WO2007045192A1

WO2007045192A1 - Glucosaminylmuramic acid derivatives

Info

Publication number: WO2007045192A1
Application number: PCT/CZ2006/000068
Authority: WO
Inventors: Miroslav Ledvina; Daniel Zyka; Martina Dzoganova
Original assignee: Ustav Organicke Chemie A Biochemie Akademie Ved Ceske Republiky
Priority date: 2005-10-18
Filing date: 2006-10-18
Publication date: 2007-04-26
Also published as: CZ301451B6; CZ2005658A3

Abstract

Disaccharide synthon I (benzyl-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-2-acetamido-3-0-[1-(R)-carboxyethyl-2-deoxy-α-D-glucopyranoside) and its use for synthesis of 2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-N-acetylmuramic acid VIII and muramyl glycopeptides based on a GMDP molecule IX are described. The synthesis of compound I starts from glycosyl acceptor, the derivative of muramic acid II, which is prepared from compound III by 3-O-alkylation with 2-bromopropionic acid, then following esterification affords compound IV that on splitting off of the isopropylidene group affords the compound V, the partial benzoylation of which affords compound II. Reaction of compound II with 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl bromide affords disaccharide VI that is then transformed by sequential removing of the protecting groups, sensitive to alkali, to compound VII, the N-acetylation of which affords compound I. Synthon I is transformed to 2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-N-ace- tylmuramic acid VIII by hydrogenolytic splitting off of benzyl protecting group. Synthon I on reaction with bis(pentafluorophenyl)carbonate affords compound X that on reaction with peptide component affords compound XI, wherein X is a peptide residue. Removing of the protecting groups from compound XI affords muramyl glycopeptide based on a GMDP molecule of general formula IX, wherein X is a peptide residue.

Description

Glucosaminylmuramic acid derivatives

Technical field of the invention

The invention deals with glucosaminylmuramic acid (2-amino-2-deoxy-β-D-gluco- pyranosyl-(l→4)-iV-acetylmuramic acid) derivatives, method of their synthesis, and their use for the synthesis of glucosaminylmuramyl glycopeptides, i.e. disaccharide analogues of muramyl glycopeptides.

Background of the invention

Immunopharmaceutics represent a new group of drugs that already have an irreplaceable role in modern clinical practice. Disorders of the organism immune capacity due to negative changes in the environment, an increased occurrence of pathogenic microorganisms resistant to antibiotics, and infections damaged immunity (e.g. AIDS) as well as a frequent application of immunosuppressive therapeutic procedures such as radio- and chemotherapy of tumor diseases, just as following transplantations, present a serious medical problem. The present tendency to replace live vaccines by safer semi-synthetic, recombinant, and synthetic ones requires new structurally defined adjuvants. Therefore, considerable attention is devoted to the development of substances with immunostimulatory activity with the aim to obtain a) safe, structurally defined adjuvants for protein, peptide and DNA vaccines, b) stimulators of nonspecific antiinfectious and antitumor immunity, and c) stimulators of hemopoiesis.

Important representatives of such compounds are muramyl glycopeptides that are derived from peptidoglycan fragments of bacterial cell wall, i.e. compounds derived from the minimal immunoadjuvant unit, "muramyl dipeptide" (MDP), and basic repeating disaccharide-dipeptide unit, "glucosaminyl-muramyl-dipeptide" (GMDP).

During the past three decades many analogues of MDP and GMDP were synthesized in order to increase the specificity of therapeutic activity and to suppress undesired side effects, especially pyrogenicity. This effort led to compounds interesting for pharmaceutical industry (for review see ref. ^M). From MDP derived compounds, therapeutic Romurtid™ (MDP-Lys-L-18), based on MDP molecule prolonged in the peptide part by Lys substituted with stearoyl residue, is in the most progressed phase of development. Romurtid™ is less pyrogenic than MDP and successfully passed clinical trials. Principal practical application is stimulation of hemopoiesis in patients after radiotherapy or chemotherapy^3"5. Acceleration of neutrophil number normalization in periphery blood is important in order to suppress the risk of infection in immunosuppressed patients⁶'⁷. Romurtid™ is also able to increase natural immunity against viral⁸ and bacterial⁹'¹⁰ infections and tumor growth¹¹.

GMDP, in comparison with MDP, has higher immunoadjuvant activity and lower pyrogenicity. Hence, the compounds derived from GMDP are more perspective potential immunotherapeutics than compounds derived from MDP². GMDP-based compound Likopid™ is the first immunotherapeutic of the muramyl glycopeptide type introduced to the clinical practice. Likopid™ was developed and registered by a Russian company Peptek as an immunotherapeutic with broad applicability, e.g. immunostimulation and prevention of infections complicating post-traumatic, post-operative, post-chemotherapeutic and post- radiotherapeutic patienthood¹². Other areas are treatment of infectious diseases, as tuberculosis, human cervical papilomavirus, ophthalmic herpetic infections, psoriasis and treatment of ulcerous and inflammation processes. Likopid™ can be used alone or in combination with antibiotics and antivirotics¹³ for treatment of infectious diseases. Side effects are associated with pyrogenicity that stems from the active component GMDP.

