CS245797B2 - Production method of muramylpeptides - Google Patents

Description

The invention relates to a process for the preparation of novel muramyl peptides of the general formula I

(DJ (L) O l

OH

II

O R 11 i

C _x

O ⁴ 0R ^S ti

RŽ

C-O-CH

CH, 1 (dream I ⁴ 0 in which

R ² represents an alkyl group having 1 to 4 carbon atoms, hydroxymethyl or phenyl,

R ⁵ represents hydrogen or an alkyl group having 1 to 3 carbon atoms, 10 β ^ιυ 0

R ³ represents hydrogen or methyl,

R ⁷ is an optionally substituted (C 1 -C 3) alkyl-substituted alkyl group,

R ⁸ is hydrogen or C 1 -C 4 alkyl,

X is oxygen or NH,

Y represents a C 1 -C 4 alkylidene group and

R ⁹ and R ¹⁰ are independently C 11 -C 17 alkyl or C 11 -C 17 alkenyl, as well as salts of these compounds, which have valuable pharmacological properties and can be used as active pharmaceutical ingredients.

The compounds of formula I have, in the case of asymmetric substitution, on the CR ³ (Dj-configuration, on the C-R ⁷ (L) -configuration, on the C-C-OR ⁸ (Dj-configuration, on the remainder Y (Lj- The C-1 D-glucose residue and / or the β-configuration, i.e. the compounds of formula I may exist as pure anomers or as mixtures of anomers.

The prefix “sn” is used to distinguish the configuration at the mid-carbon atom of the glycerol moiety according to IUPAC rules in accordance with the stereospecific numbering of the carbon atoms of the glycerin moiety. The uppermost carbon atom in the Fischer projection of the vertical carbon chain, with the hydroxyl group at the 2-carbon atom on the left, receives the number 1. In the compounds of the invention, the phosphoric acid residue is bound to the 3-carbon atom in stereospecific numbering.

The term " lower " as used herein refers to radicals having 1 to 7 carbon atoms, in particular 1 to 4 carbon atoms.

Lower alkanoyl means, for example, propionyl, butyryl or hexanoyl, in particular acetyl.

Lower alkyl is, for example, n-propyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl or n-heptyl, especially methyl, ethyl or isopropyl.

In each case, it is an alkyl radical R @ ² , especially methyl when R @ ³ is methyl, or ethyl when R @ ³ is hydrogen; an alkyl radical R ⁵ is especially methyl, an alkyl radical R ⁷ is especially methyl, ethyl or isopropyl and the alkyl radical R ⁸ is especially an alkyl radical of 1 to 4 carbon atoms, in particular a straight alkyl radical of 1 to 4 carbon atoms, in particular n- butyl.

An alkyl group having 1 to 3 carbon atoms which is substituted by a hydroxy group, in the meaning of R ^7, is in particular a hydroxymethyl group or a 1-hydroxyethyl group.

The alkylidene group of Y is a divalent radical in which both bonds start from the same carbon atom, preferably a radical bonded at the 1,1-position, such as, in particular, a methylene or ethylidene radical, a propylidene radical or an isobutylidene radical.

The alkyl group having from 11 to 17 carbon atoms in the meaning of R ⁹ or R ¹⁰ is a straight or branched, in particular a straight alkyl group, in particular a straight alkyl group with an odd number of carbon atoms, such as n-undecyl or n-tridecyl, especially a straight alkyl group having 15 to 17 carbon atoms with an odd number of carbon atoms, i.e. an n-heptadecyl group or, in particular, an n-pentadecyl group.

The C 11 -C 17 alkenyl group represented by R ⁹ or R ¹⁰ is in particular a hepta-8 (Z) -decenyl group.

The proton bonded via an oxygen atom to a phosphorus atom is acidic and can be readily cleaved by a base and replaced with another cation. Thus, at a pH of 7, the compounds of formula I are present wholly or mainly in the form of a salt. These salts as well as salt-acid mixtures are also within the scope of the invention. In particular, the invention relates to pharmaceutically usable, non-toxic salts of the compounds of formula I. They are in particular metal salts or ammonium salts, such as alkali metal or alkaline earth metal salts, for example sodium, potassium, magnesium or calcium salts, as well as ammonium salts. or salts with suitable organic amines, such as lower alkylamines, for example triethylamine. Pharmaceutically unsuitable salts may also be used for isolation or purification. However, only pharmaceutically usable, non-toxic salts are used for therapeutic purposes and are therefore preferred.

Compared to the compounds previously described, the compounds of the invention have greatly reduced undesirable side effects, in particular they are much less pyrogenic, i.e. practically non-pyirogenic at all. This finding is of great importance since the administration of the pyrogenic substance may cause a thermal shock under circumstances, so that safe use is ensured only under constant medical supervision and the use of certain dosage forms such as intravenous administration is out of the question.

Pyrogenicity testing may be carried out according to the protocol of Europaische Pharmakopoe, Vol. 2, pp. 56-59 (1971) on rabbits. For example, even by subcutaneous administration of a high dose such as 30 mg / kg N-acetylmuramyl-L-alanyl-D-glutamyl- (C _and -n-butyl ester-Cy) -L-alanine-2- ( 1,2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxyjethylamide (compound IJ shows no pyrogenic effect).

In contrast, the same dose of sodium salt is used

N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1,2-dipalmitoyl-sn-glycero-35)

-hydroxyphosphoryloxyjethylamide strongly pyrogenic.

The compounds produced by the process of the invention may be used as described in European Patent Application No. 0 025 495.

However, by virtue of their excellent tolerability, the compounds produced according to the invention are particularly suitable for the prophylaxis and therapy of infectious diseases, with a strong effect not only in the case of agents of bacterial diseases but also surprisingly in the case of agents of viral diseases. The latter finding has not been described so far and is of great practical importance since pharmaceuticals for the treatment of viral diseases have so far only been insufficiently available. Of particular note is the long prophylactic or therapeutic duration of action from a few days to several weeks after a single administration of the compounds produced according to the invention, as well as a wide range of causative agents of the viral diseases against which the compounds are effective.

Muramyl peptides of the formula I can be used, in particular, for the prophylaxis and therapy of diseases which are caused by the viruses described below [for nomenclature, cf. J.L. Melnick, Prog. copper. Virol., 28, 214-232 (1980) and 28, 208-221 (1982)]:

DNA viruses with cubic symmetry and non-enveloped nucleocapsid,

DNA viruses with enveloped virion, as well as

RNA viruses with cubic symmetry a

RNA viruses with helicoidelid symmetry of the capsid.

Preferably, the compounds of the formula I are used in the case of DNA viruses with enveloped virion and cubic symmetry of the capsid, in the case of RNA viruses with cubic symmetry of capsid with the non-enveloped virion in RNA viruses with helicoidal symmetry of the capsid. Adenoviridae, Poxviridae and Coronaviridae, as in particular in the case of human coronaviruses.

In particular, the compounds of the formula I are used in the Herpesviridae, Picornaviridae and myxoviruses, but also in the case of mastadenoviruses, in particular in the case of human adenoviruses, in the case of chordopoxviruses, in particular orthopoxviruses, in particular rotaviruses, as well as in the case of Calicivlridae and Rhabfloviridae, in particular vesicular viruses of humans as well as horses. cattle and pigs.

In particular, the compounds of the formula I are used in the case of α-herpesviruses, rhinoviruses, cardioviruses and Orthomyviridae, but also in the case of β-herpesviruses, in particular human cytomegaloviruses, in the case of afthoviruses, in particular afthoviruses. in particular pneumoviruses, for example human respiratory syncitialviruses, and, in addition, mcrbilliviruses or paramyxoviruses, such as human parainfluenzaviruses including sendaiviruses.

In particular, the compounds of formula I produced by the process according to the invention are used in the case of simplexviruses, for example human Herpes simplex viruses of types 1 and 2, furthermore in the case of human encephalomyocarditis viruses and Influenza viruses such as Influenza A and Influenza B virus, e.g. the viruses mentioned in the examples.

Muramyl peptides of formula I may be used for the prophylaxis or therapy of viral infections by administering enterally or parenterally, in particular together with suitable excipients or carriers. They are preferably applied to the mucosa, for example, intranasally, rectally, vaginally, or to the conjunctiva, or orally. However, the antiviral effect also occurs when administered by other means, for example subcutaneously, intravenously, intramuscularly or when applied to normal skin.

The most advantageous method of administration depends inter alia on the type of virus concerned. For example, in the case of respiratory viruses, intranasal administration is preferred in many cases. The dosage of the active ingredient depends, inter alia, on the species of warm-blooded animal, on the body's defense capabilities, on the mode of administration and on the type of virus.

