IE48735B1 - Muramyl-peptide ester compounds and their application in pharmaceutical compositions and laboratory reagents - Google Patents

Description

The invention relates to novel products endowed with biological and pharmacological properties of great value, which can be applied either for constituting standardized laboratory reactants for the comparative study of similar biological properties of other compounds, or for the constitution of novel medicaments for human or veterinary use.

The invention relates more particularly to novel products capable of modifying the immune responses in warm10 blooded creatures. More specifically again, it relates to novel products capable of stimulating the immune responses which bring into play at least one and preferably all of the mechanisms constituting the humoral immune response support (opsonins, antibodies) or the cell mediated immune response.

These mechanisms may be specific (their intervention depending on prior vaccination by a given antigen mixture, and more 8 7 3 δ especially derived from pathogenic agents)^ or non-specific (such as are induced by agents such as BCG, corynebacteria or endotoxins).

It is known that organisms of warm-blooded creatures comprise several types of cells constituting what could he called several lines of defense with respect to the various aggressions to which these organisms may be subject.

The first line of defense brings into play, as 1C is well-known, the leucocytes and macrophages in the blood stream, capable, when they encounter a foreign a.rent such as an antigen, of phagocyting it and often of destroying it. This phagocytosis can he facilitated notably by non-specific constituents of the serum (opsonins), even by external interventions lending to activation of the macrophages.

A second line of defense of the organism brings into play mechanisms triggered by antigenic constituents of the foreign agent. These immune mechanisms bring '0 into play cellular differentiations in at least one of two of the folloY/ing principal directions.

A first type of immune mechanism brings into action specialized cells (B lymphocytes) which are precursors of the cells which secrete immunoglobulins or antibodies. These immunoglobulins or antibodies play an important role in the battle against infection caused by bacteria or other micro-organisms which replicate in the humoral fluids, or again to neutralize toxins or the like.

With this type of action, specific to the antigen concerned, is added an indirect action by circulating mediators, which is manifested by activation of the cells responsible for phagocytosis, thus reinforcing the non-specific defenses of the organism.

The second type of immune mechanism brings into play specifically sensitized cells belonging to the lymphocyte line (T cells), which interact on the previously indicated B cells, or on the macrophages leading to activa10 tion of the latter or of other non-specific cells.

In the face of these various mechanisms presented diagrammatically below, it is possible to distinguish two types of infection according to the manner in which they develop and the mechanisms which are obliged to combat them. Thus, for certain infections, a simple phagocytosis, facilitated possibly by specific humoral or non-humoral factors, permits the destruction of the infectious agents. For other types of infections, phagocytosis is not sufficient.

The phagocyted infectious agent is not destroyed lo and even continues to act within the phagocyting cell, resulting on the contrary in the destruction of the latter.

In this case, to prevent intracellular growth of the .infectious agents, it is necessary to activate the phagocyting cells according to the above-indicated processes. .-5 When it is desired to study the anti-infectious properties of novel products, the Klebsiella, as infecting strains with extracellular replication, are particularly representative. These bacteria have in fact a capsule of large size which only permits effective phagocytosis by macrophages. This mechanism applying non-specific agents, the results obtained with Klebsiella may be extended to micro-organisms which replicate in the same manner.

An experimental procedure was studied'by CHEDID 1. et col. in Proc. Natl. Acad. Sci. USA, 1977, 74 : P089, which permits the stimulating effect or not to be observed of substances studied with regard to immun e defenses according as they protect or not the mice to which they are injected , the mice being inoculated by a dose of Klebsiella Pneumoniae which results in the death of almost all of the controls.

When it is desired to study the effects of such substances on infections whose agents multiply intracellularly, the Listeria are among the micro-organisms most used, and notably Listeria monocytogenes. The latter are the basis of biological tests which have become Ό classical, both for the study of specific and non-specific reactions.

By way of example, may be mentioned the experimental procedure described by MEDINA, VAS and ROBSON (J. Immunol., 1979, 114, 17?0), which permits the stimulant or non-stimulant '5 effect to be observed of substances studied with regard to immun e defenses, according as they protect or not mice into which they are injected against a dose of Listeria monocytogenes which results in the death of almost all of the controls, or according as they result or not in the short-term destruction of said micro-organisms.

It is more and more affirmed today that cell mediated immunity constitutes a complex defense system of organisms coming into play in numerous situations, not only with respect to intracellular micro-organisms but also with respect to neoplastic growth and the multiplication of numerous fungal, parasitic agents, etc.; it is this lu system which also is responsible in the rejection of-grafts and numerous auto-immune processes.

In a general way, reference may be made, as regards all oi the above-mentioned problems, to, for example, the articles of Priscilla A. CAMPBELL, entitled Immunocompe15 tent Cells in Resistance to Bacterial Infections, which appeared in Bacteriological Reviews, June 1976, p. 284-313, of G.B. I.1ACKANESS, entitled Cellular Immunity, which appeared in the Annals of the Pasteur Institute, 1971, 120, 421-437, of G.H. WERNER et coll., entitled Toxicological Aspects of Immunopotentiation by Adjuvants and Immunostiinulating Substances, which appeared in the bulletin of the PASTEUR INSTITUTE, volume 75, No. 1 of January 1977, and in a report of a scientific group of the World Health Organization, entitled Reponses immunitaires 2; a suDport cellulaire , which appeared in Org. mond. Sante, Ser. Rapp, techn., 1969, Ho. 423.

It results from the foregoing that the placing at the disposal of specialists, notably of the biologist ana of the clinician, of compositions or products capable of stimulating immune defenses of the aboveindicated types, can have capital importance both for the study of other substances at the research level, both fundamental and applied, and in the domain of human or veterinary therapeutics.

Certain agents are already known which are capable of stimulating non-specifically these various immune responses, both for cellular mediation and for humoral mediation. Thus, for example, bacterial lipopolysaccharides (IPS), are known for their protective activity with respect to humoral or cellular infections. The US possess, in addition, non-specific immunological adjuvant properries, Ί;. in that they facilitate an increase in the ratio of specific antibody synthesis by an organism subjected to antigen aggression, of any nature.

It is known in the same way that mycobacteria, and more particularly Calmette-Guerin bacillus (CGB), . ;< possess powerful non-specific immunostimulating properties.

However, there can be no question of contemplating the utilization of IPS in therapeutics, considering their extreme toxicity which is well known. CGB itself is not free of numerous drawbacks, which can be demonstrated, ..5 for example, in the animal, notably at the level of the increase in sensitivity of the host to endotoxins, of the production of a hypersensitivity to tuberculin, of the induction of granuloma, of hyperplasia of the lymphoid tissue and, notably in the rat, of polyarthritis.

It is known that numerous researchers are engaged in the study of extracts capable of being obtained from mycobacteria, for the purpose of obtaining purified or detoxified agents retaining the biological properties of value of CGB or LPS, whilst being free of the above-mention ed drawbacks. The development of these researches has led to small molecules, representing in themselves a C considerable contribution to the arsenal of substances which the researcher and clinician has available, which are now acceptable to chemical synthesis, which are practically devoid of toxicity and whose very powerful immunological adjuvant activity, is manifested even whan they are administered to a host, in the absence of an oily support, as was necessary as regards more particularly the fractions obtained by extraction,· notably from mycobacteria.

The above-said small molecules are, as is now well known, constituted by N-acyl-murarayl-peptides or certain Ί of their substitution derivatives, characterized by an N-acyl-muramic group to which is attached a peptide chain comprising a first aminoacyl residue directly linked to the N-acyl-muramic acid, constituted by a glycyl residue, or derivative of another levorotatoiy amino acid, preferably an i? 1-alanyl or L-seryl residue, and a second aminoacyl group, linked to the first, derived frcm D-glutamic acid (DE 2450355).

Derivatives characterised by a certain analogy of structure, particularly N-acyl-normuramyl-dipeptides, in which the second aminoacyl residue of the dipeptide chain is constituted by D-isoasparagine or D-isoglutamine, and shown as having adjuvant properties are described in French Patent Publication FR-A-2 349 600.

Furthermore French Patent Publication FR-A-2 361 902 describes similar compounds, in which the second aminoacyl residue of the dipeptide chain is constituted by glutamic acid, the carboxyl functions of which may be substituted by different functions, among which are ester functions, more particularly those resulting from the esterification of the glutamyl carboxyl groups by lower alkanols. The derivatives resulting from the substitution of the different functions include the esters of the aforesaid N-acyl-muramyl-peptides, in which the acyl group is an acetyl group, which are not embraced by the general formula which appears in French Patent Publication FR-A-2 361 902.

The most active products as immunological adjuvants are constituted by the M-acetyl-muramyl-peptides, whose first aminoacyl residue is a L-alanyl or L-seryl and the second a D-glutamyl residue whose a carboxyl function may be either free, or esterified or amidated 5 (the amide group being itself capable of carrying substitution groups) and whose γ carboxyl function may also be cither free or amidated or esterified, or again enters a longer peptide chain. The most representative product of the series of M-acyl-murarayl-peptides is 1C' constituted by M-acyl-muramyl-L-alanyl-L-isoglutamine (MDP). Various researches have however shown that the immunological adjuvant activity could he maintained, to a greater or lesser extent, by the introduction of various substitutions into the muramyl group or by the replacement of the first amino-acyl residue by others, derived from various amino acids of the L-series.

It has already been observed for certain of the muramyl-peptides of the type concerned, and more particularly as regards N-acetyl-muramyl-L-alanyl-D-isoglutamine already mentioned (ittDjP) or for the corresponding non-araidated derivative, namely M-acetyl-muramyl-L-alanyl-D-glutamic acid (MLPA) or again oC -esters of IfflPA whose ester functions contain at most 3 carbon atoms, that they possessed in addition already very considerable anti-infectious properties, which are manifested more particularly with micro-organisms. with humoral multiplication (Klebsiella) (DE-A 2710454).

It is known nevertheless that many possible derivatives of the muramyl-peptide type, carrying at most low molecular weight substituents (containing for example at most 3 carbon atoms) are completely devoid of anti5 infectious properties. As evidence of this reference can be made to the article by L. Chedid et al published in Proc. Natl. Acad. Sci. OSA, vol. 74, No. 5, pages 2089-2093 of May 1977, entitled, Enhancement of nonspecific immunity to Klebsiella pneumoniae infection by a synthetic immuno-adju10 vant (N-acetylmuramyl-L-alanyl-D-isoglutamine) and several analogs.

The invention arises from the discovery that introducing of a lipophilic chain (that is to say, an essentially non-polar hydrocarbon chain),- to the if carboxylic function of the glutamyl residue led to the production of a novel category of derivatives of the muramyl-peptide type characterized notably by powerful anti-infectious activity. It will be observed that the fixing of the lipophilic chain on to the peptide chain, which characterizes the novel products according to the invention, goes entirely contrary to what it has been possible to observe in water-soluble adjuvant natural 2(.. products which can be produced from cellular walls of micro-organisms, such as mycobacteria, and which include in their structure at least one peptidoglycan fragment. In fact, certain of these natural products can include lipo pbilic chains fixed indirectly to certain saccharide units of the peptidoglycan, but have never been found on the peptide chains of the latter.

The different properties of the products according to the invention and more particularly their antimfectious properties are in addition all the more unexpected as the short -monoesters of the KDPA do not possess anti-infectious properties with regards to the humoral immune defense mechanism.

Products according to the invention demonstrate in addition a new advance with respect to products already known, in that they show themselves to be capable not only of exerting a non-specific stimulating effect of the humoral immune defenses, but also of cell mediated immunity.

The novel compounds according to the invention correspond to the general formula R - CH - CO - X - NH - CH - CO - Y I CHI 2 CO - (A)n - Z in which the substituents R, R·^, R2, R^, Rg, X, Y and Z 5 have the following significances: - R is either a hydrogen atom or a methyl group, - R^ is a hydrogen atom, an alkyl group having at most 4 carbon atoms, a substituted or unsubstituted aryl or alkylaryl group comprising at the most 10 carbon atoms, - R2 is a methyl group, - R^ is a hydrogen atom or an acyl radical comprising at most 4 carbon atoms, - Rg is a hydrogen atcm or a substituted or unsubstituted acyl radical, which is saturated or unsaturated containing from l· to 90 carbon atoms, and optionally carrying functional groups selected from: hydroxyl, carboxyl, carbonyl, amino, cyclopropane and methoxy, - X is an aminoacyl residue selected from: L-alanyl, L-arginyl, L-asparagyl, L-aspartyl, L-cysteinyl, L-glutaminyl, L-glutamyl, glycyl, L-histidyl, L-hydroxyprolyl, L-isoleucyl, L-leucyl, L-lysyl, L-methionyl, L-ornithyl, L-phenylalanyl, L-prolyl, L-seryl, 1-threonyl, L-tryptophanyl, L-tyrosyl and L-valyl, - Y is either -OH, or an alkoxy radical comprising from to 10 carbon atoms, or -KH2, the hydrogens of the amino group being optionally substituted by alkyl residues of 1 to 10 carbon atoms, or an aminoacyl residue, - A is an aminoacyl residue of the group indicated above for X, or, for the last of the peptide chain, an aminoalcohol residue (-KK-CH-CHg-O-) corresponding to the aminoacyls (-iiH-CH-CO-), it being understood that the A groups present in the same compound may be identical or different n only representing the total number of A groups in this /U compound, - n is zero or 1, 2 or 3, - Z is a group -OR’, -NHR‘, -OCH^-CH^-COR' or -OCH2-CHOH-CH 0 COR’ when the last A residue of the peptide chain is an aminoacyl, and -COR' when this last A xs an aminoalcohol, in which R' is saturated or unsaturated alkyl and optionally containing functional groups selected from: hydroxyl, carbonyl, carboxyl, cyclopropane and optionally substituted aryl, the group Z containing at least 4 carbons and being likely to include up to 90 carbon atans with the proviso that when R is hydrogen, the (A)n - Z group is a -NH-CHCCH^l-COO-CHj-CBOH-CHjO-R° group, with R° being a mycolic acid comprising fran 80 to 90 carbon atoms.

In this formula, the second aminoacyl group of the peptide chain connected to the group of the muramyl type is the D-glutamyl residue. The first aminoacyl group (denoted by X) may, on the other hand, be selected from among the various aminoacyl groups mentioned above. Among the compounds of formula (l) those are preferred in which the first aminoacyl group is L-alanyl, a second type of preferred compound is that in which this aminoacyl is L-seryl, another type of preferred compound is that in which the aminoacyl is the glycyl group.

Also advantageous are the compounds in which the first aminoacyl group is L-prolyl, L-threonyl or L-valyl.

The substituents at the ft position of the glutamyl residue all have in common a hydrocarbon group R' of which a feature is that on the whole, whatever the exact structure of this group, it has a tendency either to confer a certain lipo philic character on the compound concerned, or to increase this character when, independently of the R* group, it is already manifested.

The lipophilic character of the hydrocarbon groups corresponding to the definition of R' increases quite obviously with the number of the carbon atoms of the group when one starts from the shortest substituents, that is to say, in the present case, the groups R' having 4 carbon atoms.

In theory, the number of carbon atoms comprised in R' is nox limited upward. 48735 Nonetheless, in practice, the increase in the number of caruon atoms, beyond a certain threshold, does not contribute advantages and necessitates the application of reactants which are difficult to obtain commercially.

In practice, the group R’ does not contain more than a certain number of carbon atoms, and preferably not more than 90. The lipophilic character reaches an advantageous level ac soon as R' contains about ten carbons.