Company ImmunoTherapeutics, Inc., North Dacota, USA, is developing lipophilic analogues of GMDP, e.g. "disaccharidetripeptide-glyceroldipalmitate" (DTP-GDP), modified in the carboxy-terminal part by glycerol-dipalmitate residue, and structurally related GMTP- N-DPG, modified in the carboxy-terminal part of the peptide chain by dipalmitoylpropylarnide residue. DTP-GDP was proposed as potential immunotherapeutic of tumor diseases¹⁴. The compound applied in liposomes is active in the therapy of liver tumors metastases ⁵. DTP-GDP passed successfully phase II of clinical trials, but is strongly pyrogenic¹⁵. Its main side effects, besides pyrogenicity, are shivers, nausea and hypotension. GMTP-N-DPG is a perspective adjuvans for cellular immunity usable for construction of new generation vaccines against some serious infectious diseases (AIDS, tuberculosis, malaria)¹⁶'¹⁷. Some of the above mentioned analogues are part of produced adjuvants. As examples can be given Gerbu Adjuvant™, containing GMDP (CC Biotech Corporation, USA), ImmTher™, containing DTP-GDP₅ and Theramide ™, containing DTP-DPP (GMTP-N- DPG) (ImmunoTherapeutics, USA); ref.¹⁸.

The accessibility of the carbohydrate component is the main problem in the synthesis of GMDP-derived murarnyl glycopeptides. The synthesis of oligosaccharides represents a challenging task, especially as for the sequences containing D-glucose units connected with β(l->4) glycosidic bond due to a low reactivity of the OH(4) group^19"23. Known synthetic approaches of disaccharide GMDP component represent a challenging process due to many synthetic steps and associated low affectivity. These syntheses can be divided into three groups (according to the glycosyl acceptor used for glycosylation reaction representing a key step in disaccharide unit synthesis): a) glycosylation with acyclic precursor of muramic acid²⁴'²⁵, b) glycosylation with cyclic precursor of muramic acid^26"28, and c) glycosylation with cyclic derivative of muramic acid^29"33. Therefore semisynthetic, in addition to de novo synthetic, approach is applied in the field of GMDP and its analogues synthesis, too. The sugar component preparation is based on a biotechnological process of preparation of bacterial mass by fermentation, by partial enzymatic hydrolysis of bacterial walls and difficult isolation of disaccharide unit by chromatographic techniques. The obtained 2-acetamido-2- deoxy-β-D-glucopyranosyl(l->4)N-acetylmuramic acid (β-D-GlcΝAc(l->4)MurΝAc) is then condensed with the peptide component¹⁷'^34"36. The semisynthetic approach is applied as by a Russian company Peptek for the preparation of the active component of Likopid™ (GMDP) and by ImmunoTherapeutics, Inc., USA, for preparation of GMDP analogues (DTP-GDP and GMTP-N-DPG) modified in carboxy terminal part of the peptide chain by lipophilic residue. In spite of illusive simple semisynthetic approach, their disadvantages are poor reproducibility and necessity of standardization of the biotechnological production process of disaccharide component. At the same time, reproducibility and standardization of production processes are the main criteria for the production of drugs and their substances.

A limiting factor for introduction of GMDP-based therapeutics into the practice is complicated accessibility of the disaccharide component, 2-acetamido-2-deoxy-β-D- glucopyranosyl(l→4)iV-acetylmuraniic acid (β-D-GlcNAc(l-»4)MurNAc), and their derivatives, respectively. Present invention solves this problem by elaboration of effective synthesis of disaccharide synthon enabling reasonable synthesis of GMDP-based muramyl glycopeptides as well as 2-acetamido-2-deoxy-β-D-glucopyranosyl(l->4)N-acetylmuramic acid itself.

Detailed description of the invention

a) One aspect of the invention relates to disaccharide synthon of the following formula I

b) Other aspect of the invention relates to synthetic pathway of preparation of disaccharide synthon of formula I.

The synthesis of the compound I starts with glycosyl donor, 3,4,6-tri-0-acetyl-2- deoxy-2-phthalimido-β-D-glucopyranosyl bromide, obtained in two steps synthesis according to art³⁷'³⁸, from commercially available D-glucosamine hydrochloride. As glycosyl acceptor, a muramic acid derivative, i.e. benzyl-2-acetamido-6-O-benzoyl-2-deoxy-3-O-[l-(R)-(metho- xycarbonyl)ethyl]-α-D-glucopyranoside of formula II

the synthesis of which is also a part of the invention, is used.

Glycosyl acceptor II synthesis starts from benzyl-2-acetamido-2-deoxy-4,6-O- isopropylidene-α-D-glucopyranoside lll

which is obtained in two synthetic steps as given in the art³⁹ from commercially available N- acetyl-D-glucosamine.

3-0-Alkylation of compound III on treatment with racemic bromopropionic acid or its (S)-stereoisomer in a polar aprotic solvent and in the presence of a strong base (with advantage in dioxan or tetrahydrofurane in the presence of sodium hydride) and the following esterification of the produced acid with diazomethane or of its salt with methyl iodide affords benzyl-2-acetamido-2-deoxy-4,6-O-isopropylidene-3-O-[l-(R)-(methoxycarbonyl)ethyl]-α- D-glucopyranoside IV.

IV

The acetal protecting group in compound IV is removed by acidic hydrolysis, advantageously by action of ion-exchanger Dowex 50 in H⁺ cycle in anhydrous methanol or by heating in aqueous acetic acid, affording benzyl-2-acetamido-2-deoxy-3-O-[l-(R)- (methoxycarbonyl)ethyl] -α-D-glucopyranoside V.

V

Partial benzoylation of compound V, in advantage with benzoylimidazole in dichloro- methane, affords glycosyl acceptor of formula II.

