NO151201B - PROCEDURE FOR THE PREPARATION OF THERAPEUTICALLY EFFECTIVE ANTI-DERIVATIVES. - Google Patents

Description

The invention relates to a method for the production of pharmacologically active antigen derivatives which consist of an antigen, to which per antigen molecule is bound at least one and a maximum of so many muramyl peptide molecules that the antigenic character is retained, as well as possibly a carrier, these components being linked to each other covalently either directly via oxycarbonyl, iminocarbonyl or carbonylthio groups or optionally indirectly via bridging residues of an aliphatic compound with at least two amino groups, at least one amino and at least one carboxyl group or at least two carboxyl groups, since the bond between bridge link and antigen as well as bridge link and muramyl peptide likewise takes place via oxycarbonyl, iminocarbonyl or carbonylthio groups, and since the muramyl peptide in unbound form has the formula Ila specified structure

in which

X means a carbonyl or carbonyloxy group,

R^, R^ and Rg independently mean hydrogen, trimethylsilyl or alkanoyl,

R2 means a phenyl radical or unsubstituted or lower alkyl substituted with hydroxy, carboxy, lower alkoxy and/or lower alkoxycarbonyl,

R^, Ry and Rg independently mean hydrogen or lower alkyl,

Rg means a phenyl radical or unsubstituted or with hydroxy, halogen, amino, mercapto, lower alkylthio or a phenyl radical, substituted lower alkyl, or

Ry and Rg together mean the alkylene with 3 or 4 carbon atoms, and

<r>esten<e> R1Q , r^ and R^2 independently of each other mean carboxy, trimethyl-silyloxycarbonyl, unsubstituted or with a thenyl residue substituted lower alkoxycarbonyl or carbamoyl, which is unsubstituted or substituted at the nitrogen atom with unsubstituted or with carboxy, lower alkoxycarbonyl or carbamoyl substituted lower alkyl, or

R^^ also means hydrogen,

with the precaution that at least one free hydroxy, amino, mercapto or carboxy group is present in a starting compound of formula Ila, the method being characterized by condensing an optionally bridged antigen with optionally bridged muramyl peptides of formula Ila, one of the two reaction components has at least one free amino, hydroxy and/or mercapto group and the other has at least one carboxylic acid group, and if desired, a compound formed analogously to the above is condensed onto a possibly bridged carrier, one of the two the reaction components have at least one free amino, hydroxy and/or mercapto group, and the other has at least one carboxylic acid group.

By antigen is understood here an organic substance which is recognized by a physiological medium, i.e. a human or animal organism, as immunologically foreign or which under suitable conditions can be recognized as foreign.

Antigens primarily include all substances that cause specific immunization of a living organism against infectious pathogens or unwanted reactions such as allergic sensitization or abstention of transplanted foreign tissue. In particular, antigen as an ingredient in vaccines is included here.

As vaccines, those that are used for specific immunological protection against infectious diseases within the framework of classical inoculation technique come into consideration. As antigen, which is contained in such vaccines, the weakened living or dead, modified or deconstructed producers of infectious diseases, toxoids or natural or synthetically produced sub-components of the producers and toxoids come into question. As a class of pathogens, the following are mentioned: viruses, chlamydia, rickettsiae, bacteria, protozoa and metazoan parasites. Preferred antigens are those which are suitable as components in vaccines which are used for treatment for e.g. influenza A and B, parainfluenza 1-3, respiratory diseases caused by respiratory syncytial virus, rhinovirus or adenovirus, cytomegaly, rubella, measles, mumps, pertussis, poliomyelitis, herpes simplex 1 and 2, varicella; and herpes zoster, rotavirus diseases, hepatitis A, B and others, rabies, foot-and-mouth disease, trachoma, caries, meningitis caused by meningococci A, B and C, diseases caused by pneumococci, H. influenzae, streptococci (especially rheumatic fever), pseudomonas and proteus, typhoid, paratyphoid and other intestinal diseases caused by enterobacteria, gonorrhea, syphilis, malaria, trypanosomiasis (sleeping sickness and Chagas disease), leishmaniasis, filariasis, schistosomiasis, ankylostomiasis and other diseases.

On the other hand, new vaccines must be mentioned

which are not directed against the producers of infectious diseases, but against body-specific components, i.e. normal and aberrant autoantigens or against sensitizing environmental antigens, i.e. against allergens.

In some cases, efforts are made to abolish the function of body-specific molecules (e.g. of hormones, of other mediators and of humoral and cellular receptors) against normal or aberrant autoantigens or to abolish the spread or persistence of aberrant, especially neoplastic cell lines . Preferred antigens as constituents of such vaccines are, for example: partial sequences of human chorionic gonadotrophin or parts of spermatozoa for immunization against mediators of fertility and thus for immunological suppression of reproductive function, mediators of inflammatory processes, and especially in pure form, including lymphokines which are secreted by lymphocytes and especially MIF (macrophage migration inhibitory factor) for immunological suppression of inflammatory diseases, immunologically specific antigen receptors from lymphocytes and antibody (fractionated antigen-specific lymphocytes, antigen receptors extracted from these lymphocytes, fractionated antigen-specific antibodies or antibody fragments) for anti-idiotype immunization (immunization against the auto-antigens that are characteristic of the antigen-receptor structures) with the aim of eliciting specific immune reactions to eliminate disease-causing immunoprocesses, such as autoimmunity (e.g. against synovial antigens and immunoglobulin in primary chronic polyarthritis, against the myelin components in degenerative diseases of the central nervous system, against TSH receptors in autoimmune thyroiditis, against acetylcholine receptors in the striated muscles in myasthenia gravis, against single cell components in juvenile diabetes etc.) or allergy (e.g. e.g. against grass pollen, dust or drugs in the case of allergic asthma, allergic rhinitis or drug hypersensitivity) or for the reduction of per se normal but unwanted immune reactions (e.g. induction of immune tolerance to prevent rejection of transplanted, foreign organs and tissues) , autologous or cross-reacting homologous or heterologous tumor cells, tumor cell fragments or tumor cell membrane components including oncornavirus-encoded glycoproteins such as GP 70, for tumor-specific immunization within the framework of prophylaxis and therapy in cancer diseases.

In all cases, it is desired by means of immunization against the allergens instead to induce the pathogenetically relevant IgE antibody response predominantly or in the surrounding mass of IgG and IgA antibodies against the sensitizing environmental antigens and thus capture the allergens in the circulation and in the secretions by specific antibodies, before they react with IgE antibodies that are bound with fat cells and can thus trigger the release of allergic mediators. In this antigen-specific desensitization, the allergens themselves, i.e. e.g. grass pollen, dust or drugs, the antigenic components of the vaccine.

