JPS6360760B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides novel lipopeptides and, in particular, the general formula [In the formula, R ₁ is an alkyl group having 13 to 17 carbon atoms; R ₂ is an alkyl group having 11 to 17 carbon atoms; R ₃ is hydrogen, or a group R ₁ -CO-O-CH ₂ −
and R ₁ has the above meaning; and X has the following formula: (1) -L-Ser-L-Ser; (2) -L-Ser-L-Ser-L-Asn; (3 ) -L-Ser-L-Ser-L-Asn-L-Ala; (4) -X ₁ -X ₂ -X ₃ -L-Ala-X ₅ (wherein, X ₁ is L-Ser, L- Phe or L-
Glu; X ₂ is L-Ser, L-Phe or L-
_{(5 )} -L-Ser-L-Ser-L-Asn-L- Ala-
X ₅ ′-L-Ile-L-Asp-L-Glu (wherein, X ₅ ′ is L-Glu or L-Lys); Compounds in substantially pure form or **epimeric R- and S-compounds (R/S) on carbon, and salts thereof; and medicaments containing said compounds. Lipopeptides have already been described in the literature as degraded fragments of lipoproteins. i.e. Hankte and Braun (Er. J. Biochem, 34,
284-296 (1973) isolated an impure lipopeptide or lipopeptide mixture by enzymatic degradation from murein-lipoproteins of the outer cell wall of Escherichia coli; The following structural formula (In this formula Y ₁ , Y ₂ and Y ₃ are acyl groups of different higher saturated or unsaturated fatty acids, such as fatty acids with 14 to 19 carbon atoms)
and the corresponding formula with a shorter polypeptide chain. Although this structure is the basis of the murein protein (see the above-mentioned literature), the absolute configuration of the bisymmetric carbon atoms in the above formula has not been studied. Murein lipoprotein and its degradation products exhibit mitotic reproductive effects on mouse lymphocytes in vitro [Z.Immun.-
Forschung, Vol. 153, pp. 11-22 (1977)]. As is clear, Eq. Glycerylcysteine is based on a degraded lipopeptide derived from murein protein. These fragments are not products whose composition is well defined as products of enzymatic degradation of natural products, but are clearly complex mixtures of condensation products of said peptide moieties and very different acyl derivatives of glycerylcysteine. be. The lipopeptides of the invention differ in many respects from the aforementioned degradation products of murein proteins. Thus, this lipopeptide is a lipopeptide of a single, precisely defined chemical composition and configuration, obtained synthetically, and therefore suitable for therapeutic use. The group of compounds constituted by this lipopeptide has an erythritol group in place of the glycerin moiety in "glycerylcysteine" and/or an amino acid sequence different from that described above for known degraded lipopeptide mixtures of the formula or It includes compounds in which only one amino acid is present, and derivatives such as amides or esters of a terminal carboxyl group. In the acyl groups R ₁ CO, R ₂ CO in the compounds of the general formula and their derivatives, R ₁ and R ₂ are saturated or unsaturated aliphatic groups of 11 to 21 carbon atoms, optionally substituted by an oxygen function. or an aliphatic-alicyclic hydrocarbon group, i.e. the acyl group is a saturated or unsaturated aliphatic or Derived from cycloaliphatic carboxylic acids. These include saturated or unsaturated fatty acids such as lauric acid, myristic acid,
palmitic acid, margaric acid, stearic acid, arachidic acid, oleic acid, elaidic acid, linoleic acid, alpha- and beta-eleostearic acid, stearolic acid, alpha-linolenic acid, also cycloaliphatic, Examples include dihydrosteruclic acid, albalic acid, hydronocarpic acid and yolmouglicic acid. Similarly the acyl groups R ₁ CO and R ₂ CO
Oxidized acids of this type which may be mentioned are, for example, those obtained by epoxidation of the aforementioned olefinic fatty acids and cycloaliphatic acids, such as d,l-epoxystearic acid, and also those containing one hydroxyl group. or higher derivatives of said acids, such as ricinoleic acid. In compounds of the general formula or diastereomeric mixtures thereof and salts or complexes thereof, the group R ₁
and R ₂ may be the same or different from each other. Preference is given to compounds in which the 3 or 4 acyl groups are identical and in particular are palmitoyl, stearoyl or oleoyl groups. The peptide sequence X in the compound of general formula or its salt consists of up to 10 natural aliphatic amino acids,
and at least half of them carry hydrophilic groups, such as in particular hydroxyl, amino, carboxyl, carbamide, guanidino or imidazoyl groups. Among the ionic amino acids that fall into this category, those with acidic groups include aspartic acid, glutamic acid and oxyglutamic acid, and those with basic properties include lysine, ornithine, arginine and histidine. Neutral amino acids are especially amides, such as asparagine, guanidine, and those with hydroxyl groups are mainly serine and threonine. When X in the formula is an amino acid as described above, it is also one that carries the hydrophilic group as described above, such as in particular serine or threonine. Amino acids that can be included in the above sequence and do not have a hydrophilic group include those that are not particularly substituted, such as glycine, alanine, valine, norvaline, leucine, and isoleucine, but also non-hydrophilic substituted 2 , 3, such as methionine. The order of the amino acids in the peptide chain Preferred are those in which the sequence is attached to the carboxyl group of the cysteine in the triacylglycerylcysteine or tetraacylerythritylcysteine moiety. An important group of lipopeptides according to the invention are those in which the peptide chain X consists of 5 amino acids. Among other things, the general formula (In this formula, R ₁ and R ₂ have the above meanings, and X has the meaning given in the general formula), and when the amino acid sequence has 2 to 5 amino acids, there are salts and complexes thereof. . Such lipopeptides include groups X
Examples include those whose amino acid sequence is that of the lipopeptide as described above obtained by decomposition of murein-lipoprotein, that is, those of the general formula, or those corresponding to short chains derived therefrom. It will be done. Particular mention may be made of the following lipopeptide types: (ACGCT = acyl-glycerylcysteine moiety of the formula) 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 form where threonine, glutamine or asparagine replaces serine; also compound type: 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 equivalents with threonine, glutamine or asparagine as replacement amino acids for serine, and terminal carbamide groups or esters amides and carboxylic acid esters having groups (herein, mainly compounds in which the acyl groups R ₁ CO and R ₂ CO of the acylglycerylcysteine moiety are the same and are palmitoyl, stearoyl or oleoyl groups), and
R ₁ CO and R ₂ CO are different, for example palmitoyl, stearoyl or oleoyl, and these groups may appear in any combinations and/or variations as shown in the table below. be. Here, the glycerylcysteine moiety is written as N-acyl-O-O-diacyl-Cys corresponding to the N- and O-acyl substituents:

【table】

[Table] Furthermore, compounds obtainable by substitution of serine by threonine, glutamic acid, glutamine, aspartic acid or asparagine, or by substitution of lysine by glutamic acid, and by simultaneous substitution of lysine by glutamic acid and asparagine by phenylalanine. Examples include compounds that can be used. Compounds with different acyl groups on the N- and O-atoms in the glycerylcysteine group include: N-myristoyl-OO-dipalmitoyl-
Cys-Phe-Phe-Asn-Ala-Lya-OH N-lauroyl-OO-dipalmitoyl-Cys
-Ser-Ser-Asn-Ala-Glu-OH N-stearoyl-OO-dipalmitoyl-
Cys-Ser-Ser-Asn-Ala-Ala-OH and compounds obtained by replacing Ser with Phe or by replacing Phe with Ser. The second group of compounds according to the invention is defined as compounds in which the configuration of the C** atom in the general formula is S instead of R, and mixtures of R- and S-diastereomers, e.g. What you can get. Also in this group, special types and compounds are those specifically mentioned in the R group (general formula). Third group of lipopeptides of the present invention
encompasses compounds in the general formula in which X is an amino acid sequence of 6 to 10 amino acids, preferably the first 5 are the above sequence. The 6 to 10 amino acids may be any of the above-mentioned natural amino acids, such as serine, asparagine, alanine, glutamic acid, lysine, phenylalanine, among others, and may have the following sequence, for example: Ile-Asp-Glu −OH Asp−Glu−OH. Special compounds are, for example: N-stearoyl-S-[2(R),3-dipalmitoyloxypropyl]-Cys-Ser-Ser-
Asn-Ala-Glu-Ile-Asp-Glu-OH N-palmitoyl-S-[2(R),3-dipalmitoyloxypropyl]-Cys-Ser-Ser-
Asn-Ala-Glu-Ile-Asp-Glu-OH N-palmitoyl-S-[2(R),3-dipalmitoyloxypropyl]-Cys-Ser-Ser-
Asn−Ala−Lys−Ile−Asp−Glu−OH. According to the invention, corresponding compounds of the type S-configuration at the C** atom can also be prepared, as well as diastereomeric mixtures of S- and R-compounds. The fourth group of lipopeptides according to the present invention has a general formula in which R ₃ is R ₁ -CO-O-CH ₂
- group, and also includes compounds in which the configuration at the C** atom is arbitrary, and in particular diastereomeric mixtures. The subgroups are general expressions. (In this formula, X has the meaning given in the general formula)
-It has a configuration. The acyl groups R ₁ CO- and R ₂ CO- are preferably the same, especially palmitoyl, stearoyl or oleoyl. However, all combinations and/or variations can be made as mentioned above for other groups. Preferred types and compounds of this group are those in which X has the specified meanings above. For this group, X is preferably an amino acid or a sequence of five amino acids, especially those listed as preferred for the other three groups of lipopeptides. X may however also be an amino acid sequence of 6 to 10 amino acids as described for the third group of lipopeptides of the invention, in particular as described for each group. The specified compounds are, for example, those shown in the table above as specific compounds of the group, and are N-acyl-O-O-acyl-
compounds in which Cys is an erythritylcysteine group rather than a glycerylcysteine moiety as described above, and in which serine is replaced by threonine, glutamic acid, glutamine, aspartic acid or asparagine, and lysine is replaced by glutamic acid; It is a compound obtained by simultaneously replacing Asn with phenylalanine, if desired. A fifth group of lipopeptides according to the invention comprises compounds of the general formula in which X is a peptide chain and the sequence of the first five amino acids differs from that present in known murein-lipoprotein degradation polypeptides. , and especially those compounds in which R ₃ is hydrogen, and also in the general formula R ₁ CO and
Compounds, and salts and complexes thereof, in which R ₂ CO is preferably an acyl group as mentioned above or below in the above combinations. Such substances are, for example, the following lipopeptide type (here
ACGCT=acyl-glycerylcysteine moiety), i.e. ACGCT-Phe-Ile-Ile-Phe-Ala-OH ACGCT-Val-Lys-Val-Tyr-Pro-OH and their amides and esters, especially those mentioned below. be. A further subgroup consists of compounds in which in the general formula X represents the said unnatural sequence of amino acids compared to the murein-lipoprotein degradation product, and from 6 to 10 amino acids are present.
