KR840001689B1 - Method of preparing for phosphoryl muramyl peptides - Google Patents

Abstract

Title compds. I[X=carbonyl, carbonyloxy; R1=optionally substituded alkyl, aryl; R2, R4,R6,R3 independently=alkyl, H; R5=H, alkyl; R7=H, carboxyl were prepd. as immune potentiators. Thus, N-acetylmuramyl-L-alanyl-D-isoglutamine was esterified with N-hydroxysuccinimide to give the active ester, which was amidated with Me(Ch2)15op(o)(OH)OCH2CH NH2 to give N-acetylmuramyl-L alanyl-D-isoglutamine-2(hexadecyloxy-hydroxyphosphoryloxy)-ethylamide.

Description

Method for preparing phosphoryl muramil peptide

The present invention relates to novel phosphorylmuramil peptides of the following general formula (I) which can be used for the promotion of immunity and a method of preparing the same.

Wherein X is carbonyl or carbonyloxy, R ₁ is optionally substituted alkyl or aryl, R ₂ , R ₄ , R ₆ and R ₃ are hydrogen or lower alkyl, R ₅ is hydrogen or lower alkyl, R ₄ is hydrogen, lower alkyl, free or modified high-hydroxylowalkyl, free or modified mercapto-lowalkyl, optionally substituted amino-lower alkyl, cycloalkyl, cycloalkyl-loweralkyl, optionally substituted aryl Or aralkyl, or nitrogen-containing heterocyclyl or heterocyclyl-lower alkyl, and R ₅ together are alkylene of 3 or 4 carbon atoms and R ₇ is hydrogen or optionally esterified or amidated carboxyl, A _{One of 1} and A ₂ is a radical of the general formula (II)

[Wherein T is NH or 0 and Y is an optionally substituted alkylene group inserted into one oxycarbonyl and / or iminocarbonyl group and W is an aliphatic radical or a cycloalkyl or cycloalk having at least 6 carbon atoms, respectively Kenyl radical.]

The other of A ₁ and A ₂ is free or etherified hydroxy, amino, or lower alkyl amino, or amino carbonyl-lower alkylamino optionally substituted in lower alkyl radicals. Alkyl is straight or branched chain alkyl of up to 18 carbon atoms, bonded at any position, but especially lower alkyl.

Substituents of an optionally substituted alkyl group are, in particular, free or modified hydroxy or mercapto groups, such as etherified or etherified hydroxy or mercapto groups (eg lower alkoxy or lower alkylthio groups), or halogens. Atom or a free or modified carboxyl group such as a lower alkoxycarbonyl or carbamoyl group. Substituted alkyl radicals, such as lower alkyl radicals, may in particular have one or two or more of the same or different substituents selected from free hydroxy groups and halogen atoms.

If the aliphatic radical W is an alkyl or alkenyl radical containing up to 30 carbon atoms, it is preferably substituted or modified as a substituent, such as an etherified or esterified hydroxy group (eg lower alkoxy or lower alkylnoyl oxy group). It may have a hydroxy group, a halogen atom, or an alkanoylamino group, such as a free or acylated amino group such as lower alkanoylamino, or a keto group. These substituents are in the patent 2-position, i.e., the β-position Bound to a phosphoryloxy group, alternatively W may be a cycloalkyl or cycloalkenyl radical, such as cholesteryl, up to 30 carbon atoms.

Arylradicals are in particular monocyclic and acyclic arylradicals, in particular phenyl and naphthyl. It may be optionally mono-, di- or polysubstituted with lower alkyl groups, free, esterified or etherified hydroxy (eg lower alkoxy or lower alkylenedioxy), or halogen atoms, and / or trifluoromethyl groups. Can be.

Aralkyl is especially aryl-loweralkyl, wherein aryl is as described above. Aryl-lower alkyl is especially benzyl or phenylethyl, wherein the phenyl nucleus can be mono-, di- or polysubstituted.

Optionally substituted aralkyl radicals are, in particular, lower alkyl, free, etherified or esterified hydroxy (eg lower alkoxy or lower alkylene dioxy) or mercapto groups, lower alkylthio or trifluoromethyl groups and or As halogen atoms, aromatic nuclei are optionally mono-, di- or polysubstituted radicals.

Cycloalkyl is cycloalkyl, in particular 5 or 6 carbon atoms, such as cyclopentyl or cyclohexyl, and cycloalkyl-lower alkyl is especially cycloalkyl radical having 5 or 6 carbon atoms and lower alkyl radicals being especially methyl or ethyl.

Nitrogen-containing heterocyclyls are especially 5- or 6-membered heterocyclic compounds which contain one or two nitrogen atoms in the ring. It may be unsaturated or saturated and may contain, for example, condensed phenyl radicals. Examples include pyrrolyl, indolyl, pyridyl and imidazolyl radicals.

In nitrogen-containing hetero cyclyl-lower alkyl the hetero cyclyl radical has the above meaning and the lower alkyl radicals are especially methyl or ethyl.

Alkylene radicals, which may consist of R ₄ and R ₅ radicals, are preferably unsubstituted and in particular trimethylene radicals.

Optionally an esterified or amidated carboxyl group itself, or a carboxyl group esterified with a lower alkane, or alternatively a nitrogen atom, unsubstituted or optionally substituted alkyl, especially lower alkyl, aryl in particular phenyl, or aralkyl such as benzyl Is a mono- or di-substituted carbamoyl group. Alternatively, however, the carbamoyl group may contain alkylene radicals such as tetra- or penta-methylene radicals.

The optionally modified hydroxy or mercapto group is in particular an etherified or esterified hydroxy or mercapto group, such as lower alkoxy or lower acyloxy (eg lower alkanoyloxy), halogen atoms, or lower alkylthio (eg Lower alkanoylthio).

The modified amino-lower alkyl is in particular mono- or di-lower alkylamino-lower alkyl such as methylamino-, ethylamino-, dimethylamino- or diethylamino-lower alkyl or alkanoylamino-lower alkyl (e.g. lower Acylated amino-lower alkyl, such as alkanoylamino-lower alkyl).

