IE46957B1 - New peptide derivatives - Google Patents

Abstract

1. Compounds of the formula see diagramm : EP0000330,P30,F1 wherein R1 and R2 each represent a saturated or unsaturated aliphatic or mixed aliphatic-cycloaliphatic hydrocarbon radical which has 11-21 C atoms and which is also optionally substituted by oxygen functions, R3 represents hydrogen or the radical R1 -CO-O-CH2 -, where R1 has the same meaning, and X represents a natural aliphatic amino acid linked by a peptide bond and having a free, esterified or amidated carboxyl group, or an amino acid sequence of 2-10 natural aliphatic amino acids, the terminal carboxyl group of which is free or in the ester or amide form, with the asymmetric centres denoted by * and ** possessing the absolute R- and S- or R-configurations, respectively ; and, possibly, mixtures of the R- and S-compounds epimeric on the C** atoms, and salts and complexes of all these compounds.

Description

The present invention relates to new lipopeptides and in particular to compounds of the formula R,-CO-O-CH x j RpCO-O-Sfl (I) * = R ** = R or S . s I C1IO L2 R2co-rh-ch-co-x wherein R^ and R2 each represent a saturated or unsaturated 5 aliphatic or mixed aliphatic-cycloaliphatic hydrocarbon radical which has 11-21 C atoms and which is also optionally substituted by oxygen functions, R^ represents hydrogen or the radical R^-CO-O-CH^-, where has the same meaning, and X represents a natural aliphatic amino acid linked by a 10 peptide bond and having a free, esterified or amidated carboxyl group, or an amino acid sequence of 2-10 natural aliphatic amino acids, the terminal carboxyl group of which is free or in the ester or amide form, with the asymmetric centres denoted by * and ** possessing the absolute R- and S- or R-configurations, respectively, and - 3 optionally to mixtures of the R- and S-compounds epimeric on the C** atoms; and optionally to salts and complexes of these compounds; as well as to processes for producing them synthetically; and to pharmaceutical preparations containing one or more of these lipopeptides together with a pharmaceutical carrier material, optionally together with other pharmaceutical compounds.

Lipopeptides as degradation fragments of lipoproteins have already been described. Thus, e.g., Hantka and Braun (Eur. J. Biochem. 34, 284 - 296 (1973) were able to isolate from the murein lipoprotein of the outer cell wall of Escherichia coli, by enzymatic degradation, lipopeptide mixtures in impure form, to which may be attributed, in accordance with the examinations carried out, the following structure Yi-°-ch2 Yo-0-CH 1 I s I CIL I 2 Y^-NH-CH-CO-Ser-Ser-Asn-Ala-Lys-OH or corresponding formulae with shortened polypeptide chain, wherein Y^, Y2 and Yg represent acyl radicals of different higher saturated and unsaturated fatty acids, for instance of those having 14-19 C atoms and of others. Also the murein protein as such is based on the same structure (loc.cit); the absolute configuration on the two asymmetrical carbon atoms of the above formula was not investigated. Both mureinlipoprotein and its stated degradation products exhibit in vitro mitogenic activity towards mouse lymphocytes [see also Z. Immun.-Forschung, Vol. 153, pp. 11-22 (1977)]. - 4 As can be seen, the degradation lipopeptides obtained from miirein protein are based on glycerylcysteine ' HO-CH, I 2 HO-CH S I CH, NH2-CH-COOH As products of an enzymatic cleavage of natural products, these fragments were not products well defined in their composition, and were obviously complicated mixtures of condensation products from the stated peptide part and very different acyl derivatives of glycerylcysteine.

The lipopeptides of the present application differ from the stated degradation products of murein proteins in many respects: they are lipopeptides of a precisely defined homogeneous chemical constitution and configuration, which are obtainable synthetically, and which are therefore suitable for therapeutic application; the group formed by them comprises compounds in which the erythrite residue takes the place of the glycerol part in the glycerylcysteine”, and/or comprises compounds in which there is an amino acid sequence different from that given above for the known degradation lipopeptide mixtures in the formula (II), or in place thereof there is just a single amino acid, and, finally, also derivatives, such as amides and esters of the terminal carboxyl group.

In the acyl groups R^CO and R2CO ·*-η t^le compounds of the formula I and derivatives thereof, and R2 are saturated or unsaturated, aliphatic or aliphatic-cycloaliphatic hydrocarbon radicals which are also optionally substituted by oxygen functions and which have 11-21 C atoms, i.e. the acyl - 5 groups are derived from saturated or unsaturated, aliphatic or cycloaliphatic carboxylic acids which are optionally oxygenated in the hydrocarbon radical and which contain 12-22, preferably 14-18, C atoms. To be mentioned as such are the saturated or unsaturated fatty acids, such as lauric acid, myristic acid, palmitic acid, margaric acid, stearic acid, arachic acid, oleic acid, elaidic acid, linoleic acid, a- and β-eleostearic acid, stearolic acid or a-linolenic acid; and among the cycloaliphatic-aliphatic acids, e.g.: dihydrosterculic acid, malvalic acid, hydnocarpic acid and chaulmoogric acid. Oxygenated acids of this type, which are likewise suitable for the acyl groups R^CO and R2CO, are, e.g., the acids obtained by epoxidation of the abovementioned olefinic fatty acids and cycloaliphatic-aliphatic acids, e.g. -epoxystearic acid, also derivatives of the above-mentioned acids, which contain, e.g., one or more hydroxyl groups, such as ricinoleic acid.

In the compounds of the formula (I) or in the diastereoisomeric mixtures and salts or complexes thereof, the groups and R2 can be identical with or different from each other. Preferred compounds are those wherein all three or four acyl groups are identical, and among these compounds in particular those in which the acyl groups are palmitoyl, stearoyl or oleoyl.

The peptide sequence X in the compounds of the formula (I) or in the salts thereof is composed of a maximum of 10 natural aliphatic amino acids, preferentially with at least half carrying a hydrophilic group, such as in particular hydroxyl, amino, carboxyl, carbamide, guanidino or imidazolyl groups. Of the ionic amino acids of this category, there may be emphasised among those carrying acid groups in particular aspartic acid and glutamic acid and oxyglutamic acid, and among those carrying basic groups especially lysine, ornithine, arginine and histidine. Amino acids having neutral character are in - 6 particular the amides, such.as asparagine and glutamine, and those carrying hydroxyl groups are above all serine and threonine.

If X in the formula (I) represents the amino acid mentioned, 5 it is likewise one of the stated amino acids, e.g. one carrying hydrophilic groups, e.g. especially serine or threonine.

Among the amino acids which can go into the above sequence and which carry no hydrophilic groups, there are to be mentioned in particular the unsubstituted amino acids, such as glycine, alanine, valine, norvaline, leucine and isoleucine; also however some substituted amino acids which are of non-hydrophilic character, such as methionine.

The sequence of the stated amino acids in the peptide chain X can be optional, but preferred sequences are those in which all the amino acids having hydrophilic groups are bound directly to each other, and among these are preferred those in which this sequence of hydrophilic amino acids is bound to the carboxyl group of the cysteine in the triacylglycerylcysteine part or tetraacyl-erythritylcysteine part. 2q An important group [Group I] of lipopeptides according to the present invention is formed by those in which the peptide chain consists of a maximum of 5 amino acids. To be mentioned among these are in particular the lipopeptides of the formula R. -CO-O-CIL 1 j z R. -C0-0-(3i '· I CII9 (IV) I 2 s I CIL I* ... R R2CO-KH-CH-CO-X ~ = k ** = R wherein R^ and R2 have the same meanings, and X has the - 7 meaning given under the formula (I), but contains 2-5 amino acids in the case of the amino acid sequence, in which, preferably, at least half carry a hydrophilic group, and also the salts and complexes thereof. Among these lipopeptides, there may be emphasised those of which the amino acid sequence in the group X corresponds to that of the aforementioned lipopeptides obtained by degradation of murein lipoprotein, i.e. corresponds to that of the formula (II), or to a shortened chain derived therefrom. There may be mentioned in particular the following lipopeptide types: (AcGCT = acyl-glycerylcysteine part according to the formula IV): AcGCT- Ser - Ser - Asn - Ala - Lys - OH AcGCT- Ser - Ser - Asn - Ala - OH AcGCT- Ser - Ser - Asn - OH AcGCT- Ser - Ser - OH ; also the corresponding types in which threonine, glutamine or asparagine replace serine; furthermore the compound types of the following kind: AcGCT- Ser - Ser - Asn - Ala - Glu - OH AcGCT- Ser - Ser - Phe - Ala - Glu - OH AcGCT- Ser - Ser - Phe - Ala - OH AcGCT- Ser - Ser - Phe - OH , and the corresponding compounds with threonine, glutamine or asparagine as exchange amino acids for serine, and the amides and carboxylic acid esters having a terminal carbamide or ester group, with particular consideration being given to compounds in which the acyl groups R^-CO, Rj-CO in the acylglycerylcysteine part are identical and are palmitoyl, stearoyl or oleoyl, and to all those compounds in which R^-CO and R2-C0 are different and are, e.g., palmitoyl, stearoyl or oleoyl, and in which these radicals occur in any chosen combination and/or variation, such as those listed in the following Table, wherein the glycerylcysteine part correspondin'? to the N- and O-acyl substituents is written in abbreviated form as N-acyl-O-O-di-acyl-cys : - 8 N-palmitoyl-O.O-di-palmitoyl-cys - Ser-Ser-Asn-Ala-Lys-ΟΗ tl II II II ii II - Ser-Ser-Asn-Ala-OH tt II 11 tl It It - Ser-Ser-Asn-OH It II II It tl II — Ser-Ser-OH It It U tl II II • - Ser-OH N-stearoyl-0.O-di- -stearoyl-cys - Ser-Ser-Asn-Ala-Lys tl Il tt It tl II - Ser-Ser-Asn-Ala-OH II It Η II tl II - Ser-Ser-Asn-OH II II It tt II tt - Ser-Ser-OH II ιϊ η ii.. II tl - Ser-OH------- N-oleoyl-0.O-di-oleoyl-cys - Ser-Ser-Asn-Ala-Lys 1» II II II If tl - Ser-Ser-Asn-Ala-OH 11 It tt If It II - Ser-Ser-Asn-OH II II ft It It It - Ser-Ser-OH It If It ll¥ II II - Ser-OH N- -lauroyl-O. O-di-lauroyl-cys - Ser-Ser-Asn-Ala-Lys-OH II II II II II It - Ser-Ser-Asn-Ala-OH It II tl tl It It - Ser-Ser-Asn-OH If II tl II »1 ft - Ser-Ser-OH II II It It It tl - Ser-OH ; and in addition compounds resulting from the substitution of serine by threonine, glutamic acid, glutamine, aspartic acid or asparagin, from the substitution of lysine by glutamic acid, and compounds resulting from the simultaneous substitution of lysine by glutamic acid and asparagine by phenylalanine.