Between the B-glutamyl residue and the group R’ may be inserted one or several additional aminoacyl residues denoted in the general formula by A.

Preferably, these aminoacyls are selected from: alanyl, leucyl, lysyl, qlycyl, valyl glutamyl and isoleucyl. □.5 In particularly preferred manner, the first aminoacyl fixed at the £ of the B-glutamyl is 1-alanyl. Also hy preference, this aminoacyl is 1-lysyl or L-gLutamyl.

The number of aminoacyls between the B-glutamyl and the R' group may vary from 0 to 3, preferably however it is either 0, or 1, or 2.

If necessary, the last aminoacyl can be replaced cy the amihoalcohol residue of the same carbon structure as the aminoacyl, the linkage with the R' group then taking the form of an ester of this aminoalcohol with an acyl -CO-R'.

At the CC position of the B-glutamyl residue, the modifications or possible substitutions are more limited than at the 3 position of this same group.

The Y substituent can firstly represent the -OH radical, that is to say that the carboxylic function of glutamic acid again occurs. It can also he the amide form of this acid, that is to say the isoglutaminyl form, Y being -KK2, one at least of the hydrogen atoms of the amino group being capable of being substituted by short alkyl residues comprising from 1 to 10 carbon atoms. It can again be esterified forms of the acid, Y then being an alkoxy, comprising from 1 to 10 carbon atoms.

In a preferred form, Y is a hydroxyl.

In another preferred form, Y is -NH2· ' Another preferred form is constituted by the case where Y is either -OCH^, or -OC^Hg.

In the most customary preferred form, that is to say that corresponding to the muramic acid structure, ti is -CHj. In another preferred form, the group H is hydrogen; the structure of the lower homologue denoted by the name nor-muramic acid is then found.

The glycoside linkage of the saccharide portion m the products according to the invention can be present in ϋ- or fi) form. The osidic residue can also receive different substituents of which the prior literature relating to adjuvant agents of muramyl-peptide type, has riven a certain number of examples. In particular, the literature describes products whose functional groups . 48735 of the ocidic residue are blocked*' by the formation of ester or ether groups on the hydroxyls, or of amide groups on the amino radical at the 2 position.

In the general formula of the products according 5 to the invention, the substituents of the glucopyranoside cycle have been denoted by R2, and Rg. The various positions do not present the same possibilities of substitution, the 6 position being that for which the greatest latitude is offered.

Preferred compounds are those in which one or several of the substituents R^, R^ and Rg, independently of one another or simultaneously, are hydrogen.

Advantageous compounds are also those for which is the succinyl group -CO-(CH2)2-C0gH or the acetyl 1£ group.

Preferred compounds are also those for which 11& is an acyl radical containing from 1 to 4 carbon·,atoms, and notably the acetyl (-COCH^), succinyl(-C0(CH2)2-C02H), radicals or again those for which Rg is the mycoloyl group (about Cgj·, to Co[)) or corynomycoloyl (C^).

Among the compounds according to the invention are particularly preferred those for which Rir R^, Rg are simultaneously a hydrogen atom, R is -CH^, X is L-alanyl, Y is -NH2 and (A)n - Z is an esterified or amidified L-alanyl residue an esterified or amidified L-lysyl residue an ester residue.

In these groups (A) ^-Z, the ester or amide residues are preferably those of the hydrocarbon groups butyl, decyl, pentadecyl, eicosyl, benzyl, or a residue derived from an acid of the mycolic type such as glyceryl-mycolate.

Particularly preferred (a)r “ z groups are those of the formulae -NH-CH(CH^)-COO-(CH2)1g-CH3 10 -Iffl-CH(CH5)-CONH-(CH2)g-CH5 -MH-CH(CH^)-COO-(CH2)g-CH5 -NH-CH(CH^)-C00-(CH2) 14~CH3 -NH-CH(CHj)-COO-(CH2)3~CH3 -RH-CH(CH3)-COO-CH2-CgH5 -NH-CH(CH3)-COO-CH2-CHOH-CH2-O-R°, with R° corresponding to the 15 micolic acid comprising from 80 to 90 carbons, -NH-CH[(CH2)4-NH2]-COO-(CH2)g-CHj -o-(ch2)3-ch3 -o-(ch2)9-ch3 Preferred compounds according to the invention are notably the following -b-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanyluecylamide of the formula CHgOH ' INH-C0CH3 h,c-ch-conh-ch-conh-ch-conh5 I .1.ch3 (ch2)2 CONH-CH-CONH-(CHg)g-CH3 ' - K-acetyl-muramyl-L-alanyl-D-isoglutaminyl-decyl-ester of the formula It .?O H,C-CH-CONH-CH-CONH-OH-CONH> ' , · , άCH3 ?H2 2 coo-(ch2)9-ch3 β -D-p.-aminophenyl-N-acetyl-muramyl-l-alanyl-D-isoglu taminyl-L-alanyl-decyl-ester of the formula ch2oh NH-COCH, H,C-CH-CONH-CH-CONH-CH-CONH„ CH, (0¾ CONH-CH-COO-(CH2)g-CHj CHj - N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-Ii-alanylpentadecyl-ester of the formula H,C-CH-CONH-CH-CONH-CH-CONH, J tit (<M CONH-CH-COO-(CH2)14-CH5 CH, CH, - N-acetyl-muran.yl-L-alanyl-D-isoglutaminyl-l-alanyleicooyl-ester of the formula H,G-GH-CONH-CH-CONH-CH-CONHn -1 ’ , · . * CH3 (?H2}2 CONH-CH-COO-(CH2)t g-CH3 - N-acetyl-muramyl-l-alanyl-D-isoglutaminyl-Ii-alanyl-j benzyl-ester of the formula HjC-CH-CONII-CH-CONH-CH-CONHg ch3 (ch2)2 CONH-CH-COO-CHo-CrH,ι <2 b 5 ch3 - N-acetyl-muramyl-l-alanyl-B-isoglutaminyl-L-alanylD-glyceryl-mycolate of the formula h3c-ch-conh-ch-conh-ch-conh2 CH3 (ch2)2 CONH-CH-COO-CH_-CHOH-GH0-OR° I <· t CH3 H° being a mycolic acid radical containing from 80 to 9U carbon atoms; - N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-lysinedecvl-ester of the formula H,C-CH-CONH-CH-COHH-CH-CONH„ ι ι 2 CH, (CH2)2 CO-NH-CH-COO-(CH2)g-CH3 (ch2)4 NH„ - 1,-acetyl-muramyl-L-alanyl-D-isoglutaminyl- butylester of the formula H,C-CH-C0NH-CH-C0NH-CH-C0NHo ? 1 ,' , * ch3 (ch2)2 coo-(ch2)3-ch3 - K-acetyl-muramyl-L-alanyl-D-glutamyl- HjC-CH-CONH-OH-CONH-CH-COO-CH, ch3 (ch3)2 coo-(ch2)3-ch3 1C - K-acetyl-muramyl-l-alanyl-D-glutamyl- oc -methyl-ester-decyl-ester of the formula H,C-CH-CONH-CH-CONH-CH-COO-CH, > , > , 7 CH3 (θΗ2}2 COO-(CH2)g-CH3 The products according to the invention are prepared by synthesis. If necessary, certain of the fragments used for the syntheses can be derived from natural products.

To arrive at the same compound, various routes are possible. In all cases, the synthesis includes a series of steps in the course of which the various fragments constituting the structure of the whole of the compoundc according to the invention are progressively assembled.

The principal differences between the possible routes is situated in the sequence selected for the assembly of the fragments. The reaction methods leading to the fastening of one fragment to the one or more contiguous fragments are on the whole little modified by the order in vzhich this integration is conducted, it being well understood that this order depends, on the one hand, on the selection of functional groups which react and which, consequently, must be freed for the step concerned, and on the other hand, the choice of the groups which must’ be blocked in order not to intervene in the course of this same step. i5 The preparation of the products according to the invention can be done from the corresponding compounds of the muramyl-peptide type. The production of the latter has been described in numerous publications. If necessary, for those whose preparation does not appear expressly in the literature, notably for the various modifications corresponding to the substitutions of the muramyl group or of similar groups, they can be obtained by following the conventional methods of preparation of corresponding derivatives of oligosaccharide chemistry.

In the same way, the constitution of the peptide chain connected to the muramic acid is carried out according to traditional -methods in the synthesis of peptides. 3elovz are given in succinct manner the main indications relating to various operations which can be applied for synthesizing the products according to the invention, first by envisaging separately each step, then by indicating some preferred typical sequences. a) Formation of muramic acid or the like To obtain the analogues of N-acetyl-muramic acid of the formula in which If has the previously indicated meaning, it is possible to start from a derivative of N-aeetyl-2-glucosaaine v/hose hydroxyls in position 1, 4 and 6 are blocked in traditional manner. The method of preparation of such a derivative, the benzyl-2~acetaraido-4,6-0-benzylidene20 2-deoxy-L-glucopyranoside, is described notably by P.K.

GROoS and If.«7. JEANLOZ (J.Org. Chem. 1967, 32, 2761).

The formation of N-acetyl-muramic acid (R = CHj) or of or.e of its analogues can be' effected in the way described in French Patent Application Nos. 74 22909 or 76 19236 (respectively, for these applications, R = CHj and R = H) taking the method described by OZAWA and JEANLOZ (J. Org. Chem., 1965, 36, 448).

This formation comprises for example the preparation of a sodium salt of the hydroxyl at the 3 position and the subsequent condensation of the sodium derivative with the salt or the ester of an f halogenated acid such as 2-chloro-propionic acid or chloroacetic acid to take up again the case of the two previously indicated patent applications. The halogen compound used in the l· form can oe prepared by the method described by SIHAY et al (J. Biol. Chem., 1972, 247, 391). By using the appropriate halogenated acids, it is possible to prepare all the derivatives corresponding to the various significances of B. Thus, to introduce an R group with 4 carbons, the salts or esters of 2-chloro-butyric acid may be used. when a halogenated acid ester is utilized, in order to be able to proceed with the subsequent peptide condensation, the carboxylic function may be freed by suitable hydrolysis. b) Substitution on the saccharide residue R - CH - CO • · · The substituents at the 1,4 and 6 positions may be effected by methods which have been described previously and which are conventional in sugar chemistry. When the contemplated substituents are different from one another, as many successive substitution reactions follow as there are distinct substituents. In the course of these reactions, the positions which do not have to be substituted or those which must subsequently be the subject of another substitution are protected temporarily by blocking groups according to the usual methods.

The blocking groups initially present, in the case where one starts, as previously indicated, from benzyl-2acetamido-4,6-0-benzylidene-2-deoxy-D-glucopyranoside, are removed, for example, by the action of the acetic acid (at 60/ for 1 hour under reflux) and catalytic hydrogenation, as described, for example, by MERSER et al. (Biochem. Biophy. Res. Coramun., 1975, 66, 1316), or by catalytic hydrogenation by the method of LEFRANCIER et al. (Int. J. Peptide Protein Res., 1977, 9, 249).

The methods of substitution are those traditionally used. To obtain the acylated derivatives, procedure with the aid of an acylating agent corresponding to the substituent that it is desired to introduce (anhydride, acyl chloride, etc.).

The 1, 4, 6 positions are not equivalent as regards their reactivity. The C6 position is easier to substitute, also, when this position must be substituted, it is possible to operate without blocking the other positions, with an amount of substitution agent equal to that necessary for the substitution in a single position.

A particular example of the method of preparation of the derivatives substituted at the 6 position is given in the article of KU3UM0T0 et al. (Tetrahedron letters, 1976, 47, 4237).

The substitutions on the oside residue may be produced before or after fixing the peptide chain or the Λ87 3 5 fragments of the latter, c) Peptide chain H-X-CH-CO-Y 5 I (CH2)2 CO - (A)n - z The fixing of a peptide chain to the N-acetylmuramic acid, or to an analogue of the latter as has been ' J indicated above, is effected by traditional methods in the field of peptide synthesis. Such methods have been amply described in the prior literature and in particular. in the previously indicated French patent applications.

In general, glycopeptide syntheses can be effected either by fixing a first amino acid to the muramyl group, then by fixing to the compound thus obtained the second amino acid, and so on. It is also possible to prepare the entire peptide chain separately amino acid by amino acid and to fix the latter on the muramyl group. It is finally possible to select intermediate methods in which fragments of the chain are prepared, and then these fragements are joined together until a complete chain is formed which is then fixed to the muramyl group, or to fix a first fragment to the muramyl group, then a second to the product thus obtained, etc.

The choice of sequence is guided mainly by reasons of convenience or of yield.

The Y and Z substitutions are advantageously effected on the glutamyl group before the synthesis of the chain.

In the same way, when n is different from 0, that is to say when one or several aminoacyl groups complete the peptide chain, the 2 group is first fixed to the terminal aminoacyl before the latter is integrated into the peptide chain.

The peptide syntheses are carried out by traditional methods. By way of example, it is possible to use methods of activating carboxyls, like the method called mixed anhydrides. Advantageously, the peptide synthesis is carried out by means of a compound of the carbodiimide type such as Η,Ν’-dicyclo-hexylcarbodiimide or equivalent carbodiimides. A review of the traditional methods of peptide synthesis is to he found in J. H. JONES, Chemistry and Industry, 723 (1974). It is also possible to refer to the already mentioned French patent applications, or again to the following applications: 75 29624, 76 06619, 76 06620, 76 O6C21, 76 21889, 77 02646 and to the article of LEFHAMC1ER et al. (Int. J. Peptide Protein Res., 1977, 9, 249 and 1978, 11, 289).

The formation of esterified or amidated derivatives corresponding to the group Y is obtained in known manner.

It is possible, in particular, to refer to the aboveindicated French patent applications, and notably to Applications 76 06820, 76 06821, 76 21889 and 77 02646.

'J'o fix the residue Z to the amino acid situated at the end of the peptide chain, one proceeds with activation of the carboxylic group of the amino acid in a manner known in itself, and it is subjected, to alcoholysis or aminolysi's by an R'OH alcohol or an R'NI^ amine. d) Synthesis seouence of glycopeptide compounds corresponding to the modification of the general formula in which n is 1 or 2 Diagram (I) shows a typical sequence of reactions iO ending in the production of peptide derivatives corresponding to the portion (A)n - Z of the general formula I.

An activated ester BOC-Ag-OR'1 (succinimide ester, p-nitrophenyl ester, etc.) of an N- protected amino acid (like for example the ter butyloxycarbonyl denoted by BOC, or indeed any other suitable temporary group for the amine function, used in peptide synthesis) is subjected, either to alcoholysis by an alcohol with a chain of more than 4 carbons in the presence of imidazole as catalyst (as indicated by 130DAHZKY et al. J. Or;;. Chem., 1977, 42, 149), however, in the case where K'^ is an alkyl group with a chain of 4 to 20 carbons, the known conventional methods of esterification of the amino acids are advantageously used, or to aminolysis by an alkyl amine with a chain of more than 4 carbons. Once the acyl-aminoacyl-alkyl ester or acyl-aminoacyl-alkyl amide respectively are obtained, freed from the Ν-protective acyl group (for example, for ter-butyloxycarbonyl, by a H solution of hydrochloric acid in glacial acetic acid), one obtains a compound or formula or (A-^-Zg). The product obtained may be coupled by known methods of peptide synthesis v;ith a second acyl amino acid to give, after removal of the II- protecting acyl group used, a dipeptide compound of the formula - Z.

To prepare the compounds according to the invention in which the last residue of (A)n corresponds to an aminoalcohol, it is possible to operate according to a modification of the preceding sequence shown by the diagram (1').

