and worked up. Benzyl-2-caprinoyl-amido-2-deoxy-4»6-0-isopropylidene-α-D-glucopyranoside is obtained by [a]<2>° = +81° +1° (chloroform, c = 1.019).

A solution of 3>8 g of benzyl-2-caprinoylamido-2-deoxy-4»6-0-isopropylidene-α-D-glucopyranoside in 40 ml of acetonitrile is mixed with 0.4 g of sodium hydride, and stirred for 2 hours at °. OC. This mixture is then cooled to -10°C, 1.4 ml of bromoacetic acid is added and it is stirred for a further hour at 0°C. Worked up as indicated in example 33, benzyl-3-0-carboxymethyl-2-caprinoylamido-4,6-0-isopropylidene-2-deoxy-α-D-glucopyranoside ethyl ester is obtained as yellow-brown oil of (a)^ = +114° + 1° (chloroform, c = 1.242).

Analogous to example 33, the ethyl ester is hydrolysed, the product is condensed with the L-alanine-D-isoglutamine benzyl ester trifluoroacetate in the presence of EEDQ and the isopropylidene ketal is cleaved by mild acid hydrolysis. Thus, benzyl-3-O-^(L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoylethyl]-carbamoylmethyl-2-caprinoyl-amido-2-deoxy-α-D-glucopyranoside benzyl ester is obtained as a yellowish resin.

Example 35

A 5% solution of benzyl-2-acetamido-2-deoxy-3-O-j(L-1-(D-1, -bis-methylcarbamoylpropyl)-carbamoylethyl]-carbamoylmethylj-α-D-glucopyranoside in distilled water/methanol l/l is hydrated with 5 percent palladium on charcoal, the catalyst is filtered off and the filtrate is evaporated. The product, 2-acetamido-2-deoxy-3-0- \CL.-1-(D-1,3-bis-methyl-carbamoylpropyl)-carbamoylethyl) -carbamoylmethylj-D-glucose is freeze-dried.

The starting material can be prepared as follows:

A solution of 9>1 g of benzyl-2-acetam£do-4,6-0-benzylidene-3-0-carboxymethyl-2-deoxy-α-D-glucopyranoside in

100 ml of N,N-dimethylformamide and 2.77 ml of triethylamine are mixed with 5.6 g of L-alanyl-D-glutamic acid bis-methylamide hydrochloride and 5.1 g of 2-ethoxy-N-ethoxycarbonyl-1,2-dihydroquinoline and leave for 48 hours at room temperature. The solvent is distilled off, the oily residue is mixed with water, the insoluble matter is filtered off, washed with water and dried. This product is stirred twice more with ether, filtered off and dried and is benzyl-2-acetamido-4,6-0-benzylidene-2-deoxy-3-0-|CL-1-(D-1,3-bis-methylcarbamoylpropyl) -carbamoylethyl)-carbamoylmethylj-α-D-glucopyranoside.

With mild acid hydrolysis as indicated in Example 29

with 60 percent acetic acid, the benzylidene group is split off and benzyl-2-acetamido-2-deoxy-3-0-|(L-1-(D-1,3-bis-methyl-carbamoylpropyl)-carbamoylethyl)-carbamoylmethylj -a-D-glucopyranoside is isolated .

Example 36

A 5 percent aqueous solution of benzyl-2-acetamido-3-0-£[L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoyl-2-hydroxyethyl)-carbamoylmethylj-2-deoxy-α-D-glucopyranoside is hydrated in presence of 5 percent palladium on charcoal, filtered and freeze-dried. 2-acetamido-3-O-[(L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoyl-2-hydroxyethyl)-carbamoylmethyl]-2-deoxy-D-glucose is thus obtained.

The starting material can be prepared as follows:

A solution of 3.7 g of benzyl-2-acetamido-3-0-carboxymethyl-2-deoxy-α-D-glucopyranoside and 1.38 ml of triethylamine in 150 ml of tetrahydrofuran is mixed with 3.26 g of L-serine-D- isoglutamine tert-butyl ester hydrochloride and 2.6 g of 2-ethoxy-N-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) and allowed to stand for 20 hours at room temperature. After evaporation of the solvent, the residue is dissolved in chloroform/methanol S/l, washed with water, ice-cold 2N hydrochloric acid, water, a saturated sodium bicarbonate solution and water, filtered and freed from solvent. Benzyl-2-acetamido-3-O-[L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoyl-2-hydroxy-ethyl)-carbamoylmethyl]-2-deoxy-α-D-glucopyranoside tert-butyl ester is thus obtained .