Amino carbonyl-lower alkylamino groups optionally substituted with lower alkyl radicals are in particular lower alkyl amino groups having amino carbonyl-radicals in the 1-position (e.g., amino carbonylethylamino, 1-amino carbonylisobutyl 1-amino or 1-amino carbonyl-3-methylbutyl amino) or, alternatively, the lower alkyl group is a 1-amino carbonyl-lower alkyl amino group having a hydroxy, carboxy or amino group (eg, 1 -Amino carbonyl-2-hydroxy ethylamino, 1-amino carbonyl-2-hydroxy propyl amino, 1,2-bis- (amino carbonyl) ethylamino or 1-amino carbonyl-5-amino-1 Pentylamino).

Alkylene radicals Y are in particular lower alkylene radicals in which one or two oxycarbonyl or NR ₈ -carbonylimino groups are inserted, in particular of formulas (III _a ), (III _b ), (III _c ) or Is a radical of (III _d ).

In the above formula,

Y ₁ and Y ₂ are each optionally substituted lower alkylene radicals, or Y ₁ and Y ₂ together are at least 2 carbon atoms and R ₈ is hydrogen or lower alkyl.

Particular mention of substituents of Y ₁ and Y ₂ are free or modified hydroxy or hydroxy-lower alkyl, free or modified capto or mercapto-lower alkyl, free or mono- or di-lower alkylated or acylated Amino-lower alkyl, amino carbonyl, alkyl, cycloalkyl, aryl or aralkyl having 5 or 6 carbon atoms (wherein general terms are as described above). Alternatively, however, the alkylene radicals Y may preferably be lower alkylene radicals having 2 to 3 carbon atoms.

The radicals and compounds represented herein as "lower" are preferably up to 7 carbon atoms, in particular up to 4 carbon atoms.

In this specification, the general term may have the following meanings.

Lower alkyl is for example n-propyl, n-butyl, isobutyl, secondary-butyl or tert-butyl, n-pentyl, n-hexyl, isohexyl or n-heptyl, in particular methyl or ethyl. In aryl-, cycloalkyl- or heterocyclyl-loweralkyl, the loweralkyl radical is especially methyl or ethyl and the aryl, cycloalkyl or heterocyclyl radical is as described above.

Lower alkoxy is for example n-propoxy, n-butoxy, isobutoxy, secondary-butoxy or tert-butoxy, in particular methoxy or ethoxy.

Lower alkylthio is, for example, n-propylthio, n-butylthio, secondary-butylthio or tert-butylthio, in particular methylthio or ethylthio.

Lower alkylenedioxy is especially methylenedioxy, ethylenedioxy or propylenedioxy.

Halogen represents fluorine or bromine, with chlorine being preferred.

Lower alkanoyl is especially propionyl or butyryl but more preferably acetyl.

The compounds of formula (I) may exist in mixtures of isomers or in pure isomeric form. When R ₃ represents lower alkyl, the radical of the general formula —CH (R ₃ ) —C (═O) —, which is linked to the oxygen atom, is preferably present in optically active form, especially in the D-form. On the other hand, when R ₅ does not represent hydrogen, the radicals of the amino acids of the formula -N (R ₄ ) -CH (R ₅ ) -C (=)-are likewise preferably present in optically active form, in particular in L-form. And the terminal α-amino glutaric acid radicals are preferably present in optically active form, especially in the D-form. And optionally substituted 1-hydroxy groups may have an α- or β-configuration; However, the novel compounds of general formula (I) may alternatively exist in the form of a mixture of 1α- and 1β-isomers.

In the compound of formula (I), both bound to the phosphorus by oxygen can be easily separated by base. The compound of general formula (I) is usually present in the form of a mixture of free compound and salts thereof. Among the muramyl peptides of formula (I) described in the examples, for example about 40-55% are in the form of sodium salts. The present invention also relates to their salts.

The present invention generally relates to salts of compounds of general formula (I) with other salt forming groups. Contemplated salt-forming groups are, for example, carboxyl groups which may be represented by the radicals COA ₁ , COA or R ₇ , or amino groups in the radicals R ₅ . The present invention relates, in particular, to pharmaceutically non-toxic salts of the compounds of general formula (I). Particular ions of carboxylate anions to be mentioned in particular are metal or ammonium ions, for example alkali metal and alkaline earth metal ions (e.g. sodium, potassium, magnesium or calcium ions), and ammonia or lower alkyl amines (e.g. triethyl Ammonium ions derived from suitable organic amines, such as amines). Compounds of general formula (I) having basic groups (eg amino groups) may form acid addition salts. This compound may preferably be present in the form of an intramolecular salt, ie in the form of zwitterions. Protons bound to the phosphorus by oxygen atoms can give protons to the amino groups in the radical R ₅ . Salts that are not pharmaceutically toxic may also be used for separation or purification. Only pharmaceutically non-toxic salts can be used therapeutically and are preferred.

The novel phosphoryl muramyl peptides of the present invention exhibit a variety of effective pharmacological features, particularly significant immunopotentiating action.

Thus, in vivo experiments, these compounds significantly increase the antibody-forming ability of mice.

10 μg of sediment-free bovine-Serum-albumin (BSA) is immunized by intraperitoneal injection into NMRI mice. After 9, 15 and 29 days, serum samples are taken and the amount of anti-BSA antibody is adjusted using manual haem aggregation. Soluble BSA at the dose used is sub-immunogenic for experimental animals, producing no or very minimal antibody production. Further treatment of experimental animals before and after antigen administration with certain immunopotentiates increases antibody titers in serum. The treatment effect is represented by a score value, ie, the sum of the log ₂ titer differences over three days from which blood samples were taken.

In this test, if the compound of formula (I) at 0.5 to 5 mg / kg was continuously intraperitoneally or subcutaneously administered for 5 days after immunization with BSA, the compound of formula (I) significantly increased antibody production against BSA. You can. In this respect the compounds are much better than conventional hydrophilic muramyl peptides.

Expression of cell-imparted immunity may be enhanced in vivo by the compound:

The sensitization of morphot with BSA in an incomplete Freund's auxiliary solution only results in humoral formation of the antibody. Delays hypersensitivity to: Three weeks after immunization, intradermal injection of BSA results in local inflammation with erythrocytosis and skin thickening, peaking within 24 to 48 hours. The delay of this response corresponds quantitatively and qualitatively to the reaction usually obtained by immunization with BSA in complete Freund's auxiliary solution (ie, addition of mycobacteria). ED ₅₀ values (in animals treated and untreated 24 hours after induction), 200 μg per animal, required to cause a difference in reaction volume of 200 μl (red blood cell area, increase in skin thickness) are 10 to 20 μ.