Among the.compounds having different acyl groups on N and. 0 atoms in the glycerylcysteine part, the following are to be emphasised: N-myristoyl-0.O-di-palmitoyl-Cys-Phe-Phe-Asn-Ala-Lys-OH -lauroyl r -Ser-Ser-Asn-Ala-Glu-OH -stearoyl - - - - -Ser-Ser-Asn-Ala-Ala-OH , and the compounds resulting from- replacement of Ser by Phe or of Phe by Ser.

A second group of compounds [Group II] according to the - 9 invention are compounds corresponding to the above formula IV in which however the configuration on the C** atom is S instead of R, and the mixtures of R- and S-diastereoisomers, such as can occur, for example, in the synthetic process of preparation. Specific types and compounds correspond in this group too to those particularly emphasised in the foregoing for the R group (formula IV).

A third group [Group III) of the new lipopeptides comprises the compounds according to the formula (IV), in which however X represents an amino acid sequence having 6-10 amino acids, with the first 5 preferably constituting the sequences already emphasised above. The amino acids 6-10 can be any of the natural amino acids mentioned above, such as in particular serine, asparagine, alanine, glutamic acid, -lysine or phenylalanine, and can occur for instance in the following sequences: Ile-Asp-Glu-OH and Asp-Glu-OH.

Specific compounds are, e.g.: N-stearoyl-S-[2(R),3-dipalmitoyloxypropyl]-Cys-Ser-Ser-AsnAla-Glu-Ile-Asp-Glu-OH, N-palmitoyl-S-[2-(R),3-dipalmitoyloxypropyl]-Cys-Ser-SerAsn-Ala-Glu-Ile-Asp-Glu-OH, and N-palmitoyl-S-[2(R),3-dipalmitoyloxypropyl]-Cys-Ser-SerAsn-Ala-Lys-Ile-Asp-Glu-OH.

It is possible according to the invention to also produce corresponding compounds of this type with the S-configuration on the C** atom, as well as diastereoisomeric mixtures of S- and R-compounds.

A fourth group [Group IV] of lipopeptides according to the invention comprises compounds of the formula (I) in which R^ represents the radical R^-CO-O-CI^··, wherein the configuration on the C atoms is R or S, particularly also - ΙΟ diastcreoisomeric mixtures. Subgroups arc compounds of the formula n _rn_n_pn.

I I R, -CO-O-CH (V) R2-C0-NH-CtI-C0-X ** = R or S especially those having the R-configuration on the C** atoms, 5 wherein X has the meaning given under the formula (IV).

The acyl groups R^-CO- and R2-CO- are preferably the same and are preferably again palmitoyl, stearoyl or oleoyl; they can. be however, In the way discussed in the foregoing with regard to the other groups, in all combinations and/or 10 variations. Preferred types and compounds of this class are again those in which X has the meanings specified above.

For this group too, X is preferably an amino acid or a sequence of 5 amino acids, particularly one of those designated above as being preferred in the case of the other three groups of lipopeptides. X can however also represent one of the amino acid sequences with 6-10 amino acids described for the third group of lipopeptides of the present application, particularly the sequences specifically mentioned for that group.

Specific compounds are, e.g., those derived from the above Table compiled for specific compounds of the Group I if, in these compounds, N-acyl-O-O-di-acyl-cys does not denote ns in that case the glycerylcysteine part but the erythritylcystcine part; and the compounds resulting from substitution of serine by 469 57 - η threonine, glutamic acid, glutamine, aspartic acid or asparagine, and of lysine by glutamic acid, and, optionally, simultaneously of Asn by phenylalanine.

A fifth group of lipopeptides according to the present invention is formed of those compounds according to the formula (I) in which X represents a peptide chain wherein the sequence of the first 5 amino acids is different from those present in the known murein-lipoprotein-degradation lipopeptides, and particularly of such compounds wherein R.g represents hydrogen, which correspond therefore to the above formula (IV), where R^-CO and represent the acyl groups particularly emphasised as being preferred in the foregoing and in the following, in the stated combination, and also of salts and complexes thereof. There may be mentioned for example the following lipopeptide types (AcGCT = acyl - glycerylcysteine part according to the formula (XV)) AcGCT - Phe - He - He - Phe - Ala - OH AcGCT - Val - Lys - Val - Tyr - Pro - OH and amides and esters thereof, particularly those especially emphasised in the following.

A further subgroup comprises compounds according to the formula (IV) in which X represents however the said unnatural sequence of amino acids in comparison with the murein-lipoprotein degradation products, and in which 6-10 amino acids are present. The first five amino acids have e.g. the sequence given above or any other desired sequence; there may be mentioned, e.g., the following lipopetides, where AcGCT has the above meaning: AcGCT - Ala - He - Gly - Val - Gly - Ala - Pro - OH AcGCT - Ser - Ser - Asn - Ala - Glu - 11c - Asp - Glu - OH and amides and esters thereof, particularly those emphasised in the following. 469S7 - 12 There are preferably produced compounds of this type in which the configuration on the C** atom of the AcGCT is R, or mixtures of R- and·S-epimers.

In the above discussed new lipopeptides according to the present invention, the terminal carboxyl group can be in the amide form. Besides the unsubstituted amide, there are applicable also those which are derived from a primary or secondary amine. Suitable in particular are amides of lower aliphatic amines having 1-7 C atoms, such as methylamine, ethylamine, propylamine, butylamine, dimethylamine, diethylamine or propyl- and isopropylamine, also aromatic amines, especially monocyclic amines, such as aniline or toluidine, araliphatic amines such as benzylamine, or heterocyclic amines, such as the aminopyridines. In the case of secondary aliphatic amines, these can be ring-closed nitrogen bases, such as pyrrolidine, piperidine or piperazine.

Specific amides are, e.g., the unsubstituted amides or the methyl-, ethyl-, dimethyl- or diethylamides pf all the aforementioned specific lipopeptides according to the present invention, or those amides of the compounds described in the illustrative Examples.

In the esterified terminal carboxyl groups of the new lipopeptides, the alcohol constituent is derived preferably from lower aliphatic alcohols having 1-7 C atoms, such as methyl alcohol, ethyl alcohol, n-propyl alcohol, isopropyl alcohol or butyl alcohols. The esterifying alcohols may however also be polyhydric alcohols, such as ethylene glycol or propylene glycol or glycerol. For esterification there can also be used araliphatic alcohols, particularly monocyclic lower aliphatic alcohols having 1-7 C atoms In the aliphatic part, such as benzyl alcohol, or heterocyclic alcohols, such as tetrahydrofuranol or tetrahydropyranol. As specific esters of this type of Lipopeptides according to the invention, there may be mentioned, for example, the methyl and ethyl - 13 esters and the ethylene glycol or propylene glycol esters of all the aforementioned specific lipopeptides, or of those which are described in the illustrative Examples.

Depending on the nature of their substituents, the present new lipopeptides are neutral, acid or basic compounds. If excess acid groups are present, they form salts with bases, such as ammonium salts, or salts with alkali metals or alkaline-earth metals, e.g. sodium, potassium, calcium or magnesium; if however excess basic groups are present, they form acid addition salts.

Acid addition salts are in particular pharmaceutically applicable, nontoxic acid addition salts, such as those with inorganic acids, e.g. hydrochloric acid, hydrobromic acid, nitric acid, sulphuric acid or phosphoric acid, or with organic acids, such as organic carboxylic acids, e.g. acetic acid, propionic acid, glycolic acid, succinic acid, maleic aeid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2~acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinic acid, or organic sulphonic acids, e.g. methanesuiphonic acid, ethanesulphonic acid, 2-hydroxyethanesulphonic acid, ethane-1,2disulphonic acid, henzenesulphonic acid, p-toluenesulphonic acid or naphthalene-2-sulphonic acid; and also other acid addition salts which can be used, e.g., as intermediates, e.g. for purification of the free compounds, or in the production of other salts, as well as for characterisation, for example those with picric acid, picrolonic acid, flavianic acid, phosphotungstic acid, phosphomolybdic acid, chloroplatinic acid, Reinecke's acid or perchloric acid.

Complexes are the compounds formed with metal salts, e.g. with heavy-metal salts, such as salts of copper, zinc, iron or cobalt. To form such complexes, there are preferably - 14 used the phosphates, pyrophosphates and polyphosphates of these metal salts, optionally in combination with acid organic substances, e.g. polysaccharides containing acid groups, such as carboxymethylcellulose, tannic acid, polyglutamic acid or partially hydrolysed gelatin, also alkali metal polyphosphates, such as Calgon N, Calgon 322, Calgon 188 or Plyron B 12.

The compounds of the present invention according to the above formula (I) and salts, complexes and mixtures thereof have valuable pharmacological properties, in particular they have a pronounced immunity-potentiating action. Thus the compounds in the dosage range of 0.5 - 320 jig/ml stimulate the proliferation of B-lymphocytes, which is determined in vitro by means of thymidine incorporation, 20-fold to 50-fold in comparison with control lymphocytes not stimulated. The extent of stimulation corresponds to that obtained with the most effective known B-cell mitogens [dextrane sulphate, E. coli Lipopolysaccharide, PPD (purified protein derivative)]; and in the case of the new compounds of the present invention even high concentrations do not have a lymphocytotoxic effect.

The new compounds of the formula (I) and the salts, complexes and mixtures thereof are moreover able, at a concentration of 0.3 - 60 yig/ml, to induce in splenocyte cultures of normal mice the formation of antibody-producing cells (propagation of the 19S-plaque-forming cells by a factor of 20 to 50 with respect to the control value (in the absence of the stimulating substances)): In the presence of the stated compounds, there are thus formed, e.g., specific antibodies against sheep erythrocytes, without there being added to the cultures sheep erythrocytes for immunisation. On the other hand, the stated substances in the same concentration range are able to increase also the immunological reactivity of splenocyte cultures depleted of T-cells (of congenitally athymic nu/nu mice) compared with a normally thymus-dependant antigen (sheep - 15 erythrocytes) [factor 10 to 40 compared with untreated control cultures]. The action of the stated compounds is however not only to induce in vitro, directly or indirectly, proliferation and synthesis performances of B-lymphocytes (i.e. of potentially antibody-forming cells), but also to induce effects on T-lymphocytes (to which belong supporting cells, suppressor cells and also cytotoxic effector cells which are active as regulators). Thus, for example, towards allogenic irradiated stimulator lymphocytes, the said compounds in a concentration range of 10-100 ug/ml are capable of substantially potentiating the reactivity of cortisoneresistant thymus cells (up to 10-fold).

The effects mentioned above are probably indirectly a result of the lipopeptides activating macrophages which, in their turn, promote the reactivity of T- and B-lymphocytes.

It can in fact be shown that the stated compounds, even at an extremely low concentration (0.01 - 10 jjg/ml), release large amounts of colony stimulating activity (CSA) from mouse macrophages (induction of up to 150 - 200 colonies within 7 days from 10 mouse marrow cells, after addition of 20% of the supernatants from macrophage cultures incubated during 24 hours with substance, in comparison with 0-5 colonies with the addition of supernatants from untreated macrophage cultures). CSA is a biological mediator which is necessary for differentiation of bone-marrow parent cells with respect to macrophages and polymorphonuclear leucocytes. The stated compounds hence effect an increased supply of cells which are of extreme importance for the non-specific resistance and for the induction amplification and expression of specific (lymphocyte-induced) immune reactions.