For this modification, an. N- protected derivative of an amino acid such as BOC-A'-COOH is selectively reduced to its aminoalcohol derivative BOC-A'-Cf^OH according to well-known methods. The amino acid can also be reduced in the same manner to its corresponding aminoalcohol, then selectively acylated at its amine function according to currently used methods in peptide synthesis. The alcohol function thus formed can then be esterified by an activated ester (succinimide ester, p-nitrophenyl ester, etc.) of a fatty acid with a chain of more than 4 carbons (R'-^-COOR) , in the presence of imidazole used as catalyst (as indicated by EODANZKY et al., J. Org. Chem., 1977, 42, 149). This ester may also be formed from the chloride, from the anhydride or from the imidazolide of the fatty acide. The sequence of the reactions is essentially that described above for the modification of diagram (i).

Diagram (II) represents the reaction sequences leading to the production of glycopeptide derivatives.

One starts with a derivative (1) with R1 a benzyl radical, as described by GROSS and JEANLOZ (J. Org. Chem., 1967, 32, 2759). To obtain the compound in which is an alkyl or aryl-alkyl group, one may use the method of preparation corresponding a - or β -glycosides also described in this same article, or any known method for such preparations in the chemistry of oligosaccharides.

The derivatives of formula (1) are coupled with a dipeptide derivative of the general formula H-X-D-Glu (OBzl)-OY, hydrochloride. In which formula X corresponds to an amino acid, and Y, for example, to an amino-, hydroxy-, methylamino-, methoxy- or glycylamide radical.

These various peptide derivatives are prepared according to methods described by LEFRANCIER et al. (Int. J. Peptide Protein Res., 1977, 3_, 249, and Int. J. Peptide Protein Res., 1978, in press). The coupling methods used to obtain the glycopeptide derivatives of formula (2) are also described in the previously cited articles. However, both in the synthesis of the dipeptide derivatives and in that of the derivatives of formula (2), any coupling method used in peptide synthesis may be used. 4873S Catalytic hydrogenation of the compounds of formula (2) is carried out in traditional manner (1EFRANCIER et al., Int. J. Peptide Protein Res., 1977, 9, 249) to produce compounds of formula (3).

The derivatives of formula (3) are coupled, for example, hy the method described below in detail for the modification (point d), (page 32) of the preparation of the decyl ester of MDP-l-alanine, by means of a carbodiimide and hydroxy-benzotriazole, with one of the derivatives 1C of the general formula H-(A)n-Z hydrochloride of which the diagram (I) gives the synthesis sequence. Compounds of formula (4) are obtained.

In a modification, the derivatives of formula (2) undergo selective debenzylidenation as described by ltLEKSEK et al. (Biochem. Biophys. Res. Commun., 1975, 66, 1316) to give the derivatives of formula (5). Selective acylation of the primary hydroxyl at the 6 position of the saccharide residue can then be done directly by the action of a slight excess of carboxylic or acyl-imidazole acid anhydride. Derivatives of formula (6) are obtained.

The derivatives of formula (6) can be synthesized by a totally different sequence (diagram IV, formula 4) similar to that developed hy KUSUMOTO et al. (Tetrahedron Letters, 1976, 47, 4237), from specific tosylation of the ?5 primary alcohol of the saccharide residue.

After catalytic hydrogenation of the compounds (6), carried out as usual in the presence of 5?» palladium on charcoal, the compounds of formula (7) are obtained, to which, as previously, may be coupled a residue to give the compound (8) according to the invention.

In another modification, the derivatives of formula (5) are diacylated on the two hydroxyls in the 4 and 6 positions of the saccharide residue by the action of an excess of carboxylic acid anhydride, then subjected to catalytic hydrogenation done as usual in the presence of 5/ palladium on carbon , to obtain compounds of formula (io). After coupling with the residue (A)nZ, as described previously, the compounds (ll) according to the invention are obtained. . e) Synthesis sequences of glycopeptide compounds corresponding to the embodiment of the general formula in ’.vhich n is zero When n = 0, the jf -carboxyl function of the li-glutamyl residue is engaged in an ester linkage with an alcohol having a chain of more than 4 carbons.

A particular method for preparing these derivatives consists of making the / -p-nitrophenyl ester of the blocked peptide fragment BOC-X-D-Glu-O-Y, then proceeding with alcoholysis by an alcohol with a chain of more than 4 carbons in the presence of imidazole as catalyst (as indicated by BODAHZkY et al., J. Org. Chem., 1977, 42, 149). After the usual acidolysis of the ter-butyloxycarbonyl group, the derivative obtained is coupled with a suitable saccharide derivative corresponding to the formula (1) (Diagram III) to result in derivatives of the formula (2) according to the method described below ,. 48735 in detail at points a), b) and c) for the preparation of the decyl ester of MDP-L-alanine or according to the methods described in MERSER et al. (Biochem. Biophys.

Res. Commun., 1974, 466, 1316), LEFRANCIER et al. (Int.

J. Peptide Protein Res., 1977, .9, 249, and Int. J.

Peptide Protein Res., 1978, 11, 289). After catalytic hydrogenation and purification carried out as described in the two last articles cited, the compounds of formula (3) are obtained. In a modification, the derivatives of formula (2) undergo selective debenzylidenation as . described by MERSER et al. (Biochem. Biophys. Res. Commun., 1975, 66, 1316) to give the derivatives of formula (4).' Selective acylation of the primary hydroxyl at the 6 position of the saccharide residue can be done directly by the action of a slight excess of the anhydride, or of the acyl-imidazole of carboxylic acid. The derivatives of formula (5) are thus prepared ( if R^ is a benzyl radical, it may be removed by catalytic hydrogenation carried out as usual in the presence of 5% palladium £0 on carbon ). These derivatives may be synthesized by a totally different sequence (Diagram IV, formula (5) ) similar to that developed by KUSU1I0T0 et al. (Tetrahedron Letters, 1976, 47, 4237).

In another embodiment, the derivatives of formula (4) tire diacylated on the hydroxyls at position 4 and 6 of the caccharide residue by the action of an excess of carbox· vlic acid anhydride to produce finally the compound of the formula (7) subjected if necessary to catalytic hydrogenation effected as usual in the presence of 5# palladium on carbon , if R^ is initially a benzyl protection radical. 487 35 αί OJ N I I <ί I (Μ I w ΓΜ O I rt tv << CM w fa > H Em C\i > h H O fa fa H Q II fa Q £ H H .£ fa +3 fa -rM fa 5 fa ο ai ι g I *4 I CM *4 ι o o m o I *4 I CM T o o A Z Ο H Eh s co < fa 1 s fa c fa S fa <4 o < fa H fa Q EM fa O fa CJ < z fa fa □ o □ fa O w fa z fa o H Em o fa fa o W O I CM o o P4 o I <4 g I <4 I o o A Ph O I <4 I o o m CM N I «Η < REACTION SEQUENCE OF THE PREPARATION OF PEPTIDE DERIVATIVES 0=0 I o I CM 0=0 I o CM I I o o PA pci ;O | H o W O I CM fcd o o -P Φ 0=0 ci I CM g I > I <4 W % g I o o PA I I o o m . 4873S w £ Η O ft H a H o H a ft H ft Q ft z o o o ft W hi w o ft ft ft o ο υ w ω w Q u a z □ ω o □ ft a s w o cn u ω H cn ft ft H Z >< cn ft Λ < ° ft -η y £ a 5 ft e? CM q •rl H o ft < E-i ft fl ft ft £ co O ft -H O Z ft G O H e* o d fM ft O £ >* f o u I ft οι ft V X I o o I ft -o I ft fM < I o u I ft fM o 4? Ο ω Μ Ν C-* α. Λ ΗΗο ftK'rt ΟΕίΛ Ο 5 ί«Ο _ β ο ·Η Ο 6(Ζ< Ο Μ Ο OS rass MOPS OfcO S5 W W hw , σ§ WWW HHO wz HSM εοζ >£· _ O fn /X *Λ Λ 113-0 a Sm 487 35 P w c-4 X A o >-* CO K O W ϊτ; < > M CO r-< Ch H O > w W P X K P w w Q CO M P A X P A w W £-1 CC P > Ph o H l-l ω Eh X P <2 O A X 2 O < o ?H CC o P cc A W M u O 2 co o < M X O P P O X >4 fc M X EM £ W Eh < O O X z O M P A mr X K O’ o 5 < W 2 O X CO Hi X o P P co 2 £ o HI O X 2 <0 P o Pl co o A X ω o E< X C 2 X w o X >- CO O Ε-» 00 in EH R-CH-CO-X-NH-CH-CO-Y R-CH-CO-X-NH-CH-CO-Y I - I (ch2)2 ioOCH,“Ph COOH The invention also relates to methods of using the compounds corresponding to the preceding definitions, notably as a reactant or as an active substance in pharmaceutical compositions.

The invention relates to biological reactants, for example standard immunological adjuvants, which can be constituted by means of the compounds according to the invention, notably in order to study the possible adjuvant properties of the substances under investigation, by comparison with such standard adjuvants or, on the contrary, as an agent capable of countering certain effects connected with the administration of immunosuppressive substances.

More particularly, the invention relates to medicaments including as active principle at least one of the compounds according to the invention, this medicament being applicable to control immune responses of the subject to which it is administered.

These medicaments are notably applicable when a reinforcement of the immune response to any immunogenic agent is sought. Such immunogenic agents may be natural or synthetic, and necessitate the use of a stimulating agent for the immune system, whether the immunogenic agent is weak in nature or whether it is strong and can be used at very small dose, or again if the immunogenic character has been reduced, for example in the course of modifications or prior purifications. 487 35 In general, the utilization of the inununoregulator compounds according to the invention is useful each time that the immunogenic agent does not permit the induction of a sufficient response.

The invention relates more particularly again to the application of the compounds concerned to the amplification of the immunogenic effect of active principles of vaccines administered to a host, animal or human, notably in the case where these vaccinating principles lu belong to the above-mentioned immunogenic categories' of agents. Consequently, the invention relates also to pharmaceutical compositions whose active principle is constituted by at least one of the compounds according to the invention, :n association with the appropriate pharmaceutical vehicle for the mode of administration required or usable having regard to the nature of the vaccinating principle used.

The invention is applied in particular to those vaccinating agents whose immunogenic character is strong 2U but which are difficult in use in normal times by reason oi too high a toxicity or undesirable side-effects. It h-s been confirmed that the adjuvant agents according io the invention are capable of effectively compensating for the loss in immunogenic effect which would result normally from dilution or from reduction of the doses used, notably for the purpose of reducing the toxicity or the side-effects of the abovesaid agents to a corresponding degree, and this without unfavorably influencing the latter phenomena.

The same effects are observed in the case of strong vaccinating agents of which the immunogenic character has been reduced, notably by extensive purifica5 tion, to the extent where this becomes necessary for the corresponding reduction of their toxic effects or injurious secondary effects. This is particularly the case for vaccinating principles constituted by bacterial anatoxins or viral anatoxins or, generally, vaccinating principles constituted by a part only of the constituents initially contained in the bacteria or virus against which protection is sought.

In general, the invention is applied to any antigen which has undergone chemical or physical transforma15 tions seeking to remove or modify the parts of the antigen which are responsible for its troublesome secondary effects whilst preserving the portions vzhich are the cause of its immunogenic properties. It is to this type of weak immunogen that are attached, for example, the principles constituted by the sub-units derived from flu virus, and vzhich retain only the hemagglutinins and the neuraminidases of the latter, to the exclusion of the nucleoproteins and other nucleotide constituents of the virus from which they are derived. This applies also to certain anatoxins, such as those, for example, of diphtheria or of tetanus, which, as is known, may be constituted by soluble substances, such as obtained by the simultaneous action of formaldehyde and of heat on bacterial toxins derived from the corresponding bacteria.

The invention also relates to the application of the compounds according to the invention for the treatment of infectious diseases. In this application, it must be noted that the products according to the invention are clearly distinguished from the customarily used antibiotics. The products according to the invention, contrary to antibiotics, do not have a bactericidal or bacteriostatic effect in vitro. On the other hand, they can activate isolated macrophages in vitro and their action in vivo ic demonstrated as will he seen in the pharmacological test examples. Unlike the antibiotics again, the-action is not limited to certain varieties of micro-organisms.

This is explained, as we have seen, by the fact that their activity is not direct hut develops through the nonspecific immunivary defense mechanisms of the host, which mechanisms their administration stimulates and amplifies. This difference in action with respect to antibiotics renders these products all the more advantageous as they can be used against pathogenic germs which have become resistant to antibiotics.

As has been seen, the mode of action of the products according to the invention approaches that of known antiinfectious compounds such as BCG or the lipopolysaccharides ana since they can be employed with success for the treatment of infections without presenting the,drawbacks, notably of toxicity, v.-hich limit or prevent the use of LI'S or of BCG.

The appl: cation of the products according to the invention in :ludes both the treatment of diseases caused by extracellular growth micro-organisms such as Klebsiella (or again notably Pseudomonas, staphylococci, streptococci) and that of micro-organisms with intracellular growth (Listeria, mycobacteria, corynobacteria...).

The applications indicated previously by way of examples are not exclusive of other applications bringing into action the iminunoregulator properties of the compounds acoording to the invention. There can also be cited by way of example, their reinforcing action at the level if this specific immunization of the host with regard to parasitic antigens, the restoration of the immunocompetence of the host, when the latter is at a lower level than normal, notably when the latter has been damaged by the antigens or parasites themselves, or under the effect of chemotherapy, radiotherapy, or any other treatment having an immunosuppressive action.

The pharmaceutical compositions according to the invention, generally, are useful for the treatment or the prevention of infectious diseases of bacterial or parasitic origin, or for the inhibition of tumoral diseases.

The adjuvants according to the invention can be £5 administered to the host -animal or human being - in any suitable manner for producing the desired effect. Administration of the immunoregulator principle, notably „ 48735 adjuvant, and of the immunogen agent,, notably vaccinating antigen, can be contemplated simultaneously or separately, in the latter case if necessary staggered in time, if necessary again by similar or different routes of adminis5 tration (for example parenteral and oral routes respectively or vice versa).

The invention relates naturally also to the various pharmaceutical compositions with which the compounds according to the invention can be incorporated, if necessary in association with other active substances.

In particular, the compounds I are advantageously associated with immunogen agents, where, for example, immunogenic1 agents used at very low doses, or weak immunogenic agents, are concerned.

Advantageous pharmaceutical compositions are constituted by injectable solutions or suspensions containing an effective dose of at least one product according to xhe invention. Preferably, these solutions or suspensions arc formed in an isotonic sterilized aqueous phase, preferably again saline or glucosed.

The invention relates more particularly to such suspensions or solutions which are suitable for administration by intradermal, intramuscular, or sub-cutaneous injection, or again hy scarification and notably pharma25 ceutical compositions in the form of liposomes whose constitution will he explained below.

It relates also to pharmaceutical compositions administerahle by other routes, notably by the oral or rectal route, or again in the form of aerosols designed to be applied to the mucous membranes, notably the ocular, nasal, pulmonary or vaginal mucous membranes.

In consequence, it relates to pharmaceutical compositions in vhich one at least of the compounds according to the invention is associated with pharmaceutically acceptable excipients, solid or liquid, adapted to the constitution of oral, ocular or nasal forms., or with excipients adapted for the constitution of rectal forms of administration, or again with gelatinous excipients for vaginal administration. It relates also to isotonic, liquid compositions containing one at least of the products according to the invention, adapted for administration to the mucous membranes, notably the ocular or nasal mucous membranes.