The first five amino acids have, for example, the above sequence or any other sequence. This is for example the following lipopeptide (where ACGCT has the meaning given above): ACGCT-Ala-Ile-Gly-Val-Gly-Ala-
Pro−OH ACGCT−Ser−Ser−Asn−Ala−Glu−Ile−
Asp-Glu-OH and amides and esters thereof, especially those described below. Particularly preferred are compounds of these types in which the configuration of the C** atom of ACGCT is R, or diastereomeric mixtures of R- and S-epimers. In the novel lipopeptides according to the invention, the terminal carboxyl group can be present in amidated form. In addition to unsubstituted amides, mention may be made of those derived from primary and secondary amines. In particular lower aliphatic amines of 1 to 7 carbon atoms, such as methylamine, ethylamine, propylamine, butylamine, dimethylamine, diethylamine or propylamine and isopropylamine, and also aromatic amines, especially monocyclic amines such as aniline or toluidine, Mention may be made of amides of araliphatic amines, such as benzylamine, or of heterocyclic amines, such as aminopyridine. In the case of secondary aliphatic amines, mention may be made of ring-closed nitrogen bases such as pyrrolidine, piperidine or piperazine. Particular amides are the unsubstituted amides or methylamides, ethylamides, dimethylamides or diethylamides of all the above-mentioned special lipopeptides of the invention, or the amides of the compounds mentioned in the examples. In the esterified terminal carboxyl group of the lipopeptides of the invention, the alcohol component is preferably a lower aliphatic alcohol of 1 to 7 carbon atoms, such as methyl alcohol, ethyl alcohol, n-propyl alcohol, isopropyl alcohol or butyl alcohol. be guided.
The alcohol to be esterified may be polyhydric, for example ethylene glycol or propylene glycol or glycerin. Aroaliphatic alcohols, especially monocyclic lower aliphatic alcohols with 1 to 7 carbon atoms in the aliphatic part, such as benzyl alcohol, or heterocyclic alcohols such as tetrahydrofuranol or tetrahydropyranol may also be used for the esterification. I can do it. Special esters of this type of lipopeptides according to the invention include, for example, the methyl, ethyl, ethylene or propylene glycol esters of all the lipopeptides specified above, or those mentioned in the Examples. The novel lipopeptides of the present invention are neutral, acidic or basic compounds depending on the type of substituents.
When an excess of acidic groups is present, the compounds form salts with bases, such as ammonium salts, or with alkali metals or alkaline earth metals such as sodium, potassium, calcium or magnesium. Furthermore, when an excess of basic groups is present, acid addition salts are formed. Acid addition salts are in particular pharmaceutically useful non-toxic acid addition salts, such as inorganic acids such as hydrochloric acid, hydrobromic acid,
salts with nitric, sulfuric or phosphoric acids, or organic acids such as organic carboxylic acids such as acetic acid, propionic acid,
Glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, phosphoric acid, tartaric acid, citric acid, benzoic acid, timtic acid, mandelic acid, salicylic acid, 4-amino-salicylic acid, 2-phenoxybenzoic acid acids, 2-acetoxybenzoic acid, embonic acid, nicotinic acid, or isonicotinic acid, or organic sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, benzenesulfonic acid acid, salts with p-toluenesulfonic acid or naphthalene, -2-sulfonic acid,
Furthermore, other acid addition salts, such as acid addition salts which can be used for purifying difficult compounds or as intermediates in making other salts or can be used to impart special properties, e.g. Salts with picric acid, picloronic acid, flavian acid, phosphotungstic acid, phosphomolybdic acid, chloroplatinic acid, Reinetske's acid or perchloric acid. The complex is a metal salt, such as a heavy metal salt, such as a copper salt,
It is a compound formed with zinc, iron or cobalt salts. To create such complexes, the phosphates, pyrophosphates and polyphosphates of these metal salts are optionally combined with acidic organic substances such as polysaccharides containing acidic groups, such as carboxymethylcellulose, tannic acid, polyglutamic acid. , or partially hydrolyzed gelatin, alkali metal polyphosphates such as "Calgon N", "Calgon 322",
It is preferred to use it in combination with "Calgon 188" or "Plyron B 12". The compounds of the general formula according to the invention, their salts and complexes and mixtures exhibit valuable pharmacological properties, in particular an excellent immunostimulating effect. That is, this compound stimulates constant cell proliferation of B-lymphocytes by thymidine injection in vitro at doses ranging from 0.5 to 320 μg/ml compared to unstimulated control lymphocytes by a factor of 20-50. This degree of stimulation is comparable to that obtained with known effective B-cell-mitotic preparations (dextran sulfate, E. coli lipopolysaccharide, PPD (purified protein derivative)); The novel compounds of the present invention are free of lymphocytotoxicity even with high precision.Compounds of the general formula, their salts and complexes and mixtures are furthermore effective in spleen cell culture of normal mice.
A concentration of 60 μg/ml can induce the formation of antibody-producing cells (comparative value (in the absence of stimulants)
20-50 times the proliferation of 19S-plaque-forming cells).
Thus, even if sheep erythrocytes are not added to the medium for immunization, antibodies against eg sheep erythrocytes are generated in the presence of said compound. On the other hand, at the same concentration, the substance is able to increase the immunoreactivity of T-cell depleted splenocyte cultures towards antigens (sheep erythrocytes) normally derived from the thymus (comparable to the untreated control medium).
10-40 times). Directly or indirectly in vitro, said compounds not only promote the cell proliferation and synthesis efficiency of B-lymphocytes (i.e., latent antibody-forming cells), but also promote the cell proliferation and synthesis efficiency of T-lymphocytes (which include regular mediate effects on active accessory cells and inhibitory cells and cytotoxic effect cells). That is, for example, the compound may be 10' to 100μ
At concentrations of g/ml, it is possible to significantly (up to 10-fold) enhance the reactivity of anticortisone thymocytes to allogen-irradiated stimulated lymphocytes. The above-mentioned effects probably occur indirectly through lipopeptide activation of macrophages that promote the activity of T- and B-lymphocytes. In fact, it can be shown that the compound releases large amounts of "colony stimulating activity (CSA)" from murine macrophages at very low concentrations (0.01-10 μg/ml) (addition of untreated macrophage medium supernatant Macrophage medium inoculated with the substance compared to 0 to 5 cells.
By adding 20% supernatant in 24 hours, 150-200 colonies can be induced in 7 days from 10 ⁵ mouse bone marrow cells). CSA is a biological intermediary value necessary to distinguish myeloid cells from maquiphages and polymorphonuclear leukocytes. The compounds thus act to increase the recruitment of cells that are critical for the induction, amplification and expression of specific (lymphocyte-mediated) immune responses and against unspecified resistance. The immunoenhancing effect of the compound of the present invention represented by the general formula, its salt, complex, or mixture can also be demonstrated in vivo. That is, when the lipopeptide according to the present invention is injected, serum CSA decreases within 3 to 9 hours.
The concentration becomes much higher (10 ⁵ colonies of mouse bone marrow cells after addition of chloroform-extracted serum (5% final concentration) compared to 0-5 colonies in the case of untreated animals).