The immunity-potentiating action of the new compounds of the formula (1) and of salts, complexes or mixtures thereof can be verified also in vivo. Thus the injection of a lipopeptide according to the invention leads within 3-9 hours to a high 46357 - 16 , increase of the CSA concentration in the serum (up to 120 colonies per 10^ mouse marrow cells after the addition of serum extracted with chloroform [5% final concentration], compared with 0-5 colonies in the case of untreated animals). Accordingly, the antibody-forming capacity of mice _ is greatly potentiated by administration of the same compounds in vivo. , NMRI mice are immunised by intraperitoneal injection of 10 jug of precipitate-free BSA on day 0. Serum samples are 10 taken 9, 15 and 29 days later and tests are carried out, using a passive haemagglutination technique, to determine their content of anti-BSA antibodies. In the employed dosage amount, soluble BSA is subimmunogenic for the receiver animals, that is to say it is not able to initiate any production of antibodies, or is able to initiate only a very slight production of antibodies. Additional treatment of the mice with specific immunity-potentiating substances before or after administration of the antigen leads to a rise in the antibody titre in the serum. The effect of the treatment is expressed by the score value attained, that is to say by the sum of the log2 titre differences on the three days when blood is sampled. In this test, the compounds of the formula (I) and salts, complexes or mixtures thereof are able, on intraperitoneal or subcutaneous administration of 0.03 - 3 mg/kg of. animal weight on five consecutive days, before or after immunisation with BSA to significantly increase the production of antibodies against BSA.

’’Also manifestations of the cell-transmitted immunity can be potentiated by the said compounds in vivo.

Whereas sensibilisatioii of guinea pigs with BSA in incomplete Freund's adjuvant leads only to humoral antibody formation, the addition of' the lipopeptides according to the present invention in a dosage range of 1-15 jig, to the aqueous phase of the antigen-oil emulsion induces delayed-type - 17 hypersensitivity towards BSA. Three weeks after immunisation, the intracutaneous injection of BSA in the case of these animals leads to a local inflammatory reaction with erythema and thickening of the skin, which attains its maximum within 24 to 48 hours. These delayed-type reactions correspond quantitatively and qualitatively to those which are usually obtained by immunisation with BSA in complete Freund's adjuvant (i.e. with addition of mycobacteria). The values (required jug/animal for the induction of a difference in the reaction volume (surface area of the erythema x increase in thickness of skin) in the case of treated and untreated animals of 200 pi,. 24 hours after induction) are 2-3 ^ig.

The lipopeptides according to the present invention are moreover negligibly toxic. Even intraperitoneal administration five times in a dosage amount of 10 mg/kg/day on five consecutive days was tolerated by the mice without there being any apparent symptoms . Since the doses necessary for immunitystimulation are very low, the therapeutic range of the new compounds is extremely wide.

The new lipopeptides according to the present invention can therefore substantially increase the cellular and, in particular, the humoral immunity, both when administered in admixture with the antigen itself (adjuvant effect in the narrower sense) and when supplied at times and places which differ from those of the antigen injection (systemic immunity potentiation).

.. The new lipopeptides according to the present invention can hence be used as adjuvants in admixture with vaccines to improve the success rate of the vaccination, and to improve the protection imparted by humoral antibodies and/or cellular immunity against infection by bacterial, viral or parasitic pathogens.

Finally, the compounds· described are suitable, in admixture 469 57 . - 18 with various antigens, as adjuvants for the experimental ' and industrial manufacture of antisera for therapy and diagnosis, and for inducing immunologically activated lymphocyte populations for cell-transfer processes.

Furthermore, the new lipopeptides can be used, even without the simultaneous administration of an antigen to promote immune reactions which are already taking place subliminally in humans and animals. Accordingly, the compounds are especially suitable for stimulating the body's own Iq resistance, e.g. in the case of chronic and acute infections, or in the case of selective (antigen-specific) immunological defects; and also in the case of general (that is to say not antigen-specific) immunological defective states which are congenital or acquired, such as arise in old age, in the course of severe primary diseases and, above all, after therapy with ionising rays or with hormones having an immunosuppressive action. The said substances can thus be administered, preferably also in combination with antibiotics, chemotherapeutic agents or in combination with other therapeutical 2q treatments, in order to counteract immunological damage.

Finally, the substances described are suitable also for the general prophylaxis of infectious diseases in humans and in animals . - 19 The new lipopeptides can be produced by methods known per se, or by new processes which are described herein.

The compounds of the formula (I) and diastereoisomeric mixtures, salts and complexes thereof are produced by a preferred process which is characterised in that a) in a compound of the formula b R, -CO-O-CH 1 Ιλη, -CO-O-CH 1 I CH„ S I chq I 2 R2CO-NH-CH-CO-Y (VI), * = R ** = R, S or mixture (R,S) wherein Rp R2 and have the meanings given under the formula (I), and Y represents an amino acid or amino acid 1q sequence corresponding to X in the formula (I), wherein however at least one of the hydrophilic groups substituting the amino acids and/or the terminal carboxyl group is (are) protected by a protective group which can be split off under neutral or mild acid conditions, or in a salt of such a compound, the protective group(s) is (or are) split off; or b) a compound of the formula R.-C0-0-CH 1 CH, (VII), l2 * = R ** = R, S or fTT mixture (R,S) r2-co-nh-ch~co-w wherein R^, R^ and R^ have the meanings given under the formula (I), and W represents OH or an amino acid or an incomplete sequence of amino acids according to X in the formula (I), is reacted with a compound of the formula nh2 - zx (VIII) where Z^ represents a group corresponding to the group X in the formula (I), or an amino acid sequence or amino acid complementary to the stated amino acid or incomplete sequence of amino acids according to X, in which sequence however no free amino groups are present, or with a salt thereof; or c) a compound of the formula HO-CH HO-CH' CH, (IX) CH.

* = R ** = R, S or mixture (R,S) r2co-hh-ck-co-z2 wherein R2 has the meaning given under the formula (I), and R^ represents hydrogen or the group HO-CH^-, and - 21 46957 Z2 represents a group corresponding to X in the formula (I), wherein however no free hydroxyl groups are present, or a salt of this compound, is acylated in a manner known per se; and, if required, in resulting compounds having a free terminal carboxyl group this group is converted into an amide group or ester group, and/or the compounds are converted into salts or complexes thereof.

The protective groups in the process according to variant a) are, in particular, those known from the synthesis of peptides.

Thus, for example, protective groups for amino groups are acyl or aralkyl groups such as formyl, trifluoroacetyl, phthaloyl, benzenesulphonyl, p-toluenesulphonyl, o-nitrophenylsulphenyl and 2,4-dinitrophenylsulphenyl groups (these sulphenyl groups can also be split off by reaction with nucleophilic reagents, e.g. sulphites or thiosulphates); benzyl, or diphenyl- or triphenylmethyl groups which are optionally substituted, e.g. by lower alkoxy groups, especially by o- or p-methoxy groups; or groups derived from carbonic acid, such as aryImethyloxycarbonyl groups optionally substituted in the aromatic rings, e.g. by halogen atoms such as chlorine or bromine, nitro groups, lower alkyl or lower alkoxy groups or by chromophoric groups, e.g. azo groups, in which the methylene group can be substituted by a further aryl group and/or by one or optionally two alkyl groups, such as benzyl-, benzhydrylor 2-phenyl-isopropyloxycarbonyl groups, e.g. carbobenzoxy, p-bromo- or p-chlorocarbobenzoxy, p-nitrocarbobenzoxy or p-methoxycarbobenzoxy, p-phenylazo-benzyloxycarbonyl and p-(p1-methoxy-phenylazo)-benzyloxycarbonyl, 2-tolyl-isopropyloxycarbonyl and, in particular, 2-(p-biphenylyl)-isopropyloxycarbonyl, as well as aliphatic oxycarbonyl groups such as adamantyloxycarbonyl, cyclopantyloxycarbonyl, trichloroethyloxycarbonyl, tert-amyloxycarbonyl or, in particular, tert-butyloxycarbonyl, - 22 The amino groups can also be protected by the formation of enamines, obtained by reaction of the amino group with 1.3- diketones, e.g. benzoylacetone, acetylacetone or dimedone.

Carboxyl groups are protected for example by amide 5 or hydrazide formation or by esterification. The amide and hydrazide groups can be optionally substituted : the amide group e.g. by the 3,4-dimethoxybenzyl or bis-(p-methoxyphenyl)methyl group; the hydrazide group e.g. by the carbobenzoxy group, the trichloroethyloxycarbonyl group, the trifluoro10 acetyl group, the trityl group, the tert-butyloxycarbonyl group or the 2-(p-biphenylyl)-isopropyloxycarbonyl group. Suitable for esterification are, e.g., lower optionally substituted alkanols such as methanol, ethanol, cyanomethyl alcohol, benzoylmethyl alcohol or, in particular, tert-butanol, also aralkanols such as aryl-lower-alkanols, e.g. benzyl alcohols, p-nitrobenzyl alcohol, p-methoxybenzyl alcohol, 2,4,6-triraethylbenzyl. alcohol, or benzhydrols such as benzhydrol, all optionally substituted by lower alkyl or lower alkoxy groups or halogen atoms; phenols and thiophenols 20 optionally substituted by electron-attracting substituents, such as thiophenol, thiocresol, p-nitrothiophenol, 2,4,5and 2,4,6-trichlorophenol, pentachlorophenol, p-nitrophenol, 2.4- dinitrophenol, p-cyanophenol or p-methanesulphonylphenol; also, e.g., N-hydroxysuceinimide, N-hydroxyphthalimide, N-hydroxypiperidine and 8-hydroxyquinoline.

The hydroxyl groups of the serine and threonine groups can be protected for example by esterification or etherification -· Suitable acyl groups in the case of esterification are in particular radicals derived from carbonic acid, such as 3o benzoyloxycarbonyl or ethyloxycarbonyl. Groups suitable for etherification are, e.g., benzyl, tetrahydropyranyl or tertbutyl groups. Suitable for protecting the hydroxyl groups are also the 2,2,2-trifluorO-l-tert-butyloxycarbonylamino or -1-benzyloxycarbonylaminoethyl groups (Weygand) described in Ber. 100 (1967), 3838 - 3849. - 23 Preferably, the tert-butyl ester group or the benzhydrol group is used for protecting the carboxyl group of the side chains and optionally the terminal carboxyl group; the tert-buty3oxycarbonyl group for protecting the amino groups of the side chains; the tert-butyl ether group for protecting the hydroxyl groups of serine or threonine; and, optionally, the 2,2,2-trifluoro-l-tert-butyloxycarbonylaminoethyl group for protecting the imino group of histidine.