Of particular emphasis is the ability of the phosphoryl muramil peptide to delay hypersensitivity to BSA in morphot by administering it without toxic mineral oil components with BSA in liposomes (nanlecithin: cholesterol 4: 1: 4 mg / animal). This delayed response is quantitatively and qualitatively identical to the response obtained by immunization with BSA in complete Freund's auxiliary solution.

ED ₅₀ values are between 100 and 300 μg per animal. Compared to the hydrophilic Muramil dipeptide, the new compound of general formula (I) has another improvement in quality:

Balb / c mice were immunized with intraperitoneal injection of 2 × 10 ⁴ p815 mast cell tumor cells. After 15 days the splenocytes of the immunized animals are tested in vitro for the presence of T-lymphocyte cells of cytotoxicity against p815 mastoma cells. The p815 target cells were labeled with 51Cr and the cytotoxic response was confirmed by measuring the radioactivity of the culture supernatant. At the dose, p815 mastoma cells induce very minimal or no production of cytotoxic T-cells as sub-immunogenic to mice. Simultaneous intraperitoneal administration of 1-50 μg of the Muramyl peptide of Formula (I) significantly increases the production of cytotoxic T-cells (increased by a factor of 10-30 compared to untreated mice).

Immunopotentiating features of the novel compounds of general formula (I) may appear in mice in the case of induction of specific immunotolerance to graft antigens by immunization of autoblasts with adjuvant:

In mixed lymphocyte cell culture, future lymphocyte cells of recipient graft recipients (C 57 B 1/6 J) are cultured with irradiated splenocytes of the future graft feeder. T-lymphocyte cells with a specific receptor for histocompatability antigens of the feeder proliferate and become blast cells; Can be isolated from other cells by sedimentation. In particular, blasts exhibit the related genotype specificity of the membrane receptor and are mixed with complete Freund's Supplement (CFA) and injected into the autoimmune for induction of specific resistance to the associated graft antigen in the future graft recipient (57 B1 / 6J). . Four immunizations with self anti-CBA / JT-lymphocytes are performed at four week intervals. T-autoblast adsorbate with a novel compound of formula (I) (109 blasts are suspended in 20 mg of a solution of substance in 20 ml of PBS; after 2 hours of incubation, the cells are centrifuged and washed twice with PBS. Can induce specific immunotolerance in the absence of CFA, and the adsorbate is as effective as lymphoblasts in CFA.

In addition, the novel compounds of general formula (I) can induce antibody-producing cell production, usually at concentrations of 0.5-100 μg / ml in mouse non-cell cultures (19 s-plaque compared to control in the absence of stimulants). ) -Producing cells increased by a factor of 10 to 30): Thus, in the presence of the compound, for example, antibodies specific for red blood cells in sheep are produced even without the addition of positive red blood cells to the immune culture. On the other hand, when compared with conventional thymus-dependent antigens (positive erythrocytes), the substance was immunologically expressed in T-cell-deficient non-cell cultures (cultures of nu / nu mice innate athymic) at the same concentration. Reactivity can also be increased (with a factor of 10 to 30 compared to untreated control culture). However, the compounds not only directly or indirectly induce the proliferation and synthesis of B-lymphocytes (i.e., potent antibody-producing cells) in in vitro tests, but also T-lymphocyte cells (herein, regulatory promoters and suppressors and Cytotoxic effect cells belong to). Thus, for example, the compound at a concentration of 1-20 μg / ml can significantly (up to 10 times) enhance the responsiveness of cortisone-resistant thymic cells as compared to irritant lymphocyte cells.

This effect occurs indirectly as a result of phosphoryl muramil peptide activating macrophage, which enhances the reactivity of T- and B-lymphocyte cells. In fact, it can be seen that the compound induces a large amount of "colony stimulating activity (CSA)" from rat macrophages even at low concentrations (0.5 to 10 µg / ml) (when supernatant of untreated macrophage culture medium is added). Compared with 0-5 colonies, 150-200 colonies are induced within 10 days from 10 bone marrow cells of rats after addition of 20% supernatant of macrophage cultures incubated with the material for 24 hours). CSA is a biological mediator necessary to distinguish myeloid cells from macrophages and polymorphonuclear leukocytes. The compound increases cells that are very important for inducing, enhancing and expressing nonspecific resistance and specific (lymphocyte cell-induced) immune responses.

The immunopotentiating action of the novel compounds can be seen in vivo tests. Injection of phospholipid derivatives of muramyl peptides according to the present invention significantly increases serum CSA concentrations within 3 to 9 hours (serum extracted with chloroform [5% final concentration], compared to 0-5 colonies in untreated animals). Increase to 120 colonies per 10 ⁵ bone marrow cells after addition), thus administration of the same compound significantly enhances the antibody-producing ability of mice in vivo.

The immunopotentiating properties of the novel compounds of general formula (I) may also be present in tumor motels, such as the Erhrlich ascites tumor in rats.

Intraperitoneal injection of 10 ⁶ congenital Yerlich ascites tumor cells into Balb / c mice causes animals to die within 18 days on average. If intraperitoneal injection of 10 ⁷ (Group 1), 10 ⁶ (Group 2) and 10 ⁵ (Group 3) multiple tumor cells filled in vitro with a novel compound of formula (I) (20 ml phosphate-buffered) 10 ⁹ ascites tumor cells were suspended in a 40 mg test substance solution in physiological common salt solution (PBS), incubated at 37 ° C. for 2 hours, and the cells were centrifuged and washed twice with PBS. Compounds bind to the cells to form membrane cells), and tumor growth does not occur even after 18 days. On day 19, 10 ⁶ natural Yerlich ascites tumor cells are administered intraperitoneally to each animal. The following is the result.

Group 1: 8 out of 10 survived on day 80

Group 2: Six out of 10 survived on day 80

Group 3: After 18 days, died with control animals.