The antigens can, especially when they are low molecular weight, consistently also advantageously, be covalently bound to a high molecular weight carrier. As carriers, particular mention should be made of polymers of lactic acid and its derivatives which have a free carboxyl group at the chain end, such as its esters with glycolic acid, polylactic acid amides or lactic acid polyesteramides such as those which e.g. is described in English patent no. 9 32 382, further alginic acid, poly-galacturonic acid, pectinic acid, carboxymethyl cellulose or agarose. Furthermore, basic, neutral or acidic polyamino acids which are not themselves immunogenic, such as polyaspartic acid, polyglutamic acid, polylysine or polyornithine, are used as carriers. As carriers, other suitable antigens within the scope of the definition given above or reproduced in the given examples also come into consideration, to the extent that an immune reaction against these is elicited or desired. Thus, e.g. an HCG peptide be covalently bound to tetanus toxoid as a carrier.

The different parts of the new antigen derivatives

are bound covalently to each other, i.e. that the antigen is connected to at least one of its functional groups directly or through a bridge link (Spacer) connected to the rest of the muramyl peptide by such a connection that is common in peptide chemistry.

As bridging links, mention should first be made of α,β-diamino-alkanes, especially α,β-diamino-lower alkanes,

such as ethylenediamine, propylenediamine, tetramethylenediamine, alkyl dicarboxylic acids, such as succinic acid or glutaric acid, α,8 or γ-aminoalkanecarboxylic acids, preferably α-amino-lower alkanecarboxylic acids and especially the natural α-amino-loweralkanecarboxylic acids such as glycine, 3-alanine, L -alanine, α-amino-iso-butyric acid, valine or leucine.

The aforementioned antigen derivatives can, e.g. have the following formula where A means the rest of the antigen, T means the rest of the carrier, MP means the rest of the muramyl peptide, Z' and Z"<1> bivalent bridges and where X<1>,X",X"<1> and X »<v> are covalent compound types, q, r, and t independently mean 0 or 1, and m and n are whole numbers greater than 0.

Alkyl is straight or branched alkyl with up to

18 carbon atoms which are bound in a suitable position, but primarily lower alkyl.

Phenyl, can e.g. be mono-, di- or poly-substituted, e.g. with lower alkyl groups, hydroxy, lower alkoxy, lower alkylenedioxy, halogen atoms and/or with trifluoromethyl groups.

In the first place, phenyl lower alkyl is benzyl or phenylethyl, where the phenyl ring can be mono-, di- or poly-substituted.

Alkanoyl has above all 2-18 carbon atoms and is primarily lower alkanoyl.

The residues which in connection with the present description and the patent claims are referred to as "lower" are residues and compounds which preferably contain up to 7 and primarily up to 4 carbon atoms.

In the above and in the following, the general terms have the following meaning: Lower alkyl is, for example, n-propyl, n-butyl, isobutyl, sec.-butyl or tert.-butyl, further n-pentyl, n-hexyl, isohexyl or n-heptyl and primarily methyl or ethyl. Phenyl-lower alkyl is the lower alkyl radical, especially methyl or ethyl, where the phenyl radical has the above meaning.

Low-area coke is e.g. n-propoxy, n-butoxy, iso-butoxy, sec.-butoxy or tert.-butoxy and primarily methoxy or ethoxy.

Lower alkyl mercapto is e.g. n-propyl-, n-butyl-, isobutyl, sec.-butyl or tert.-butyl mercapto and primarily methyl mercapto or ethyl mercapto.

Lower alkylene dioxy is in particular methylenedioxy, ethylene or propylenedioxy.

Halogen is fluorine or bromine, preferably chlorine.

Laverealkanoyl is especially propionyl or

butyryl, and primarily, however, acetyl.

The new antigen derivatives according to the invention can exist in the form of mixtures of isomers or of pure isomers.

It is known that muramyl peptides are good additives which, in a suitable mixture with antigens, can increase their immunogenicity. However, it has been shown that they only have a very short period of activity, as they are relatively quickly excreted by the human or animal organism. Above all, they can only be used under certain conditions which are not particularly suitable for clinical purposes, namely in admixture with antigen in an emulsion with mineral oil to induce an in vivo cell-mediated immunity against soluble antigens. The new antigen derivatives according to the invention bring not only a marked increase in the immunity of the antigen, but also a cell-mediated immunity under clinically acceptable conditions, which will be shown in the following examples.

1. Potentiation of cell-mediated immunity in vivo: Enhancement of late hypersensitivity such as bovine serum albumin (BSA)

and against schaferythrocytes (SRBC) in guinea pigs.

Pifbright guinea pigs are immunized on day 0 with 1 mg BSA or 1 mg SRBC-"Ghosts" (SRBCG) (outer membrane of SRBC)

in complete Freund's adjuvant by injection in each of 0.1 ml of an antigen-adjuvant mixture into both hind paws. 3 weeks later, the skin reaction is triggered by intracutaneous injection of 100

ug BSA or 100 pg SRBCG in 0.1 ml buffered physiological salt solution and quantified on the basis of the reaction volume calculated from the erythema surface and the thickness increase in the skin 24 hours later. The observed antigen-specific increase in the reaction volume after 24 hours (the late reaction) applies as a measure of the cell-mediated immunity. BSA and SRBCG are too weakly immunogenic for alone or in a water-in-oil emulsion with incomplete Freund's adjuvant (10 parts BSA solution or SRBCG suspension in 0.9% NaCl mixed with 8.5 parts "Bayol F" ( paraffin oil, hydrocarbon fraction from petroleum) and 1.5 parts "Arlacel A" (manniton monooleate) to induce a late reaction, but must, for an effective immunization in complete

adjuvant is added to mycobacteria (5 mg killed and lyophilised M, butyricum per 10 ml "Bayol F"/"Arlacel A").

Instead of mycobacteria, the new antigen derivatives containing as antigen BSA (1 mg BSA, 60 ug MDP per animal) or as antigen SRBCG (1 mg SRBCG, 25 ug per animal) are added either as an antigen-oil mixture or suspended in carboxymethyl cellulose (CMC). The new antigen derivatives induce late-type reactions in the absence of mycobacteria in the described experimental method.

A significant potentiation of the late-type reactivity against BSA and against SRBCG can also be achieved if the new antigen derivatives are not incorporated in complete Freund's adjuvant, but are added suspended in CMC. Intramuscular administration proves to be particularly effective in this case. Under these circumstances, an equivalent amount of free muramyl peptide added to the CMC mixture was substantially less active than the new active substance. Thus, it has been shown that the new compounds are capable of inducing cell-mediated immunity under clinically acceptable conditions,

i.e. when supplied with body-friendly byproducts, even against a soluble protein antigen. 2. Potentiation of cell-mediated immunity in vivo: Increase of late hypersensitivity to BSA and to SRBCG in mice.