(increased to 120 colonies per individual). Accordingly, by administering the same compound to living organisms, the ability of mice to produce antibodies is significantly enhanced. NMRI mice are immunized by intraperitoneal injection of 10 μg/day of non-precipitated BSA. Serum tests are taken after 9, 15, and 29 days, and the anti-BSA antibody content is examined by passive blood cell collection reaction technique. Soluble BSA at the doses used is a co-immunogen to the treated animals. That is, they do not produce antibodies or only produce them in very small amounts. Additional treatment of mice with certain immune-enhancing substances before or after challenge increases the antibody titer in the serum. The effect of the treatment is the result obtained, namely bleeding for 3 days.
It is expressed by the sum of log titer differences. In this test, a compound of the general formula, a salt or complex or mixture thereof, produced antibodies against BSA when administered intraperitoneally or subcutaneously in an amount of 0.03 to 3 mg/Kg of the substance 5 days before or after immunization with BSA. production can be significantly increased. The signature of cell-mediated immunity can be enhanced by said compounds in vivo. The above test results are expressed in terms of ED ₅₀ as follows.

Table: Sensitizing guinea pigs with BSA in incomplete Freund's aid results in only humoral antibody production, whereas lipopeptides of the invention are mixed into the aqueous phase of an antigen-oil emulsion at doses ranging from 1 to 15 μg. This causes delayed hypersensitivity to BSA. Intradermal injection of BSA into these animals 3 weeks after immunization produced a local inflammatory reaction with redness and thickening of the skin;
It reaches its peak state in 24-48 hours. This delayed response is both quantitative and qualitative and is usually obtained by immunization with BSA in incomplete Freund's supplement (ie, with addition of filamentous fungi). ED ₅₀ value (μ required for the difference in reaction volume (red x skin thickness increase) to be 200μ in treated and untreated animals 24 hours after onset
g/animal) is 2-3 μg. The lipopeptides according to the invention are even less toxic.
No symptoms appear in mice even after intraperitoneal administration 5 times at a dose of 10 mg/Kg/day every 5 days. Since the doses required for immune stimulation are extremely low, the therapeutic range of the compounds of this invention is extremely large. The novel lipopeptides according to the invention therefore significantly increase the immunity of cells, especially humoral toxicity, and can be mixed with the antigen itself (adjuvant action in the narrow sense) and supplied in a time and place separate from antigen injection (systematic immunization). strengthen immunity). The novel lipopeptides according to the invention therefore improve the inoculum effect and inhibit humoral antibodies and/or
They can be used as adjuvants in combination with inoculants to improve the protection against bacterial, viral or parasitic pathogens mediated by cellular immunity. Finally, the above compounds can be used as adjuncts in the laboratory and industrial production of therapeutic and diagnostic antisera in combination with various antigens and in the attraction of immune activated lymphocyte populations for cell exchange methods. It is suitable as Furthermore, the compounds of the invention can be used to stimulate an already essentially existing immune response in humans and animals, without at the same time supplying antigens. This compound is therefore particularly effective in stimulating body defenses.
Congenital or acquired diseases that appear, for example, during chronic and acute infections or selective (antibody-specific) immune deficiencies, and during the course of severe initial illness, for example in the elderly, and especially after treatment with ionic irradiation or immunosuppressive hormones. Suitable for stimulating the body's defenses during conditions of general (ie non-antigen-specific) immune deficiency. This substance can therefore be processed in combination with antibiotics, chemotherapeutic agents or other treatments to counteract immune disorders. Finally, the substance is also suitable for the general prevention of infectious diseases in humans and animals. The lipopeptides of the invention can be made by methods known per se or by the methods described herein. According to a preferred method, compounds of the general formula, diastereomeric mixtures thereof and salts and complexes thereof are of the general formula a (In this formula, R ₁ , R ₂ and R ₃ have the meanings described in the general formula, Y is an amino acid or amino acid sequence corresponding to X in the general formula,
At least one of the hydrophilic group and/or terminal carboxyl group to be substituted for the amino acid is protected by a protecting group that can be removed under neutral or mild acidic conditions) or The protecting group of the salt of the compound is removed, or b General formula (In this formula, R ₁ , R ₂ and R ₃ have the meanings given in the general formula, and W is OH or an amino acid or an incomplete sequence of amino acids according to X in the general formula) , according to known methods of peptide compounding, using the general formula NH ₂ -Z ₁ (in this formula, Z ₁ is a group corresponding to an amino acid sequence or an amino acid, in which no attracting amino acid is present), or with a compound represented by the general formula c or a salt thereof; (In this formula, R ₂ has the meaning described in the general formula, R ₃ ' is hydrogen or a H-O-CH ₂ - group, and Z ₂ is a group corresponding to X in the general formula, and therein (assuming that no free hydroxyl group is present) or a salt thereof is acylated by a method known per se and, if desired, the carboxyl group of the resulting compound having a free terminal carboxyl group is acylated. or ester group, and/or
made by converting the compound into its salt or complex. The protecting groups used in method a are those known in particular in peptide synthesis. Protecting groups for amino groups are thus, for example, acyl or aralkyl groups, such as formyl, trifluoroacetyl, phthaloyl, benzenesulfonyl, p-toluenesulfonyl, o-nitrophenylsulfenyl, 2,4- dinitrophenylsulfenyl groups (these sulfenyl groups can also be eliminated by the action of nucleophilic reagents such as sulfites, thiosulfates), optionally e.g. lower alkoxy groups, in particular o- or p-methoxy groups; a benzyl group, diphenyl group or triphenylmethyl group, thus substituted;
or groups derived from carbonic acid, such as arylmethyl optionally substituted in the aromatic ring by a halogen atom such as chlorine or bromine, a nitro group, a lower alkyl group or a lower alkoxy group or a colored group such as an azo group. an oxycarbonyl group (wherein the methylene group may be further substituted by an aryl group and/or one or optionally two lower alkyl groups), such as a benzyl group, a benzhydryl group or a 2-phenylisopropyl group; oxycarbonyl groups, such as carbobenzoxy, p-bromo- or p-chlorocarbobenzoxy, p-nitrocarbobenzoxy or p-methoxycarbobenzoxy,
p-phenylazobenzyloxycarbonyl groups and p-(p'-methoxyphenylazo)-benzyloxycarbonyl groups, 2-tolylisopropyloxycarbonyl groups and especially 2-(p-biphenylyl)-isopropyloxycarbonyl groups, and Aliphatic oxycarbonyl groups are, for example, adamantyloxycarbonyl, cyclopentyloxycarbonyl, trichloroethyloxycarbonyl, tertiary amyloxycarbonyl or especially tertiary butyloxycarbonyl. Amino groups are obtained by formation of enamines and can be protected by reaction of amino groups with 1,3-diketones such as benzoylacetone, acetylacetone or dimedone. Carboxyl groups are protected, for example, by amidation or hydrazide formation, or by esterification. The amide and hydrazide groups may be optionally substituted, ie the amide group may be substituted, for example by a 3,4-dimethoxybenzyl group or a bis-
By the (p-methoxyphenyl)-methyl group, the hydrazide group can be defined, for example, by the carbobenzoxy, trichloroethyloxycarbonyl, trifluoroacetyl, trityl, tert-butyloxycarbonyl or 2-(p- (biphenylyl)-isopropyloxycarbonyl group. For example, optionally substituted lower alcohols such as methanol, ethanol,
cyan methyl alcohol, benzyl methyl alcohol or especially tertiary butanol, also aralkanols such as aryl lower alkanols, such as benzyl alcohols or benzhydrols optionally substituted by lower alkyl or lower alkoxy groups or halogen atoms, such as benzhydro; roll, p-nitrobenzyl alcohol, p-methoxybenzyl alcohol, 2,4,
6-Trimethylbenzyl alcohol, phenols and thiophenols optionally substituted by electrophilic substituents, such as thiophenol, thiocresol, p-nitrothiophenol, 2,4,5- and 2,4,6 -Trichlorophenol, pentachlorophenol, p-nitrophenol, 2,4-dinitrophenol, p
Suitable for the esterification are cyanophenol or p-methanesulfonylphenol, as well as, for example, N-hydroxysuccinimide, N-hydroxyphthalimide, N-hydroxypiperidine, 8-hydroxyquinoline. The hydroxy groups of serine and threonine groups can be protected, for example, by esterification or etherification. Particularly suitable acyl groups in the esterification are groups derived from carbonic acid, such as benzoyloxycarbonyl or ethyloxycarbonyl groups. Groups suitable for etherification are, for example, benzyl, tetrahydropyranyl or tertiary-butyl. Furthermore, 2,2,2-trifluoro-1- described in Ber. 100 (1967), 3838-3849
tert-butyloxycarbonylamino- or -1
-Benzyloxycarbonylaminoethyl groups are suitable for protecting hydroxyl groups (Weygand). For the protection of side chain carboxyl groups and optionally terminal carboxyl groups, it is preferred to use tertiary butyl ester groups or benzhydrol groups; for the protection of side chain amino groups, tertiary butyloxycarbonyl groups are also used. A tert-butyl ether group is used for the hydroxyl group of serine or threonine and, if desired, a 2,2,2-trifluoro-1-tert-butyloxycarbonylaminoethyl group for protection of the imino group of histidine. Removal of the protecting group in the process of the invention by acidic agents under mild conditions is carried out by one of the known methods of releasing the peptide compound, for example by treatment with trifluoroacetic acid. A special protecting group for a carboxyl group that can be removed under neutral conditions is, for example, the general formula described in German Patent Publication No. 2706490. (In this formula, R ¹ , R ² and R ³ are each a hydrocarbon group, and these groups have a single bond with each other.