Reaction of 3,4,6-tri-(9-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl bromide, as glycosyl donor, with glycosyl acceptor II in aprotic solvent promoted by heavy metal salts (in advantage in dichloromethane in the presence of silver trifluoromethanesulfonate as promotor) afforded benzyl-3 ,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl- (1 →4)-2-acetamido-6-(9-benzoyl-2-deoxy-3 -O- [ 1 -(R)-(methoxycarbonyl)ethyl] -α-D-gluco- pyranoside VI.

Vl

Sequential removing of protecting groups sensitive to alkali (i.e. acetyl, benzoyl, methyl ester and phthalimide), in advantage on treatment with sodium methoxide in methanol, sodium hydroxide in water, and n-butylamine in methanol, respectively, affords benzyl-2- amino-2-deoxy-β-D-glucopyranosyl-(l-→4)-2-acetamido-2-deoxy-3-O-[l-(R)-carboxyethyl]- α-D-glucopyranoside VII.

VIl

iV-Acetylation of this compound VII with acetic acid anhydride in water affords disaccharide synthon of formula I. c) The next aspect of the invention relates to the use of disaccharide synthon I for synthesis of 2-acetamido-2-deoxy-β-D-glucopyranosyl-(l→4)-JV-acetyl muramic acid VIII.

2-Acetamido-2-deoxy-β-D-glucopyranosyl-(l→4)-iV-acetyl muramic acid VIII is synthesized on hydrogenolytic removing of ben2yl protecting groups, in advantage with palladium catalyst on charcoal in acetic acid, from disaccharide synthon of formula I.

d) The next aspect of the invention relates to the use of disaccharide synthon I for the synthesis of muramyl glycopeptides based on GMDP molecule of general formula IX

in which X is peptide residue.

Reaction of disaccharide synthon I with bis(pentafluorophenyl)carbonate in polar aprotic solvent in the presence of base, with advantage in N,iV-dimethylformamide in the presence of N-methylmorpholine, leads to benzyl-2-acetamido-2-deoxy-β-D-glucopyranosyl- (l→-4)-2-acetamido-2-deoxy-3-O-[l-(R)-(pentafluorophenoxycarbonyl)ethyl]-α-D-glucopyra- noside X.

Reaction of pentafluorophenyl ester X with peptide component in polar aprotic solvent in the presence of base, in advantage in N,N-dimethylformamide in the presence of tri- ethylamine or JV-methylmorpholine, affords protected glycopeptide XI

in which X is a peptide residue. For example, reaction with benzyl ester of L-alanyl-D-isoglu- tamine (available according to the ref.⁴⁰) affords benzyl ester of iV-{2-O-[benzyl-2-acetamido- 2-deoxy-β-D-glucopyranosyl-(l-→4)-2-acetamido-2,3-dideoxy-α-D-glucopyranosid-3-yl]-(R)- lactoyl}-L-alanyl-D-isoglutamine (XI wherein X is L-Ala-D-iso-Gln-OBn).

Hydrogenolytic removing of benzyl protecting group from reducing end of the saccharide part and protecting groups cleavable under these conditions from the peptide chain (benzyl or benzyloxycarbonyl groups) of glycopeptide of general formula XI (with advantage with the use of palladium catalyst on charcoal in acetic acid), affords glycopeptide IX. For example, hydrogenolysis of benzyl groups of benzyl ester of iV-{2-O-[benzyl-2-acetamido-2- deoxy-β-D-glucopyranosyl-(l— >4)-2-acetamido-2,3-dideoxy-α-D-glucopyranosid-3-yl]-(R)- lactoyl}-L-alanyl-D-isoglutamine (XI, wherein X is L-Ala-D-iso-Gln-OBn) with palladium catalyst on charcoal in acetic acid affords 2-acetamido-2-deoxy-β-D-glucopyranosyl-(l→4)- iV-acetylmuramyl-L-alanyl-D-isoglutamine (GMDP) (IX, wherein X is L-Ala-D-iso-Gln-OH).

The present invention is further illustrated by the following examples that are only illustrative and are not meant as any limitation of this invention. Examples

Example 1

Sodium hydride (9.0 g, 375 mmol) was added to a stirred solution of benzyl-2- acetamido-2-deoxy-4,6-0-isopropyliden-a-D-glucopyranoside III (24.6 g, 70 mmol) in anhydrous dioxane (270 ml) at room temperature and the mixture was heated for 2 h at 90 ⁰C under stirring. The mixture was cooled down to room temperature, 2-bromopropionic acid (9.0 ml, 100 mmol) is added and the mixture is heated under stirring for 6 h at 65 °C. Excess hydride after cooling down to room temperature was decomposed with water; solid carbon dioxide was added to the mixture under Stirling followed by adding of water (20 ml). The mixture was evaporated in vacuum. The remaining residue is dissolved in water (450 ml), and the solution obtained was with ice neutralized with 5% water solution of sodium hydrogen sulphate under stirring and cooling. Separated product was extracted with ethyl acetate (3 x 200 ml), the extract was dried with anhydrous magnesium sulphate and evaporated in vacuum to about 100 ml. A solution of diazomethane in diethyl ether was added to evaporated residue under stirring and cooling with ice up to permanent yellow colour. After 30 minutes, the excess diazomethane was decomposed with acetic acid, the mixture was evaporated in vacuum, and evaporated residue was chromatographed on silica gel column in ethyl acetate-toluen mixture (2:1).