The splitting-off of the protective groups according to the process with acid agents under mild conditions can be effected in a manner known from peptide chemistry, e.g. by treatment with trifluoroacetic acid.

A particular protective group for carboxyl groups, which group can be split off under neutral conditions, is the group described e.g. in the German Offenlegungsschrift No. 06 490, this group being of the general formula fl R2 - Si - CH, - CH, I 2 2 R3 3 wherein R , R and R each represent a hydrocarbon radical, whereby the radicals may also be linked with each other by a simple C-C bond, in particular alkyl groups having 1-5 C atoms. Protective groups of this type are, e.g., the 2-(dimethyl-tert.-butylsilyl)-ethyl group, the 2-(dibutylmethylsilyl)-ethyl group and particularly the 2-(trimethylsilyl) -ethyl group. Although these protective groups can also be split off basically, it is in particular the splitting-off of these groups under neutral conditions which is of interest, for instance by reaction with a salt of hydrofluric acid.

The protective group is advantageously split off in an aprotic oi'ganic solvent; it is preferable in this respect to avoid the presence of those solvents which are able to solvate the fluoride anion, such as water or lower aliphatic alcohols. - 24 The condensation according to the variant b) of the compound of the formula (VII) with the compound of the formula (VIII) is performed, for example, by reacting the compound (VII) in the form of the activated carboxylic acid with (VIII); or by reacting the acid-(VII) with the compound (VIII) of which the amino group is in the activated form.

The compounds (VII) and (VIII) are used optionally in the form of their salts for the condensation reaction.

The carboxyl group of the compound (VII) can be activated for example by conversion into an acid azide, acid anhydride, acid imidazolide or acid isoxazolide, or into an activated ester, such as cyanomethyl ester, carboxymethyl ester, thiophenyl ester, p-nitrothiophenyl ester, thiocresyl ester, p-methanesulphonylphenyl ester, p-nitrophenyl ester, 2,415 dinitrophenyl ester, 2,4,5- or 2,4,6-trichlorophenyl ester, pentachlorophenyl ester, N-hydroxysuccinimide ester, Nhydroxyphthalimide ester, 8-hydroxyquinoline ester, 2-hydroxy1,2-dIhydro-l-carbethoxy-quinoline ester, N-hydroxypiperidine ester or an enol ester, which is obtained with N-ethyl-52o phenyl-isoxazolium-3-sulphonate [Woodward reagent]; or by reaction by means of a carbodiimide (optionally with the addition of N-hydroxysuccinimide), or by means of a l-hydroxybenzotriazole, 3-hydroxy-4-oxo-3,4-dihydrobenzo[d]1,2,3-triazine or N,Ν'-carbonyldiimidazole, each of which is unsubstituted or substituted, e.g., by halogen, methyl or methoxy.

The amino group of the compound (VIII) can be activated for example by reaction with a phosphite.

The most customary methods of condensation to be mentioned 30 are: the method according to Weygand-Wiinsch (carbodiimide in the presence of N-hydroxysuccinimide), the azide method, the N-carboxyanhydride or N-thiocarboxyanhydride method, the activated ester method and the anhydride method. These condensation reactions can be performed in particular by - 25 the Merrifield method.

It is also possible to perform the condensation of the compound (VII), using the above-mentioned methods, with a compound corresponding to the formula (VIII) in which the terminal carboxyl group is blocked by a protective group which is. different from an amide or ester group as defined for X, such as by one of the aforementioned groups, and/or one or more of the hydrophilic groups present is in the protected form, as described above. There are thus obtained condensation products which belong to the group of starting materials of the formula (VI) used for the variant a) described in the foregoing, and which are likewise subject matter of the present invention.

According to the above variant c), there are acylated in compounds of the formula (IX), which have not to contain free hydroxyl groups in the Z2 part, the free hydroxyl groups in the glyceryl or erythrityl group. This acylation can be performed in a manner known per se, e.g. by reaction with a reactive functional derivative of the acid corresponding to the radical to be introduced, such as with the anhydride or an acid halide, preferably in the presence of a tertiary base, such as pyridine or collidine.

This process is particularly suitable for producing products according to the formula I in which the acyl groups in the glyceryl or erythrityl group and on the cysteine group are different.

” If this variant is used in the case of compounds according to the formula (IX) in which however in the Z2 part the terminal carboxyl group is protected by protective groups other than an amide or ester group as defined for X, and/or in which one or more of the hydrophilic groups present is in the protected form as described above, there are likewise obtained starting compounds for the above variant a). 69 S'? - 26 In the lipopeptides according to the formula I which are obtained by any one of the process variants described and in which there is a free terminal carboxyl group in the X part, this group can be converted in a manner known per se, e.g. by one of the methods customary in peptide chemistry, into the amide or ester group.

An N-acyl- or N,O,O-triacyl-S-(2,3-dihydroxypropyl)-cysteine according to the formula (VII), which is to be used as starting material, can, if W=OH and R^H, be obtained in the following manner: starting with I.i,5.6-D-mannitol diacetonide, there is obtained, by periodate oxidation, 2-0,3-0-isopropylidene-D-glycerol aldehyde; the aldehyde group is then reduced to the carbinol group, and this is esterified with p-toluenesulphonic acid. The l-0-tosyl-2(R)-0,3-015 isopropylidene glycerol thus obtained is condensed with N-acyl-(R)-cysteine in the presence of a basic agent, e.g. potassium carbonate, and there is thus obtained N-acyl-S[2(R),3-isOpropylenedioxy-propyl]-(r)-cysteine, which is saponified by an acid reaction, e.g. with acetic acid. The resulting N-acyl-S-[2(R),3-dihydroxypropyl]-(R)-cysteine can, if required, be further acylated in the 2- and 3-positions of the glycerol moiety, preferably with temporary protection of the carboxyl group, e.g. by means of the above-described trimethylsilylethyl group, or preferably the benzhydryl ester group.

To produce an N-acyl- or N,O,O,O-tetraacyl-S-(2,3,4trihydroxybutyl)-cysteine to be used as starting material, according to the formula (VII), the method employed can be analogous to the method of Example 7.

The amino acids and peptides to be used for producing by the processes of the invention the new lipopeptides are known, or they can be produced by methods known per se.

The above starting compounds (VII) for the variant b) in ’ the case where W -- OH, and also diasterioisomeric mixtures, 6 9 5 7 - 27 ' salts and complexes thereof, themselves have also an immunity-potentiating action, and this action occurs in vitro in the same dosage range as that in which it occurs oe application of the compounds of the formula I and derivatives thereof. The stated compounds produce in vivo in the abovedescribed test for potentiation of the humoral immunity, with intraperitoneal administration 5 days before administration of the antigen, an increase of the antibody titres in the serum in the dosage range of 1.0 to 3 mg/kg of animal, weight.

These compounds can be obtained by acylation of a compound according to the formula (IX) in which, however, a hydroxyl group is present in place of Z2, as described above for the variant c). The production of the starting materials required, i.e. the glyceryl- and erithrityl.-N-acyl-cysteines, is described in the foregoing. Compounds of the formula (IX) in which has a meaning corresponding to that of group X in the formula (I), and in which however no free hydroxyl groups are present, can be obtained by reaction of the N-acyl-cysteine derivatives just mentioned with a compound of the formula , according to the same principles as for the above-described variant b) of the general process for production of the lipopeptides according to the invention.

The lipopeptides obtained can be converted into their salts in a manner known per se, e.g. by reaction of resulting acid compounds with alkali metal hydroxides or alkaline-earth metal hydroxides, or of resulting basic compounds with acids .

..By virtue of the close relationship between the new compounds in the free form and in the form of salts and complexes thereof, it is to be understood, in the? foregoing and in the following, that by the term 'free compounds' is meant, where, the case applies and with the appropriate modifications, also the corresponding salts.

By virtue of the physical-chemical differences in their 469S7 - 28 constituentsj isomeric mixtures obtained can be separated in a known manner, for example by chromatography and/or by fractional crystallisation. Advantageously, the more active of the isomers is isolated.

The processes described above are performed e.g. by methods known per se, in the absence or preferably in the presence of diluting agents or solvents, if necessary with cooling or heating, under elevated pressure and/or in an inert gas, such as in a nitrogen atmosphere. With due regard being given to all substituents present in the molecule, there are to be used if necessary, especially where readily hydrolysable O-acyl groups are present, particularly gentle reaction conditions, such as short reaction times, use of mild acid agents at low concentration, stoichiometric quantity ratios, and choice of suitable catalysts, solvents, temperature and/or pressure conditions.

The invention relates also to those modifications of the process whereby a compound occurring as an intermediate at some stage of the process is used as the starting material 2o and the uncompleted steps are performed,· or whereby the process is interrupted at some stage; or whereby a starting material is formed under the reaction conditions, or is used in the form of a reactive derivative or salt. There are preferably used those starting materials by which are obtained, according to the process of the present invention, the compounds which have been described in the foregoing as being particularly valuable.

The present invention relates likewise to pharmaceutical preparations which contain the described new lipopeptides according to the invention, both those of the formula (I) and those of the formula (VII), and to mixtures, salts or complexes thereof. The pharmaceutical preparations according to the invention are preparations which are for enteral administration, such as oral or rectal as well as parenteral - 29 administration, to warm-blooded animals, and which contain the pharmacological active substance on its own or together • with a pharmaceutically applicable carrier material. The dosage of active substance depends on the species, on the age and on the individual condition of the warm-blooded animal concerned, and also on the mode of administration.

The new pharmaceutical preparations contain from about 10% to about 95%, preferably from about 20% to about 90%, of active substance. Pharmaceutical preparations according to the invention can be, e.g., in the form of dosage units, such as dragees, tablets, capsules, suppositories or ampoules.

The pharmaceutical preparations of the present invention are produced in a manner known per se, e.g. by means of conventional mixing, granulating, coating, solution or lyophilising processes.

Suitable carrier substances are in particular fillers, such as sugar, e.g. lactose, saccharose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, e.g. tricalcium phosphate or calcium hydrogen phosphate; also binders such as starch mucilage, with the use e.g. of maize starch, wheat starch, rice starch or potato starch, gelatine, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone, and/or, if desired, effervescent agents, such as the afore25 mentioned starches, also carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, alginic acid or a salt thereof, such as sodium alginate. Auxiliaries are in particular flow-regulating agents and lubricants, e.g. silicic acid, talcum, stearic acid or salts thereof, such as magnesium stearate or calcium stearate, and/or polyethylene glycol.

DriJgee cores are provided with suitable coatings, optionally resistant to gastric juices; for this purpose there are used, - 30 inter alia, concentrated sugar solutions which optionally contain gum arabic, talcum, polyvinylpyrrolidone, polyethylene glycol and/or titanium oxide, lacquer solutions in suitable organic solvents or solvent mixtures, or, for producing coatings resistant to gastric juices, solutions of suitable cellulose preparations, such as acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate. Dyes or pigments may be added to the tablets or dragee coatings, e.g. for identification or for indication of the various doses of active substance.