The compounds produced by the process of the invention have another very interesting property of inhibiting metastasis formation in some tumors, particularly lung tumors, as demonstrated experimentally in the B16-BL6 melanoma model and in Lewis lung cancer, with liposome administration being particularly preferred.

In particular, the invention relates to a process for the preparation of compounds of the formula I in which R ² represents a C 1 -C 2 alkyl or hydroxymethyl group, R ³ represents hydrogen or a methyl group, R ⁵ represents hydrogen or a methyl group, R 5 represents an alkyl group C 1 -C 3 or hydroxymethyl. R ³ is hydrogen or C 1 -C 4 alkyl; X is oxygen or a group. NI-I. Y represents an alkylidene group having 1 to 3 carbon atoms and R ⁹ and R ¹⁰ independently represent a direct alkyl. a group having 11 and 17 carbon atoms or a desarbonyloleoyl group, and salts thereof.

In particular, the invention relates to a process for the preparation of compounds of the formula I in which R ² is methyl or ethyl, R ³ is hydrogen or methyl, R ⁵ is hydrogen or methyl, R ⁷ is C 1 -C 3 alkyl , R ⁸ is hydrogen or C 1 -C 4 alkyl, X is oxygen or NH, Y is C 1 -C 2 alkylidene, and R ⁹ and R ¹⁰ are independently C 11 -C 17 alkyl; an odd number of carbon atoms or a desarbonyl oloyl group, and salts thereof.

In particular, the invention relates to a process for the preparation of the aforementioned compounds of the general formula I, in which R ⁸ represents any of said alkyl radicals, and salts thereof.

In particular, the invention relates to a process for the preparation of compounds of formula I wherein R ² is methyl or ethyl, R ³ is hydrogen when R ² is ethyl, or is methyl when R ² is methyl, R ⁵ is hydrogen or methyl, R ⁷ is C 1 -C 2 alkyl, R ⁸ is C 1 -C 4 alkyl, X is oxygen or NH, Y is ethylidene and R ⁹ and R ⁴⁰ are independently each having a straight alkyl group having 15 to 17 carbon atoms with an odd number of carbon atoms and salts thereof.

In particular, the invention relates to a process for the preparation of the aforementioned compounds of formula I wherein R ³ is methyl, R ⁵ is hydrogen, R ⁸ is n-butyl, X is NH and / or R ⁹ as well as R ¹⁰ are n- pentadecyl group, and salts thereof, as well as the compounds of formula I exemplified.

The novel compounds of the formula I and their salts are prepared according to the invention by the production of a compound of the formula Ia

(L) fl 0 «

X-γ-C-NH-CH 3 CH 3 O-P-O-CH 3.

θ? ' ¹

In-O-CH $ CH?

O

I in which

R ¹ , R ⁴ and R ® are hydrogen, and the other symbols have the same meaning as in formula I, with at least one of R ¹ , R ² , R ⁴ , R ⁸ , R ⁷ , R ³ or a hydroxyl group on the The phosphorus atom is present in a protected form, cleaves the protecting group or protecting groups, whereupon the compound of the formula I obtained is optionally converted into a salt or the salt obtained is converted into another salt.

Protective groups, their introduction and cleavage are described, for example, in "Protective Groups in Órganic Chemístry", Plenum Press London, New York 1973, and in "Methoden der organischen Chemie", Houben-Weyl, 4th edition, Vol. 15/1, Georg-Thieme Verlag, Stuttgart 1974. Characteristic of protecting groups is that they are readily cleaved (i.e., without undesirable side reactions);

solvolytically, reductively, photolytically or also under physiological conditions.

Protecting groups of the liydroxy group are, for example, acyl groups, such as, for example, halogen-substituted lower alkanoyl, such as 2,2-dichloroacetyl, or acyl radicals of carbonic acid semi-esters, especially tert-butyloxycarbonyl, optionally substituted benzyloxycarbonyl, for example 4-nitrobenzyloxy or carbonyl; diphenylmethoxycarbonyl, or 2-halo (lower) alkoxycarbonyl, such as 2,2,2-trichloroethoxycarbonyl, further trityl or formyl, or organic silyl or stannyl groups, further readily cleavable etherifying groups such as target.

(lower alkyl), for example tert-butyl, 2-oxa- or 2-thiaaliphatic or -cycloaliphatic hydrocarbon radicals, in particular 1- (lower) alkoxy (lower) alkyl or 1- (lower) alkylthio (lower) alkyl, for example methoxymethyl, 1-methoxyethyl, 1-ethoxyethyl, 1-methylthiomethyl, 1-methylthioethyl or 1-ethylthioethyl, or 2-oxa- or 2-thiocycloalkyl of 5 to 6 ring atoms, for example tetrahydrofuryl or 2-tetrahydropyranyl or the corresponding thiaanogens, as well as optionally substituted 1-phenyl (lower alkyl, such as optionally substituted benzyl or diphenylmethyl), such as phenyl substituents being, for example, halogen such as chlorine, lower alkoxy such as methoxy and / or nitro.

The acid group of the phosphoric acid may be protected in esterified form, with those preferred as esterifying moieties which may be regloselectively cleaved, for example a methyl ester group which may be cleaved for example by alkaline saponification, or in particular hydrogenolytically cleavable groups, for example benzyl or phenylester groups wherein the benzyl ester groups can be cleaved, for example, in the presence of palladium catalysts such as palladium. the carbon and phenyl ester groups can be cleaved, for example, in the presence of platinum catalysts or mixed catalysts containing pileitin and palladium.

In particular, divalent hydroxy protecting groups are optionally substituted alkylidene or cycloalkylidene groups. In particular, the alkylidene group is a lower alkylidene group such as isopropylidene and the cycloalkylldene group is preferably a cyclopentylidene or cyclohexylidene group. The substituents of the alkylidene radicals are, in particular, aromatic radicals, for example phenyl radicals.

The cleavage of the protecting groups mentioned in the description of the invention is accomplished in a manner known per se by solvolysis, in particular by hydrolysis, alcoholysis or acidolysis, or in some cases also by careful reduction, optionally sequentially or simultaneously. The silyl protecting groups are preferably removed by treatment with fluorides, for example tetraethylammonium fluoride.

The cleavage of the aforementioned divalent hydroxyl protecting groups is carried out, for example, with dilute acid, preferably at pH 2-4, e.g. at pH 3 in a manner known per se, for example by an acid ion exchanger, in particular an ion exchanger with sulfonic acid residues such as Amberlite IR-120 (styrene resin). with strongly acidic sulfo groups) or Dowex 50 (polystyrenesulfonic acid) or by treatment with a strong inorganic or organic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid or sulfonic acid, for example methanesulfonic acid, or with phenylsulfonic acid optionally substituted in the aromatic ring , such as p-toluenesulfonic acid, or trifluoroacetic acid.

The starting materials required for carrying out the above processes are known or can be prepared by methods known per se.

The salts of the compounds of the formula I are generally formed already during processing, for example by dialysis against a buffer solution having a pH of 7. These salts can be prepared in a manner known per se. Thus, salts of the compounds of formula I can be prepared, for example, by reaction with metal compounds, such as alkali metal salts of suitable organic carboxylic acids, for example sodium α-ethylcaproic acid, or with inorganic alkali or alkaline earth metal compounds such as corresponding hydroxides, carbonates or bicarbonates, such as sodium or potassium hydroxide, carbonate or bicarbonate, or with the appropriate calcium or ammonia compounds or with suitable organic amines, preferably using stoichiometric amounts or a small excess of the salt-forming agent.

Mixtures of isomers may be separated in a manner known per se, for example, by fractional crystallization, chromatography, etc., into the individual isomers, racemates, for example to form derivatives with optically active compounds and the separation of the diastereomer mixtures thus obtained into optically active alpha-lipioids.

The methods described above, including the processes for the deprotection and additional measures of these methods, are carried out in a manner known per se, for example in the presence or absence of solvents and diluents and, if necessary, in the presence of condensing agents or catalysts, at reduced or elevated temperatures. , for example in a temperature range of about -20 to 150 ° C, in a closed vessel and / or under an inert gas atmosphere, for example under a nitrogen atmosphere.

Taking into account all substituents present in the molecule, the work is carried out, if necessary, for example in the presence of readily hydrolyzable residues, in particular under gentle reaction conditions such as short reaction times, using mildly acidic basic agents at low concentrations using stoichiometric relative ratios, as well as the choice of suitable catalysts, solvents, temperature and / or pressure conditions.

The invention also relates to those process embodiments wherein the starting material is a compound obtained as an intermediate at any stage of the process and the missing process steps or the process is interrupted at any stage, or the starting material is formed under the reaction conditions or used. in the form of a reactive derivative or a salt. The starting materials are preferably those which give the compounds of the invention which have been described as being particularly valuable.