It may be connected by C′-C, especially 1 carbon atom
~5 alkyl groups). Protecting groups of this type include, for example, the 2-(dimethyl-tert-butylsilyl)-ethyl group,
2-(dibutylmethylsilyl)-ethyl and especially 2-(trimethylsilyl)-ethyl. These protecting groups can be removed with basicity, but
Particularly important are the removability under neutral conditions and the action of hydrofluoric acid salts. The removal of the protective group is advantageously carried out with an aprotic organic solvent. At this time, it is preferable to avoid the presence of a solvent that can solubilize the fluoride anion, such as water or lower aliphatic alcohol. The condensation of a compound of general formula with a compound of general formula by method b can be carried out, for example, by reacting a compound of general formula in activated form with a compound of general formula, or by reacting an acid of general formula with a compound of general formula whose amino group is activated. It is carried out by reacting with a compound of the general formula existing in the form. Optionally, compounds of the general formula or are subjected to the condensation in the form of their salts. The carboxyl group of the compound of the general formula is, for example,
Acid azides, acid anhydrides, acid imidazolides, acid isoxazolides or activated esters, such as cyan methyl ester, carboxymethyl ester, thiophenyl ester, p-nitrothiophenyl ester, thiocresyl ester, p-methanesulfonylphenyl ester , p-nitrophenyl ester, 2,4-dinitrophenyl ester, 2,
4,5- or 2,4,6-trichlorophenyl ester, pentachlorophenyl ester, N-
Hydroxyquinoline ester, 2-hydroxy-
1,2-dihydro-1-carboethoxyquinoline ester, N-hydroxypiperidine ester or enol ester obtained with N-ethyl-5-phenyl isoxazolium-3-sulfonate (Woodward reagent), or carbodiimide 1-hydroxybenzotriazole, 3-hydroxy-4-oxo-3, (with optional addition of N-hydroxysuccinimide) or unsubstituted or substituted, for example by halogen, methyl or methoxy groups;
Activation can be achieved by reaction with 4-dihydrobenzo[d]-1,2,3-triazine or N,N'-carbonyldiimidazole. The amino groups of compounds of the general formula can be activated, for example, by reaction with phosphites. The most common condensation method is Weygand-
Wunsch method (using carbodiimide in the presence of N-hydroxysuccinimide), azide method, N-
There are carboxy anhydride or N-thiocarboxy anhydride methods, activated ester methods and anhydride methods.
In particular, these condensations can be carried out by the Merrifield method. It is also possible to carry out condensation of compounds of the general formula with compounds of the general formula by the method described above, in which the terminal carboxyl group is protected by a protective group different from the amide or ester group defined for X, for example by those mentioned above. and/or
It is provided that one or more of the hydrophilic groups present are in a protected form as described above. In this way, condensation products belonging to the group of raw materials of the general formula used in process a) above are obtained, which are likewise the object of the present invention. According to method c, the free hydroxyl group of the glyceryl group or erythrityl group of the compound of the general formula (wherein the Z ₂ moiety does not need to contain a free hydroxyl group) is acylated. This acylation is carried out by methods known per se, for example by reaction with a reactive functional derivative of the acid corresponding to the group to be introduced, such as an anhydride or an acid halide, preferably in the presence of a tertiary base such as pyridine or collidine. This can be done by This method is particularly suitable for structures of compounds in which the acyl group in the glyceryl or erythrityl group is different from the acyl group in the cysteine group in the general formula. In the general formula, the terminal carboxyl group of the Z ₂ moiety is protected by an amide or ester group as mentioned for X and also by other protecting groups and/or one or more hydrophilic protecting groups present are When this method is applied to compounds that exist in a protected form such as, the starting material for method a) is obtained in the same way. This carboxyl group of a lipopeptide of the general formula (where a free terminal carboxyl group is assumed to be present in the It can be converted into an amide group or an ester group by a method. General formula used as raw material (where W=
OH, N-acyl- or N (where R ₃ = H),
O,O-triacyl-S-(2,3-dihydroxypropyl)-cysteine is obtained as follows: starting from 1,1,5,6-D-mannitodiacetonide and converting to periodate oxide. 2-0,3-O-isopropylidene-D-glyceraldehyde is obtained, the aldehyde group is reduced to a carbinol group, and this is esterified with p-toenesulfonic acid. 1-0-tosyl- thus obtained
2(R)-0,3-0-isopropylidene glycerin is condensed with N-acyl-(R)-cysteine in the presence of a basic agent such as potassium carbonate, thus forming N-acyl-S-[2(R)-cysteine. ), 3-isopropylenedioxypropyl]-(R)-cysteine,
This is saponified with an acid such as acetic acid. The carboxyl group of the N-acyl-S-[2(R),3-dihydroxypropyl]-(R)-cysteine thus obtained is optionally protected in advance, preferably by the aforementioned trimethylsilylethyl group or preferably by the benzhydryl ester group. and glycerin part 2
Further acylation at the - and 3-positions is possible. N-acyl- or N,O,O,O-tetraacyl-S-(2,
The production of 3,4-trihydroxybutyl)-cysteine can be carried out analogously to the method of Example 7. The amino acids or peptides used for the production of the lipopeptides of the invention are known or made by methods known per se. The above raw material of the general formula of method b, where W=OH,
The diastereomeric mixtures, salts and complexes themselves have an immunopotentiating effect, which is manifested in vitro in the same dosage range as the compounds of the general formula and their derivatives. This compound shows an increase in serum antibody titer in the above-mentioned test for fluid immunity enhancement in vivo when administered intraperitoneally, 5 days before antigen delivery, in the dose range of 1.0 to 3 mg/Kg heavy body. This compound can be obtained by acylating a compound in which a hydroxyl group is present in place of Z ₂ in the general formula as described in method c). The preparation of the raw materials necessary for this, namely glyceryl- or erythrityl-N-acylcysteine, has been described above. Compounds in which Z ₂ in the general formula has a meaning corresponding to X in the general formula and there is no free hydroxyl group are the above N-
The acylcysteine derivative is prepared according to the same principle as the above-mentioned method b) of the general production method of the lipopeptide of the present invention,
It can be obtained by reaction with a compound of formula _NH2 - _Z2 . The obtained lipopeptides can be prepared by methods known per se, for example by reacting the obtained acidic compounds with alkali metal hydroxides or alkaline earth metal hydroxides or by reacting the obtained basic compounds with acids. It can be converted into salt by Because of the close relationship between the free form of the compounds of the invention and their salt and complex forms, statements made herein regarding the free compounds may also apply to the corresponding salts. This is true. The isomer mixtures obtained can be separated in known manner, for example by chromatography and/or fractional crystallization, according to the differences in the physicochemical properties of the components. It is advantageous to isolate the effective isomer. The process is carried out according to known methods, in the presence or preferably in the absence of diluents or solvents, if necessary with cooling or heating, and under elevated pressure (or) in an atmosphere of an inert gas, for example nitrogen. In this case, care is taken to ensure that all substituents present in the molecule are particularly easily hydrolyzed, if necessary. - reaction conditions with special care when acyl groups are present;
For example, short reaction times, use of low stoichiometric proportions of mild acidic agents, selection of appropriate catalyst, solvent, temperature and/or pressure conditions should be used. The present invention also describes a method for carrying out the remaining process steps starting from a compound obtained as an intermediate product at any stage of the manufacturing process, or for interrupting the manufacturing process at any stage, or for introducing raw materials under reaction conditions. It also relates to methods of making or using raw materials in the form of reactive derivatives or salts. In this case, it is preferable to start from raw materials from which the compounds mentioned above are particularly valuable. The invention likewise relates to pharmaceutical preparations containing the above-mentioned compounds of the invention, compounds of the general formula and, mixtures, salts or complexes thereof. The pharmaceutical preparations according to the invention contain a pharmacologically active substance, alone or together with a pharmaceutically usable carrier substance, which can be administered enterally, e.g. orally or rectally.
or for parenteral administration to warm-blooded animals. The amount of active substance administered depends on the species of warm-blooded animal, its age, its individual condition, and the method of administration. The pharmaceutical formulations of the invention contain from about 10 to about 95 active ingredients.