Homogenous fraction with higher RF was evaporated in vacuum, and the obtained crystalline residue (19.6 g, 64,0 %), benzyl-2-acetamido-2-deoxy-4,6-O-isopropyliden-3-O- [l-(R)-(methoxykarbonyl)ethyl]-α-D-glucopyranoside IV₅ is without next purification deprotected (isopropylidendioxy group is removed; see Examples 3 and 4). Crystallization of the residue from toluene-diethyl ether-petrolether mixture afforded an analytical sample of compound IV in the yield 76.5 %; m.p. 123 °C, [α]_D + 139 (c 0.5, chloroform). For C₂₂H₃₁NOg calculated: relative molecular mass 437.5, monoisotopic mass 437.2. FAB MS, mlz: 438.4 [M + H]⁺, 460.3 [M + Na]⁺. For C₂₂H₃iNO₈ (437.5) calculated: 60.40 % C; 7.14 % H; 3.20 % N; found: 60.23 % C₅ 7.28 % H, 3.27 % N.

Evaporation of homogenous fraction with lower Rp value afforded 9.0 g (29.4 %) treacly residue, benzyl-2-acetamido-2-deoxy-4,6-<9-isopropyliden-3-0-[ 1 -(S)-(methoxycar- bonyl)ethyl]-α-D-glucopyranoside, that crystallized on standing; m.p. 83 °C ; [α]o + 77 (c 0.5, chloroform). For C₂₂H₃INOs calculated: relative molecular mass 437.5, monoisotopic mass 437. FAB MS, mlz: 438.3 [M + H]⁺, 460.3 [M + Na]⁺. For C₂₂H₃₁NO₈ (437.5) calculated: 60.40 % C; 7.14 % H; 3.20 % N; found: 60.51 % C, 7.23 % H₅ 3.18 % N.

Example 2

Sodium hydride suspended in mineral oil (60%, 1.2 g, 30 mmol) under argon is washed with anhydrous hexane (3 x 5 ml,) and then suspended in anhydrous tetrahydrofuran (10 ml). A solution of benzyl-2-acetamido-2-deoxy-4,6-(9-isopropyliden-α-D- glucopyranoside III (3.5 g, 10 mmol) in anhydrous tetrahydrofuran (20 ml) is added to the mixed suspension at room temperature and the mixture is heated for 2.5 h at 50 °C under stirring. A solution of (S)-2-bromopropionic acid (1.1 ml, 12 mmol) in anhydrous tetrahydrofuran (10 ml) is added to the mixture during 2 hours and the mixture is heated for the next 8 h at 50 ⁰C under stirring. The mixture is cooled down to room temperature, methyl iodide (1.0 ml, 16 mmol) is added under stirring, and the mixture is stirred 24 h at room temperature. An excess hydride is decomposed with anhydrous methanol (2.0 ml) and solid carbon dioxide and the resulted mixture is evaporated in vacuum. Evaporated residue is extracted with ethyl acetate (60 ml) and water (20 ml), organic phase is separated, washed with water (2 x 20 ml), dried over anhydrous magnesium sulphate, and evaporated in vacuum. The evaporated residue is filtered through silica gel column in a mixture of ethyl acetate-toluene (2:1) and filtrate is evaporated in vacuum. Crystallization of the product from toluene-diethyl ether-petrolether mixture afforded 3.3 g (75.4 %) of benzyl-2-acetamido-2- deoxy-4,6-+O-isopropyliden-3-O-[l-(R)-(methoxycarbonyl)ethyl]-α-D-glucopyranoside IV, identical with compound IV prepared in Example 1.

Example 3

Ion exchanger Dowex 50 in H⁺ cycle (35 g) is added to a solution of benzyl-2- acetamido-2-deoxy-4,6-O-isopropyliden-3-O-[l-(R)-(methoxycarbonyl)ethyl]-α-D-glucopy- ranoside IV (87.5 g, 200 mmol) in anhydrous methanol (500 ml) and this mixture is stirred 2 h at room temperature. Reaction is monitored by TLC in chloroform-methanol system (10:1). Ion exchanger is filtered off, washed with methanol (2 x 400 ml), and the filtrate was f evaporated in vacuum. Yield 78.7 g (99 %) of crystalline benzyl-2-acetamido-2-deoxy~3-<9- [l-(R)-(methoxycarbonyl)ethyl]-α-D-glucopyranoside V that was without any purification treated with benzoylimidazol as given in Example 5. Crystallization of the product from ethyl acetate-diethyl ether afforded an analytical sample of compound V; m.p. 134 - 138 °C, [α]π + 152 (c 0.5, chloroform); lit.⁴¹ m.p. 130 - 132 ⁰C, [αfo + 145 (c 1.0, chloroform); lit.⁴² m.p. 120 - 122 °C, [α]_D + 137 (c 0.9, chloroform); lit.⁴³ [α]_D + 127 (c 1.0, chloroform). For C₁₉H₂₇NO₈ calculated: relative molecular mass 397.4, monoisotopic mass 397.2. ESI MS, m/z: 420.3 [M + Na]⁺. For C₁₉H₂₇NO₈ (397.4) calculated: 57.42 % C₅ 6.85 %H, 3.52 % N; found: 57.17 % C, 6.88 % H, 3.49 % N.