The compounds produced according to the invention may be used in the form of pharmaceutical preparations containing a pharmacologically effective amount of the active ingredient, optionally together with pharmaceutically acceptable carriers which are suitable for enteral, for example oral or rectal, or parenteral administration, and which may be of inorganic or organic origin. in solid or liquid state. Thus, tablets or gelatin capsules are used which contain the active ingredient together with diluents, for example lactose, dextrose, sucrose, mammoth, sorbitol, cellulose and / or glycerine, and / or lubricating agents, for example diatomaceous earth, talc, stearic acid or the like. salts thereof, such as the magnesium or calcium salt of stearic acid, and / or with polyethylene glycol. The tablets may also contain binding agents, for example magnesium aluminum silicate, starches such as corn starch, wheat starch or rice starch, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone and optionally disintegrating agents, such as starches, agar, alginic acid, or a salt thereof, such as sodium alginate and / or effervescent mixtures, or adsorbents, colorants, flavors, and sweeteners. It is also possible to use the pharmacologically active compounds produced according to the invention in the form of parenterally administrable preparations or in the form of infusion solutions. Such solutions are preferably isotonic aqueous solutions or suspensions, which preparations may be prepared prior to use, as is the case, for example, with lyophilized preparations which contain the active substance alone or together with a carrier, for example mannitol. The pharmaceutical preparations may be sterilized and / or contain adjuvants, for example preservatives, stabilizers, wetting agents and / or emulsifiers, co-solvents, salts for regulating the osmotic pressure and / or buffers. Said pharmaceutical preparations which, if desired, may contain other pharmacologically active substances, such as antibiotics, are prepared in a manner known per se, for example by conventional mixing, granulating, dragee, dissolution or lyophilization processes and contain about 0.001 to 99%, in particular 0, The active compound (s) are preferably present at a concentration of from 1% to 10%, in particular from 0.1% to 5%, with a concentration of active substance below 1% being particularly suitable for topical preparations.

The following locally applicable dosage forms are particularly suitable for the prophylaxis and therapy of viral infections:

creams, ointments or pastes having an active ingredient content of from 0.001 to 1%, in particular 0.01 to 0.1%, for example 0.05%, for example ointments for intranasal application or lipstick, or aqueous solutions containing an active ingredient content of 0.001 up to 1%, in particular 0.05 to 0.5%, for example 0.1 percent, preferably isotonic, sterile and physiologically usable solutions, for example eye drops, preferably in microspheres for jelly use, or sprays for oral and pharyngeal cavities.

Particularly suitable are the pharmaceutical compositions described in the Examples.

Creams are represented by oil-in-water emulsions containing more than 50% water. The oily base used is preferably a fatty alcohol such as lauryl alcohol, cetyl alcohol or stearyl alcohol, fatty acids such as palmltic acid or stearic acid, liquid to solid waxes such as isopropyl myristate, sheep's wax or beeswax and / or hydrocarbons such as petrolatum (paraffin oil) or paraffin oil. Suitable emulsifiers are surfactants having predominantly hydrophilic properties, such as the corresponding non-ionic emulsifiers, for example fatty acid esters of polyalcohols or their adducts with ethylene oxide, such as fatty acid esters of polyglycerine or fatty acid esters of polyoxyethylene sorbitan (Tweensj, hereinafter polyoxyethylene ethers of fatty acids). alcohols or polyoxyethylene esters of fatty acids, or the corresponding ionic emulsifiers, such as alkali metal salts of sulfated fatty alcohols, for example natriumauryl sulphate, sodium trimethylsulphate or sodium stearyl sulphate, which are usually used in the presence of fatty alcohols, for example cetyl alcohol or stearyl alcohol. which prevent the drying of creams, for example polyalcohol, such as glycerin, sorbitol, propylene glycol and / or polyethylene glycols, furthermore preservatives, fragrances; etc.

Ointments are water-in-oil emulsions containing up to 70%, preferably from about 20 to 50%, water or aqueous phase. Suitable fatty phases are, in particular, hydrocarbons, for example petroleum jelly, paraffin oil and / or hard paraffin, and the fatty phase preferably contains suitable hydroxy compounds, such as fatty alcohols or esters thereof, for example cetyl alcohol or sheep's wax alcohols to improve water binding capacity. or sheep wool wax. The emulsifiers are represented by corresponding lipophilic substances such as a fatty acid ester of sorbitan (Spans), for example sorbitan oleate and / or sorbitan isostearate. For example, moisture-retaining agents such as polyalcohols such as glycerine, propylene glycol, sorbitol and / or polyethylene glycol, preservatives, fragrances, etc. are used as aqueous phase additions.

Ointments with a fatty component are anhydrous and contain, in particular, hydrocarbons such as paraffin, all and / or liquid paraffins, natural or semi-synthetic fats such as coconut fat triglyeeride or preferably a hardened oil such as hydrogenated peanut oil or castor oil, and partial esters glycerol with fatty acids, for example glycerol monostearate and glycerine distearate, as well as, for example, in relation to ointments said fatty alcohols which increase the ability to absorb water, emulsifiers and / or additives.

Pastes are creams and ointments with secretion-absorbing powder components, such as metal oxides, for example titanium dioxide or zinc oxide, as well as talc and / or aluminum silicates, which are intended to bind the moisture or secretions present.

Foams are applied from pressure vessels and are liquid oil in water emulsions present in aerosol form using halogenated hydrocarbons such as chlorofluoride (lower alkanes such as dichlorodifluoromethane and dichlorotetrafluoroethane as propellant). For example, paraffin oil, fatty alcohols such as cetyl alcohol, fatty acid esters such as isopropyl myristate and / or other waxes Emulsifiers used include, but are not limited to, mixtures of emulsifiers with predominantly hydrophilic properties such as polyoxyethylene esters of fatty acid and sorbitan (TweensJ) and properties such as sorbitan fatty acid esters (Spans). In addition, conventional additives such as preservatives, etc. are added.

Tinctures and solutions usually contain an aqueous-ethanolic base to which, inter alia, polyalcohols, such as glycerin, glycols and / or polyethylene glycol, are added as moisture evaporation reducing agents and fat returning agents, such as fatty acid esters of lower polyethylene glycols, e.g. water-soluble, lipophilic substances as a replacement for fatty substances that have been removed from the skin with ethanol, and optionally other excipients and additives.

For the preparation of topical pharmaceutical preparations, the process is carried out in a manner known per se, for example by dissolving or suspending the active substance in the base substance or part thereof, if necessary. When the active ingredient is applied in the form of a solution, the active ingredient is usually dissolved in either of these two phases prior to emulsification. When applied as a suspension, the active ingredient is admixed after emulsification with a portion of the base and then the remainder of the formulation is added.

The dosage of the active ingredient depends on various factors such as the mode of administration, the species, the defensive capacity of the organism and, to a large extent, the nature of the disease being treated. Thus, daily dosages for oral administration with a warm-blood weight of about 70 kg range from 0.0001 to 0.1 g, with doses less than 0.001 g being used primarily to prevent the formation of metastasis after removal of the primary tumor.

For the prophylaxis of viral infections, a single dose of about 0.5 mg to 50 mg, preferably 25 to 15 mg, for example 7 mg, of an active substance to a warm-blooded animal of about 70 kg, for example to humans, is administered. For times of increased risk of infection, the dose is repeated at intervals of 1 to 3 weeks, for example 2 weeks.

The therapeutic dose for warm-blooded bodies of about 70 kg in the case of a viral infection is in the range of 1 mg to 50 milligrams, preferably 5 to 20 mg, for example 10 mg, especially when administered orally. The intranasal dose is reduced by a factor of 10. If necessary, the administration of the hepoxypyranose derivatives of formula I is repeated at intervals of several days, for example 1 to 3 days, until the onset of the disease.

The following examples illustrate the invention in more detail without limiting its scope. R _f values are determined on silica gel plates for thin layer chromatography. The ratio of solvents in the solvent mixture used is expressed in parts by volume.

Example 1

1.0 g (0.7 mmol) of the sodium salt of 4,6-O-isopropylidene-N-propionyldesmethylmuramyl-L-alanyl-D-glutamyl- (C 1 H-n-butyl ester- (Cy 1 -L-alanine-2) - (1,2-Dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) ethylamide, which still contains 0.8 moles of water, is stirred in 10 ml of 50% acetic acid at room temperature for 2 hours, then 40 ml of water is added and The crude product is then purified by column chromatography on 50 g of silica gel (type 60, pure, particle size 0.063-0.200 mm) using a 70: 30: 2 to 5 mixture of chloroform, methanol and water as eluent. The fractions containing the desired product were combined and evaporated in vacuo at 30 ° C.

The residue is taken up in 100 ml of double-distilled water, the solution is filtered through a milliporous filter (0.2 gm) and the solution thus obtained is again lyophilized.