%, preferably about 20 to about 90%. The pharmaceutical formulations of the invention can be presented in the form of dosage units such as dragees, tablets, capsules, suppositories or ampoules. The pharmaceutical formulations of the invention can be prepared by methods known per se, such as conventional mixing methods, granulation methods, dragee methods,
Produced by melting or freeze-drying. Suitable carrier materials include in particular fillers, such as sugars,
lactose, sucrose, mannite or sorbitol, cellulose preparations and/or calcium phosphates, such as tricalcium phosphate or calcium hydrogen phosphate, as well as binders such as corn starch, wheat starch, rice starch or potato starch. starch paste used,
gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone, and/or optionally dusting agents such as the aforementioned starches, carboxymethylstarch, reticulated polyvinylpyrrolidone, agar, alginic acid or its salts, such as alginic acid It is sodium. Auxiliaries are primarily flow control agents and lubricants, such as silicic acid, talc, stearic acid or its salts, such as magnesium or calcium stearate, and/or polyethylene glycol. The dragee cores are optionally provided with a suitable coating resistant to gastric juices, in which case in particular a concentrated sugar solution optionally containing gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, suitable Lacquer solutions in organic solvents or solvent mixtures are used, and solutions of suitable cellulose preparations such as acetyl cellulose phthalate or hydroxypropyl methyl cellulose phthalate are used to produce gastric juice-resistant coatings. Dyestuffs or pigments can be added to the tablets and dragee coatings, for example for identification or indication of different active substance doses. The present invention will be explained in more detail with reference to the following examples, but these examples are not intended to limit the scope of the present invention. Each symbol in the examples has the following meaning. Z=carbobenzoxy group, ^Butt =tertiary butyl ether group, ^OBut =tertiary butyl ester group, BOC=tertiary butoxycarbonyl group, DMF=dimethylformamide, ONp=p-nitrophenyl ester, HOBt=N- Hydroxybenzotriazole, DC = thin layer chromatography. In thin layer chromatography, silicic acid gel was used as an adsorbent and the following system was used as a developing agent. System 3: Acetic acid, pyridine-water (65:20:15). System 157: Chloroform-methanol-water-acetic acid (70:42:10:0.5) System 157c: Chloroform-methanol-water-acetic acid (75:25:5:0.5). Example 1 [N-palmitoyl-S-2(R),3-dipalmitoyloxypropyl]-Cys-Ser-( ^But )-
1.13 g of Ser( ^But )-Asn-OBut are dissolved in 5 ml of 90% trifluoroacetic acid and after 45 minutes at 20 DEG C. the solution is concentrated to about 2 ml. The product is precipitated by addition of 75 ml of peroxide-free ether, filtered and dried over potassium hydroxide. The thus obtained lipopeptide [N-palmitoyl-S-2(R),3-dipalmitoyloxypropyl]-Cys-Ser-Ser
-Asn-OH shows the following RF value in a thin layer chromatogram on silica gel: Rf (chloroform-methanol-acetic acid-water 79:25:0.5:4.5) =
0.25. The starting material can be produced as follows. [N-Palmitoyl-S-2(R),3-dihydroxypropyl]-Cys-Ser( ^But )-Ser( ^But )-
Dissolve 0.89 g of Asn-OBu ^t in 10 ml of pyridine, add 0.725 ml of palmitic acid chloride, and keep the solution at 45° C. for 24 hours. 200 ml of chloroform are then added, the solution is extracted with 1N citric acid, 1N sodium bicarbonate and water, the organic phase is dried over sodium sulfate and the solvent is evaporated. The residue is purified by chromatography on silicic acid gel using chloroform or chloroform-methanol (98:2) system. Single lipopeptide [N-palmitoyl-S-2(R),3-dipalmitoyloxypropyl]-Cys-Ser-( ^But ) by thin-layer chromatography
-Ser(Bu ^t )-Asn-OBu was prepared on silica gel using a chloroform-methanol (9:1) system with an Rf of 0.75.
Show value. [N-Palmitoyl-S-2(R),3-dihydroxypropyl] in 20 ml of dimethylformamide
- Cysteine 0.90g and H-Ser( ^But )-Ser
Add 0.99 g of ( ^But )-Asn-OBu, 0.472 g of dicyclohexylcarbodiimide and 0.309 g of N-hydroxybenzotriazole. After 2 hours at 0°C and 15 hours at 20°C, it is filtered and the filtrate is evaporated. The residue is purified by chromatography on a silica gel column using a chloroform-methanol (98:2) system. The thus obtained lipopeptide derivative [N-
Palmitoyl-S-2(R),3-dihydroxypropyl]-Cys-Ser-( ^But )-Ser( ^But )-Asn-
^OBut shows an Rf value of 0.60 (chloroform-methanol=8:2). Dissolve 6.0 g of Z-Ser(Bu ^t )-Ser(Bu ^t )-Asn-OBu ^t in 60 ml of methanol, and add 0.5 g of Pd-charcoal (10%).
After adding , hydrogenation is carried out at room temperature for 3 hours. Filter the catalyst and evaporate the filtrate. The product remains as a white foam. This peptide H-Ser( ^But )-
Ser( ^But )-Asn-OBu ^t shows an Rf value of 0.41 in a chloroform-methanol (8:2) system in a thin layer chromatogram on silica gel. Z-Ser( ^But )-OH4.134g and H-Ser
4.64 g of (Bu ^t )-Asn-OBu ^t is dissolved in 50 ml of dimethylformamide and 3.172 g of dicyclohexylcarbodiimide and 1.89 g of N-hydroxybenzotriazole are added at 0°C. After 2 hours at 0°C and 15 hours at 20°C, the precipitated dicyclohexylurea is filtered off with suction, the filtrate is evaporated, the residue is dissolved in acetic acid ester, and this solution is extracted with 1N citric acid, 1N sodium bicarbonate and water. , dried over sodium sulfate and evaporated. The residue is converted into acetate ester.
Recrystallize from hexane. The obtained peptide Z-
Ser( ^But )-Ser( ^But )-Asn- ^OBut has a melting point of 96-98°C. Rf value on silica gel (chloroform methanol = 95:5) = 0.25. 4.65 g of Z-Ser(Bu ^t )-Asn-OBu ^t are hydrogenated in 50 ml of methanol in the presence of 0.4 g of Pd-charcoal (10%). After filtering off the catalyst and evaporating the filtrate, the peptide H-Ser( ^But )-Asn- ^OBut is obtained as a white foam. Rf value on silica gel (chloroform-methanol = 7:3) = 0.48. Z-Ser( ^But )-OH5.9g and H-Asn-
Dissolve 3.76 g of ^OBut in 60 ml of dimethylformamide and add N-hydroxybenzotriazole at 0°C.
3.06g and dicyclohexylcarbodiimide
Add 4.53g. 2 hours at 0℃ and 15 hours at 20℃
After a period of time it is filtered, the filtrate is evaporated and the residue is crystallized from acetate-petroleum ether. The resulting peptide Z-Ser(Bu ^t )-Asn-OBu ^t was heated to 114-115°C.
to melt. [α] ²⁰ _D = +4° (c = 1.5, in methanol). Rf value on silica gel (chloroform-methanol = 9:1) = 0.48. Example 2 [N-palmitoyl-S-2(R),3-dipalmitoyloxypropyl]-Cys-Ser( ^But )-Ser
1.6 g of ( ^But )-Asn-Ala-OBu ^t is dissolved in 7.5 ml of 90% trifluoroacetic acid and the solution is concentrated to about 2 ml after 15 minutes at 20°C. This product is ether 100
ml, filter and dry over sodium hydroxide. A white powder with a melting point of 215-217°C (decomposes) is obtained, which is composed of the lipopeptide [N-palmitoyl-S-2(R),3-dipalmitoyloxypropyl]-Cys-Ser-Ser-Asn-Ala- It's OH.
Rf value on silica gel (system 157) = 0.55. Example 3 [N-palmitoyl-S-2(R),3-dipalmitoyloxypropyl]-Cys-Ser( ^But )-Ser
(Bu ^t ) - 0.95 g of OBu ^t in 4 ml of 90% trifluoroacetic acid
dissolve in After 15 minutes at 20° C. 100 ml of ether are added, filtered and the residue is dried over sodium hydroxide. The resulting lipopeptide [N-palmitoyl-S-2(R),3-dipalmitoyloxypropyl]-Cys-Ser-Ser-OH is obtained as a colorless, non-hygroscopic powder. Rf value on silica gel (chloroform-methanol-acetic acid, water = 74:25:0.5:
4.5) = 0.53. Example 4 N-Palmitoyl used as raw material in Example 1
S-[2(R),3-dihydroxypropyl]-(R)
- Cysteine can be made as follows: N-palmitoyl-(R)-cysteine 13,9
g, potassium carbonate 5.5 g and 1-0-tosyl-
15 g of 2(R)-0-3-0-isopropylidene glycerin was dissolved in 250 ml of ethanol in a nitrogen atmosphere for 80 min.
Heat at ℃ for 8 hours. The reaction mixture obtained after evaporation was chromatographed on 400 g of Merck silicic acid gel, first with chloroform/acetone (8:
2) and then by elution with chloroform/methanol (8:2). The potassium salt of N-palmitoyl-S-[2(R),3-isopropylidenedioxypropyl]-(R)-cysteine is thus obtained, which is heated to 80° C. for 4 hours in 80% aqueous acetic acid. Saponify by. Evaporate to dryness and dissolve the residue in chloroform and shake with water. After drying and evaporating the chloroform phase, a colorless residue was obtained, which was recrystallized from cyclohexane to give N-palmitoyl-S-[2
(R),3-dihydroxypropyl]-(R)-cysteine is obtained. Melting point 110°C, [α] ²⁰ _D = -25° (c = 0.9, in MeOH). N-palmitoyl-(R)-cysteine can be produced as follows. 45 g (0.37 mol) of (R)-cysteine was suspended in 350 ml of pyridine, and 140 ml of palmitic acid chloride was stirred well at room temperature in a nitrogen atmosphere.