By mild acid hydrolysis as stated in example 11, the tert-butyl ester is hydrolysed and benzyl-2-acet-amido-3-0-[fL-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoyl-2-hydroxyethyl is obtained ]-carbamoylmethylj -2-deoxy-α-D-glucopyranoside.

Example 37

A 5 percent solution of benzyl-2-acetamido-3-0-£(L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoylbutylj-carbamoylmethylj-2-deoxy-α-D-glucopyranoside in methanol/water l/l is hydrated in the presence of 5 percent palladium on charcoal, filtered and evaporated The residue is dissolved in distilled water and freeze-dried.

One thus obtains 2-acetamido-3-O-£(L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoylbutylj-carbamoylmethylj-2-deoxy-D-glucose.

The starting material can be prepared as follows:

A solution of 3>7 g of benzyl-2-acetamido-3-O-carboxymethyl-2-deoxy-α-D-glucopyranoside and 1.38 ml of triethylamine in 100 ml of tetrahydrofuran is mixed with 3.28 g of L-Norvaline-D- isoglutamine tert-butyl ester hydrochloride and 2.6 g of 2-ethoxy-N-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) and allowed to stand for 24 hours at room temperature. The reaction mixture is worked up as in example j8. Benzyl-2-acetamido-3-0-|(.L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoyl-butyl)-carbamoylmethyl-2-deoxy-α-D-glucopyranoside tert-butyl ester is obtained.

The tert-butyl ester is cleaved by mild acid hydrolysis.

Example 38

A solution of 8.3 g of benzyl-3-O-[(L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoylethyl)-carbamoylmethyl]-2-deoxy-2-stearoylamido-α-D-glucopyranoside benzyl ester in 200 ml of glacial acetic acid is hydrated in the presence of 5 percent palladium on charcoal at normal pressure and room temperature. After work-up, J-O-£[L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoylethyl)-carbamoylmethyl]-2-deoxy-2-stearoylamido-D-glucose is obtained as a white powder.

The starting material used can be prepared as follows: A solution of 6.2 g of benzyl-4,6-0-benzylidene-2-deoxy-2-stearoylamido-α-D-glucopyranoside in 150 ml of N,N-dimethylformamide is mixed with 0.75 g sodium hydride, and stir for 1 l/2 hours at 40°C. The mixture is now cooled to -5°C, mixed with 2.2 ml of bromoacetic acid and stirred for 4 hours at room temperature. After adding 10 ml of glacial acetic acid and 400 ml of distilled water, extract with chloroform, the organic phase is washed with water, dried and evaporated to dryness. The benzyl-4,6-0-benzylidene-3-0-carboxymethyl-2-deoxy-2-stearoylamido-α-D-glucopyranoside ethyl ester formed is crystallized from ethanol, melting point 151-154°C, 0)<2>° <= ><+>04<0> +1° (chloroform, c = 1,186).

A solution of 23.4 g of benzyl-4,6-0-benzylidene-3-0-carboxymethyl-2-deoxy-2-stearoylamido-α-D-glucopyranoside ethyl ester in 334 ml of methanol and 334 ml of tetrahydrofuran is mixed with 50.1 ml IN caustic soda, stir $ 0 minutes at ^ 0°C and evaporate. The residue is stirred in ice water, the product is filtered off with suction, washed with water, dried and recrystallized from ethanol. Benzyl-4,6-0-benzylidene-3-0-carboxymethyl-2-deoxy-2-stearoylamido-α-D-glucopyranoside sodium salt.lHgO melts at 225-242°C (under decomposition),

(aj^<0> = +45° +1° (chloroform, c = 1.097).

A solution of 14.4 g (20 mmol) of benzyl-4,6-0-benzylidene-3-0-carboxymethyl-2-deoxy-2-stearoylamido-α-D-glucopyranoside sodium salt monohydrate in 120 ml of tetrahydrofuran is mixed with 20 mmol L-alanine-D-isoglutamine benzyl ester trifluoroacetate and 4.95 g (20 mmol) of 2-ethoxy-N-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) are left for 20 hours at room temperature and evaporated to dryness. The residue is dissolved in chloroform, this solution is washed with water, ice-cold 2N hydrochloric acid, water, a saturated sodium bicarbonate solution and water, dried over magnesium sulfate and evaporated. The product, benzyl-4,6-0-benzylidene-3~0-|(L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoylethylj-carbamoyl-methylj -2-deoxy-2-stearoylamido-α-D-glucopyranoside benzyl ester is recrystallized from ethanol, [αj^ = +62° +1° (N,N-dimethylformamide, c = 1.148).