The compounds according to the invention are also less toxic: no symptoms were seen even after five consecutive intraperitoneal administrations at a 100 mg / kg / day dose. Since the dose required for immunostimulation is very small, the therapeutic range of the novel compounds is very broad.

The novel compounds according to the invention, when administered separately from antigen injection sites and other sites (systemic immunopotentiation), as a mixture with the antigen itself (adjuvant effect of consultation), and at different times, with cellular immunity and especially humoral immunity. To increase.

The novel compounds according to the invention are used as adjuvant in a mixture with waxy to improve the success rate of vaccination and to improve the prevention of infection by liquid antibodies and / or cellular immunity against bacteria, viruses or parasitic corrosive microorganisms.

Finally, the compounds in admixture with other antigens are suitable as adjuvants in the laboratory and industrial preparation of therapeutic and diagnostic serum, and in immunologically activated lymphocyte induction of cell metastasis processes.

The novel compounds can also be used to promote the immune response in humans and animals without the simultaneous administration of antigens, which is potentially progressing. The compounds are therefore particularly suitable for stimulating the defense mechanisms of the human body, for example during acute infections or during selective (antigen-specific) immunity, congenital and severe primary diseases, and especially with ionizing irradiation or immunosuppressive action. After treatment with hormones, it is an acquired general (non-antigen-specific) immunodeficiency condition as occurs in older age. Thus, the substance may be administered in combination with an antibiotic, chemotherapy or other drug. Finally, the material is also suitable for the general prevention of infectious diseases in humans and animals.

The present invention also relates to the combination of muramyl peptides according to the invention with antibiotics, which results in an increase in antibiotic activity. To this end, an effective dose of antibiotics, for example about 20 to about 750 mg, is used, depending on the nature of the latter.

Muramilpeptide of formula (I) is used in about 5 mg to about 1/2 dose of antibiotic. Muramil peptide derivatives may be administered 24 hours before or after antibiotic administration, but are preferably administered at about the same time as the antibiotic.

Antibiotics are administered by conventional methods, such as subcutaneously, intravenously or orally, while muramyl peptides are usually administered subcutaneously, especially if administered separately from antibiotics.

In this way, individual antibiotics and antibiotic mixtures can be used. Antibiotics characterized by containing at least one of the above antibiotics and at least one muramyl peptide of general formula (I), may be used in the usual doses of antibiotics, for example 20-100 mg, preferably about 200-500 mg and general Muramil femide of formula (I) contains from 5 mg to half the dose of antibiotic. In particular, when these agents are to be administered orally, they may contain conventional doses of pharmacological carriers, extenders and / or diluents.

The high antimicrobial effects of the new formulations and novel formulations can be represented by in vivo testing of various types of animals, particularly mammals such as mice. To this end, animals are infected with lethal or sub-lethal pathogenic microorganisms, followed by administration of the new agents or separate doses of muramyl femide and antibiotics. The effect is confirmed as ED ₅₀ , the dose at which 50% of the animals survive.

Surprisingly, infections with pathogenic bacillus, in particular gram-negative bacteria that are difficult to treat, such as aerobacter strains, Brucella, Escherichia, Klebsiella, Maleomyces, Neisseria, Pasteurella, Proteus, Pseudomonas, Shigella and Vibrio, and Gram-positive bacteria, for example Actinomycetis, Clostridia, Corynebacteria, Diplococci, Mycobacteria or Staphylococcus, or Fungal Candida albicans, Cryptococcus neoformans, Flacci It was found that Stomachis dematatides or histoplasma capsules were inhibited and eradicated to an increased limit.

Among the antibiotics suitable for use with muramyl femide according to the invention, mention may be made in particular of those having the following groups: β-lactam antibiotics, amino glycosides, tetracyclines, macrolides, lincomycin, polyene antibiotics , Polypeptide antibiotics, anthracyclines, chloramphenicol, thiamphenicol, cycloserine, fusidic acid or rifamycin.

Penicillins, cephalosporins, penems, nocardisin, thienamycins and clavulanic acids may be preferred β-lactam antibiotics.

Penicillin antibiotics include, in particular, amoxicillin, ampicillin, carbenicillin, clocksacillin, cyclacillin, dicloxacillin, mesilinam, methicillin, penicillin G, penicillin V, pibampicillin, sulfenicillin, azoles Rocillin, ticarcillin, mezlocillin, fibresilinam or 6- [4-endo-azatricyclo [5. 2. 2. 0 2, 6] undec-8-enyl) methyleneamino waste nisilic acid.

In the cephalosporin group, for example, cephachlor, cephazafluoro, cephazoline, cephadoxyl, cefaxitine, cepuroxime, cephacetyl, cephalexin, cephalglycine, cephalodine, cephalotin, Sephamandol, Sephanon, Sephapirin, Sephatrizin, Sephadin, Ceproxadine (7β- [D-2-amino-2- (1,4-cyclohexadienyl) -acetamido] -3- Methoxy-3-cefe-4-carboxylic acid = GCP 9000), ceftsulodine, cephataxime, cethithiam, cepresol or cefazedone.

Examples of nocardycin include nocardicin A, and examples of thienamycin and clavulanic acid include thienamycin and clavulanic acid.

In aminoglycosides, in particular streptomycins such as streptomycin and streptomycin A, neomycins, for example neomycin B, tobramycins, for example tobramycin or dibecacin, kanamycin Amicasins, gentamicins (eg, mixtures of gentamycin A, C ₁ , C ₂ or C _1a ), or sisomycin or netyl, as well as amines (eg, mixtures of kanamycin A, B and C) Sisomycins such as mycin, and ribidomycin, ribocamycin, and paromomycin.

Specific examples of the tetracyclines include tetracycline, doxycycline, chlorotetracycline, oxytetracycline and metacycline.

Examples of macrolides include, for example, spiramycins such as maridomycin and spiramycin I, II and III, erythromycin, for example erythromycin, oleandomycin, for example oleomycin and tetera. Acetyloleandomycin lean and commycins such as lincomycin and clindamycin.

Polyene antibiotics include, in particular, amphotericin B and its methyl esters or nystatins.

Polypeptide antibiotics include, in particular, colistin, gramicidine S, polymyxin B, vernamycin, tyrotricin, biomycin or vancomycin.