(1.67 eq.) in 550 ml of methylene chloride dropwise. After stirring for 20 hours, acidify with 2N hydrochloric acid.
Partition between chloroform and water. After drying and evaporating the chloroform phase, a crystalline mixture consisting of approximately 1/3 dipalmitoylcysteine and approximately 2/3 monopalmitoylcysteine is obtained. hot acetone 1
and from which a 1:1 mixture of di- and mono-palmitoylcysteine is crystallized. N-palmitoylcysteine is obtained from this acetone solution by evaporation and is recrystallized from benzine. Melting point 65-67℃, [α] ²⁰ _D = +1° (c =
0.8, methanol). A mixture of di- and mono-palmitoylcysteine is treated in methanolic solution with 2 ml of concentrated caustic soda solution for 15 minutes at room temperature while adding an aqueous solution of 12 g of sodium sulfite. After evaporation of the methanol and acidification of the residue with 2N hydrochloric acid, chloroform extraction yields the remaining N-palmitoylcysteine, which is likewise recrystallized from benzine. Example 5 N-palmitoyl-S-[2(R),3-dipalmitoyloxypropyl]-(R)-cysteine
0.80g and H-Ser( ^But )-Phe-Ala-Glu
0.67 g of (OBu ^t ) ₂ is dissolved in 10 ml of dimethylformamide and 0.22 g of dicyclohexylcarbodiimide and 0.16 g of N-hydroxybenzotriazole are added. Brief heating to 40° C. gives a clear solution which solidifies to a glue-like consistency at 20° C. in about 1 hour. This was heated to 40°C to make a solution again after 24 hours, and from this N-palmitoyl-S-[2-(R), 3.dipalmitoyloxypropyl]-Cys-Ser( ^But )-Phe-Ala-
Precipitate Glu(OBu ^t ) ₂ with water. The product is purified by chromatography on silicic acid gel in chloroform or chloroform-methanol (98:2) system and has an Rf value of 0.4 in chloroform-methanol-water (95:5:). . 540 mg of the compound thus obtained was dissolved in 30 ml of 90% trifluoroacetic acid, the solution was concentrated to about half after 30 minutes at 20°C, and N-palmitoyl-S-[2(R),3-dipalmitoyloxy] was dissolved in petroleum ether. Propyl]-Cys-Ser-Ser-Phe-Ala-Glu-OH
precipitate. The compound is centrifuged, washed three times with petroleum ether and dried over potassium hydroxide. The product is a white powder with an Rf value (system 157) = 0.63. Similarly, the following lipopeptide is made: N-palmitoyl-S-[2(R,S). 3-dipalmitoyloxypropyl]-Cys-Phe-Phe
-Asn-Ala-Lys-OH, Rf (system 157c) = 0.18; N-myristoyl-S-[2(R),3-dipalmitoyloxypropyl]-Cys-Glu-Glu-
Asn-Ala-Lys-OH, Rf (system 157c) = 0.12; N-lauroyl-S-[2(R), 3. dipalmitoyloxypropyl]-Cys-Ser-Ser-Asn-
Ala-Glu-OH, Rf (system 157c) = 0.20; N-stearoyl-S-[2(R),3-dipalmitoyloxypropyl]-Cys-Ser-Ser-
Asn-Ala-Ala-OH, Rf The above peptide with the above protecting group (this one is suitable for condensation with N-palmitoyl-S-[2(R),3-dipalmitoyloxypropyl]-(R)-cysteine) ) can be made as follows; Z−Ala−ONp17.22g, HCl・HGlu(OBu ^t )
₂ 14.8 g and 6.3 ml of N-ethylmorpholine are left overnight at 20° C. in 40 ml of dimethylformamide.
The solution is evaporated and the residue is dissolved in acetic ester and extracted with 1N sodium bicarbonate and water. This acetic acid solution was evaporated to give Z-Ala-Glu with an Rf value of 0.60 (chloroform-methanol = 95:5).
(OBu ^t ) ₂ is stored as a foam, which is immediately used further. 8.5g of this stuff in 60ml of methanol
After adding 0.8 mg of Pd-charcoal (10%) dissolved in 20
Hydrogenate for 2 hours at °C. Filter the catalyst and evaporate the filtrate. H-Ala-Glu(OBu ^t ) ₂ is obtained as a white foam with Rf=0.36 in chloroform-methanol (9:1). Z-Phe- in 50 ml of dimethylformamide
OH11.46g and H-Ala-Glu(OBu ^t ) ₂ 12.64g
Dicyclohexylcarbodiimide at 0℃ 8.68
g and -N-hydroxybenzotriazole
Add 5.86g. After 15 hours at 4° C. it is filtered, the filtrate is evaporated and the residue is recrystallized from acetic acid-hexane. Z-Phe-Ala-Glu with melting point 161-163℃
(OBu ^t ) ₂ is obtained. Rf (toluene-acetone=
1:1)=0.63. Dissolve 6.4 g of this product in 50 ml of methanol,
Add 0.5 g of Pd-charcoal (10%) and hydrogenate at 20°C for 20 minutes. When the catalyst is filtered and the filtrate is evaporated, H-
Phe-Ala-Glu(OBu ^t ) ₂ is obtained as a white foam. Rf (chloroform-methanol = 7:
3)=0.75. Z-Ser(Bu ^t ) in 30 ml of dimethylformamide
−OH4.43g and HCl・H−Ser(Bu ^t )−
1.89 N-ethylmorpholine in 3.18 g of OMe at 0℃
ml, 2.29 g HOBt and 3.4 g dicyclohexylcarbodiimide are added. 2 hours at 0℃ and
After 15 hours at 20° C. it is filtered, the filtrate is evaporated and dissolved in acetic acid ester. When this solution is extracted with sodium bicarbonate, dilute hydrochloric acid and water and then evaporated, Z
-Ser( ^But )-Ser( ^But )OMe is obtained as an oil. Rf (toluene-acetone=1:1)=
0.70. 7.5 ml of hydrazine hydrate are added to a solution of 6.8 g of this product in 40 ml of methanol and the volume is evaporated to approximately half after 24 hours at 20.degree. Acetate ester Dissolve in 250 ml of acetate ester and extract with water. This acetate ester solution was concentrated and the obtained Z-Ser
( ^But )-Ser( ^But )-NH- _NH2 is precipitated with petroleum ether. Rf (toluene-acetone=1:1)
=0.50. Add 2.26 g of this product to 20 ml of dimethylformamide.
1.78N− in acetate at −10 °C.
HCl7.022ml and tertiary butyl nitrite 0.635
Add ml. After 10 minutes at -10℃ H-Phe-Ala-
Glu(OBu ^t ) ₂ 2.38g and N-ethylmorpholine
2.52 ml of solution was added dropwise and incubated at -10°C for 1 hour and then at 0.
Keep at °C for 15 hours. The solution is then evaporated and the residue is dissolved in acetate and washed with sodium bicarbonate, dilute hydrochloric acid and water. Z-Ser( ^But )-Ser( ^But )-Phe-Ala- obtained after evaporating this solution
Glu(OB ^t ) ₂ is recrystallized from acetate-hexane. Melting point: 213-215°C, Rf (toluene-acetone = 1:1) = 0.70. Dissolve 2.30 g of this product in 40 ml of methanol,
After adding 0.5 g of Pd-charcoal (10%), hydrogenation is carried out at 20° C. for 2 hours. When the catalyst is filtered and the filtrate is evaporated, H-Ser( ^But )-Ser( ^But )-Phe-Ala-Glu
(OBu ^t ) ₂ is obtained as a white foam. Rf (toluene-acetone=1:1)=0.23. N-palmitoyl-S- used as raw material
[2(R),3-dipalmitoyloxypropyl]-
(R)-cysteine and 2 corresponding to this compound
(S)-compound (this is abbreviated as 2(R,S))
A diastereomeric mixture of can be made as follows. 6.3 g (2.78 mmol) of N-palmitoyl-S-[2(R),2-dipalmitoyloxypropyl]-(R)-cysteine benzhydryl ester are dissolved in a mixture of 12 ml of trifluoroacetic acid and 48 ml of methylene chloride. . After 1 hour at room temperature, the solution is evaporated and the oily residue is triturated with water. The precipitated N-palmitoyl-S-[2(R),3-dipalmitoyloxypropyl]-(R)-cysteine is filtered off with suction, dried and triturated three times with benzine to remove the benzhydrol. When the residue is crystallized from acetone-water, the product has a melting point of 71~
At 75°C, colorless crystals of [α] ²⁰ _D = -1° (dioxane) are obtained. The above ester is obtained as follows: 12 g (20 mmol) of N-palmitoyl-S-[2(R),3-dihydroxypropyl]-(R)-cysteine benzhydryl ester in 60 ml of pyridine.