It relates lastly to compositions formed of pharmaceutically acceptable liquified ga3es, of the propellant type, in which the products according to the invention are dissolved or held in suspension, and of which the release causes the dispersion in an aerosol.

The invention consists also of a method of treatment of a non-human animal aimed at reinforcing the inmune defenses of the host, consisting of administering to said host an effective dose of one at least of the products according to the invention, in one of the adrainisterable forms which have been mentioned above. By way of example of doses capable of inducing an effect, may be mentioned doses of to 1,000 yg per kg of body weight, for example of 50 fig, when the administration is effected by the parenteral route, or again of a dose of 200 to 20,000 fig per kg body weight, for example of 1,000 jig, for other methods of administration, such as for example the oral route.

Besides the compounds themselves, the full importance of the form in which the compounds are used for exerting their immunoregulator properties is already known. Water in oil emulsions have been the first vehicles proposed for the administration of this type of product. However, the preparation, and especially the utilization of these emulsions raises difficulties. Thus, when the oily phase is not metabolizable, the injection of the composition can lead to undesirable local reactions.

The discovery of the activity of the compounds in the absence of an oily phase has constituted a remarkable advance by the suitability of administrati op which results therefrom in the absence of troublesome side-effects. However, the passage from the water in oil emulsion forms into forms characteristic of the active compounds in the absence of an oily phase, and notably aqueous solutions, can modify a certain aspect of the activity of the products concerned.

In this order of ideas, the active compounds in the absence of an oily phase present notably very interesting adjuvant properties as regards immunitary protection with humoral mediation.

It seems at the present time that the administration of these products in the absence of an oily phase does not permit the same results to be produced as in their administration in emulsion as regards the immunitary responses of the type of those that have been demonstrated, for example, by delayed hypersensitivity reactions.

Studies have shown that a suitable means for modifying the activity spectrum of the products of the muramyl-peptide type is to use them in the form of liposomes (which technique has been described notably for the administration of enzymatic preparations).

! The liposomes, as is known, are generally produced from phospholipids or other lipid substances and are formed by mono or multilamellar hydrated liquid crystals. They are customarily used in dispersion in an aqueous medium.

It has thus been observed that the liposome forms could result in an increase in the anti-infectious activity in the tests carried out according to the procedure described below to detect activity with respect to Klebsiella infection. It has also been possible, in certain cases, to obtain an increase in the immunitary responses of the humoral and/or cellular mediation type.

The variations which result through the utilization of the liposome form Eire not necessarily oriented in the direction of a general increase in the activities of the product concerned. The essential point is, from a given product, to be able to obtain a whole range of 487 35 properties as a function of the desired result, by selecting the most appropriate form.

As has been indicated above, pharmaceutical compositions according to the invention which are particular ly advantageous are in the form of liposomes.

To form the liposomes, procedure is conventional.

Any non-toxic, physiologically acceptable and metabolizable lipid, capable of forming liposomes, can be used.

The most usual lipids are the phospholipids, and notably the phosphatidyl-cholines (lecithins) both natural and synthetic. Phospholipids may also be used and among the latter notably are the phosphatidyl-seriiisi the phosphatidyl-inositides or the sphingomyelines.

Other lipids can also be used, which have been described notably by W.R. HARGREAVES and D.V/. DEAMER (Conference on liposomes and their Uses in Biology and Medicine, Sept. 14-16, 1977, New York Acad. Sci.) and in the article of 3iochem., 1978, 18, p. 3759.

Traditional techniques and apparatus can be employed to form the liposomes according to the invention. These techniques have been described notably in Chapter IV of the work entitled Methods in cell biology, edited by David M. PRESCOTT, Volume XIV,· 1976 Academic Press, New York, page 33 et seq.

Advantageously, the initial dispersion is obtained from a thin film of lipid formed on the wall of a container from a solution of these lipids which had previously been introduced into this container and after evaporation of the solvent. The aqueous phase is then introduced into the container and the dispersion of the lipids into the midst of the aqueous solution is produced by stirring the medium, and generally by resorting to ultrasonic treatment.

In the first step there is obtained a suspension of liposomes having a milky appearance. It seems that, at this stage, the liposomes are formed of a series of concentric lipidic double layers alternating with aqueous compartments.

The liposomes thus obtained may be separated from the aqueous medium, for example by centrifugation. The liposomes contained in the centrifugation culot can then be washed, so as to remove any active substances not IL incorporated with the liposomes. The latter may then be resuspended in the buffer solution, in which they can be preserved, notably in the cold, preferably at a temperature of +4°C. Such liposomes are stable for long periods.

Preferably, the initial lipid composition contains also a stabilizing agent, for example cholesterol. If necessary, recourse may also be had to an araphiphile agent, which may be added to the initial lipid dispersion in order to permit the final production of electrical charge carrying liposomes. Such agents comprise for example dicetyl25 phosphate or phosphatidyl-serine, to the extent that it is desired to obtain negatively charged liposomes, or stearylaraine, if it is desired to obtain positively charged liposomes.

Advantageously one starts from lecithin and from cholesterol. Generally, after having formed a film of lecithin-cholesterol in a container, a buffer aqueous solution is added and it is shaken to obtain a dispersion of the lipid film in the aqueous phase resulting in the formation of liposomes. The muramyl-peptide type compound, according to its solubility characteristics, is introduced either with the lipid phase (lecithin-cholesterol), or in the buffer aqueous solution.

The molar proportions of the constituents of· the lipid phase are advantageously comprised between 8:1 and 1:1 (lecithin/cholesterol).

Advantageously, the aqueous phase is buffered so that its pH is close to neutrality. A buffer solution, for example, phosphate-KaCl 0.9% is used.

The liposome form compositions according to the invention can include other substances compatible with this particular form. They can notably, constitute vaccinating compositions, contain immunogen agents.

Generally, the compositions in the form of the liposomes can contain, in addition to the compound of the muramyl-peptide type, any constituents: stabilizers, preservatives, excipients or other active substances capable of being used in the injectable solutions or emulsions presented previously for administration of muramyl-peptide compounds, provided that they are compatible with this liposome form. 4873 Contents of the order of 1 mg of muraniyl-peptide per 35 mg of lipids are advantageously used.

The reasons for which the liposome forms lead to different results from those that are obtained from the preparations in the form of water in oil emulsions or in the form of solutions are not fully known. One may offer some hypotheses on the more precise nature of this. Thus the absence of troublesome reactions, which appear at the point of injection when water in oil emulsions (mineral oil) are used and this in spite of the presence of lipid constitutents, may reasonably be explained by the fact that 1 ! the latter are metabolizable by nature.

The invention is described in more detail in the examples which follow relating to the preparation of products according to the invention, of a method of preparing the liposome pharmaceutical form, and various tests relating to the pharmacological properties of these products and of this particular form.

PREPARATION OF PRODUCTS ACCORDING TO THE INVENTION 1) Decyl ester of N-acetyl-muramyl-D-alanyl-D-isoglutaminyl h-alanine a) Uec^l-ester-Of-L-alanine^ paratoluene sulphonate g of 1-alanine, 30 ml of n-decanol and 6.54 g of paratoluene sulphonic acid (monohydrate) are mixed in a 250 ml flask. This flask is heated carefully over a bare flame of a bunsen burner until boiling of the n-decanol and the production of a homogeneous solution. To this still warm solution is then added, carefully, 100 ml of boiling benzene. The resulting solution is refluxed for 55 hours in a Soxhlet apparatus whose cartridge contains magnesia. The solution is then concentrated to remove the benzene, then the excess decanol is evaporated under high vacuum at 100°C. The reaction mixture is then cooled to 0°C, and the crystalline paste obtained is triturated with cold ether. The crystalline precipitate is rapidly drained and rinsed with cold ether. 1.71 g of paratoluene sulphonate of the decyl ester of 1-alanine is obtained. This product has the following characteristics: m.p. 65-66°C The elementary analysis is: for C2OK35KO5S (401.567) C H N 15 Calculated; 59.82 8.79 3.49 Pound: 59.97 9.02 3-36 Evaporation of the mother liquor to dryness under vacuum provides 6.66 g of the same product probably still containing n-decanol. b) p£cyl_ester_of_L-al^yl-D2isoj^ut^inyl2l-alanineA hydrochloride To 350 mg of the product prepared at a), dissolved in 5 ml of dimethylformamide a't -10°C, are added 100 jil of M-methylmorpholine. To this solution, are successively added 317 mg of t-butyloxycarbonyl-L-alanyl-D-isoglutamine, obtained by the method described by Lefrancier P. and Bricas Ii., (Bull. Soc. Chim. Biol., 1967, 49, 1257), 135 mg of hydroxy benzotriazole and 178 mg of dicyclohexylcarbo48735 diimide, The reaction mixture is stirred for 24 hours at room temperature. The latter is then evaporated to dryness, and then the dicyclohexylurea is precipitated with dichloromethane. The solution is then filtered, the filtrate is washed successively with a solution of citric acid (10$) with water, with an N solution of sodium bicarbonate and with water. After evaporation to dryness of the organic phase, 307 mg of raw product are obtained.

This product is dissolved in a minimum volume of dimethylformamide, and then passed through a column (2 x 10 cm) of Amberlyst 15 (H+) previously equilibrated with dimethylformamide. After evaporation to dryness of the eluate, 275 mg of decyl ester of BOC-L-alanyl-D-isoglutaminylΙι-alanine are obtained.

The whole of this product is then treated with 2 ml of hydrochoric acid (l N solution in acetic acid) for 30 minutes. The solution is evaporated to dryness under vacuum, the residue is rinsed several times with acetone and the acetone is evaporated until the disappearance of the acetic odor. The resulting product is taken up again with water, ultrafiltered, then freeze-dried. 206 mg of the hydrochloride of the decyl ester of L-alanylD-isoglutaminyl-L-alanine are obtained. This product has a rotatory power of ioflp = -14.7° (water).

Its elementary analysis is: for C-.Η..Ν.ΟςΟΙ (465.04) 41 4 5 calculated 54.23 found 54.15 8.89 8.93 12.05 11.31 c) Decyl ester of N-acetyl-muramyl-D-alanyl-D-isoglutaminyl L-alanine__(Mur-NAc-L-Ala-D-isoGln- ft -L-Ala-decyl ester) 100 mg of the product obtained at b) are dissolved in 2 ml of dimethylformamide. This solution is cooled to -15°C, and 23 jil of N-methylmorpholine added thereto.

In addition, in a thermostatic cell at -15°C, is prepared a solution of 81 mg of benzy-0-henxylidene-4,6-N-aoetyl muramic acid, prepared according to the method described by Ozawa T. and Jeanloz R. W. (J.Org. Chem., 1965, 30, 448), in 2 ml of dimethylformamide, then to this solution is added 20 jil of N-methylmorpholine, 23 pi of isobutyl chloroformate and, 5 minutes later, the previously obtained solution. After 4 hours, the temperature is allowed to rise again to 0°C. There is then added 0.18 ml of a 2.5 M KHCO3 solution and it is stirred again for 30 minutes About 40 ml of water is then added to this solution and the resulting precipitate is drained, washed with 2.5 II KHCO^, then with water. 162 mg of the decyl ester of (benzyl-0-benzylidene-4,6-N-acetyl muramyl)-l-alanyl-Disoglutaminyl-L-alanine are obtained. This precipitate io disoolved in about 10 ml of glacial acetic acid. 150 mg of 5% palladized carbon are added and the mixture is hydrogenated for 40 hours. The carbon is then filtered, the residue is evaporated, then taken up again in a water/ Η 7.88 7.77 Ν 8.5 8.97 acetic acid mixture and freeze-dried. 90 mg of the decyl ester of N-acetyl-muramyl-L-alanyl-D-iBOglutaminyl-L-alanine are obtained. Ito rotatory power is C oc] D = +13.1 (glacial acetic acid) The elementary analysis is: for C32H57N5°12» 2 θΗ3°θθΗ (823-92) C calculated: 52.48 found: 52.30 d) In thq method which has just been described, the complete peptide chain is first synthesized, then fixed to the muramyl residue. Another method consists of starting from N-acetyl-muramyl-L-alanyl-D-isoglutamine, to fix the decyl ester derivative of L-alanyl prepared as has been described in a).

To a mixture of N-acetyl-muramyl-L-alanyl-Disoglutamine (0.5 m mole, 246.25 mg), hydroxybenzotriazole (0.5 mmole, 67.5 mg) and N-cyclohexyl-N’ [ ft (N-methylmorpholino)ethyl ] carbodiimide, ^-toluene sulphonate (0.5 mmole, 211.8 mg) in 5 ml of dimethylformamide, are added, after an hour, the £-toluene sulphonate of the decyl ester of L-alanine (0.6 mmole, 241 mg) and N-methylmorpholine (0.6 mmole, 0.066 ml), in solution in 5 ml of dimethylmorpholine. After 48 hours of stirring at ambient temperature, the reaction mixture is diluted with an equal volume of 0.1 N acetic acid and the solution obtained is passed through a column of AG50 WX2, previously equilibrated in the mixture 0.1 N acetic acid-dimethylformamide (50/50).

After concentration of the fractions containing the product, the syrupy residue obtained was taken up again in a volume of 5 to 10 ml of dimethylformamide to which is added an equal volume of 2.10-¾ acetic acid.

The solution was then passed through a column of AG1 X2, previously equilibrated in 2.10-^ M acetic acid - dimethylformamide (50/50).

After concentration of the fraction containing the product, the latter was obtained by lyophilization of its acetic solution.