0.91 g (86.8% of theory) of pure N-propionyldesmethylmuramyl-L-alanyl-D-glutamyl- (C _and N-n-butyl ester-Cyj-L-aloginine-2- ( l, 2-d _i iipia | lmltio _{tolyl-an.-glycero-pyrrolo-3-hydroxyphosphoryloxy)} ethylamide as a colorless powder, which still contained 4.41 mol of water.

[α] ₀ = ^2θ + 6.9 ° (c = 0.504, methylene chloride j.

R _f = 0.65 (solvent system: a mixture of chloroform chlo245797 methanol: water = 70: 30: 5) the -0.63 (solvent system: chloroform: methanol = 7: 3).

The starting material is obtained as follows:

13.0 g of 4,6-O-isopropylidene-N-propionyldesmethylmuramyl-L-alanyl-D-glutamyl- (C _H ) -n-butyl- (C) -benzyl ester are catalytically hydrogenated in a mixture of dimethoxyethane and water in a ratio of 20 1 in the presence of 2.5 g of palladium on carbon (10% palladium) as catalyst at room temperature and atmospheric pressure. After completion of the hydrogenation (2 hours), the catalyst was filtered off and the filtrate thus obtained was evaporated under high vacuum at 20 ° C.

The obtained 4,6-O-isopropylidene-N-propionyldesmethylmuramyl-L-alanyl-D-glutamic acid (C) - n-butyl ester is converted in a 7: 3 mixture of chloroform and isopropyl alcohol to which a small amount of dimethylformamide is added, the d-cyclohexylcarbodiimide and N-hydroxysuccinimide to the N-hydroxysuccinimide ester, which is added in portions in solid form to a solution of a mixture of L alanine-2- (1,2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) ethylamide and triethylamine in chloroform, Isopropyl alcohol and water in a ratio of 70: 30: 2. The reaction mixture was stirred at room temperature for 3 hours.

The turbid reaction solution thus obtained is evaporated under vacuum at 30 ° C. The crude product was then suspended in doubly distilled water (pH 5.5) and then adjusted to pH 6.0 by addition of triethylamine. Insoluble matter (dicyclohexylurea) was filtered off and the filtrate was then lyophilized under high vacuum.

The lyophilisate thus obtained is dissolved in doubly distilled water and the solution is then concentrated to a volume of 75 ml in a dialysis chamber (PM 10/76 mm O ultrafilter) under a pressure of 0.3 MPa (positive pressure). The product thus concentrated is filtered against 250 ml of a mixture of phosphate buffer and sodium chloride (0.1 molar each, 1: 1 ratio, pH 7) and 1750 ml of double-distilled water until the chloride ions are removed. The solution remaining in the chamber (about 75 mL) is then successively filtered through two milliporous filters (0.45 µm and 0.2 µm) and the clear, colorless solution so obtained is then lyophilized under high vacuum.

The lyophilisate was further purified by Craig separation using a 7: 17: 18: 6 mixture of carbon tetrachloride, chloroform, methanol and 0.5% sodium chloride solution. The fractions containing the product were combined and evaporated to dryness under high vacuum without heating (cooling). . A colorless powder is obtained which still contains sodium chloride. To remove sodium chloride, the product is dissolved in 600 ml of double distilled water and filtered in a dialysis chamber (PH 30/76 millimeter 0 ultrafilter) at 0.3 MPa first up to about 50 ml of internal volume and then using a total of 1,750 ml twice distilled water.

The solution remaining in the chamber (about 50 ml) is then sterile filtered using two milliporous filters (0.45 μΐη and 0.2 μΐη, respectively) and then lyophilized under high vacuum.

The sodium salt of 4,6-O-isopropylidene-N-propionyldemethylmuramyl-L-alanyl-D-glutamyl- _(K C j n-butyl ester- (C _Y) -L-alanine-2- (l, 2-dipalmitoyl -sn-glycero-3-hydroxyphosphoryloxyjethylamide in the form of a colorless powder which still contains 1.2 moles of water.

In an analogous manner to that described in Example 1, from 1.0 g (0.8 mmol) of the sodium salt of 4,6-O-isopropylidene-N-acetylmuramyl-L-alanyl-D-glutamyl- (Cn) -methyl ester- ( C _Y ) -L-alanine-2- (1,2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxyjethylamide, which still contains 1 mole of water), yields 0.92 g (91.5%) of pure N acetylmuramyl-L sodium salt -alanyl-D-glutamyl- (C ₍₁ ) -methyl- (C _Y ) -L-alanine-2- (1,2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) ethylamide, which still contains 2, 96 mol of water, in the form of a colorless powder.

[α] _D 20 = + 11 + 1 ° (c = 0.285, 10% acetic acid).

R f = 0.39 (solvent system: mixture of ethyl acetate, n-butanol, pyridine, acetic acid and water in a ratio of 42: 21: 21: 6: 10).

R _f = 0.55 (solvent system: acetonitrile: water = 3: 1).

R _f = 0.25 (solvent system: chloroform: methanol: water = 70:: 30: 5).

In like manner, 1.0 g (0.7 mmol) of sodium 4,6-O-isopropylidene-N-acetyl-muramyl-L-alanyl-D-glutarayl- _(C) -terc.butylester- (G _Y ) -L-alanine-2- (1,2-dipalmitoyl-sn-glycero-3 hydroxyphosphoryloxy) ethylamide, which still contains 0.5 moles of water, yields

0.94 g (93.8% of theory) of pure sodium salt of N-acetylmuramyl-L-alanyl-D-glutamyl- (C _a ) -tert-butyl ester- (C _Y ) -L-alanine-2- (1,2 dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) ethylamide in the form of a colorless powder, which still contains 3.38 moles of water.

[α] _D ²⁰ = + 14 + 1 ° (C = 0.817, 10% acetic acid).

R _f = 0.29 (solvent system: chloroform: methanol: water = 70:

: 30: 5.

245737

R _f = 0.53 (solvent system: ethyl acetate: n-butanol: pyridine: acetic acid: water = 42: 21: 21: 6: 10).

In an analogous manner, from 1.0 g (0.7 mmol) of the sodium salt of 4,6-O-isopropylidene-N-acetylmuramyl-L-alanyl-D-glutamic acid 1- (C _a ) -n-butyl ester (C _Y) ) -L-alanine-2- (1,2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) ethylamide, which still contains 1.0 mol of water, yields

0.95 g (94.5% of theory) of pure sodium salt of N-acetylmuramyl-L-alanyl-D-glutamyl- (C _a ) -n-butyl ester- (C _Y ) -L-alanine-2- (1,2 -dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) ethylamide, which still contains 3.61 moles of water.

_[A] 20 = + i4 + i ° _(C = 0.440, water).

R _f = 0.37 (solvent system: chloroform: methanol: water = 70:

: 30: 5.

R _f = 0.26 (solvent system: chloroform: methanol = 7: 3).

Rf = 0.64 (solvent system: ethyl acetate / n-butanol / pyridine / acetic acid / water = 42: 21: 21: 6: 10).

In an analogous manner to that described above, 1-L-alanyl-1-D-glutamyl- (Cn) -methyl ester of 4,6-O-isopropylidene-N-propionyldesmethylmuram 1-L-alanyl- (C _Y ) -L-ailianil-2- (1,2-dipaillmitolyl, 1-sin-glycero-3-hydroxyphosphoryloxy) ethylamide, which still contains 1.2 moles of water, yields 0.93 g (93%). 5% of theory) of pure sodium salt of _{N-propionyldemethylmuramyl-L-alanyl-D-glutamyl-C)} methyl ester- (C _Y) -L-alanine-2- (1,2-dipalmitoyl-sn-glycero-3- hydroxyphosphoryloxy) ethylamide in the form of a colorless powder, which still contains 3.09 mol of water.

[.alpha.] D @ ²⁰ = + 3.7 DEG (c = 0.672, in chloroform).

R _f = 0.62 (solvent system: ethyl acetate: n-butanol: pyridine: acetic acid: water = 42: 21: 21: 6: 10).

R _f = 0.25 (solvent system: chloroform: methanol = 7: 3).

R _f = 0.42 (solvent system: ethyl-chloroform: methanol: water = 70: 30: 5).

In an analogous manner to that described above, from 1.0 g (0.7 mmol) of the sodium salt of 4,6-O-isopropylidene-N-propionyldesmethylmuramyl-L-alanyl-D-glutamyl- (C _a ) -tert-butyl ester- ( C _Y ) -L-alanine-2- (1,2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) ethylamide, which still contains 0.7 moles of water, yields 0.96 g (96.9% of theory). pure sodium salt N18

-propionyldesmethylmuramyl-L-alanyl-D-glutamyl- (C 6) -tert-butyl ester- (C _Y ) -L-alanine-2- (1,2-dipalmitoyl) -sn-glycero-3-hydroxyphosphoryloxy) ethylamide in the form of a colorless powder, which still contains 2.64 moles of water. _{[?] D} 20 = + 2.5 ± 0.1 ° (c = 0.649 in water).