Example 4

Benzyl-2-acetamido-2-deoxy-4,6-(9-isopropyliden-3-0-[l-(R)-(methoxycarbonyl)- ethyl]-α-D-glucopyranoside IV (8.8 g, 20 mmol) is heated under stirring in a mixture acetic acid-water (1:1, 80 ml) for 30 min at 90 °C. The mixture was evaporated in vacuum. Evaporated residue was crystallized from ethyl acetate-diethyl ether mixture. Yield 6.2 g (78.0 %) of benzyl-2-acetamido-2-deoxy-3-O-[l-(R)-(methoxycarbonyl)ethyl]-α-D-glucopy- ranoside V, identical with compound V prepared according to example 3

Example 5

A solution of benzimidazol (37,9 g, 220 mmol) in anhydrous dichlormethane (150 ml) was added to a mixed suspension of benzyl-2-acetamido-2-deoxy-3-0-[l-(R)-(methoxy- carbonyl)ethyl]-α-D-glucopyranoside V (79.5 g, 200 mmol) in anhydrous dichlormethane (500 ml) at 0 ⁰C. A mixture is mixed for 30 min at 0 °C and then left to stand for at least 24 h at room temperature. The reaction process is monitored with TLC in chloroform-methanol system (10:1) and in toluene-ethyl acetate system (1:1). The mixture is diluted with chloroform (1000 ml) and the obtained solution is washed with 5% sodium hydrogen sulphate in water (700 ml), water (1000 ml) and saturated sodium chloride solution in water (1000 ml), dried over anhydrous magnesium sulphate and evaporated in vacuum. Yield 99.3 g (99 %) of crystalline benzyl-2-acetamido-6-0-benzoyl-2-deoxy-3-O-[l-(R)-(methoxycarbonyl)ethyl-α-D-glucopy- ranoside II which is possible to use without any purification as glycosyl acceptor for disacharide synthesis (see Example 6). Crystallization of the product from toluene-heptane mixture afforded an analytical sample of compound II in the yield of 90 %; m.p. 133-135 ⁰C, [α]_D + 85 (c 0,3, chloroform). Lit.⁴⁴ gives m.p. 98 - 100 ⁰C, [α]_D + 81 (c 1.0, chloroform). For C₂₆H₃₁NO₉ calculated: relative molecular mass 501.5, monoisotopic mass 501.2. FAB MS, m/z: 502.1 [M + H]⁺, 524.3 [M + Na]⁺. For C₂₆H₃₁NO₉ (501.5) calculated: 62.27 % C, 6.23 % H, 2.79 % N; found: 62.58 % C, 6.45 % H, 2.66 % N.

Example 6

A mixture of benzyl-2-acetamido-6-O-benzoyl-2-deoxy-3-O-[l-(R)-(methoxycarbo- nyl)ethyl]-α-D-glucopyranoside II (50.2 g, 100 mmol) and silver trifluoromethansulfonate (46.3 g, 180 mmol) is dried for 6 h at room temperature at 1.32 Pa. Anhydrous dichlorniethan (300 ml) is added to the mixture under argon. The mixture is cooled down to -30 °C and during 1 h under stirring a solution of 3,4,6-tri-O-acetyl-2-deoxy-2-ftalimido-β-D-glucopyi-a- nosyl bromide (89.7 g, 180 mmol) in anhydrous dichlormethane (300 ml) is added. The mixture is stirred for 1 h at -30 °C and for 2 h at -20 ⁰C. Pyridine (50 ml) is then added to the mixture under stirring at -20 ⁰C and after rising of temperature to room temperature the mixture is diluted with chloroform (1000 ml) and filtered. The filtrate is washed with cool (+4 °C) saturated aqueous sodium hydrogen carbonate (1000 ml), water (1000 ml) and saturated aqueous sodium chloride solution (1000 ml), dried over anhydrous magnesium sulphate, evaporated at vacuum a co-distilled with toluene (2 x 500 ml) in vacuum. Column chromatography of the residue in ethyl acetate-toluene (2:1) system afforded 78.1 g (85.0 %) of benzyl-3 ,4,6-tri-O-acetyl-2-deoxy-2-ftalimido-β-D-glucopyranosyl-( 1 →4)-2-acetamido-6-0- benzoyl-2-deoxy-3-O-[l-(R)-(methoxycarbonyl)ethyl]-α-D-glucopyranoside VI as solid foam; [OC]_D +37 (c 0.1, chloroform). For C₄₆H₅₀N₂O₁₈ calculated: relative molecular mass 918.9, monoisotopic mass 918.3. ESI MS, m/z: 941.5 [M + Na]⁺. For C₄₆H₅₀N₂O₁₈ (918.9) calculated: 60.13 % C, 5.48 % H, 3.05 % N; found: 60.33 % C, 5.45 % H, 2.86 % N.