A purification was finally carried out on a silica gel column in the solvent methanol-chloroform (l—3 v/v). 113 mg of the product were obtained, namely a yield of 32$. Its rotatory power was [= +20.9° (glacial acetic acid) The elementary analysis is: C32H57N5°12’ 1H2° (721-85) C H N calculated: 53.19 8.23 9.66 found: 53.11 7.9 9.45 In the same way, by resorting to the same technique the following products were prepared: - eicosyl ester of N-acetyl-muramyl-l-alanyl-D-isoglutamine L-alanine whose rotatory power and elementary analysis were respectively = +20° (glacial acetic acid) for C42H77N5°12» °-75 CH^OH, 0.25 CHC13 C Η Ν calculated: 57.52 9.00 7.80 found: 57.53 8.74 7.79 - butyl ester of N-acetyl-rauramyl-L-alanyl-D-isoglutamineL-alanine whose rotatory power and elementary analysis were respectively E OC] +6.4° (methanol-water 1-3) for C26H45K5°12’ °15 CliC13’ 0.34 h2) C H calculated: 48.79 7.17 found: 48.77 7.11 N .88 .88 2) Becylamide of h-acetyl-muramyl-L-alanyl-D-isoglutamineL-alanine a) hecylamide_of_the_B0C-I,-alanine 333 mg (1.16 mmoles) of BOC-Ala-OSu and 189 mg (1.2 mmoles) of decylamine were dissolved in 10 ml of dimethylformamide. The reaction mixture was allowed to stand for 1 hour at ordinary temperature, then concentrated to dryness. The product crystallized in needles when the ether added to the syrup obtained was evaporated. It was dried under vacuum in the presence of P2°5· mg product were obtained, namely a yield of 96%. Its physical constants are: m.p. 60-62°C [CC l/ = -24 °1 (chloroform) The elementary analysis is: for ClgH36N203 C Η N calculated: 65.81 11.04 8.52 found: 65.67 10.8 8.28 b) Hydrochloride of the decylamide of alanine 330 mg (1 mmole) bf BOC-L-Ala-decylamide were treated with 4 ml of a normal solution of HCl in glacial acetic acid. After 30 minutes, the reaction mixture was concentrated to dryness and the product suspended in ether. 231 mg of product were obtained, namely a yield of 87.5 ¢. The determination of the melting point shows a change in appearance at 95°C. m.p. 157-158°C = +4.8C (methanol) The elementary analysis is: for C13H2gN2OCl, 0.25 H20 C Η N calculated: 58.18 10.70 10.44 found: 58.24 10.53 10.26 c) Decylamide of N-acetyl-muramyl-D-alanyl-D-isoglutaminyl l-alanyl The experimental method followed for the synthesis of this product is essentially the same as that used for the synthesis of the decyl ester of N-acetyl-muramyl-Lalanyl-D-isoglutarainyl-L-alanyl (second method of preparation d) ). The rotatory power and the elementary analysis of the product were: [Η57^6θ11’ θ*^ ^2θ calculated: found: c H N 53.99 8.35 11.80 53.94 8.10 11.83 3) n-butyl ester of N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanine > a) p-toluene sulphonate_of the n-butyl ester of l-alanine 891 mg (10 mmoles) of l-alanine and 2 g (10.5 mmoles) of ^-toluene sulphonic acid are dissolved in 10 ml of nbutanol apd 2 ml of benzene. The reaction mixture is heated under reflux (110-120°C) for 3 hours in a Soxhlet apparatus, the oenzene being added regularly. The nbutanol and the benzene having been evaporated, the product is precipitated with ether, then crystallized in the methanol-ether mixture. 2.65 g of product are obtained, namely a yield of 83$. The characteristics of the product are: m.p. 98-99°C [¢6¾20 = 0°C (absolute methanol) for C14H23NO5S C$ H$ N$ calculated: 52.97 7.30 4.41 found: 52.58 7.05 4.23 b) n-butyl ester of N-acetyl-muramyl-L-alanyl-D-isoglutaminjrl-L^alanine In solution in 8 ml of dimethylformamide, 493 mg (1 mmole) of N-acetyl-muramyl-I-alanyl-D-isoglutamine, 170 mg (1 mmole) of N-hydroxybenzotriazole, and 424 mg (1 mmole) of N-cyclohexyl-N'- [β (N-methyl-morpholino) 8 735 ethyl 1 carbodiimide, p-toluene sulphonate, are allowed to stand for 1 hour at room temperature, then added to a solution in 7 ml of dimethylformamide of 224 mg (0.7 mmole) of the p-toluene sulphonate of N-butyl ester of L-alanine and of 0.077 ml'(0.7 mmole) of N-methylmorpholine. After 4 days, the reaction mixture is concentrated to dryness, taken up again in an 0.1 M acetic acid solution and passed through a column of ion exchange resin marketed by the BIORAD Company under the name AG-5O-W-X2 (8 to 10 ml). The interesting fractions are combined, freeze-dried, taken up again in a 2.10-^ M acetic acid solution and passed through a column of ion exchange, resin marketed ty the BIORAD Company under the name AG-1-X2. The interesting fractions 'are combined and lyophilized. The product was then chromatographed on a silica gel column (8 gj in methanol-chloroform (1:4) mixture. After freeze-drying, 158 mg of product are obtained, namely a yield of 36.4$· The characteristics of the product are: [ Cf ] D20 = +6.4° (methanol-water, 1-3) for C26H45N5O12, 0.15 CHClg, 0.34 HgO C$ H$ N$ calculated: 48.79 7.17 10.58 found: 48.77 7.11 10.88 4) Decyl ester of N-acetyl-muramyl-Ii-alanyl-D-isoglutaminyl25 L-lysine a) p-toluene sulphonate of decyl ester of N__benzyloxy-_ carbonyl-L-lysine c 561 mg ·2 mmoles) of N -benzyloxycarbonyl-1lysine and 418 mg (2.2 mmoles) of £-toluene sulphonic acid are dissolved in 2 ml (10 mmoles) of decanol. The reaction mixture is heated under reflux (12O°C) for 5 hours in a Soxhlet apparatus, benzene being added regularly. The product was precipitated from this reaction mixture, then crystallized in the methanol-ether mixture. 941 mg of product were obtained, namely a yield of 77.55=.

The characteristics of the product were: m.p. 99-100°C C CC 3 20 = + 1.8° (chloroform) for C31H48N2O7S N% calculated: 62.80 8.16 4.72 found: 62.88 8.25 4.79 b) Decyl ester of N-acetyl-muramyl-L-alanyl-D-isoglutaminyl_-N^_-benzyloxj!rcarbonyl-L-;ljrsine In solution in 5 ml of dimethylformamide, 492.5 mg (1 mmole) of N-acetyl-muramyl-Ii-alanyl-D-isoglutamine, 170 mg (1 mmole) of N-hydroxybenzotriazole and 424 mg (1 mmole) of N-cyclohexyl-N’ [$ -(N-methylmorpholino) ethyl] -carbodiimide, £-toluene sulphonate, are allowed to stand for one hour at room temperature, then added to a solution in 5 ml of dimethylformamide of 415 mg (0.7 mmole) of the £-toluene sulphonate of the decyl ester of -benzyloxycarbonyl-L-lysine and of 0.077 ml (0.7 mmole) of Nmethylmorpholine. After 48 hours at room temperature, the product was precipitated from the reaction mixture by the addition of 250 ml of iced water. It was then chromatographed on a silica gel column (20 g) in methanol-chloroform (1-5) mixture. 393 mg of product were obtained, namely a yield of 62.7%. The characteristics of the product were: m.p. 174-185°C [ oC ] = +21° (glacial acetic acid) for θ43ΗγΟΝ6Ο14 C% H% K% calculated: 57.69 7.88 9.39 10 found: 56.8 7.79 9.14 c) decyl ester of N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-lysine 279 mg (0.31 mmole) of the decyl ester of the N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-Nf -benzyl15 oxycarbonyl-L-lysine, were hydrogenated, in solution in 25 ml of glacial acetic acid, in the presence of 200 mg of 5% palladium on carbon. After 2 hours, the catalyst was filtered and the product was obtained by freeze-drying. 255 mg of product were obtained, namely a yield of 100%, the characteristics of the product were: [<Γ]ρθ = +19° (glacial acetic acid) for C37H6gN6014, 1 H20 C% H% calculated: 52.91 8.41 10.02 found: 52.78 7-99 10.02 ) Pentadecyl aster of H-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanine a ) Βζί°Aiiene_sulphonate_of_the_gentadecyl_ester_of_L-alanine 400 ng (4.46 mmoles) of L-alanine, 937 mg (4.92 mmoles) of £-toluene sulphonic acid, and 4.4 g (19 mmoles) of pentadecanol were heated under reflux (120°C) for 2 hours in a Soxhlet apparatus, in the presence of benzene. The product wa& precipitated with ether from the reaction mixture, then recrystallized in ether. 2.076 g of product were obtained, namely a yield of 98$.

The characteristics of the product were: m.p. 73°C [ CC ] ρθ = 0° (chloroform) for C25H45N5S C$ H$ N$ calculated: 63.65 9.61 2.96 found: 63.27 9.26 3.25 b) Pentadec^l_ester o^N-acet^l-muram^l-l^alan^l-D·is oglutaminjl-L-alanine_ In solution in 5 ml of dimethylformamide, 492.5 mg (l mmole) of N-acetyl-muramyl-L-alanyl-D-isoglutamine, 170 mg (l mmole) of N-hydroxybenzotriazole and 424 mg (1 mmole) of N-cyclohexyl-N’[ β -(N-methylmorpholino) ethyl ] -carbodiimide, £-toluene sulphonate, were allowed to stand for one hour at room temperature, then added to a solution in 5 ml dimethylformamide of 330.2 mg (0.7 mmole) of the £-toluene sulphonate of the pentadecyl ester of l-alanine and 0.77 ml (0.7 mmole) of N-methylmorpholine. After 48 hours, the reaction mixture was evaporated to dryness and chromatographed on a silica gel column (40 g) in methanol-chloroform (1-4). The interesting fractions were combined, concentrated and the product .. 48735 was precipitated in methanol-water. 125 mg of this product were obtained, namely a yield of 23$. After passage over a new column of silica gel (MERCK - type A) in n-butanol-acetic acid-water mixture (4:1:5 upper phase), the product was recovered hy freeze-drying. mg of produc- were obtained whose characteristics were: [ci] = +19.5° (glacial acetic acid) for C’ 0,75 CH3C00H« 0,5 H2° C/o Hf. 10 calculated: 55.84 8.64 8.45 found: 55.60 8.10 8.47 6) Benzyl ester of N-acetyl-muramyl-Ii-alanyl-D-isoglutaminy1-L-alanine In solution in 8 ml of dimethylformamide, 493 mg (lmmole) of N-acetyl-muramyl-L-alanyl-D-isoglutamine, 170 mg (l mmole) of N-hydroxybenzotriazole and 424 mg (l m mole) of H-cyclohexyl-N’[/6 -(N-methylmorpholino)ethyl 1 carbodiimide, o-toluene sulphonate, were allowed to stand for an hour at room temperature,then added to a solution in 7 ml of dimethylformamide of 248 mg (0.7 mmole) of the ^-toluene sulphonate of the benzyl ester of L-alanine and of 0.77 ml (0.7 mmole) of N-methylmorpholine. After 5 days, the reaction mixture was concentrated to dryness, taken up again in a 0.1 M acetic acid solution and passed through a column of AG-50-W-X2 resin. The interesting fractions were combined, freeze-driei, taken up in a 2.10 3 M acetic acid solution and passed through a column of AG-1-X2 resin. The fractions containing the product were combined, freeze-dried, then chromatographed over a column of silica gel (MERCK - type a) in the mixture n-hutanol-acetic acid-water (150:5:25). The product was recovered by freeze-drying. 151 mg of product were obtained, namely a yield of 33%· The characteristics of the product were: [OClJO = +5>2o (Η2θ) for C29H43N5°12 C% H% N% calculated: 53-28 6.63 10.71 found: 52.29 6.68 10.21 7) Decyl ester of N-acetyl-muramyl-L-alanyl-D-isoglutamine a) Hydrochlorine of the decyl ester of D-isoglutamine A solution of 490 mg (2 mmoles) of BOC-D-isoglutamine (BOG - butyloxycarbonyl) dossolved in 10 ml of methanox and 1 ml of water was adjusted to pH 7 with an aqueous solution of 20% Cs2CO3 .

After evaporation to dryness and drying to the residue, 5 ml of dimethylformamide and 0.5 ml (2.2 mmoles) of 1-bromodecane were added. After 24 hours at ordinary temperature, the reaction mixture was concentrated to dryness, taken up in aqueous ethyl acetate and washed with water. The ethyl acetate phase was drieu over lla2S0^, then evaporated. The product was crystallized in an ethyl acetate-petroleum ether mixture. 719 mg of product were obtained, namely a yield of 93%· The characteristics of the product were: m.p. 106-107°C - 48735 72 ECC 3 2° = +4.5° (chloroform)12OH38N2°5 CJ» H/$ 1$ calculated: 62.09 9.90 7.24 found: 62.35 9.96 7.26 The debutyloxycarbonylation, carried out by the action of a normal HCl solution in glacial acetic acid, enabled the product mentioned in the title to be obtained. b) Decyl ester of the hydrochloride of l-alanyl-D-iso10 glutamine 284 mg (1.5 mmole) of BOC-D-alanine are dissolved in 7 ml of dimethylformamide. To this solution cooled to -15°C are successively added 0.165 ml (1.5 mmole) of N-methylmorpholine and 0.195 ml (1.5 mmole) of isobutyl chloroformate. After 3 minutes, a solution, cooled to -15°C of 485 mg (1.5 mmole) of the hydrochloride of the decyl ester of the D-isoglutamine and 0.165 ml (1.5 mmole) of N-methylformamide, in 5 ml of dimethylformamide, was added. After 4 hours, the reaction mixture was brought to 0°C, and 2.5 ml of a 2.5 M KHCOj solution were added, then ml of water. The product was extracted with ethyl acetate, and the extract was successively washed with 10/» citric acid, with water, with a IM NaHCO^ solution, and then with water.

The ethyl acetate phase was dried, then concentrated The product was precipitated by the addition of petroleum ether. 632 mg of product were obtained, namely a yield of 92/°. The characteristics of the product were: 73 m.p. 92-93°C COC] = +2.5° (chloroform) for C23H43N3O6 C$ H$ N$ calculated: 60.36 9.47 9.18 found: , 60.25 9.0 9.0 The debutyloxycarbonylation, carried out by the action of a normal HC1 solution in glacial acetic acid, enabled the procuct mentioned in the heading to be obtained, c) De cyl_ester_of _N;;ace tyl-nniramyl-Xl-^^benzyl-^x^benzyli^eneJ-l^alanyl-D-isoelutaroine 471 mg (1 mmole) of (1- Cf -benzyl-4,6-0-benzylidene) -N-acetyl-muramic acid was dissolved in 5 ml of dimethylformamide. To this solution, cooled to -15°0, were successively added 0.11 ml (1 mmole) of N-methylmorpholine and 0.13 ml (l mmole) of isobutyl chloroformate. After 3 minutes, a solution, cooled to -15°C, of 394 mg (l mmole) of the hydrochloride of the decyl ester of 1-alanyl-Disoglutamine and 0.11 ml (1 mmole) of N-methylmorpholine, in 5 ml of dimethyIfomamide, were added.

At the end of 4 hours, the reaction mixture was brought to 0°C, and 1.65 ml of a 2.5 M KHCO^ solution was added and, after 30 minutes, 100 ml of water. The precipitated product was crystallized in methanol. 768 mg of product were obtained, namely a yield of 94.7$. The characteristics of the product were: m.p. 235-241°C [CC] ρθ = +83.7° (dimethylformamide) for C43H62N4°11 C% H% N% calculated: 55.04 8.28 8.692 found: 54.52 8.12 8.7 „ 48735 d) Decyl ester_of N-acetyl-muramyl-l-alanyl-D-isoglutamine 400 mg (0.5 mmole) of decyl ester of N-acetylmuramyl-(l- cr -benzyl-4,6-0-benzylidene)-L-alanyl-D-isoglutamine were hydrogenated in glacial acetic acid, in the presence of 5$ Pd on charcoal (400 mg).

After 48 hours, the catalyst was filtered, the acetic acid evaporated, and the product precipitated in methanol-ethyl acetate (twice). 200 mg of product were obtained. The characteristics of the product were: m.p. 185-19O°C 20 = +38>?o (glacial acetic acid) 29H52N4°11 C$ H$ N$ Calculated: 55.04 8.28 8.692 found: 54.52 8.12 8.7 ) (£ -methyl, ϋ -decyl N-acetyl-muramyl-l-alanylD-glutamic diester a θϊϊΐ °ίίέθ_ of_the___ff_-meΐ1ΐ2ΐχ__γ_2άβcyl_diester_ °ΐ D-glutamic acid 884.6 mg (2 mmoles) of the dicyclohexylamine sal351 of the c( -methyl ester of BOC-D-glutamic acid were dissolv ed in 5 ml of water. This solution was supplemented with 3-26 ml of an aqueous solution of 20$ Cs2CO3 (namely 2 mmoles), then concentrated to dryness and dried. The residue was taken up in 20 ml of dimethylformamide and 0.46 ml (2.2 mmoles) of bromodecane were added. After one night the reaction mixture was concentrated and taken up again in aqueous ethyl acetate. The organic phase was washed with 4873S 100 citric acid, with water, with a molar solution of NaHCOp and then with water. It was dried over McSO^, filtered and concentrated. 791 mg of product were obtained, namely a yield of 98.50.