Male MAG mice are immunized on day 0 with graded doses of antigen not conjugated with muramyl peptide (BSA-agarose or SRBCG) or with antigens conjugated with muramyl peptide (BSA-agarose or SRBCG). BSA-agarose preparations are administered subcutaneously in a dosage of from 0.1 to 100 vig (equivalent in the case of the new compounds with muramyl peptides to a dosage of the active substance of from 0.0029-6 pg per animal) in a volume of 0, 2 ml buffered physiological saline solution. The SRBCG preparation was added in a dosage of from 0.01 - 3 mg (corresponding to the new antigen derivatives with muramyl peptides a dosage of the active substance of from 0.25 - 75 pg per animal) in a volume of 0.5 ml in buffered physiological saline solution intraperitoneally or 0.05 ml intradermally or distributed in 3 paws.

4 to 20 days later, the late-type reaction is triggered

by injection of 100 µg BSA or 10 Q SRBCG in 20 µg buffered physiological saline into the left hindpaw and the reaction volume was quantified after 24 and 48 hours due to the absorption in the paw. The observed, antigen-specific increase in the volume in the paw is a measure of the cell-mediated immunity.

From day 14 after immunization with BSA-agarose-MDP compound, even the very low dosage (0.1 µg, corresponding to 0.006 µg of active substance) shows pronounced late-type reactions. In contrast, free BSA-agarose is not able to sensitize for late-type reactions SRBCG-MDP compounds (MDP = muramyl peptide), and above all intradermal delivery, are able to induce late-type activity that is significantly more pronounced than that obtained after sensitization with SRBCG not associated with muramyl peptide.

Thus, it has been shown that the new antigen derivatives are able to significantly increase the cell-based immunity.

3. Potentiation of humoral immunity in vivo:

Increase in antibody production against BSA in mice.

NMRI mice are immunized by intraperitoneal injection of 0.1 to 100 ug BSA linked to muramyl peptides (0.0014 to 6.0 ug active substance) on day 0. 10, 17 and 28 days later, serum samples are taken and the content of anti-BSA antibody is examined with a passive hemagglutination technique. In the dose used, free BSA is subimmunogenic for the experimental animals, i.e. one manages to trigger either no or only a rather small production of antibody. The antigenic derivative of BSA with a muramyl peptide enables a 2-3-fold increase in the antibody number in serum (the sum of the log2 number difference in three blood samples). Furthermore, it is noteworthy that the new antigen derivatives attached only to the agarose carrier,

is even more immunogenic after intraperitoneal or subcutaneous administration.

Due to the stated tests, it has been shown that the antigen derivatives produced according to the invention are also capable of significantly increasing humoral immunity.

The new antigen derivatives are new or improved known vaccine substances and serve as new vaccine agents or to simplify the methods used (as, for example, the number of injections required to maintain protection over a longer period of time can be lowered).

The invention relates in particular to the production of

new antigen derivatives that contain antigen as an ingredient in vaccines against parasites, bacteria, viruses, tumor cells, physiologically relevant components, their modified forms or subunits thereof which are optionally covalently bound via a bridge link to a muramyl peptide of formula II, where R^, R^ , R^,

Rg and Ry are hydrogen, X is carbonyl and R2 is optionally lower alkyl which is substituted by hydroxy or lower alkoxy or optionally phenyl which is substituted by hydroxy, lower alkoxy, lower alkyl or halogen and where Rg, R^, R1Q/R^i' Ri2 and R13 has the meaning given above.

The invention relates in particular to the production of

new antigen derivatives that contain antigen as an ingredient in vaccines against parasites, bacteria, viruses, tumor cells, physiological body components, their modified forms or subunits that are covalently linked via a bridge link to a muramyl peptide of formula II, where R^, R^, Rg and R? is hydrogen,

X is carbonyl, R2 is lower alkyl which is optionally substituted with hydroxy or lower alkoxy or phenyl which is optionally substituted with hydroxy, lower alkoxy, lower alkyl or halogen and in which R3 is methyl and Rg, Rg, R1Qf Rn' R12 and R13 have the above meaning .

The invention primarily relates to the production of new antigen derivatives which contain antigen as an ingredient in vaccines against parasites, bacteria, viruses or tumor cells, physiologically suitable components, their modified forms and subunits and which are optionally covalently bound via a bridge link to muramyl peptide of formula II , where R^, R4,

Rg and R13 are hydrogen, X is carbonyl, R2 is optionally lower alkyl which is substituted with hydroxy or methoxy and phenyl which is optionally substituted with hydroxy/methoxy, methyl, ethyl or halogen, R is hydrogen or methyl, Ry and Rg are hydrogen, Rg is lower alkyl, lower alkyl mercapto-lower alkyl, hydroxy-lower alkyl, benzyl, p-hydroxybenzyl or phenyl and where R-^q/ R1^<p>g R^2 is carboxyl, lower alkoxycarbonyl or carbamoyl and where R^1 is also hydrogen.

The invention relates above all to the production of new antigen derivatives containing antigens as ingredient f in vaccines against parasites, bacteria, viruses, tumor cells, physiologically relevant components, their modified forms and subunits, which are possibly linked via a bridge link to the muramyl peptide of formula II where R .. , R 4 , R 8 and R 13 are hydrogen,

X is carbonyl, R2 is lower alkyl which is optionally substituted with hydroxy or methoxy or phenyl which is optionally substituted with hydroxy, methoxy, methyl, ethyl or halogen,

R3 and Rg are hydrogen or methyl, Rg is methyl, ethyl, n-propyl, i-propyl, 2-methylpropyl, methylmercaptomethyl, hydroxymethyl, hydroxyethyl, phenyl, benzyl, or p-hydroxybenzyl and where <R>10' <R> ll°g R 12 is carboxyl lower alkoxycarbonyl or carbamoyl, and where R 1 is also hydrogen.

The invention also relates to the production of new antigen derivatives that contain antigen as an ingredient in vaccines against bacteria, viruses, tumor cells, physiologically appropriate components, their modified forms and subunits of these covalently bound possibly via a bridge link to the muramyl peptide of formula II where R^, R^, Rg and R13 are hydrogen, X is carbonyl, Ry and Rg together are propylene or butylene and where R2, R3, Rg, R"lq/ R^^°9 Rj2 ^are ^a above stated meaning.

Bridging links in the above definitions are especially 01,0,-diamino-lower alkanes, lower alkyl-dicarboxylic acid, natural α-amino-lower alkane carboxylic acids.

In particular, the invention relates to a method for producing the new antigen derivatives described in the examples.

The new compounds can be produced in a manner known per se.