The following Examples illustrate the invention described in the foregoing; they are however not intended in any way to limit the scope of this invention. Temperatures are given in degrees Centigrade.

The abbreviations used are as follows: Z = carbobenzoxy Bu1 = tert-butyl ether 0 But= tert-butyl ester BOC = tert-butoxycarbonyl DMF = dimethylformamide ONp = p-nitrophcnyl ester HOBt = N-hydroxybenzotriazole DC = thin-layer chromatography In DC there is used silica gel as adsorbent, and the following systems are used as eluants: system 3 : ethyl acetate/pyridine/water (65:20:15), system 157 : chloroform/methanol/water/glacial acetic acid (70:42:10:0.5), system 157c: chloroform/mcthanol/water/glacial acetic acid (75:25:5:0.5) . - 31 46937 • Example 1 1.13 g of [N-palmitoyl-S-2(R),3-dipalmitoyloxypropyl]Cys-Ser-(But)-Ser(But)-Asn-OBut is taken up in 5 ml of 90% trifluoroacetic acid, and after 45 minutes at 20 the solution is concentrated by evaporation to about 2 ml.

The product is precipitated by the addition of 75 ml of peroxide-free ether, filtered off, and dried over potassium hydroxide. The li.popeptide[N-palmitoyl-S-2(R),3-dipaImitoy1oxypropyl]-Cys~Ser-Ser-Asn-OH thus obtained gives in the thin-layer chromatogram on silica gel the following Rf values: Rf (chloroform/methanol/glacial acetic acid/water 79:25: 0.5:4.5) = 0.25. 0.89 g of (N-palinitoyl-S-2(R),3-dihydroxypropylj-Cys-Ser (But)-Ser(But)-Asn-OBu1 is taken up in 10 ml of pyridine; 0.725 ml of palmitic acid chloride is added, and the solution is kept at 45° for 24 hours. There is then added 200 ml of chloroform, and the solution is extracted with IN citric acid, IN sodium bicarbonate and water; the organic phase is dried over sodium sulphate, and the solvent is evaporated off.

The residue is purified by chromatography on silica gel in the system chloroform, or chloroform/methanol (98:2). The thin-layer-chromatographically homogeneous lipopeptide[N-palmitoyl-S-2(R),3-dipalmitoyloxypropyl]-Cys-Ser-(Bu )t t Ser(Bu )-Asn-0Bu gives in the system chloroform/methanol (9:1) on silica gel an Rf value of 0.75.

To 0.90 g of [N-palmitoyl-S-2(R),3-dihydroxypropyl]-cysteine and 0.99 g of H-Ser (But)-Ser(BuL')-Asn-OBut in 20 mi of dimethylformamide are added at 0° 0.472 g of dicyclohexylcarbodiimide and 0.309 g of N-hydroxybenzotriazole. After 2 hours at 0° and 15 hours at 20°, the product is filtered off and the filtrate is concentrated by evaporation. The residue is purified by chromatography on a column of silica gel in the system chloroform/methanol (98:2), In the thin-layer chromatogram on silica gel, the resulting lipopeptide derivative - 32 [N-palmitoyl-S-2(R),3-dihydroxypropyl]-Cys-Ser-But)-Ser (Bu*·)-Asn-OBu1' gives an Rf value of 0.60 (chloroform/ methanol 8:2). 6.09 g of Z-Ser(But)-Ser(But)-Asn-OBut is dissolved in ml of methanol and the solution, after the addition of 0.5 g of Pd-charcoal (10%), is hydrogenated at room temperature for 3 hours. The catalyst is filtered off, and the filtrate is concentrated by evaporation. The product is obtained as white foam. In the thin-layer chromatogram on silica gel, t t t the resulting peptide H-Ser(Bu )-Ser(Bu )-Asn-0Bu gives an Rf value of 0.41 in the system chloroform/methanol (8:2). 4.134 g of Z-Ser (Bu4)-OH and 4.64 g of H-Ser(But)-Asn0Buc are dissolved in 50 nil of dimethylformamide, and at 0° there are added 3.172 g of dicyclohexylcarbodiimide and 1.89 g of N-hydroxybenzotriazole. After 2 hours at 0° and hours at 20°, the dicyclohexylurea which has precipitated is filtered off with suction, the filtrate is concentrated by evaporation, the residue is taken up In ethyl acetate, and the solution is extracted with IN citric acid, IN sodium bicarbonate and water, dried over sodium sulphate, and then concentrated by evaporation. The residue is recrystallised £ from ethyl acetate/hexane to yield peptide Z-Ser(Bu )-Ser t t (Bu )-Asn-0Bu having a melting point of 96-98°; on silica gel: Rf (chloroform/methanol 95:5) = 0.25. f-* t* 4.65 g of Z-Ser(Bu )-Asn-0Bu in 50 ml of methanol is hydrogenated in the presence of 0.4 g of Pd-charcoal (10%).

The catalyst is filtered off and the filtrate Is concentrated by evaporation to yield peptide H-Ser(But)Asn-0But in the form of white foam; on silica gel: Rf (chloroform/methanol 7:3)=0.48. .9 g of Z-Ser (Bu1)-OH and 3.76 g of H-Asn-0BuL are dissolved in 60 ml of dimethylformamide, and at 0° there are added 3.06 g of N-hydroxybenzotriazole and 4.53 g of dieyclo4 69 371 - 33 hexylcarbodiimide. After 2 hours at 0° and 15 hours at 20°, the resulting product is filtered off, the filtrate is concentrated by evaporation, and the residue is crystallised from ethyl acetate/petroleum ether. The peptide Z-SeriBu1')Asn-OBu1 obtained melts at 114-115°. ία]^θ = (c ~ 1.5 in methanol); on silica gel: Rf (chloroform/methanol 9:1) = 0.48.

Example 2 1.6 g of lN-palmitoyl-S-2(lt),3-dipalmitoyloxypropyl]-Cyst ϋ t Ser(Bu )-Ser(Bu )-Asn-Ala-OBu is dissolved in 7.5 ml of 90% trifluoroacetic acid, and after 15 minutes at 20° the solution is concentrated hy evaporation to about 2 ml. The product is precipitated xvith 100 ml of ether; it is then filtered off and subsequently dried over sodium hydroxide to yield, in the form of a white powder having a melting point of 215-217° (decomposition), lipopeptide [N-palmitoyl£ 2(R),3-dipalmitoyloxypropylJ-Cys-Ser-Ser-Asn-Ala-OH; on silica gel: Rf (157) = 0.55.

Example 3 0.95 g of [N-palmitoyl-S-2(R),3-dipalmitoyloxypropyl]Cys-Ser(But)-Ser(But)-0But is dissolved in 4 ml of 90% trifluoroacetic acid. After 15 minutes at 20°, there is added 100 ml of ether; the product obtained is filtered off, and the residue is dried over sodium hydroxide. The lipopeptide [N-palmitoyl-S-2(R),3-dipalmitoyloxypropyl]-Cys-Ser-Ser-OH is thus obtained as a colourless, non-hygroscopic powder; on silica gel: Rf (chloroform/methanol/glacial acetic acid/ water 79:25:0.5:4.5) = 0.53.

Example 4 The N-palmitoyl-S-[2(R),3-dihydroxypropyl]-(R)-cysteine used as starting material in Example 1 can be produced in the following manner: - 34 13.9 g of N-palmitoyl-(.R)-cysteine, 5.5 g of potassium carbonate and 15 g of l-0-tosyl-2(R)-0-3-0-isopropyl.idene• glycerol in 250 ml of ethanol are heated under nitrogen for 8 hours at 80°. After concentration by evaporation, the reaction mixture obtained is purified by chromatography on 400 g of silica gel(Merck,) by elution firstly with chloroform/acetone (8:2) and subsequently with chloroform/ methanol (8:2). There is thus obtained the potassium salt of N-palmitoyl-S-[2(R),3-isopropylidenedioxypropyl]-(R)10 * cysteine, which is saponified by being heated for 4 hours at 80° in 80% aqueous acetic acid. The resulting product is evaporated to dryness; the residue is dissolved in chloroform and extracted with water. After drying and evaporating off the chloroform phase, there remains a colourless residue; this is recrystallised from cyclohexane to yield N-palroitoyl-S-(2(R),3-dihydroxypropyl]-(r)-cysteine, m.p. 110°, [a]y° = -25° (c = 0.9, MeOll).

N-Palmitoyl-(R)-cysteine can be produced in the following manner: 45 g of (r)-cysteine (0.37 mole) is suspended in 350 ml of pyridine, and there is then added dropwise, with thorough stirring and in a nitrogen atmosphere, at room temperature a solution of 140 ml (1.67 equivalents) of palmitic acid chloride in 550 ml of methylene chloride. The reaction mixture is stirred for 20 hours; it is subsequently acidified with 2N hydrochloric acid, and distributed between chloroform and waver. The chloroform phase is dried and evaporated to dryness to yield a crystal mixture consisting to the extent' of about a third of dipalmitoylcysteine and to the extent of about 3q two thirds of monopalmitoylcysteine. Extraction is performed three times with 1 litre of hot acetone, from which crystallises a 1 : 1 mixture of di- and mono-palmitoylcysteine. From the acetone solution is obtained, by evaporating to dryness, N-palmitoylcysteine, which is reerystallised from ligroin; 469S7 - 35 90 m.p. 65 - 67°. [α]β =+1° (c -- 0.8; methanol).

The mixtures of di- and mono-palmitoylcysteine are treated in a methanolic solution, with the addition of an aqueous solution of 12 g of sodium sulphide with 2 ml of cone, sodium hydroxide solution, for 15 minutes at room temperature. After evaporating off the methanol and acidifying the residue with 2N hydrochloric acid there is obtained, by chloroform extraction, the remaining Npalmitoylcysteine, which is likewise recrystallised from ligroin.