Rifamycins include, in particular, rifamycin S, rifamycin SV, or rifamycin B or a semi-synthetic derivative thereof, in particular rifampicin.

In the present invention, in particular, X represents carbonyl and R ₁ represents optionally substituted alkyl or aryl, R ₂ , R ₃ , R ₄ and R ₆ represent hydrogen or lower alkyl, and R ₅ is hydrogen; Lower alkyl optionally substituted with hydroxy, lower alkoxy, mercapto, lower alkylthio or halogen; Cycloalkyl radicals each having 4 to 6 carbon atoms or cycloalkyl-lower alkyl; Optionally substituted phenyl or phenyl-lower alkyl: or heterocyclyl-lower alkyl each containing one or two nitrogen atoms, or alternatively R ₄ and R ₅ together represent alkylene of 3 or 4 carbon atoms, R ₇ represents hydrogen, one of A ₁ and A ₂ represents a radical of the following general formula (II), the other represents hydroxy, lower alkoxy, amino or lower alkylamino, or lower alkyl radical is hydroxy, It relates to a compound of formula (I) and a salt thereof, which represents an aminocarbonyl-lower alkylamino optionally substituted with carboxy and / or amino.

Wherein T represents -NH or -O and Y represents optionally substituted alkylene inserted with oxycarbonyl or iminocarbonyl and W represents hydroxy, lower alkanoyloxy, amino, alkanoylamino or oxy Substituted alkyl group having 6 or more carbon atoms.

In the present invention, in particular, X represents carbonyl, R ₁ represents lower alkyl optionally substituted with hydroxy, lower alkoxy or halogen, or phenyl optionally substituted with hydroxy, lower alkoxy, lower alkyl or halogen, R ₂ , R ₄ and R ₆ represent hydrogen, R ₃ represents hydrogen or lower alkyl, which is hydrogen; Lower alkyl having 1 to 3 carbon atoms optionally substituted with hydroxy, lower alkoxy, mercapto, lower alkylthio or halogen; Cycloalkyl or cycloalkyl-lower alkyl having a lower alkyl radical having 1 to 3 carbon atoms and each cycloalkyl having 4 to 6 carbon atoms; Phenyl-lower alkyl having 1 to 3 carbon atoms or phenyl optionally substituted with hydroxy, lower alkoxy or halogen, respectively; Or lower alkyl is heterocyclyl or heterocyclyl-loweralkyl having 1 to 3 carbon atoms, each containing one or two nitrogen atoms, and having 5 or 6 members, alternatively R ₄ and R ₅ together are alkylene having 3 or 4 carbon atoms; R ₇ represents hydrogen, one of A ₁ and A ₂ is a radical of the general formula (II) and the other represents hydroxy, lower alkoxy, amino or lower alkylamino, or lower alkyl radical is hydroxy. It relates to compounds of formula (I) and salts thereof, which represent amino carbonyl-lower alkyl amino optionally substituted by oxy, carboxy or amino group.

In the above formula

T represents -NH or -0,

Y represents a radical of the following general formula (III _a ), (III _b ), (III _c ) or (III _d ),

[Wherein Y ₁ and Y ₂ each represent optionally substituted lower alkylene and R ₈ represents hydrogen.]

W represents an alkyl group having 10 to 25 carbon atoms and having a hydroxy group, an alkanoyloxy group, an amino group or an alkanoylamino group at the 2-position.

In the present invention, in particular, X represents carbonyl, R ₁ represents lower alkyl or phenyl having 1 to 3 carbon atoms, R ₂ , R ₄ represents R ₆ hydrogen, and R ₃ represents hydrogen or C 1-3 Lower alkyl and R ₅ is hydrogen; Lower alkyl having 1 to 3 carbon atoms optionally substituted with hydroxy, methoxy, mercapto, methylthio or halogen; Phenyl or phenyl methyl, each optionally substituted with hydroxy, methoxy or halogen; Or heterocyclyl or heterocyclyl-methyl, each containing one or two nitrogen atoms, or alternatively R ₄ and R ₅ together represent trimethylene, R ₇ represents hydrogen, and in A ₁ and A ₂ One relates to a compound of formula (I) and a salt thereof, which represents a radical of the general formula (II), the other representing hydroxy, lower alkoxy, amino, lower alkylamino or amino carbonyl-lower alkylamino .

In the above formula

T represents -NH or -0

Y represents a radical of the general formula (III _a ) or (III _c ),

[In the above formula

R ₈ represents hydrogen,

Y ₁ and Y ₂ are each lower alkylene having 1 to 3 carbon atoms optionally substituted with hydroxy, lower alkoxy, mercapto or lower alkylthio, or optionally hydroxy, methoxy- or halogen-substituted phenyl lower alkyl- Lower alkyl; Or lower alkylene substituted by heterocyclyl or heterocyclyl-lower alkyl having 1 to 3 carbon atoms, each containing one or two nitrogen atoms, and having 5 or 6 members;

W represents an alkyl group having 10 to 25 carbon atoms in which the 2-position is substituted with hydroxy, lower alkanoyloxy, amino or alkanoylamino.

The present invention particularly relates to the novel muramyl peptides described in the Examples.

The novel compound of general formula (I) can be obtained by the following method.

That is, the compound of general formula (I) is obtained by condensing the compound of the following general formula (XIV) with the compound of the following general formula (XIV), and then removing any protecting group.

In the above formula,

One of A ₁ ′ and A ₂ ′ represents a group of the general formula —TY ₁ -M ₁ (XV), and the other represents etherified hydroxy or amino, lower alkylamino or aminocarbonyl-lower alkylamino .

Wherein X, R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , Y ₁ , Y ₂ and W are as described above, and the hydroxy group present in the starting material is easily Optionally protected with a removable group, one of M ₁ and M ₂ represents a free amino or hydroxy group or an active derivative thereof, and the other represents a carboxylic acid group or an activation derivative thereof.)