The condensation according to the invention in the preparation of the antigen derivatives takes place by e.g. reacts one reaction component in the form of an activated carbonic acid with the other reaction component as a free amino, hydroxy or mer-capto compound. The activated carboxyl group can e.g. be an acid anhydride, preferably 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: cyanomethyl ester, carboxymethyl ester, p-nitrophenylthioester, methoxyethylthioester, acetyl-aminoethylthioester, p-nitrophenylester, 2,4,5-trichlorophenylester, N-hydroxysuccinimide ester, N-hydroxyphthalimide ester, 8-hydroxyquinoline ester, N-hydroxypiperidine ester. Active esters can also optionally be provided with a carbodiimide with the addition of N-hydroxysuccinimide or an unsubstituted N-hydroxybenzotriazole or 3-hydroxy-4-oxo-3, 4-dihydro-benzoi/~d7-1, 2, 3-triazone or these compounds substituted with e.g. halogen, methyl or methoxy.

The leaving groups used during the condensation must be non-toxic or also easily removable, in order to prevent the most high-molecular compounds from retaining toxic parts during adsorption.

One prefers for this reason as active esters those with N-hydroxysuccinimide and their C-substitution products such as N-hydroxy-methyl- or -dimethylsuccinimide or reaction with carbodiimide, such as carbodiimide itself or 1-ethyl-3-(3-dimethylaminopropyl) -carbodiimide.

The above-mentioned reaction is carried out in a known manner in the absence or presence of diluting, condensing and/or catalytic agents, and if necessary, by heating or cooling. Preferably, you work in an aqueous environment in a pH range from 6-9, and primarily from 7-8 in order not to break down the antigens.

The invention also covers such embodiments

for the process where the process is interrupted at any stage and starts from a compound that arises as an intermediate and carries out the subsequent reaction steps, or where the starting materials are prepared under the reaction conditions or where reaction components are used in the form of isomer mixtures or pure isomers .

To carry out the condensation according to the invention, starting materials are preferably used which immediately lead to specially desired groups of end substances and, above all, to the specially described or preferred end substances.

The starting materials used are known or can be prepared in a manner known per se.

The new muramyl peptides of formula IIa which are used as starting substances, where X is a carbonyl group, at least one of the residues, Rg and R^^ are lower alkyl, and primarily methyl and the others hydrogen, and in which the other substituents have the above meaning, can is obtained when a compound with the formula is condensed in a manner known per se

where X, R2, R3 and R^ have the meaning given above and where R°, R° and R° are the residues R^, R^ or Rg or is an easily removable protecting group or a derivative thereof with a compound of the formula

wherein Rg, R-|.o' Rll and R12 * iar ^a sairane meaning as Rg, R-^ q/ <R>ll and R12 mec^ it ti1!6?? that present carboxy and, if desired, free hydroxyl groups in these residues are protected with easily cleavable protective groups and optionally cleaved protective groups present.

Condensation takes place by e.g. reacts the compound of formula IV in the form of the activated carboxylic acid with the amino compound V or by reacting the acid IV with the compound V, where the amino group is present in activated form. The activated carboxyl group can e.g.

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 activated esters, special mention should be made of: cyanomethyl ester, carboxymethyl ester, p-nitrophenylthioester, p-nitrophenylester, 2,4,5-trichlorophenylester, pentachlorophenylester, N-hydroxysuccinimide ester, N-hydroxyphthalimide ester, 8-hydroxyquinoline ester, 2-hydroxy-1,2-di -hydro-1-carbethoxy-quinoline esters, N-hydroxypiperidine esters or enol esters, which are prepared with N-ethyl-5-phenyl-isoxazolium-3<1->sulfonate. Activated esters can also be provided with a carbodiimide with the addition of N-hydroxysuccinimide or an unsubstituted 1-hydroxybenzotriazole or this compound substituted with halogen, methyl or methoxy and 3-hydroxy-4-oxo-3,4-dihydro-benzo/d/- 1,2,3-triazine.

The amino group is e.g. activated by reaction with a phosphitamide.

Of these methods, the reaction with activated esters and especially those with N-ethyl-5-phenyl-isoxazolium-31-sulfonate (Woodward Reagent K) or 2-ethoxy-1,2-dihydro-1-carbethoxy-quinoline or carbodiimide is preferred.

Easily cleavable protecting groups are those known from peptide or sugar chemistry. For carboxy groups, tertiary butyl, benzyl or benzhydryl should be mentioned in particular and for hydroxy groups especially acyl residues, e.g. lower alkanoyl residues, such as acetyl, aroyl residues such as benzoyl and above all residues that can be derived from carboxylic acid such as benzyloxycarbonyl or lower alkoxycarbonyl or alkyl, especially tert-butyl, benzyl or tetrahydropyranyl which are optionally substituted with nitro, lower alkoxy or halogen or optionally substituted alkylidene residues connecting the oxygen atoms in the 4- and 6-position. Such alkylidene residues are in particular a lower alkylidene residue and primarily ethylidene, isopropylidene or propylidene residues or also a benzylidene residue which is optionally substituted and preferably in the p-position.

These protections can be detached on the inside and outside

known way. Thus, they can be removed hydrogenol ytically, 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.

Another method for producing these

the new starting materials consist of condensing a compound

with formula VI

where R2, R°, R3, R°, R°, R^ and Rg have the above meaning with a compound of the formula

wherein R-^Q/ R-^° and R^° have the above meaning with the addition that carboxyl groups and, if desired, free hydroxy groups present in the residues R?, R, ° and Rn° and R12 are protected with easy cleavable protective groups

and optionally cleaved protecting groups.

Condensation takes place by e.g. e.g. reacts the compounds VI in the form of the activated carboxylic acid with the amino compound of formula VII or that one reacts the acid VI with the compound VII, where the amino group is present in activated form. The activated carboxyl group can optionally 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 primarily mentioned. The amino group is preferably activated, e.g. by a reaction with a phosphitamide.

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

The starting materials can be produced in a manner known per se. Thus, e.g. a sugar unsubstituted in the 3-position is reacted with a halogen-R^-acetamido-R^g-acetic acid or a compound of formula IV with an amino-R,0-acetic acid, where the carboxyl group is protected in the manner indicated above and the protecting group is removed .

Another method for removing a side chain that sits in the 3-position on the sugar residue consists in reacting a compound of formula VIII. where X, R2, R°, R°, R° and R. , have the above meaning and where any hydroxy groups present are protected with an easily removable protecting group with a compound of the formula

where Z is a reactive, esterified hydroxy group and R^, Ry, Rg, Rg, R-^Q» R-^° and R^2 have ^a above stated meaning,

and optionally cleaved protecting groups present.

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

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

The new muramyl peptides of formula IIa which are used as starting materials where X is a carbonyl group, and R^,

R^ and Rg are trimethylsilyl, and the other substituents have the above meaning, can be obtained as indicated in Norwegian application 793993.

The following examples illustrate the invention described above, and the temperature is given in degrees Celsius.