Mild acid hydrolysis of this benzylidene deriv. i

65 percent acetic acid leads to benzyl-3-O-£[L-l-(D-l-carbamoyl-3-carboxypropyl)-carbamoylethylj-carbamoylmethylj-2-deoxy-2-

o-

stearoylamido-α-D-glucopyranoside benzyl ester, mp 188-189°C, fj^<0> = +59° j;10 (N,N-dimethylformamide, c = 1.022).

Example 39

A 5 percent aqueous solution of benzyl-2-acetamido-3-0-£(L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoyl-2-methylpropyl)-carbamoylmethylj -2-deoxy-α-D-glucopyranoside is hydrated in the presence of 5 percent palladium on charcoal, filtered and the filtrate lyophilized. 2-acetamido-3-0-|(L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoyl-2-methylpropyl)-carbamoylmethylj-2-deoxy-D-glucose is thus obtained.

The starting material is produced as follows:

A solution of 3>7 g of benzyl-2-acetamido-3-O-carboxymethyl-2-deoxy-α-D-glucopyranoside and 1.38 ml of triethylamine in 100 ml of tetrahydrofuran is mixed with 3.38 g of L-Valine-D- isoglutamine tert-butyl ester hydrochloride and 2.6 g of 2-ethoxy-N-ethoxycarbonyl-1,2-dihydroquinoline, are allowed to stand for 24 hours at room temperature and worked up as indicated in example 3. Thus, benzyl-2-acetamido- 3-0-|(L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoyl-2-methylpropyl)-carbamoylmethylj-2-deoxy-α-D-glucopyranoside tert-butyl ester which is mildly acid hydrolyzed to benzyl-2-acetamido- 3-O-£(L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoyl-2-methylpropyl)-carbamoylmethylj-2-deoxy-α-D-glucopyranoside. ,

Example 40

A 5 percent solution of benzyl-2-acetamido-3-0-|(L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoyl-^-methyl-butylj-carbamoylmethylj-2-deoxy-α-D-glucopyranoside in 50 percent aqueous methanol is hydrated in the presence of 5 percent palladium on

charcoal, filtered and evaporated. The residue is dissolved in distilled water, filtered again and freeze-dried. 2-acetamido-3-O-[(L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoyl-3-methyl-butyl}-carbamoylmethyl]-2-deoxy-D-glucose is thus obtained.

The starting material used can be prepared as follows: A solution of 3>7 g of benzyl-2-acetamido-3-0-carboxymethyl-2-deoxy-α-D-glucopyranoside and 1.58 ml of triethylamine in 100 ml of tetrahydrofuran is mixed with 3> 52 g of L-Leucine-D-isoglutamine-tert-butyl ester hydrochloride and 2.6 g of 2-ethoxy-N-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) are left for 24 hours at room temperature and worked up as indicated in example 3^ * 'One obtains benzyl-2-acetamido-3-0-|(L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoyl-3-methylbutylJ-carbamoylmethylj-2-deoxy-α-D-glucopyranoside-tert-butyl ester , whose mild acid hydrolysis leads to benzyl-2-acetamido-3-0-£(L-l-(D-l-carbamoyl-3-carboxypropyl)-carbamoyl-3-methylbutyl)-carbamoylmethylj-2-deoxy-α-D-glucopyranoside.

Example 41

A 5 percent solution of benzyl-2-acetamido-3-O-£XL-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoylpropyl)-carbamoylmethylj-2-deoxy-α-D-glucopyranoside in 50 percent aqueous methanol is hydrated in the presence of 5 percent palladium on charcoal at normal pressure and room temperature, the catalyst is filtered off and it is evaporated. The residue is dissolved in distilled water and freeze-dried. 2-acetamido-3-O-[(L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoylpropyl)-carbamoylmethyl]-2-deoxy-D-glucose is thus obtained.

The starting material can be prepared as follows:

A solution of 3.7 g of benzyl-2-acetamido-3-0-carboxymethyl-2-deoxy-a-D-glucopyranoside and 1.38 ml of triethylamine in 100 ml of tetrahydrofuran is mixed with 3>24 g of N-a-L-aminobutyryl-D-isoglutamine- tert.•-butyl ester hydrochloride and 2.6 g of 2-ethoxy-N-ethoxycarbonyl-1,2-dihydroquinoline and worked up after 24 hours at room temperature as described in example jS. The formed benzyl-2-acetamido-3-0-|(L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoylpropylJ-carbamoylmethylj-2-deoxy-α-D-glucopyranoside-tert-butyl ester is then mildly acid hydrolyzed to benzyl-2 -acetamido-3-O-|(,L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoyl-propyl)-carbamoylmethylj -2-deoxy-α-D-glucopyranoside.