and 60 ml of methylene chloride, and 14.5 ml (13.2 g) of palmitic acid chloride are added dropwise at 0-10°C. The reaction is complete by thin layer chromatography after 2 hours at room temperature. 10 ml of methanol are added to the suspension (pyridine hydrochloride) and stirred for 20 minutes at 25°C, then the reaction mixture is further evaporated. The residue is dissolved in chloroform, shaken twice each with 0.5N hydrochloric acid, 10% sodium bicarbonate and water, the chloroform phase is dried over sodium sulfate and evaporated to a syrup. The syrup is chromatographed on silicic acid gel in chloroform to give the pure ester. This chromatographically single fraction (DC in chloroform:acetate (98:2) with silicic acid gel - comparison; Rf = 0.5) is evaporated and recrystallized from acetone. Melting point 69-71℃
Colorless crystals of N-palmitoyl-S-[2(R),3-dipalmitoyloxypropyl]-(R)-cysteine benzhydryl ester are obtained. N-palmitoyl-S-[2(R),
The benzdolyl ester of 3-dihydroxypropyl]-(R)-cysteine is obtained, for example, as follows: 23.4 g of N-palmitoyl-S-[2(R),3-dihydroxypropyl]-(R)-cysteine.
(54 mmol) in chloroform-acetone mixture 150
ml and to this is added dropwise at room temperature a solution of 13.6 g (70 mmol) of diphenyldiazomethane in 50 ml of chloroform. After 3 hours at room temperature, the initially red solution fades and the reaction is completed by thin layer chromatography (chloroform:methanol=9:1; using silicic acid gel). Evaporate to dryness in vacuo and extract the residue cold with petroleum ether. The residue is filtered through silicic acid gel with methylene chloride as solvent. Colorless crystals of benzhydryl ester with a melting point of 88-91°C are thus obtained. To prepare N-palmitoyl-S-[2(R,S)3-dihydroxypropyl]-(R)-cysteine, the following procedure is performed. N-palmitoyl-R-cysteine 50g
(139 mmol), 12.4 g of glycide and 45 g of potassium carbonate are heated in 375 ml of ethanol to 80° C. for 16 hours with good stirring in a nitrogen atmosphere. After cooling to room temperature, the pH is made acidic to about 4 with 2N hydrochloric acid and water is added. At this time the product precipitates. Filter with suction and wash with water until the filtrate is free of chloride ions. The suction filter cake is dried in vacuo and then recrystallized from acetic ester. N-Palmitoyl-
S-[2(R),3-dihydroxypropyl]-
(R)-cysteine and N-palmitoyl-S-[2
Colorless crystals (melting point 76 DEG -155 DEG C.) of a diastereomeric mixture with (S),3-dihydroxypropyl]-(R)-cysteine are obtained. N-palmitoyl-S-[2(R,S),3-dihydroxypropyl]-(R)-cysteine was reacted with diphenyldiazomethane and then with palmitenoyl chloride as described above for the R-compound. N-palmitoyl-S-[2(R,S),
3-Dipalmitoyloxypropyl]-(R)-cysteine benzhydryl ester is obtained. This diastereomeric mixture (19.8 g) was chromatographed on a column of 250 g of silicic acid gel (Merck) using methylene chloride as solvent, and the ester moiety (1.55 g) contained the R-diastereomer (Rf = 0.61 ; methylene chloride:acetic acid ester = 98:2, thin layer, silicic acid gel) 12.7 g of R,S-diastereomer mixture in the second part and S-diastereomer (Rf =
0.53; methylene chloride:acetic acid ester = 98.2) 1.1g
is obtained. N-palmitoyl-S-[2(S),3-dipalmitoyloxypropyl]-(R)-cysteine is obtained from the (S)-diastereomer as described above; colorless crystals, melting point 64-65 °C, [α] ²⁰ _D =
+1° (dioxane). N-palmitoyl-S-[2R,
3-dipalmitoyloxypropyl]-Cys-Ser
-Ser-Phe-Ala-Glu-OH The raw material necessary for the production of the novel lipopeptide of the present invention prepared in the same manner as for example N-myristoyl-S-[2(R),3-dihydroxypropyl]-(R) - cysteine, melting point
Colorless crystals at 140-150℃; N-stearoyl-S-
[2(R),3-dihydroxypropyl]-(R)-cysteine, colorless crystals, melting point 140-150°C; N-lauroyl-S-[2(R),3-dihydroxypropyl]-(R)-cysteine , melting point 140-150℃, colorless crystals. These can be made as described in Example 4. The benzhydryl esters prepared as described above are, for example, as follows: N-myristoyl-S-[2(R),3-dihydroxypropyl]-
(R)-cysteine benzhydryl ester, melting point
Colorless crystals at 90-92°C; N-stearoyl-S-
[2-[2(R),3-dihydroxypropyl]-
(R)-cysteine benzhydryl ester, melting point
Colorless crystals at 95-97℃; N-lauroyl-S-[2
(R),3-dihydroxypropyl]-(R)-cysteine benzhydryl ester, colorless crystals, melting point 95-97°C. In the same manner as above, the following N-amyl-S-
[2(R),3-diacyloxypropyl]-(R)-
Cysteine benzhydryl ester is obtained:
N-Lauroyl-S-[2(R),3-dipalmitoyloxypropyl]-(R)-cysteine benzhydryl ester, melting point 65-68°C; Palmitoyloxypropyl]-(R)-cysteine benzhydryl ester, melting point 68-70°C; N-stearoyl-S-
[2(R),3-dipalmitoyloxypropyl]-
(R)-cysteine benzhydryl ester, melting point
70-72℃. Other compounds which can be made as described above and are used as intermediates are N-lauroyl-S-[2(R),3-dipalmitoyloxypropyl]-(R)-cysteine, melting point 70-72 N-Myristoyl-S-[2(R),3-dipalmitoyloxypropyl]-(R)-cysteine, colorless crystals with a melting point of 71-73°C; N-stearoyl-S-[2(R) ), 3-dipalmitoyloxypropyl]-(R)-cysteine, colorless crystals with a melting point of 74-77°C. Example 6 The reaction described in Example 5 produces the following raw material for producing the lipopeptides of the invention: N-palmitoyl-S-[2(R),3-dilauroyloxypropyl]-(R) -Cysteine benzhydryl ester, non-waxy substance; N-palmitoyl-
S-[2R,3-distearoyloxypropyl-
R-cysteine benzhydryl ester, melting point 80
Colorless crystals at ~82°C; N-palmitoyl-S-[2
(R),3-dioleoyloxypropyl]-(R)
-Cysteine benzhydryl ester, colorless oil; N-palmitoyl-S-[2(R),3-dibehenoyloxypropyl]-(R)-cysteine benzhydryl ester, colorless crystals with a melting point of 85-88°C; N -Palmitoyl-S-[2(R),3-di-
(dihydrosterculoyloxypropyl)]-
(R)-Cysteine benzhydryl ester, colorless wax. Corresponding substituted isteines that are not esterified are, for example: N-palmitoyl-
S-(2(R),3-dilauroyloxypropyl)
-(R)-cysteine, colorless oil, Rf=0.27; N
-Palmitoyl-S-[2(R),3-disaroyloxypropyl]-R-cysteine, melting point 82~
Colorless crystals at 85°C; N-palmitoyl-S-[2R,
Rf = 3-dioleoyloxypropyl]-(R)-cysteine, colorless oil, chloroform:methanol = 9:1 (thin layer - silicate gel - Merck)
0.35; N-palmitoyl-S-[2(R),3-dibehenoyloxypropyl]-(R)-cysteine - colorless crystals with melting point of 87-90°C; N-palmitoyl-
S-[2(R),3-(dihydrosterculoyloxypropyl)]-(R)-cysteine, colorless waxy substance, Rf = 0.38 (same conditions as N-palmitoyl-2,3-oleoyloxy derivative) . These cysteine derivatives are condensed with the following peptides as described in Example 5: H-Phe-
Phe−Asn−Ala−Lya−OH, H−Glu−Glu−
Asn−Ala−Lys−OH, H−Ser−Ser−Asn−
Ala−Glu−OH. The corresponding lipopeptide of the invention is thus obtained. Example 7 The raw material compound for producing the compound described in Example 8 can be produced as follows. N-palmitoyl-S-2(R),3(R),4-
0.9 g of tripalmitoyloxybutyl-(R)-cysteine benzhydryl ester is left in a mixture of 3 ml of trifluoroacetic acid and 12 ml of methylene chloride for 2 hours at room temperature. Then evaporate to dryness, mash the residue with ice water, filter with suction, and dry the solid.