Example 1

To a solution of 1 g of bovine serum albumin i

100 ml of a 0.1 M sodium bicarbonate-0.5 M sodium bicarbonate solution is added with stirring to 500 mg of 2-acetamido-3-0- \^~ L- 1-(D-1-carbamoyl-3-succinimidooxycarbonyl-propyl)-carbamoyl -ethyl/-carbamoyl-methylJ -2-deoxy-D-glucose. The solution is stirred for 4 hours at room temperature and then sterile filtered (MF-Millipore, 0.45 µm filter). The conjugated bovine serum albumin is separated in the dialfiltration method with an Amikon UM-10 filter from low-molecular reaction products or from salts and freeze-dried.

The quantitative determination of the muramyl peptide that is bound to bovine serum albumin takes place according to the Morgan-Elson reaction following the modification of J.M. Ghuysen

et al (1 "Methods in Enzymology" 8, (1966) 629). On average, 6 µg of muramyl peptide are found in 1 mg of bovine serum albumin compound.

The succinimido ester used as starting

substance f must be produced in the following way:

1 mmol of 2-acetamido-3-0-[/~L-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl-7-carbamoyl-methyl)-2-deoxy-D-glucose, 1 mmol of dicyclohexylcarbodiimide and 1.1 mmol of N-hydroxysuccinimide is dissolved in 3 ml of absolute dimethylformamide and stirred in a closed vessel to the exclusion of water for 20 hours. The precipitated dicyclohexylurea is separated, the solvent is evaporated in high vacuum and the residue is treated with ether, filtered off with suction and dried. The provided succinimidoxy ester can be stored under the exclusion of moisture, e.g. in a nitrogen ampoule.

Example 2

The condensation of the antigen derivative from Example 1 on activated agarose; thereby using a commercially available agarose derivative from BIO-RAD, Affi-Gel 10. On an agarose skeleton containing ether side chains ("spacer arms")

of N-alkyl succinamides which are esterified with N-hydroxysuccinimide.

120 mg of bovine serum albumin derivative according to example 1 is dissolved in 12.5 ml of 0.1 M phosphate buffer, pH 7.3 at 4°C. 500 mg Affi-Gel 10 is then suspended in the solution by shaking and shaken for 4 hours at 4°C. The still remaining, active ester groups are converted by treatment for 30 minutes with a 1 M ethanolamine.HCl-0.1 M phosphate buffer solution (pH 7.3). The gel is then filled into a column and washed first with 200 ml of 0.1 M phosphate buffer-1 M sodium chloride solution (pH 7.3) then with 20 ml of physiological sodium chloride solution.

The determination of bovine serum albumin-muramyl peptide compounds that are condensed to Affi-Gel 10 takes place by quantitative analysis of representative bovine serum albumin amino acids in total hydrolysates of defined gel samples. On average, 9-10 mg of bovine serum albumin muramyl peptide derivatives were bound to 1 ml of swollen Affi-Gel 10.

Example 3

Scaferythrocyte membranes are prepared from fresh Schaf blood according to the method of Dodge, J.I. et al (Arch. Biochem. Biophys. 100, (1963) p. 114-180). To a suspension of 500 mg of Schaferytrocyte membranes in 50 ml of 0.1 M sodium bicarbonate - 0.1 M sodium bicarbonate solution, 400 mg of 2-acetamido-3-0-L-l-(D-l-carbamoyl-3-succinimido-oxycarbonyl-propyl) is added with stirring )-carbamoyl-ethyl 7-carbamoyl-methyl j - 2-deoxy-D-glucose and the mixture is stirred for 4 hours at room temperature. The erythrocyte membrane conjugate is then sedimented by one hour of ultracentrifugation at 90,000 g and 4°C. The sediment is washed three times - each time followed by suspension and ultracentrifugation - with phosphate-buffered saline and once with distilled water. The washed erythrocyte membrane conjugate is suspended in 100 ml of distilled water and freeze-dried.

The quantitative determination of muramyl peptide

which is bound to schaferythrocytes takes place with the Morgan-Elson reaction and gives 25 ug of muramyl peptide per 1 mg erythrocyte membrane.

Example 4

100 mg group C polysaccharide of Neisseria meningitidis and 110 mg (0.2 mol) 2-acetamido-3-0-[/~L-1-(D-1-carbamoyl-3-N-aminoethyl-carbamoyl-propyl)- carbamoyl-ethyl/-carbamoyl-methylJ -2-deoxy-D-glucose-HCl is dissolved in 10 ml of distilled water because the pH value in the solution is set to 5 with dilute hydrochloric acid.

19.2 mg of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. HC1 is added with stirring to the solution. The mixture is stirred for one hour at room temperature, the pH value is maintained at 5 with the addition of dilute caustic soda, then 5 ml of 2 M sodium acetate buffer solution, pH 5, is added and the mixture is stirred for a further 30 minutes. The pH value is thereby set to 7 with caustic soda. The solution is sterile filtered and dialyzed at 4°C against distilled water and then freeze-dried.

The quantitative determination of desmethylmuramyl dipeptide which is linked to C-polysaccharide takes place as in example 1 with the Morgan-Elson reaction and gives 80 µg of desmethylmuramyl dipeptide per 1 mg polysaccharide.

The 2-acetamino-3-O-L-1-(D-1-carbamoyl-3-N-aminoethyl-carbamoyl-propyl)-carbamoyl-ethyl/-methyl J-2-deoxy-D-glucose hydrochloride used can e.g. produced on

the following way:

1 mmol 2-acetamlno-3-0-[/~L-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl7-carbamoyl-methylJ-2-deoxy-D-glucose and 2 mmol N-ethyl N 1 -(3-Dimethyl-aminopropyl)-carbodiimide. HC1 is dissolved in 10 ml of water and the pH is adjusted to 5.0 with hydrochloric acid, then a solution of 5 mmol of ethylene diamine dihydrochloride in 10 ml of water is added. The mixture is stirred at pH 5.0 and room temperature for 6 hours. The mixture is taken up on a weakly acidic cation-ion exchange column-Amberlite CG 50 II,

2.5 x 45 cm- and eluted with c;n linear gradient of 0.05 M pyridine acetate, pH 6 (300 ml) to 0.5 M pyridine acetate, pH 3.7 (300 ml). The fractions containing 2-acetamino-3-O-f/~L-1-(D-1-carbamoyl-3-N-aminoethyl-carbamoyl-propyl)-carbamoyl-ethyl7-carbamoyl-methylJ -2-deoxy-D-glucose-acetate - these fractions are tested and characterized against ninhydrin or high-voltage electrophoresis - freeze-dried. The conversion to hydrochloride form takes place in the following way: The lyophilisate is dissolved in 6 ml of 0.2 N HCl and chromatographed on a Bio-Gel P2 column, 2.5 x 90 cm, with water as eluent. Fractions containing 2-acetamino-3-O-2-L-1-(D-1-carbamoyl-3-N-aminoethyl-carbamoyl-propyl)-carbamoyl-ethyl-7-carbamoyl-methyl-2-deoxy-D-glucose hydrochloride are freeze-dried. The preparation is uniform in the high-voltage electrophoresis. When determining the continuities in the total hydrolysates, the molar ratio is 1 muramyl acid : 1 L-alanine : 1 D-glutamic acid : 1 ethylenediamine.