Example 42

A 5 percent solution of benzyl-2-acetamido~3-0-£[L-l-(D-l-carbamoyl-3-carboxypropyl)-carbamoylphenyl-methyl)-carbamoylmethylj -2-deoxy-α-D-glucopyranoside in $ 0 percent aqueous methanol is hydrated in the presence of 5 percent palladium on charcoal at normal pressure and room temperature, the catalyst is filtered off and it is evaporated. The residue is dissolved in distilled water and freeze-dried and is 2-acetamido~3-0-|(L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoylphenyl-methyl)-carbamoylmethyl-2-deoxy-D-glucose.

The starting material can be prepared as follows:

A solution of 3>7 g of benzyl-2-acetamido-3-0-carboxymethyl-2-deoxy-α-D-glucopyranoside and 1.38 ml of triethylamine in 100 ml of tetrahydrofuran is mixed with 3»72 g of 1-phenylglycine-D-isoglutamine- tert-butyl ester hydrochloride and 2.6 g of 2-ethoxy-N-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ), are left for 24 hours at room temperature and worked up as indicated in example j6. One thus obtains benzyl-2-acetamido-3-0-£(L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoyl-phenylmethylj-carbamoylmethylj-2-deoxy-α-D-glucopyranoside-tert-butyl ester which is hydrolyzed mildly acid to benzyl-2-acetamido-3-O- (L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoylphenylmethyl)-carbamoylmethyl-2-deoxy-α-D-glucopyranoside.

Example 43

A solution of 3>5 g of benzyl-2-acetamido-3-0-£(L-1-(D-1,3-bis-carbamoylpropyl)-carbamoylethyl)-carbamoylmethylj-2-deoxy-4>6-0-isopropylidene- B-D-glucopyranoside in 50 ml of glacial acetic acid is mixed with stirring at ^ 0°C dropwise over 15 minutes with 50 ml of distilled water and stirred for one hour at this temperature. After cooling, another 100 ml of distilled water is added and the solvent is evaporated in a water jet vacuum. The residue is mixed with 20 ml of water and freeze-dried. One thus obtains benzyl-2-acetamido-3-0-|(L-1-(D-1,3-bis-carbamoylpropyl)-carbamoylethyl)-carbamoylmethylj-2-deoxy-B-D-glucopyranoside of fa) n =

-44° +1° (N,N-dimethylformamide, c = 0.989).

The starting material used can be prepared as follows: A solution of 30 g of benzyl-2-acetamido-2-deoxy-B-D-glucopyranoside in 100 ml of N,N-dimethylformamide is mixed with stirring, cooling and moisture exclusion with 0.1 ml of methanesulfonic acid. Now a solution of 20 ml of isopropenyl methyl ether in 60 ml of N,N-dimethylformamide is added dropwise over the course of an hour, then stirred for 2 hours at room temperature and made alkaline with triethylamine. After evaporation of the solvent, benzyl-2-acetamido-2-deoxy-4>6-0-isopropylidene-B-D-glucopyranoside is crystallized from acetic ester, melting point 194°C, LaJ d° = -104° l1-<0> (chloroform, ' c = 0.850).

A solution of 36.2 g of benzyl-2-acetamido-2-deoxy-4,6-0-isopropylidene-B-D-glucopyranoside in 400 ml of acetonitrile and 100 ml of N,N-dimethylformamide is mixed with 4.95 g of sodium hydride, stirred 2 hours at 40°C. This mixture is now cooled to -10°C, mixed with 17.2 ml of bromoacetic acid and stirred for 3^ minutes at 0°C. One now adds /\. 0 ml of ethanol, neutralize with glacial acetic acid and evaporate to dryness. The residue is distributed between ether and distilled water, the ether phase is washed with water, dried over magnesium sulphate and evaporated. The product, benzyl-2-acetamido-3-0-carboxy-methyl-2-deoxy-4,6-0-isopropylidene-B-D-glucopyranoside ethyl ester is crystallized from ether, mp 93-94°C, (aj^<0> = -49° <+>1°

(CHCl 2 , c = 1.001).