Example 5 0.80 g of N-palmitoyl-S-[2(R),3-dipalmitoyloxypropyl](R)-cysteine and 0.67 g of H-Ser(But)-Phe-Ala-Glu(0But)2 are taken up in 10 ml of dimethylformamide, arid 0.22 g of dicyclohexylcarbodiimide and 0.16 g of N-hydroxybenzotriazole are added. There is obtained by heating for a short time at 40° a clear solution which, after about one hour at 20°, solidifies into the form of a jelly. After 24 hours, this is again converted into a solution by heating at 40°, and from the solution obtained there is subsequently precipitated with water N-palmitoyl-S-(2(R),3-dipalmitoyloxypropyl]-Cyst t Ser(Bu )-Phe~Ala-Glu(OBu )2- The product is purified by chromatography on silica gel in the system chloroform or chloroform/methanol 98:2, and gives the Rf value of 0.64 in chloroform/methanol/water 95:5. 540 mg of the compound thus obtained is dissolved in 30'ml of 90% trifluoroacetic acid, and the solution, after 30 minutes at 20°, is concentrated by evaporation to about a half, and N-palmitoyl-S-(2(R),3-dipalmitoyloxypropyl]Cys-Ser-Ser-Phe-Ala-Glu-OH is precipitated with petroleum ether. The compound is centrifuged off, washed three times with petroleum ether, and dried over potassium hydroxide; the product obtained is a white powder having the Rf value (system 157) of 0.63. 69 57 - 36 The following lipopeptides are produced in an analogous manner: N-palmitoyl-S-[2(R,S),3-dipalmitoyloxypropyl]-Cys-Phe~PheAsn-Ala-Lys-OH, Rf (system 157 c) = 0.18; N-myristoyl-S-[2(R),3-dipalmitoyloxypropylJ-Cys-Glu-GlnAsn-Ala-Lys-OH, Rf (system 157 c) = 0.12; N-lauroyl-S-[2(R),3-dipalmitoyloxypropyl]-Cys-Ser-SarAsn-Ala-Glu-OH, Ef (system 157 c) - 0.20; and N-stearoyl-S-[2(R) ,3-dipalmitoyloxypropyl]-Cys-Ser-Ser10 Asn-Ala-Ala-OH, Rf The above described peptide provided with the stated protective groups, which peptide is used as starting material for the condensation reaction with N-palmitoyl-S-[2(R),3dipalmitoyloxypropyl ]-(R) -cysteine, can be produced in the following manner: 17.22 g of Z-Ala-ONp, 14.8 g of HCl-HGlu(OBut)2 and 6.3 ml of N-ethylmorpholine in 40 ml of dime thy Iformann.de are left to stand overnight at 20°. The solution is concentrated by evaporation, the residue is taken up in ethyl acetate, and extracted with i N sodium bicarbonate and water. The ethyl acetate solution is concentrated by evaporation, and it leaves, in the form of foam, Z-Ala-Glu(OBu )2 having the Rf value of 0.60 (in chloroform/methanol 95:5), which is directly further processed: 8.5 g thereof is dissolved in 60 ml of methanol, and the solution, after the addition of 0.8 mg of Pd-charcoal (10%) is hydrogenated at 20° for 2 hours. The catalyst is filtered off, and the filtrate is concentrated by evaporation to yield Η-ΑΙη-ΟΙοζΟΒη1^ as a white foam giving the Rf value of 0.36 in chloroform/methanol 9:1.

To 11.46 g of Z-Phe-OH and 12.64 g of H-Ala-GluiOEu*)? in 50 ml of dimethylformamide are added at 0° 8.68 g of dicyclohexylcarbodiimide and 5.86 g of N-hydroxybenzotriazole.

After standing for 15 hours at 4°, the resulting product is - 37 filtered off, the filtrate is concentrated by evaporation, and the residue is recrystallised from ethyl acetate/hexane. There is thus obtained Z-Phe-Ala-GluiOBu1)2, m.p. 161-163°; Rf (toluene/acetone 1:1) = 0.63. 6.4 g of this product is dissolved in 50 ml of methanol and, after the addition of 0.5 g of Pd-charcoal (10%), the solution is hydrogenated at 20° for 20 minutes. The catalyst is filtered off, and the filtrate is concentrated by evaporation to yield H-Phe-Ala-Glu(OBut)2 as white foam; Rf (chloroform/methanol 7:3) = 0.75.

To 4.43 g of Z-Ser(But)-0H and 3.18 g of HCl-H-SeriBu^-OMe in 30 ml of dimethylformamide are added at 0° 1.89 ml of N-ethylmorpholine, 2.29 g of HOBt and 3.4 g of dicyclohexylcarbodiimide. After standing for 2 hours at 0° and for 15 hours at 20°, the resulting product is filtered off, the filtrate is concentrated by evaporation and taken up in ethyl acetate. After extraction of the solution with sodium bicarbonate, dilute hydrochloric acid and water, the solution is concentrated by evaporation to leave as oil Z-Ser(Bu^)-Ser(Bu^)-OMe; Rf (toluene/acetone 1:1) - 0.70.

To a solution of 6.8 g of this product in 40 ml of methanol is added 7.5 ml of hydrazine hydrate, and after 24 hours at 20° the mixture is evaporated to about half the volume. It is then taken up in 250 ml of ethyl acetate, and extracted with water. The ethyl acetate solution is concentrated by evaporation and the Z-Ser(Bu^)-Ser (Bu^)-NH-NH2 obtained is precipitated with petroleum ether; Rf (toluene/acetone 1:1) = 0.50. 2.26 g of this product is dissolved in 20 ml of dimethylformamide, and at -10° are added 7.022 ml of 1.78 N HCl in ethyl acetate, and 0.635 ml of tert-butyl nitrite. After 10 minutes at -10°, there is added dropwise a solution of 2.38 g of H-Phe-Ala-Glu(OBut)2 and 2.52 ml of N-ethylmorpholine, and the whole is allowed to stand for 1 hour at -10° and for - 38 15 hours at 0°. The solution is then concentrated by evaporation; the residue is subsequently taken up in ethyl acetate, and washed with sodium bicarbonate, dilute hydrochloric acid and water. The solution is concentrated by evaporation to leave Z-Ser (Bu1)-Ser (But)-Phe-Ala-Glu(OBut)2, which is then recrystallised from ethyl acetate/hexane; m.p. 213-215°, Rf (toluene/acetone 1:1) = 0.70. 2.30 g of this product is dissolved in 40 ml of methanol and, after the addition of 0.5 g of Pd-charcoal (10%), the 10 solution is hydrogenated at 20° for 2 hours. The catalyst is filtered off and the filtrate is concentrated by evaporation to yield H-Ser (Bu1)-Ser (But)-Phe-Ala-Glu(OBt)2 in the form of a white foam; Rf (toluene/acetone 1:1) = 0.23.

The N-palmitoyl-S-[2(R),3-dipalmitoyloxypropyl]-(H)15 cysteine used as starting material and the diasteriomeric mixture of this compound with the corresponding 2(S) compound, which is denoted here in an abbreviated form as 2(R,S), can be produced as follows: 6.3 g (2.78 mMols) of N-palmitoyl-S-[2(R),3-dipalmitoy120 oxypropyl]-(R)-cysteine-benzhydryl ester is dissolved in a mixture of 12 ml of trifluoroacetic acid and 48 ml of methylene chloride. After one hour at room temperature, the solution is concentrated by evaporation, and the oily residue is triturated with water. The N-palmitoyl-S-[2(R),3-dipalmitoy125 oxypropyll-(R)-cysteine which has crystallised out is filtered off with suction, dried and, to remove the benzhydrol, triturated three times with ligroin. The residue is crystallised from acetone/water, and there are thus obtained colourless crystals of this product, m.p. 71-75°; [a]p = -1° (dioxane).

The above ester is obtained in the following manner: g (20 niMols) of N-palmitoyl-S[2(R),3-dihydroxypropyl](R)-cysteine-benzhydryl ester is dissolved in a mixture of 60 ml of pyridine and 60 ml of methylene chloride, and there - 39 > is then added dropwise at 0-10° 14.5 ml (13.2 g) of palmitic acid chloride. After 2 hours at room temperature, the reaction has finished according to the thin-layer chromatogram. 10 ml of methanol is added to the suspension (pyridine hydrochloride); the reaction mixture is stirred for 20 minutes at 25°, and is then extensively concentrated by evaporation. The residue is_taken up in chloroform; extraction is performed twice with 0.5 N hydrochloric acid, 10% sodium bicarbonate solution and water each time, the chloroform phase is dried with sodium sulphate, and subsequently evaporated to a syrup.

The pure ester is obtained hy chromatography of this syrup through silica gel. in chloroform. The chromatographically homogeneous fractions (thin-layer chromatographical test on silica gel in chloroform : ethyl acetate 98:2, Rf -- 0.5) are concentrated by evaporation and recrystallised from acetone. There are thus obtained colourless crystals of N-palmitoyl-(S)-I2(R),3-di palmitoyloxypropyl]-(S)-cysteinebenzhydryl ester, m.p. 69-71°.

The benzhydryl ester of N-palmitoyl-S/2(r), 3-dihydroxypropyl/(R)-cysteine used above is obtained» e.g., as follows: 23.4 g (54 mMols) of N-palmitoyl-S-[2(R),3-dihydroxypropyl](R)-cysteine is dissolved in 150 ml of a ehloroform/ethanol mixture 1:1, and to this solution is added dropwise a solution of 13.6 g (70 mMols) of diphenyl diazomethane in 50 ml of chloroform. After 3 hours at room temperature, the initially red solution is decolorised and, according to thin-layer chromatography (chloroform : methanol - 9:1, silica gel), the reaction is complete. The reaction mixture is concentrated in vacuo to dryness, and the residue is extracted cold with petroleum ether. The residue is filtered through silica gel with methylene chloride as solvent to thus obtain colourless crystals of benzhydryl ester, m.p. 88-91° To produce N-palmitoyl-2-[2(R,S),3-dihydroxypropyl]-(R)cysteine, the procedure is carried out as follows: - 40 50 g (139 mMols) of N-palmitoyl-R-cysteine, 12.4 g of glycide and 45 g of potassium carbonate in 375 ml of ethanol are heated under nitrogen, with thorough stirring, for 16 hours at 80°. After cooling to room temperature, the reaction mixture is acidified with 2 N. hydrochloric acid to about pH 4, and water is added; the product then precipitates. It is filtered off with suction, and washing with water is performed until the filtrate is free from chlorine ions. After drying the filter residue in vacuo, it is recrystallised from 10 ethyl acetate to yield colourless crystals of the diastcreoisomeric mixture of N-palmitoyl-S-[2(R),3-dilnydroxypropyll(R)-cysteine and N-palmitoyl-S-[2(S),3-dihydroxypropyl]-(R)cysteine, m.p. 76-155°.

By reaction of N-palmitoyl-S-[2(R,S),3-dihydroxypropyl}15 (R)-cysteine with diphenyldiazomethane, as described above for the R-compound, there is obtained N-palmitoyl-S-[2(R,S), 3-dipalmitoyloxypropyl]-(R)-cysteinebenzhydryl ester: colourless crystals, m.p. 84-85°.

If this diastereoisometric mixture (19.8 g) is chroraalo20 graphed on a column with 250 g of silica gel (Merck) with methylene chloride as solvent, there is obtained in a first fraction 1.55 g of the R-diastcreoisomer (Rf - 0.61, methylene chloride/ethyl acetate = 98:2, thin-layer, silica gel), in a second layer there is obtained 12.7 g of an R,S-diastereoisomeric mixture, and in a third fraction 111 g of the S-diastereoisomer (Rf = 0.53 in methylene chloride/ ethyl acetate = 98:2).

From the (S)-diastereoisomer there is obtained, in the manner described above, N-palmitoyl-S-[2(S),3-dipalmitoyloxypropyl]-(r)-cysteine; colourless crystals, m.p. 64-65° [a]]3 = + 1° (dioxane).