Example 7

A solution of ben2yl-3,4,6-tri-O-aceryl-2-deoxy-2-ftalimido-β-D-glucopyranosyl-(l→4)- 2-acetamido-6-O-benzoyl-2-deoxy-3-6>-[l-(R)-(methoxycarbonyl)ethyl-α-D-glucopyranoside VI (18.4 g, 20 mmol) in 0.01M sodium methoxide solution in anhydrous methanol (500 ml) is left to stand for 48 h at laboratory temperature and the mixture is neutralized by adding of ion exchanger Dowex 50 in pyridine cycle. Ion exchanger is filtered off, washed with methanol (200 ml) and filtrate was evaporated in vacuum. Evaporated residue is heated under stirring in 0,25M aqueous sodium hydroxide solution (550 ml) at 60 ⁰C for 3.5 h. After cooling down to room temperature the mixture is neutralized with ion exchanger Dowex 50 in pyridine cycle. Ion exchanger is filtered off, washed with water (100 ml), the eluate was evaporated in vacuum, and the evaporated residue is co-distilled at vacuum with anhydrous methanol (3 x 80 ml). The evaporated residue is dissolved in methanol and n-butylamine mixture (4:1, 300 ml) and the solution is heated in pressure tank for 11 h at 85-90 ⁰C. After cooling down to room temperature, the mixture is evaporated in vacuum and the residue is extracted with diethyl ether (3 x 150 ml). Insoluble residue is dissolved in water (250 ml), pH solution is adjusted with formic acid to pH 4 and the solution is applied to ion exchanger Dowex 50 column in ammonium cycle (800 ml). The column is washed with water (1500 ml) and product is liberated from ion exchanger washing with 5% aqueous ammonia (1000 ml). The eluted fraction was evaporated in vacuum. The obtained benzyl-2-amino-2-deoxy-β-D-glucopyranosyl-(l→4)-2-acetamido-2-deoxy-3-O- [l-(R)-carboxyethyl]-α-D-glucopyranoside VII is N-acetylated without any next purification (see Example 8). An analytical sample of compound VII is prepared on crystallization from ethanol; m.p. 183-185 °C; [α]_D +109 (c 0.2, water). For C₂₄H₃₆N₂O₁₂ calculated: relative molecular mass 544.6, monoisotopic mass 544.2. ESI MS, m/z: 545.0 [M + H]⁺, 567.3 [M + Na]⁺.

Example 8

Acetic acid anhydride (20 ml) is stepwise added to a stirred benzyl-2-amino-2-deoxy-β- D-glucopyranosyl-(l-→4)-2-acetamido-2-deoxy-3-O-[l-(R)-carboxyethyl-α-D-glucopyranosi- de VII (obtained in Example 7) solution (160 ml) at room temperature during 15 min. After 30 min the next portion of acetic acid anhydride is added (20 ml), and the stirring continues for 2 h. The solution was evaporated in vacuum, the evaporated residue is dissolved in water and this solution is applied to ion exchanger Dowex 50 column in pyridinium cycle (300 ml). The column is washed with water (700 ml), and eluted portion was evaporated in vacuum. ^' The residue is lyofilized from water. The yield is 7.1 g (60.5 %) of benzyl-2-acetamido-2- deoxy-β-D-glucopyranosyl-(l-→4)-2-acetamido-2-deoxy-3-O-[l-(R)-carboxyethyl]-α-D- glucopyranoside I, in relation to compound VI; [α]p + 70 (c 0.2, water). For C₂₆H₃₈N₂O₁₃ calculated: relative molecular mass 586.6, monoisotopic mass 586.2. ESI MS, m/z: 609.4 [M + Na]⁺. For C₂₆H₃₈N₂O₁₃ (586.6) calculated: 53.24 % C, 6.53 %H, 4.78 % N; found: 53.05 % C₅ 6.82 % H₅ 4.55 % N.

Example 9

Bis(pentafluorophenyl)carbonate (434 mg, 1.1 mmol) and 4-methylmorfoline (0.11 ml, 1 mmol) were added to a stirred solution of benzyl-2-acetamido-2-deoxy-β-D-gluco- pyranosyl-(l →4)-2-acetamido-2-deoxy-3-0-[l -(R)~carboxyethyl-α-D-glucopyranoside I (587 mg, 1 mmol) in iV,iV-dimethylformamide (4 ml) at room temperature, and the mixture is stirred for one hour. Reaction process is monitored by TLC in ethanol. Dioxan (20 ml) is then added and the mixture is lyofilized. A repeated lyofilization of the product from dioxan afforded 723 mg (96 %) benzyl-2-acetamido-2-deoxy-β-D-glucopyranosyl-(l-→4)-2-acetami- do-2-deoxy-3-O-[l-(R)-(pentafluorophenoxycarbonyl)ethyl]-α-D-glucopyranoside X that is, without any purification, condensed with a peptide component (see Example 10). For (C₃₂H₃₇F₅N₂O_1S) calculated: relative molecular mass 752.6, monoisotopic mass 752.2. ESI MS, m/z: 775.6 [M + Na]⁺.

Example 10

A solution of L-Ala-D-iso-Gln-OBn (615 mg, 2 mmol) and iV-methylmorfoline (0.30 ml, 2.73 mmol) in pyridine (6 ml) at 0 ⁰C is added to a stirred solution of ben2yl-2-acetamido- 2-deoxy-β-D-glucopyranosyl-(l→-4)-2-acetamido-2-deoxy-3-O-[l-(R)-(pentafluorofenoxycar- bonyl)ethyl]-α-D-glucopyranoside X (753 mg, 1 mmol) and the stirring continues for 20 h at +5 ⁰C. The mixture was evaporated in vacuum, the evaporated residue is co-distilled in vacuum with acetic acid (10 ml), and the residue is chromatographed on silica gel Cl 8 column in water-methanol system (linear gradient: 0→30 %, 60 min). Lyofilization of evaporated residue of homogenous fraction from water afforded 744 mg (85.0 %) benzyl ester of N-{2-0-[benzyl-2-acetamido-2-deoxy-β-D-glucopyranosyl-(l→4)-2-acetamido-2,3- dideoxy-α-D-glucopyranosid-3-yl]-(R)-lactoyl}-L-alanyl-D-isoglutamine of formulae IX (wherein X is L-Ala-D-iso-Gln-OBn); [α]_D +50 (c 0.4, 50% methanol in water). For (C₄₁H₅₇N₅O₁₆) calculated: relative molecular mass 875.9, monoisotopic mass 875.4. ESI MS, m/z: 898.4 [M + Na]⁺. For C₄IH₅₇N₅O₁₆ (875.9) calculated: 56.22 % C₅ 6.56 % H, 8.00 % N; found: 55.81 % C₅ 6.60 % H₅ 7.90 % N. Example 11