A solution of 6.56 g of benzyl-2-acetamido-3-0-carbamoylmethyl-2-deoxy-4'' 6-0-isopropylidene-B-D-glucopyranoside ethyl ester in 70 ml of methanol is mixed with 22.5 nil of 1N caustic soda. After completion of ester hydrolysis, 7.5 ml of 1N hydrochloric acid is added and evaporated to dryness. The sodium salt formed is dissolved in 50 ml of N,N-dimethylformamide and mixed with 3.7 g of L-alanyl-D-glutamic acid diamide hydrochloride and 3.72 g of 2-ethoxy-N-ethoxycarbonyl-. 1,2-dihydroquinoline (EEDQ). After standing for 24 hours at room temperature, the mixture is evaporated to dryness, the residue is dissolved in water and extracted with methylene chloride and twice with n-butanol. The product that remains after distilling off n-butanol is benzyl-2-acetamido-3-0-^{L-l-(D-l,3-bis-carbamoylpropyl)-carbamoylethyl)-carbamoylmethylj-2-deoxy-4 > 6-0- isopropylidene-B-D-glucopyranoside.

Example 44

A solution of 5.1 g of benzyl-2-acetamido-3-O-[(L-1-(D-1,3-bis-carboxypropyl)-carbamoylethyl)-carbamoylmethyl]-2-deoxy-4'6-0-isopropylidene-B-D- glucopyranoside dimethyl ester in 120 ml of glacial acetic acid is mixed dropwise with 80 ml of distilled water while stirring at 50°C and stirred for one hour at this temperature. After evaporation of the acetic acid, benzyl-2-acetamido-3-0-

I [Ij-1-(D-1,3-bis-carboxypropyl)-carbamoylethyl)-carbamoylmethylj-2-deoxy-8-D-glucopyranoside dimethyl ester.

The starting material1 used can be prepared as follows: 6.56 g of benzyl-2-acetamido-3-0-carboxymethyl-2-deoxy-4 16-0-isopropylidene-B-D-glucopyranoside ethyl ester is hydrolyzed as indicated in example 43 with caustic soda and condensed with 4>24 g

L-alanyl-D-glutamic acid dimethyl ester hydrochloride in the presence of

3.72 g of 2-ethoxy-N-ethoxycarbonyl-1,2-dihydroquinoline. After evaporation of the solvent, the residue is taken up in chloroform,

washed with water, ice-cold 2N hydrochloric acid, water, a saturated sodium bicarbonate solution and water, dried over magnesium sulfate and evaporated to dryness. The residue is benzyl-2-acetamido-3-0-1(L-1-(D-1,3-bis-carboxypropyl)-carbamoylethyl]-carbamoylmethylj-2-deoxy-4,6-0-isopropylidene-B-D-glucopyranoside dimethyl ester.

Example 45

A 5 percent solution of benzyl-2-acetamido-3-0-£(L-1-(D-1-carbamoylmethylcarbamoyl-3-carboxypropyl)-carbamoylethyl)-carbamoylmethylj-2-deoxy-α-D-glucopyranoside benzyl ester in tetrahydrofuran/water 2 /l is hydrated in the presence of 5 percent palladium on charcoal at normal pressure and room temperature. After approx. 50% of the theoretical amount of hydrogen, the catalyst is filtered off and evaporated. The residue is now dissolved in distilled water and further hydrated. After the theoretical amount of hydrogen has been absorbed, the catalyst is filtered off and the filtrate is freeze-dried. 2-acetamido-3-0-|(L-1-(D-1-carbamoyl-methylcarbamoyl-3-carboxypropyl)-carbamoylethyl)-carbamoylmethyl-2-deoxy-D-glucose is thus obtained as a white powder.

The starting material can be prepared as follows:

4.8 g of N-tert.-butoxycarbonyl-L-alanine-D-glutamine-y.-benzyl ester glycinamide monohydrate are dissolved in a mixture of 5 ml of trifluoroacetic acid and 5 ml of 1,2-dichloroethane and allowed to stand under moisture exclusion for 16 hours at room temperature. This solution is diluted with 50 ml of tetrahydrofuran, cooled in an ice bath and neutralized with triethylamine. After adding the solution of 3.7 g of benzyl-2-acetamido-3-carboxymethyl-2-deoxy-α-D-glucopyranoside and 1.38 ml of triethylamine in 100 ml of tetrahydrofuran, the whole is mixed with 2.6 g of 2-ethoxy- N-ethoxycarbonyl-1,2-dihydroquinoline is allowed to stand for 24 hours at room temperature and worked up as indicated in example 36. Benzyl-2-acetamido-3-0-£(L-1-(D-1-carbamoylmethylcarbamoyl-3-carboxypropyl) is thus obtained -carbamoylethyl]-carbamoylmethyl]-2-deoxy-α-D-glucopyranoside benzyl ester.