It is then extracted with benzene and recrystallized from acetone. N-palmitoyl-S-2 with melting point 76-76.5℃
Colorless crystals of (R),3(R),4-tripalmitoyloxybutyl-(R)-cysteine are obtained. Specific optical rotation [α] ²⁰ _D = -5° (c = 0.819, dioxane). Benzhydryl ester used as a raw material is obtained as follows. N-Palmitoyl-S-2(R), 3(R). 4-
1 g of trihydroxybutyl-(R)-cysteine benzhydryl ester was mixed with palmitic acid chloride in a mixture of 6 ml of pyridine and 5 ml of methylene chloride.
Acylate with 1.83ml. After 18 hours at room temperature 1 ml of methanol is added and evaporated to dryness. The residue is filtered through 100 g of silicic acid (Merck) with chloroform as solvent. The pure portion by thin layer chromatography (CHCl ₃ :acetic acid ester = 98.2) gives colorless crystals after evaporation with a melting point of 60-62°C. The raw material for the above reaction is obtained as follows: N-palmitoyl-S-2(R),3(R),4-
Trihydroxybutyl-(R)-cysteine 2.53g
is reacted with 1.37 g of diphenyldiazomethane in a mixture of 12 ml of ethanol and 32 ml of chloroform at room temperature for 15 hours. Evaporate to dryness and purify the corresponding benzhydryl ester with 150 g of silicic acid gel using chloroform as solvent. Rf=0.3 ( _CHCl3 :acetone=95:5, silicic acid gel (Merck), thin layer). N-palmitoyl-S-2(R),3(R),4-
Trihydroxybutyl-(R)-cysteine is obtained as follows: 20 g of N-palmitoylcysteine, 27.6 g of 1-tosyl-2,4-diethylidene-D-erythrite.
g and 17 g of caustic potash are stirred in 240 ml of ethanol at 80° C. for 15 hours in a nitrogen atmosphere. The salts are then filtered off and the filtrate is evaporated to dryness. Residue _H2O :
Dissolved in 200ml of tetrahydrofuran (1:1),
Make acidic to PH=3 with 2N-hydrochloric acid. Next, shake with ether three times and adjust the pH of the ether phase with pyridine.
4.5, shake with water, dry the ether phase,
Evaporate to dryness. This residue is silicic acid gel (Merck)
200g with chloroform:acetone=8:2(1) as eluent, then _CHCl3 :MeOH
Purify by =6:4 (1.2). The pure portion is evaporated, triturated with hexane and recrystallized from acetate-petroleum ether. N-Palmitoyl-S- with melting point 62-65℃
[(2,4-ethylidene)-erythrityl]-(R)-
Colorless crystals of cysteine are obtained. Hydrolysis of this compound was carried out at 80°C with 3% _HBF4 .
It will take 4.5 hours. 8.2 g of ethylidene derivative and 100 g of dimethoxyethane
Treat with 100 ml of 3% HBF ₄ in ml. Then, upon cooling to 60°C, the hydrolysis products precipitate. After suction filtration and drying, it is recrystallized from acetic acid ester. Melting point 160-165℃, sintering from 93℃, Rf=0.285
(CHCl ₃ :MeOH=6:4, thin layer, silicic acid gel (Merck). Example 8 N-stearoyl-S was prepared as described in Example 5.
-[2(R),3-dipalmitoyloxypropyl]
-(R)-cysteine and N-palmitoyl-S
-[2(R),3(R),4-tripalmitoyloxybutyl]-(R)-cysteine produces the following lipopeptide: N-stearoyl-S-[3
(R),3-dipalmitoyloxypropyl]-
Cys−Ser−Ser−Asn−Ala−Glu−Ile−Asp−
Glu-OH, Rf (157) = 0.32: N-palmitoyl-
S-[2(R),3(R),4-tripalmitoyloxybutyl]Cys-Ser-Ser-Asn-Ala-Lys-
OH, Rf (157) = 0.55; N-palmitoyl-S-
[2(R),3(R),4-tripalmitoyloxybutyl]-Cys-Ser-Ser-Asn-Ala-Lys-Ile
-Asp-Glu-OH, Rf(157) = 0.38.

Claims (1)

[Claims] 1. Next, the general formula (): [In the formula, R ₁ is an alkyl group having 13 to 17 carbon atoms; R ₂ is an alkyl group having 11 to 17 carbon atoms; R ₃ is hydrogen or a group R ₁ -CO-O- CH ₂
and X has the following formula: ( ₁ ) -L-Ser-L-Ser; (2) -L-Ser-L-Ser-L-Asn; 3) -L-Ser-L-Ser-L-Asn-L-Ala; (4) -X ₁ -X ₂ -X ₃ -L-Ala-X ₅ (wherein, X ₁ is L-Ser, L -Phe or L-
Glu; X ₂ is L-Ser, L-Phe or L-
_{(5 )} -L-Ser-L-Ser-L-Asn-L- Ala-
X ₅ ′-L-Ile-L-Asp-L-Glu (wherein, X ₅ ′ is L-Glu or L-Lys); compounds in substantially pure form, and their salts. 2. The compound according to claim 1, wherein R ₁ CO- is a palmitoyl group, and a salt thereof. Claim 1 in which 3 R ₂ CO- is a palmitoyl group, myristoyl group, or lauroyl group
Compounds described in Section 1, and salts thereof. 4. The compound according to any one of claims 1 to 3, wherein the acyl groups R ₁ CO and R ₂ CO are the same. 5. The compound according to any one of claims 1 to 3, wherein the acyl groups R ₁ CO and R ₂ CO are different from each other. 6 X is the following amino acid sequence, i.e. -Ser-Ser-Asn-Ala-Lys-Ile-Asp-Glu
−OH −Ser−Ser−Asn−Ala−Lys−OH −Ser−Ser−Asn−Ala−OH −Ser−Ser−Asn−OH −Ser−Ser−OH −Ser−Ser−Asn−Ala−Glu−Ile −Asp−Glu
−OH −Ser−Ser−Asn−Ala−Glu−OH −Ser−Ser−phe−Ala−Glu−OH Glu−Gln−Asn−Ala−Lys−OH −Phe−Phe−Asn−Ala−Lys−OH The compound according to any one of claims 1 to 5, which is any one of claims 1 to 5. 7. The compound according to any one of claims 1 to 6, wherein _R3 is hydrogen, and a salt thereof. 8. A compound according to any one of claims 1 to 7, having the (R)-configuration at the C ^** atom, and a salt thereof. 9 Lipopeptide [N-palmitoyl-S-2
(R), 3-dipalmitoyloxypropyl]-
The compound according to claim 1, which is Gys-Ser-Ser-Asn-OH. 10 Lipopeptide [N-palmitoyl-S-2
(R), 3-dipalmitoyloxypropyl]-
The compound according to claim 1, which is Cys-Ser-Ser-Ala-OH. 11 Lipopeptide N-palmitoyl-S-[2
(R), 3-dipalmitoyloxypropyl]-
The compound according to claim 1, which is Cys-Ser-Ser-Phe-Ala-Glu-OH. 12 Lipopeptide N-palmitoyl-S-[2
(R,S), 3-dipalmitoyloxypropyl]
The compound according to claim 1, which is -Cys-Phe-Phe-Asn-Ala-Lys-OH. 13 Lipopeptide N-myristoyl-S-[2
(R), 3-dipalmitoyloxypropyl]-
The compound according to claim 1, which is Cys-Glu-Gln+Asn-Ala-Lys-OH. 14 Lipopeptide N-lauroyl-S-[2
(R),3-dipalmitoyloxypropyl]-
The compound according to claim 1, which is Cys-Ser-Ser-Asn-Ala-Glu-OH. 15 Lipopeptide N-stearoyl-S-[2
(R),3-dipalmitoyloxypropyl]-
Cys−Ser−Ser−Asn−Ala−Glu−Ile−Asp−
The compound according to claim 1, which is Glu-OH. 16 Lipopeptide N-palmitoyl-S-[2
The compound according to claim 1, which is (R),3(R),4-tripalmitoyloxybutyl]-Cys-Ser-Ser-Asn-Ala-Lys-OH. 17 Lipopeptide N-palmitoyl-S-[2
(R),3(R),4-tripalmitoyloxybutyl]-Cys-Ser-Ser-Asn-Ala-Lys-Ile-
The compound according to claim 1, which is Asp-Glu-OH. 18 Lipopeptide N-palmitoyl-S-[2
(R),3-dipalmitoyloxypropyl]-
Claim 1 which is Cys-Ser-Ser-OH
Compounds described in Section. 19. An ammonium salt, alkali metal salt or alkaline earth metal salt of the acidic compound according to any one of claims 1 to 18. 20. A non-toxic acid addition salt of the basic compound according to any one of claims 1 to 18 that can be used as a medicine. 21 The following general formula (), [wherein R ₁ is an alkyl group having 15 carbon atoms; R ₂ is an alkyl group having 15 carbon atoms; R ₃ is hydrogen; and X is of the following formula: (1) - L-Ser-L-Ser; (2) -L-Ser-L-Ser-L-Asn; (3) -L-Ser-L-Ser-L-Asn-L-Ala; (4) -L- Ser-L-Ser-L-Phe-L-Ala-
L-Glu; or a pharmaceutically acceptable salt thereof in substantially pure form, together with a pharmaceutical carrier.