R _f = 0.60 (solvent system: chloroform: methanol: water = 70:: 30: 5).

Example 3

In an analogous manner to that described in Example 1, the following compounds are obtained:

N-acylmuramyl-N-methyl-L-alanyl-D-glutamyl- (C _H ) -ethyl ester- ( _Y ) -L-alanine-2- (1,2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) ethylamide sodium salt, N-acetyldesmethylmuramyl-L-alanyl-D-glutamyl- (C _a ) methyl (C _Y ) -L-alanine-2- (1,2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) ethylamide, sodium salt, N-acetylmuramyl-L-seryl-D-glutamyl- (C _H ) -n-butyl ester- (C _Y ) -L-alanine-2- (1,2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) sodium salt ) ethylamide, the sodium salt of N-L-acetylmiuiramyl cystelnyl-D-glutamyl _(K C j n-butyl ester- (C j _Y -L-alanine-2- (l, 2-dipalmitoyl-sn-glycero-3- hydroxyphosphoryloxy) ethylamide, N-acetylmuramyl-L-methionyl-D-glutamyl- (C _a ) -n-butyl ester- (C _Y ) -L-alanine-2- (1,2-dipalmitoyl-sn-glycero) sodium salt -3-hydroxy-phosphoryloxy) ethylamide, the sodium salt of N-acetyl-L-alanyl-D-glutamyl- (C _K) -n-butyl _C-Y) -L-alanine-2- (l, 2-sn-dlmyristoyl glycero-3-hydroxyphosphoryloxy) ethylamide, N-acetylmuramyl-L-alanyl-D-glutamyl- (C _c ) -n-b sodium salt (C _Y ) -L-alanine-2- (1,2-distearoyl-sn-glycero-3-hydroxyphosphoryloxy) ethylamide, N-acetylmuramyl-L-alanyl-D-glutamyl- (C _a ) sodium ester (C _Y ) -L-alanine-2- (1,2-dioleoyl-sn-glycero-3-hydroxyphosphoryloxy) ethylamide-N-butyl ester, N-benzoylmuramyl-L-alanyl-D-glutamyl- ( ( _K ) -n-butyl ester- (C _Y ) -L-alanine-2- (1,2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) ethylamide, N-glycolmuramyl-L-alanyl-D-glutamyl sodium salt - (C) -n-butyl ester- (C _Y ) -L-alanine-2- (1,2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) ethylamide and N-acetyl-1,4,6- O-Triacetylmuramyl-L-alanyl-D-glutamyl- (C _a ) -n-butyl ester (C _Y ) -L-alanine-2- (1,2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) ethylamide .

Example 4

Eye drops

Ingredients:

N-acetylmuramyl-N-aminobutyryl-D-glutamyl- (C _(R ) -n-butyl ester- (C _Y ) -L-alanine-2- (1,2-dipalmitoyl-sn-glycero-3-hydroxyphosphonate) sodium salt oryloxy) ethylamide (compound II) 0.10 mg boric acid 30.00 mg sodium tetraborate (10 HsO) 0.10 mg benzalkonium chloride 0.20 mg water for injections into 1.0 ml

Production

Boric acid, sodium tetraborate and benzalkonium chloride are dissolved at room temperature in one part of the above amount of water for injection purposes under aseptic conditions and with stirring. Thereafter, compound II is dissolved in the solution thus obtained and the solution is made up to a final volume of 1.0 ml with water for injection purposes.

This solution or a portion or multiple thereof is filtered through a membrane filter and filled into clean containers. Suitable containers are, for example:

flexible plastic container (5 ml or 10 ml) with dropper, glass container (5 ml or 10 ml) with dropping pipette of glass or plastic and elastic pipette holder or disposable plastic pipette (containing 1 to 2 drops).

Example 5

Non-aqueous single dose for nasal administration

Ingredients:

N-propionyldesmethylmuramyl-L-alanyl-D-glutamyl- (Cn) -n-butyl ester (C _Y -L-alanine-2- (1,2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) ethylamide (compound) III) 0.03 mg miglyol 812 to 30.00 mg

Production

0.3 mg of compound III is dissolved under aseptic conditions in 29.97 mg of miglyol 812.

This solution is filled by a commercially available single-shot nasal application device, for example according to U.S. Pat. No. 3,739,951, which is applied to a container of proipelenite prior to use.

Example 6

Nasal drops

Ingredients:

N-acetylmuramyl-L-valyl-D-glutamyl- (C _and N-butyl ester- (C _Y ) -L-alanine-2- (1,2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) sodium) ethylamide (compound IV) thiomersal sodium monohydrogen phosphate H2O sodium dihydrogen phosphate 12 H2O benzalkonium chloride disodium ethylenediaminetetraacetic acid sodium chloride demineralized water pH value lowering the freezing temperature Δ ¹

I II

0.15 mg 0,10 mg 0.02 mg - 0.30 mg 0.30 mg 10.10 mg 10.10 mg - 0,10 mg 0.50 mg 0.50 mg 3,70 mg 4,50 mg 988.30 mg 987.60 mg 5.0 + 0.3 5.0 + 0.3 - 0.51 ° C 0.56 ° C

Production

In one portion of the aforesaid amount of demineralized water is dissolved inaitirium hydride disulfate, di-sodium hydrogen phosphate, sodium chloride, thiomersal and disodium ethylenediaminetetraacetic acid disodium at room temperature with stirring.

Compound IV is then dissolved in this solution and the solution is brought to final volume with the remaining demineralized water.

This solution, or a portion or multiple thereof, is filtered through a membrane filter and filled into clean containers. Suitable containers are, for example

(a) a glass or plastic container (5 ml or 10 ml), fitted with a glass or plastic pipette with an elastic pipette tip adapted to be sucked in

(b) plastic squeeze bottles with plastic tube and spray head

(c) single dose plastic containers (2 to 3 drops in volume); or

(d) glass or plastic bottles fitted with a standard mechanical plastic sprayer (without propellant).

Example 7

Gel

Ingredients:

sodium N-acetyl-muramyl-L-alanyl-D-glutamate 1- (C ") -n-butyl ester- _(C-F) -L-alanine-2- (1,2-dilauroyl-sn-glycero-3-hyroxyf (phosphoryloxy) ethylamide

(Compound V) 0.01 g glycerin 85% 10,00 g méthylparaben 0,12 g propylparaben 0,03 g sodium carboxymethylcellulose (high viscosity) 2,50 g demineralized water 87.34 g

Production

Methyl- and propylparaben are dissolved in 1 part of hot demineralized water. Sodium methoxylmethylcellulose is then added to the obtained solution with vigorous stirring. The mucilaginous product obtained is swollen with stirring. After cooling, glycerin and a solution of the active compound (compound V) in the remainder of the water are added.

Example 8

Cream

Ingredients:

N-acetylmuramyl-L-alanyl-D-glutamyl- (C _a ) -n-butyl ester (C _Y ) -L-alanine-2- (1-palmitoyl-2-oleyl-sn-glycero-3-hydroxyf) sodium salt Ophthyloxy) ethylamide (compound VI) 0.10 g sorbitan monosulfate 0.60 g cetyl alcohol 3.00 g isopropyl palmitate 2.00 g methylparaben 0.12 g viscous paraffin oil 10.00 g

PEG (20) -sorbitan monostearate 4.40 g propylparaben 0.03 g

70% solution of crystalline sorbitol in demineralized water 6,00 g sulfuric acid 9,00 g demineralized water 64,67 g

Production

The fatty phase consisting of sorbitan monostearate, cetyl alcohol, stearic acid, PEG (20) sorbitan monostearate, isopropyl palminate and paraffin oil is melted. Then methyl and propylparaben are dissolved in one part of hot demineralized water. Sorbitol solution is added to the aqueous phase. The aqueous phase is then added to the fatty phase with stirring at about 75 ° C. The cream base is then allowed to cool with stirring. At about 40 ° C, a solution of the active ingredient (Compound VI) is added to the cream base in the rest of the water.

Example 9

Nasal ointment

Ingredients:

N-acetylmuramyl-L-alanyl-D-glutamyl- (Cn) -methyl ester- (C _Y ) -L-alanine-2- (1,2-dlpalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) ethylamide sodium salt (compound VII) 0.03 g viscous paraffin oil 20.00 g white petrolatum 30.00 g wool grease, anhydrous 40.00 g demineralized water 19.97 g

Production

The oily phase, consisting of paraffin oil, vasellna and sheep wool fat, melts. An aqueous solution of the active ingredient is incorporated into the fatty phase at a temperature of about 50 ° C.