The debutyloxycarbonylation was carried out by the action of a normal HCl solution in glacial acetic acid, enabling the product mentioned in the heading to be obtained. 987 mg of product were produced, namely a yield of 1000. The characteristics of the product were: m.p. 95-97°C [Of] P° = -13.3° (absolute methanol) forC16H32NO4C1 C0 H0 N0 calculated: 56.98 9-55 4.15 found: 56.49 . 9.34 4.25 b) Hydrochloride of L-alanyl-D-glutami c oc -methyl S'- decyl ester 593 mg (3.13 mmoles) of BOC-l-alanine were dissolved in 5 ml of dimethylformamide. To this solution, cooled to -15°C, was successively added 0.35 ml (3·13 mmoles) of N-methylmorpholine and 0.4 ml (3.13 mmoles) of isobutyl chloroformate. After 3 minutes, a solution of 962 mg (2.85 mmoles) of hydrochloride of methyl cC -ester, decyl £ -ester of D-glutamic acid and 0.3 ml (2.85 mmoles) of N-methylmorpholine in 5 ml of dimethylformamide, cooled to -15°C, was added.

At the end of one night at -15°C, the reaction mixture was brought to 0°C, then supplemented with 3 ml of a 2.5 M KHCO^ solution. After 1 hour, 100 ml of water 487 35 were added and the product was extracted with ethyl acetate, the organic phase was washed with 10$ citric acid, with water, with a molar solution of hHCOj, and then with water. It was dried over MgSO^, filtered, concentrated to give a non-crystalline.residue. 1.22 g of product were produced, namely a yield of 90.6$.

The debutyloxycarbonylation carried out by the action of a normal hydrochloric acid solution in glacial acetic acid enabled the production of the derivative given in the heading. c) Of-methyl, Tf-decyl____N-acetyl-muramyl(£-_K__-benzyl-Z, 6-0-benzylidene )-Ii-alanyl-D-glutamic diester 1.216 g (2.8 mmoles) of 1- OC -benzyl-4,6-0benzylidene-N-acetyl-muramic acid were dissolved in 5 ml of dimethylformamide. To this solution, cooled to -15°C, were successively added 0.31 ml (2.8 mmoles) of N-methylmorpholine and 0.37 ml (2.8 mmoles) of isobutyl chloroformate. After 3 minutes, a solution of 1.15 g (2.58 mmoles) of the hydrochloride of the oc -methyl, γ -decyl .>· 120 alanyl-D-glutamic ester and 0.28 ml (2.58 mmoles) of N-methylformamide, in 5 ml of dimethylformamide, cooled to -15°C, is added.

At the end of 4 hours, the reaction mixture was brought to 0°C and supplemented with 2.8 ml of a 2.5 M KHCO^ solution. After 30 minutes, the product was precipitated by the addition of water. 1.87 g of product, namely a yield of 87.7$ was obtained. The characteristics of . 48735 the product were: m.p. 207-211°C [CC ]2θ _ +79° (dimethylformamide) for C44H63N3°12 C% H% N% calculated: .63.98 7.69 5.09 found: 63.81 7.73 5.07 d) ¢-methyl, y -decyl N-acetyl-murainyl-L-alanylD-glutamic diester 1.6 g (2.3 mmoles) of the OC -methyl, Y decyl K-acetyl-muramyl-(l-OC -benzyl-4,6O-benzylidene)-l-alanyl-D-glutamic diester were hydrogenated in 100 ml of glacial acetic acid, for 41 hours, in the presence of 5% Pd on charcoal (1.9g). After filtration of the catalyst, the acetic acid is evaporated and the residue is taken up in 4 ml of chloroform, chromatographed on a silica gel column (MERCK - type C) in methanol-chloroform-acetic acid mixture (7:1:0.2).

The fractions containing the product were combined, concentrated, taken up with water and freeze-dried.

The product obtained is again purified on a silica gel column (silica 60-80 g) in the same mixture of solvents as previously, and finally freeze-dried. 929 mg of product were obtained whose characteristics were: [*]2° = +26.3° (glacial acetic acid) for C^qH^N^O ; 0. 5 CHjCOOH, 1 H20 C% H% calculated: 53.51 8.26 6.04 found: 54.14 7.96 6.14 -, 48735 9) <£-methyl, y -n-butyl_N-acetyl-muramyl-l·alanyl-D-glu-bamic diester a) Hydrochloride of the__oC__-.methjrl_j._y -n-butyl__D-____ glutamic diester 1-33 g (3 mmoles)' of the dicyclohexylamine salt of the OC -methyl ester of BOC-D-glutamic acid were dissoled in 5 ml of water. This solution was supplemented with 4.9 ini of an aqueous solution of 20% of Cs2CO3 (namely 3 mmoles), then concentrated to dryness and dried. The residue was taken up in 50 ml of dimethylformamide, and 0.36 ml (3.3 mmoles) of bromobutane were added. After 20 hours, the reaction mixture was concentrated to dryness and taken up in aqueous ethyl acetate. The organic phase was washed with 10% citric acid, with water, with a molar solution of KHCO^ and with water. It was dried over MgSO^, filtered and concentrated. 884 mg of product were obtained, namely a yield of 93%.

The debutyloxycarbonylation, carried out by the action of a normal HCl solution in glacial acetic acid, led to the product given in the heading. 587 mg of product were obtained, namely a yield of 83%. The characteristics of the product were: m.p. 84-88°C t Cf] = -20° (absolute methanol) for C1OH2()NO4C1 C% H% N% calculated: 47.34 7.95 5.52 found: 47.39 7.49 5.15 4873S b) Hydrochloride of the X -methyl, Ίί -n-butyl__1alanyl-D-glutamic diester 473 mg (2.5 mmoles) of BOC-Ir-alanine were dissolved in 5 ml dimethylformamide. To this solution, cooled to -15°C, were successively added 0.28 ml (2.5 mmoles) of H-methylmorpholine and 0.33 ml (2.5 mmoles) of isobutyl chloroformate. After 3 minutes, a solution of 558 mg (2.2 mmoles) of hydrochloride of the cc -methyl, tf n-butyl diester of D-glutamic acid and 0.24 ml (2.2 mmoles) of H-methylmorpholine, in 5 ml of dimethylformamide, cooled to -15°C, was added.

At the end of one night at -15°C, the reaction mixture was brought to 0°C, then supplemented with 3 .ml of a 2.5 M KHCO-j solution. After one hour, 100 ml of water were added and the product was extracted with ethyl acetate. The organic phase was washed with 10% citric acid, with water, with a molar solution of KHCOp then with water. It was dried over MgSO^, filtered, concentrated to give a non-crystalline residue. 804 mg of product, namely a yield of 94% was obtained.

The debutyloxycarbonylation, carried out by the action of normal hydrochloric acid solution in glacial acetic acid enabled the obtaining of the derivative given in the heading. c) -methyl,__ϋΓ -n-butyl__N-acetyl-muramy1-(1- cc -benzyl4±6-0-benzyl idene}-L-almy l-D-^utami e_di eater 1,037 g (2.2 mmoles) of 1-cC -benzyl-4,6-0-benzylidene-N-acetyl-muramic acid were dissolved in 5 ml of Λ87 35 dimethylformamide. To this solution, cooled to -15°C, were successively added 0.24 ml (2.2 mmoles) of N-methylmorpholine and 0.29 ml (2.2 mmoles) of isobutyl chloroformate. After 3 minutes, a solution of 700 mg (2.1 mmoles) of the hydrochloride of the Of -methyl, if -nbutyl L-alanyl-D-glutamic diester and 0.24 ml (2.1 mmoles) of N-methylmorpholine in 5 ml of dimethylformamide, cooled to -15°C, was added.

At the end of 4 hours, the reaction mixture was brought to 0°C and supplemented with 2.5 ml of a 2.5 11 KHCO^ solution. After 30 minutes, the product was precipitated by the addition of water. 1.365 g of product, namely a yield of 89-3% was obtained. The characteristics of the product were: m.p. 195-2O3°C [(X ] +92.3° (dimethylformamide for C^gH^H^O^ C$ H$ N$ calculated: 61.52 6.93 5.66 found: 61.47 7.02 5.41 d) -n-but^l K-ace tyl-muramyl-L-alanj;l-p-_ glutamic diester 1.34 g (1.8 mmoles) of the tt -methyl, If -n-butyl N-acetyl-muramy1-(1- κ -benzyl-4,6-0-benzylidene)-l-alanylD-glutamic diester were hydrogenated in 50 ml of glacial acetic acid, for 40 hours, in the presence of 5$ Pd on charcoal (1.35 g). After filtration of the catalyst and evaporation of the acetic acid, the product was chromato25 4873S graphed on a silica gel column (silica 60 - 80 g) in chloroform-methanol-acetic acid mixture (6:1:0.2). The fractions containing the product were combined, concentrated, taken up in water, then freeze-dried. 658.4 mg of product, namely a yield of 660 was obtained. The characteristics of the product were: [(X]p° = +30.8° (glacial acetic acid) for θ24Η41Ν3Ο1ρ ; 0.3 CH3COOH, 1 Ηρ0 calculated: C0 49.44 7.12 N0 7.03 found: 49.01 7.03 7.12 ) N-ac e ty1-muramyl-1-alany1-Ώ-iso glutaminy1-1-alany1glyceryl-mycolate a) t o sy l-^ly eery 1-^ colate To 3.5 g of glycerol monomycolate, in solution in ml of dried pyridine, were added 6 portions of 126 mg of tosyl chloride. At the end of 72 hours, the reaction mixture was concentrated and the residue was tritrated in toluene, 'l’he latter was collected and concentrated after filtration. The product was purified hy chromatography on a silica gel column (silica 60) in batches of 1.8 g each, in the chloroform-ether mixture (95-5)· 1.719 g of product were obtained, namely a yield of 460. b) 2 o-LBiS-iffidrochlor ide 1.45 g (1 mmole) of tosyl-glyceryl-mycolate dissolved in 20 ml of dry benzene were added to 250 mg of the potassium salt of BOC-L-alanine and to 140 mg of 18crown-6 in solution in 15 ml of dry benzene. The reaction mixture was heated under reflux for 6 hours under strictly anhydrous conditions, then, after cooling, it was filtered and concentrated to dryness. The product was chromatrographed on a silica gel column (silica 60) in the benzene-ether mixture (65r35). 958 mg of product were obtained, namely a yield of 65%.

The debutyloxycarhonylation by the action of a normal HCl solution in glacial acetic acid enabled the production of 970 mg of product given in the heading. c) K^acetyl-muramyl-L-alanyl-p-isoglutaminyl-L-alanylgly c ery1-mycolate In solution in 15 ml of dimethylformamide, 352 mg of H-acetyl-muramyl-L-alanyl-D-isoglutamine, 121.5 mg of N— hydroxybenzotriazole and 303 mg of N-cyclohexyl-K’[ft 15 (N-methylmorpholino)ethyl 3 -carhodiimide, ^-toluene sulphonate, were allowed to stand for 1 hour at room temperature, then added to a solution in 10 ml of dry benzene of 970 mg of the hydrochloride of L-alanyl-glycerylmycolate and of 0.075 ml of N-methylmorpholine.

After 28 hours at room temperature, the reaction mixture was brought to dryness. The residue was taken up in a benzene-methanol (50-1) mixture, and filtered over silica to remove all traces of dimethylformamide. The concentrated eluate was purified by the passage over a silica gel column in benzene-methanol (5-1) mixture. The interesting fractions were combined, concentrated, taken up in hot acetic acid (60°C) and freeze-dried. 664.6 mg of product were obtained, namely a yield of 57$. The characteristics of the product were: [ ot] 2° = +12° (benzene) for cxogH2O5Ol6N5 ’ OH^COOH C$ H$ i;$ calculated: 69.63 11.01 3.62 found: 69.90 10.91 3.34 METHOD OF PREPARATION OF LIPOSOMES jimoles (7.3 mg) of DL-dipalmitoyl- a phosphatidyl-choline (PM 734, grade 1, approximately 99$, marketed by SIGMA) and 10 jimoles (3·9 mg) of cholesterol (PM 386, 6, 99 +$, SIGMA) were dissolved in about 5 ml of chloroform (pure grade) in a round bottom 10 ml flask.

The lipophile adjuvants, in the proportion of 1.0 mg, were introduced with a chloroform phase.

The solution was evaporated in a rotary evaporator under vacuum at a temperature below 30°C so as to obtain a fine film of lecithin + cholesterol on the inner wall of the flask. ml of a buffer solution of 0.013 M sodium phosphate, pH 7.0, containing 9 °/oo of NaCl are brought to 55°C. The water-soluble adjuvants are introduced into this solution. In addition, the flask containing the above lipid film is also heated to 55°C.

The buffer solution is slowly poured into the flask keeping 1 he temperature at 55°C. The contents are then subjected to gentle stirring at 55°C so as to obtain a suspension oi liposomes containing the envisaged adjuvant. It is left to stand for about 1 hour. 8'4 The preparation is then subjected to sonication at 0°C under a nitrogen or argon atmosphere for 30 seconds (0.3 kW).

When the adjuvants are water-soluble, the sus5 pension is also centrifuged at 100,000 g, the culot is taken up with the phosphate buffer -NaCl. The operation is repeated six times. The culot finally obtained is taken up with a phosphate buffer-NaCl solution, to the selected volume.

PHARiJACOLOOICAL PROPERTIES The tests whose results are given below relate to various products corresponding to the general formula (I). For convenience, for these products, the group N-acetyl -muramyl-L-alanyl-D-isoglutaminyl is denoted by MDP, in the same way as the group N-acetyl-murarayl-Lalanyl-D-glutamyl is denoted by IfflPA. 1) Toxicity The toxicity of the products according to the invention was investigated by parenteral administration in mice. It was observed that the toxic doses were of an order of magnitude very much higher than that of the doses at which these products manifest their activity. Thus the lethal dose 50 of the products according to the invention tested was higher than 5 mg/kg of animal in the adrenaleetomised mouse, whose sensitivity to endotoxins is well-known.

The tested products were notably: - MDP decyl-ester - MDP-L-Ala-decyl-amide - MDP-L-Ala-eicosyl-ester - MDP-L-Ala-glyceryl-mycolate - MDP-L-Ala-benzyl-ester - MDP-L-Lys-decyl-ester .

It is possible to demonstrate the very favorable properties of the products according to the invention, notably by resorting to the tests described below. 2) limulus test To show the absence of activity of the endotoxic type, products according to the invention were subjected to the Limulus test. For this purpose, 0.1 ml of the Limulus amoebocyte lysate preparation (marketed by the MALL1NCKR0DT Company, at SAINT-LOUIS, U.S.A.) was mixed with an equal volume of the tested product at various concentrations in solution in apyrogenic distilled water. The vessels used were also made apyrogenic by dry heating in the oven, at 18O°C, for 2 hours.

After incubating the mixture for 20 minutes at 37°C in tubes, the formation of gel characteristic of the presence of endctoxin was estimated.

A test is positive when the presence of a firm gel is observed which remains adherent to the bottom of the tube when the latter is inverted (technique described by Elin R.J. and Wolff S.M., J. Infect. Dis., 1973, 128; 349). The results of these tests are as follows; 100 fig/ml MDP-L-Ala-butyl-ester 8735 MDP-L-Ala-decyl-ester II MDP-L-A1a-de cy1-amid e fifljP-L-Ala-eicosyl-et ter - lV£UP-L-Ala- gly c o syl-r.y colat e 100 100 II Under the same conditions, the IPS extract of Escherichia coli by way of comparison is positive at 0.01 jig/ml.