Easily removable protective groups include those known in the art of peptide and sugar chemistry. Of particular note are the protecting groups of the carboxy group which are substituted triphenylmethyl or benzhydryls of tertiary butyl, benzyl or lower alkoxykines such as halogen or methoxy, respectively, and the lower alkanoyl radicals as the protecting group of the hydroxy group. Acyl radicals such as acetyl, aroyl radicals such as benzoyl and radicals derived from patented carbonic acid derivatives such as benzyloxycarbonyl or lower alkoxycarbonyl, alkyl (especially tertiary butyl) nitro, lower Benzyl or tetrahydropyranyl optionally substituted with alkoxy or halogen, or optionally substituted alkylidene radicals which bond with oxygen atoms at the 4- and 6-positions of the glu-cosane group. Such alkylidene radicals are in particular lower alkylidene radicals such as ethylidene, isopropylidene or propylidene radicals, or alternatively are benzylidene radicals optionally substituted at the p-position.

The protecting group can be removed by a method known per se. Therefore, they can be removed by acid hydrolysis, and can be removed by hydrocracking in the presence of a noble metal catalyst such as benzyl or benzylidenedipotassium palladium or platinum catalyst.

Starting materials are known or can be prepared by methods known per se.

When one of the radicals M ₁ and M ₂ represents an amino group or an activation derivative thereof, the condensation reaction may for example react compound (XIV) in the form of an activated carboxylic acid with an amino compound (XVI) or the acid (XVI) ) Is reacted with a compound (XVI) in which an amino group is present in activated form. Activated carboxyl groups include, for example, acid anhydrides, preferably mixed acid anhydrides with carboxylic acid lower alkyl esters (e.g. carboxylic acid ethyl esters or isobutyl esters), acid azides, acid amides (e.g. Isoxoxazide) or activated esters. Examples of the activated esters are cyanomethyl ester, carboxymethyl ester, p-nitrophenyl dio ester, p-nitrophenyl ester, 2,4,5-trichlorophenyl ester pentachlorophenyl ester and N-hydroxysuccinimide Esters, N-hydroxy phthalimide esters, 8-hydroxyquinoline esters, 2-hydroxy-1,2-dihydroxy-1-ethoxy carbonylquinoline esters or N-hydroxypiperidine esters, or There is the obtained enol ester reacted with N-ethyl-5-phenylisooxazolium 3'-sulfonate. The activating ester is, if necessary, carbodiimide produced by the addition of N-hydroxysuccinimide, or 1-hydroxybenztriazole or 3-hydroxy-4 unsubstituted or substituted with halogen, methyl or methoxy. It can also be obtained with -oxo-3,4-dihydrobenz [d] -1,2,3-triazine.

The amino group is activated by, for example, reacting with phosphite.

Among the methods for reacting with activated acids, in particular N-ethyl-5-phenylisooxazolium 3'-sulfonate (woodward reagent K) or 2-ethoxy-1,2-dihydro-1-ethoxycarbonylquinoline or carbodii Reaction with mead is important.

If one of the radicals M ₁ and M ₂ represents a hydroxy group or an active derivative thereof, the reaction is carbo-imide, such as dicyclohexylcarbodiimide, and pyridine or dimethylaminopyridine, or trialkylami (e.g. trimethyl) In the presence of a dehydrating agent of an amine, such as an amine). In contrast, carboxylic acids are, for example, in the form of salts, with strong inorganic acids or organic acids such as hydrochloric acid (eg hydrochloric acid, bromic acid or iodide) or organic sulfonic acids such as p-toluenesulfonic acid or methanesulfonic acid or ethanesulfonic acid It can react with the reaction ester of the alcohol which is an ester.

The alcohol may also be reacted optionally with the activated carboxylic acid in the form of a salt such as a sodium salt or potassium salt. Activated carboxylic acids are especially anhydrides, inter alia mixed acid anhydrides, acid azides, halides or activated esters (e.g. cyano methyl esters, carboxymethyl esters, p-nitrophenylthio esters, p-nitrophenyl esters, 2,4,5- Trichlorophenyl ester, pentachlorophenyl ester, N-hydroxysuccinimide ester, N-hydroxy phthalimide ester, 8-hydroxy quinoline ester, 2-hydroxy-1,2-dihydro-1- Oxycarbonyl quinoline ester or N-hydroxy piperidine ester, or enol ester obtained from N-ethyl-5-phenylisooxazolium 3'-sulfonate). Activated esters may also be optionally substituted with 1-hydroxybenztriazole or 3-hydroxy-4-oxo-3,4-dihydrobenz [d] -1, each unsubstituted or substituted with halogen, methyl or methoxy. It can be obtained from 2,3-triazine.

The methods are carried out according to methods known per se under elevated pressure and / or inert air pressure, for example nitrogen pressure, with diluent in the absence, or preferably in the presence of solvent, cooling or heating, if necessary.

In the presence of all substituents present in the molecule, especially 0-acyl radicals, which are readily hydrolyzed, take into account these specially relaxed reaction conditions, eg short reaction times, use of weak acids or weak bases at low concentrations, stoichiometric ratios, and Selection of suitable catalysts, solvents, temperature and / or pressure conditions should be made.

The present invention also relates to a reaction process in which the starting material can be used in the form of a reactive derivative or salt. The starting materials used are preferably described as of particular importance, according to the above method.

The present invention also provides a medicament containing the compound of general formula (I). The medicament according to the present invention is an oral, nasal or rectal oral or parenteral agent for warm-blooded animals, containing the pharmacologically active ingredient alone or in combination with a pharmaceutically nontoxic carrier. The dose of active substance depends on the type of warm-blooded animal, the age and condition of the patient, and the method of administration.

The new pharmaceutical formulations contain from about 10 to about 95%, preferably from about 20 to about 90%, of the active substance. The pharmaceutical preparations according to the invention may be in unit dosage form, ie dragees, tablets, capsules, suppositories or ampoules.

The pharmaceutical preparations of the invention are prepared by methods known per se, i.e. by means of conventional mixing, granulating, coating, dissolving or lyophilizing. In addition to the dosage forms, in particular the pharmaceutical preparations for oral administration mix the active substance with a solid carrier, granulate the mixture obtained if necessary, and if necessary or necessarily add a suitable adjuvant, Can be obtained by purification or by processing into dragee nuclei. These agents can also be incorporated into synthetic carriers which release or diffuse the actives upon administration.