Example 46

A solution of 5.2 g of benzyl-4,6-0-benzylidene-3~0-£(L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoylethyl}-carbamoyl-methylj -2-deoxy-2-p- Tolylsulfonylamino-cc-D-glucopyranoside benzyl ester in 120 ml of 70 per cent aqueous acetic acid is hydrogenated in the presence of palladium on charcoal at normal pressure and room temperature. After the theoretical amount of hydrogen has been taken up, the catalyst is filtered off and evaporated to dryness. The residue is dissolved in JO ml of distilled water, and freeze-dried. Thus 3-0-|(L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoylethyl)-carbamoyl-methylj-2-deoxy-2-p-tolylsulfonylamino-D-glucose is obtained as white powder.

The starting material used can be prepared as follows: A solution of 6.0 g of benzyl-2-amino-4,6-0-benzylidene-3-0-carboxymethyl-2-deoxy-α-D-glucopyranoside in 100 ml of methylene chloride and 6 .3 ml of triethylamine is mixed dropwise at 5°C with a solution of 3.1 g of p-toluenesulphonic acid chloride in 30 ml of methylene chloride and allowed to stand for 16 hours at room temperature. This solution is then washed with water, 2N caustic soda, water, cold 2N hydrochloric acid and water, dried over magnesium sulphate and evaporated to dryness. The residue is extracted several times with ether, dried and is benzyl-4,6-0-benzylidene-3-0-carboxymethyl-2-desoxy-2-tolylsulfonylamino-α-D-glucopyranoside of melting point 185-187°C, (<x){ j° = +75° +1° (chloroform, c = 0.855).

A solution of 5.7 g of benzyl-4,6-0-benzylidene-3-0-carboxymethyl-2-deoxy-2-tolylsulfonylamino-α-D-glucopyranoside and 1.01 g of triethylamine in 80 ml of N,N-dimethylformamide is mixed with 10 mmol of L-alanyl-D-isoglutamine benzyl ester trifluoroacetate and 2.5 g of 2-ethoxy-N-ethoxycarbonyl-1,2-dihydroquinoline and allowed to stand for 24 hours at room temperature. It is then evaporated to dryness, mixed with water, filtered off undissolved, washed with water and ether and dried in vacuum. One thus obtains benzyl-4,6-0-benzylidene-3-0-|Q,-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoylethyl)-carbamoyl-methylj -2-deoxy-2-tolylsulfonylamino-a-D- glucopyranoside benzyl ester as white powder.

Example 47

20 g of 2-amino-2-deoxy-3-(carboxymethyl)-D-glucopyranose (30 per cent, mixed with KCl) has been finely pulverized in 100 ml of absolute acetonitrile. With good stirring and slight cooling, it is first mixed with 26.6 ml of trimethylchlorosilane, then with 1.5 ml of triethylamine. After 4 hours at room temperature, a thin-layer chromatography (silica gel, Merck) in the eluant chloroform:methanol = J:1 shows practically only one ninhydrin-positive spot (Rp = 0.7) of persilylated amino acid. A further 3.4 ml of triethylamine and 42.5 ml of a 0.62N solution of 2-methyloxazolinone-(5) in absolute dimethylformamide are mixed with and allowed to stand for 15 hours at room temperature. The solution of 2-methyloxazolinone-(5) is prepared from 5>^5 g of N-acetylglycine and 11.4 6 dicyclohexylcarbodiimide in 40 ™1 dimethylformamide, filtering off 85% (9.6 g) of the dicyclohexylurea formed.

After filtering off the salts, partitioning between methylene chloride and water and hydrolysis of the silyl ether in CH2C12-

phase with a few drops of acetic acid gives 2-(acetaminomethyl-carbonylamino)-2-deoxy-3-carboxymethyl-D-glucopyranose. as a colorless powder when extracted with vinegar.

Reaction with L-alanyl-D-glutamic acid-α-amide-d'-benzyl ester-trifluoroacetate and 2-ethoxy-N-carbethoxy-1,2-dihydroquinoline in DMFrmethylene chloride = 1:1 takes place analogously to example 33. After hydration with 5 f ° <p>d/C gives 2-(acetaminomethylcarbonyl-amino)-2-deoxy-3.0-|(L-1-(D-1-carbamoyl-3-carboxy-propyl)-1-carbamoyl-ethyl)-carbamoylmethylj- a,B-D-glucose as lyophilisate.