These results show, taking into account the effective anti-infectious doses, that the properties 10 demonstrated in the following tests could not arise from endotoxic contamination. 3) Adjuvant character in the aqueous phase and in emulsion a) In_the agueous^ghase Groups of 8 Swiss mice aged two months received, by sub-cutaneous injection (SC), 0.5 mg of antigen constituted by bovine serum albumin (BSA) with 0.1 mg or without the tested substance in an isotonic saline solution. This high dose of antigen, because it is situated at the limit of the paralysing dose with respect to the immuni20 tary response, results, for this reason, in a weak or zero response to the antigen only in the controls: It constitutes therefore a severe criterien to establish the activity of an adjuvant substance. Thirty days later, the mice received, by the same administrative route, a booster containing 0.1 mg of the same antigen.

The antibody level was determined, six days after the booster, by passive hemagglutination using sheep’s red blood corpuscles treated with formalin and covered >48735 8? with the antigen studied according to the method described by A.A. HIKATA and M.W. BEANDISS (J. Immunol., 100, 641648, 1968).

The antibody titer, represented by the maximum serum dilution agglutinating a given amount of sheep corpuscles, reaches a maximum at the 36th day and is established in the following manner: Log? of hemagglutinating titer Controls MDP MLP-decyl-ester MDP-L-Ala-butyl-ester MDP-L-Ala-decyl-ester I£DP-L-Ala-de cyl-amide MDP-L-Ala-penta-d e cyl-es ter HDP-L-Ala-eicosyl-ester MDP-L-Ala-glyceryl-mycolate MDP-L-Ala-benzyl-ester MUP-L-Lys-decyl-ester 3.15 - 1.72 8.24 ί 1.27 6.64 - 1.31 10.31 - 0.49 6.23 - 1.23 8.64 - 0.95 8.39 - 0.89 7.11 - 2.03 7.47 - 1.17 7.47 - 0.89 9.64 ί 0.70 The titers are expressed in base 2 logarithms.

It is seen that all the compounds permitted the level of the humoral response to be considerably increased (at least8 times and up to more than 100 times). b) ln emulsion The tests were carried out on batches of 6 male Hartley guinea pigs of 350 g. The administration was done by intradermal injection into the plantar pad of each of the rear paws. Ovalbumin (constituting the antigen) in the amount of 1 mg is prepared in 0.1 ml of an emulsion of saline isotonic solution, in an oily phase constituted either by the Freund imcomplete adjuvant (F1A), or by the complete·adjuvant (FCA) formed by the FIA to which is added 0.1 mg of whole Mycobacterium smegmatis cells. The compound according to the invention was administered in the amount of 0.05 mg added in the emulsion containing the FIA.

Eighteen days after this immunization, possible delayed hypersensitivity reactions to the antigen were sought by injecting by the intradermal route 0.01 mg of ovalbumin in the side of the animals, and 48 hours later, the reaction of the point of. injection was observed.

The diameter in millimeters of the reaction thus caused was measured.

Twenty-one days after the injection, the animals were bled. On the collected serum, the content of specific antibodies of the ovalbumin was measured by pre20 cipitation of the antibody-antigen complex in the equivalence zone. The amount of protein nitrogen contained in the precipitate was estimated by the Folin method.

The average values of the contents of antibodies are indicated in the table of results. These values express the amount, in micrograms, of nitrogen precipitatable by the antigen, per milliliter of the serum.

The results of these tests are as follows. 4873S HSH Log# of the hemazalut inatinr titer Control (AIF) 0 10.29- 0.75 AIP + 50 pg MDP 14-4 12.58± 1.02 AIP + 50 pg MLP-L-Ala-butyl-ester 18 - 2 13-31- 0.52 AIP+ 50 pg MDP-L-Ala-decyl-ester 17- 2.5 10.81-1.47° AIP+50pg MDP-L-Ala-decyl-ester 5 12.31- 0.82 AIP + 50 pg MDP-L-Ala-penta-decyl-ester 5 10.97-1.04 All·'+50 pg MDP-L-Ala-eicosyl-ester 6-4 11.25-1.14+ AIP + 50 pg MDP-l-Ala-glyceryl-mycolate 2li 3 12.77-0.83 AIP+ 50 pg MDP-L-Ala-benzyl-ester 12 !.5-3-5 12.31± 0.82 A1P+ 50 pg HDP-I-Ala-decyl-ester 13- 2 13-4 ί 0.55 These results show that the tested products caused, to various degrees, an increase in the level of the antibodies formed.

The administration of the product also generates a delayed type of hypersensitization in the treated subject with respect to the antigen, which hypersensitization is revealed by the cutaneous test. 4) Anti-infectious activity with respect to Klebsiella The testing procedure is described in the article CHED1D L. et col., Proc. Natl. Acad. Sci. USA, 74:2059.

In this way there was previously established an experimental method permitting the anti-infectious character of the product to be demonstrated. It was 4 shown that a dose of about 10 Klebsiella pneumoniae, injected by the intramuscular route in mice, results in 487 3 5 the gradual death of a considerable part, if not all, of the animals in the week following the inoculation. After 8 days, the survival of the animals was definitely achieved The survival of groups of inoculated mice under 5 the above conditions and treated by means of the products according to the invention was followed.

For these tests, hybrid mice (C57B1/6 χ AKE) FI bred at the PASTEUR INSTITUTE, from strains derived from the CNRS breeding station at ORLEANS, were used.

The infection by Klebsiella pneumoniae, a strain of the capsular 2, biotype d type, wets done from a culture of 16 hours in a medium for pneumococci (No. 53515, PASTEUR INSTITUTE). The infecting dose was 2.104 Klebsiella ; it was administered by,the intramuscular route.

The administration of the tested product was carried out by the intravenous route in 0.2 ml of apyrogenic physiological solution, the controls receiving the solution alone. It was carried out 24 hours before the inoculation.

The results of these tests are reported in the following table. The percentage protection indicated is a difference of the percentages of survivors of the treated group with respect to the control group. /4-8 735 Dose per i.v. Treatment 24h before Number’ Number of of survivors of protec- ! 2 x 10^ bacteria i.m. ’mouse. 1 mice f I 5 ; 58 ί tion ! Controls 1 24 i 8 ! 3 3 ! ! MDP-decyl-ester ! 10 8 1 ! 0 0 ! ! 100 24 24 ! 21 21 '! 75 ! Controls 24 13 ! 5 4 ! ! MDP-L-Ala-butyl-ester i ioo 24 23 ! 18 17 ! 54 ι Controls 32 13 ! 9 8 ! ! MDP-L-Ala-decyl-ester ! 100 32 30 ! 22 22 ! 44 ! Controls 24 13 ! 5 4 ! ! MDP-L-Ala-decyl-amide ! 100 24 21 ! 17 16 ! 50 ! Controls 24 8 ! 3 3 i ! MDP-L-Ala-penta-decyl-ester ! 100 1 . . . 1 24 24 ! 1615 ; 50 ! Controls t M 24 12 ! 4 3 ! ! MDP-L-Ala-eicosyl-ester ! 10 24 18 ! 14 14 ·! 46 ! 100 - I __ 24 23 ! 23 20 ! 70 ! Controls 24 7 ! 4 4 ! ! MDP-L-Ala-glyceryl-mycolate ! 10 8 5 ! 4 4 ! 33 ! 100 24 23 ! 23 23 ! 79 ! Controls 24 8 ! 3 3 ! ! MDP-L-Ala-benzyl-ester ! 100 . 1 __ 24 24 ! 14 14 ! 46 ! Controls 24 8 ! 3 3 ! ! MDP-L-lys-decyl-ester ! 100 ....1. . 24 22 ! 20 20 ! 71 ! Controls 16 7 ! 3 3 ! ! MDPA(butyl-ester)OCH3 ! 100 16 12 ! 11 9 ! ____ 1. 38 ! Controls 16 7 ! 3 3 ! ! MDPA(decyl-ester)OCH3 ! 100 t 16 13 Ϊ 1 11 10 ! 1 44 The results show significantly increased protection in applying the Student t test, for the animals which had received the products according to the invention with respect to the control animals. ..487 3 5 ) Anti-infectious acbivity with respect to Listeria Under similar conditions to those tests No. 4), the influence oi the administration of Mur-NAc-L-Ala-DisoGln- $ -L-Ala-decyl-ester was determined on the mortality of mice in which a dose of Listeria monocytogenes was inoculated, known for causing typically a cellular type infection.

As previously, the action of the product according to the invention was compared with that of BCG whose anti-infectious properties are well-known in this field. There were also determined, still by way of comparison, the action of LPS, Corynebacterium granulosum, and Mur-NAc-L-Ala-D-isoGln (IffiP).

The treatment of the mice and the inoculation was carried out by the intravenous route. The injected products were in solution or suspension in 0.2 ml of apyrogenic physiological solution. The controls only received the solution.

The dose of Listeria administered was, in all tests, 1.10^ units.

The tests whose results are given below were carried out by varying the doses of tested products and the time intervals separating the treatment from the inoculation of the Listeria monocytogenes (either 1, or 7 days). The number of surviving mice at the 5th and 10th day following the inoculation, was watched and the percentage protection as for the preceding tests was determined.

Dose INumbef NumIntravenous treatment ι per !of dayfe ibeford Controls LPS !raouse!infec! ! tion ! ι ber of mice Number of surviving animals at day 5 ! 10 of proteoj ‘ tion 0,3 '1 Controls BCG BCG Controls C. granulosum C. granulosum Controls MDP MDP MDP MDP Controls Mur-NAc-LAla-D^sgG^-^Alg100 100 300 300 100 : 1000 : 1000 J 100 : These results show, on the one hand, the absence of practically all protection by means of MDP under the conditions of the experiment and, on the contrary, very significant protection in the case of the products according to the invention, and this even at a very low dose of product (10 fig). 6) Tests of the liposome forms 5 Pharmacological tests similar to the preceding ones were reproduced with HDP, MDP-L-Ala-eicosyl-ester and MDP-L-Ala-glyceryl-mycolate comparing the effects of administration in liposome form.with those obtained for products in solution and with liposomes without muramyl-peptide.

The liposomes were prepared in the manner described above. a) Toxicity in the adrenalectomized_mouse The products, the doses administered and their 15 effect on the mortality of groups of six mice are indicated below.

Number of dead mice Bare liposomes 1/20 ml 0/6 SUP 100 pg 0/6 MDP liposomes 1,3 pg (=1/20 ml) 0/6 MbP-L-Ala-eicosyl-ester 100 pg 0/6 iilbP-L-Ala-eicosyl-ester liposomes 100 us (= 1/20 ml) 0/6 IsiDP-l-Ala-glyceryl-mycolate 100 pg 0/6 MDP-L-Ala-glyceryl-mycolate liposomes 25 100 pg (= 1/20 ml) 0/6 It was observed, as previously, that the lethal dose 50 is situated well beyond 5 mg/kg, whether the product was administered in solution or in liposome form. b) Adjuvant character The series of tests was carried out on mice under the same conditions as indicated in 3)a), using BSA as antigen.

The results of these tests are the following.

Logo of the hemagglutinating titer Controls 1.64 Bare liposomes 1.64 MDP 100 jig 8.21 - 0.53 10 jig 6.89 - 2.05 MDP-L-Ala-eicosyl-ester 10 jig 3.64 - 1.15 IfflP-L-Ala-glyceryl-mycolate 10 jig 2.07 - 1.13 MDP-L-Ala-eicosyl-esterliposomes (10 yg of the MDP derivative) 2.78 ί 1.35 MDP-l-Ala-glyceryl-mycolate liposomes (10 jig of the MDP derivative) 2.07 - 1.13 A second series of tests was carried out on Wistar rats, under the following conditions.

The antigen was ovalbumin injected by the subcutaneous route in male Wistar rats in a volume of 0.5 ml at the dose of 0.5 mg. The adjuvant preparations were injected simultaneously, at the dose indicated. At day 20, the animals received a booster of 0.5 mg of antigen alone by the same route. The serums were drawn and tested under the same conditions as in the mouse with the appropriate antigen.

The results obtained were: Log» of the hemagglutinating titer Controls 5.14 ί 1.38 Bare liposomes 5-31 - 2.8 MDP 200 jig 4.97 0.82 MDP liposomes 2.5 jig 7.64 ί 1.24 MDP-L-Ala-glyceryl-mycolate (200 pg) 7.64 ί 1.90 MDP-L-Ala-glyceryl-mycolate liposomes 6.47 - 2.32 Eicosyl-ester liposomes (200 jig) 8.64 - 1.26 A third series of tests was carried out on guinea pigs. The conditions of these tests are as follows.

The antigen was ovalbumin injected in aqueous solution in the foot pad of Hartley male guinea pigs at a volume of 0.1 ml in each rear- paw, at the dose of 1 mg per guinea pig. The adjuvant preparations were injected simultaneously at the indicated doses. After three weeks, the animals received by the dermal route, in a volume of 0.1 ml, 0.025 mg of antigen. The diameter of the cutaneous reactions was measured after 48 hours. The animals were bled and the titers measured.

The results obtained were the following; Controls MDP 200 jig MDP liposomes 2.5 Jig MDP-L-Ala-eicosyl-ester 200 pg HSR Logg of 'the hemag glutinating titer 6.64 - 0.82 9.19 ± 1.37 4(5)*0(6) 7.37 ί 1.19 7-33 ί 1.51 -ft 4-8735 MDP-L-Ala-eicosyl-ester liposomes 200 jig MDP-L-Ala-glyceryl-mycolate 8(3) 0(3) 9.64 t 0.63 8.64 - 1.63 MDP-L-Ala-glyceryl-mycolate liposomes 200 |ig ' 9-2 9.64 ί 1.55 * between parentheses, number of animals showing the reaction indicated.

The whole of these results shows that the administration in the form of liposomes preserves generally the adjuvant activity of the products and can even sometimes improve it. The most sensitive effect is manifested at the level of the delayed hypersensitivity of phenomena. c) An ti.2Klebsiella_ac tivity Under the previously described conditions, with the exception of the infection produced by the intravenous injection of 1.5.10^ Klebsiella, the tests are renewed to determine the anti-infectious action. The administrations in the form of solution and of liposome were prepared.

The results regrouped in the following table show, for an equal content of the tested compound, an appreciable improvement in the effectiveness for the liposome form with respect to the solution. . 48735 i i.v. Treatment at D-1 dumber of mice Number of survivors at 1 1 %of ; oroteo-J tion ; D+3 D+5 D+ 8 ! Controls 24 8 6 6 _ I J Bare liposomes 1/20000 ml 16 3 3 2 ° ; i 1/2000 ml 16 5 4 4 θ ί J 1/200 ml 24 10 7 60 i ! MDP 1 pg 16 5 3 2 0 ! ! 10 pg 16 9 5 5 6 ! ! 100 pg 24 21 15 15 38 ! J Liposomes MDP 0,001 pg 16 0 0 0 ° ; 'ι 0,01 pg 16 1 1 10 i i 0,13 pg = 1/200 ml 16 11 10 10 38 ; ! MDP-L-Ala-eicosyl-ester 1 pg 8 4 4 4 25 ! ! 10 pg 16 13 9 9 31 ! ! 100 pg 16 14 14 14 63 ! J liposomes MDP-L-Ala-eicosyl-ester i 0,1 ps 24 9 8 88 i i 1 pg 24 10 8 88 : I 10 pg = 1/200 ml 24 19 19 1954 ! ! MDP-L-Ala-glyceryl-mycolate 1 pg 8 2 2 2 0 ! ! 10 pg 16 9 8 8 25 ! ! 100 pg 8 8 7 7 63 ! j Liposomes MDP-L-Ala-glyceryl- J mycolate 0,1 pg 24 5 4 3 o ; i 1 ρε 24 10 10 10 17 j ; 10 pg ! 24 15 15 14 33 ; d) Anti-Listeria_Activity The tests were carried out under the conditions of 5), using a dose of Listeria monocytogen, known to cause typically a cellular type infection.

