Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K9/00—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof
  • C07K9/001—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof the peptide sequence having less than 12 amino acids and not being part of a ring structure
  • C07K9/005—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof the peptide sequence having less than 12 amino acids and not being part of a ring structure containing within the molecule the substructure with m, n > 0 and m+n > 0, A, B, D, E being heteroatoms; X being a bond or a chain, e.g. muramylpeptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H5/00—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium
  • C07H5/04—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium to nitrogen
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
  • Y02P20/00—Technologies relating to chemical industry
  • Y02P20/50—Improvements relating to the production of bulk chemicals
  • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

• the invention deals with glucosaminylmuramic acid (2-amino-2-deoxy- ⁇ -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.
• 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.
• 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).
• MDP minimal immunoadjuvant unit
• GMDP basic repeating disaccharide-dipeptide unit
• GMDP basic repeating disaccharide-dipeptide unit
• 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 MDP 2 .
• GMDP-based compound LikopidTM is the first immunotherapeutic of the muramyl glycopeptide type introduced to the clinical practice. LikopidTM 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 patienthood 12 .
• LikopidTM can be used alone or in combination with antibiotics and antivirotics 13 for treatment of infectious diseases. Side effects are associated with pyrogenicity that stems from the active component GMDP.
• DTP-GDP 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 diseases 14 .
• 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 pyrogenic 15 .
• 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.
• Gerbu AdjuvantTM containing GMDP (CC Biotech Corporation, USA), ImmTherTM, containing DTP-GDP 5 and Theramide TM, 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 ⁇ (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.
• 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- ⁇ -D-glucopyranosyl(l->4)N-acetylmuramic acid ( ⁇ -D-Glc ⁇ Ac(l->4)Mur ⁇ Ac) is then condensed with the peptide component 17 ' 34"36 .
• a limiting factor for introduction of GMDP-based therapeutics into the practice is complicated accessibility of the disaccharide component, 2-acetamido-2-deoxy- ⁇ -D- glucopyranosyl(l ⁇ 4)iV-acetylmuraniic acid ( ⁇ -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- ⁇ -D-glucopyranosyl(l->4)N-acetylmuramic acid itself.
• glycosyl donor 3,4,6-tri-0-acetyl-2- deoxy-2-phthalimido- ⁇ -D-glucopyranosyl bromide, obtained in two steps synthesis according to art 37 ' 38 , from commercially available D-glucosamine hydrochloride.
• glycosyl acceptor a muramic acid derivative, i.e. benzyl-2-acetamido-6-O-benzoyl-2-deoxy-3-O-[l-(R)-(metho- xycarbonyl)ethyl]- ⁇ -D-glucopyranoside of formula II
• Glycosyl acceptor II synthesis starts from benzyl-2-acetamido-2-deoxy-4,6-O- isopropylidene- ⁇ -D-glucopyranoside lll
• 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] - ⁇ -D-glucopyranoside V.
• Sequential removing of protecting groups sensitive to alkali i.e. acetyl, benzoyl, methyl ester and phthalimide
• 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- ⁇ -D-glucopyranosyl-(l- ⁇ 4)-2-acetamido-2-deoxy-3-O-[l-(R)-carboxyethyl]- ⁇ -D-glucopyranoside VII.
• 2-Acetamido-2-deoxy- ⁇ -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.
• 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
• X is a peptide residue.
• reaction with benzyl ester of L-alanyl-D-isoglu- tamine affords benzyl ester of iV- ⁇ 2-O-[benzyl-2-acetamido- 2-deoxy- ⁇ -D-glucopyranosyl-(l- ⁇ 4)-2-acetamido-2,3-dideoxy- ⁇ -D-glucopyranosid-3-yl]-(R)- lactoyl ⁇ -L-alanyl-D-isoglutamine (XI wherein X is L-Ala-D-iso-Gln-OBn).
• 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.
• 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]- ⁇ -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.
• Benzyl-2-acetamido-2-deoxy-4,6-(9-isopropyliden-3-0-[l-(R)-(methoxycarbonyl)- ethyl]- ⁇ -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.
• 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.
• 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 0 C 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 0 C. 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.
• Acetic acid anhydride (20 ml) is stepwise added to a stirred benzyl-2-amino-2-deoxy- ⁇ - D-glucopyranosyl-(l- ⁇ 4)-2-acetamido-2-deoxy-3-O-[l-(R)-carboxyethyl- ⁇ -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).
• Ben2yl-2-acetamido-2-deoxy- ⁇ -D-glucopyranosyl-(l— >4)-2-acetamido-2-deoxy-3-O- [l-(R)-carboxyethyl]- ⁇ -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 compounds of the present invention can be used in basic research, pharmaceutical industry and both in human and veterinary medicine.
• Azuma L Vaccine 1992, 10, 1000.
• Garegg PJ. Advances in Carbohydrate Chemistry and Biochemistry, VoI 52 1997, 52, 179.

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• 2005
  • 2005-10-18 CZ CZ20050658A patent/CZ301451B6/cs not_active IP Right Cessation
• 2006
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