Suitable carriers are in particular sugars (eg lactose, saccharose, mannitol or sorbitol), cellulose preparations and or fillers such as calcium phosphate (eg tricalcium phosphate or potassium hydrogen phosphate); Binders such as starch waste yeast using millet, wheat, rice or potato starch, gelatin, tragacanth, methylcellulose, hydroxy propylmethyl cellulose, sodium carboxymethylcellulose and or polyvinylpyridone; And disintegrants such as starch, carboxymethyl starch, crosslinked polyvinylpyrrolidone, agar, alginic acid or salts thereof, such as sodium alginate, as described above. Auxiliaries are in particular flow regulators and lubricants, for example silica, talcum, stearic acid or salts thereof (eg magnesium stearate or calcium), and or polyethylene glycol. Dragee nuclei may optionally be coated with a suitable coating to be resistant to gastric fluids, among which are a thick sugar solution containing gum arabic, talcum, polyvinylpyrrolidone, polyethylene glycol and or titanium dioxide, and a suitable organic solvent or solvent mixture. Lacca solution is used, or a suitable cellulose formulation solution such as acetylcellulose phthalate or hydroxypropyl methylcellulose phthalate is used for the coating formulation which is resistant to placebo. Colorants or pigments may be added to the tablets or dragee coatings to identify the actives or indicate different dosages.

The following examples illustrate the invention and are not intended to limit the scope of the invention. Temperature is Celsius.

Compounds of formula (I) according to the invention cannot be characterized by melting points or spectroscopic values such as NMR and IR spectra.

In addition, the R _f value is also not suitable for accurate identification because of the predominant nature of the lipid site.

However, since the starting material structure is clearly known and the bonds between the atoms thereof are also distinct, the order of block formation of the final product and its structure are also clear.

Example 1

2 mmol of N-acetylmurayl-L-alanyl-D-isoglutaminyl-L-alanine N-hydroxysuccinimide S-ter solution in 6.5 ml of dimethyl acetamide was added to 1.4 mmol of 2- ( Hexadecyloxyhigh-hydroxy phosphoryloxy) ethylamide and 3 mmol of triethylamine are added dropwise to a solution dissolved in 25 ml of a mixture of chloroform, methanol and mol (65: 25: 4). After stirring at 20 ° C. for 18 hours, the solution is concentrated under reduced pressure to about 15 ml to form an emulsion. Dilute with 100 ml of these and lyophilize. The residue is suspended in 25 ml of water and dialyzed at 4 ° C. in the following order: 18 h for water, 24 h for 0.1 M NaCl solution in 0.1 M sodium phosphate buffer pH 7 and 48 h for water. The final dialysis results show that there is no forest in the internal dialysate. The internal dialysate containing the desired product is centrifuged at 10000 g and 20 ° C. for 30 minutes and the top layer is freeze-dried. The liberated product is chromatographed to give pure N-acetylmurayl-L-alanyl-D-isoglutaminyl-L-alanyl 2- (hexadecyloxyhydroxy-phosphoryloxy) ethylamide, which gives Na ⁺ Im present in the form. When thin layer chromatography on silica gel, the compound has the following R _f value: 0.22 (in chloroform / methanol / water, 65: 25: 4). The novel compounds are characterized analytically by N-acetylmuramic acid, hexadecane by quantitatively measuring the block formation of phosphate, Na ⁺ , L-alanine-D-glutamic acid.

N-acetylmuramic acid is determined spectroscopically using the Morgan-Elson reaction. See J.M. Ghuyson et al., "Methods in Enzymology" 9,629 (1966).

Phosphoric acid is determined politically according to the method of Lowry et al. [J. Biol. Chem. 207, 1 (1954).

Amino acids and hexadecanol are quantitatively determined in total hydrolysates (6N HCl, 24 h, 110 ° C.) using an amino acid analyzer or gas chromatography using norleucine or pentadecanol as internal standard reagents.

The molar ratio calculated from phosphoric acid is:

PO ₄ '''': N-acetylmuramic acid: L-alanine: D-glutamic acid: hexadecanol: Na ⁺ = 1: 0.93: 1.92: 0.91: 1.1: 0.94.

Example 2

In a similar manner to Example 1, the following compounds are obtained:

N-acetyldesmethylmurayl-L-alanyl-D-isoglutaminyl-L-alanine 2- (hexadecyloxyhigh-hydroxyphosphoryloxy) ethylamide-sodium salt, R _f = 0.17 (chloroform: Methanol: Water = 65: 25: 4), N-acetylmurayl-L-alanyl-D-isoglutaminyloxymethyl carboxylic acid 2- (hexadecyloxyhydroxyphosphoryloxy) ethylamide-sodium salt, N Acetylmuramil-L-alanyl-D-isoglutaminyl-L-alanyl-2- (tetradecyloxyhydroxyphosphoryloxy) ethylamide-sodium salt, R _f = 0.17 (chloroform: methanol: water = 65: 2: 4).

Example 3

In a method similar to Example 1, 2-[(3'R) -hydroxy- (2'S) -palmitoylamino-4't-octadecenyloxyhydroxyphosphoryloxy] ethylamine and the corresponding muramyl peptide -N-hydroxy succinimide ester is used to obtain the following compound: N-acetyldesmethylmuramil-L-alanyl-D-isogluramiminyl-L-alanine 2-[(3'R) hydr Roxy- (2'S) -palmitoylamino-4't-octadecenyloxyhydroxyphosphoryloxy] ethylamide-sodium salt, N-acetylmurayl-L-alanyl-D-isoglutaminyl-L- Alanine-2-[(3'R) -hydroxy- (2'S) -palmitoylamino-4't-octadecenyloxyhydroxyphosphoryloxy] ethylamide-sodium salt, N-acetylmurayl-L- Alanyl-D-isoglutaminyloxymethyl carboxylic acid 2-[(3'R) -hydroxy- (2'S) palmitoylamino-4't-octadecenyloxyhydroxyphosphoryloxy] ethylamide-sodium salt .