Example 5

Immediately after extraction, merozoites of the malaria agent Plasmodium knowlesi - the total yield from the blood of an infected Rhesus monkey (Method for extraction of merozoites: see G.H. Mitchel et al. (1975), Immunology 29, 397) - are suspended in a solution of 100 mg (0.17 mmol) 2-acet-amino-3-O-L-1-(D-1-carbamoyl-3-succinimidoxy-carbonyl-propyl)-carbamoyl-ethyl7-carbamoyl-methylJ -2-deoxy-D- glucose in 15 ml physiological buffer solution, pH 7.2. The suspension is incubated for one hour at 37°C. The conjugate is then sedimented by centrifugation. The sediment is washed by resuspension in physiological buffer solution, followed by new centrifugation.

The washed merozoite conjugate is suspended according to G.H. Mitchel (ref. see above) in 10% autologous Rhesus serum and freeze-dried.

The quantitative determination of muramyl dipeptide which is coupled to merosite takes place with the Morgan-Elson reaction and gives 40-60 ug of muramyl peptide per 1 mg merozoite.

Example 6

In a solution of 200 mg (0.34 mmol) of 2-acetamino-3-0-\l_ L-l-(D-l-carbamoyl-3-succinimidooxy-carbonyl-propyl)-carbamoyl-ethyl7-carbamoyl-methylj -2- deoxy- 10 qT lymphoblasts from CBA/J mice are suspended in D-glucose in 20 ml of phosphate-buffered physiological saline solution, pH 7.2. (The T-lymphoblasts are recovered in a "mixed lymphocyte culture" against C57BL/6 mouse stimulator cells according to L.C. Anderson et al., (1977), The Journal and Experimental Medicine, 146, 1124). The suspension is incubated for 90 minutes at room temperature. The lymphoblast conjugate is then sedimented by centrifugation and washed by resuspension in phosphate-buffered, physiological saline solution, followed by new centrifugation.

The quantitative determination of muramyl dipeptide which is coupled to T-lymphoblasts follows from the Morgan-Elson reaction (see example 1) and gives 60-70 ug of muramyl peptide per IO<7> T lymphoblasts.

Example 7

In a similar way as in example 1, bovine serum albumin is obtained coupled with

2-acetamido-3-O-\ D-1-^L-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl7-carbamoyl-ethyl J -2-deoxy-D-glucose,

2-benzoylamino-3-0-[D-l-^~L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbaryl)yl-ethyl-7-carbaryl-ethyl J -2-deoxy-a, B-D-glucose,

2-benzoylamino-3-0-[ /~L-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl-7-carbamoyl-methyl}-2-deoxy-α,6-D-glucose,

2-acetamido-3-0-|^ L-1-(D-1-carbamoylmethyl-carbamoyl-3-carboxypropyl)-carbamoylethyl7-carbamoylmethylJ - 2-deoxy-D-glucose,

2- benzmido-2-deoxy-3-O- (L-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoyl-propyl/-carbamoylmethyl J -D-glucopyranose,

3- 0-[/~L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoylethyl/-carbamoylmethyl]-2-oxy-2-propionamido-D-glucose and

2-acetamido-3-O-L-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoylpropyl-carbamoylmethyl J -2-deoxy-D-glucose. 2-acetamido-3-0- f/ L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoyl-2-hydroxyethyl/-carbamoylmethyl J -2-deoxy-D-glucose, 2-propionylamino-3-0- ^/ _ L-1-(D-1,3-dicarboxy-propyl)-carbamoylethyl7-carbamoylmethylJ -2-deoxy-D-glucose,

2-benzoylamino-3-O-£ L-1-(D-1-N-carbamoylmethyl-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl/-carbamoylmethylJ -2-deoxy-D-glucose,

2-acetamino-3-0-{D-1-^ L-1-carbamoyl-3-carboxy-propyl7~carbamoyl-2'-methyl-propyl7_carbamoylethyl J-2-deoxy-D-glucose, 2-benzoylamino-3-0-f/ ~L-1-(d,1,3-dicarboxy-propyl)-carbamoyl-ethyl-7-carbamoylmethyl J -2-deoxy-D-glucose,

2-acetamido-3-0- L-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoyl-21-methyl-propyl7-carbamoylmethyl^-2-deoxy-D-glucose, 2-acetamino-3-0- { D-1-/~L-1-(D-1-carbamoyl-3-(L-1-carboxy-ethyl)-carbamoyl-propyl)-carbamoylethyl7_carbamoylethyl ^-2-deoxy-glucose,

2-acetamino-3-0- \/_ L-l-(D-l,3-dicarboxy-propyl)-carbamoyl-2'-methyl-propyl7-carbamoyl-methylj -2-deoxy-D-glucose, 2-acetamino-3- 0- \ /~L-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoyl-N,N-tetramethylene7-carbamoyl-methyl J*-2-deoxy-D-glucose ,

2-acetamino-3-O-£ /~L-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoyl-methyl7-N-methyl-carbamoyl-methyl]-2-deoxy-D-glucose, or 2 -benzoylamino-3-0- [ £ L-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoyl-2<1->methyl-propyl7-carbamoyl-methylJ-2-deoxy-D-glucose.

With the Morgan-Elson reaction, each time you find approx. 60 vg of the relevant muramyl peptide in 1 mg of the relevant bovine serum albumin compound.

Example 8

In a similar way as in example 3, sheep erythrocyte membranes are obtained coupled with

2-acetamino-3-O-\D-l-/L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl/-carbamoyl-ethyl J -2-deoxy-D-glucose, with the Morgan-Elson reaction you find 25 yg of muramyl peptide per 1

mg erythrocyte membrane.

Example 9

In a solution of 100 mg of 2-acetamido-3-O-£D-l-/ L-l-(D-l-carbamoyl-3-succinimidoxycarbonyl-propyl)-carbamoyl-ethyl)-carbamoyl-ethyl J-2-deoxy-D-glucose in In 10 ml of phosphate-buffered physiological saline solution, pH 7.2, 10 g of canine mammary carcinoma cells are suspended. (the tumor cells are extracted after H,H. Sedlacek, H. Messmann and F.R. Seiler, Behring Inst. Mitt. no. 55, 349-355 (1974)). The suspension is incubated

for 90 minutes at room temperature. The tumor cell-muramyldipeptide conjugate is then sedimented by centrifugation and washed by resuspension in phosphate-buffered physiological saline solution, followed by centrifugation.

The quantitative determination of muramyl dipeptide linked to tumor cells follows the Morgan-Elson reaction (see example 1) and gives 60-80 µg of muramyl peptide per 10 7-mammary carcinoma cells.