Example 48

3.2 g of 2-amino-2-deoxy-3-carboxymethyl-D-glucopyranose, 2.5 g of KgCO 3 and 4.5 g of pivalic acid-8-nitrophenyl ester are stirred in 100 ml of dimethylformamide at 100°C for 15 hours. The salts are then filtered off, the filtrate is evaporated in an oil vacuum and dissolved in an aqueous solution over an ion exchanger IR-120. The eluate is shaken with CHCl^ and evaporated in vacuo to syrup. When expelled with acetic acid, a faint yellow powder is formed 2-pivaloylamido-2-desoxy-3-carboxymethyl-D-glucopyranose, melting point = 89-93°^•

The starting material, 2-amino-2-deoxy-3-carboxymethyl--D-glucopyranose, is obtained as follows: 100 g of 2-phenyl-4,5-(3,0-carboxymethyl-5,6-isopropylidene-D- glucofurano)-A-oxazoline in 1000 ml of 2.6N hydrochloric acid is boiled for 4 hours under reflux. The precipitated benzoic acid is filtered off, the filtrate is extracted three times with ether. The water phase is set on a pH meter with 3N K0H exactly to pH 7 and managed with activated carbon. It is then evaporated in a vacuum to approx. 500 ml and mix with 3 liters of acetone, cool with ice and filter off any precipitate. After drying at 4-0°C in vacuum, 247 g of a yellowish powder is obtained, which consists to 85% of KCl and to 15% of the title compound.

From the mother liquor, you also get 22 g of a mixture of the title compound and the associated lactam.

The reaction of 2-pivaloylamido-2-desoxy-3-carboxy-methyl-D-glucopyranose with L-alanyl-D-glutamic acid-oc-amide-3'-benzyl ester-trifluoroacetate takes place with 2-ethoxy-N-carbethoxy-1, 2-dihydroquinoline in DMF/CHpCl2 =1:1. After hydration with 5 percent Pd-C, 2-trimethylacetamido-2-deoxy-3,0-1[L-1-(D-1-carbamoyl-3-carboxy-propyl)-1-carbamoyl-ethyl)-carbamoylmethylj -a,8 -D-glucose as lyophilisate.

Example 49

4.1 g of benzyl-2-acetamido-2,-deoxy-4,6-benzylidene-α-D-glucopyranoside is dissolved in 30 ml of N,N-dimethylformamide and first stirred at 40°C with 0.5 g of sodium hydride mineral oil dispersion under exclusion of moisture, and then at room temperature until a homogeneous solution is formed. Then, while cooling at 10°C, a solution of 4.76 g of N-iodo-acetyl-L-alanyl-D-isoglutamine-γ-benzyl ester in 15 ml of N,N-dimethylformamide is added dropwise and allowed to stir for 24

hours at room temperature. It is filtered, neutralized with acetic acid and evaporated in a vacuum to a syrup.

A solution of 8 g of this compound in 400 ml of 60% acetic acid is kept for 1 hour at 80°C. After cooling, the solution is evaporated to dryness, the residue is mixed twice more with 50 ml of water and evaporated each time to dryness. The formed crystalline residue is dried with a little water, the crystals are sucked off and dried. One thus obtains benzyl-2-acetamido-3-O-£-/" L-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl-7-carbamoyl-ethyl^ -2-deoxy-ct-D-glucopyranoside- benzyl ester with mp 200-202°C, { qj^ ° = + 77° in 1° (dimethylformamide, c = 0.599).

A solution of 4 g of benzyl-2-acetarado-3-O-[L-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl-7-carbamoyl-, methyl-2-deoxy-α-D-glucopyranoside benzyl ester in 80 ml of methanol is hydrogenated with 0.4 g of 5% palladium on charcoal as a catalyst at normal pressure and room temperature until standstill. The catalyst is filtered off, washed with a little methanol, and the filtrate is evaporated to dryness in a water jet vacuum. The residue is dissolved in 50 ml of distilled water and further hydrogenated with 1 g of 5% palladium on charcoal as a catalyst at normal pressure and room temperature until standstill. The catalyst is filtered off, washed with a little water and the filtrate is evaporated to dryness. The 2-acetamido-3-0-£/L-1-(D-1-carbamoyl-3-carboxyl-propyl)-carbamoyl-ethylj^-carbamoyl-ethylj-2-deoxy-D-glucose is dried in high vacuum over phosphorus pentoxide, fqj< 2>^ -10°

- 1° (water, c= 0.930), yield 75%.