The starting materials required according to the invention for the production of the new lipopeptides which are mentioned - 41 in this Example and which arc to be produced in a manner analogous to that in which N-palmitoyl-S[2(R),3-dipalmltoyloxypropyll-Cys-Ser-Ser-Phe-Ala-Glu-OH is produced are, for example: N-myristoyl-S-[2(R),3-dihydroxypropyl]-(r)-cysteine, colourless crystals, m.p. 140-150°; N--stearoyl-S- [2(R) ,3-dihydroxypropyl] - (r) -cysteine, colourless crystals, m.p. 140-150; and N-lauroyl-S-[2(R),3-dihydroxypropyl]-(r)-cysteine, colourless crystals, m.p. 140-150°.

They can be produced according to the data given in Example 4.

The benzhydryl esters to be produced according to the information given above have the following characteristics: N-myristoyl-S-[2(R),3-dihydroxypropyl]-(R)-cysteine-benzhydryl ester, colourless crystals, m.p. 90-92°; N-stearoyl-S-[2(R),3-dihydroxypropyl]-(R)-cysteine-benzhydryl ester, colourless crystals, m.p. 95-97°; and N-lauroyl-S-[2(R),3-dihydroxypropyl]-(R)-cysteine-benzhydryl ester, colourless crystals, m.p. 95-97°.

In a manner analogous to that described, it is possible to obtain the following N-acyl-S-[2(R),3-diacyloxypropyl](R)-cysteine-benzhydryl esters having various acyl groups on the N atom and on the 0 atom: N-lauroyl-S-[2(R),3-dipalmitoyloxypropyl]-(R) -cysteinebenzhydryl ester, m.p. 65-68°; N-myristoyl-S-[2(R),3-dipalmitoyloxypropyl]-(R)-cysteinebenzhydryl ester, m.p. 68-70°; and N-stearoyl-S-(2(R),3-dipalmitoyloxypropyl]-(R)-cysteinebenzhydryl ester, m.p. 70-72°. 46θ57 - 42 ' Further compounds which are to be used as intermediates and which can be produced according to the above data are: N-lauroyl-S-[2(R),3-dipalmitoyloxypropyl. ]-(R)-cysteine, colourless crystals, m.p. 70-72°; N-myristoyl-S-[2(R),3-dipalmitoyloxypropyl]-(R)-cysteine, colourless crystals, m.p. 71-73°; and N-stearoyl-S“[2(R),3-dipalmitoyloxypropyl]-(r)-cysteine, colourless crystals, m.p. 74-77°.

Example 6 By means of the reactions described in Example 5, there are produced the following starting materials for the production of lipopeptides according to the present application N-palmitoyl-S-[2(R),3-dilauroyloxypropyl]-(R)-cysteinebenzhydryl ester, colourless wax; N-palmitoyl-S-[2(R),3-distearoyloxypropyl]-(r)-cysteinebenzhydryl ester, colourless crystals, m.p. 80-82°; N-palmitoyl-S-[2(R),3-dioleoyloxypropyl]-(R)-cysteinebenzhydryl ester, colourless oil; N-palmitoyl-S-[2(R),3-dibehenoyloxypropyl]-(R)-cysteine20 benzhydryl ester, colourless crystals, m.p. 85-88°; and N-palmitoyl-S-[2(R),3-di-(dihydrosterkuloyloxypropyl]-(R)cysteine-benzhydryl ester, colourless wax.

The corresponding unesterified substituted cysteines have the following characteristics: N-palmitoyl-S-[2(R),3-dilauroyloxypropyl]-(R)-cysteine, colourless oil, Rf = 0.27; N-palmitoyl-S-[2(R),3-distearoylo::ypropyl]-(R)-cysteine, colourless crystals, m.p. 82-85°; N-palmitoyl-S-[2(R),3-dioleoyloxypropyl]-(r)-cysteine, colourless oil, Rf in chloroform/methanol = 9:1 (thin-layer - 43 silica gel (Merck)) = 0.35; N-palmitoyl-S-[2(R),3-dibehenoyloxypropyl]-(R)-cysteine, colourless crystals, m.p. 87-90; and N-palmitoyl-S-[2(R),3-di-(dihydrosterkuloyloxypropyl]-(R)5 cysteine, colourless wax, R£ = 0.38 (identical conditions as given above for the N-palmitoyl-2.,3-dioleoyloxy derivative) .

These cysteine derivatives are condensed with the following peptides according to the data given in Example 5: H-Phe-Phe-Asn-Ala-Lys-OH, H-Glu-Gln-Asn-Ala-Lys-OH and 11-Ser-Ser-Asn-Ala-Glu-OH.

There are thus obtained the corresponding lipopeptides according to the invention.

Example 7 0.9 g of N-palmitoyl-S-2(R),3(R),4-trihydroxybutyl-(R)cysteine-benzhydryl ester in a mixture of 3 ml of trifluoroacetic acid and 12 ml of methylene chloride is allowed to stand for 2 hours at room temperature. The reaction mixture is then evaporated to dryness; the residue is triturated with ice-water, 2o filtered off with suction and the solid substance is dried.

It is then extracted with ligroin, and recrystallised from acetone to yield colourless crystals of N-palmitoyl-S-2(R), 3(R),4-trihydroxy-butyl-(R)-cysteine, m.p. 76 - 76.5°, having 20 the specific rotation [a]p «= -5° (c = 0.819, dioxane).

The benzhydryl ester used as starting material is obtained in the following manner: g of N-palmitoyl-S-2(R),3(R),4-trihydroxybutyl-(R)-cysteinebenzhydryl ester is acylated in a mixture of 6 ml of pyridine in’5 ml of methylene chloride with 1.83 ml. of palmitic acid chloride. Z.fter 18 hours at room temperature, there is added 1 ml of methanol, and the mixture is evaporated to dryness.

The residue is filtered through 100 g of silica gel (Merck) 46S57 - 44 with chloroform as solvent. The fractions that are pure according to thin-layer chromatography (CHCl^:ethyl acetate = 98:2) yield, after being concentrated by evaporation, .colourless crystals, m.p. 60-62°.

The starting material for the above reaction is obtained as follows: 2.53 g of N-palmitoyl-S-2(R) ,3 (R) ,4-trihydroxybutyl-(R)cysteine is reacted in a mixture of 12 ml of ethanol and 32 ml of chloroform with 1.37 g of diphenyldiazomethane for 15 hours at room temperature. The reaction mixture is subsequently evaporated to dryness, and the formed benzyIhydryl ester is purified on 150 g of silica gel' in chloroform as solvent; Rf = 0.3 (CHCl^:acetone = 95:5) silica gel (Merck), thin layer.

N-Palmitoyl-S-2(R),3(R),4-trihydroxybutyl-(R)-cyst:eine is obtained in the following manner: g of N-palmitoyl-cysteine, 27.6 g of l-tosyl-2,4ethylidene-D-erythrite and 17 g of potassium carbonate in 240 ml of ethanol are stirred under nitrogen for 15 hours at 80°. The salts are then filtered off, and the filtrate is evaporated to dryness. The residue is taken up in 200 ml of H^O:tetrahydrofuran = 1:1, and the solution is acidified with 2 N hydrochloric acid to pH = 3. It is then extracted three times with ether; the pH value of the ether phase is adjusted with pyridine to 4.5; the ether phase is subsequently extracted with water, dried, and evaporated to dryness. This residue is purified through 200 g of silica gel (Merck) with chloroform:acetone = 8:2 (1 1) and then with CHCl^MeOH = 6:4 (1.2 1) as eluant.

The pure fractions are concentrated by evaporation, triturated with hexane and recrystallised from ethyl acetate/ petroleum ether.

There are obtained colourless crystals of N-palmitoyl-S[(2,4-ethylidene)-erythrityl]-(R)-cysteine, m.p. 62-65°. 6 9 5 7 - 45 ' The hydrolysis oi this compound is performed with 3% HBF, at 80° for 4.5 hours. 8.2 g of the etbylidcne derivative ia t) t ated in iO(J nil of dimethoxyethane and 100 ml of 3% HBF^. The temperature is then lowered to 0”, whereupon the hydrolysis product precipitates. It is filtered off with suction, dried, and recrystallised from ethyl acetate, m.p. 160-165°, sintering from 93°; Rf = 0.285 (CHCJyMeOH = 6:4), thin layer, silica gel (Merck) .

Example 8 By a procedure analogous to that given in Example 5, there are produced from N-stearoyl-S-[2(R),3-dipalroitoyloxypropyl]-(R)-cysteine and from N-palmitoyl-S-[2(R),3(R), 4-tripalmitoyloxypropyl]-(R)-cysteine the following lipopeptides: N-stearoyl-S-[2(R),3-dipalmitoyloxypropy1]-Cys-Ser-SerAsn-Ala-Gl.u-Ile-Asp-Clu-011, Rf (157) = 0.32; N-palmitoyl-S-[2(R),3(R),4-tripalmitoyloxybutyl]-Cys-ScrSer-Asn-Ala-Lys-OH, Rf (157) = 0.55; and 2o N-palmitoyl-S-(2(R),3(R),4-tripalmitoyloxybutyl]-Cys-SerSer-Asn-Ala-Lys-Ile-Asp-Glu-OH, Rf (157) = 0.38. - 46 Example 9 In. a manner analogous to that of Example .5, there are produced from N-palmitoyl-2(R),3-dipalmitoyioxyprapyl-(R)cysteine or N-palmitoyl.-2(R,S),3-dipalmitoyloxypropyl cysteine the lipopeptides listed below; in this case the radicals of the cysteine derivatives just mentioned are denoted by PC(R) and PC(R,S), respectively: PC(R,S)-Ser-Ser-Asn-OH PC(R)“Ser-Ser-Asn-Ala-Lys-OIl PC(R,S)-Ser-Ser-Asn-Ala-Lys-OH PC(R)-Ser-OH PC(R,S)-Ser-OH PC(R,S)-Ser-Ser-Asn-Ala-OII PC(R)-Phe-Ile-Ile-Phe-Ala-OII PC(R,S)-Phe-Ile-Ile-Phe~Ala-OH PC(R)-Val~Lys-Val-Tyr-Pro-OH PG(R,S)-Val-Lys-Val-Tyr-Pro-OH PC(R)-Ala-Ile-Gly-Val-Gly-Ala-Pro-NH2 PC.(R, S )-Ala-Il e-Gly-Val-Gly--Ala-Pro~Nll2 PC(R)-Ser-Ser-Asn~Ala-Lys-lle-Asp-Glu-01l 2Q PC(R,S)-Ser-Ser-Asn-Ala-Lys-Ile-Asp-Glu-Oil Furthermore, there are produced the R,S-epimeric mixtures of all lipopeptides described in the preceding Examples.