Example 10

In a similar way as in example 6, T-lymphoblasts from CBA/J mice are obtained coupled with

2-acetamino-3-O-£d-1-/ L-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl 7-carbamoyl-ethyl J-2-deoxy-D-glucose.

With the Morgan-Elson reaction, there are 60-70 µg of muramyl peptide per

7

10 T-lymphoblasts.

In mice, rats and baboons, their immunization with the antigen derivative consisting of 2-acetylamino-3-0-£ D-1-/ L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl7-carbamoyl-ethyl J -2-deoxy-D-glucose and certain T-lymphoblasts a specific transplant tolerance, i.e. that thereby e.g. after organ transplantation, the casting of the transplanted organ is prevented.

Example 11

To 2 ml of a solution of tetanus toxoid in phosphate-buffered physiological saline solution is added 10 mg of 2-acetamido-3-0-£ L-1-(D -1-carbamoyl-3-succinimidoxy-carbonyl-propyl)-carbamoyl-ethyl7- carbamoyl-methylJ-2-deoxy-D-glucose. The toxoid concentration amounts to 3 mg/ml. The solution is stirred at 4°C for 4 hours. Low molecular weight reaction products are then separated from the tetanus toxoid-muramylpeptide conjugate by ultrafiltration, the ultrafiltered solution is frozen and stored before use at -20°C. The quantitative determination (Morgan-Elson reaction) gives 50 ug of muramyl peptide per 1 mg of tetanus toxoid-muramyl peptide conjugate.

Example 12

To 10 ml of a solution of cholera toxoid of Vibrio cholera in phosphate-buffered physiological saline solution, 50 mg of 2-acetamido-3-0-[/"l-1-(D-l-carbamoyl-3-succinimidooxycarbonyl-propyl)-carbamoyl-ethyl7 are added -carbamoyl-methyl J -2-deoxy-D-glucose. The protein concentration in the solution is 0.6 mg/ml. The solution is stirred for 4 hours at 4°C. The low molecular weight reaction products are then separated from the cholera toxoid-muramylpeptide conjugate by ultrafiltration,

the ultrafiltered solution is frozen and stored before use at -20°C. The quantitative determination (Morgan-Elson reaction) gives 40-50 ug of muramyl peptide per 1 ml cholera toxoid-muramyl dipeptide conjugate.

Albino guinea pigs are immunized once subcutaneously with 22.2 µg/ml cholera toxoid-(2-acetylamino-3-0-f/"L-1-^D-1-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl-7-carbamoylmethylJ -2- desoxy-D-glucose) conjugate and then charged with 4.8 times the amount of the Blauungs dose i.c. Out of a group of 12 immunized animals, 3 are shown to be protected. In contrast, under the same conditions, none of 10 animals of a group that has only been vaccinated is protected with 22.2 ug/ml cholera toxoid.

Example 13

20 mg of a synthetic eicosa peptide, which is identical with the C-terminal sequence to human chorionic gonadotropin (HCG) reference: C.H. Schneider, K. Blaser, Ch. Pfeuti and E. Gruden, Febs Lettecs 50, 272 (1975)) and 30 mg of 2-acet-amido-3-O-£/~L-1-(D-1-carbamoyl-3-succinimidoxy-carbonyl-propyl)-carbamoyl-ethyl /-carbamoyl-methylJ -2-deoxy-D-glucose is dissolved in 2 ml of sodium phosphate buffer solution, pH 7.2. The solution is stirred for 6 hours at room temperature. The eicosapeptide-muramyldipeptide conjugate is then isolated by chromatography on a Sephadex G-25 column (1.4 x 40 cm) with water as eluent and freeze-dried. The quantitative amino acid analysis shows that the 1-muramyldipeptide molecule is bound to the 1-eicosapeptide molecule.

Example 14

In a similar way as in example 13, you get

the C-terminal eicosapeptide fragment of human chorionic gonadotropin linked to

2-acetamino-3-O-[D-1-[L-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl-7-carbamoyl-ethyl]-2-deoxy-D-glucose. The quantitative amino acid analysis shows that the 1-muramyldipeptide molecule is bound to the 1-eicosapeptide molecule.

Various of the above-mentioned muramyl peptides or forms thereof coupled with spacers are new. They can be produced according to the following examples:

Example A

A solution of 3.4 g of benzyl-2-acetamino-3-0-{D-l-l L-l-/D-l-carbamoyl-3-(L-l-carboxy-ethyl-carbamoyl)-propyl/-carbamoyl-ethylJ -carbamoyl-ethylj -2-deoxy-α-D-glucopyranoside benzyl ester in 100 ml of methanol/distilled water 2/1 is hydrated in the presence of 0.3 g of 10% palladium on charcoal at normal pressure and 45°C for 24 hours. The catalyst is filtered off, and the filtrate is evaporated. The residue is dissolved in 40 ml of water and this solution is extracted 3 times with 40 ml of water saturated with secondary butanol. The organic phase is washed a further 3 times with 40 ml of water saturated with secondary butanol. The water solutions are evaporated after being combined, the remainder is dissolved in some distilled water and freeze-dried. This gives 2-acetamino-3-0-£ D-l-£ L-l-/~D-l-carbamoyl-3-(L-l-carboxy-ethyl-carbamoyl )-propyl7-carbamoyl-ethyl j-carbamoyl-ethylJ -2-deoxy-D -glucose as a white powder with / <*7n^ = + 9° + 1°

(distilled water, c = 1.090).

The starting material used is prepared as follows: A solution of 6.1 g of benzyl-2-acetamino-3-0- [ D-l-/ L-l-(D-l-carbamoyl-3-carboxypropyl)-carbamoyl-ethyl_/- carbamoyl-ethylj -2-deoxy-α-D-glucopyranoside monohydrate and 3.5 g of L-alanine benzyl ester p-toluenesulfonate in 30 ml of N ,N-dimethylformamide is added to 1.4 ml of triethylamine, 1.1 g of N-hydroxysuccinimide and 2.3 g of dicyclohexylcarbodiimide and stirred for 48 hours at room temperature. The crystallized dicyclohexylurea is filtered off and washed with 10 ml of N,N-dimethylformamide and the filtrate is evaporated to dryness. The residue is suspended in 100 ml of water, stirred for one hour at 0°C, the insoluble parts are suctioned off, washed with some ice water and dried. The product is dissolved in methanol, precipitated with twice the amount of acetic acid, filtered off with suction, waxed with some acetic acid and dried:

/"a/^0 = + 72° + 1° (methanol, c = 0.998).