Benzyl ester of N-{2-O-[benzyl-2-acetamido-2-deoxy-β-D-glucoρyranosyl-(l→4)-2- acetamido-2,3-dideoxy-α-D-glucopyranosid-3-yl]-(R)-lactoyl}-L-alanyl-D-isoglutamine XI (876 mg, 1 mmol) is hydrogenolyzed with hydrogen on 5% palladium catalyst on charcoal (300 mg) in acetic acid (14 ml) at room temperature 48 h. After washing out of apparatus with argon, the catalyst is filtered off, washed with 50% acetic acid (30), and filtrate is evaporated in vacuum. The evaporated residue is chromatographed at silica gel Cl 8 column in water- methanol system (linear gradient: 0→20 %, 60 min). Lyofilization of the residue of homogenous fraction from water afforded 612 mg (88 %) 2-acetamido-2-deoxy-β-D- glucopyranosyl-(l-→4)-iV-acetylmuramyl-L-alanyl-D-isoglutamm (GMDP) IX (in which X is L-Ala-D-iso-Gln-OH); [α]_D -3 (c 0.3, water); lit.²⁷ [α]_D -2 (c 1.0, water). For (C₂₇H₄₅N₅O₁₆) calculated: relative molecular mass 695.7, monoisotopic mass 695.3. ESI MS, m/z: 718.5 [M + Na]⁺. For C₂₇H₄₅N₅O₁₆ (695.7) calculated: 46.62 % C, 6.52 % H, 10.07 % N; found: 46.21 % C, 6.85 % H, 9.96 % N.

Example 12

Ben2yl-2-acetamido-2-deoxy-β-D-glucopyranosyl-(l— >4)-2-acetamido-2-deoxy-3-O- [l-(R)-carboxyethyl]-α-D-glucopyranoside I (5.9 g, 10 mmol) is hydrogenolyzed with hydrogen over 5% palladium catalyst on charcoal (3 g) in acetic acid (140 ml) at room temperature for 48 h. The apparatus was washed out with argon, the catalyst was filtered off, washed with 50% acetic acid (300 ml), and filtrate was evaporated in vacuum. The evaporated residue is cliromatographed on silica gel Cl 8 column in water-methanol system (linear gradient: 0→20 %, 60 min). Lyofilization of the evaporated residue of homogenous fraction afforded 4667 mg (94 %) of 2-acetamido-2-deoxy-β-D-glucopyranosyl-(l-→4)-iV- acetylmurarnic acid VIII; [α]_D +5 (c 0.3, water); lit.²⁹ [α]_D +4 (c 1.0, water). For (C_1PHs₂N₂O₁₃) calculated: relative molecular mass 496.5, monoisotopic mass 496.2. ESI MS, m/z: 519,4 [M + Na]⁺. For C₁₉H₃₂N₂O₁₃ (496,5) calculated: 45.97 % C₅ 6.50 % H, 5.64 % N; found: 45.57 % C, 6.72 % H, 5.43 % N.

The structures of all above given compounds are unambiguously proved by their ¹H a ¹³C NMR spectra. Industrial utility

The compounds of the present invention can be used in basic research, pharmaceutical industry and both in human and veterinary medicine.

Formulae:

IV

V

VII

Xl

REFERENCES

1. Adam A.Adam A., in: Synthetic Adjuvants, p. 1. John Wiley and Sons, New York 2004.

2. Andronova T.M., Ivanov V.T.: Sov. Nedical. Review D. Immunology 1991, 4, 1.

3. Azuma L: Vaccine 1992, 10, 1000.

4. Baschang G.: Tetrahedron 1989, 45, 6331.

5. Hiraoka A., Masaoka T., Satoh T., Ota K., Oyama A., Horiuchi A., Hasegawa H., Nagai K., Kosaki M., Kanemaru A.: Arzneimittel-Forschung 1988, 55,1499.

6. Furuse K., Sakuma A. : Arzneimittel-Forschung 1989, 39,915.

7. Khaidukov S., V, Komaleva R.L., Nesmeyanov V.A. : International Journal of Immunopharmacology 1995, 77,903.

8. Yoo Y.C., Yoshimatsu K., Koike Y., Hatsuse R., Yamanishi K., Tanishita O., Arikawa J., Azuma L: Vaccine 1998, 16, 216.

9. Namba K., Nakajima R., Otani T., Azuma I. : Vaccine 1996, 7^,1149.

10. Niki Y., Tatara O.: Advances in Experimental Medicine and Biology 1992, 319,185.

11. Yoo Y.C., Park S. Y., Lee K.B., Azuma L: Journal of Microbiology and Biotechnology 2000, 10, 399.

12. Aston R. ((Biokine Technology Ltd., UK): WO 92-GB2137.9310148;

13. Aston R., Maitchouk L, Andronova T.M. ((Peptech (Uk) Limited, UK): WO 95- GB2237.9609063;

14. Vosika GJ., Cornelius D. A., Bennek J.A., Sadlik J.R., Gilbert CW. : MoI. Biother. 1990, 2, 50.

15. Vosika G. J., Cornelius D. A., Gilbert C. W., Sadlik J.R., Bennek J. A., Doyle A., Hertsgaard D.: J. Immunother. 1991, 10, 256.