Example_50

To 5.31 g of benzyl-2-acetamido-3-o-carboxymethyl-2-deoxy-α-D-glucopyranoside dissolved in 40 ml of absolute dimethylformamide, add 3.87 g of L-alanine benzyl ester p-toluenesulfonate, 1.07 ml of N-methylmorpholine and 3.00 g of 2-ethoxy-N-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) and the whole is stirred for 24 hours with stirring at room temperature. _ The oily residue remaining after the evaporation of the solvent is worked up analogously to example 31. After evaporation of the solvent and recrystallization from acetic acid to ester, 4.5 g of benzyl-2-acetamido-3-0-[(L-1-carboxyethyl -carbamoylmethyl]-2-deoxy-a-D-glucopyranoside benzyl ester with melting point 180-181°C [ a]^ ° = +82°C (97)+ 1° (C = 1, methanol), R. = 0.61 (system: acetonitrile/water 3:1) and R^ = 0.81 (system: acetic acid ethyl ester/n-butanol/pyridine/acetic acid/

water 4 2:21:21:6:10.

4.32 g of benzyl-2-acetamido-3-0-[(L-1-carboxy-ethyl)-carbamoylmethyl]-2-deoxy-α-D-glucopyranoside benzyl ester dissolved in 40 ml of ethylene glucodimethyl ester/water 9:1 is treated for 2 hours in presence of 0.4 g of palladium on charcoal (10%-ig) with hydrogen. The catalyst is filtered off and the filtrate is evaporated to dryness. That over phosphorus pentoxide

dried residue (2.84 g, Rf = 0.47 (system: acetic acid ethyl ester/n-butanol/pyridine/acetic acid/water = 4 2:21:21:6:10)) is taken up in 40 ml of absolute dimethylformamide, mixed with 2.18 g of D-isoglutamine-γ-benzyl ester hydrochloride and 0.89 g of N-methylmorpholine and finally 2.16 g of EEDO and the whole is stirred for 23 hours at room temperature Preparation analogously to example 31 in 3.95 g of benzyl-2 -acetamido-3-0-{[L-1-carbamoyl-3-carboxypropyl)-carbamoyl-ethyl]-carbamoylmethyl}-2-deoxy-α-D-glucopyranoside benzyl ester, melting point 20 3-20 5°C, [a]p° = +66° (90)+ 1° (c = 1, dimethylformamide), Rf 0.59 (system: acetonitrile/water 3:1), R^ = 0.66 (system: methylene chloride/methanol/acetic acid).

0

Claims (1)

Analogy development method for the production of immune potentiating glucosamine derivatives of the general formula I in which X means a carbonyl or sulfonyl group, R means an alkyl group or a phenyl group optionally substituted with a lower alkyl group and if X means the carbonyl group, R can also mean an alkoxy or benzyloxy group, R-^ means hydrogen, alkyl or a benzyl group, R2 means hydrogen or a lower alkyl group, R^ and Rg means hydrogen, alkyl, a benzyl or an alkanoyl group, R^ means hydrogen, lower alkyl or hydroxymethyl, Rg means COOH, CONH2, CONH-lower alkyl, COO-lower alkyl, CONH-benzyl or CONH-CH2-CONH2, and Rg means COOH, CONH2, CONHCH3 or COO -lower alkyl, with the precaution that when R2 means a methyl group and X is a carbonyl group, the alkyl group R has more than one carbon atom, and if X means a carbonyl group, R2 means hydrogen or methyl, R^ methyl or phenyl, and R8 and Rg each a carboxyl group, as well as their salts, characterized by a) condensing a compound of formula in which X, R and R2 have the above meaning and , R4° and Rg° mean the residues R1, R4 or Rg or an easily cleavable protecting group or a derivative thereof, with a compound of formula: in which Ry0f Rg° and Rg° have the meaning of R7, Rg and Rg and furthermore that in these residues present carboxyl and, if desired, free hydroxy groups are protected with easily removable protective groups, and optionally remove protective groups present, or b) reacts a compound with formula: wherein X, R, R^°, R^° and Rg° have the above meaning and any hydroxy groups present therein are protected with an easily cleavable protecting group, with a compound of formula: in which Z means a reactive, esterified hydroxy group and R7 , Rg° and Rg° have the above-mentioned meaning, and optionally cleaves' present protective groups, or c) in a compound of formula: in which R, R2, , Rg° and Rg° have the above meaning oe? R^ means an alkylidene or cycloalkylidene residue acid cleaves the oxazoline or dioxolane ring, optionally cleaves off any protective groups present and in the possibly freed amino group in the 2-position of the sugar molecule the X-R residue is introduced or d) condenses a compound of formula: in which R^ , R^°, R2 / R4°' R6° 0<? ^7° *in the above meaning with'a compound of formula: in which Rg and Rg have the above-mentioned meaning with the precaution that in the residues R7°/ Rg° and Rg° present carboxyl and, if desired, free hydroxyl groups are protected with easily cleavable protective groups, and optionally cleave off the protective groups present, and transfer formed compounds, if desired, in their salts. in in in