Example B

A 5% solution of benzyl-2-acetamido-3-0-£(L-1-/ D-1-(L-1-carboxy-ethyl)-carbamoyl-3-carboxypropyl-7~ carbamoyl-ethylJ -carbamoyl-methylj -2-desoxy- α-D-glucopyranoside in tetrahydrofuran/water 2/1 is hydrogenated in the presence of 10% 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 the filtrate is freeze-dried. This gives 2-acetamido -3-O-£{l-1-/ D-l-(L-l-carboxy-ethyl)-carbamoyl-3-carboxypropyl/-carbamoyl-ethylJ -carbamoyl-methyl^-2-deoxy-D-glucose as a white powder. The Rf value in the thin-layer chromatogram = 0.21 (ethyl acetate/n-butanol/pyridine/acetic acid/water 42:21:21:6:10).

In a similar way, the derivative with the real muramic acid is obtained.

The starting material is prepared in the following way: 5.68 g of N-tert.-butoxycarbonyl-L-alanyl-D-y-benzyl-glutamyl-L-alanine benzyl ester is 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.

The solution is diluted with 50 ml of tetrahydrofuran, cooled

in an ice bath and neutralized with triethylamine. After addition to the solution of 3.7 g of benzyl-2-acetamido-3-carboxymethyl-2-deoxy-ot-D-glucopyranoside and 1.38 ml of triethylamine in 100 ml of tetrahydrofuran, the mixture 2,6 9 2-ethoxy-N -ethoxycarbonyl-1,2-dihydroquinoline and the mixture is allowed to stand for 24 hours at room temperature. After evaporation of the solvent, the residue is dissolved in chloroform/methanol 9/1, washed with water, ice-cold 2N hydrochloric acid, water, a saturated sodium hydrogen carbonate solution and water, filtered and freed from solvent. This gives benzyl-2-acetamido-3-O-£(1-1-/~D-1-(L-1-carboxy-ethyl)-carbamoyl-3-carboxy-propyl7-carbamoylethyl^-carbamoyl-methyl} -2- Desoxy-α-D-glucopyranoside as a colorless powder Rf value =0.48 (in an identical system).

The starting material used can be manufactured

in the following way:

7.15 g of N-tert.-butoxycarbonyl-L-alanyl-D-glutamic acid-γ-benzyl ester and 6.15 g of L-alanine benzyl ester p-toluenesulfonate are dissolved in 100 ml of anhydrous dimethylformamide. The mixture is cooled in an ice bath and, with stirring, 4.03 g of N-hydroxysuccinimide, 3.61 g of dicyclohexylcarbodiimide and finally 1.95 ml of N-methylmorpholine are successively added. After 6 hours of stirring at 0°C and 15 hours at room temperature, the suspension is cooled, the precipitate (dicyclohexylurea and morpholine hydrochloride) is filtered off and the filtrate is evaporated. The residue is taken up in acetic acid ethyl ester, washed several times with water, 1N citric acid and 1N sodium bicarbonate and finally

equal to water. The ethyl acetate solution is dried, evaporated, and the crystalline residue is recrystallized from ethyl acetate/petroleum ether 1/1. Sm.p. 135-136°C, Z~a/D° = "9° + 1° (c = 1, methanol), Rf value = 0.82 (in the above system) and 0.86 (acetonitrile/water 3: 1).

Instead of alanine, dipeptide can be used

similarly extended with other natural L-amino acids.

Example 15

7.5 x 10 g of killed trypanosoma cruzi parasites (causing agent of Chagas disease) are suspended in a solution of 50 mg of 2-acetamino-3-0-L-1-(D-carbamoyl-3-carboxypropyl)-carbamoylethyl/-carbamoylmethylJ- 2-deoxy-D-glucose-N-hydroxysuccinimide ester in 6 ml physiological buffer solution. The suspension is incubated for two hours at 37°C. The parasites conjugated with muramyl dipeptide are then sedimented by centrifugation. The sediment is washed with a new suspension in physiological buffer solution and a new centrifugation. The waxy parasite-muramyl dipeptide conjugate is suspended in physiological buffer solution and used for immunization.

The quantitative determination of muramyl dipeptide coupled to trypanosomes takes place as indicated in example 1 with the Morgan-Elson reaction and gives 50-70 mg of muramyl dipeptide per mg trypanosomes.

Claims (2)

1. Method for the production of therapeutically effective antigen derivatives consisting of an antigen, to which per antigen molecule is bound at least one and at most so many muramyl peptide molecules that the antigenic character is retained, as well as optionally a carrier, these components being linked together covalently either directly via oxycarbonyl, iminocarbonyl or carbonylthio groups or optionally indirectly via bridging residues of an aliphatic compound with at least two amino groups, at least one amino and at least one carboxy group or at least two carboxy groups, since the bond between bridge link and antigen as well as bridge link and muramyl peptide likewise takes place via oxycarbonyl, iminocarbonyl or carbonylthio groups, and since the muramyl peptide in unbound form has the structure given in formula Ila X means a carbonyl or carbonyloxy group, R1', R4 and R6 independently mean hydrogen, trimethylsilyl or alkanoyl, R2 means a phenyl radical or unsubstituted or lower alkyl substituted with hydroxy, carboxy, lower alkoxy and/or lower alkoxycarbonyl, R^, Ry and Rg independently mean hydrogen or lower alkyl, Ro0 means a phenyl radical or unsubstituted or with hydroxy, halogen, amino, mercapto, lower alkylthio or a phenyl radical substituted lower alkyl, or R_ and R0 together mean the alkylene with 3 or 4 carbon atoms, and the radicals R-^gf R-^ and R.^ independently mean carboxy, trimethylsilyl-silyloxycarbonyl, unsubstituted or with a phenyl residue substituted lower alkoxycarbonyl or carbamoyl, which is unsubstituted or substituted on the nitrogen atom with unsubstituted or with carboxy, lower alkoxycarbonyl or carbamoyl substituted lower alkyl or R^^ also means hydrogen, with the precaution that at least one free hydroxy, amino, mercapto or carboxy group is present in a starting compound of formula Ila, characterized by condensing an optionally bridged antigen with optionally bridged muramyl peptides of formula Ila, one of the two reaction components have at least one free amino, hydroxy and/or mercapto group and the other has at least one carboxylic acid group, and if desired, a compound formed analogously to the above is condensed onto an optionally bridged carrier, one of the two reaction components having at least one free amino, hydroxy and/or mercapto group, and the other having at least one carboxylic acid group. 2. Process according to claim 1, characterized in that 2-acetamine-3-0-(d-1-/L-l-(D-l-carbamoyl-3-carboxylpropyl)-carbamoylethyl/-carbamoylethyl)-2-deoxy-D-glucose is used or 2-acetamido-3-0-£/~L-1-(D-1-carbamoyl-3-carboxypropyl)-carbamoylethyl,7-carbamoylmethyl J -2-deoxy-D-glucose as the mura-mylpeptide component, synthetic eicosapeptide, which is identical to the C-terminal sequence of the human chorionic gonadotropin (HCG) as an antigenic component and possibly tetanus toxoid as a carrier.