Example 10

Skin ointment

Ingredients:

sodium N-propionyldemethylmuramyl-L-alanyl-D-glutamyl- (C ') -n-butyl ester- (C j _Y -glycine-2- (l, 2-dipalmltoyl-sn-glycero-3-yloxy or hydroxyphosphinyl) Ethyl amide (compound VIII) 0.25 g sorbitan quioleate 10.00 g white beeswax 5.00 g cetyl alcohol 2.50 g methylparaben 0.15 g viscous paraffin oil 20.00 g propylparaben 0.02 g stearyl alcohol 2.50 g white vaseline 40,00 g demineralized water 19,58 g

Production

The oily phase, which consists of sorbitan sesquioleate, white beeswax, cetyl alcohol, paraffin oil, stearyl alcohol and white vaseline, will melt. Then methyl- and propylparaben are dissolved in the main amount of water while hot. At about 80 ° C, the aqueous phase is incorporated into the fatty phase. A solution of the active compound (compound VIII) in the remaining amount of water is added to the butter base thus obtained at a temperature of about 40 ° C.

Example 11

Lipstick

Ingredients:

N-acetylmuramyl-N-methyl-L-alanyl-D-glutamyl- (C _a ) -n-butyl ester- (C _Y ) -L-alanine-2- (1,2-dipalmitoyl-sn-glycero) salt -3-Hydroxy24-xyphosphoryloxy ethylamide (Compound IX) 1.00 g polyethylene glycol Mw 400 15.00 g polyethylene glycol Mw 1,000 83.00 g polyethylene glycol Mw 4000 1.00 g

Production

The active ingredient is gently swallowed in the melt of polyethylene glycols. The viscous melt is then poured into suitable lipstick molds and allowed to solidify.

Example 12

0.6 g (0.5 mmol) of N-propionyldesmethylmuram 1-L-alanyl-D-glutamyl- (C _a ) -methyl ester- (C _Y ) -L-alanine-2- (1,2-diimidoimitoyl-sn- o-3-methoxyphosphoryloxy) ethylamide glycerate is suspended in 100 ml of acetone, 0.45 g (3.0 mmol) of sodium iodide is added and the mixture thus obtained is heated to reflux. Initially, a clear solution is formed from which the desired product is gradually precipitated. After reaction under the above reaction conditions (reaction time 4 hours), the reaction mixture is cooled in an ice bath, the precipitate is filtered off and washed with acetone. The crude product thus obtained is dissolved in methylene chloride to remove residual sodium iodide, then the product is precipitated by the addition of acetone and purified by column chromatography on silica gel 60 (particle size 0.063-0.200 mm) using a solvent system consisting of chloroform / methanol / water 70: 30: 2 to 5. Fractions containing the desired product (5 ml each) were combined and evaporated in vacuo at 30 ° C.

The residue is taken up in 100 ml of double-distilled water, filtered through a millipore filter (0.2 µm) and the solution thus obtained is freeze-dried. 0.62 g (91.9%) of chromatographically pure sodium salt of N-propionyldemethylmuramyl-L-alanyl-D-glutamyl- (C _K) methyl ester- (C _Y) -L-alanyl-2- (l, 2 dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) ethylamide in the form of a colorless powder, which still contains 2.87 moles of water.

[.alpha.] D @ ²⁰ = -11.7 DEG (c = 0.672, chloroform).

_Rf = 0.42 (chloroform: methanol: water = 70: 30: 5).

The starting material is obtained as follows:

To a solution of 1.0 g (0.8 mmol) 4,6-O-isopro245797 pylidene-N-propionyldesmethylmuramyl-L-alanyl-D-glutamyl- (C _a ) -methyl ester- (C _Y ) -L-alanine-2 - (1,2-Dipamitoyl-sn-glycero-3-methoxyphosphoryloxy) ethylamide in 10 ml of acetic acid is added with stirring over 10 minutes 6 ml of water. After 12 hours, the gel formed from the solution is taken up in a mixture of 12 ml of chloroform, methanol and water, the organic phase is separated and then evaporated in vacuo at 30 [deg.] C. The residue is purified by column chromatography on 50 g of silica gel 60, particle size 0.063-0.200 mm], eluting with 5: 1 methylene chloride: methanol, collecting 5 ml fractions. The fractions containing the desired product were combined and evaporated in vacuo at 30 ° C. There were obtained 0.92 g (95.4%) of N-propionyldemethylmuramyl-L-alanyl-D-glutamyl- (C _G) methyl ester- (C _Y) -L-alanine-2- (1,2-dipalmitoyl-sn -glycero-3-methoxyphosphoryloxyethyl amide.

The starting material is obtained as follows:

1,1-mol of 4,6-O-isopropylidene-N-proplonyldesomethylmuramic acid is suspended in 20 ml of absolute methylene chloride. Then successively added 0.17 mL (1.2 mmol), triethylamine (anhydrous, d = 0.73), 1.0 mM L-alanyl-D-glutamyl- (C _K) methyl ester- (C _Y) -L -alanine-2- (1,2-dipalmitoyl-sn-glycero-3-methoxyphosphoryloxy) ethylamide hydrochloride and 0.12 g (1.0 mmol) of N-hydroxysuccinimide to give a clear solution. To this solution was added a solution of 0.25 g (1.2 mmol) of dicyclohexylcarbodiimide in 5 mL of absolute methylene chloride over 10 minutes, and the reaction mixture was stirred at room temperature for 12 hours. The precipitated dicyclohexylurea is then filtered off and the filtrate is extracted with water. The organic phase is separated, dried over magnesium sulphate and evaporated under cooling at 20 ° C under vacuum. The crude product thus obtained was purified by column chromatography on 80 g of silica gel 60, (particle size 0.083-0.200 mm), eluting with methylene chloride: methanol (8: 1). The fractions containing the desired product (10 ml each) were combined and evaporated in vacuo under cooling at 20 ° C.

To afford 4,6-O-isopropylidene-N-propionyldemethylmuramyl) -L-alanyl-D-glutamyl- (C _K) methyl ester- (C _Y) -L-alanine-2- (1,2-dipalmitoyl-sn glycero-3-methoxyphosphoryloxy) ethylamide.

The sodium salt of the starting material is obtained as follows:

A solution of 34.4 g (73.8 mmol, 2.14 mmol / g, still containing sodium chloride) of sodium 1α-O-benzyl-4,6-O-isopropylidene-N-propionyldesmethylmuramic acid in 340 mL of water is hydrogenated at constant temperature. pH 7.1 in the presence of 6.0 g of 10% palladium on carbon catalyst for 23 hours at room temperature and atmospheric pressure.

The catalyst is then filtered off, the filtrate is evaporated at pH 7.1 under vacuum at 30 ^DEG C. and the residue thus obtained is dried under high vacuum with phosphorus pentoxide.

There was obtained 4,6-O-isopropylidene-N-propionyldesmethylmuramic acid sodium salt as a colorless powder.

Rf = 0.50 (70: 30: 5 chloroform / methanol / water) a

Rf = 0.66 (3: 1 acetonitrile / water).

The starting material is obtained as follows:

To a solution of 14.3 g (32.7 mmol) of methyl 1a-O benzyl-4,6-O-isopropylidene-N-propionyldesmethylmuramic acid in 130 ml of methanol is added 24.4 ml (48.7 mmol) of 2N sodium hydroxide solution. and the mixture is stirred at room temperature for one and a half hours.

The clear, slightly yellow solution thus obtained was adjusted to pH 7.0 by addition of 1 N hydrochloric acid solution and evaporated in vacuo at 30 ° C.

After drying over phosphorus pentoxide, 1α-O-benzyl-4,6-O-isopropylidene-N-propionyldesmethylmuramic acid sodium salt is obtained as colorless crystals.

_Rf = 0.67 (chloroform: methanol: water = 70: 30: 5)

R _f = 0.74 (acetonitrile: water = 3: 1).

The starting material is obtained as follows:

To a suspension of 6.75 g (224.7 mmol) of sodium hydride in 120 ml of absolute tetrahydrofuran was added dropwise a solution of 31.3 g (85.65 mmol) of 1a-O- at 5 ° C under nitrogen atmosphere with stirring over 5 minutes. benzyl-2-esoxy-4,6-O-isopropylidene-2-propionamido-α-D-glucopyranoside in 200 ml of absolute tetrahydrofuran at a temperature of 20 ^° C.

The suspension thus obtained was stirred at 40 DEG C. for a further 2 hours, then cooled to 0 DEG C. and added dropwise to a solution of 20.7 g (135.77 mmol) of bromoacetic acid methyl ester in 95 ml under stirring and under nitrogen. absolute tetrahydrofuran, which is cooled to -15 ° C.