Example 4

a) 0.8 mmol of 1 mmol of 1α-benzyl-N-acetyl-4,6-isopropylidene desmethyl muramil-L-alanyl-D-isoglutamine 2-hydroxyethylamide according to the method of Steglich et al. Esterification with hexadecyloxyhydroxyphosphoryl oxyacetic acid (see B. Neises and W. Steglich, Amgew. Chem. 90, 556 (1978). After 20 hours at room temperature, a little water is added dropwise to filter the resulting dicyclohexylurea and the filtrate is evaporated to dryness in vacuo. Chromatography on silica gel (Merck) using chloroform / methanol (7: 3) affords a muramyl peptide / phospholipid conjugate from which the protecting group is liberated and purified by dialysis. N-Acetylnormurayl-L-alanyl-D-isoglutamine 2- (hexadecyloxyhydroxyphosphoryloxymethylcarbonyloxy) ethylamide is R _f = 0.27 (CHCl ₃ / methanol / H ₂ O = 65: 25: 4, obtained in the form of the semi-sodium salt of thin silica gel (merch plate).

b) 1α-benzyl-N-acetyl-4,6-isopropylidenenormuramyl-L-alanyl-D-isoglutamine 2-hydroxyethylamide used as starting material is 1α-benzyl-N-acetyl Obtained as a colorless amorphous material by condensation of -4, 6-isopropylidene normuramyl-L-alanyl-D-isoglutamine with 2-aminoethanol; [α] _D ²⁰ = + 85 ° (CHCl ₃ , C = 1), R _f = 0.38 (CHCl / methanol / H ₂ O = 70: 30: 5, thin silica gel (made by Merck))

The adduct 1α-benzyl-N-acetyl-4,6-isopropylidenenormutamyl-L-alanyl-D-isoglutamine shows an R _f value of 0.34 in the eluent. The corresponding R _f value at 42: 21: 21: 0.6: 10 phosphorus in ethyl acetate / n-butanol / pyridine / acetic acid / water is 0.50 adduct and 0.64 hydroxyethylamide.

Example 5

The following compounds are prepared analogously to the process described in Example 1:

N-Acetylmuramil-L-valyl-D-isoglutaminyl-L-alanine 2- (cholest-5-ene-3β-oxyhigh- hydroxyphosphotyloxy) ethylamide-sodium salt, N-propy Onildesmethylmuramil-L-alanyl-D-isoglutaminyl-L-alanine 2- (tetradecyloxyhydroxyphosphoryloxy) ethylamide-sodium salt, N-acetylmurayl-L-prolyl- D-isoglutaminyl-L-alanine 2- (tetradecyloxyhydroxyphosphoryloxy) ethylamide-sodium salt, N-acetyldesmethyl muramil-L-seryl-D-glutaminyl-L-alanine 2 -(Cholester-5-ene-3β-oxyhydroxyphosphoryloxy) ethylamide-sodium salt, N- (4-methylbenzoyl) muramyl glycyl-D-glutamyl-α-glycinamide γ-L-alanine 2- (tetradecyloxyhydroxy phosphoryloxy) ethylamide-sodium salt.

Example 6

Similar to the method of Example 1, N-acetylmuramil-L-alanyl-D-isoglutaminyl-L-alanine 2- [3'R] -hydroxy- (2'S) -palmitoyl amino octade Siloxy hydroxyphosphoryloxy] ethylamide and N-acetylmurayl-L-α-aminobutyryl-D-isoglutaminyl-L-alanine 2-[(3'R) -hydroxy- (2'S) Palmitoylaminooctadecyloxyhydroxyphosphoryloxy] ethylamide is obtained.

Example 7

Preparation of foodstuffs containing 0.005% of active ingredient:

Premix:

4.5 kg of vitamin premix contains: 16,000,000 IU Vitamin A, 1,000,000 IU Vitamin D ₃ , 5000 IU Vitamin E Acetate, 6g Vitamin K ₃ , 6mg Vitamin B ₁₂ , 3g Riboflavin, 30g Niacin, 4.5 kg of 5 g calcium pantothenate and 100 g ethoxyquine (1,2-dihydro-6-ethoxy-2,2,4-trimethyl-quiline), and corn flour.

Manufacturing method

The active substance and sugar are passed through a sieve of 0.6 mm mesh width and thoroughly mixed before being mixed with soy flour. The premix is added to the foodstuff in the amount corresponding to the desired concentration and homogenized with a horizontal drum mixer.

Example 8

Formulations are obtained by the addition of adjuvants and carriers per capsule of the active ingredients in the amounts specified below: 500 mg of cephalexin and 5 mg of N-acetylmuramil-L-alanyl-D-isoglutami Nyloxymethylcarboxylic acid 2- (hexadecyloxyhydroxyphosphoryloxy) ethylamide.

Claims (1)

A method for producing a muramyl peptide of the general formula (I) and salts thereof, characterized by condensing a compound of the general formula (XIV) with a compound of the general formula (XIV) and then removing any protective group.

Wherein X represents carbonyl or carbonyloxy, R ₁ represents optionally substituted alkyl or aryl, R ₂ , R ₄ and R ₆ represent hydrogen or lower alkyl, and R ₃ represents hydrogen or lower alkyl R ₅ represents hydrogen, lower alkyl, free or modified hydroxy-lower alkyl, free or modified mercapto-lower alkyl, optionally substituted amino-lower alkyl, cycloalkyl, cycloalkyl-lower alkyl, optionally substituted Aryl or aralkyl, or nitrogen-containing heterocyclyl or heterocyclyl-lower alkyl, or R ₄ and R ₅ together are alkylene having 3 or 4 carbon atoms, R ₇ is hydrogen or optionally esterified or amidated carboxyl One of A ₁ and A ₂ is a radical of the general formula (II)

[Wherein T is -NH or -0, Y is an optionally substituted alkylene group inserted into one oxycarbonyl and or aminocarbonyl group, and W is an aliphatic radical, or is each cycloalkyl having 6 or more carbon atoms or Cycloalkenyl radical] The other of A ₁ and A ₂ is free or etherified hydroxy, amino or lower alkylamino, or Among the lower alkyl radicals is optionally substituted aminocarbonyl-lower alkylamino, one of A ₁ ′ and A ₂ ′ is a radical of the general formula (XV), -TY ₁ -M ₁ (XV) The other is etherified hydroxy or amino, lower alkyl amino or aminocarbonyl-lower alkylamino, one of M ₁ and M ₂ represents a free amino or hydroxy group or an active derivative thereof, and the other is carboxylic acid Y ₁ and Y ₂ each represent an optionally substituted lower alkylene, wherein the hydroxy groups present in the starting material can be easily removed and optionally protected with a protecting group.