Claims (33)

1. Compounds of the formula CO—0—CH Rj—C ** H CH„ S I CH„ R 2 CO—NHH-CO--X *=R **=R or S wherein R^ and R 2 each represent a saturated or unsaturated 5 aliphatic or mixed aliphatic-cycloaliphatic hydrocarbon radical which has 11—21 C atoms and which is also optionally substituted by oxygen functions, R 3 represents hydrogen or the radical Rj—CO—0—CH 2 —, where R^ has the same meaning, and X represents a natural aliphatic amino acid linked by a 10 peptide bond and having a free, esterified or amidated carboxyl group, or an amino acid sequence of 2—10 natural aliphatic amino acids, the terminal carboxyl group of which is free or in the ester or amide form, with the asymmetric Λ it centres denoted by and possessing the absolute R- and 15 S- or R-configurations, respectively; and, possibly, mixtures of the R- and S-compounds epimeric on the C atoms, and salts and complexes of all these compounds. - 48

2. Compounds according to Claim 1, wherein the acyl groups R^CO and R 2 C0 are derived from saturated or unsaturated fatty acids having 14-18C atoms, and wherein R^ and R 2 can be identical or different. 5

3. Compounds according to Claim 1, wherein the acyl groups are derived from palmitic, stearic, oleic, lauric, myristic, behenic, dihydrosterculic, malvalic, hydnocarpic or chaulmoogric acid.

4. Compounds according to any one of Claims 1—3, wherein 10 amino acids carrying hydrophilic groups in X are the ionic amino acids: aspartic acid, glutamic acid, and/or oxyglutamic acid, or: lysine, ornithine, arginine and/or histidine, or asparagine, glutamine, serine and/or threonine, and amino acids carrying no hydrophilic groups in X are glycine, 15 alanine, valine, norvaline, leucine, isoleucine and/or methionine.

5. Compounds according to any one of Claim 1—4, wherein in the peptide chain X the amino acids having hydrophilic character are linked directly with each other. 20

6. Compounds according to Claim 5, wherein the sequence of the amino acids having hydrophilic character is bound directly to the carboxyl group of the cysteine in the triacylglycerylcysteine part or tetraacyl-erythritylcysteine part. 46937 - 49

7. Compounds according to any one of Claims 1-6, wherein X in the formula 1 represents an amino acid, or a peptide chain having 2-5 amino acids.

8. Compounds according to Claim 1 of the formula R,-CO——0-CH, I I ** D -co-o-CH 1 I CH, (IV) I 2 s I CH, I* R,CO-NH-CH—CO—X *=R **=R their salts and complexes, wherein X represents a natural aliphatic amino acid linked by a peptide bond and having a hydrophilic group and a free, esterified or amidated carboxyl group, or an amino acid sequence of 2-5 natural 10 aliphatic amino acids of which half carry at least one hydrophilic group, and of which the terminal carboxyl group is free or in the ester or amide form, with the asymmetic centres denoted by * and ** possessing the R-configuration.

9. Compounds according to any one of Claims 1—6, 15 wherein X in formula 1 of Claim 1 represents an unnatural amino acid sequence compared with that of the known mureinlipoprotein-degradation lipopeptides. - 50

10. Compounds corresponding to those of Claim 8, in which however the configuration on the C** atom is S instead of R, and the mixtures of the R- and S-diastereomers.

11. Compounds according to any one of Claims 1-6 of the 5 formula CH„ I* R CO—NH-CH-CO—X *=R **=R wherein R^ and R 2 have the meanings given under the formula X, and X represents a peptide chain having 6—10 amino acids, with the asymmetric centres denoted by * and ** possessing 10 the R-configuration, and also the compounds having on the C** atom the (S)-configuration, as well as R- and S-diastereomers mixtures, and salts and complexes thereof.

12. Compounds according to any one of Claims 1-6 of the formula R,-CO—0-CH * **=R or S wherein R^ and R 2 have the meanings given in any one of Claims 1—6, and X represents an amino acid, or a peptide chain having 2—5 amino acids, and mixtures of the compounds 2. Stereoisomeric on the C** atoms, and salts and complexes thereof.

13. The amides and esters of the compounds according to Claims 7—12, wherein an ester radical in the terminally esterified carboxyl group of X is derived from a lower 10 aliphatic alcohol having 1-7C atoms, an ethylene glycol or glycerin, and a terminal amide group in X is the amide group CONH 2 , or an amide group derived from a lower aliphatic amine having 1—7C atoms, or from pyrrolidine, piperidine or piperazine. 4 69 51 - 52

14. The lipopeptides N-palmitoyl-S-/?(R),3-dipalmitoyloxypropyl7-Cys-Ser-Ser-Asn-Ala-Lys-0H, according to Claim 1.

15. . The lipopeptide: N-palmitoyl-S-/5(R),3-dipalmitoyloxypropyl7-Cys-Ser-Ser-Phe-Ala-Glu-0H,. according to Claim 1.

3. 5 16. N - palmitoyl - S - /?(R),3 - di - (dihydrosterculoyloxypropyl7-Cys-Ser-Ser-Asn-Ala-Glu~0H, according to Claim 1.

17. The lipopeptide: N-lauroyl-S-/2(R),3-dipalmitoyloxypropyiy-Cys-Ser-Ser-Asn-Ala-Glu-OH, according to Claim 1.

18. N-Myristoyl-S-/?-(R),3-dipalmitoyloxypropyl7-Cys-Glu10 Gln-Asn-Lys-OH, according to Claim 1.

19. N-Palmitoyl-S-/2(R,S),3-dilauroyloxypropyl7-Cys-PhePhe-Asn-Ala-Lys-OH, according to Claim.1.

20. N-palmitoyl-S-/T(R),3-dilauroyloxy7~Cys-Ser-Ser-AsnAla-Glu-OH, according to Claim 1. 15

21. N-palmitoyl-S-ZTiR),3-dipalmxtoyloxypropyl7-Cys-Alalle-Gly-Val-Gly-Ala-Pro-Ni^, according to Claim 1.

22. N-palmitoyl-S-/?(R),3-dioleoyloxypropyl/-Cys-Ser-SerAsn-Ala-Glu-OH, according to Claim 1. - 53

23. N-Palmitoyl-S-/2(R,S),3-dipalmitoyloxypropyl/-CysPhe-Phe-Asn-Ala-Lys-OH, according to Claim 1.

24. N-Stearoyl-S-/2(R),3-dipalmitoyloxypropyl/-Cys-SerSer-Asn-Ala-Glu-lle-Asp-Glu-OH, according to Claim 1. 5 25. The lipopeptides corresponding to the compounds of

Claims 14-15, in which instead of palmitoyl radicals being present in the diacycloxypropyl radical, there are present lauroyl, stearoyl, oleoyl, behenoyl or dihydrosterculoyl radicals. 4. 10 26. A compound of Claims 14-—18,21 and 23—24 having the

R-configuration on the 2-C atom of the propyl group, a corresponding epimeric compound having the S-configuration, or R- and S-diastereomers mixtures.

27. Ammonium salts, alkali metal or alkaline-earth metal j_5 salts of acid compounds, and pharmaceutically applicable nontoxic acid addition salts of basic compounds, and copper-, zinc-, iron- or cobalt-complex salts of compounds according to any one of Claims 1-26.

28. Compound of the formula 469S7 CH -CO—ΟΕ χ -CO---Ο CH ** S CH. 30-NH— CH-COOH *=R **=R,S or mixture (R,S) wherein R^ and R 2 each represent a saturated or unsaturated, aliphatic or mixed aliphatic-cycloaliphatic hydrocarbon radical which has XI—21 C atoms and which is also optionally 5 substituted by oxygen functions, and R g represents hydrogen or the radical R^—CO—0CH 2 -, where R^ has the same meaning, with the asymmetric centres denoted by * and ** having the absolute R- and S- or R-configurations, respectively, and mixtures of the compounds stereoisomeric on the C** atoms. 10

29. A compound according to Claim 28, wherein R^ represents hydrogen, and wherein the acyl groups R^ and R 2 are derived from palmitic, stearic, oleic, lauric, myristic, behenic, dihydrosterculic, malvalic, hydnocarpic or chaulmoogric acid.

30. Pharmaceutical preparations containing a compound

5. 15 according to any one of Claims 1—28. - 55 31. A compound according to any one of Claims 1——28 for application in a process for the therapeutic treatment of the non-human animal body.

32. Compounds according to any one of Claims 1-28 for 5 application as immunity-stimulating therapeutic preparations, especially as adjuvants in the production of antisera for therapy and diagnosis, and in the induction of immunologically activated lymphocyte populations for cell-transfer processes, or as prophylactics or therapeutics against XO infectious diseases in non-human animals.

33. A process for producing compounds of the formula I** -CH R 1— C0 R 2 CO-NHc3——CO-X *=R **=R or S wherein R^ and R 2 each represent a saturated or unsaturated aliphatic or mixed aliphatic-cycloaliphatic hydrocarbon 15 radical which has 11—21 C atoms and which is also optionally substituted by oxygen functions, R 3 represents hydrogen or - 56 wthe radical -CO—0—CH^—, where R^ has the same meaning, and X represents a natural aliphatic amino acid linked by a peptide bond and having a free, esterified or amidated carboxyl group, or an amino acid sequence of 2—10 5 natural aliphatic amino acids, the terminal carboxyl.group of which is free or in the ester or amide form, with the asymmetric centres denoted by * and ** possessing the absolute R- and S- or R-configurations, respectively; and optionally mixtures of the R- and S-oompounds epimeric on 10 the C** atoms, and salts and complexes thereof, whereby a) in a compound of the formula R-,-CO—0-CH I*= R.-CO—0-CH 1, 1 ' s R,CO-NH-CH-CO—Y *=R **=R,S or mixture R,S wherein R^, R 2 and R^ have the meanings given under the formula (I) of Claim 1, and Y represents an amino acid or 15 amino acid sequence corresponding to X in the said formula 1, wherein however at least one of the hydrophilic substituting the amino acids and/or the terminal carboxyl group is - 57 (are) protected by a protective group which can be split off under neutral or mild acid conditions, or in a salt of such a compound, the protective group(s) is or are split off; or b) a compound of the formula Rj—COR^-C0 R 3 I J f ** -CH | ** -CH CH, I s CH R 2 -CO—NH-CH—CO—W *=R **=R,S or mixture (R,S) wherein R^, R 2 and R 3 have the meanings given under the said formula (I), and W represents OH or an amino acid or an incomplete sequence of amino acids according to X in the said formula I, is reacted with a compound of the formula NH 2 —4 where represents a group corresponding to the group X in the said formula (I), or an amino acid sequence or amino acid complementary to the stated amino acid or incomplete sequence of amino acids according to X, in which sequences however no free amino groups are present, or with a salt thereof; or - 58 c)a compound of the formula HO—CH I ** HO—CH I CH„ i 2 s f CH„ I. R CO-NH—CH-CO-Z ? *=R z z **=R,S or mixture (R,S) wherein R 2 has the meaning given under the said formula (I), and Rj represents hydrogen or the group H—0—CH 2 —, and Z 2 represents a group corresponding to X in the said formula (I), wherein however no free hydroxyl groups are present, or a salt of this compound, is acylated in a manner known per se? and, if required, in resulting compounds having a free terminal carboxyl group this group is converted into an amide group or ester group, and/or the compounds are converted into salts or complexes thereof.