16. Fast DJ., Vosika G.J.: Vaccine 1997, 15, 1748. W

17. Vosika GJ. ((Endorex Corporation, USA): WO 97-US8146.9743308;

18. M.F.Powell, M. J. Newman Eds.Vaccine design, (M.F.Powell, M. J. Newman Eds.), p. 141. Plenum Press, New York 1995.

19. Banoub J., Boullanger P., Lafont D. : Chemical Reviews 1992, 92, 1167.

20. Garegg PJ.: Advances in Carbohydrate Chemistry and Biochemistry, VoI 52 1997, 52, 179.

21. Paulsen H.: Angewandte Chemie-International Edition in English 1982, 21, 155.

22. Schmidt R.R., Kinzy W.: Advances in Carbohydrate Chemistry and Biochemistry, VoI 50 1994, 50, 21.

23. Sinay P. : Pure and Applied Chemistry 1991 , 63, 519.

24. Inage M., Imoto M., Kambayashi Y., Kusumoto S., Shiba T.: Tetrahedron Letters 1980, 21, 3767.

25. Kusumoto S., Yamamoto K., Imoto M., Inage M., Tsujimoto M., Kotani S., Shiba T.: Bulletin of the Chemical Society of Japan 1986, 59, 1411.

26. Durette P.L., Meitzner E.P.: Carbohydr. Res. 1981, 89, 279.

27. Kiso M., Kaneda Y., Shimizu R., Hasegawa A., Azuma L: Carbohydr. Res. 1982, 104,

253.

28. Durette PX. ((Merck and Co., Inc. USA): EP 80-400566.18901 ;

29. Kantoci D., Keglevic D., Derome A.E.: Carbohydr. Res. 1987, 162, 227.

30. Kusumoto S., Imoto M., Ogiku T., Shiba T.: Bulletin of the Chemical Society of Japan 1986, 59, 1419.

31. Termin A., Schmidt R.R. : Liebigs Annalen der Chemie 1989,789.

32. Termin A., Schmidt R.R.: Liebigs Annalen der Chemie 1992,527.

33. Vosika GJ. ((Endorex Corporation, USA): WO 96-US 15391.9712894; 34. Bezrukova EJ. ((Bezrukov, Mikhail Vasilievich Russia and Bezrukova, Elena Jurievna): WO 96-RU254.9710259;

35. Bistricenko A. ((Bistricenko, Andrejs Latvia and Jerniolajevs, Jevgenijs): LV 94- 940018.10963;

36. Vosika GJ. ((ImmunoTherapeutics, Inc. USA): WO 89-US302.9000396;

37. Farkas J., Ledvina M., Brokes J., Jezek J., Zajicek J., Zaoral M.: Carbohydr. Res. 1987, 163, 63.

38. Vesely J., Ledvina M., Jindfich J., Saman D., Trnka T.: Collect. Czech. Chem. Commun. 2003, 68, 1264.

39. Ledvina M., Farkas J., Zajicek J., Jezek J., Zaoral M.: Collect. Czech. Chem. Commun. 1989, 54, 2784.

40. Zaoral M., Jezek J., Straka R., Masek K.: Collect. Czech. Chem. Commun. 1978, 43, 1977.

41. Keglevic D., Pongracic M.: Carbohydr. Res. 1984, 135, 85.

42. Flowers H.M., Jeanloz R.: Jouranal of Organic Chemistry 1963, 28, 2983.

43. Arita H., Fukukawa K., Matsushima Y. : Bulletin of the Chemical Society of Japan 1972, 45, 3611.

44. Hasegawa A., Kaneda Y., Amano M., Kiso M., Azuma L: Agricultural and Biological Chemistry 1978, 42, 2187 '.

Claims

1. Disaccharide synthon I

2. Method of synthesis of compound I according to the claim 1, comprising as glycosyl acceptor the derivative of muramic acid of formula II

which is prepared from compound III

which is 3-O-alkylated on treatment with 2-bromopropionic acid and then transformed on esterifϊcation to compound IV

IV

which is then converted by splitting off of the isopropylidene group to compound V

which is then transformed on partial benzoylation with benzoylimidazole to glycosyl acceptor of formula II

Il

which on reaction with 3,4,6-tri-(9-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl- bromide, as glycosyl donor, affords disaccharide VI

Vl

which is then transformed by gradual removing of protecting groups, sensitive to alkali, e.g. acetyl, benzoyl, methyl ester and phthalimide groups, to compound VII

VII

which on N^~-acetylation affords disaccharide synthon of formula I.

3. Use of disaccharide synthon I according to the claims 1 and 2 for the synthesis of 2- acetamido-2-deoxy-β-D-glucopyranosyl-(l→4)--V-acetylniuramic acid VIII

which is obtained on hydrogenolytic removing of benzyl protecting group.

4. Use of disaccharide synthon I according to the claims 1 and 2 for the synthesis of muramyl glycopeptides based on a GMDP molecule of general formula IX

in which X is a peptide residue, wherein the disaccharide synthon I is transformed on treatment with bis(pentafluoro- phenyl)carbonate to compound X

which is then on treatment with peptide component transformed to compound of general formula XI

Xl

in which X is a peptide residue, and this compound on removing of protecting groups, under conditions given by their character, affords compound X

in which X is a peptide residue.