The suspension thus obtained was stirred for a further 3 hours at 0 to 5 ° C and then 20 ml of methanol and 20 ml of tetrahydrofuran were added. The pH is adjusted to 6 with 4.5 ml of glacial acetic acid and then concentrated in vacuo at 30 ° C.

The residue is dissolved in 200 ml of methylene 2,445,797 chloride and the solution thus obtained is washed three times with 125 ml of water each time. The methylene chloride phases were combined, dried over sodium sulfate, filtered and then evaporated in vacuo at 30 ° C.

The crude product was obtained as pale yellow crystals which were purified by column chromatography on 500 g of neutral alumina (Super 1) using ethyl acetate as eluent (10 ml fractions were collected). Fractions 14 to 36 are combined and evaporated in vacuo.

There was obtained 1α-O-benzyl-4,6-O-isopropylidene-N-propionyldesmethylmuramic acid methyl ester as colorless crystals, m.p. 121-123 ° C (from diethyl ether / petroleum ether 1: 2).

[.alpha.] D @ ²⁰ = + 146.9 + 0.1 DEG (c = 0.849, chloroform).

Rf ₌ 0.67 (9: 1 chloroform: methanol).

Example 1 d

To a solution of 2.85 g (2.09 mmol) of the sodium salt of N-propionyldemethylmuramyl-L-alanyl-D-glutamyl- (C _K) -terc.butylester- (C _s) -L-alanine-2- (l, 2 -dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxyjethylamide in 112 ml of absolute methylene chloride was added 28 ml of trifluoroacetic acid and the mixture was stirred at room temperature for 3 hours.

The colorless, clear solution is then evaporated in vacuo at 30 DEG C. and the residue is taken up several times with methylene chloride and the solution is evaporated again.

3.0 g of a colorless oil are obtained, which is dissolved in 350 ml of a mixture of phosphate buffer and sodium chloride (0.1 M each, in a ratio of 1: 1, pH 7) and the solution obtained is filtered in a dialysis filter (PM 30 / filter). 76 mm 0) at an overpressure of 0.3 MPa. Then it is filtered against a total of 2.1 liters of double-distilled water until the chloride ions are removed. The solution remaining in the dialyser chamber (about 50 ml) was lyophilized under high vacuum.

A colorless lyophilisate is obtained, which is further purified by column chromatography on 260 g of silica gel (silica gel 60, pure, particle size 0.063-0.200 mm) using a 70: 30: 5 mixture of chloroform, methanol and water as eluent. collect fractions of 10 ml. Fractions 90-260 were combined and evaporated without heating under high vacuum (cooling) to dryness. The residue is dissolved in 250 ml of double-distilled water and first concentrated to a volume of about 50 ml at a pressure of 0.3 MPa in a dialysis filter (PM 30/76 mm O ultrafilter) and then filtered successively against a 250 ml mixture of phosphate buffer and sodium chloride. (in each case 0.1 M, 1: 1, pH 7) and 1750 ml of double-distilled water until the chloride ions are removed. The solution remaining in the dialysis chamber is then sterilized by filtration with two milliporous filters (0.45 μτα and 0.2 μΐη, respectively) and then lyophilized under high vacuum.

Give the disodium salt (C _s) -L-alanine-2- (l, 2-dipalmitoyl-sn-glycero-3-hydroxy-phosphoryloxy) ethylamide N propionyldemethylmuramyl-L-alanyl-D-glutamic acid as a hygroscopic powder , which still contains 4.80 moles of water.

[ «] ₌ + _ _{6) 1} 4 0.1 DEG (c = 0.489, methylene chloride to ethanol in a ratio 1: 1).

Rf = 0.28 (42: 21: 21: 6: 10 mixture of ethyl acetate, n-butanol, pyridine, acetic acid and water), and

R _f = 0.62 (acetonitrile: water = 3: 1).

Example 14

4.5 g (34 mmol) of N-acetylmuramyl-L-alanyl-D-glutamyl- (C _a ) -tert-butyl ester- (C _y ) -L-alanine-2- (1,2-dipalmitoyl-sn) sodium salt (glycero-3-hydroxyphosphoryloxy) ethylamide, which was dried under high vacuum over phosphorus pentoxide, was dissolved in 75 ml of absolute dichloromethane. The solution was cooled to 0C and then 25 ml of anhydrous trifluoroacetic acid was added under stirring and moisture excluded, and the solution was allowed to rise to room temperature. After two and a half hours, the clear, colorless solution was strongly concentrated on a rotary evaporator at room temperature (about 10 ml), added several times with 100 ml of dichloromethane each time and the chloroethane was evaporated again. The residual oil is taken up in 100 ml of tert-butanol and the solution is lyophilized. The crude product was purified by chromatography twice on 400 g of silica gel (silica gel 60), eluting with chloroform: methanol: water (70: 30: 5). The product contained in fractions 92 to 172 is dissolved in 100 ml of double-distilled water, and this solution is purified by dialysis filtration in an analogous manner to that described in the example.

1. Filter the solution remaining in the dialyser chamber through a milliporous filter (0.2 tons) and lyophilize the filtrate.

N-Acetylmuramyl-L-alanyl-D-glutamic acid disodium salt of ( _Cγ ) -L-alanine-2- (1,2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) ethylamide, which still contains 4, 89 mol of water, in the form of a colorless fluffy powder.

[Α] D = + 10 + 1 ° (c = 0.675, methanol).

_Rf = 0.08 (chloroform: methanol: water = 70: 30: 5).

R 1 = 0.15 (mixture of ethyl acetate, n-butanol, pyridine, acetic acid and water in a ratio of: 21: 21: 6: 10) and

Rf = 0.30 (70: 40: 9: 1: chloroform / methanol / water / acetic acid).

Example 15

The alveolar rat macrophages obtained by washing the lungs are plated and incubated in vitro for 24 hours with either the active ingredient-containing liposomes or the active ingredient in saline (phosphate buffered saline). Iodine ( ¹²⁵ J) -labeled tumor cells are then added and incubation is continued for a further 72 hours. Thereafter, the killed tumor cells are removed by washing and the number of still living tumor cells is determined based on their radioactivity. Activation of macrophages is assessed on the basis of their cytotoxicity; according to the proportion of tumor cells killed at the end of the experiment. The specific cytotoxicity (in percent) is calculated as follows:

100 number of tumor cells incubated with macrophages and drug in 1 ml [cpm]

1 ------------------------------------------- number of tumor cells incubated by macrophages a active substance in saline, which is buffered with phosphate buffer, in 1 ml (cpmj)

Further details of the above assay are described in I. J. Fidler et al., J. Biol. Response Modifiers 1, 43-55 (1982). The following table shows some of the results of the experiment that have been achieved when testing the following active substances:

N-acetylmuramyl-L-alanyl-D-glutamyl- (Cn) -n-butyl ester- (Cn) -L-alanine-2- (1,2-dipalmitoyl-sn-glycero-3-hydroxyf) (phosphoryloxy) ethylamide,

II sodium N-propionylnormuramyl-L-alanyl-D-glutamyl- (C _K) -n-butyl ester- (C _Y) -L-alanine-2- (1,2-dipalmitoyl-sn-glycero-3 hydroxyphosphate thiophosphoryloxy ) ethylamide,

III N-propionylnormuramyl-L-alanyl-D-glutamyl- (C _a } -n-methyl ester-C _Y ) -L-alanine-2- (1,2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) ethylamide sodium salt and

IV N-acetylmuramyl-L-alanyl-D-glutamyl- (C _a ) -tert-butyl ester- (C _Y ) -L-ailiaidin-2- (1,2-dipialmitoyl-sn-glycero-3-hydroxyphosphoryloxy) sodium salt j of ethylamide.

active specific cytotoxicity (%) active ingredient in 0.2 ml PBS active ingredient in 100 µg liposomes per 0.2 ml / µg / culture ^ g / culture

0.02 0.2 2 20 0.02 0.2 2 20

AND 39 59 56 51 44 II 21 48 74 10 23 11 III 3 81 47 64 76 79 IV 23 18 20 May 64 68 52

PBS = phosphate buffered saline

Example 16

Production of 1,000 tablets containing 0.5% active ingredient

Composition for 1000 tablets:

N-acetylmuramyl-L-alanyl-D-glutamyl- (C _a ) -methyl ester- (C _Y ) -L-alanine-2- (1,2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) ethylamide 0, 5 g ground lactose 43.0 g corn starch 52.0 g hydroxypropylmethylcellulose containing 28 to 30% methoxy groups (Pharmatcoat 603 ^(R) 3.0 g colloidal silica (Aerosil ^(R)) 1.0 g magnesium stearate 0.5 G

Production

First, the active ingredient and 15 g of lactose are mixed. The masterbatch thus obtained is further mixed with 28 g of lactose and 47 g of corn starch.