The treatment of the mice and the inoculation were carried out by the intravenous route. The products were injected in the liposome form at the indicated doses. The controls only received isotonic solution.

The dose of Listeria administered was, in all the .10 cclIs.

The tests whose results are given below were carried out by varying the interval of time separating the treatment of inoculation from the Listeria monocytogenes (either 1, either 4, or 8 days). The number of surviving mice at the 5th and at the 10th day following inoculation was followed, and the percentage protection determined as for the preceding tests. i.v. Treatment ;Tim| Product ®°^·ΠΪΟΘ·0+ 5 ;D+1 Option· ; Controls 1 ; 16 ; 6 ί 2 ; i Bare liposomes (1/20 ml) ! - ! 16 ! 6 ! 3 ! - J Liposomes MDP Η,- >; 16 ! θ ί 7 ! 25 ! D-1 ! J MDP-L-Ala-eicosyl-ester ;ioo; 16 ί 3 ΐ0 ί 0 ! Liposomes MDP-L-Ala-eicosyl-ester 1100! 16 ! 2 ! 0 ! 0 j MDP-L-Ala-glyceryl-mycolate ;ioo; 8 ! 3 ! 2 ΐ 0 Liposomes MDP-L-Ala-glycdryl-myco- ! late - 1 ! 100! 16 ! 8 ! 6 ! 19 ! Controls 16 ! 6 ! 2 ! - ; Bare liposomes (1/20 ml) I 1 16 ;8; 4 ; ! Liposomes MDP !1,3! 16 ! 14 ! 5 ! 6 D- _4; MDP-L-Ala-eicosyl-ester ; 100;16 ΐ 4 ; 4 ; 0 ! Liposomes MDP-L-Ala-eicosyl-ester 1100! 16 ! 9 i 2 ! 0 J MDP-L-Ala-glyceryl-mycolate •ioo;8 ; 1 · o : 0 Liposomes MDP-L-Ala-glyceryl-myco- ! late 1 .... .. . _ ! 100! 16 ! 14 9 ! 31 ! Controls 16 ! 5 2 ! - ; Bare liposomes (1/20 ml) 1 1 16 1 i 4 3 ; - ! Liposomes MDP !1,3! 16 ! 6 3 ! 0 i MDP-L-Ala-eicosyl-ester boo; 8 ΐ 2 2 i 0 D—8 * ! Liposomes MDP-L-Ala-eicosyl-ester Ϊ100! 16 ! 12 7 j 25 i MDP-L-Ala-glyceryl-mycolate ; ioo; 8 i 10 ! .0 1 Liposomes MDP-L-Ala-glyc6ryl-myco· 1 late 1 -! ! Hoo! 1 1 16 1 ! 11 1 10 ! 44 ί 00 These results show that the protection by liposome, under the conditions of the experiment, of the products according to the invention is a function of the moment at which they are administered with respect to the infection.

They also show that administration in liposome form is capable of generating a non-negligible protection under the conditions for which the product administered in solution does not manifest anti-Listeria activity.

Claims (10)

1. To 10 carbon atoms, or -NH 2 , the hydrogens of the amino 10 group being optionally substituted by alkyl residues of 1 to 10 carbon atoms, or an aminoacyl residue, - A is an aminoacyl residue of the group indicated above for X, or, for the last of the peptide chain, an aminoalcohol residue (-NH-CH-CH .,-0-) corresponding to these 15 aminoacyls (-NH-CH-C0-) it being understood that the A groups present in a same compound may be identical or different, n only representing the total number of A groups in this compound, - n is zero or 1,

1. A compound of the general formula: CH 2 ° R 6 CO - (A) - Z in which

2. Or 3, 20 - Z is a group -OR’, -NHR', -OCHj-d^O-COR' or -0CH 2 -CH0HCH 2 0-C0R' when the last A residue of the peptide chain is an aminoacyl, and -COR' when this last A is an aminoalcohol, in which R' is saturated or unsaturated alkyl and optionally containing functional groups selected from: 25 hydroxyl, carbonyl, carboxyl, cyclopropane and optionally substituted aryl, the group Z containing at least 4 carbons 103 and being likely to include up to 90 carbon atoms, with the proviso that when R is hydrogen, the (A) n ~Z group is a -NH-CH(CH 3 )-COO-CH 2 -CHOH-CH 2 -0-R° group, with R° being a mycolic acid comprising from 80 to 90 carbon atoms.

3. A compound according to Claim 1, wherein X is an L-seryl residue. 4. -8735 Η,C-CH-CON H-CH-CONH-CH-CONH „ 3 I I 2 CH 3 (ch 2 ) 2 CONH-CH-COO-CH_CHOH-CH_-OR° l 2 L R° being a mycoJ ic acid radical containing from 80 to 90 carbon atoms: - N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-lysine-decyl5 ester of formula H,C-CH-CONH-CH-CONH-CH-CONH, 3 I I 2 CHg 2 CO-NH-CH-COO-(CHg)g-CHg ( ? H 2>4 NHg -N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-butyl-ester of formula no H q C-CH-CONH-CH-CONH-CH-CONH_ 3 I I 2 ch 3 (ch 2 ) 2 COO-(CH 2 ) 3 -CH 3 - N-acetyl-muramyl-L-alanyl-D-glutamyl-a-methyl-ester butyl-ester of fonnula H-.C-CH-CONH-CH-CONH-CH-COO-CH, 3 I I 3 ch 3 (ch 2 ) 2 COO-(CH 2 ) 3 -CH 3 4-8 7 35 107 H,C-CH-C0NH-CH-C0NH-CH-C0NII o 3 f I 2 ch 3 (ch 2 ) 2 COO-(CH 2 ) g -CH 3 -β-D-p-aminophenyl-N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanyl-decyl-ester of formula CH -N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanyl-pentadecyl-ester of formula . 48735 H,C -CH-CONH-CH-CONH-CH-CONH, 3 I I 2 CH 3 ^H 2 ) 2 CONH-CH-COO-(CH 2 ) 14 ~CH 3 ch 3 - N-acetyl-muranp-l-L-alanyl-D-isoglutaminyl-L-alanyl-benzylester of formula H,C-CH-CONH-CH-CONH-CH-CONH_ 3 . ι 2 CH 3 (ch 2 ) 2 CONH-CH-COO-CH 2 -C g H 5 CH,

4. A compound according to Claim 1, wherein X is a 10 glycyl residue. 4 carbon atoms, - r 6 is a hydrogen atom, or a substituted or unsubstituted acyl radical, which is saturated or unsaturated, containing from 1 to 90 carbon atoms, and optionally carrying functional 15 groups selected from: hydroxyl, carboxyl, carbonyl, amino, 102 cyclopropane and methoxyl, - X is an aminoacyl residue selected from: L-alanyl, L-arginyl, L-asparagyl, L-aspartyl, L-cysteinyl, L-glutaminyl, L-glutamyl, glycyl, L-histidyl, L-hydroxyprolyl, 5. 52. A laboratory reagent according to Claim 46, substantially as hereinbefore described. 53. A process ior the preparation of a compound of the general formula I given and defined in Claim 1, substantially as hereinbefore described. 5 by lecithin and cholesterol. 33. A pharmaceutical composition according to Claim 32, wherein the constituents of the lipid phase are in molar ratios between 81 and 1:1 lecithin/cholesterol. 34. A pharmace itical composition according to any one of 10 Claims 30 to 33, wherein the aqueous phase is buffered to a pH close to neutrality. 35. A pharmaceutical compositon according to Claim 34, wherein the aqueous phase is buffered with phosphate. 36. A pharmaceutical composition according to Claim 29, 15 wherein the injectable medium is constituted by an agueous solution for injection. 37. A pharmaceutical composition according to Claim 36, wherein the aqueous solution for injection is an isotonic and sterile saline or glucose solution. 38. A pharmaceutical composition according to Claim 27 or Claim 28, wherein the compound (I) is associated with excipients adapted for oral administration. 39. A pharmaceutical composition accordirg to Claim 27 or 5 Claim 28, wherein the compound (I) is associated with excipients permitting administration by application to the ocular mucous membranes or the respiratory tract. 40. A pharmaceutical composition according to Claim 27 or Claim 28, wherein the compound (I) is associated with 10 excipients for vaginal administration, 41. A pharmaceutical composition according to Claim 27 or Claim 28, wherein the compound (I) is associated with excipients for rectal administration. 42. A pharmaceutical composition according to any one of 15 Claims 29 or Claims 36 and 37, wherein, for an antiinfectious treatment and parenteral administration, the unit doses contain from 10 to 1,000 ug of compound (I). 43. A pharmaceutical composition according to Claim 38, wherein, for an anti-infectious treatment and oral 20 administration, the unit doses contain from 200 to 20,000 ug of compound (X). 114 44. A pharmaceutical composition according to any one of Claims 27 to 43, dosed and packaged for use in human medicine. 45. A pharmaceutical composition according to any one of 5 Claims 27 to 43, dosed and packaged for use in veterinary medicine. 46. A laboratory reagent comprising at least one compound according to any one of Claims 1 to 26. 47. A compound of the general formula (1) given and defined 10 in Claim 1 substantially as hereinbefore described with particular reference to the Examples. 48. A pharmaceutical ccmposition according to claim 27 substantially as hereinbefore described with reference to the Examples. 49. A method of modifying the immune response in a 15 non-human, warm-blooded creature comprising administering an effective amount of a compound according to any one of Claims 1 to 26 to said creature. 50. A method of studying the immunological properties of a substance comprising comparing the immune response of 20 a warm-blooded creature to said substance with those of a reagent according to Claim 46. * 51. A method of treating infectious diseases in a nonhuman, warm-blooded creature comprising administering an effective amount of a compound according to any one of Claims 1 to 26 to said creature. 5 28. A pharmaceutical composition according to Claim 27, wherein the one or more compounds (I) are associated with pharmaceutically accej table vehicles or excipients. 29. A pharmaceutical composition according to Claim 27, constituted by a solution or suspension of the compound (I) 10 in an injectable medium. 30. A pharmaceutical composition according to any one of Claims 27 to 29, wherein the composition is in the form of liposomes. 31. A composition according to Claim 30, wherein the „ 48735 112 lipid portion of the liposomes is constituted by a phospholipid. 32. A pharmaceutical composition according to Claim 31, wherein the lipid portion of the liposomes is constituted 5 - N-acetyl-muramyl-L-alanyl-D-glutamyl-a-methyl-ester' decyl-ester of fonnula HO ch 2 oh |/F\ OH Inh-coch 3 H 3 C-CH-CONH-CH-CONH-CH-COO“CH. COO-(CH 2 )g-CH 3 27. A pharmaceutical composition, containing an effective dose of at least one compound of formula (I) according to any one of Claims 1 to 26. 5 - N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanyl-Dglyceryl-mycolate of formula 5 25. A compound according to Claim 23, wherein the residue (A) n ~Z is one of the following: -NH-CH(CH 3 )-coo-(ch 2 ) -ch 3 , -NH-CH(CH 3 )-CONH-(CH 2 ) g -CH 3 , -NH-CH (CH 3 ) -COO- (CH 2 ) g -CH 3 , io -NH-CH(ch 3 )-coo-(:h 2 ) 14 -ch 3 , -NH-CH (CH 3 ) -COO- ( ;h 2 ) 3 -CH 3 , -NH-CH(CH,)-COO-CH,-C_-H c , -NH-CH(CH 3 )-COO-CH 2 -CHOH-CH 2 -O-R°, with R° corresponding to a mycolic acid comprising 80 to 90 carbon atoms, 15 -NH-CH [ (CH 2 ) 4 -NH 2 ] -C00-(CH 2 ) g -CH 3 , -0-(CH 2 , 3 -CH 3 or -0-(CH 2 ) 9 -ch 3 26. A compound according to Claim 1, which is one of the following: 20 - N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-decyl-ester of the formula

5. A compound according to Claim 1, wherein X is an aminoacyl residue selected from: L-prolyl, L-threonyl and L-valyl. 5 2. A compound according to Claim 1, wherein X is an L-alanyl residue. 5 L-isoleucyl, L-leucyl, L-lysyl, L-methionyl, L-orninthyl, L-phenylalanyl, L-prolyl, L-seryl, L-threonyl, L-tryptophanyl, L-tyrosyl and L-valyl, - Y is either -CH, or an alkoxy radical comprising from 5 - R is either a hydrogen atom or a methyl group, - R^ is a hydrogen at; m, an alkyl group having at most 4 carbon atoms, a substituted or unsubstituted aryl or alkyl-aryl group comprising at most 10 carbon atoms, - R 2 is a methyl grouy, 10 - R 4 is a hydro- en atom, an acyl radical comprising at most

6. A compound according to any preceding claim wherein 15 one or more A groups are aminoacyl residues selected from: alanyl, leucyl, lysyl, glycyl, valyl and isoleucyl.

7. A compound according to Claim 6, wherein the first aminoacyl group A fixed to the α-carboxyl function of the D-glutamyl residue is an L-alanyl residue. 104

8. A compound according to Claim 6, wherein the first aminoacyl group Λ fixed to the γ-carboxyl function of the D-glutamyl residue is an L-lysyl residue or an L-glutamyl residue. 5

9. A compound iccording to any one of claims 6 to 8 wherein n = 1 or 2. 10. A compound according to any one of the preceding claims wherein Y is an -OH group. 11. A compound according to any one of Claims 1 to 9, 10 wherein Y is an -OCHg group. 12. A compound according to any one of Claims 1 to 9, wherein Y is an -OC^Hg group. 13. A compounc according to any one of Claims 1 to 9, wherein Y is an -NHg group. 15 14. A compound according to any one of the preceding claims wherein < 4 is a hydrogen atom. 15. A compound according to any one of Claims 1 to 13, wherein is a -CO-(CHg)g-COgH group. 105 16, A compound according to any one of Claims 1 to 13, wherein R 4 is a -COCH^ group. 17. A compound according to any preceding claim, wherein Rg is a hydrogen atom. 5 18. A compound according to any one of Claims 1 to 16, wherein R g is an acyl radical containing from 1 to 4 carbon atoms. 19. A compound according to any one of Claims 1 to 16, wherein Rg is a -COCH 3 group. 10 20. A compound according to any one of Claims 1 to 16, wherein Rg is a -CO(Cfl 2 ) 2 -CO 2 H group. 21. A compound according to any one of Claims 1 to 16, wherein R g is a mycoloyl group (Οθθ to Cg Q ) or corynomycoloyl group (C 32 ) . 15 22. A compound according to any preceding Claim, wherein R is a hydrogen atom. 23. A compound according to any one of Claims 1 to 21, wherein R and R 2 are -CHj. 106 24. A compound according to Claim 23, wherein R' is an alkyl group comprising from 5 to 90 carbon atoms, optionally bearing hydroxyl, carboxyl, carbonyl, cyclopropane and methoxyl functional groups.

10. 54. A compound of the general formula X given and defined in Claim 1, whenever prepared by a process claimed in Claim 53.