From this mixture and an aqueous solution of hylroxypropylmethylcellulose, a granulation mass is obtained, which is granulated, the granulate is dried and ground. 5 g of 5 g of corn starch, colloidal silicon dioxide and magnesium stearate are then admixed and 1,000 tablets of 100 mg in weight are compressed.

The compressed tablets may be coated in a manner known per se with a coating which is resistant to the action of gastric juice.

Example 17

The active ingredient in the form of a lyophilized dry substance mg of sodium N-acetyl-L-alanyl-D-glutamyl- (C _c) methyl ester- (C _Y) -L-alanine-2- (l, 2-dipalmitoyl-sn glycero-3-hydroxyphosphoryloxy) ethylamide and 500 mg of mannitol (pyrogen-free) are dissolved in water for injection and the solution is sterilized by filtration through a membrane filter, under aseptic conditions, the sterile solution is filled into sterile glass ampoules or glass floles and dried After liophilization, the ampoules are closed and the vials are closed with a rubber stopper and covered with aluminum foil.

Claims (18)

A process for the preparation of miuramyl peptides of the general formula I II I S I x II OR R (L) O κ OH CN-CH-C-NH-CH-CH, CH, CX ~ Y ~ C ~ NH CH CH _CH-Z OPO O OR II O R ⁹ C-O-CH CH * 1 (dream l * O in which. R ² represents an alkyl group having 1 to 4 carbon atoms, hydroxymethyl or phenyl, R ³ represents hydrogen or methyl, R ⁶ represents hydrogen or alkyl having 1-3 carbon atoms; R ⁷ is an optionally substituted (C 1 -C 3) alkyl-substituted alkyl group, R ⁸ is hydrogen or C 1 -C 4 alkyl, X is oxygen or NH, Y represents a C 1 -C 4 alkylidene group and R ⁹ and R ¹⁰ are independently C 11 -C 17 alkyl or C 11 -C 17 alkenyl, and / or salts thereof, characterized in that in the compound of formula Ia CH 2 OR 3,, (D) NH-R ^2, R-CH _v . (L) (D) (ϋ 9 C-N-CH-C-NH-CH-CH-CH-CH-C-X-Y-C-NH-CH-CH-O-P-O-CHo II l _r ι, II I II / o ^ CO-CH $ (- ^ 2. 1 '' ( ^Si2 -) II 1f I, II QO ⁷ O io £. R 'in which R ¹ , R ¹ and R ⁶ are hydrogen and the other symbols have the same meaning as in formula I, with at least one of R ¹ , R ² , R ⁴ , R ⁶ , R ⁷ , R ⁸ or a hydroxyl group on the atom The phosphorus is present in a protected form, cleaving the protecting group or protecting groups, whereupon the compound of formula I obtained is optionally converted into a salt or the salt obtained is converted into another salt. 2. A method according to claim 1, characterized in that starting from the corresponding compound of formula Ia to produce the compounds of formula I wherein R ² represents an alkyl group having 1-2 carbon atoms or a hydroxymethyl group, R ³ denotes hydrogen or methyl, R ⁵ represents a dialect or methyl group, R ⁷ represents a C 1 -C 3 alkyl or hydroxymethyl group, R ³ represents hydrogen or a C 1 -C 4 alkyl group, X represents oxygen or NH, Y represents an alkylidene group having 1 to 3 carbon atoms and R ⁹ and R ¹⁰ independently represent a straight alkyl group having 11 to 17 carbon atoms or a descarbonyloleoyl group and the compounds of the formula I are then optionally converted into their salts. 3. The method according to claim 2, characterized in that starting from the corresponding compound of formula Ia to produce the compounds of formula I wherein R ² represents methyl group or ethyl group, R ³ represents hydrogen or methyl, R ³ is hydrogen or methyl, R ⁷ is C 1 -C 3 alkyl, R ⁸ is C 1 -C 4 alkyl, X is oxygen or NH, Y is C 1 -C 2 alkylidene and R ⁹ and R ¹⁰ are, independently of one another, a straight-chain alkyl group having 11 to 17 carbon atoms with an odd number of carbon atoms or a descarbonyloleoyl group, and the compounds of formula I are then optionally converted into their salts. 4. Method according to one of claims 1 to 3, characterized in that starting from the corresponding compound of formula Ia to produce the compounds of formula I wherein ^R8 represents the alkyl groups mentioned in points 1 to 3 and the other substituents are the meaning given in one of the 243797, points 1 to 3, and the compounds of formula I are then optionally converted into their salts. 5. Method according to claim 3, characterized in that starting from a corresponding compound of formula Ia to produce the compounds of formula I wherein R ² represents methyl group or ethyl group, R ³ is hydrogen when R ² represents ethyl, or represents methyl group, when R ² is methyl, R ⁵ is hydrogen or methyl group, R ⁷ represents an alkyl group having 1-2 carbon atoms, R ³ represents a linear alkyl group having 1 to 4 carbon atoms, X is oxygen or NH, Y is ethylidene and R ⁹ and R ¹⁰ are independently straight alkyl of 15 to 17 carbon atoms with an odd number of carbon atoms, and the compounds of formula I are then optionally converted into their salts. 6. Method according to one of claims 1 to 5, characterized in that starting from the corresponding compound of formula Ia or to give the compounds of formula I wherein R ³ is methyl and the other substituents are as defined in In one of the steps 1 to 5, the compounds of formula I are then optionally converted into their salts. 7. Method according to one of claims 1 to 6, characterized in that starting from a corresponding compound of formula Ia to produce the compounds of formula I wherein R ⁵ is hydrogen and the other substituents are as defined in one of 1 to 6, and the compounds of formula I are then optionally converted into their salts. 8. Method according to one of claims 1 to 7, characterized in that starting from a corresponding compound of formula Ia to produce the compounds of formula I wherein R ⁸ is n-butyl, and the other substituents are as defined in one of items 1 to 7, and the compounds of formula I are then optionally converted into their salts. 9. A process according to any one of claims 1 to 8, wherein the corresponding compounds of formula Ia are used as starting materials to give compounds of formula I wherein X is NH and the other substituents are as defined in one of the following: 1 to 8 and the compounds of formula I are optionally converted into their salts. Process according to one of Claims 1 to 9, characterized in that the corresponding compounds of the formula Ia are used as starting materials, to give compounds of the formula I in which R ⁹ as well as R ¹⁰ represent n-pentadecyl and other substituents are as defined in one of items 1 to 9 and the compounds of formula I are then optionally converted into their salts. 11. The method according to claim 1, characterized in that as starting materials, the corresponding compounds of formula Ia to give N-acetyl-muramyl-L-alanyl-D-glutamyl- (C «] -t utylester- (C _f 1-L-alanine-2- (1,2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) ethylamide, which is then optionally converted to its salt. 12. A process according to claim 1, wherein the corresponding compound of formula (Ia) is used as the starting material to give (C ,,) -L-alanine-2- (1,2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy). N-acetylmuramyl-L-alanyl-D-glutamic acid ethylamide, which is optionally also fed to its salt. 13. The method of claim 1, characterized in that as starting materials, the corresponding compounds of formula Ia to give N-acetyl-muramyl-L-alanyl-D-glutamyl - (C _K) -n-butyl ester- (C _s) - L-alanine-2- (1,2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxyethyl) amide, which is then optionally converted to its salt. 14. The method according to claim 1, characterized in that as starting materials, the corresponding compounds of formula Ia to give N-propionyldemethylmuramyl-L-alanyl-D-glutamyl- _(C) -n-butyl ester- (C _Y) - L-alanine-2- (1,2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) ethylamide, which is then optionally converted to its salt. 15. A process according to claim 1, wherein the corresponding compound of formula (Ia) is used as the starting material to produce N-acetylmuramyl-L-alanyl-D-glutamyl- ( _CH ) -methyl ester- (C _Y ) -L -alanine-2- (1,2-dipalmltoyl sn-glycero-3-hydroxyphosphoryloxyjethylamide), which is then optionally converted to its salt. 16. The method of claim 1, characterized in that as starting materials, the corresponding compounds of formula Ia to give N-propionyldemethylmuramyl-L-alanyl-D-glutamyl- (C _C! J methyl ester- (C _Y] -L- -olanine-2- (1,2-dipslmitoyl-sn-glycero-3-hydroxyphosphoryloxy) ethylamide, which is then optionally converted to its salt. Process according to one of Claims 1 to 16, characterized in that the corresponding compounds of the formula Ia are used as starting materials to give the compounds of the formula I in the form of pharmaceutically usable salts. 18. A process according to claim 17, wherein the corresponding compounds of the formula Ia are used as starting materials to give the compounds of the formula I in the form of sodium salts.